A source of artifact in the lacZ reversion assay in Escherichia coli.

PubMed

Hoffmann, George R; Gray, Carol L; Lange, Paulina B; Marando, Christie I

2015-06-01

The lacZ reversion assay in Escherichia coli measures point mutations that occur by specific base substitutions and frameshift mutations. The tester strains cannot use lactose as a carbon source (Lac(-)), and revertants are easily detected by growth on lactose medium (Lac(+)). Six strains identify the six possible base substitutions, and five strains measure +G, -G, -CG, +A and -A frameshifts. Strong mutagens give dose-dependent increases in numbers of revertants per plate and revertant frequencies. Testing compounds that are arguably nonmutagens or weakly mutagenic, we often noted statistically significant dose-dependent increases in revertant frequency that were not accompanied by an absolute increase in numbers of revertants. The increase in frequency was wholly ascribable to a declining number of viable cells owing to toxicity. Analysis of the conditions revealed that the frequency of spontaneous revertants is higher when there are fewer viable cells per plate. The phenomenon resembles "adaptive" or "stress" mutagenesis, whereby lactose revertants accumulate in Lac(-) bacteria under starvation conditions in the absence of catabolite repression. Adaptive mutation is observed after long incubation and might be expected to be irrelevant in a standard assay using 48-h incubation. However, we found that elevated revertant frequencies occur under typical assay conditions when the bacterial lawn is thin, and this can cause increases in revertant frequency that mimic chemical mutagenesis when treatments are toxic but not mutagenic. Responses that resemble chemical mutagenesis were observed in the absence of mutagenic treatment in strains that revert by different frameshift mutations. The magnitude of the artifact is affected by cell density, dilution, culture age, incubation time, catabolite repression and the age and composition of media. Although the specific reversion assay is effective for quickly distinguishing classes of mutations induced by potent mutagens, its utility for discerning effects of weak mutagens may be compromised by the artifact. Copyright © 2015 Elsevier B.V. All rights reserved.

The in vitro and in vivo genotoxicity of benzocaine: a brief communication.

PubMed

Brock, William J; Bell, Thomas A

2012-06-01

Benzocaine has a long history of use in human medicine. However, benzocaine also has been used in aquaculture with finfish for more than 40 years for sedating fish for marking, transport, surgery, and so on, although benzocaine does not have a current Food and Drug Administration (FDA) approval for this application in the United States. As part of a FDA approval for use as an animal drug, the genotoxicity of benzocaine was evaluated in the in vitro bacterial reverse mutation assay and the forward mutation assay and in vivo in the mouse micronucleus assay. These studies were conducted in compliance with Good Laboratory Practice regulations and according to Veterinary International Conference on Harmonisation guidelines. Based on the results of these studies, benzocaine was determined not to be genotoxic.

A Conservative Amino Acid Mutation in the Master Regulator FleQ Renders Pseudomonas aeruginosa Aflagellate

PubMed Central

Jain, Ruchi; Kazmierczak, Barbara I.

2014-01-01

Flagellar-based motility plays a critical role in Pseudomonas aeruginosa pathogenesis, influencing both the establishment of bacterial infection and the host's response to the pathogen. Nonetheless, aflagellate clinical strains are often isolated from acutely and chronically infected patients and include the virulent laboratory strain PA103. We determined that PA103's aflagellate phenotype is the result of a single amino acid change (G240V) in the master flagellar regulator, FleQ. This mutation, which lies just outside the Walker B box of FleQ, abrogates the ability of FleQ to positively regulate flagellar gene expression. Reversal of this seemingly conservative amino acid substitution is sufficient to restore swimming motility to PA103, despite the presence of mutations in other flagellar genes of PA103. We also investigated the consequences of restoring flagellar assembly on PA103 virulence. Although a negative correlation between flagellar assembly and Type 3 secretion system (T3SS) expression has been reported previously, we did not observe downregulation of T3SS expression or function in Fla+ PA103. Restoration of flagellar assembly did, however, amplify IL-1 signals measured during murine pulmonary infection and was associated with increased bacterial clearance. These experiments suggest that loss of flagellar motility may primarily benefit PA103 by attenuating pathogen recognition and clearance during acute infection. PMID:24827992

Bioleaching of Arsenic-Rich Gold Concentrates by Bacterial Flora before and after Mutation

PubMed Central

Xie, Xuehui; Yuan, Xuewu; Liu, Na; Chen, Xiaoguang; Abdelgadir, Awad; Liu, Jianshe

2013-01-01

In order to improve the bioleaching efficiency of arsenic-rich gold concentrates, a mixed bacterial flora had been developed, and the mutation breeding method was adopted to conduct the research. The original mixed bacterial flora had been enrichedin acid mine drainage of Dexing copper mine, Jiangxi Province, China. It was induced by UV (ultraviolet), ultrasonic, and microwave, and their combination mutation. The most efficient bacterial flora after mutation was collected for further bioleaching of arsenic-rich gold concentrates. Results indicated that the bacterial flora after mutation by UV 60 s combined with ultrasonic 10 min had the best oxidation rate of ferrous, the biggest density of cells, and the most activity of total protein. During bioleaching of arsenic-rich gold concentrates, the density of the mutant bacterial cells reached to 1.13 × 108 cells/mL at 15 days, more than 10 times compared with that of the original culture. The extraction of iron reached to 95.7% after 15 days, increased by 9.9% compared with that of the original culture. The extraction of arsenic reached to 92.6% after 12 days, which was increased by 46.1%. These results suggested that optimum combined mutation could improve leaching ability of the bacterial flora more significantly. PMID:24381948

Practical aspects of mutagenicity testing strategy: an industrial perspective.

PubMed

Gollapudi, B B; Krishna, G

2000-11-20

Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon.

Genotoxicity assessment of some cosmetic and food additives.

PubMed

Di Sotto, Antonella; Maffei, Francesca; Hrelia, Patrizia; Di Giacomo, Silvia; Pagano, Ester; Borrelli, Francesca; Mazzanti, Gabriela

2014-02-01

α-Hexylcinnamaldehyde (HCA) and p-tert-butyl-alpha-methylhydrocinnamic aldehyde (BMHCA) are synthetic aldehydes, characterized by a typical floral scent, which makes them suitable to be used as fragrances in personal care (perfumes, creams, shampoos, etc.) and household products, and as flavouring additives in food and pharmaceutical industry. The aldehydic structure suggests the need for a safety assessment for these compounds. Here, HCA and BMHCA were evaluated for their potential genotoxic risk, both at gene level (frameshift or base-substitution mutations) by the bacterial reverse mutation assay (Ames test), and at chromosomal level (clastogenicity and aneuploidy) by the micronucleus test. In order to evaluate a primary and repairable DNA damage, the comet assay has been also included. In spite of their potential hazardous chemical structure, a lack of mutagenicity was observed for both compounds in all bacterial strains tested, also in presence of the exogenous metabolic activator, showing that no genotoxic derivatives were produced by CYP450-mediated biotransformations. Neither genotoxicity at chromosomal level (i.e. clastogenicity or aneuploidy) nor single-strand breaks were observed. These findings will be useful in further assessing the safety of HCA and BMHCA as either flavour or fragrance chemicals. Copyright © 2013 Elsevier Inc. All rights reserved.

Highlights of the DNA cutters: a short history of the restriction enzymes

PubMed Central

Loenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.; Murray, Noreen E.

2014-01-01

In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine. PMID:24141096

Highlights of the DNA cutters: a short history of the restriction enzymes.

PubMed

Loenen, Wil A M; Dryden, David T F; Raleigh, Elisabeth A; Wilson, Geoffrey G; Murray, Noreen E

2014-01-01

In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.

Live Attenuated Influenza Vaccine Enhances Colonization of Streptococcus pneumoniae and Staphylococcus aureus in Mice

PubMed Central

Mina, Michael J.; McCullers, Jonathan A.; Klugman, Keith P.

2014-01-01

ABSTRACT Community interactions at mucosal surfaces between viruses, like influenza virus, and respiratory bacterial pathogens are important contributors toward pathogenesis of bacterial disease. What has not been considered is the natural extension of these interactions to live attenuated immunizations, and in particular, live attenuated influenza vaccines (LAIVs). Using a mouse-adapted LAIV against influenza A (H3N2) virus carrying the same mutations as the human FluMist vaccine, we find that LAIV vaccination reverses normal bacterial clearance from the nasopharynx and significantly increases bacterial carriage densities of the clinically important bacterial pathogens Streptococcus pneumoniae (serotypes 19F and 7F) and Staphylococcus aureus (strains Newman and Wright) within the upper respiratory tract of mice. Vaccination with LAIV also resulted in 2- to 5-fold increases in mean durations of bacterial carriage. Furthermore, we show that the increases in carriage density and duration were nearly identical in all aspects to changes in bacterial colonizing dynamics following infection with wild-type (WT) influenza virus. Importantly, LAIV, unlike WT influenza viruses, had no effect on severe bacterial disease or mortality within the lower respiratory tract. Our findings are, to the best of our knowledge, the first to demonstrate that vaccination with a live attenuated viral vaccine can directly modulate colonizing dynamics of important and unrelated human bacterial pathogens, and does so in a manner highly analogous to that seen following wild-type virus infection. PMID:24549845

Evaluation of in vitro and in vivo genotoxicity of single-walled carbon nanotubes.

PubMed

Kim, Jin Sik; Song, Kyung Seuk; Yu, Il Je

2015-08-01

Single-walled carbon nanotubes (SWCNTs) have extensive potential industrial applications due to their unique physical and chemical properties; yet this also increases the chance of human and environment exposure to SWCNTs. Due to the current lack of hazardous effect information on SWNCTs, a standardized genotoxicity battery test was conducted to clarify the genetic toxicity potential of SWCNTs (diameter: 1-1.2 nm, length: ∼20 μm) according to Organization for Economic Cooperation and Development test guidelines 471 (bacterial reverse mutation test), 473 (in vitro chromosome aberration test), and 474 (in vivo micronuclei test) with a good laboratory practice system. The test results showed that the SWCNTs did not induce significant bacterial reverse mutations at 31.3-500 μg/plate in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 or in Escherichia coli strain WP2uvrA, with and without a metabolic activation system. Furthermore, the in vitro chromosome aberration test showed no significant increase in structural or numerical chromosome aberration frequencies at SWCNT dose levels of 12.5-50 μg/ml in the presence and absence of metabolic activation. However, dose-dependent cell growth inhibition was found at all the SWCNT dose levels and statistically significant cytotoxic effects observed at certain concentrations in the presence and absence of metabolic activation. Finally, the SWCNTs did not evoke significant in vivo micronuclei frequencies in the polychromatic erythrocytes of an imprinting control region mice at 25-100 mg/kg. Thus, according to the results of the present study, the SWCNTs were not found to have a genotoxic effect on the in vitro and in vivo test systems. © The Author(s) 2013.

Aspect ratio has no effect on genotoxicity of multi-wall carbon nanotubes.

PubMed

Kim, Jin Sik; Lee, Kyu; Lee, Young Hee; Cho, Hyun Sun; Kim, Ki Heon; Choi, Kyung Hee; Lee, Sang Hee; Song, Kyung Seuk; Kang, Chang Soo; Yu, Il Je

2011-07-01

Carbon nanotubes (CNTs) have specific physico-chemical and electrical properties that are useful for telecommunications, medicine, materials, manufacturing processes and the environmental and energy sectors. Yet, despite their many advantages, it is also important to determine whether CNTs may represent a hazard to the environment and human health. Like asbestos, the aspect ratio (length:diameter) and metal components of CNTs are known to have an effect on the toxicity of carbon nanotubes. Thus, to evaluate the toxic potential of CNTs in relation to their aspect ratio and metal contamination, in vivo and in vitro genotoxicity tests were conducted using high-aspect-ratio (diameter: 10-15 nm, length: ~10 μm) and low-aspect-ratio multi-wall carbon nanotubes (MWCNTs, diameter: 10-15 nm, length: ~150 nm) according to OECD test guidelines 471 (bacterial reverse mutation test), 473 (in vitro chromosome aberration test), and 474 (in vivo micronuclei test) with a good laboratory practice system. To determine the treatment concentration for all the tests, a solubility and dispersive test was performed, and a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) solution found to be more suitable than distilled water. Neither the high- nor the low-aspect-ratio MWCNTs induced any genotoxicity in a bacterial reverse mutation test (~1,000 μg/plate), in vitro chromosome aberration test (without S9: ~6.25 μg/ml, with S9: ~50 μg/ml), or in vivo micronuclei test (~50 mg/kg). However, the high-aspect-ratio MWCNTs were found to be more toxic than the low-aspect-ratio MWCNTs. Thus, while high-aspect-ratio MWCNTs do not induce direct genotoxicity or metabolic activation-mediated genotoxicity, genotoxicity could still be induced indirectly through oxidative stress or inflammation.

Reversible Congenital Hypogonadotropic Hypogonadism in Patients with CHD7, FGFR1 or GNRHR Mutations

PubMed Central

Laitinen, Eeva-Maria; Tommiska, Johanna; Sane, Timo; Vaaralahti, Kirsi; Toppari, Jorma; Raivio, Taneli

2012-01-01

Background Congenital hypogonadotropic hypogonadism (HH) is a rare cause for delayed or absent puberty. These patients may recover from HH spontaneously in adulthood. To date, it is not possible to predict who will undergo HH reversal later in life. Herein we investigated whether Finnish patients with reversal of congenital hypogonadotropic hypogonadism (HH) have common phenotypic or genotypic features. Methods and Findings Thirty-two male HH patients with anosmia/hyposmia (Kallmann Syndrome, KS; n = 26) or normal sense of smell (nHH; n = 6) were enrolled (age range, 18–61 yrs). The patients were clinically examined, and reversal of HH was assessed after treatment withdrawal. KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11, GNRHR, GNRH1, KISS1R, KISS1, TAC3, TACR3, and LHβ were screened for mutations. Six HH patients (2 KS, 4 nHH) were verified to have reversal of HH. In the majority of cases, reversal occurred early in adulthood (median age, 23 yrs; range, 21–39 yrs). All had spontaneous testicular growth while on testosterone replacement therapy (TRT). One nHH subject was restarted on TRT due to a decline in serum T. Two reversal variants had a same GNRHR mutation (R262Q), which was accompanied by another GNRHR mutation (R139H or del309F). In addition, both of the KS patients had a mutation in CHD7 (p.Q51X) or FGFR1 (c.91+2T>A). Conclusions Considerable proportion of patients with HH (8% of KS probands) may recover in early adulthood. Spontaneous testicular enlargement during TRT was highly suggestive for reversal of HH. Those with the GNRHR mutation R262Q accompanied by another GNRHR mutation may be prone to reversal, although even patients with a truncating mutation in CHD7 or a splice-site mutation in FGFR1 can recover. We recommend that all adolescents and young adults with congenital HH should be informed on the possibility of reversal. PMID:22724017

A Mutator Phenotype Promoting the Emergence of Spontaneous Oxidative Stress-Resistant Mutants in Campylobacter jejuni.

PubMed

Dai, Lei; Sahin, Orhan; Tang, Yizhi; Zhang, Qijing

2017-12-15

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerophilic organism, C. jejuni must be able to defend against oxidative stress encountered both in the host and in the environment. How Campylobacter utilizes a mutation-based mechanism for adaptation to oxidative stress is still unknown. Here we present a previously undescribed phenotypic and genetic mechanism that promotes the emergence of oxidative stress-resistant mutants. Specifically, we showed that a naturally occurring mutator phenotype, resulting from a loss of function mutation in the DNA repair enzyme MutY, increased oxidative stress resistance (OX R ) in C. jejuni We further demonstrated that MutY malfunction did not directly contribute to the OX R phenotype but increased the spontaneous mutation rate in the peroxide regulator gene perR , which functions as a repressor for multiple genes involved in oxidative stress resistance. Mutations in PerR resulted in loss of its DNA binding function and derepression of PerR-controlled oxidative stress defense genes, thereby conferring an OX R phenotype and facilitating Campylobacter survival under oxidative stress. These findings reveal a new mechanism that promotes the emergence of spontaneous OX R mutants in bacterial organisms. IMPORTANCE Although a mutator phenotype has been shown to promote antibiotic resistance in many bacterial species, little is known about its contribution to the emergence of OX R mutants. This work describes the link between a mutator phenotype and the enhanced emergence of OX R mutants as well as its underlying mechanism involving DNA repair and mutations in PerR. Since DNA repair systems and PerR are well conserved in many bacterial species, especially in Gram positives, the same mechanism may operate in multiple bacterial species. Additionally, we developed a novel method that allows for rapid quantification of spontaneous OX R mutants in a bacterial population. This method represents a technical innovation and may also be applied to other bacterial species. These findings significantly advance our understanding of bacterial mechanisms for survival under oxidative stress. Copyright © 2017 American Society for Microbiology.

Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.

PubMed Central

Kögel, D; Aboud, M; Flügel, R M

1995-01-01

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. Images PMID:7544460

Coexistence of BRAF V600E and TERT Promoter Mutations in Low-grade Serous Carcinoma of Ovary Recurring as Carcinosarcoma in a Lymph Node: Report of a Case.

PubMed

Tavallaee, Mahkam; Steiner, David F; Zehnder, James L; Folkins, Ann K; Karam, Amer K

2018-04-03

Low-grade serous carcinomas only rarely coexist with or progress to high-grade tumors. We present a case of low-grade serous carcinoma with transformation to carcinosarcoma on recurrence in the lymph node. Identical BRAF V600E and telomerase reverse transcriptase promoter mutations were identified in both the original and recurrent tumor. Given that telomerase reverse transcriptase promotor mutations are thought to play a role in progression of other tumor types, the function of telomerase reverse transcriptase mutations in BRAF mutated low-grade serous carcinoma deserves investigation.

40 CFR 79.53 - Tier 2.

Code of Federal Regulations, 2013 CFR

2013-07-01

..., Salmonella typhimurium Reverse Mutation Assay). (b) Manufacturer determination. Manufacturers shall determine... Sister Chromatid Exchange Assay, and § 79.68 Salmonella typhimurium Reverse Mutation Assay. Teratogenic...

40 CFR 79.53 - Tier 2.

Code of Federal Regulations, 2012 CFR

2012-07-01

..., Salmonella typhimurium Reverse Mutation Assay). (b) Manufacturer Determination. Manufacturers shall determine... Sister Chromatid Exchange Assay, and § 79.68 Salmonella typhimurium Reverse Mutation Assay. Teratogenic...

40 CFR 79.53 - Tier 2.

Code of Federal Regulations, 2011 CFR

2011-07-01

..., Salmonella typhimurium Reverse Mutation Assay). (b) Manufacturer Determination. Manufacturers shall determine... Sister Chromatid Exchange Assay, and § 79.68 Salmonella typhimurium Reverse Mutation Assay. Teratogenic...

40 CFR 79.53 - Tier 2.

Code of Federal Regulations, 2014 CFR

2014-07-01

..., Salmonella typhimurium Reverse Mutation Assay). (b) Manufacturer Determination. Manufacturers shall determine... Sister Chromatid Exchange Assay, and § 79.68 Salmonella typhimurium Reverse Mutation Assay. Teratogenic...

40 CFR 79.53 - Tier 2.

Code of Federal Regulations, 2010 CFR

2010-07-01

..., Salmonella typhimurium Reverse Mutation Assay). (b) Manufacturer Determination. Manufacturers shall determine... Sister Chromatid Exchange Assay, and § 79.68 Salmonella typhimurium Reverse Mutation Assay. Teratogenic...

Gamma-irradiated bacterial preparation having anti-tumor activity

DOEpatents

Vass, Arpad A.; Tyndall, Richard L.; Terzaghi-Howe, Peggy

1999-01-01

A bacterial preparation from Pseudomonas species isolated #15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

Bacterial mutation affecting plasmid maintenance in Pseudomonas aeruginosa.

PubMed Central

Chang, B J; Holloway, B W

1977-01-01

A bacterial mutation, risA, in Pseudomonas aeruginosa caused growth inhibition at 43 degrees C of risA strains containing P2 plasmids. Incubation at 43 degrees C resulted in selection for clones that had lost P2 plasmids. PMID:122513

Roles of s3 site residues of nattokinase on its activity and substrate specificity.

PubMed

Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

2007-09-01

Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

PubMed Central

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. The present article focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor-intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. PMID:28645368

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.

PubMed

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. This chapter focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here, we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the chimeric ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. © 2017 Elsevier Inc. All rights reserved.

Gamma-irradiated bacterial preparation having anti-tumor activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vass, A.A.; Tyndall, R.L.; Terzaghi-Howe, P.

1999-11-16

This application describes a bacterial preparation from Pseudomonas species isolated {number{underscore}sign}15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

Genotoxicity assessment of nanomaterials: recommendations on best practices, assays and methods.

PubMed

Elespuru, Rosalie; Pfuhler, Stefan; Aardema, Marilyn; Chen, Tao; Doak, Shareen H; Doherty, Ann; Farabaugh, Christopher S; Kenny, Julia; Manjanatha, Mugimane; Mahadevan, Brinda; Moore, Martha M; Ouédraogo, Gladys; Stankowski, Leon F; Tanir, Jennifer Y

2018-04-26

Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.

Trade-offs with stability modulate innate and mutationally acquired drug-resistance in bacterial dihydrofolate reductase enzymes.

PubMed

Matange, Nishad; Bodkhe, Swapnil; Patel, Maitri; Shah, Pooja

2018-06-05

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In this study, we show that trimethoprim-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harbouring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild type making them prone to aggregation and proteolysis. This destabilization is associated with lower expression level resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis we show that perturbation of critical stabilizing hydrophobic interactions in wild type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial DHFRs. Mutational and computational analyses in EcDHFR as well as in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues, modulates TMP-resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to trimethoprim. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme. ©2018 The Author(s).

Genetic Instability at the Agouti Locus of the Mouse (Mus Musculus). I. Increased Reverse Mutation Frequency to the A(w) Allele in a/a Heterozygotes

PubMed Central

Sandulache, R.; Neuhauser-Klaus, A.; Favor, J.

1994-01-01

We have compiled the reverse mutation rate data to the white bellied agouti (A(w)) allele in heterozygous A/a mice and shown it to be increased by a factor of at least 350 in comparison to the reverse mutation rate in homozygous a/a mice. Employing tightly linked flanking restriction fragment length polymorphism DNA markers, we have shown that reversion to A(w) is associated with crossing over in the vicinity of the agouti locus. The non-agouti (a) allele has been recently shown to contain an 11-kb insert within the first intron of the agouti gene. Together with our present results, these observations suggest possible mechanisms to explain the reversion events. PMID:7982562

Cell-Based Genotoxicity Testing

NASA Astrophysics Data System (ADS)

Reifferscheid, Georg; Buchinger, Sebastian

Genotoxicity test systems that are based on bacteria display an important role in the detection and assessment of DNA damaging chemicals. They belong to the basic line of test systems due to their easy realization, rapidness, broad applicability, high sensitivity and good reproducibility. Since the development of the Salmonella microsomal mutagenicity assay by Ames and coworkers in the early 1970s, significant development in bacterial genotoxicity assays was achieved and is still a subject matter of research. The basic principle of the mutagenicity assay is a reversion of a growth inhibited bacterial strain, e.g., due to auxotrophy, back to a fast growing phenotype (regain of prototrophy). Deeper knowledge of the ­mutation events allows a mechanistic understanding of the induced DNA-damage by the utilization of base specific tester strains. Collections of such specific tester strains were extended by genetic engineering. Beside the reversion assays, test systems utilizing the bacterial SOS-response were invented. These methods are based on the fusion of various SOS-responsive promoters with a broad variety of reporter genes facilitating numerous methods of signal detection. A very important aspect of genotoxicity testing is the bioactivation of ­xenobiotics to DNA-damaging compounds. Most widely used is the extracellular metabolic activation by making use of rodent liver homogenates. Again, genetic engineering allows the construction of highly sophisticated bacterial tester strains with significantly enhanced sensitivity due to overexpression of enzymes that are involved in the metabolism of xenobiotics. This provides mechanistic insights into the toxification and detoxification pathways of xenobiotics and helps explaining the chemical nature of hazardous substances in unknown mixtures. In summary, beginning with "natural" tester strains the rational design of bacteria led to highly specific and sensitive tools for a rapid, reliable and cost effective ­genotoxicity testing that is of outstanding importance in the risk assessment of compounds (REACH) and in ecotoxicology.

An alternative derivation of the stationary distribution of the multivariate neutral Wright-Fisher model for low mutation rates with a view to mutation rate estimation from site frequency data.

PubMed

Schrempf, Dominik; Hobolth, Asger

2017-04-01

Recently, Burden and Tang (2016) provided an analytical expression for the stationary distribution of the multivariate neutral Wright-Fisher model with low mutation rates. In this paper we present a simple, alternative derivation that illustrates the approximation. Our proof is based on the discrete multivariate boundary mutation model which has three key ingredients. First, the decoupled Moran model is used to describe genetic drift. Second, low mutation rates are assumed by limiting mutations to monomorphic states. Third, the mutation rate matrix is separated into a time-reversible part and a flux part, as suggested by Burden and Tang (2016). An application of our result to data from several great apes reveals that the assumption of stationarity may be inadequate or that other evolutionary forces like selection or biased gene conversion are acting. Furthermore we find that the model with a reversible mutation rate matrix provides a reasonably good fit to the data compared to the one with a non-reversible mutation rate matrix. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Risk of population extinction from fixation of deleterious and reverse mutations.

PubMed

Lande, R

1998-01-01

A model is developed for alternate fixations of mildly deleterious and wild-type alleles arising by forward and reverse mutation in a finite population. For almost all parameter values, this gives an equilibrium load that agrees closely with the general expression derived from diffusion theory. Nearly neutral mutations with selection coefficient a few times larger than 1/(2N(e)) do the most damage by increasing the equilibrium load. The model of alternate fixations facilitates dynamical analysis of the expected load and the mean time to extinction in a population that has been suddenly reduced from a very large size to a small size. Reverse mutation can substantially improve population viability, increasing the mean time to extinction by an order of magnitude or more, but because many mutations are irreversible the effects may not be large. Populations with initially high mean fitness and small effective size, N(e) below a few hundred individuals, may be at serious risk of extinction from fixation of deleterious mutations within 10(3) to 10(4) generations.

Transgenic expression of Map3k4 rescues T-associated sex reversal (Tas) in mice

PubMed Central

Warr, Nick; Siggers, Pam; Carré, Gwenn-Aël; Bogani, Debora; Brixey, Rachel; Akiyoshi, Mika; Tachibana, Makoto; Teboul, Lydia; Wells, Sara; Sanderson, Jeremy; Greenfield, Andy

2014-01-01

Disorders of sex development in the human population range in severity from mild genital defects to gonadal sex reversal. XY female development has been associated with heterozygous mutations in several genes, including SOX9, WT1 and MAP3K1. In contrast, XY sex reversal in mice usually requires complete absence of testis-determining gene products. One exception to this involves T-associated sex reversal (Tas), a phenomenon characterized by the formation of ovotestes or ovaries in XY mice hemizygous for the hairpin-tail (Thp) or T-Orleans (TOrl) deletions on proximal mouse chromosome 17. We recently reported that mice heterozygous for a null allele of Map3k4, which resides in the Thp deletion, exhibit XY ovotestis development and occasional gonadal sex reversal on the sensitized C57BL/6J-YAKR (B6-YAKR) genetic background, reminiscent of the Tas phenotype. However, these experiments did not exclude the possibility that loss of other loci in the Thp deletion, or other effects of the deletion itself, might contribute to Tas. Here, we show that disruption to Sry expression underlies XY gonadal defects in B6-YAKR embryos harbouring the Thp deletion and that a functional Map3k4 bacterial artificial chromosome rescues these abnormalities by re-establishing a normal Sry expression profile. These data demonstrate that Map3k4 haploinsufficiency is the cause of T-associated sex reversal and that levels of this signalling molecule are a major determinant of the expression profile of Sry. PMID:24452333

Mutation—The Engine of Evolution: Studying Mutation and Its Role in the Evolution of Bacteria

PubMed Central

Hershberg, Ruth

2015-01-01

Mutation is the engine of evolution in that it generates the genetic variation on which the evolutionary process depends. To understand the evolutionary process we must therefore characterize the rates and patterns of mutation. Starting with the seminal Luria and Delbruck fluctuation experiments in 1943, studies utilizing a variety of approaches have revealed much about mutation rates and patterns and about how these may vary between different bacterial strains and species along the chromosome and between different growth conditions. This work provides a critical overview of the results and conclusions drawn from these studies, of the debate surrounding some of these conclusions, and of the challenges faced when studying mutation and its role in bacterial evolution. PMID:26330518

CRISPR: a Versatile Tool for Both Forward and Reverse Genetics Research

PubMed Central

Gurumurthy, Channabasavaiah B.; Grati, M'hamed; Ohtsuka, Masato; Schilit, Samantha L.P.; Quadros, Rolen M.; Liu, Xue Zhong

2016-01-01

Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples. PMID:27384229

Role of Reverse Divalent Cation Diffusion in Forward Osmosis Biofouling.

PubMed

Xie, Ming; Bar-Zeev, Edo; Hashmi, Sara M; Nghiem, Long D; Elimelech, Menachem

2015-11-17

We investigated the role of reverse divalent cation diffusion in forward osmosis (FO) biofouling. FO biofouling by Pseudomonas aeruginosa was simulated using pristine and chlorine-treated thin-film composite polyamide membranes with either MgCl2 or CaCl2 draw solution. We related FO biofouling behavior-water flux decline, biofilm architecture, and biofilm composition-to reverse cation diffusion. Experimental results demonstrated that reverse calcium diffusion led to significantly more severe water flux decline in comparison with reverse magnesium permeation. Unlike magnesium, reverse calcium permeation dramatically altered the biofilm architecture and composition, where extracellular polymeric substances (EPS) formed a thicker, denser, and more stable biofilm. We propose that FO biofouling was enhanced by complexation of calcium ions to bacterial EPS. This hypothesis was confirmed by dynamic and static light scattering measurements using extracted bacterial EPS with the addition of either MgCl2 or CaCl2 solution. We observed a dramatic increase in the hydrodynamic radius of bacterial EPS with the addition of CaCl2, but no change was observed after addition of MgCl2. Static light scattering revealed that the radius of gyration of bacterial EPS with addition of CaCl2 was 20 times larger than that with the addition of MgCl2. These observations were further confirmed by transmission electron microscopy imaging, where bacterial EPS in the presence of calcium ions was globular, while that with magnesium ions was rod-shaped.

The microbiology of mutability.

PubMed

Sundin, George W; Weigand, Michael R

2007-12-01

Bacteria possessing elevated spontaneous mutation rates are prevalent in certain environments, which is a paradox because most mutations are deleterious. For example, cells with defects in the methyl-directed mismatch repair (MMR) system, termed mutators or hypermutators, are overrepresented in populations of bacterial pathogens, with the mutator trait hypothesized to be advantageous in the changing host enviroments faced during colonization and establishment of chronic infections. Error-prone DNA polymerases, such as polIV and polV, function in translesion DNA synthesis, a DNA damage response that ensures genome integrity with a cost of increased mutation. While the biochemical aspects of these mutability pathways are well understood, the biological impacts have received less attention. Here, an examination of bacterial mutability systems and specifically the ecological and evolutionary context resulting in the selection of these systems is carried out.

A pivot mutation impedes reverse evolution across an adaptive landscape for drug resistance in Plasmodium vivax.

PubMed

Ogbunugafor, C Brandon; Hartl, Daniel

2016-01-25

The study of reverse evolution from resistant to susceptible phenotypes can reveal constraints on biological evolution, a topic for which evolutionary theory has relatively few general principles. The public health catastrophe of antimicrobial resistance in malaria has brought these constraints on evolution into a practical realm, with one proposed solution: withdrawing anti-malarial medication use in high resistance settings, built on the assumption that reverse evolution occurs readily enough that populations of pathogens may revert to their susceptible states. While past studies have suggested limits to reverse evolution, there have been few attempts to properly dissect its mechanistic constraints. Growth rates were determined from empirical data on the growth and resistance from a set of combinatorially complete set of mutants of a resistance protein (dihydrofolate reductase) in Plasmodium vivax, to construct reverse evolution trajectories. The fitness effects of individual mutations were calculated as a function of drug environment, revealing the magnitude of epistatic interactions between mutations and genetic backgrounds. Evolution across the landscape was simulated in two settings: starting from the population fixed for the quadruple mutant, and from a polymorphic population evenly distributed between double mutants. A single mutation of large effect (S117N) serves as a pivot point for evolution to high resistance regions of the landscape. Through epistatic interactions with other mutations, this pivot creates an epistatic ratchet against reverse evolution towards the wild type ancestor, even in environments where the wild type is the most fit of all genotypes. This pivot mutation underlies the directional bias in evolution across the landscape, where evolution towards the ancestor is precluded across all examined drug concentrations from various starting points in the landscape. The presence of pivot mutations can dictate dynamics of evolution across adaptive landscape through epistatic interactions within a protein, leaving a population trapped on local fitness peaks in an adaptive landscape, unable to locate ancestral genotypes. This irreversibility suggests that the structure of an adaptive landscape for a resistance protein should be understood before considering resistance management strategies. This proposed mechanism for constraints on reverse evolution corroborates evidence from the field indicating that phenotypic reversal often occurs via compensatory mutation at sites independent of those associated with the forward evolution of resistance. Because of this, molecular methods that identify resistance patterns via single SNPs in resistance-associated markers might be missing signals for resistance and compensatory mutation throughout the genome. In these settings, whole genome sequencing efforts should be used to identify resistance patterns, and will likely reveal a more complicated genomic signature for resistance and susceptibility, especially in settings where anti-malarial medications have been used intermittently. Lastly, the findings suggest that, given their role in dictating the dynamics of evolution across the landscape, pivot mutations might serve as future targets for therapy.

Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing.

PubMed

Zhou, Katherine I; Clark, Wesley C; Pan, David W; Eckwahl, Matthew J; Dai, Qing; Pan, Tao

2018-05-11

The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

Mutagenicity and genotoxicity studies of aspartame.

PubMed

Otabe, Akira; Ohta, Fumio; Takumi, Asuka; Lynch, Barry

2018-02-08

Two studies were conducted to further assess its mutagenic and genotoxic potential. In a bacterial reverse mutation pre-incubation study, Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2 uvrA were treated with aspartame at concentrations of up to 5000 μg/plate with or without metabolic activation and showed no mutagenic potential. Similarly, in vivo micronucleus testing of aspartame following gavage administration (500-2000 mg/kg body weight) to Crlj:CD1(ICR) strain SPF male mice showed no increase in the proportion of micronucleated polychromatic erythrocytes in bone marrow cells collected and evaluated 24 or 48 h post administration. Overall, aspartame had no potential for mutagenic or genotoxic activity. Copyright © 2018 Elsevier Inc. All rights reserved.

HIV type 1 genotypic variation in an antiretroviral treatment-naive population in southern India.

PubMed

Balakrishnan, Pachamuthu; Kumarasamy, Nagalingeswaran; Kantor, Rami; Solomon, Suniti; Vidya, Sundararajan; Mayer, Kenneth H; Newstein, Michael; Thyagarajan, Sadras P; Katzenstein, David; Ramratnam, Bharat

2005-04-01

Most studies of HIV-1 drug resistance have examined subtype B viruses; fewer data are available from developing countries, where non-B subtypes predominate. We determined the prevalence of mutations at protease and reverse transcriptase drug resistance positions in antiretroviral drug-naive individuals in southern India. The pol region of the genome was amplified from plasma HIV-1 RNA in 50 patients. All sequences clustered with HIV-1 subtype C. All patients had at least one protease and/or RT mutation at a known subtype B drug resistance position. Twenty percent of patients had mutations at major protease inhibitor resistance positions and 100% had mutations at minor protease inhibitor resistance positions. Six percent and 14% of patients had mutations at nucleoside reverse transcriptase inhibitor and/or nonnucleoside reverse transcriptase inhibitor resistance positions, respectively. Larger scale studies need to be undertaken to better define the genotypic variation of circulating Indian subtype C viruses and their potential impact on drug susceptibility and clinical outcome in treated individuals.

Structure-based methods to predict mutational resistance to diarylpyrimidine non-nucleoside reverse transcriptase inhibitors.

PubMed

Azeem, Syeda Maryam; Muwonge, Alecia N; Thakkar, Nehaben; Lam, Kristina W; Frey, Kathleen M

2018-01-01

Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a leading cause of HIV treatment failure. Often included in antiviral therapy, NNRTIs are chemically diverse compounds that bind an allosteric pocket of enzyme target reverse transcriptase (RT). Several new NNRTIs incorporate flexibility in order to compensate for lost interactions with amino acid conferring mutations in RT. Unfortunately, even successful inhibitors such as diarylpyrimidine (DAPY) inhibitor rilpivirine are affected by mutations in RT that confer resistance. In order to aid drug design efforts, it would be efficient and cost effective to pre-evaluate NNRTI compounds in development using a structure-based computational approach. As proof of concept, we applied a residue scan and molecular dynamics strategy using RT crystal structures to predict mutations that confer resistance to DAPYs rilpivirine, etravirine, and investigational microbicide dapivirine. Our predictive values, changes in affinity and stability, are correlative with fold-resistance data for several RT mutants. Consistent with previous studies, mutation K101P is predicted to confer high-level resistance to DAPYs. These findings were further validated using structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results confirm that changes in affinity and stability for mutant complexes are predictive parameters of resistance as validated by experimental and clinical data. In future work, we believe that this computational approach may be useful to predict resistance mutations for inhibitors in development. Published by Elsevier Inc.

Use of bacterial artificial chromosomes in generating targeted mutations in human and mouse cytomegaloviruses.

PubMed

Borst, Eva Maria; Benkartek, Corinna; Messerle, Martin

2007-05-01

Cloning of cytomegalovirus (CMV) genomes as bacterial artificial chromosomes (BAC) in E. coli and their manipulation using the techniques of bacterial genetics has greatly facilitated the construction of CMV mutants. This unit describes easily applicable procedures that allow rapid introduction of any kind of targeted mutation into BAC-cloned CMV genomes. Protocols for the reconstitution of virus from isolated BAC DNA, preparation of a virus stock, and isolation and characterization of viral DNA are also included. Special emphasis is laid on description of critical steps and thorough characterization of the altered BACs.

Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii

USDA-ARS?s Scientific Manuscript database

Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2) using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/ CRISPR-associated) hom...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryo, H.; Yoo, M.A.; Fujikawa, K.

Somatic reversion of strains with the ivory (wi) allele, a mutation associated with a tandem duplication of a DNA sequence at the white locus, increased with the age of larvae at the time of X-irradiation as expected from the increase in the number of target cells. In contrast, two independently isolated strains with unstable w+ loci associated with insertion of transposable elements showed higher reversion frequencies after treatment with X rays or ethyl methanesulfonate (EMS) at early larval stages than at late stages. Nevertheless, both the wi strain and the two unstable w+ strains reverted at nearly equal rates aftermore » treatment with X rays or EMS at early larval stages. Possible similarity in hot spot structure for the high reversibility of the two types of mutations is discussed in relation to production of presumed mutator-type cofactors specific to the transposon-caused mutations at early larval stages.« less

An Examination of Adaptive Reversion in Saccharomyces Cerevisiae

PubMed Central

Steele, D. F.; Jinks-Robertson, S.

1992-01-01

Reversion to Lys(+) prototrophy in a haploid yeast strain containing a defined lys2 frameshift mutation has been examined. When cells were plated on synthetic complete medium lacking only lysine, the numbers of Lys(+) revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. An examination of the distribution of the numbers of early appearing Lys(+) colonies from independent cultures suggests that the mutations to prototrophy occurred randomly during nonselective growth. In contrast, an examination of the distribution of late appearing Lys(+) colonies indicates that the underlying reversion events occurred after selective plating. No accumulation of Lys(+) revertants occurred when cells were starved for tryptophan, leucine or both lysine and tryptophan prior to plating selectively for Lys(+) revertants. These results indicate that mutations accumulate more frequently when they confer a selective advantage, and are thus consistent with the occurrence of adaptive mutations in yeast. PMID:1398066

Reverse Engineering Field Isolates of Myxoma Virus Demonstrates that Some Gene Disruptions or Losses of Function Do Not Explain Virulence Changes Observed in the Field

PubMed Central

Liu, June; Cattadori, Isabella M.; Sim, Derek G.; Eden, John-Sebastian; Read, Andrew F.

2017-01-01

ABSTRACT The coevolution of myxoma virus (MYXV) and wild European rabbits in Australia and Europe is a paradigm for the evolution of a pathogen in a new host species. Genomic analyses have identified the mutations that have characterized this evolutionary process, but defining causal mutations in the pathways from virulence to attenuation and back to virulence has not been possible. Using reverse genetics, we examined the roles of six selected mutations found in Australian field isolates of MYXV that fall in known or potential virulence genes. Several of these mutations occurred in genes previously identified as virulence genes in whole-gene knockout studies. Strikingly, no single or double mutation among the mutations tested had an appreciable impact on virulence. This suggests either that virulence evolution was defined by amino acid changes other than those analyzed here or that combinations of multiple mutations, possibly involving epistatic interactions or noncoding sequences, have been critical in the ongoing evolution of MYXV virulence. In sum, our results show that single-gene knockout studies of a progenitor virus can have little power to predict the impact of individual mutations seen in the field. The genetic determinants responsible for this canonical case of virulence evolution remain to be determined. IMPORTANCE The species jump of myxoma virus (MYXV) from the South American tapeti to the European rabbit populations of Australia and Europe is a canonical example of host-pathogen coevolution. Detailed molecular studies have identified multiple genes in MYXV that are critical for virulence, and genome sequencing has revealed the evolutionary history of MYXV in Australia and Europe. However, it has not been possible to categorically identify the key mutations responsible for the attenuation of or reversion to virulence during this evolutionary process. Here we use reverse genetics to examine the role of mutations in viruses isolated early and late in the Australian radiation of MYXV. Surprisingly, none of the candidate mutations that we identified as likely having roles in attenuation proved to be important for virulence. This indicates that considerable caution is warranted when interpreting the possible role of individual mutations during virulence evolution. PMID:28768866

Genetic stability of Rift Valley fever virus MP-12 vaccine during serial passages in culture cells.

PubMed

Lokugamage, Nandadeva; Ikegami, Tetsuro

2017-01-01

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa which affects both ruminants and humans. RVF causes serious damage to the livestock industry and is also a threat to public health. The Rift Valley fever virus has a segmented negative-stranded RNA genome consisting of Large (L)-, Medium (M)-, and Small (S)-segments. The live-attenuated MP-12 vaccine is immunogenic in livestock and humans, and is conditionally licensed for veterinary use in the U.S. The MP-12 strain encodes 23 mutations (nine amino acid substitutions) and is attenuated through a combination of mutations in the L-, M-, and S-segments. Among them, the M-U795C, M-A3564G, and L-G3104A mutations contribute to viral attenuation through the L- and M-segments. The M-U795C, M-A3564G, L-U533C, and L-G3750A mutations are also independently responsible for temperature-sensitive (ts) phenotype. We hypothesized that a serial passage of the MP-12 vaccine in culture cells causes reversions of the MP-12 genome. The MP-12 vaccine and recombinant rMP12-ΔNSs16/198 were serially passaged 25 times. Droplet digital PCR analysis revealed that the reversion occurred at L-G3750A during passages of MP-12 in Vero or MRC-5 cells. The reversion also occurred at M-A3564G and L-U533C of rMP12-ΔNSs16/198 in Vero cells. Reversion mutations were not found in MP-12 or the variant, rMP12-TOSNSs, in the brains of mice with encephalitis. This study characterized genetic stability of the MP-12 vaccine and the potential risk of reversion mutation at the L-G3750A ts mutation after excessive viral passages in culture cells.

Persistent induction of somatic reversions of the pink-eyed unstable mutation in F1 mice born to fathers irradiated at the spermatozoa stage.

PubMed

Shiraishi, Kazunori; Shimura, Tsutomu; Taga, Masataka; Uematsu, Norio; Gondo, Yoichi; Ohtaki, Megu; Kominami, Ryo; Niwa, Ohtsura

2002-06-01

Untargeted mutation and delayed mutation are features of radiation-induced genomic instability and have been studied extensively in tissue culture cells. The mouse pink-eyed unstable (p(un)) mutation is due to an intragenic duplication of the pink-eyed dilution locus and frequently reverts back to the wild type in germ cells as well as in somatic cells. The reversion event can be detected in the retinal pigment epithelium as a cluster of pigmented cells (eye spot). We have investigated the reversion p(um) in F1 mice born to irradiated males. Spermatogonia-stage irradiation did not affect the frequency of the reversion in F1 mice. However, 6 Gy irradiation at the spermatozoa stage resulted in an approximately twofold increase in the number of eye spots in the retinal pigment epithelium of F1 mice. Somatic reversion occurred for the paternally derived p(un) alleles. In addition, the reversion also occurred for the maternally derived, unirradiated p(un) alleles at a frequency equal to that for the paternally derived allele. Detailed analyses of the number of pigmented cells per eye spot indicated that the frequency of reversion was persistently elevated during the proliferation cycle of the cells in the retinal pigment epithelium when the male parents were irradiated at the spermatozoa stage. The present study demonstrates the presence of a long-lasting memory of DNA damage and the persistent up-regulation of recombinogenic activity in the retinal pigment epithelium of the developing fetus.

De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.

PubMed

Hamilton, P J; Campbell, N G; Sharma, S; Erreger, K; Herborg Hansen, F; Saunders, C; Belovich, A N; Sahai, M A; Cook, E H; Gether, U; McHaourab, H S; Matthies, H J G; Sutcliffe, J S; Galli, A

2013-12-01

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

Stabilizing multicellularity through ratcheting

PubMed Central

Libby, Eric; Conlin, Peter L.; Kerr, Ben; Ratcliff, William C.

2016-01-01

The evolutionary transition to multicellularity probably began with the formation of simple undifferentiated cellular groups. Such groups evolve readily in diverse lineages of extant unicellular taxa, suggesting that there are few genetic barriers to this first key step. This may act as a double-edged sword: labile transitions between unicellular and multicellular states may facilitate the evolution of simple multicellularity, but reversion to a unicellular state may inhibit the evolution of increased complexity. In this paper, we examine how multicellular adaptations can act as evolutionary ‘ratchets’, limiting the potential for reversion to unicellularity. We consider a nascent multicellular lineage growing in an environment that varies between favouring multicellularity and favouring unicellularity. The first type of ratcheting mutations increase cell-level fitness in a multicellular context but are costly in a single-celled context, reducing the fitness of revertants. The second type of ratcheting mutations directly decrease the probability that a mutation will result in reversion (either as a pleiotropic consequence or via direct modification of switch rates). We show that both types of ratcheting mutations act to stabilize the multicellular state. We also identify synergistic effects between the two types of ratcheting mutations in which the presence of one creates the selective conditions favouring the other. Ratcheting mutations may play a key role in diverse evolutionary transitions in individuality, sustaining selection on the new higher-level organism by constraining evolutionary reversion. This article is part of the themed issue ‘The major synthetic evolutionary transitions’. PMID:27431522

Silent mutations at codons 65 and 66 in reverse transcriptase alleviate indel formation and restore fitness in subtype B HIV-1 containing D67N and K70R drug resistance mutations

PubMed Central

Telwatte, Sushama; Hearps, Anna C.; Johnson, Adam; Latham, Catherine F.; Moore, Katie; Agius, Paul; Tachedjian, Mary; Sonza, Secondo; Sluis-Cremer, Nicolas; Harrigan, P. Richard; Tachedjian, Gilda

2015-01-01

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or ‘silent’ mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65–67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65–67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses. PMID:25765644

Biological safety concepts of genetically modified live bacterial vaccines.

PubMed

Frey, Joachim

2007-07-26

Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis- or trans-complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.

Clavibacter michiganensis ssp. michiganensis: bacterial canker of tomato, molecular interactions and disease management.

PubMed

Nandi, Munmun; Macdonald, Jacqueline; Liu, Peng; Weselowski, Brian; Yuan, Ze-Chun

2018-03-12

Bacterial canker disease is considered to be one of the most destructive diseases of tomato (Solanum lycopersicum), and is caused by the seed-borne Gram-positive bacterium Clavibacter michiganensis ssp. michiganensis (Cmm). This vascular pathogen generally invades and proliferates in the xylem through natural openings or wounds, causing wilt and canker symptoms. The incidence of symptomless latent infections and the invasion of tomato seeds by Cmm are widespread. Pathogenicity is mediated by virulence factors and transcriptional regulators encoded by the chromosome and two natural plasmids. The virulence factors include serine proteases, cell wall-degrading enzymes (cellulases, xylanases, pectinases) and others. Mutational analyses of these genes and gene expression profiling (via quantitative reverse transcription-polymerase chain reaction, transcriptomics and proteomics) have begun to shed light on their roles in colonization and virulence, whereas the expression of tomato genes in response to Cmm infection suggests plant factors involved in the defence response. These findings may aid in the generation of target-specific bactericides or new resistant varieties of tomato. Meanwhile, various chemical and biological controls have been researched to control Cmm. This review presents a detailed investigation regarding the pathogen Cmm, bacterial canker infection, molecular interactions between Cmm and tomato, and current perspectives on improved disease management. © 2018 AGRICULTURE AND AGRI-FOOD CANADA. MOLECULAR PLANT PATHOLOGY © 2018 JOHN WILEY & SONS LTD.

Bacterial genes mutL, mutS, and dcm participate in repair of mismatches at 5-methylcytosine sites.

PubMed Central

Lieb, M

1987-01-01

Certain amber mutations in the cI gene of bacteriophage lambda appear to recombine very frequently with nearby mutations. The aberrant mutations included C-to-T transitions at the second cytosine in 5'CC(A/T)GG sequences (which are subject to methylation by bacterial cytosine methylase) and in 5'CCAG and 5'CAGG sequences. Excess cI+ recombinants arising in crosses that utilize these mutations are attributable to the correction of mismatches by a bacterial very-short-patch (VSP) mismatch repair system. In the present study I found that two genes required for methyladenine-directed (long-patch) mismatch repair, mutL and mutS, also functioned in VSP mismatch repair; mutH and mutU (uvrD) were dispensable. VSP mismatch repair was greatly reduced in a dcm Escherichia coli mutant, in which 5-methylcytosine was not methylated. However, mismatches in heteroduplexes prepared from lambda DNA lacking 5-methylcytosine were repaired in dcm+ bacteria. These results indicate that the product of gene dcm has a repair function in addition to its methylase activity. PMID:2959653

Rapid Evolution of Culture-Impaired Bacteria During Adaptation to Biofilm Growth

PubMed Central

Penterman, Jon; Nguyen, Dao; Anderson, Erin; Staudinger, Benjamin J.; Greenberg, Everett P.; Lam, Joseph S.; Singh, Pradeep K.

2014-01-01

Summary Biofilm growth increases the fitness of bacteria in harsh conditions. However, bacteria from clinical and environmental biofilms can exhibit impaired growth in culture, even when the species involved are readily cultureable, and permissive conditions are used. Here we show that culture-impaired variants of Pseudomonas aeruginosa rapidly and abundantly evolve in laboratory biofilms. The culture-impaired phenotype is caused by mutations that alter the outer-membrane lipopolysaccharide structure. Within biofilms, the lipopolysaccharide mutations markedly increase bacterial fitness. However, outside the protected biofilm environment, the mutations sensitize the variants to killing by a self-produced antimicrobial agent. Thus, a biofilm-mediated adaptation produces a stark fitness trade off that compromises bacterial survival in culture. Trade offs like this could limit the ability of bacteria to transition between biofilm growth and the free-living state, and produce bacterial populations that escape detection by culture-based sampling. PMID:24412364

CRISPR/Cas9 Editing of the Bacillus subtilis Genome

PubMed Central

Burby, Peter E.; Simmons, Lyle A.

2017-01-01

A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with unparalleled facility. CRISPR/Cas9 has allowed for genome edits to be more precise, while also increasing the efficiency of transferring mutations into a variety of genetic backgrounds. As a result, the advantages are realized in tractable organisms and organisms that have been refractory to genetic manipulation. Here, we describe our method for editing the genome of the bacterium Bacillus subtilis. Our method is highly efficient, resulting in precise, markerless mutations. Further, after generating the editing plasmid, the mutation can be quickly introduced into several genetic backgrounds, greatly increasing the speed with which genetic analyses may be performed. PMID:28706963

Genotoxicity profile of erinacine A-enriched Hericium erinaceus mycelium.

PubMed

Li, I-Chen; Chen, Yen-Lien; Chen, Wan-Ping; Lee, Li-Ya; Tsai, Yueh-Ting; Chen, Chin-Chu; Chen, Chin-Shuh

2014-01-01

Hericium erinaceus ( H. erinaceus ) has a long history of usage in traditional Chinese medicine for the treatment of gastric disorders. Recently, it has become a well-established candidate in causing positive brain and nerve health-related activities by inducing nerve growth factor (NGF) from its bioactive ingredient, erinacine A. This active compound, which exists only in fermented mycelium but not in its fruiting body, increases NGF levels in astroglial cells in vitro as well as catecholamine and NGF levels in vivo . With increasing recognition of erinacine A in H. erinaceus (EAHE) mycelium improving neurodegenerative diseases, numerous products are being marketed based on these functional claims. To our knowledge, there have been no reports on the mutagenicity of EAHE prior to this paper. Hence, the present study was undertaken to determine the mutagenicity and genotoxicity effects of EAHE mycelium conducted in three standard battery of tests (reverse mutation, chromosomal aberration, and micronuclei tests) according to the latest guidelines in order to meet all international regulatory requirements and provide information on the safety of this new and promising natural remedy. Our results have indicated that EAHE mycelium did not significantly increase the number of revertant colonies in the bacterial reverse mutation test nor induce higher frequency of aberrations in the chromosome aberration test. Moreover, no statistically significant EAHE mycelium-related increase was observed in the incidence of reticulocytes per 1000 red blood cells and micronucleated reticulocytes per 1000 reticulocytes. In conclusion, the three standard battery of tests suggested that EAHE mycelium was devoid of mutagenicity and genotoxicity in the tested doses and experimental conditions.

Molecular Genetic Analysis of Chlamydia Species.

PubMed

Sixt, Barbara S; Valdivia, Raphael H

2016-09-08

Species of Chlamydia are the etiologic agent of endemic blinding trachoma, the leading cause of bacterial sexually transmitted diseases, significant respiratory pathogens, and a zoonotic threat. Their dependence on an intracellular growth niche and their peculiar developmental cycle are major challenges to elucidating their biology and virulence traits. The last decade has seen tremendous advances in our ability to perform a molecular genetic analysis of Chlamydia species. Major achievements include the generation of large collections of mutant strains, now available for forward- and reverse-genetic applications, and the introduction of a system for plasmid-based transformation enabling complementation of mutations; expression of foreign, modified, or reporter genes; and even targeted gene disruptions. This review summarizes the current status of the molecular genetic toolbox for Chlamydia species and highlights new insights into their biology and new challenges in the nascent field of Chlamydia genetics.

Impact of human immunodeficiency virus type 1 reverse transcriptase inhibitor drug resistance mutation interactions on phenotypic susceptibility.

PubMed

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R; Shell, Elisabeth J; Parkin, Neil; O'Brien, William A; Ferguson, Monique R

2008-10-01

The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.

Impact of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor Drug Resistance Mutation Interactions on Phenotypic Susceptibility

PubMed Central

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R.; Shell, Elisabeth J.; Parkin, Neil; O'Brien, William A.

2008-01-01

Abstract The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals. PMID:18844463

Comparative analysis of drug resistance mutations in the human immunodeficiency virus reverse transcriptase gene in patients who are non-responsive, responsive and naive to antiretroviral therapy.

PubMed

Misbah, Mohammad; Roy, Gaurav; Shahid, Mudassar; Nag, Nalin; Kumar, Suresh; Husain, Mohammad

2016-05-01

Drug resistance mutations in the Pol gene of human immunodeficiency virus 1 (HIV-1) are one of the critical factors associated with antiretroviral therapy (ART) failure in HIV-1 patients. The issue of resistance to reverse transcriptase inhibitors (RTIs) in HIV infection has not been adequately addressed in the Indian subcontinent. We compared HIV-1 reverse transcriptase (RT) gene sequences to identify mutations present in HIV-1 patients who were ART non-responders, ART responders and drug naive. Genotypic drug resistance testing was performed by sequencing a 655-bp region of the RT gene from 102 HIV-1 patients, consisting of 30 ART-non-responding, 35 ART-responding and 37 drug-naive patients. The Stanford HIV Resistance Database (HIVDBv 6.2), IAS-USA mutation list, ANRS_09/2012 algorithm, and Rega v8.02 algorithm were used to interpret the pattern of drug resistance. The majority of the sequences (96 %) belonged to subtype C, and a few of them (3.9 %) to subtype A1. The frequency of drug resistance mutations observed in ART-non-responding, ART-responding and drug-naive patients was 40.1 %, 10.7 % and 20.58 %, respectively. It was observed that in non-responders, multiple mutations were present in the same patient, while in responders, a single mutation was found. Some of the drug-naive patients had more than one mutation. Thymidine analogue mutations (TAMs), however, were found in non-responders and naive patients but not in responders. Although drug resistance mutations were widely distributed among ART non-responders, the presence of resistance mutations in the viruses of drug-naive patients poses a big concern in the absence of a genotyping resistance test.

Update on HIV-1 acquired and transmitted drug resistance in Africa.

PubMed

Ssemwanga, Deogratius; Lihana, Raphael W; Ugoji, Chinenye; Abimiku, Alash'le; Nkengasong, John; Dakum, Patrick; Ndembi, Nicaise

2015-01-01

The last ten years have witnessed a significant scale-up and access to antiretroviral therapy in Africa, which has improved patient quality of life and survival. One major challenge associated with increased access to antiretroviral therapy is the development of antiretroviral resistance due to inconsistent drug supply and/or poor patient adherence. We review the current state of both acquired and transmitted drug resistance in Africa over the past ten years (2001-2011) to identify drug resistance associated with the different drug regimens used on the continent and to help guide affordable strategies for drug resistance surveillance. A total of 161 references (153 articles, six reports and two conference abstracts) were reviewed. Antiretroviral resistance data was available for 40 of 53 African countries. A total of 5,541 adult patients from 99 studies in Africa were included in this analysis. The pooled prevalence of drug resistance mutations in Africa was 10.6%, and Central Africa had the highest prevalence of 54.9%. The highest prevalence of nucleoside reverse transcriptase inhibitor mutations was in the west (55.3%) and central (54.8%) areas; nonnucleoside reverse transcriptase inhibitor mutations were highest in East Africa (57.0%) and protease inhibitors mutations highest in Southern Africa (16.3%). The major nucleoside reverse transcriptase inhibitor mutation in all four African regions was M184V. Major nonnucleoside reverse transcriptase inhibitor as well as protease inhibitor mutations varied by region. The prevalence of drug resistance has remained low in several African countries although the emergence of drug resistance mutations varied across countries. Continued surveillance of antiretroviral therapy resistance remains crucial in gauging the effectiveness of country antiretroviral therapy programs and strategizing on effective and affordable strategies for successful treatment.

Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity.

PubMed

Blanca, Giuseppina; Baldanti, Fausto; Paolucci, Stefania; Skoblov, Alexander Yu; Victorova, Lyubov; Hübscher, Ulrich; Gerna, Giuseppe; Spadari, Silvio; Maga, Giovanni

2003-05-02

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

Genotoxicity evaluation of So-ochim-tang-gamibang (SOCG), a herbal medicine.

PubMed

Lee, Mi Young; Park, Yang-Chun; Jin, Mirim; Kim, Eunseok; Choi, Jeong June; Jung, In Chul

2018-02-02

So-ochim-tang-gamibang (SOCG) is a traditional Korean medicine frequently used for depression in the clinical field. In this study, we evaluated the potential genotoxicity of SOCG using three standard batteries of tests as part of a safety evaluation. SOCG was evaluated for potential genotoxic effects using the standard three tests recommended by the Ministry of Food and Drug Safety (MFDS) of Korea. These tests were the bacterial reverse mutation test (Ames test), in vitro mammalian chromosomal aberration test using Chinese hamster lung cells, and in vivo micronucleus test using ICR mice. The Ames test with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and the Escherichia coli strain WP2uvrA(pKM101) showed that SOCG did not induce gene mutations at any dose level in all of the strains. SOCG did not induce any chromosomal aberrations in the in vitro chromosomal aberration test (for both the 6 and 24 h test) and the in vivo micronucleus test. Based on the results of these tests, it was concluded that SOCG does not exhibit any genotoxic risk under the experimental conditions of this study.

nr0b1 (DAX1) mutation in zebrafish causes female-to-male sex reversal through abnormal gonadal proliferation and differentiation.

PubMed

Chen, Sijie; Zhang, Hefei; Wang, Fenghua; Zhang, Wei; Peng, Gang

2016-09-15

Sex determinations are diverse in vertebrates. Although many sex-determining genes and pathways are conserved, the mechanistic roles of these genes and pathways in the genetic sex determination are not well understood. DAX1 (encoded by the NR0B1 gene) is a vertebrate specific orphan nuclear receptor that regulates gonadal development and sexual determination. In human, duplication of the NR0B1 gene leads to male-to-female sex reversal. In mice, Nr0b1 shows both pro-testis and anti-testis functions. We generated inheritable nr0b1 mutation in the zebrafish and found the nr0b1 mutation caused homozygous mutants to develop as fertile males due to female-to-male sex reversal. The nr0b1 mutation did not increase Caspase-3 labeling nor tp53 expression in the developing gonads. Introduction of a tp53 mutation into the nr0b1 mutant did not rescue the sex-reversal phenotype. Further examination revealed reduction in cell proliferation and abnormal somatic cell differentiation in the nr0b1 mutant gonads at the undifferentiated and bi-potential ovary stages. Together, our results suggest nr0b1 regulates somatic cell differentiation and cell proliferation to ensure normal sex development in the zebrafish. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Deletion of Citrate Synthase Restores Growth of Sinorhizobium meliloti 1021 Aconitase Mutants▿

PubMed Central

Koziol, Uriel; Hannibal, Luciana; Rodríguez, María Cecilia; Fabiano, Elena; Kahn, Michael L.; Noya, Francisco

2009-01-01

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 1021 encodes only one predicted aconitase (AcnA) in its genome. AcnA has a significant degree of similarity with other bacterial aconitases that behave as dual proteins: enzymes and posttranscriptional regulators of gene expression. Similar to the case with these bacterial aconitases, AcnA activity was reversibly labile and was regained upon reconstitution with reduced iron. The aconitase promoter was active in root nodules. acnA mutants grew very poorly, had secondary mutations, and were quickly outgrown by pseudorevertants. The acnA gene was stably interrupted in a citrate synthase (gltA) null background, indicating that the intracellular accumulation of citrate may be deleterious for survival of strain 1021. No aconitase activity was detected in this mutant, suggesting that the acnA gene encodes the only functional aconitase of strain 1021. To uncover a function of AcnA beyond its catalytic role in the tricarboxylic acid cycle pathway, the gltA acnA double mutant was compared with the gltA single mutant for differences in motility, resistance to oxidative stress, nodulation, and growth on different substrates. However, no differences in any of these characteristics were found. PMID:19820082

Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens

PubMed Central

Geier, Christoph B.; Piller, Alexander; Linder, Angela; Sauerwein, Kai M. T.; Eibl, Martha M.; Wolf, Hermann M.

2015-01-01

Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency. PMID:26186701

Genome-based approaches to develop vaccines against bacterial pathogens.

PubMed

Serruto, Davide; Serino, Laura; Masignani, Vega; Pizza, Mariagrazia

2009-05-26

Bacterial infectious diseases remain the single most important threat to health worldwide. Although conventional vaccinology approaches were successful in conferring protection against several diseases, they failed to provide efficacious solutions against many others. The advent of whole-genome sequencing changed the way to think about vaccine development, enabling the targeting of possible vaccine candidates starting from the genomic information of a single bacterial isolate, with a process named reverse vaccinology. As the genomic era progressed, reverse vaccinology has evolved with a pan-genome approach and multi-strain genome analysis became fundamental for the design of universal vaccines. This review describes the applications of genome-based approaches in the development of new vaccines against bacterial pathogens.

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

PubMed

Aulicino, Paula C; Rocco, Carlos A; Mecikovsky, Debora; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

2010-01-01

Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.

Mutations in the PCCA gene encoding the {alpha} subunit of propionyl-CoA carboxylase in patients with propionic acidemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campeau, E.; Leon-Del-Rio, A.; Gravel, R.A.

Propionic acidemia is a rare autosomal recessive disorder characterized by a deficiency of the mitochondrial biotin-dependent enzyme, propionyl-CoA carboxylase (PCC). PCC has the structure {alpha}{sub 4}{beta}{sub 4}, with the {alpha} subunit containing the biotin prosthetic group. This study is concerned with defining the spectrum of mutations occurring in the PCCA gene encoding the {alpha} subunit. Mutations were initially assigned to this gene through complementation experiments done after somatic fusion of patient fibroblasts. The analyses were performed on PCR-amplified reverse transcripts of fibroblast RNA. The mutations were identified by single strand conformational polymorphism analysis and direct sequencing of PCR products. Threemore » candidate disease-causing mutations and one DNA polymorphism were identified in the {alpha} subunit sequence in different patients: (1) a 3 bp deletion {triangle}CTG{sub 2058-2060}, which eliminates Cys687 near the biotin binding site (Lys669); (2) T{sub 611}{r_arrow}A which converts Met204 to Lys in a highly conserved region matching that of an ATP binding site; (3) An {approximately}50 bp deletion near the 3{prime} end of the cDNA which likely corresponds to the loss of an exon due to a splicing defect; and (4) a 3 bp insertion, +CAG{sub 2203}, located downstream of the stop codon, which is likely a DNA polymorphism. In order to determine the effect of the Cys687 deletion on the biotinylation of PCC, we expressed the mutation in a 67 amino acid C-terminal fragment of the PCC {alpha} subunit in E. coli in which biotinylation is directed by the bacterial biotin ligase. While the mutant peptide was expressed at about half-normal levels, the biotinylation of the peptide that was present was reduced to only {approximately}20% normal. We suggest, therefore, that the absence of PCC activity due to {triangle}Cys687 results at least in part from defective biotinylation of an unstable protein.« less

The public health approach to identify antiretroviral therapy failure: high-level nucleoside reverse transcriptase inhibitor resistance among Malawians failing first-line antiretroviral therapy

PubMed Central

Hosseinipour, Mina C.; van Oosterhout, Joep J.G.; Weigel, Ralf; Phiri, Sam; Kamwendo, Debbie; Parkin, Neil; Fiscus, Susan A.; Nelson, Julie A.E.; Eron, Joseph J.; Kumwenda, Johnstone

2010-01-01

Background Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi. Methods Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed. Results Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine. Conclusion When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART. PMID:19417582

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

Detection of 98. 5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuppens, H.; Marynen, P.; Cassiman, J.J.

1993-12-01

The authors have previously shown that about 85% of the mutations in 194 Belgian cystic fibrosis alleles could be detected by a reverse dot-blot assay. In the present study, 50 Belgian chromosomes were analyzed for mutations in the cystic fibrosis transmembrane conductance regulator gene by means of direct solid phase automatic sequencing of PCR products of individual exons. Twenty-six disease mutations and 14 polymorphisms were found. Twelve of these mutations and 3 polymorphisms were not described before. With the exception of one mutant allele carrying two mutations, these mutations were the only mutations found in the complete coding region andmore » their exon/intron boundaries. The total sensitivity of mutant CF alleles that could be identified was 98.5%. Given the heterogeneity of these mutations, most of them very rare, CFTR mutation screening still remains rather complex in the population, and population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available. 24 refs., 1 fig., 2 tabs.« less

Carcinogens induce reversion of the mouse pink-eyed unstable mutation

PubMed Central

Schiestl, Robert H.; Aubrecht, Jiri; Khogali, Fathia; Carls, Nicholas

1997-01-01

Deletions and other genome rearrangements are associated with carcinogenesis and inheritable diseases. The pink-eyed unstable (pun) mutation in the mouse is caused by duplication of a 70-kb internal fragment of the p gene. Spontaneous reversion events in homozygous pun/pun mice occur through deletion of a duplicated sequence. Reversion events in premelanocytes in the mouse embryo detected as black spots on the gray fur of the offspring were inducible by the carcinogen x-rays, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, benzo[a]pyrene, trichloroethylene, benzene, and sodium arsenate. The latter three carcinogens are not detectable with several in vitro or in vivo mutagenesis assays. We studied the molecular mechanism of the carcinogen-induced reversion events by cDNA analysis using reverse transcriptase–PCR method and identified the induced reversion events as deletions. DNA deletion assays may be sensitive indicators for carcinogen exposure. PMID:9114032

FGFR2 mutation in 46,XY sex reversal with craniosynostosis

PubMed Central

Bagheri-Fam, Stefan; Ono, Makoto; Li, Li; Zhao, Liang; Ryan, Janelle; Lai, Raymond; Katsura, Yukako; Rossello, Fernando J.; Koopman, Peter; Scherer, Gerd; Bartsch, Oliver; Eswarakumar, Jacob V.P.; Harley, Vincent R.

2015-01-01

Patients with 46,XY gonadal dysgenesis (GD) exhibit genital anomalies, which range from hypospadias to complete male-to-female sex reversal. However, a molecular diagnosis is made in only 30% of cases. Heterozygous mutations in the human FGFR2 gene cause various craniosynostosis syndromes including Crouzon and Pfeiffer, but testicular defects were not reported. Here, we describe a patient whose features we would suggest represent a new FGFR2-related syndrome, craniosynostosis with XY male-to-female sex reversal or CSR. The craniosynostosis patient was chromosomally XY, but presented as a phenotypic female due to complete GD. DNA sequencing identified the FGFR2c heterozygous missense mutation, c.1025G>C (p.Cys342Ser). Substitution of Cys342 by Ser or other amino acids (Arg/Phe/Try/Tyr) has been previously reported in Crouzon and Pfeiffer syndrome. We show that the ‘knock-in’ Crouzon mouse model Fgfr2cC342Y/C342Y carrying a Cys342Tyr substitution displays XY gonadal sex reversal with variable expressivity. We also show that despite FGFR2c-Cys342Tyr being widely considered a gain-of-function mutation, Cys342Tyr substitution in the gonad leads to loss of function, as demonstrated by sex reversal in Fgfr2cC342Y/− mice carrying the knock-in allele on a null background. The rarity of our patient suggests the influence of modifier genes which exacerbated the testicular phenotype. Indeed, patient whole exome analysis revealed several potential modifiers expressed in Sertoli cells at the time of testis determination in mice. In summary, this study identifies the first FGFR2 mutation in a 46,XY GD patient. We conclude that, in certain rare genetic contexts, maintaining normal levels of FGFR2 signaling is important for human testis determination. PMID:26362256

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

PubMed Central

Gregory, John J.; Wagner, John E.; Verlander, Peter C.; Levran, Orna; Batish, Sat Dev; Eide, Cindy R.; Steffenhagen, Amy; Hirsch, Betsy; Auerbach, Arleen D.

2001-01-01

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815–2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein–Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA. PMID:11226273

A novel ion-beam-mutation effect application in identification of gene involved in bacterial antagonism to fungal infection of ornamental crops

NASA Astrophysics Data System (ADS)

Mahadtanapuk, S.; Teraarusiri, W.; Nanakorn, W.; Yu, L. D.; Thongkumkoon, P.; Anuntalabhochai, S.

2014-05-01

This work is on a novel application of ion beam effect on biological mutation. Bacillus licheniformis (B. licheniformis) is a common soil bacterium with an antagonistic effect on Curcuma alismatifolia Gagnep. and Chrysanthemum indicum Linn. In an attempt to control fungal diseases of local crops by utilizing B. licheniformis, we carried out gene analysis of the bacterium to understand the bacterial antagonistic mechanism. The bacterial cells were bombarded to induce mutations using nitrogen ion beam. After ion bombardment, DNA analysis revealed that the modified polymorphism fragment present in the wild type was missing in a bacterial mutant which lost the antifungal activity. The fragments conserved in the wild type but lost in the mutant bacteria was identified to code for the thioredoxin reductase (TrxR) gene. The gene analysis showed that the TrxR gene from B. licheniformis had the expression of the antagonism to fungi in a synchronous time evolution with the fungus inhibition when the bacteria were co-cultivated with the fungi. The collective results indicate the TrxR gene responsible for the antagonism of bacteria B. licheniformis to fungal infection.

Genome-Wide Biases in the Rate and Molecular Spectrum of Spontaneous Mutations in Vibrio cholerae and Vibrio fischeri

DTIC Science & Technology

2016-10-15

Mutation rates may be nonuniform because of greater rates of damage, asymmetric nucleotide pools, structural differences affecting polymerase fidelity...strains of these two significant bacterial species. In the MMR-deficient strains, mutation rates were nonuniform among genome regions and varied in pat

Bacteria Facilitate Enteric Virus Co-infection of Mammalian Cells and Promote Genetic Recombination.

PubMed

Erickson, Andrea K; Jesudhasan, Palmy R; Mayer, Melinda J; Narbad, Arjan; Winter, Sebastian E; Pfeiffer, Julie K

2018-01-10

RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections. Copyright © 2017 Elsevier Inc. All rights reserved.

Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution

PubMed Central

Chursov, Andrey; Frishman, Dmitrij; Shneider, Alexander

2013-01-01

Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the ‘long-term evolution experiment’. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins. PMID:23783573

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex.

PubMed

Harris, Kimberly A; Zhou, Zhiyuan; Peters, Michelle L; Wilkins, Sarah G; Breaker, Ronald R

2018-06-18

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans , OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA ( ∆ole ) or OapA ( ∆oapA ) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans .

Assessment of antimutagenic and genotoxic potential of senna (Cassia angustifolia Vahl.) aqueous extract using in vitro assays.

PubMed

Silva, C R; Monteiro, M R; Rocha, H M; Ribeiro, A F; Caldeira-de-Araujo, A; Leitão, A C; Bezerra, R J A C; Pádula, M

2008-02-01

Senna (Cassia angustifolia Vahl.) is widely used as a laxative, although potential side effects, such as toxicity and genotoxicity, have been reported. This study evaluated genotoxic and mutagenic effects of senna aqueous extract (SAE) by means of four experimental assays: inactivation of Escherichia coli cultures; bacterial growth inhibition; reverse mutation test (Mutoxitest) and DNA strand break analysis in plasmid DNA. Our results demonstrated that SAE produces single and double strand breaks in plasmid DNA in a cell free system. On the other hand, SAE was not cytotoxic or mutagenic to Escherichia coli strains tested. In effect, SAE was able to avoid H(2)O(2)-induced mutagenesis and toxicity in Escherichia coli IC203 (uvrA oxyR) and IC205 (uvrA mutM) strains, pointing to a new antioxidant/antimutagenic action of SAE.

A Comprehensive Toxicological Safety Assessment of an Extract of Olea Europaea L. Leaves (Bonolive™).

PubMed

Clewell, Amy E; Béres, Erzsébet; Vértesi, Adél; Glávits, Róbert; Hirka, Gábor; Endres, John R; Murbach, Timothy S; Szakonyiné, Ilona Pasics

2016-01-01

A battery of toxicological studies was conducted to investigate the genotoxicity and repeated-dose oral toxicity of Bonolive™, a proprietary water-soluble extract of the leaves of the olive tree (Olea europaea L.), in accordance with internationally accepted protocols. There was no evidence of mutagenicity in a bacterial reverse mutation test and in an vitro mammalian chromosomal aberration test nor was any genotoxic activity observed in an in vivo mouse micronucleus test at concentrations up to the limit dose of 2000 mg/kg bw/d. Bonolive™ did not cause mortality or toxic effects in Crl:(WI)BR Wistar rats in a 90-day repeated-dose oral toxicity study at doses of 360, 600, and 1000 mg/kg bw/d. The no observed adverse effect level in the 90-day study was 1000 mg/kg bw/d for both male and female rats, the highest dose tested. © The Author(s) 2015.

Mutagenic and genotoxic potential of direct electric current in Escherichia coli and Salmonella thyphimurium strains.

PubMed

Gomes, Marina das Neves; Cardoso, Janine Simas; Leitão, Alvaro Costa; Quaresma, Carla Holandino

2016-05-01

Direct electric current has several therapeutic uses such as antibacterial and antiprotozoal action, tissues scarring and regeneration, as well as tumor treatment. This method has shown promising results in vivo and in vitro, with significant efficacy and almost no side effects. Considering lack of studies regarding direct electric current mutagenic and/or genotoxic effects, the present work evaluated both aspects by using five different bacterial experimental assays: survival of repair-deficient mutants, Salmonella-histidine reversion mutagenesis (Ames test), forward mutations to rifampicin resistance, phage reactivation, and lysogenic induction. In these experimental conditions, cells were submitted to an approach that allows evaluation of anodic, cathodic, and electro-ionic effects generated by 2 mA of direct electric current, with doses ranging from 0.36 to 3.60 Coulombs. Our results showed these doses did not induce mutagenic or genotoxic effects. © 2016 Wiley Periodicals, Inc.

Optimal descriptor as a translator of eclectic data into endpoint prediction: mutagenicity of fullerene as a mathematical function of conditions.

PubMed

Toropov, Andrey A; Toropova, Alla P

2014-06-01

The experimental data on the bacterial reverse mutation test on C60 nanoparticles (TA100) is examined as an endpoint. By means of the optimal descriptors calculated with the Monte Carlo method a mathematical model of the endpoint has been built up. The model is the mathematical function of (i) dose (g/plate); (ii) metabolic activation (i.e. with S9 mix or without S9 mix); and (iii) illumination (i.e. dark or irradiation). The statistical quality of the model is the following: n=10, r(2)=0.7549, q(2)=0.5709, s=7.67, F=25 (Training set); n=5, r(2)=0.8987, s=18.4 (Calibration set); and n=5, r(2)=0.6968, s=10.9 (Validation set). Copyright © 2013 Elsevier Ltd. All rights reserved.

Determining mutation density using Restriction Enzyme Sequence Comparative Analysis (RESCAN)

USDA-ARS?s Scientific Manuscript database

The average mutation density of a mutant population is a major consideration when developing resources for the efficient, cost-effective implementation of reverse genetics methods such as Targeting of Induced Local Lesions in Genomes (TILLING). Reliable estimates of mutation density can be achieved ...

5-Azacytidine Can Induce Lethal Mutagenesis in Human Immunodeficiency Virus Type 1▿ †

PubMed Central

Dapp, Michael J.; Clouser, Christine L.; Patterson, Steven; Mansky, Louis M.

2009-01-01

Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2′-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription. PMID:19726509

The Application of a Homologous Recombination Assay Revealed Amino Acid Residues in an LTR-Retrotransposon That Were Critical for Integration

PubMed Central

Atwood, Angela; Choi, Jeannie; Levin, Henry L.

1998-01-01

Retroviruses and their relatives, the LTR-retrotransposons, possess an integrase protein (IN) that is required for the insertion of reverse transcripts into the genome of host cells. Schizosaccharomyces pombe is the host of Tf1, an LTR-retrotransposon with integration activity that can be studied by using techniques of yeast genetics. In this study, we sought to identify amino acid substitutions in Tf1 that specifically affected the integration step of transposition. In addition to seeking amino acid substitutions in IN, we also explored the possibility that other Tf1 proteins contributed to integration. By comparing the results of genetic assays that monitored both transposition and reverse transcription, we were able to seek point mutations throughout Tf1 that blocked transposition but not the synthesis of reverse transcripts. These mutant versions of Tf1 were candidates of elements that possessed defects in the integration step of transposition. Five mutations in Tf1 that resulted in low levels of integration were found to be located in the IN protein: two substitutions in the N-terminal Zn domain, two in the catalytic core, and one in the C-terminal domain. These results suggested that each of the three IN domains was required for Tf1 transposition. The potential role of these five amino acid residues in the function of IN is discussed. Two of the mutations that reduced integration mapped to the RNase H (RH) domain of Tf1 reverse transcriptase. The Tf1 elements with the RH mutations produced high levels of reverse transcripts, as determined by recombination and DNA blot analysis. These results indicated that the RH of Tf1 possesses a function critical for transposition that is independent of the accumulation of reverse transcripts. PMID:9445033

Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1.

PubMed

Castagna, M; Soragna, A; Mari, S A; Santacroce, M; Betté, S; Mandela, P G; Rudnick, G; Peres, A; Sacchi, V F

2007-10-01

KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K(+) and Li(+) in addition to Na(+). We have previously demonstrated that Asp338 is essential for KAAT1 activation by K(+) and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis, and expression in Xenopus laevis oocytes, we identified Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both Na(+) and K(+). However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na(+)-dependent transport and the block in K(+)-dependent transport that characterize the D338E mutant. K(+)-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)(3), we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus both structural and functional evidence suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na(+) binding site in the recently solved crystal structure of the NSS transporter LeuT(Aa) (41). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway.

S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation.

PubMed

Groome, James R; Winston, Vern

2013-05-01

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1-S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1-S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I-III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

Rift Valley Fever Virus MP-12 Vaccine Is Fully Attenuated by a Combination of Partial Attenuations in the S, M, and L Segments

PubMed Central

Hill, Terence E.; Smith, Jennifer K.; Zhang, Lihong; Juelich, Terry L.; Gong, Bin; Slack, Olga A. L.; Ly, Hoai J.; Lokugamage, Nandadeva; Freiberg, Alexander N.

2015-01-01

ABSTRACT Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine. IMPORTANCE Rift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine. PMID:25948740

Rift Valley Fever Virus MP-12 Vaccine Is Fully Attenuated by a Combination of Partial Attenuations in the S, M, and L Segments.

PubMed

Ikegami, Tetsuro; Hill, Terence E; Smith, Jennifer K; Zhang, Lihong; Juelich, Terry L; Gong, Bin; Slack, Olga A L; Ly, Hoai J; Lokugamage, Nandadeva; Freiberg, Alexander N

2015-07-01

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine. Rift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

An approximate stationary solution for multi-allele neutral diffusion with low mutation rates.

PubMed

Burden, Conrad J; Tang, Yurong

2016-12-01

We address the problem of determining the stationary distribution of the multi-allelic, neutral-evolution Wright-Fisher model in the diffusion limit. A full solution to this problem for an arbitrary K×K mutation rate matrix involves solving for the stationary solution of a forward Kolmogorov equation over a (K-1)-dimensional simplex, and remains intractable. In most practical situations mutations rates are slow on the scale of the diffusion limit and the solution is heavily concentrated on the corners and edges of the simplex. In this paper we present a practical approximate solution for slow mutation rates in the form of a set of line densities along the edges of the simplex. The method of solution relies on parameterising the general non-reversible rate matrix as the sum of a reversible part and a set of (K-1)(K-2)/2 independent terms corresponding to fluxes of probability along closed paths around faces of the simplex. The solution is potentially a first step in estimating non-reversible evolutionary rate matrices from observed allele frequency spectra. Copyright © 2016 Elsevier Inc. All rights reserved.

IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity

PubMed Central

Chiang, Sarah; Weigelt, Britta; Wen, Huei-Chi; Pareja, Fresia; Raghavendra, Ashwini; Martelotto, Luciano G.; Burke, Kathleen A.; Basili, Thais; Li, Anqi; Geyer, Felipe C.; Piscuoglio, Salvatore; Ng, Charlotte K.Y.; Jungbluth, Achim A.; Balss, Jörg; Pusch, Stefan; Baker, Gabrielle M.; Cole, Kimberly S.; von Deimling, Andreas; Batten, Julie M.; Marotti, Jonathan D.; Soh, Hwei-Choo; McCalip, Benjamin L.; Serrano, Jonathan; Lim, Raymond S.; Siziopikou, Kalliopi P.; Lu, Song; Liu, Xiaolong; Hammour, Tarek; Brogi, Edi; Snuderl, Matija; Iafrate, A. John; Reis-Filho, Jorge S.; Schnitt, Stuart J.

2017-01-01

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. PMID:27913435

Adaptive mutation: has the unicorn landed?

PubMed

Foster, P L

1998-04-01

Reversion of an episomal Lac- allele during lactose selection has been studied as a model for adaptive mutation. Although recent results show that the mutations that arise during selection are not "adaptive" in the original sense, the mutagenic mechanism that produces these mutations may nonetheless be of evolutionary significance. In addition, a transient mutational state induced in a subpopulation of starving cells could provide a species with a mechanism for adaptive evolution.

Next-generation sequencing for targeted discovery of rare mutations in rice

USDA-ARS?s Scientific Manuscript database

Advances in DNA sequencing (i.e., next-generation sequencing, NGS) have greatly increased the power and efficiency of detecting rare mutations in large mutant populations. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach for identifying gene mutations resulting fro...

The perturbation of tryptophan fluorescence by phenylalanine to alanine mutations identifies the hydrophobic core in a subset of bacterial Ig-like domains.

PubMed

Raman, Rajeev; Ptak, Christopher P; Hsieh, Ching-Lin; Oswald, Robert E; Chang, Yung-Fu; Sharma, Yogendra

2013-07-09

Many host-parasite interactions are mediated via surface-exposed proteins containing bacterial immunoglobulin-like (Big) domains. Here, we utilize the spectral properties of a conserved Trp to provide evidence that, along with a Phe, these residues are positioned within the hydrophobic core of a subset of Big_2 domains. The mutation of the Phe to Ala decreases Big_2 domain stability and impairs the ability of LigBCen2 to bind to the host protein, fibronectin.

Bending patterns of chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant.

PubMed

Brokaw, C J; Luck, D J

1985-01-01

Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the "wild-type" uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.

Network-Based Identification of Adaptive Pathways in Evolved Ethanol-Tolerant Bacterial Populations

PubMed Central

Swings, Toon; Weytjens, Bram; Schalck, Thomas; Bonte, Camille; Verstraeten, Natalie; Michiels, Jan

2017-01-01

Abstract Efficient production of ethanol for use as a renewable fuel requires organisms with a high level of ethanol tolerance. However, this trait is complex and increased tolerance therefore requires mutations in multiple genes and pathways. Here, we use experimental evolution for a system-level analysis of adaptation of Escherichia coli to high ethanol stress. As adaptation to extreme stress often results in complex mutational data sets consisting of both causal and noncausal passenger mutations, identifying the true adaptive mutations in these settings is not trivial. Therefore, we developed a novel method named IAMBEE (Identification of Adaptive Mutations in Bacterial Evolution Experiments). IAMBEE exploits the temporal profile of the acquisition of mutations during evolution in combination with the functional implications of each mutation at the protein level. These data are mapped to a genome-wide interaction network to search for adaptive mutations at the level of pathways. The 16 evolved populations in our data set together harbored 2,286 mutated genes with 4,470 unique mutations. Analysis by IAMBEE significantly reduced this number and resulted in identification of 90 mutated genes and 345 unique mutations that are most likely to be adaptive. Moreover, IAMBEE not only enabled the identification of previously known pathways involved in ethanol tolerance, but also identified novel systems such as the AcrAB-TolC efflux pump and fatty acids biosynthesis and even allowed to gain insight into the temporal profile of adaptation to ethanol stress. Furthermore, this method offers a solid framework for identifying the molecular underpinnings of other complex traits as well. PMID:28961727

The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations.

PubMed

Flores-Rozas, H; Kolodner, R D

1998-10-13

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.

A reversion of an IL2RG mutation in combined immunodeficiency providing competitive advantage to the majority of CD8+ T cells

PubMed Central

Kuijpers, Taco W.; van Leeuwen, Ester M.M.; Barendregt, Barbara H.; Klarenbeek, Paul; aan de Kerk, Daan J.; Baars, Paul A.; Jansen, Machiel H.; de Vries, Niek; van Lier, René A.W.; van der Burg, Mirjam

2013-01-01

Mutations in the common gamma chain (γc, CD132, encoded by the IL2RG gene) can lead to B+T−NK− X-linked severe combined immunodeficiency, as a consequence of unresponsiveness to γc-cytokines such as interleukins-2, -7 and -15. Hypomorphic mutations in CD132 may cause combined immunodeficiencies with a variety of clinical presentations. We analyzed peripheral blood mononuclear cells of a 6-year-old boy with normal lymphocyte counts, who suffered from recurrent pneumonia and disseminated mollusca contagiosa. Since proliferative responses of T cells and NK cells to γc -cytokines were severely impaired, we performed IL2RG gene analysis, showing a heterozygous mutation in the presence of a single X-chromosome. Interestingly, an IL2RG reversion to normal predominated in both naïve and antigen-primed CD8+ T cells and increased over time. Only the revertant CD8+ T cells showed normal expression of CD132 and the various CD8+ T cell populations had a different T-cell receptor repertoire. Finally, a fraction of γδ+ T cells and differentiated CD4+CD27− effector-memory T cells carried the reversion, whereas NK or B cells were repeatedly negative. In conclusion, in a patient with a novel IL2RG mutation, gene-reverted CD8+ T cells accumulated over time. Our data indicate that selective outgrowth of particular T-cell subsets may occur following reversion at the level of committed T progenitor cells. PMID:23403317

A reversion of an IL2RG mutation in combined immunodeficiency providing competitive advantage to the majority of CD8+ T cells.

PubMed

Kuijpers, Taco W; van Leeuwen, Ester M M; Barendregt, Barbara H; Klarenbeek, Paul; aan de Kerk, Daan J; Baars, Paul A; Jansen, Machiel H; de Vries, Niek; van Lier, René A W; van der Burg, Mirjam

2013-07-01

Mutations in the common gamma chain (γc, CD132, encoded by the IL2RG gene) can lead to B(+)T(-)NK(-) X-linked severe combined immunodeficiency, as a consequence of unresponsiveness to γc-cytokines such as interleukins-2, -7 and -15. Hypomorphic mutations in CD132 may cause combined immunodeficiencies with a variety of clinical presentations. We analyzed peripheral blood mononuclear cells of a 6-year-old boy with normal lymphocyte counts, who suffered from recurrent pneumonia and disseminated mollusca contagiosa. Since proliferative responses of T cells and NK cells to γc -cytokines were severely impaired, we performed IL2RG gene analysis, showing a heterozygous mutation in the presence of a single X-chromosome. Interestingly, an IL2RG reversion to normal predominated in both naïve and antigen-primed CD8(+) T cells and increased over time. Only the revertant CD8(+) T cells showed normal expression of CD132 and the various CD8(+) T cell populations had a different T-cell receptor repertoire. Finally, a fraction of γδ(+) T cells and differentiated CD4(+)CD27(-) effector-memory T cells carried the reversion, whereas NK or B cells were repeatedly negative. In conclusion, in a patient with a novel IL2RG mutation, gene-reverted CD8(+) T cells accumulated over time. Our data indicate that selective outgrowth of particular T-cell subsets may occur following reversion at the level of committed T progenitor cells.

In vitro cross-resistance profile of nucleoside reverse transcriptase inhibitor (NRTI) BMS-986001 against known NRTI resistance mutations.

PubMed

Li, Zhufang; Terry, Brian; Olds, William; Protack, Tricia; Deminie, Carol; Minassian, Beatrice; Nowicka-Sans, Beata; Sun, Yongnian; Dicker, Ira; Hwang, Carey; Lataillade, Max; Hanna, George J; Krystal, Mark

2013-11-01

BMS-986001 is a novel HIV nucleoside reverse transcriptase inhibitor (NRTI). To date, little is known about its resistance profile. In order to examine the cross-resistance profile of BMS-986001 to NRTI mutations, a replicating virus system was used to examine specific amino acid mutations known to confer resistance to various NRTIs. In addition, reverse transcriptases from 19 clinical isolates with various NRTI mutations were examined in the Monogram PhenoSense HIV assay. In the site-directed mutagenesis studies, a virus containing a K65R substitution exhibited a 0.4-fold change in 50% effective concentration (EC50) versus the wild type, while the majority of viruses with the Q151M constellation (without M184V) exhibited changes in EC50 versus wild type of 0.23- to 0.48-fold. Susceptibility to BMS-986001 was also maintained in an L74V-containing virus (0.7-fold change), while an M184V-only-containing virus induced a 2- to 3-fold decrease in susceptibility. Increasing numbers of thymidine analog mutation pattern 1 (TAM-1) pathway mutations correlated with decreases in susceptibility to BMS-986001, while viruses with TAM-2 pathway mutations exhibited a 5- to 8-fold decrease in susceptibility, regardless of the number of TAMs. A 22-fold decrease in susceptibility to BMS-986001 was observed in a site-directed mutant containing the T69 insertion complex. Common non-NRTI (NNRTI) mutations had little impact on susceptibility to BMS-986001. The results from the site-directed mutants correlated well with the more complicated genotypes found in NRTI-resistant clinical isolates. Data from clinical studies are needed to determine the clinically relevant resistance cutoff values for BMS-986001.

Predictable Phenotypes of Antibiotic Resistance Mutations.

PubMed

Knopp, M; Andersson, D I

2018-05-15

Antibiotic-resistant bacteria represent a major threat to our ability to treat bacterial infections. Two factors that determine the evolutionary success of antibiotic resistance mutations are their impact on resistance level and the fitness cost. Recent studies suggest that resistance mutations commonly show epistatic interactions, which would complicate predictions of their stability in bacterial populations. We analyzed 13 different chromosomal resistance mutations and 10 host strains of Salmonella enterica and Escherichia coli to address two main questions. (i) Are there epistatic interactions between different chromosomal resistance mutations? (ii) How does the strain background and genetic distance influence the effect of chromosomal resistance mutations on resistance and fitness? Our results show that the effects of combined resistance mutations on resistance and fitness are largely predictable and that epistasis remains rare even when up to four mutations were combined. Furthermore, a majority of the mutations, especially target alteration mutations, demonstrate strain-independent phenotypes across different species. This study extends our understanding of epistasis among resistance mutations and shows that interactions between different resistance mutations are often predictable from the characteristics of the individual mutations. IMPORTANCE The spread of antibiotic-resistant bacteria imposes an urgent threat to public health. The ability to forecast the evolutionary success of resistant mutants would help to combat dissemination of antibiotic resistance. Previous studies have shown that the phenotypic effects (fitness and resistance level) of resistance mutations can vary substantially depending on the genetic context in which they occur. We conducted a broad screen using many different resistance mutations and host strains to identify potential epistatic interactions between various types of resistance mutations and to determine the effect of strain background on resistance phenotypes. Combinations of several different mutations showed a large amount of phenotypic predictability, and the majority of the mutations displayed strain-independent phenotypes. However, we also identified a few outliers from these patterns, illustrating that the choice of host organism can be critically important when studying antibiotic resistance mutations. Copyright © 2018 Knopp and Andersson.

Adaptive mutation: has the unicorn landed?

PubMed Central

Foster, P L

1998-01-01

Reversion of an episomal Lac- allele during lactose selection has been studied as a model for adaptive mutation. Although recent results show that the mutations that arise during selection are not "adaptive" in the original sense, the mutagenic mechanism that produces these mutations may nonetheless be of evolutionary significance. In addition, a transient mutational state induced in a subpopulation of starving cells could provide a species with a mechanism for adaptive evolution. PMID:9560365

Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity

NASA Astrophysics Data System (ADS)

Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

2016-10-01

Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.

Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity.

PubMed

Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

2016-10-01

Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.

Evidence for Mitotic Recombination in W(ei)/+ Heterozygous Mice

PubMed Central

Panthier, J. J.; Guenet, J. L.; Condamine, H.; Jacob, F.

1990-01-01

A number of alleles at coat color loci of the house mouse give rise to areas of wild-type pigmentation on the coats of otherwise mutant animals. Such unstable alleles include both recessive and dominant mutations. Among the latter are several alleles at the W locus. In this report, phenotypic reversions of the W(ei) allele at the W locus were studied Mice heterozygous in repulsion for both W(ei) and buff (bf) [i.e. W(ei)+/+bf] were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khaki colored coat of nonagouti buff homozygotes (a/a; bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a/a; W(ei)+/+bf mice: either solid black or khaki colored. Furthermore phenotypic reversions of W(ei)/+ were enhanced significantly following X-irradiation of 9.25-day-old W(ei)/+ embryos (P < 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. In addition these results rise the intriguing possibility that some W mutations may enhance mitotic recombination in the house mouse. PMID:2341029

Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

PubMed

Long, Katherine S; Poehlsgaard, Jacob; Hansen, Lykke H; Hobbie, Sven N; Böttger, Erik C; Vester, Birte

2009-03-01

Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

Safety Assessment and Botanical Standardization of an Edible Species from South America.

PubMed

Traesel, Giseli Karenina; Machado, Camila Dias; Tirloni, Cleide Adriane Signor; Menetrier, Jacqueline Vergutz; Dos Reis Lívero, Francislaine Aparecida; Lourenço, Emerson Luiz Botelho; Oesterreich, Silvia Aparecida; Budel, Jane Manfron; Junior, Arquimedes Gasparotto

2017-05-01

Tropaeolum majus L. (Tropaeolaceae), commonly known as nasturtium, is an important edible plant native to the Andean States and widely disseminated throughout South America. Despite the use of this species is quite widespread, there are no minimum quality control standards or data on its genotoxicity. So, the aim of this study was to present a detailed anatomical and histochemical study for Tropaeolum majus and provide genotoxicity parameters of a preparation routinely used in South American countries. First, three different Tropaeolum majus aqueous extracts (TMAEs) at concentrations of 1.5%, 7%, and 15% were prepared according to the popular use. Then, genetic toxicity of TMAE was evaluated on bacterial reverse mutation, genomic lesions, and micronucleus formation in male rats. Furthermore, a detailed anatomical and histochemical study of the leaves and stems of Tropaeolum majus were performed. No revertant colonies were found in any bacterial cultures examined. In the comet assay, TMAE showed no significant DNA damage in all tested doses. Micronucleus assay showed no significant increases in the frequency of inducing micronuclei in any dose examined. Light and electron microscope images of cross-section of leaves and stems from Tropaeolum majus revealed useful diagnostic features. The presented data showed significant safety parameters for the use of TMAE and provided important data for the quality control of this plant species.

Mutations That Affect the Efficiency of Translation of mRNA for the cII Gene of Coliphage Lambda

PubMed Central

Dul, Ed; Mahoney, Michael E.; Wulff, Daniel L.

1987-01-01

Starting with the λ pRE- strain λctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, λdya2 ctr1 cy3008 and λ dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations of cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'.—The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII- mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A → G mutation four bases before the initial AUG codon, and cII3059 , a GUU → GAU (Val2 → Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested. However neither the ctr1 mutation nor the dya2 mutation has much effect on translation efficiency in an otherwise cII+ background, indicating that other factors must limit the rate of translation of cII mRNA under these conditions. PMID:2953647

Rapamycin prevents, but does not reverse, aberrant migration in Pten knockout neurons.

PubMed

Getz, Stephanie A; DeSpenza, Tyrone; Li, Meijie; Luikart, Bryan W

2016-09-01

Phosphatase and tensin homolog (PTEN) is a major negative regulator of the Akt/mammalian target of rapamycin (MTOR) pathway. Mutations in PTEN have been found in a subset of individuals with autism and macrocephaly. Further, focal cortical dysplasia (FCD) has been observed in patients with PTEN mutations prompting us to examine the role of Pten in neuronal migration. The dentate gyrus of Pten(Flox/Flox) mice was injected with Cre- and non-Cre-expressing retroviral particles, which integrate into the dividing genome to birthdate cells. Control and Pten knockout (KO) cell position in the granule cell layer was quantified over time to reveal that Pten KO neurons exhibit an aberrant migratory phenotype beginning at 7.5days-post retroviral injection (DPI). We then assessed whether rapamycin, a mTor inhibitor, could prevent or reverse aberrant migration of granule cells. The preventative group received daily intraperitoneal (IP) injections of rapamycin from 3 to 14 DPI, before discrepancies in cell position have been established, while the reversal group received rapamycin afterward, from 14 to 24 DPI. We found that rapamycin prevented and reversed somal hypertrophy. However, rapamycin prevented, but did not reverse aberrant migration in Pten KO cells. We also find that altered migration occurs through mTorC1 and not mTorC2 activity. Together, these findings suggest a temporal window by which rapamycin can treat aberrant migration, and may have implications for the use of rapamycin to treat PTEN-mutation associated disorders. Mutations in phosphatase and tensin homolog (PTEN) have been linked to a subset of individuals with autism and macrocephaly, as well as Cowden Syndrome and focal cortical dysplasia. Pten loss leads to neuronal hypertrophy, but the role of Pten in neuronal migration is unclear. Here we have shown that loss of Pten leads to aberrant migration, which can be prevented but not reversed by treatment with rapamycin, a mTor inhibitor. These results are important to consider as clinical trials are developed to examine rapamycin as a therapeutic for autism with PTEN mutations. Our findings show that some abnormalities cannot be reversed, and suggest the potential need for genetic screening and preventative treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Telomerase reverse transcriptase (TERT) promoter mutation analysis of benign, malignant and reactive urothelial lesions reveals a subpopulation of inverted papilloma with immortalizing genetic change.

PubMed

Cheng, Liang; Davidson, Darrell D; Wang, Mingsheng; Lopez-Beltran, Antonio; Montironi, Rodolfo; Wang, Lisha; Tan, Puay-Hoon; MacLennan, Gregory T; Williamson, Sean R; Zhang, Shaobo

2016-07-01

To understand more clearly the genetic ontogeny of inverted papilloma of urinary bladder, we analysed telomerase reverse transcriptase (TERT) promoter mutation status in a group of 26 inverted papillomas in comparison with the mutation status of urothelial carcinoma with inverted growth (26 cases), conventional urothelial carcinoma (36 Ta non-invasive urothelial carcinoma, 35 T2 invasive urothelial carcinoma) and cystitis glandularis (25 cases). TERT promoter mutations in inverted papilloma, urothelial carcinoma with inverted growth, urothelial carcinoma and cystitis glandularis were found in 15% (four of 26), 58% (15 of 26), 63% (45 of 71) and 0% (none of 25), respectively. C228T mutations were the predominant mutations (97%) found in bladder tumours, while C250T aberrations occurred in approximately 3% of bladder tumours. In the inverted papilloma group, TERT mutation occurred predominantly in female patients (P = 0.006). Among urothelial carcinomas, TERT promoter mutation status did not correlate with gender, histological grade or pathological stage. TERT promoter mutations were found in 15% of inverted papillomas. Our data suggest that there is a subpopulation of inverted papilloma that shares a carcinogenetic pathway with urothelial carcinoma with inverted growth and conventional urothelial carcinomas. Caution is warranted in exploring TERT promoter mutation status as a screening or adjunct diagnostic test for bladder cancer. © 2015 John Wiley & Sons Ltd.

Agent Based Modeling of Human Gut Microbiome Interactions and Perturbations.

PubMed

Shashkova, Tatiana; Popenko, Anna; Tyakht, Alexander; Peskov, Kirill; Kosinsky, Yuri; Bogolubsky, Lev; Raigorodskii, Andrei; Ischenko, Dmitry; Alexeev, Dmitry; Govorun, Vadim

2016-01-01

Intestinal microbiota plays an important role in the human health. It is involved in the digestion and protects the host against external pathogens. Examination of the intestinal microbiome interactions is required for understanding of the community influence on host health. Studies of the microbiome can provide insight on methods of improving health, including specific clinical procedures for individual microbial community composition modification and microbiota correction by colonizing with new bacterial species or dietary changes. In this work we report an agent-based model of interactions between two bacterial species and between species and the gut. The model is based on reactions describing bacterial fermentation of polysaccharides to acetate and propionate and fermentation of acetate to butyrate. Antibiotic treatment was chosen as disturbance factor and used to investigate stability of the system. System recovery after antibiotic treatment was analyzed as dependence on quantity of feedback interactions inside the community, therapy duration and amount of antibiotics. Bacterial species are known to mutate and acquire resistance to the antibiotics. The ability to mutate was considered to be a stochastic process, under this suggestion ratio of sensitive to resistant bacteria was calculated during antibiotic therapy and recovery. The model confirms a hypothesis of feedbacks mechanisms necessity for providing functionality and stability of the system after disturbance. High fraction of bacterial community was shown to mutate during antibiotic treatment, though sensitive strains could become dominating after recovery. The recovery of sensitive strains is explained by fitness cost of the resistance. The model demonstrates not only quantitative dynamics of bacterial species, but also gives an ability to observe the emergent spatial structure and its alteration, depending on various feedback mechanisms. Visual version of the model shows that spatial structure is a key factor, which helps bacteria to survive and to adapt to changed environmental conditions.

Appearance of drug resistance-associated mutations in human immunodeficiency virus type 1 protease and reverse transcriptase derived from drug-treated Indonesian patients.

PubMed

Khairunisa, Siti Qamariyah; Kotaki, Tomohiro; Witaningrum, Adiana Mutamsari; Yunifiar M, Muhammad Qushai; Sukartiningrum, Septhia Dwi; Nasronudin; Kameoka, Masanori

2015-02-01

Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.

A novel Met-to-Thr mutation in the YMDD motif of reverse transcriptase from feline immunodeficiency virus confers resistance to oxathiolane nucleosides.

PubMed Central

Smith, R A; Remington, K M; Lloyd, R M; Schinazi, R F; North, T W

1997-01-01

Variants of feline immunodeficiency virus (FIV) that possess a unique methionine-to-threonine mutation within the YMDD motif of reverse transcriptase (RT) were selected by culturing virus in the presence of inhibitory concentrations of (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC]. The mutants were resistant to (-)-FTC and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and additionally exhibited low-level resistance to 2',3'-dideoxycytidine (ddC). DNA sequence analysis of the RT-encoding region of the pol gene amplified from resistant viruses consistently identified a Met-to-Thr mutation in the YMDD motif. Purified RT from the mutants was also resistant to the 5'-triphosphate forms of 3TC, (-)-FTC, and ddC. Site-directed mutants of FIV were engineered which contain either the novel Met-to-Thr mutation or the Met-to-Val mutation seen in oxathiolane nucleoside-resistant HIV-1. Both site-directed mutants displayed resistance to 3TC, thus confirming the role of these mutations in the resistance of FIV to beta-L-3'-thianucleosides. PMID:9032372

Network-Based Identification of Adaptive Pathways in Evolved Ethanol-Tolerant Bacterial Populations.

PubMed

Swings, Toon; Weytjens, Bram; Schalck, Thomas; Bonte, Camille; Verstraeten, Natalie; Michiels, Jan; Marchal, Kathleen

2017-11-01

Efficient production of ethanol for use as a renewable fuel requires organisms with a high level of ethanol tolerance. However, this trait is complex and increased tolerance therefore requires mutations in multiple genes and pathways. Here, we use experimental evolution for a system-level analysis of adaptation of Escherichia coli to high ethanol stress. As adaptation to extreme stress often results in complex mutational data sets consisting of both causal and noncausal passenger mutations, identifying the true adaptive mutations in these settings is not trivial. Therefore, we developed a novel method named IAMBEE (Identification of Adaptive Mutations in Bacterial Evolution Experiments). IAMBEE exploits the temporal profile of the acquisition of mutations during evolution in combination with the functional implications of each mutation at the protein level. These data are mapped to a genome-wide interaction network to search for adaptive mutations at the level of pathways. The 16 evolved populations in our data set together harbored 2,286 mutated genes with 4,470 unique mutations. Analysis by IAMBEE significantly reduced this number and resulted in identification of 90 mutated genes and 345 unique mutations that are most likely to be adaptive. Moreover, IAMBEE not only enabled the identification of previously known pathways involved in ethanol tolerance, but also identified novel systems such as the AcrAB-TolC efflux pump and fatty acids biosynthesis and even allowed to gain insight into the temporal profile of adaptation to ethanol stress. Furthermore, this method offers a solid framework for identifying the molecular underpinnings of other complex traits as well. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Gastrointestinal health effects associated with the consumption of drinking water produced by point-of-use domestic reverse-osmosis filtration units.

PubMed Central

Payment, P; Franco, E; Richardson, L; Siemiatycki, J

1991-01-01

During a prospective epidemiological study of gastrointestinal health effects associated with the consumption of drinking water produced by reverse-osmosis domestic units, a correlation was demonstrated between the bacterial counts on R2A medium incubated at 35 degrees C and the reported gastrointestinal symptoms in families who used these units. A univariate correlation was found with bacterial counts on R2A medium at 20 degrees C but was confounded by the bacterial counts at 35 degrees C. Other variables, such as family size and amount of water consumed, were not independently explanatory of the rate of illness. These observations raise concerns for the possibility of increased disease associated with certain point-of-use treatment devices for domestic use when high levels of bacterial growth occur. PMID:2059052

Prevalence of HIV-1 Subtypes and Drug Resistance-Associated Mutations in HIV-1-Positive Treatment-Naive Pregnant Women in Pointe Noire, Republic of the Congo (Kento-Mwana Project).

PubMed

Bruzzone, Bianca; Saladini, Francesco; Sticchi, Laura; Mayinda Mboungou, Franc A; Barresi, Renata; Caligiuri, Patrizia; Calzi, Anna; Zazzi, Maurizio; Icardi, Giancarlo; Viscoli, Claudio; Bisio, Francesca

2015-08-01

The Kento-Mwana project was carried out in Pointe Noire, Republic of the Congo, to prevent mother-to-child HIV-1 transmission. To determine the prevalence of different subtypes and transmitted drug resistance-associated mutations, 95 plasma samples were collected at baseline from HIV-1-positive naive pregnant women enrolled in the project during the years 2005-2008. Full protease and partial reverse transcriptase sequencing was performed and 68/95 (71.6%) samples were successfully sequenced. Major mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 4/68 (5.9%), 3/68 (4.4%), and 2/68 (2.9%) samples, respectively. Phylogenetic analysis of HIV-1 isolates showed a high prevalence of unique recombinant forms (24/68, 35%), followed by CRF45_cpx (7/68, 10.3%) and subsubtype A3 and subtype G (6/68 each, 8.8%). Although the prevalence of transmitted drug resistance mutations appears to be currently limited, baseline HIV-1 genotyping is highly advisable in conjunction with antiretroviral therapy scale-up in resource-limited settings to optimize treatment and prevent perinatal transmission.

Bayesian network analyses of resistance pathways against efavirenz and nevirapine

PubMed Central

Deforche, Koen; Camacho, Ricardo J.; Grossman, Zehave; Soares, Marcelo A.; Laethem, Kristel Van; Katzenstein, David A.; Harrigan, P. Richard; Kantor, Rami; Shafer, Robert; Vandamme, Anne-Mieke

2016-01-01

Objective To clarify the role of novel mutations selected by treatment with efavirenz or nevirapine, and investigate the influence of HIV-1 subtype on nonnucleoside reverse transcriptase inhibitor (nNRTI) resistance pathways. Design By finding direct dependencies between treatment-selected mutations, the involvement of these mutations as minor or major resistance mutations against efavirenz, nevirapine, or coadministrated nucleoside analogue reverse transcriptase inhibitors (NRTIs) is hypothesized. In addition, direct dependencies were investigated between treatment-selected mutations and polymorphisms, some of which are linked with subtype, and between NRTI and nNRTI resistance pathways. Methods Sequences from a large collaborative database of various subtypes were jointly analyzed to detect mutations selected by treatment. Using Bayesian network learning, direct dependencies were investigated between treatment-selected mutations, NRTI and nNRTI treatment history, and known NRTI resistance mutations. Results Several novel minor resistance mutations were found: 28K and 196R (for resistance against efavirenz), 101H and 138Q (nevirapine), and 31L (lamivudine). Robust interactions between NRTI mutations (65R, 74V, 75I/M, and 184V) and nNRTI resistance mutations (100I, 181C, 190E and 230L) may affect resistance development to particular treatment combinations. For example, an interaction between 65R and 181C predicts that the nevirapine and tenofovir and lamivudine/emtricitabine combination should be more prone to failure than efavirenz and tenofovir and lamivudine/emtricitabine. Conclusion Bayesian networks were helpful in untangling the selection of mutations by NRTI versus nNRTI treatment, and in discovering interactions between resistance mutations within and between these two classes of inhibitors. PMID:18832874

Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability.

PubMed

Lapadat-Tapolsky, M; Gabus, C; Rau, M; Darlix, J L

1997-05-02

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.

Circulating type 1 vaccine-derived poliovirus may evolve under the pressure of adenosine deaminases acting on RNA.

PubMed

Liu, Yanhan; Ma, Tengfei; Liu, Jianzhu; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Xu, Ruixue; Wang, Shujing

2015-01-01

Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of the Picornaviridae family. An effective live-attenuated poliovirus vaccine strain (Sabin 1) has been developed and has protected humans from polio. However, a few cases of vaccine virulence reversion have been documented in several countries. For instance, circulating type 1 vaccine-derived poliovirus is a highly pathogenic poliovirus that evolved from an avirulent strain, but the mechanism by which vaccine strains undergo reversion remains unclear. In this study, vaccine strains exhibited A to G/U to C and G to A/C to U hypermutations in the reversed evolution of Sabin 1. Furthermore, the mutation ratios of U to C and C to U were higher than those of other mutation types. Dinucleotide editing context was then analyzed. Results showed that A to G and U to C mutations exhibited preferences similar to adenosine deaminases acting on RNA (ADAR). Hence, ADARs may participate in poliovirus vaccine evolution.

Bacterial Group II Introns: Identification and Mobility Assay.

PubMed

Toro, Nicolás; Molina-Sánchez, María Dolores; Nisa-Martínez, Rafael; Martínez-Abarca, Francisco; García-Rodríguez, Fernando Manuel

2016-01-01

Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.

Somatic mosaicism caused by monoallelic reversion of a mutation in T cells of a patient with ADA-SCID and the effects of enzyme replacement therapy on the revertant phenotype.

PubMed

Moncada-Vélez, M; Vélez-Ortega, A; Orrego, J; Santisteban, I; Jagadeesh, J; Olivares, M; Olaya, N; Hershfield, M; Candotti, F; Franco, J

2011-11-01

Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T-B- SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in Peripheral blood lymphocytes (PBL), as a result of somatic mosaicism caused by monoallelic reversion of the causative mutation in the ADA gene. He was not eligible for haematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore he was placed on enzyme replacement therapy (ERT) with bovine PEG-ADA. The follow-up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, and sustained expansion of TCRγδ+ T cells. This was accompanied by the disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient's clinical condition improved marginally, he later developed a germinal cell tumour and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

Somatic mosaicism due to monoallelic reversion of a mutation in T cells of an ADA-SCID patient and the effects of enzyme replacement therapy on the revertant phenotype

PubMed Central

Moncada-Vélez, Marcela; Vélez-Ortega, Alejandra C.; Orrego, Julio C.; Santisteban, Inés; Jagadeesh, Jayashree; Olivares, Margarita; Olaya, Natalia; Hershfield, Michael S.; Candotti, Fabio; Franco, Jose L.

2011-01-01

Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T-B-NK- SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in PBL, as a result of somatic mosaicism due to monoallelic reversion of the causative mutation in the ADA gene. Our patient was not eligible for hematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore enzyme replacement therapy (ERT) with bovine PEG-ADA was initiated. The follow up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, with sustained expansion of TCRγδ+ T cells. This was accompanied by disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient’s clinical condition improved marginally, he later developed a germinal cell tumor and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion. PMID:21671975

Determining Mutation Rates in Bacterial Populations

PubMed Central

Rosche, William A.; Foster, Patricia L.

2010-01-01

When properly determined, spontaneous mutation rates are a more accurate and biologically meaningful reflection of the underlying mutagenic mechanism than are mutation frequencies. Because bacteria grow exponentially and mutations arise stochastically, methods to estimate mutation rates depend on theoretical models that describe the distribution of mutant numbers among parallel cultures, as in the original Luria-Delbrück fluctuation analysis. An accurate determination of mutation rate depends on understanding the strengths and limitations of these methods, and how to design fluctuation assays to optimize a given method. In this paper we describe a number of methods to estimate mutation rates, give brief accounts of their derivations, and discuss how they behave under various experimental conditions. PMID:10610800

Genetic screens for mutations affecting development of Xenopus tropicalis.

PubMed

Goda, Tadahiro; Abu-Daya, Anita; Carruthers, Samantha; Clark, Matthew D; Stemple, Derek L; Zimmerman, Lyle B

2006-06-01

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Occurrence of etravirine/rilpivirine-specific resistance mutations selected by efavirenz and nevirapine in Kenyan patients with non-B HIV-1 subtypes failing antiretroviral therapy.

PubMed

Crawford, Keith W; Njeru, Dorothy; Maswai, Jonah; Omondi, Milton; Apollo, Duncan; Kimetto, Jane; Gitonga, Lawrence; Munyao, James; Langat, Raphael; Aoko, Appolonia; Tarus, Jemutai; Khamadi, Samoel; Hamm, Tiffany E

2014-01-28

Resistance to efavirenz and nevirapine has not been associated with mutations at position 138 of reverse transcriptase. In an evaluation of virologic suppression rates in PEPFAR (President's Emergency Plan For AIDS Relief) clinics in Kenya among patients on first-line therapy (RV288), 63% (617/975) of randomly selected patients on antiretroviral therapy were suppressed (HIV RNA<400 copies/ml). Among those with non-nucleoside reverse transcriptase inhibitor resistance (n = 101), 14 (13.8%) had substitutions at 138 (A, G, K or Q), mutations selected only by etravirine and rilpivirine in subtype B viruses. All 14 patients received efavirenz or nevirapine, not etravirine or rilpivirine, and were predominantly subtype A1. This may be the first report of efavirenz and nevirapine selecting these mutations in these subtypes.

Genetic analysis of an Escherichia coli syndrome.

PubMed

Lennette, E T; Apirion, D

1971-12-01

A mutant strain of Escherichia coli that fails to recover from prolonged (72 hr) starvation also fails to grow at 43 C. Extracts of this mutant strain show an increased ribonuclease II activity as compared to extracts of the parental strain, and stable ribonucleic acid is degraded to a larger extent in this strain during starvation. Ts(+) transductants and revertants were tested for all the above-mentioned phenotypes. All the Ts(+) transductants and revertants tested behaved like the Ts(+) parental strain, which suggests that all the observed phenotypes are caused by a single sts (starvation-temperature sensitivity) mutation. The reversion rate from sts(-) to sts(+) is rather low but is within the range of reversion rates for other single-site mutations. Three-point transduction crosses located this sts mutation between the ilv and rbs genes. The properties of sts(+)/sts(-) merozygotes suggested that the Ts(-) phenotype of this mutation is recessive.

Reverse cascade screening of newborns for hereditary haemochromatosis: a model for other late onset diseases?

PubMed

Cadet, E; Capron, D; Gallet, M; Omanga-Léké, M-L; Boutignon, H; Julier, C; Robson, K J H; Rochette, J

2005-05-01

Genetic testing can determine those at risk for hereditary haemochromatosis (HH) caused by HFE mutations before the onset of symptoms. However, there is no optimum screening strategy, mainly owing to the variable penetrance in those who are homozygous for the HFE Cys282Tyr (C282Y) mutation. The objective of this study was to identify the majority of individuals at serious risk of developing HFE haemochromatosis before they developed life threatening complications. We first estimated the therapeutic penetrance of the C282Y mutation in people living in la Somme, France, using genetic, demographic, biochemical, and follow up data. We examined the benefits of neonatal screening on the basis of increased risk to relatives of newborns carrying one or two copies of the C282Y mutation. Between 1999 and 2002, we screened 7038 newborns from two maternity hospitals in the north of France for the C282Y and His63Asp (H63D) mutations in the HFE gene, using bloodspots collected on Guthrie cards. Family studies and genetic counselling were undertaken, based on the results of the baby's genotype. In la Somme, we found that 24% of the adults homozygous for the C282Y mutation required at least 5 g iron to be removed to restore normal iron parameters (that is, the therapeutic penetrance). In the reverse cascade screening study, we identified 19 C282Y homozygotes (1/370), 491 heterozygotes (1/14) and 166 compound heterozygotes (1/42) in 7038 newborns tested. The reverse cascade screening strategy resulted in 80 adults being screened for both mutations. We identified 10 previously unknown C282Y homozygotes of whom six (four men and two women) required venesection. Acceptance of neonatal screening was high; parents understood the risks of having HH and the benefits of early detection, but a number of parents were reluctant to take the test themselves. Neonatal screening for HH is straightforward. Reverse cascade screening increased the efficiency of detecting affected adults with undiagnosed haemochromatosis. This strategy allows almost complete coverage for HH and could be a model for efficient screening for other late onset genetic diseases.

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

PubMed Central

Limmathurotsakul, Direk; Max, Tamara L.; Sarovich, Derek S.; Vogler, Amy J.; Dale, Julia L.; Ginther, Jennifer L.; Leadem, Benjamin; Colman, Rebecca E.; Foster, Jeffrey T.; Tuanyok, Apichai; Wagner, David M.; Peacock, Sharon J.; Pearson, Talima; Keim, Paul

2010-01-01

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum. PMID:20090837

Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

PubMed Central

Döring, Jessica

2017-01-01

Abstract Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication. PMID:28160599

A lysozyme and magnetic bead based method of separating intact bacteria.

PubMed

Diler, Ebru; Obst, Ursula; Schmitz, Katja; Schwartz, Thomas

2011-07-01

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO(3)-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage

PubMed Central

Guerrini, Valentina; Subbian, Selvakumar; Santucci, Pierre; Canaan, Stéphane; Pozzi, Gianni

2016-01-01

Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10−9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth. PMID:27959952

Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage.

PubMed

Guerrini, Valentina; Subbian, Selvakumar; Santucci, Pierre; Canaan, Stéphane; Gennaro, Maria Laura; Pozzi, Gianni

2016-01-01

Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.

Standardized comparison of the relative impacts of HIV-1 reverse transcriptase (RT) mutations on nucleoside RT inhibitor susceptibility.

PubMed

Melikian, George L; Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V; Zolopa, Andrew; Robbins, Gregory K; Kagan, Ron; Israelski, Dennis; Shafer, Robert W

2012-05-01

Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation.

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation.

PubMed

Huang, Dong-Sheng; Wang, Zhaohui; He, Xu-Jun; Diplas, Bill H; Yang, Rui; Killela, Patrick J; Meng, Qun; Ye, Zai-Yuan; Wang, Wei; Jiang, Xiao-Ting; Xu, Li; He, Xiang-Lei; Zhao, Zhong-Sheng; Xu, Wen-Juan; Wang, Hui-Ju; Ma, Ying-Yu; Xia, Ying-Jie; Li, Li; Zhang, Ru-Xuan; Jin, Tao; Zhao, Zhong-Kuo; Xu, Ji; Yu, Sheng; Wu, Fang; Liang, Junbo; Wang, Sizhen; Jiao, Yuchen; Yan, Hai; Tao, Hou-Quan

2015-05-01

Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mutation exposed: a neutral explanation for extreme base composition of an endosymbiont genome.

PubMed

Wernegreen, Jennifer J; Funk, Daniel J

2004-12-01

The influence of neutral mutation pressure versus selection on base composition evolution is a subject of considerable controversy. Yet the present study represents the first explicit population genetic analysis of this issue in prokaryotes, the group in which base composition variation is most dramatic. Here, we explore the impact of mutation and selection on the dynamics of synonymous changes in Buchnera aphidicola, the AT-rich bacterial endosymbiont of aphids. Specifically, we evaluated three forms of evidence. (i) We compared the frequencies of directional base changes (AT-->GC vs. GC-->AT) at synonymous sites within and between Buchnera species, to test for selective preference versus effective neutrality of these mutational categories. Reconstructed mutational changes across a robust intraspecific phylogeny showed a nearly 1:1 AT-->GC:GC-->AT ratio. Likewise, stationarity of base composition among Buchnera species indicated equal rates of AT-->GC and GC-->AT substitutions. The similarity of these patterns within and between species supported the neutral model. (ii) We observed an equivalence of relative per-site AT mutation rate and current AT content at synonymous sites, indicating that base composition is at mutational equilibrium. (iii) We demonstrated statistically greater equality in the frequency of mutational categories in Buchnera than in parallel mammalian studies that documented selection on synonymous sites. Our results indicate that effectively neutral mutational pressure, rather than selection, represents the major force driving base composition evolution in Buchnera. Thus they further corroborate recent evidence for the critical role of reduced N(e) in the molecular evolution of bacterial endosymbionts.

A substitution in the transmembrane region of the glycoprotein leads to an unstable attenuation of Machupo virus.

PubMed

Patterson, Michael; Koma, Takaaki; Seregin, Alexey; Huang, Cheng; Miller, Milagros; Smith, Jennifer; Yun, Nadezhda; Smith, Jeanon; Paessler, Slobodan

2014-09-01

Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing a reverse-genetics system recently developed, we report the rescue of a rationally modified recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice, we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild-type genotype. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A novel, de novo mutation in the PRKAG2 gene: infantile-onset phenotype and the signaling pathway involved.

PubMed

Xu, Yanchun; Gray, A; Hardie, D Grahame; Uzun, Alper; Shaw, Sunil; Padbury, James; Phornphutkul, Chanika; Tseng, Yi-Tang

2017-08-01

PRKAG2 encodes the γ 2 -subunit isoform of 5'-AMP-activated protein kinase (AMPK), a heterotrimeric enzyme with major roles in the regulation of energy metabolism in response to cellular stress. Mutations in PRKAG2 have been implicated in a unique hypertrophic cardiomyopathy (HCM) characterized by cardiac glycogen overload, ventricular preexcitation, and hypertrophy. We identified a novel, de novo PRKAG2 mutation (K475E) in a neonate with prenatal onset of HCM. We aimed to investigate the cellular impact, signaling pathways involved, and therapeutic options for K475E mutation using cells stably expressing human wild-type (WT) or the K475E mutant. In human embryonic kidney-293 cells, the K475E mutation induced a marked increase in the basal phosphorylation of T172 and AMPK activity, reduced sensitivity to AMP in allosteric activation, and a loss of response to phenformin. In H9c2 cardiomyocytes, the K475E mutation induced inhibition of AMPK and reduced the response to phenformin and increases in the phosphorylation of p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Primary fibroblasts from the patient with the K475E mutation also showed marked increases in the phosphorylation of p70S6K and 4E-BP1 compared with those from age-matched, nondiseased controls. Moreover, overexpression of K475E induced hypertrophy in H9c2 cells, which was effectively reversed by treatment with rapamycin. Taken together, we have identified a novel, de novo infantile-onset PRKAG2 mutation causing HCM. Our study suggests the K475E mutation induces alteration in basal AMPK activity and results in a hypertrophy phenotype involving the mechanistic target of rapamycin signaling pathway, which can be reversed with rapamycin. NEW & NOTEWORTHY We identified a novel, de novo PRKAG2 mutation (K475E) in the cystathionine β-synthase 3 repeat, a region critical for AMP binding but with no previous reported mutation. Our data suggest the mutation affects AMP-activated protein kinase activity, activates cell growth pathways, and results in cardiac hypertrophy, which can be reversed with rapamycin. Copyright © 2017 the American Physiological Society.

Suppression of Beneficial Mutations in Dynamic Microbial Populations

NASA Astrophysics Data System (ADS)

Bittihn, Philip; Hasty, Jeff; Tsimring, Lev S.

2017-01-01

Quantitative predictions for the spread of mutations in bacterial populations are essential to interpret evolution experiments and to improve the stability of synthetic gene circuits. We derive analytical expressions for the suppression factor for beneficial mutations in populations that undergo periodic dilutions, covering arbitrary population sizes, dilution factors, and growth advantages in a single stochastic model. We find that the suppression factor grows with the dilution factor and depends nontrivially on the growth advantage, resulting in the preferential elimination of mutations with certain growth advantages. We confirm our results by extensive numerical simulations.

Reverse genetics of Newcastle disease virus

USDA-ARS?s Scientific Manuscript database

Reverse genetics allows the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique allows genetic manipulation and cloning of viral genomes, mutation through site-directed mutagenesis, and gene insertion or deletion, among othe...

[Bacterial biofilms on PVC tubing's inner surface of hemodialysis water treatment system].

PubMed

Yang, Sha; Jia, Ke; Peng, Youming; Liu, Hong; Liu, Yinghong; Chen, Xing; Liu, Fuyou

2009-10-01

To determine the morphology, bacteria and endotoxin content of biofilms on the inner surface of PVC tubes in hemodialysis water treatment system. We dissolved biofilms of segments before and after reverse osmosis machine for bacterial count and identification. We studied biofilm structure of segments before and after reverse osmosis machine with eyes and scanning electron microscope. Biofilms of all 7 segments were dissolved for qualitative and quantitative assay of endotoxin. The inner surface of segment before reverse osmosis machine was homogeneously distributed with activated carbon powder deposition. The segment after reverse osmosis machine was normal. With scanning electron microscope, biofilm with successive surface and sandwich was found on the inner surface of segment before reverse osmosis machine, formed by clustering bacillus, activated carbon powder and some coccus. Bacteria of the same shape and length were found on segment after reverse osmosis machine, but fewer and looser. Bacterial culture and identification showed the former was mostly gram-negative bacillus, the latter was only a few micrococcus. Endotoxin of biofilm was between 2.0 EU/mL and 4.0 EU/mL. Quantitative assay showed: segment after softener (2.821+/-0.807) EU/mL; segment after active charcoal canister(3.635+/-0.427) EU/mL; segment before reverse osmosis machine (3.687+/-0.271) EU/mL; segment after reverse osmosis machine (2.041+/-0.295) EU/mL; exit of power pump (1.983+/-0.390)EU/mL;the 1st dead space (2.373+/-0.535) EU/mL; and the 2nd dead space (2.858+/-0.690)EU/mL. Biofilms are found on the inner surface of segment before and after reverse osmosis machine. Endotoxin level from high to low is as follows: segment before reverse osmosis machine, segment after active charcoal canister, the 2nd dead space, segment after softener, the 1st dead space, segment after reverse osmosis machine, exit of power pump. The character of the bacteria and endotoxin of the biofilm can help us find better ways to control them.

An integrated molecular dynamics, principal component analysis and residue interaction network approach reveals the impact of M184V mutation on HIV reverse transcriptase resistance to lamivudine.

PubMed

Bhakat, Soumendranath; Martin, Alberto J M; Soliman, Mahmoud E S

2014-08-01

The emergence of different drug resistant strains of HIV-1 reverse transcriptase (HIV RT) remains of prime interest in relation to viral pathogenesis as well as drug development. Amongst those mutations, M184V was found to cause a complete loss of ligand fitness. In this study, we report the first account of the molecular impact of M184V mutation on HIV RT resistance to 3TC (lamivudine) using an integrated computational approach. This involved molecular dynamics simulation, binding free energy analysis, principle component analysis (PCA) and residue interaction networks (RINs). Results clearly confirmed that M184V mutation leads to steric conflict between 3TC and the beta branched side chain of valine, decreases the ligand (3TC) binding affinity by ∼7 kcal mol(-1) when compared to the wild type, changes the overall conformational landscape of the protein and distorts the native enzyme residue-residue interaction network. The comprehensive molecular insight gained from this study should be of great importance in understanding drug resistance against HIV RT as well as assisting in the design of novel reverse transcriptase inhibitors with high ligand efficacy on resistant strains.

Cooperative Bacterial Growth Dynamics Predict the Evolution of Antibiotic Resistance

NASA Astrophysics Data System (ADS)

Artemova, Tatiana; Gerardin, Ylaine; Hsin-Jung Li, Sophia; Gore, Jeff

2011-03-01

Since the discovery of penicillin, antibiotics have been our primary weapon against bacterial infections. Unfortunately, bacteria can gain resistance to penicillin by acquiring the gene that encodes beta-lactamase, which inactivates the antibiotic. However, mutations in this gene are necessary to degrade the modern antibiotic cefotaxime. Understanding the conditions that favor the spread of these mutations is a challenge. Here we show that bacterial growth in beta-lactam antibiotics is cooperative and that the nature of this growth determines the conditions in which resistance evolves. Quantitative analysis of the growth dynamics predicts a peak in selection at very low antibiotic concentrations; competition between strains confirms this prediction. We also find significant selection at higher antibiotic concentrations, close to the minimum inhibitory concentrations of the strains. Our results argue that an understanding of the evolutionary forces that lead to antibiotic resistance requires a quantitative understanding of the evolution of cooperation in bacteria.

In Vitro Resistance Profile of the Candidate HIV-1 Microbicide Drug Dapivirine

PubMed Central

Schader, Susan M.; Oliveira, Maureen; Ibanescu, Ruxandra-Ilinca; Moisi, Daniela; Colby-Germinario, Susan P.

2012-01-01

Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries. PMID:22123692

In vitro resistance profile of the candidate HIV-1 microbicide drug dapivirine.

PubMed

Schader, Susan M; Oliveira, Maureen; Ibanescu, Ruxandra-Ilinca; Moisi, Daniela; Colby-Germinario, Susan P; Wainberg, Mark A

2012-02-01

Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries.

A Toxicological Evaluation of a Standardized Hydrogenated Extract of Curcumin (CuroWhite™)

PubMed Central

Ravikumar, Alastimmanahalli Narasimhiah; Jacob, Joby

2018-01-01

A series of toxicological investigations were conducted in order to evaluate the genotoxic potential and repeated-dose oral toxicity of CuroWhite, a proprietary extract of curcumin that has been hydrogenated and standardized to not less than 25% hydrogenated curcuminoid content. All tests were conducted in general accordance with internationally accepted standards. The test item was not mutagenic in the bacterial reverse mutation test or in vitro mammalian chromosomal aberration test, and no in vivo genotoxic activity was observed in rat bone marrow in the micronucleus test. A 90-day repeated-dose study was conducted in male and female Sprague-Dawley rats. Two mortalities occurred in the main and satellite high-dose groups and were determined due to gavage error. No organ specific or other toxic effects of the test item were observed up to the maximum dose of 800 mg/kg bw/day, administered by gavage. NOAEL was, therefore, estimated as 800 mg/kg bw/day. PMID:29610573

A Toxicological Evaluation of a Standardized Hydrogenated Extract of Curcumin (CuroWhite™).

PubMed

Ravikumar, Alastimmanahalli Narasimhiah; Jacob, Joby; Gopi, Sreeraj; Jagannath, Tumkur Subbarao

2018-01-01

A series of toxicological investigations were conducted in order to evaluate the genotoxic potential and repeated-dose oral toxicity of CuroWhite, a proprietary extract of curcumin that has been hydrogenated and standardized to not less than 25% hydrogenated curcuminoid content. All tests were conducted in general accordance with internationally accepted standards. The test item was not mutagenic in the bacterial reverse mutation test or in vitro mammalian chromosomal aberration test, and no in vivo genotoxic activity was observed in rat bone marrow in the micronucleus test. A 90-day repeated-dose study was conducted in male and female Sprague-Dawley rats. Two mortalities occurred in the main and satellite high-dose groups and were determined due to gavage error. No organ specific or other toxic effects of the test item were observed up to the maximum dose of 800 mg/kg bw/day, administered by gavage. NOAEL was, therefore, estimated as 800 mg/kg bw/day.

Common structural changes accompany the functional inactivation of HPr by seryl phosphorylation or by serine to aspartate substitution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wittekind, M.; Klevit, R.E.; Reizer, J.

1989-12-26

Although many proteins are known to be regulated via reversible phosphorylation, little is known about the mechanism by which the covalent modification of seryl, threonyl, or tyrosyl residues alters the activities of the target systems. To address this question, modified versions of bacillus subtilus HPr, a protein component of the bacterial phosphotransferase system, have been studied by {sup 1}H NMR spectroscopy. Phosphorylation at Ser{sub 46} or a Ser to Asp substitution at this position inactivates HPr. Two-dimensional spectra of these two modified proteins display nearly identical proton chemical shifts that differ significantly from those observed in the spectra of themore » unphosphorylated, wild-type protein and of functionally active HPr mutants. These results demonstrate that the functional inactivation of HPr brought about by the serine to aspartate mutation is accompanied by the same structural changes that occur when HPr is phosphorylated at Ser{sub 46}.« less

Genotoxicity, acute oral and dermal toxicity, eye and dermal irritation and corrosion and skin sensitisation evaluation of silver nanoparticles.

PubMed

Kim, Jin Sik; Song, Kyung Seuk; Sung, Jae Hyuck; Ryu, Hyun Ryol; Choi, Byung Gil; Cho, Hyun Sun; Lee, Jin Kyu; Yu, Il Je

2013-08-01

To clarify the health risks related to silver nanoparticles (Ag-NPs), we evaluated the genotoxicity, acute oral and dermal toxicity, eye irritation, dermal irritation and corrosion and skin sensitisation of commercially manufactured Ag-NPs according to the OECD test guidelines and GLP. The Ag-NPs were not found to induce genotoxicity in a bacterial reverse mutation test and chromosomal aberration test, although some cytotoxicity was observed. In acute oral and dermal toxicity tests using rats, none of the rats showed any abnormal signs or mortality at a dose level of ∼ 2000 mg/kg. Similarly, acute eye and dermal irritation and corrosion tests using rabbits revealed no significant clinical signs or mortality and no acute irritation or corrosion reaction for the eyes and skin. In a skin sensitisation test using guinea pigs, one animal (1/20) showed discrete or patchy erythema, thus Ag-NPs can be classified as a weak skin sensitiser.

Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication.

PubMed Central

Kim, T; Mudry, R A; Rexrode, C A; Pathak, V K

1996-01-01

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively. PMID:8892879

Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution

PubMed Central

Lambowitz, Alan M.; Belfort, Marlene

2015-01-01

SUMMARY This review focuses on recent developments in our understanding of group II intron function, the relationships of these introns to retrotransposons and spliceosomes, and how their common features have informed thinking about bacterial group II introns as key elements in eukaryotic evolution. Reverse transcriptase-mediated and host factor-aided intron retrohoming pathways are considered along with retrotransposition mechanisms to novel sites in bacteria, where group II introns are thought to have originated. DNA target recognition and movement by target-primed reverse transcription infer an evolutionary relationship among group II introns, non-LTR retrotransposons, such as LINE elements, and telomerase. Additionally, group II introns are almost certainly the progenitors of spliceosomal introns. Their profound similarities include splicing chemistry extending to RNA catalysis, reaction stereochemistry, and the position of two divalent metals that perform catalysis at the RNA active site. There are also sequence and structural similarities between group II introns and the spliceosome’s small nuclear RNAs (snRNAs) and between a highly conserved core spliceosomal protein Prp8 and a group II intron-like reverse transcriptase. It has been proposed that group II introns entered eukaryotes during bacterial endosymbiosis or bacterial-archaeal fusion, proliferated within the nuclear genome, necessitating evolution of the nuclear envelope, and fragmented giving rise to spliceosomal introns. Thus, these bacterial self-splicing mobile elements have fundamentally impacted the composition of extant eukaryotic genomes, including the human genome, most of which is derived from close relatives of mobile group II introns. PMID:25878921

Antiretroviral drug susceptibility among drug-naive adults with recent HIV infection in Rakai, Uganda

PubMed Central

Eshleman, Susan H.; Laeyendecker, Oliver; Parkin, Neil; Huang, Wei; Chappey, Colombe; Paquet, Agnes C.; Serwadda, David; Reynolds, Steven J.; Kiwanuka, Noah; Quinn, Thomas C.; Gray, Ronald; Wawer, Maria

2009-01-01

Objective To analyze antiretroviral drug susceptibility in HIV from recently infected adults in Rakai, Uganda, prior to the availability of antiretroviral drug treatment. Methods Samples obtained at the time of HIV seroconversion (1998–2003) were analyzed using the GeneSeq HIV and PhenoSense HIV assays (Monogram Biosciences, Inc., South San Francisco, California, USA). Results Test results were obtained for 104 samples (subtypes: 26A, 1C, 66D, 9A/D, 1C/D, 1 intersubtype recombinant). Mutations used for genotypic surveillance of transmitted antiretroviral drug resistance were identified in six samples: three had nucleoside reverse transcriptase inhibitor (NRTI) surveillance mutations (two had M41L, one had K219R), and three had protease inhibitor surveillance mutations (I47V, F53L, N88D); none had nonnucleoside reverse transcriptase inhibitor (NNRTI) surveillance mutations. Other resistance-associated mutations were identified in some samples. However, none of the samples had a sufficient number of mutations to predict reduced antiretroviral drug susceptibility. Ten (9.6%) of the samples had reduced phenotypic susceptibility to at least one drug (one had partial susceptibility to didanosine, one had nevirapine resistance, and eight had resistance or partial susceptibility to at least one protease inhibitor). Fifty-three (51%) of the samples had hypersusceptibility to at least one drug (seven had zidovudine hypersusceptibility, 28 had NNRTI hypersusceptibility, 34 had protease inhibitor hypersusceptibility). Delavirdine hyper-susceptibility was more frequent in subtype A than D. In subtype D, efavirenz hypersusceptibility was associated with substitutions at codon 11 in HIV-reverse transcriptase. Conclusion Phenotyping detected reduced antiretroviral drug susceptibility and hypersusceptibility in HIV from some antiretroviral-naive Ugandan adults that was not predicted by genotyping. Phenotyping may complement genotyping for analysis of antiretroviral drug susceptibility in populations with nonsubtype B HIV infection. PMID:19276794

Antiretroviral drug susceptibility among drug-naive adults with recent HIV infection in Rakai, Uganda.

PubMed

Eshleman, Susan H; Laeyendecker, Oliver; Parkin, Neil; Huang, Wei; Chappey, Colombe; Paquet, Agnes C; Serwadda, David; Reynolds, Steven J; Kiwanuka, Noah; Quinn, Thomas C; Gray, Ronald; Wawer, Maria

2009-04-27

To analyze antiretroviral drug susceptibility in HIV from recently infected adults in Rakai, Uganda, prior to the availability of antiretroviral drug treatment. Samples obtained at the time of HIV seroconversion (1998-2003) were analyzed using the GeneSeq HIV and PhenoSense HIV assays (Monogram Biosciences, Inc., South San Francisco, California, USA). Test results were obtained for 104 samples (subtypes: 26A, 1C, 66D, 9A/D, 1C/D, 1 intersubtype recombinant). Mutations used for genotypic surveillance of transmitted antiretroviral drug resistance were identified in six samples: three had nucleoside reverse transcriptase inhibitor (NRTI) surveillance mutations (two had M41L, one had K219R), and three had protease inhibitor surveillance mutations (I47V, F53L, N88D); none had nonnucleoside reverse transcriptase inhibitor (NNRTI) surveillance mutations. Other resistance-associated mutations were identified in some samples. However, none of the samples had a sufficient number of mutations to predict reduced antiretroviral drug susceptibility. Ten (9.6%) of the samples had reduced phenotypic susceptibility to at least one drug (one had partial susceptibility to didanosine, one had nevirapine resistance, and eight had resistance or partial susceptibility to at least one protease inhibitor). Fifty-three (51%) of the samples had hypersusceptibility to at least one drug (seven had zidovudine hypersusceptibility, 28 had NNRTI hypersusceptibility, 34 had protease inhibitor hypersusceptibility). Delavirdine hypersusceptibility was more frequent in subtype A than D. In subtype D, efavirenz hypersusceptibility was associated with substitutions at codon 11 in HIV-reverse transcriptase. Phenotyping detected reduced antiretroviral drug susceptibility and hypersusceptibility in HIV from some antiretroviral-naive Ugandan adults that was not predicted by genotyping. Phenotyping may complement genotyping for analysis of antiretroviral drug susceptibility in populations with nonsubtype B HIV infection.

A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants.

PubMed

DebRoy, Sruti; Thilmony, Roger; Kwack, Yong-Bum; Nomura, Kinya; He, Sheng Yang

2004-06-29

Salicylic acid (SA)-mediated host immunity plays a central role in combating microbial pathogens in plants. Inactivation of SA-mediated immunity, therefore, would be a critical step in the evolution of a successful plant pathogen. It is known that mutations in conserved effector loci (CEL) in the plant pathogens Pseudomonas syringae (the Delta CEL mutation), Erwinia amylovora (the dspA/E mutation), and Pantoea stewartii subsp. stewartii (the wtsE mutation) exert particularly strong negative effects on bacterial virulence in their host plants by unknown mechanisms. We found that the loss of virulence in Delta CEL and dspA/E mutants was linked to their inability to suppress cell wall-based defenses and to cause normal disease necrosis in Arabidopsis and apple host plants. The Delta CEL mutant activated SA-dependent callose deposition in wild-type Arabidopsis but failed to elicit high levels of callose-associated defense in Arabidopsis plants blocked in SA accumulation or synthesis. This mutant also multiplied more aggressively in SA-deficient plants than in wild-type plants. The hopPtoM and avrE genes in the CEL of P. syringae were found to encode suppressors of this SA-dependent basal defense. The widespread conservation of the HopPtoM and AvrE families of effectors in various bacteria suggests that suppression of SA-dependent basal immunity and promotion of host cell death are important virulence strategies for bacterial infection of plants.

Comprehensive evaluation of the flavonol anti-oxidants, alpha-glycosyl isoquercitrin and isoquercitrin, for genotoxic potential.

PubMed

Hobbs, Cheryl A; Koyanagi, Mihoko; Swartz, Carol; Davis, Jeffrey; Kasamoto, Sawako; Maronpot, Robert; Recio, Leslie; Hayashi, Shim-Mo

2018-03-01

Quercetin and its glycosides possess potential benefits to human health. Several flavonols are available to consumers as dietary supplements, promoted as anti-oxidants; however, incorporation of natural quercetin glycosides into food and beverage products has been limited by poor miscibility in water. Enzymatic conjugation of multiple glucose moieties to isoquercitrin to produce alpha-glycosyl isoquercitrin (AGIQ) enhances solubility and bioavailability. AGIQ is used in Japan as a food additive and has been granted generally recognized as safe (GRAS) status. However, although substantial genotoxicity data exist for quercetin, there is very little available data for AGIQ and isoquercitrin. To support expanded global marketing of food products containing AGIQ, comprehensive testing of genotoxic potential of AGIQ and isoquercitrin was conducted according to current regulatory test guidelines. Both chemicals tested positive in bacterial reverse mutation assays, and exposure to isoquercitrin resulted in chromosomal aberrations in CHO-WBL cells. All other in vitro mammalian micronucleus and chromosomal aberration assays, micronucleus and comet assays in male and female B6C3F1 mice and Sprague Dawley rats, and Muta™ Mouse mutation assays evaluating multiple potential target tissues, were negative for both chemicals. These results supplement existing toxicity data to further support the safe use of AGIQ in food and beverage products. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency,more » the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.« less

COTIP: Cotton TILLING Platform, a Resource for Plant Improvement and Reverse Genetic Studies

PubMed Central

Aslam, Usman; Cheema, Hafiza M. N.; Ahmad, Sheraz; Khan, Iqrar A.; Malik, Waqas; Khan, Asif A.

2016-01-01

Cotton is cultivated worldwide for its white fiber, of which around 90% is tetraploid upland cotton (Gossypium hirsutum L.) carrying both A and D genome. Since centuries, yield increasing efforts for the cotton crop by conventional breeding approaches have caused an extensive erosion of natural genetic variability. Mutation based improvement strategies provide an effective way of creating new allelic variations. Targeting Induced Local Lesions IN Genomes (TILLING) provides a mutation based reverse genetic strategy to create and evaluate induced genetic variability at DNA level. Here, we report development and testing of TILLING populations of allotetraploid cotton (G. hirsutum) for functional genomic studies and mutation based enrichment of cotton genetic resources. Seed of two cotton cultivars “PB-899 and PB-900” were mutagenized with 0.3 and 0.2% (v/v) ethyl methanesulfonate, respectively. The phenotyping of M1 and M2 populations presented numerous mutants regarding the branching pattern, leaf morphology, disease resistance, photosynthetic lesions and flower sterility. Molecular screening for point mutations was performed by TILLING PCR aided CEL1 mismatch cleavage. To estimate the mutation frequency in the mutant genomes, five gene classes were TILLed in 8000 M2 plants of each var. “PB-899” and “PB-900.” These include actin (GhACT), Pectin Methyl Esterase (GhPME), sucrose synthase (GhSUS), resistance gene analog, and defense response gene (DRGs). The var. PB-899 was harboring 47% higher mutation induction rate than PB-900. The highest rate of mutation frequency was identified for NAC-TF5 (EU706348) of DRGs class, ranging from 1/58 kb in PB-899 to 1/105 kb in PB-900. The mutation screening assay revealed the presence of significant proportion of induced mutations in cotton TILLING populations such as 1/153 kb and 1/326 kb in var. “PB-899” and “PB-900,” respectively. The establishment of a cotton TILLING platform (COTIP) and data obtained from the resource TILLING population suggest its effectiveness in widening the genetic bases of cotton for improvement and utilizing it for subsequent reverse genetic studies of various genes. PMID:28082993

A novel homozygous Fas ligand mutation leads to early protein truncation, abrogation of death receptor and reverse signaling and a severe form of the autoimmune lymphoproliferative syndrome.

PubMed

Nabhani, Schafiq; Hönscheid, Andrea; Oommen, Prasad T; Fleckenstein, Bernhard; Schaper, Jörg; Kuhlen, Michaela; Laws, Hans-Jürgen; Borkhardt, Arndt; Fischer, Ute

2014-12-01

We report a novel type of mutation in the death ligand FasL that was associated with a severe phenotype of the autoimmune lymphoproliferative syndrome in two patients. A frameshift mutation in the intracellular domain led to complete loss of FasL expression. Cell death signaling via its receptor and reverse signaling via its intracellular domain were completely abrogated. In vitro lymphocyte proliferation induced by weak T cell receptor stimulation could be blocked and cell death was induced by engagement of FasL in T cells derived from healthy individuals and a heterozygous carrier, but not in FasL-deficient patient derived cells. Expression of genes implicated in lymphocyte proliferation and activation (CCND1, NFATc1, NF-κB1) was increased in FasL-deficient T cells and could not be downregulated by FasL engagement as in healthy cells. Our data thus suggest, that deficiency in FasL reverse signaling may contribute to the clinical lymphoproliferative phenotype of ALPS. Copyright © 2014 Elsevier Inc. All rights reserved.

Novel nonnucleoside inhibitors that select nucleoside inhibitor resistance mutations in human immunodeficiency virus type 1 reverse transcriptase.

PubMed

Zhang, Zhijun; Walker, Michelle; Xu, Wen; Shim, Jae Hoon; Girardet, Jean-Luc; Hamatake, Robert K; Hong, Zhi

2006-08-01

Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.

Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

PubMed Central

Zhang, Zhijun; Walker, Michelle; Xu, Wen; Shim, Jae Hoon; Girardet, Jean-Luc; Hamatake, Robert K.; Hong, Zhi

2006-01-01

Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies. PMID:16870771

Evaluation of the systemic toxicity and mutagenicity of OLIGOPIN®, procyanidolic oligomers (OPC) extracted from French Maritime Pine Bark extract.

PubMed

Segal, L; Penman, M G; Piriou, Y

2018-01-01

The potential systemic toxicity of Oligopin®, a French Maritime Pine Bark extract (FMPBE) rich in procyanidolic oligomers, was evaluated in an acute oral limit test and a 90-day repeated dose oral toxicity study with Sprague Dawley rats. The potential mutagenicity was assessed in a bacterial reverse mutation assay and in vitro mammalian chromosome aberration assay with human lymphocytes. The results indicate that Oligopin® was nongenotoxic in both bacterial and human cell assays, was not acutely toxic via oral administration at up to 2000 mg/kg and was well tolerated following 90 days of oral administration to SD rats, with a no observed adverse effect level of 1000 mg/kg/day. The lack of significant adverse systemic effects in the 90 day study is concordant with findings from several human clinical trials. The acute toxicity and mutagenicity data are consistent with data reported by AFSSA in a summary of FMPBE safety, in which a NOAEL of 100 mg/kg/day was established. In contrast, the NOAEL derived from the 90-day study with Oligopin® was 1000 mg/kg/day, suggesting that it is less systemically toxic than other FMPBE previously evaluated in subchronic studies, and comparable to proanthocyanidins extracted from grape seeds, which are widely used as nutritional supplement ingredients.

Ultra high performance liquid chromatography with tandem mass spectrometry screening and mutagenicity evaluation of photodegradation products of Carmoisine (E122) dye in a beverage.

PubMed

Yamjala, Karthik; Nainar, Meyyanathan Subramania; Ramisetti, Nageswara Rao

2016-06-01

The present study deals with the separation and identification of the photodegradation products formed when a commercial soft drink containing Carmoisine (E122) dye was exposed to natural sunlight. An ultra high performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry method was developed and validated to identify the unknown species of E122. During the study, it was observed that the dye decolourizes rapidly in beverage when compared to model standard solutions. The sunlight irradiation of beverage containing E122 resulted in four photodegradation products as identified by nontarget screening using high-resolution tandem mass spectrometry. Accurate mass measurements were used to identify the elemental composition, and to elucidate the structures of degradation products a software tool was employed. The degradation products (P1-P4) were formed from the interactions of the dye with other ingredients present in the beverage. The toxicity of the degradation products was evaluated on five bacterial strains (TA98, TA100, TA1535, TA1537, and WP2 uvrA pKM101) through an in vitro bacterial reverse mutation assay. The photodegradation products showed strong mutagenic potential in strain TA 100 (without S9) as detected by the Ames assay. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Di-epoxides of the three isomeric dicyclopenta-fused pyrenes: ultimate mutagenic active agents.

PubMed

Otero-Lobato, María José; Kaats-Richters, Veronica E M; Havenith, Remco W A; Jenneskens, Leonardus W; Seinen, Willem

2004-11-14

To rationalize the high bacterial mutagenic response recently found for the (di-) cyclopenta-fused pyrene congeners, viz. cyclopenta[cd]-(1), dicyclopenta[cd,mn]-(2), dicyclopenta[cd,fg]-(3) and dicyclopenta[cd,jk]pyrene (4), in the presence of a metabolic activation mixture (S9-mix), their (di-)epoxides at the externally fused unsaturated five-membered rings were previously proposed as the ultimate mutagenic active forms. In this study, cyclopenta[cd]pyrene-3,4-epoxide (5) and the novel dicyclopenta[cd,mn]pyrene-1,2,4,5-di-epoxide (6), dicyclopenta[cd,fg]pyrene-5,6,7,8-di-epoxide (7) and dicyclopenta[cd,jk]pyrene-1,2,6,7-di-epoxide (8) were synthesised from 1 to 4, respectively, and subsequently assayed for bacterial mutagenicity in the standard microsomal/histidine reverse mutation assay (Ames-assay with Salmonella typhimurium strain TA98). The di-epoxides 6-8 are present as a mixture of their cis- and trans-stereo-isomers in a close to 1:1 ratio ((1)H NMR spectroscopy and ab initio IGLO/III//RHF/6-31G** calculations). The direct-acting mutagenic activity and the strong cytotoxicity exerted by 5-8 both in the absence or presence of an exogenous metabolic activation system (+/-S9-mix) demonstrate that the ultimate mutagenic active forms are the proposed (di-)epoxides of 1-4.

Genome-Wide Biases in the Rate and Molecular Spectrum of Spontaneous Mutations in Vibrio cholerae and Vibrio fischeri.

PubMed

Dillon, Marcus M; Sung, Way; Sebra, Robert; Lynch, Michael; Cooper, Vaughn S

2017-01-01

The vast diversity in nucleotide composition and architecture among bacterial genomes may be partly explained by inherent biases in the rates and spectra of spontaneous mutations. Bacterial genomes with multiple chromosomes are relatively unusual but some are relevant to human health, none more so than the causative agent of cholera, Vibrio cholerae Here, we present the genome-wide mutation spectra in wild-type and mismatch repair (MMR) defective backgrounds of two Vibrio species, the low-%GC squid symbiont V. fischeri and the pathogen V. cholerae, collected under conditions that greatly minimize the efficiency of natural selection. In apparent contrast to their high diversity in nature, both wild-type V. fischeri and V. cholerae have among the lowest rates for base-substitution mutations (bpsms) and insertion-deletion mutations (indels) that have been measured, below 10 - 3 /genome/generation. Vibrio fischeri and V. cholerae have distinct mutation spectra, but both are AT-biased and produce a surprising number of multi-nucleotide indels. Furthermore, the loss of a functional MMR system caused the mutation spectra of these species to converge, implying that the MMR system itself contributes to species-specific mutation patterns. Bpsm and indel rates varied among genome regions, but do not explain the more rapid evolutionary rates of genes on chromosome 2, which likely result from weaker purifying selection. More generally, the very low mutation rates of Vibrio species correlate inversely with their immense population sizes and suggest that selection may not only have maximized replication fidelity but also optimized other polygenic traits relative to the constraints of genetic drift. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Intermediate-type vancomycin resistance (VISA) in genetically-distinct Staphylococcus aureus isolates is linked to specific, reversible metabolic alterations.

PubMed

Alexander, Elizabeth L; Gardete, Susana; Bar, Haim Y; Wells, Martin T; Tomasz, Alexander; Rhee, Kyu Y

2014-01-01

Intermediate (VISA-type) vancomycin resistance in Staphylococcus aureus has been associated with a range of physiologic and genetic alterations. Previous work described the emergence of VISA-type resistance in two clonally-distinct series of isolates. In both series (the first belonging to MRSA clone ST8-USA300, and the second to ST5-USA100), resistance was conferred by a single mutation in yvqF (a negative regulator of the vraSR two-component system associated with vancomycin resistance). In the USA300 series, resistance was reversed by a secondary mutation in vraSR. In this study, we combined systems-level metabolomic profiling with statistical modeling techniques to discover specific, reversible metabolic alterations associated with the VISA phenotype.

Antimutagenic activity of extracts of leaves of four common edible vegetable plants in Nigeria (west Africa).

PubMed

Obaseiki-Ebor, E E; Odukoya, K; Telikepalli, H; Mitscher, L A; Shankel, D M

1993-06-01

Organic solvent extracts of leaves of 4 common edible vegetable plants--Bryophyllum pinnatum, Dialium guincense, Ocimum gratissimum and Vernonia amygdalina--had inhibitory activity for His- to His+ reverse-mutations induced by ethyl methanesulfonate acting on Salmonella typhimurium TA100. The concentrated ethyl acetate, methanol and petroleum ether extracts were heat-stable when dissolved in dimethyl sulfoxide. The Bryophyllum ethyl acetate extract was fractionated into alkaloidal/water-soluble, acids, polar lipid and non-polar lipid fractions. The polar and non-polar lipid fractions inhibited reversion mutations induced by ethyl methanesulfonate acting on TA100 or TA102, and were also active against reversions induced by 4-nitro-O-phenylenediamine and 2-aminofluorene in TA98. The alkaloidal/water-soluble and the acid fractions had no appreciable antimutagenic activities.

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

PubMed Central

Murakami, Tetsuro; Qamar, Seema; Lin, Julie Qiaojin; Schierle, Gabriele S. Kaminski; Rees, Eric; Miyashita, Akinori; Costa, Ana R.; Dodd, Roger B.; Chan, Fiona T.S.; Michel, Claire H.; Kronenberg-Versteeg, Deborah; Li, Yi; Yang, Seung-Pil; Wakutani, Yosuke; Meadows, William; Ferry, Rodylyn Rose; Dong, Liang; Tartaglia, Gian Gaetano; Favrin, Giorgio; Lin, Wen-Lang; Dickson, Dennis W.; Zhen, Mei; Ron, David; Schmitt-Ulms, Gerold; Fraser, Paul E.; Shneider, Neil A.; Holt, Christine; Vendruscolo, Michele; Kaminski, Clemens F.; St George-Hyslop, Peter

2015-01-01

Summary The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins. PMID:26526393

Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria.

PubMed

Cui, Longzhu; Neoh, Hui-min; Iwamoto, Akira; Hiramatsu, Keiichi

2012-06-19

Genome inversions are ubiquitous in organisms ranging from prokaryotes to eukaryotes. Typical examples can be identified by comparing the genomes of two or more closely related organisms, where genome inversion footprints are clearly visible. Although the evolutionary implications of this phenomenon are huge, little is known about the function and biological meaning of this process. Here, we report our findings on a bacterium that generates a reversible, large-scale inversion of its chromosome (about half of its total genome) at high frequencies of up to once every four generations. This inversion switches on or off bacterial phenotypes, including colony morphology, antibiotic susceptibility, hemolytic activity, and expression of dozens of genes. Quantitative measurements and mathematical analyses indicate that this reversible switching is stochastic but self-organized so as to maintain two forms of stable cell populations (i.e., small colony variant, normal colony variant) as a bet-hedging strategy. Thus, this heritable and reversible genome fluctuation seems to govern the bacterial life cycle; it has a profound impact on the course and outcomes of bacterial infections.

Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli

PubMed Central

Brandl, Maria T.; Kudva, Indira T.; Katani, Robab; Moreau, Matthew R.; Kapur, Vivek

2017-01-01

ABSTRACT Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro: it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the autotransporter protein Cah confers on E. coli DH5α cells a strong autoaggregative phenotype that is inversely correlated with its ability to form biofilms and plays a strain-specific role in plant and animal colonization by STEC. Although cah is widespread in the STEC population, we detected a mutation rate of 31.3% in cah, which is similar to that reported for rpoS and fimH. The formation of cell aggregates due to increased bacterium-to-bacterium interactions may be disadvantageous to bacterial populations under conditions that favor a planktonic state in STEC. Therefore, a loss-of-function mutation in cah is likely a selective trait in STEC when autoaggregative properties become detrimental to bacterial cells and may contribute to the adaptability of STEC to fluctuating environments. PMID:29054868

Mutation spectrum and differential gene expression in cystic and solid vestibular schwannoma.

PubMed

Zhang, Zhihua; Wang, Zhaoyan; Sun, Lianhua; Li, Xiaohua; Huang, Qi; Yang, Tao; Wu, Hao

2014-03-01

We sought to characterize the mutation spectrum of NF2 and the differential gene expression in cystic and solid vestibular schwannomas. We collected tumor tissue and blood samples of 31 cystic vestibular schwannomas and 114 solid vestibular schwannomas. Mutation screening of NF2 was performed in both tumor and blood DNA samples of all patients. cDNA microarray was used to analyze the differential gene expression between 11 cystic vestibular schwannomas and 6 solid vestibular schwannomas. Expression levels of top candidate genes were verified by quantitative reverse transcription PCR. NF2 mutations were identified in 34.5% of sporadic vestibular schwannomas, with all mutations being exclusively somatic. No significant difference was found between the mutation detection rates of cystic vestibular schwannoma (35.5%) and solid vestibular schwannoma (34.2%). cDNA microarray analysis detected a total of 46 differentially expressed genes between the cystic vestibular schwannoma and solid vestibular schwannoma samples. The significantly decreased expression of four top candidate genes, C1orf130, CNTF, COL4A3, and COL4A4, was verified by quantitative reverse transcription PCR. NF2 mutations are not directly involved in the cystic formation of vestibular schwannoma. In addition, the differential gene expression of cystic vestibular schwannoma reported in our study may provide useful insights into the molecular mechanism underlying this process.

Genetic characterization and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China.

PubMed

Guo, Jinlei; Yan, Yong; Zhang, Jiafeng; Ji, Jimei; Ge, Zhijian; Ge, Rui; Zhang, Xiaofei; Wang, Henghui; Chen, Zhongwen; Luo, Jianyong

2017-03-14

The aim of this study was to characterize HIV-1 genotypes and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China. The HIV-1 partial polymerase (pol) genes in 93 of the 99 plasma samples were successfully amplified and analyzed. Phylogenetic analysis revealed the existence of five HIV-1 genotypes, of which the most prevalent genotype was CRF01_AE (38.7%), followed by CRF07_BC (34.4%), CRF08_BC (16.1%), subtype B/B' (5.4%), and CRF55_01B (2.1%). Besides, three types of unique recombination forms (URFs) were also observed, including C/F2/A1, CRF01_AE/B, and CRF08_BC/CRF07_BC. Among 93 amplicons, 46.2% had drug resistance-associated mutations, including 23.7% for protease inhibitors (PIs) mutations, 1.1% for nucleoside reverse transcriptase inhibitors (NRTIs) mutations, and 20.4% for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations. Six (6.5%) out of 93 treatment-naive subjects were identified to be resistant to one or more NNRTIs, while resistance to NRTIs or PIs was not observed. Our study showed the genetic diversity of HIV-1 strains circulating in Jiaxing and a relative high proportion of antiretroviral resistance mutations among treatment-naive patients, indicating a serious challenge for HIV prevention and treatment program.

A case report of reversible generalized seizures in a patient with Waardenburg syndrome associated with a novel nonsense mutation in the penultimate exon of SOX10.

PubMed

Suzuki, Noriomi; Mutai, Hideki; Miya, Fuyuki; Tsunoda, Tatsuhiko; Terashima, Hiroshi; Morimoto, Noriko; Matsunaga, Tatsuo

2018-05-23

Waardenburg syndrome type 1 (WS1) can be distinguished from Waardenburg syndrome type 2 (WS2) by the presence of dystopia canthorum. About 96% of WS1 are due to PAX3 mutations, and SOX10 mutations have been reported in 15% of WS2. This report describes a patient with WS1 who harbored a novel SOX10 nonsense mutation (c.652G > T, p.G218*) in exon 3 which is the penultimate exon. The patient had mild prodromal neurological symptoms that were followed by severe attacks of generalized seizures associated with delayed myelination of the brain. The immature myelination recovered later and the neurological symptoms could be improved. This is the first truncating mutation in exon 3 of SOX10 that is associated with neurological symptoms in Waardenburg syndrome. Previous studies reported that the neurological symptoms that associate with WS are congenital and irreversible. These findings suggest that the reversible neurological phenotype may be associated with the nonsense mutation in exon 3 of SOX10. When patients of WS show mild prodromal neurological symptoms, the clinician should be aware of the possibility that severe attacks of generalized seizures may follow, which may be associated with the truncating mutation in exon 3 of SOX10.

Genotoxicity evaluation of the naturally-derived food colorant, gardenia blue, and its precursor, genipin.

PubMed

Hobbs, Cheryl A; Koyanagi, Mihoko; Swartz, Carol; Davis, Jeffrey; Maronpot, Robert; Recio, Leslie; Hayashi, Shim-Mo

2018-06-04

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans. Copyright © 2018. Published by Elsevier Ltd.

Ca2+ toxicity due to reverse Na+/Ca2+ exchange contributes to degeneration of neurites of DRG neurons induced by a neuropathy-associated Nav1.7 mutation

PubMed Central

Estacion, M.; Vohra, B. P. S; Liu, S.; Hoeijmakers, J.; Faber, C. G.; Merkies, I. S. J.; Lauria, G.; Black, J. A.

2015-01-01

Gain-of-function missense mutations in voltage-gated sodium channel Nav1.7 have been linked to small-fiber neuropathy, which is characterized by burning pain, dysautonomia and a loss of intraepidermal nerve fibers. However, the mechanistic cascades linking Nav1.7 mutations to axonal degeneration are incompletely understood. The G856D mutation in Nav1.7 produces robust changes in channel biophysical properties, including hyperpolarized activation, depolarized inactivation, and enhanced ramp and persistent currents, which contribute to the hyperexcitability exhibited by neurons containing Nav1.8. We report here that cell bodies and neurites of dorsal root ganglion (DRG) neurons transfected with G856D display increased levels of intracellular Na+ concentration ([Na+]) and intracellular [Ca2+] following stimulation with high [K+] compared with wild-type (WT) Nav1.7-expressing neurons. Blockade of reverse mode of the sodium/calcium exchanger (NCX) or of sodium channels attenuates [Ca2+] transients evoked by high [K+] in G856D-expressing DRG cell bodies and neurites. We also show that treatment of WT or G856D-expressing neurites with high [K+] or 2-deoxyglucose (2-DG) does not elicit degeneration of these neurites, but that high [K+] and 2-DG in combination evokes degeneration of G856D neurites but not WT neurites. Our results also demonstrate that 0 Ca2+ or blockade of reverse mode of NCX protects G856D-expressing neurites from degeneration when exposed to high [K+] and 2-DG. These results point to [Na+] overload in DRG neurons expressing mutant G856D Nav1.7, which triggers reverse mode of NCX and contributes to Ca2+ toxicity, and suggest subtype-specific blockade of Nav1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in small-fiber neuropathy. PMID:26156380

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

Characterization of Nucleoside Reverse Transcriptase Inhibitor-Associated Mutations in the RNase H Region of HIV-1 Subtype C Infected Individuals.

PubMed

Ngcapu, Sinaye; Theys, Kristof; Libin, Pieter; Marconi, Vincent C; Sunpath, Henry; Ndung'u, Thumbi; Gordon, Michelle L

2017-11-08

The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher's exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation.

New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

PubMed Central

Collins, Peter L.; Murphy, Brian R.

2005-01-01

Development of a live pediatric vaccine against human respiratory syncytial virus (RSV) is complicated by the need to immunize young infants and the difficulty in balancing attenuation and immunogenicity. The ability to introduce desired mutations into infectious virus by reverse genetics provides a method for identifying and designing highly defined attenuating mutations. These can be introduced in combinations as desired to achieve gradations of attenuation. Attenuation is based on several strategies: multiple independent temperature-sensitive point mutations in the polymerase, a temperature-sensitive point mutation in a transcription signal, a set of non–temperature-sensitive mutations involving several genes, deletion of a viral RNA synthesis regulatory protein, and deletion of viral IFN α/β antagonists. The genetic stability of the live vaccine can be increased by judicious choice of mutations. The virus also can be engineered to increase the level of expression of the protective antigens. Protective antigens from antigenically distinct RSV strains can be added or swapped to increase the breadth of coverage. Alternatively, the major RSV protective antigens can be expressed from transcription units added to an attenuated parainfluenza vaccine virus, making a bivalent vaccine. This would obviate the difficulties inherent in the fragility and inefficient in vitro growth of RSV, simplifying vaccine design and use. PMID:16113487

Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity

PubMed Central

Butler, Kimberly S.; Peeler, David J.; Casey, Brendan J.; Dair, Benita J.; Elespuru, Rosalie K.

2015-01-01

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed. PMID:25964273

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

PubMed Central

Soparkar, Ketaki; Kinana, Alfred D.; Weeks, Jon W.; Morrison, Keith D.; Nikaido, Hiroshi

2015-01-01

ABSTRACT The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity. PMID:26240069

An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus.

PubMed

Du, Ruikun; Wang, Manli; Hu, Zhihong; Wang, Hualin; Deng, Fei

2015-10-01

Japanese encephalitis virus (JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus-host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope (E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.

Trends of drug-resistance-associated mutations in the reverse transcriptase gene of HIV type 1 isolates from North India.

PubMed

Azam, Mohd; Malik, Abida; Rizvi, Meher; Rai, Arvind

2014-04-01

A major cause of failure of antiretroviral therapy (ART) is the presence of drug-resistance-associated mutations in the polymerase gene of HIV-1. The paucity of data regarding potential drug resistance to reverse transcriptase inhibitors (RTIs) prompted us to carry out this study. This information will shed light on the extent of drug resistance already present in HIV strains and will give future directions in patient treatment and in drug design. Drug resistance genotyping of a partial reverse transcriptase gene was done in 103 HIV-1-infected patients, including the ART-naive and ART-experienced population. The drug resistance pattern was analyzed using the Stanford HIV-DR database, the IAS-USA mutation list and the REGA algorithm-v8.0. Subtyping was done using the REGA HIV-1 subtyping tool-v2.01. The majority of our sequences (96 %) were found to be subtype C, and four (3.8 %) were subtype A1. Significant prevalence of DR mutations (28 %) was observed in the RT gene. Major amino acid substitutions were seen at positions 41, 90, 98, 103, 106, 108, 138, 181, 184, 190, 215, and 219, which confer high/intermediate levels of resistance to most RTIs, independently or together. Our results show that there is an urgent need to tailor ART drug regimens to the individual to achieve optimum therapeutic outcome in North India.

The waaL gene mutation compromised the inhabitation of Enterobacter sp. Ag1 in the mosquito gut environment.

PubMed

Pei, Dong; Jiang, Jinjin; Yu, Wanqin; Kukutla, Phanidhar; Uentillie, Alejandro; Xu, Jiannong

2015-08-27

The mosquito gut harbors a variety of bacteria that are dynamically associated with mosquitoes in various contexts. However, little is known about bacterial factors that affect bacterial inhabitation in the gut microbial community. Enterobacter sp. Ag1 is a predominant Gram negative bacterium in the mosquito midgut. In a mutant library that was generated using transposon Tn5-mediated mutagenesis, a mutant was identified, in which the gene waaL was disrupted by the Tn5 insertion. The waaL encodes O antigen ligase, which is required for the attachment of O antigen to the outer core oligosaccharide of the lipopolysaccharide (LPS). The waaL(-) mutation caused the O antigen repeat missing in the LPS. The normal LPS structure was restored when the mutant was complemented with a plasmid containing waaL gene. The waaL(-) mutation did not affect bacterial proliferation in LB culture, the mutant cells grew at a rate the same as the wildtype (wt) cells. However, when waaL(-) strain were co-cultured with the wt strain or complemented strain, the mutant cells proliferated with a slower rate, indicating that the mutants were less competitive than wt cells in a community setting. Similarly, in a co-feeding assay, when fluorescently tagged wt strain and waaL(-) strain were orally co-introduced into the gut of Anopheles stephensi mosquitoes, the mutant cells were less prevalent in both sugar-fed and blood-fed guts. The data suggest that the mutation compromised the bacterial inhabitation in the gut community. Besides, the mutant was more sensitive to oxidative stress, demonstrated by lower survival rate upon exposure to 20 mM H₂O₂. Lack of the O antigen structure in LPS of Enterobacter compromised the effective growth in co-culture and co-feeding assays. In addition, O-antigen was involved in protection against oxidative stress. The findings suggest that intact LPS is crucial for the bacteria to steadily stay in the gut microbial community.

Antiviral Activity of MK-4965, a Novel Nonnucleoside Reverse Transcriptase Inhibitor▿

PubMed Central

Lai, Ming-Tain; Munshi, Vandna; Touch, Sinoeun; Tynebor, Robert M.; Tucker, Thomas J.; McKenna, Philip M.; Williams, Theresa M.; DiStefano, Daniel J.; Hazuda, Daria J.; Miller, Michael D.

2009-01-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the mainstays of therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, the effectiveness of NNRTIs can be hampered by the development of resistance mutations which confer cross-resistance to drugs in the same class. Extensive efforts have been made to identify new NNRTIs that can suppress the replication of the prevalent NNRTI-resistant viruses. MK-4965 is a novel NNRTI that possesses both diaryl ether and indazole moieties. The compound displays potency at subnanomolar concentrations against wild-type (WT), K103N, and Y181C reverse transcriptase (RT) in biochemical assays. MK-4965 is also highly potent against the WT virus and two most prevalent NNRTI-resistant viruses (viruses that harbor the K103N or the Y181C mutation), against which it had 95% effective concentrations (EC95s) of <30 nM in the presence of 10% fetal bovine serum. The antiviral EC95 of MK-4965 was reduced approximately four- to sixfold when it was tested in 50% human serum. Moreover, MK-4965 was evaluated with a panel of 15 viruses with NNRTI resistance-associated mutations and showed a superior mutant profile to that of efavirenz but not to that of etravirine. MK-4965 was similarly effective against various HIV-1 subtypes and viruses containing nucleoside reverse transcriptase inhibitor or protease inhibitor resistance-conferring mutations. A two-drug combination study showed that the antiviral activity of MK-4965 was nonantagonistic with each of the 18 FDA-licensed drugs tested vice versa in the present study. Taken together, these in vitro data show that MK-4965 possesses the desired properties for further development as a new NNRTI for the treatment of HIV-1 infection. PMID:19289522

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

PubMed

Wang, Hye-young; Kim, Hyunjung; Kim, Yeun; Bang, Hyeeun; Kim, Jong-Pill; Hwang, Joo Hwan; Cho, Sang-Nae; Kim, Tae Ue; Lee, Hyeyoung

2015-10-01

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

a/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

PubMed

Martin, P; Prakash, L; Prakash, S

1981-05-01

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.

Circularization of the HIV-1 genome facilitates strand transfer during reverse transcription

PubMed Central

Beerens, Nancy; Kjems, Jørgen

2010-01-01

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer involves a jump from the 5′ to the 3′ terminal repeat (R) region positioned at each end of the viral genome. The process depends on base pairing between the cDNA synthesized from the 5′ R region and the 3′ R RNA. The tertiary conformation of the viral RNA genome may facilitate strand transfer by juxtaposing the 5′ R and 3′ R sequences that are 9 kb apart in the linear sequence. In this study, RNA sequences involved in an interaction between the 5′ and 3′ ends of the HIV-1 genome were mapped by mutational analysis. This interaction appears to be mediated mainly by a sequence in the extreme 3′ end of the viral genome and in the gag open reading frame. Mutation of 3′ R sequences was found to inhibit the 5′–3′ interaction, which could be restored by a complementary mutation in the 5′ gag region. Furthermore, we find that circularization of the HIV-1 genome does not affect the initiation of reverse transcription, but stimulates the first strand transfer during reverse transcription in vitro, underscoring the functional importance of the interaction. PMID:20430859

Mutant p53 expression in fallopian tube epithelium drives cell migration.

PubMed

Quartuccio, Suzanne M; Karthikeyan, Subbulakshmi; Eddie, Sharon L; Lantvit, Daniel D; Ó hAinmhire, Eoghainín; Modi, Dimple A; Wei, Jian-Jun; Burdette, Joanna E

2015-10-01

Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates. © 2015 UICC.

Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities

PubMed Central

Boyden, Lynn M.; Choi, Murim; Choate, Keith A.; Nelson-Williams, Carol J.; Farhi, Anita; Toka, Hakan R.; Tikhonova, Irina R.; Bjornson, Robert; Mane, Shrikant M.; Colussi, Giacomo; Lebel, Marcel; Gordon, Richard D.; Semmekrot, Ben A.; Poujol, Alain; Välimäki, Matti J.; De Ferrari, Maria E.; Sanjad, Sami A.; Gutkin, Michael; Karet, Fiona E.; Tucci, Joseph R.; Stockigt, Jim R.; Keppler-Noreuil, Kim M.; Porter, Craig C.; Anand, Sudhir K.; Whiteford, Margo L.; Davis, Ira D.; Dewar, Stephanie B.; Bettinelli, Alberto; Fadrowski, Jeffrey J.; Belsha, Craig W.; Hunley, Tracy E.; Nelson, Raoul D.; Trachtman, Howard; Cole, Trevor R. P.; Pinsk, Maury; Bockenhauer, Detlef; Shenoy, Mohan; Vaidyanathan, Priya; Foreman, John W.; Rasoulpour, Majid; Thameem, Farook; Al-Shahrouri, Hania Z.; Radhakrishnan, Jai; Gharavi, Ali G.; Goilav, Beatrice; Lifton, Richard P.

2012-01-01

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. A rare Mendelian syndrome, pseudohypoaldosteronism type II (PHAII), featuring hypertension, hyperkalemia, and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption versus K+ and H+ excretion1. We used exome sequencing to identify mutations in Kelch-like 3 (KLHL3) or Cullin 3 (CUL3) in 41 PHAII kindreds. KLHL3 mutations are either recessive or dominant, while CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-Kelch proteins such as KLHL3 are components of Cullin/RING E3 ligase complexes (CRLs) that ubiquitinate substrates bound to Kelch propeller domains2–8. Dominant KLHL3 mutations are clustered in short segments within the Kelch propeller and BTB domains implicated in substrate9 and Cullin5 binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter (NCC) in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3/CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite combined complexities of locus heterogeneity, mixed models of transmission, and frequent de novo mutation, and establish a fundamental role for KLHL3/CUL3 in blood pressure, K+, and pH homeostasis. PMID:22266938

Tenofovir-based regimens associated with less drug resistance in HIV-1-infected Nigerians failing first-line antiretroviral therapy.

PubMed

Etiebet, Mary-Ann A; Shepherd, James; Nowak, Rebecca G; Charurat, Man; Chang, Harry; Ajayi, Samuel; Elegba, Olufunmilayo; Ndembi, Nicaise; Abimiku, Alashle; Carr, Jean K; Eyzaguirre, Lindsay M; Blattner, William A

2013-02-20

In resource-limited settings, HIV-1 drug resistance testing to guide antiretroviral therapy (ART) selection is unavailable. We retrospectively conducted genotypic analysis on archived samples from Nigerian patients who received targeted viral load testing to confirm treatment failure and report their drug resistance mutation patterns. Stored plasma from 349 adult patients on non-nucleoside reverse transcriptase inhibitor (NNRTI) regimens was assayed for HIV-1 RNA viral load, and samples with more than 1000 copies/ml were sequenced in the pol gene. Analysis for resistance mutations utilized the IAS-US 2011 Drug Resistance Mutation list. One hundred and seventy-five samples were genotyped; the majority of the subtypes were G (42.9%) and CRF02_AG (33.7%). Patients were on ART for a median of 27 months. 90% had the M184V/I mutation, 62% had at least one thymidine analog mutation, and 14% had the K65R mutation. 97% had an NNRTI resistance mutation and 47% had at least two etravirine-associated mutations. In multivariate analysis tenofovir-based regimens were less likely to have at least three nucleoside reverse transcriptase inhibitor (NRTI) mutations after adjusting for subtype, previous ART, CD4, and HIV viral load [P < 0.001, odds ratio (OR) 0.04]. 70% of patients on tenofovir-based regimens had at least two susceptible NRTIs to include in a second-line regimen compared with 40% on zidovudine-based regimens (P = 0.04, OR = 3.4). At recognition of treatment failure, patients on tenofovir-based first-line regimens had fewer NRTI drug-resistant mutations and more active NRTI drugs available for second-line regimens. These findings can inform strategies for ART regimen sequencing to optimize long-term HIV treatment outcomes in low-resource settings.

Genomic Insight into Mechanisms of Reversion of Antibiotic Resistance in Multidrug Resistant Mycobacterium tuberculosis Induced by a Nanomolecular Iodine-Containing Complex FS-1.

PubMed

Ilin, Aleksandr I; Kulmanov, Murat E; Korotetskiy, Ilya S; Islamov, Rinat A; Akhmetova, Gulshara K; Lankina, Marina V; Reva, Oleg N

2017-01-01

Drug induced reversion of antibiotic resistance is a promising way to combat multidrug resistant infections. However, lacking knowledge of mechanisms of drug resistance reversion impedes employing this approach in medicinal therapies. Induction of antibiotic resistance reversion by a new anti-tuberculosis drug FS-1 has been reported. FS-1 was used in this work in combination with standard anti-tuberculosis antibiotics in an experiment on laboratory guinea pigs infected with an extensively drug resistant (XDR) strain Mycobacterium tuberculosis SCAID 187.0. During the experimental trial, genetic changes in the population were analyzed by sequencing of M. tuberculosis isolates followed by variant calling. In total 11 isolates obtained from different groups of infected animals at different stages of disease development and treatment were sequenced. It was found that despite the selective pressure of antibiotics, FS-1 caused a counter-selection of drug resistant variants that speeded up the recovery of the infected animals from XDR tuberculosis. Drug resistance mutations reported in the genome of the initial strain remained intact in more sensitive isolates obtained in this experiment. Variant calling in the sequenced genomes revealed that the drug resistance reversion could be associated with a general increase in genetic heterogeneity of the population of M. tuberculosis . Accumulation of mutations in PpsA and PpsE subunits of phenolpthiocerol polyketide synthase was observed in the isolates treated with FS-1 that may indicate an increase of persisting variants in the population. It was hypothesized that FS-1 caused an active counter-selection of drug resistant variants from the population by aggravating the cumulated fitness cost of the drug resistance mutations. Action of FS-1 on drug resistant bacteria exemplified the theoretically predicted induced synergy mechanism of drug resistance reversion. An experimental model to study the drug resistance reversion phenomenon is hereby introduced.

Modeling cystic fibrosis disease progression in patients with the rare CFTR mutation P67L.

PubMed

MacKenzie, Isobel E R; Paquette, Valerie; Gosse, Frances; George, Sheenagh; Chappe, Frederic; Chappe, Valerie

2017-05-01

The progression of cystic fibrosis (CF) in patients with the rare mutation P67L was examined to determine if it induced a milder form of CF compared to the common severe ΔF508 mutation. Parameters of lung function, level of bacterial infection, nutritional status and hospitalization were used to represent CF progression. Age at diagnosis and pancreatic status were used to assess CF presentation. Analysis of data from the CF Canada Registry collected over a 15-year period included 266 ΔF508/ΔF508 homozygote patients from CF clinics in Atlantic Canada and 26 compound heterozygote patients with the rare P67L mutation from clinics across Canada. Late age at diagnosis, high incidence of pancreatic sufficiency, maintained Body Mass Index (BMI) with age, delayed life-threatening bacterial infection, and fewer days in hospital were observed for P67L heterozygote patients included in this study. Although the decline of lung function did not differ from ΔF508 homozygotes, the fact that a greater proportion of P67L heterozygotes live to an older age suggests that lung function is not the primary factor determining CF progression for P67L heterozygote patients. The P67L mutation is associated with a mild disease, even when combined with the severe ΔF508 mutation. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Asymptomatic Carriage of Group A Streptococcus Is Associated with Elimination of Capsule Production

PubMed Central

Jewell, Brittany E.; Olsen, Randall J.; Shelburne, Samuel A.; Fittipaldi, Nahuel; Beres, Stephen B.; Musser, James M.

2014-01-01

Humans commonly carry pathogenic bacteria asymptomatically, but despite decades of study, the underlying molecular contributors remain poorly understood. Here, we show that a group A streptococcus carriage strain contains a frameshift mutation in the hasA gene resulting in loss of hyaluronic acid capsule biosynthesis. This mutation was repaired by allelic replacement, resulting in restoration of capsule production in the isogenic derivative strain. The “repaired” isogenic strain was significantly more virulent than the carriage strain in a mouse model of necrotizing fasciitis and had enhanced growth ex vivo in human blood. Importantly, the repaired isogenic strain colonized the mouse oropharynx with significantly greater bacterial burden and had significantly reduced ability to internalize into cultured epithelial cells than the acapsular carriage strain. We conducted full-genome sequencing of 81 strains cultured serially from 19 epidemiologically unrelated human subjects and discovered the common theme that mutations negatively affecting capsule biosynthesis arise in vivo in the has operon. The significantly decreased capsule production is a key factor contributing to the molecular détente between pathogen and host. Our discoveries suggest a general model for bacterial pathogens in which mutations that downregulate or ablate virulence factor production contribute to carriage. PMID:25024363

Effect of different laser irradiation on the dysentery bacilli

NASA Astrophysics Data System (ADS)

Ou, Lin; Chen, Rong; Chen, Yanjiao; Li, Depin; Wen, Caixia

1998-08-01

The S. flexnesi, which have high drug-resistance especially in Cm, Sm, Tc, SD, were irradiated by Ar+ laser at 488 nm and semiconductor laser at 808 nm. The experiment results have shown that both Ar+ laser and semiconductor laser with power density of 1.7 w/cm2 and irradiation dose of 2000 J/cm2 can conduce to the bacterial lethality and increase the mutation rates of the bacterial drug-sensitivity, and 'Colony Count' method have the superiority over the 'Inhibacteria Ring' method. At the mean time it further indicate that the high power semiconductor laser would play an important role in the sciences of laser biological medicine. But the effect of the near infrared semiconductor laser is far lower than that of Ar+ laser of shorter wavelength at the same irradiation dose. It is clear that the output and irradiation dose of near infrared semiconductor laser shall be increased in order to get the same rates of the bacterial lethality and the drug-sensitivity mutation as Ar+ laser's.

Mutations in y-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence

USDA-ARS?s Scientific Manuscript database

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid '-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome h...

Evaluation of bacterial kill when modelling the bronchopulmonary pharmacokinetic profile of moxifloxacin and levofloxacin against parC-containing isolates of Streptococcus pneumoniae.

PubMed

Deryke, C Andrew; Du, Xiaoli; Nicolau, David P

2006-09-01

The increasingly recognized prevalence of first-step parC mutants in Streptococcus pneumoniae and the development of de novo resistance while on fluoroquinolone therapy are of concern. Previous work by our group demonstrated the ability of moxifloxacin, but not levofloxacin, to eradicate parC mutants. The objective of this experiment was to determine whether these fluoroquinolone antibiotics provided equivalent bacterial kill when similar AUC/MICs were examined. An in vitro pharmacodynamic model was used to simulate the epithelial lining fluid (ELF) concentrations following oral administration of levofloxacin 500 mg once daily and moxifloxacin 400 mg once daily in older adults. In addition, a range of AUC/MICs were also modelled, including levofloxacin 750 mg once daily. Five different S. pneumoniae containing first-step parC mutations and one isolate without mutations were tested for 48 h and time-kill curves were constructed. Samples at 0, 24 and 48 h were collected for phenotypic and genotypic profiling. HPLC was used to verify that target exposures were achieved. The isolate without a parC mutation displayed a 4 log reduction in cfu after treatment with levofloxacin 500 mg and did not select for resistance. In all five isolates containing first-step parC mutations, resistance emerged within 48 h with a > or =16-fold increase in MIC and the acquisition of a gyrA mutant. Increasing the exposure of levofloxacin to approximately 750 mg dose still led to > or =16-fold increase in MIC at 48 h in two of the four isolates containing parC mutations. On the other hand, moxifloxacin 400 mg sustained bacterial killing against the two isolates tested without the selection of resistant mutants. It appears that the critical AUC/MIC necessary to prevent the acquisition of resistance for levofloxacin is 200 and approximately 400 for moxifloxacin. Due to suboptimal exposures, once-daily oral regimens of levofloxacin at both 500 and 750 mg inconsistently led to bactericidal activity and the frequent acquisition of a second-step gyrA mutation in S. pneumoniae isolates already containing a first-step parC mutation. Conversely, once-daily moxifloxacin 400 mg provides exposures that vastly exceed the apparent efficacy breakpoint and did not select for second-step mutants until exposures were decreased 4-fold. As a result of these data and the emerging literature involving mutations in the pneumococcus, caution should be exercised when the respiratory fluoroquinolones are used to treat patients infected with S. pneumoniae suspected of having parC mutations.

Mutation covariation of HIV-1 CRF07_BC reverse transcriptase during antiretroviral therapy.

PubMed

Li, Zhenpeng; Huang, Yang; Ouyang, Yabo; Xing, Hui; Liao, Lingjie; Jiang, Shibo; Shao, Yiming; Ma, Liying

2013-11-01

To understand the effect of HIV-1 drug resistance mutations in the context of antiretroviral therapy (ART), we compared the prevalence of protease (PR) and reverse transcriptase (RT) mutations in HIV-1 CRF07_BC sequences from blood samples of treatment-naive and ART-treated patients. Mutation covariation in the RT and PR of HIV-1 CRF07_BC viruses from 542 treatment-naive patients and 261 patients treated with lamivudine/zidovudine/nevirapine or lamivudine/zidovudine/efavirenz was analysed. Stratified networks were used to display the mutation covariation. Based on the comparison between treatment-naive and ART-treated patients, three types of featured mutations for RT and PR were initially identified: treatment-associated mutations, treatment-agonistic mutations and overlapping polymorphisms. Twelve significant covariation pairs were found between five treatment-associated mutations (K103N, M184V, Q197K, G190A and Y181C) and nine overlapping polymorphisms (A36E, D39N, Y121H, D123E, R135I, T200A, R277K, L283I and D291E). Meanwhile, three covariation pairs between three treatment-associated mutations (I132L and M184V for RT and I15V for PR) and three overlapping polymorphisms (L10I, L36M and A71V) for PR were also detected. Finally, the overlapping polymorphisms for RT and PR were both found to have significant correlations with treatment-associated mutations, indicating a possible association between polymorphisms and drug resistance. When compared with HIV-1 subtype B under the same regimens as CRF07_BC, the mutation covariations of CRF07_BC showed a distinct pattern of RT and PR mutation covariation. The role of polymorphisms in the development of drug resistance has been widely reported. Here, we found a significant correlation between overlapping polymorphisms for RT and PR and treatment-associated mutations, indicating that polymorphisms exert a global influence on treatment-associated mutations. Polymorphism mutations might therefore be considered before initiating ART to improve the efficacy of drug combinations.

Coinheritance of hereditary spherocytosis and reversibility of cirrhosis in a young female patient with hereditary hemochromatosis.

PubMed

Höblinger, A; Erdmann, C; Strassburg, C P; Sauerbruch, T; Lammert, F

2009-04-16

Here we report a 33-years-old woman with hereditary spherocytosis and hemochromatosis due to homozygosity for the C282Y mutation of the HFE gene. The coinheritance of both conditions led to severe iron overload and liver cirrhosis at young age. The patient was treated by repeated phlebotomy, and reversibility of cirrhosis was documented by transient elastography. This report discusses the pathophysiology of iron accumulation in patients with hemolytic anemia combined with HFE C282Y homozygosity. The case indicates that patients with hematological disorders characterized by increased erythropoetic activity should be screened for HFE mutations.

Active PI3K Pathway Causes an Invasive Phenotype Which Can Be Reversed or Promoted by Blocking the Pathway at Divergent Nodes

PubMed Central

Wallin, Jeffrey J.; Guan, Jane; Edgar, Kyle A.; Zhou, Wei; Francis, Ross; Torres, Anthony C.; Haverty, Peter M.; Eastham-Anderson, Jeffrey; Arena, Sabrina; Bardelli, Alberto; Griffin, Sue; Goodall, John E.; Grimshaw, Kyla M.; Hoeflich, Klaus P.; Torrance, Christopher; Belvin, Marcia; Friedman, Lori S.

2012-01-01

The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. Introduction of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP3 production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis. PMID:22570710

Survival and reversion of a stable L form in soil

NASA Technical Reports Server (NTRS)

Horwitz, A. H.; Casida, L. E., Jr.

1978-01-01

The stable L form of Agromyces ramosus reverted to a bacterial form when incubated in sterilized soil. The cellular and colonial morphology of this bacterial form resembled that of the original parent bacterial form. The two forms differed, however, in that the revertant maintained its bacterial form when transferred onto a low-salt (NaCl) medium but was virtually completely induced into the L-form state on a high-salt medium. The original parent bacterial form was not sensitive to salt. The possibility is discussed that an L-form - bacterial-form cycle for this bacterium might occur naturally in soil. This cycle would be mediated by fluctuations in local salt concentrations in the soil.

Hypermutation in derepressed operons of Escherichia coli K12

PubMed Central

Wright, Barbara E.; Longacre, Angelika; Reimers, Jacqueline M.

1999-01-01

This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation. Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established between leuB mRNA abundance and leuB− reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter. When the leu operon was induced by isopropyl-d-thiogalactoside, both leuB mRNA abundance and leuB− reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon. PMID:10220423

Molecular alterations in endometrial and ovarian clear cell carcinomas: clinical impacts of telomerase reverse transcriptase promoter mutation.

PubMed

Huang, Hsien-Neng; Chiang, Ying-Cheng; Cheng, Wen-Fang; Chen, Chi-An; Lin, Ming-Chieh; Kuo, Kuan-Ting

2015-02-01

Recently, mutations of telomerase reverse transcriptase (TERT) promoter were found in several types of cancer. A few reports demonstrate TERT promoter mutations in ovarian clear cell carcinomas but endometrial clear cell carcinoma has not been studied. The aims of this study were to compare differences of molecular alterations and clinical factors, and identify their prognostic impact in endometrial and ovarian clear cell carcinomas. We evaluated mutations of the TERT promoter and PIK3CA, expression of ARID1A, and other clinicopathological factors in 56 ovarian and 14 endometrial clear cell carcinomas. We found that TERT promoter mutations were present in 21% (3/14) of endometrial clear cell carcinomas and 16% (9/56) of ovarian clear cell carcinomas. Compared with ovarian clear cell carcinomas, endometrial clear cell carcinomas showed older mean patient age (P<0.001), preserved ARID1A immunoreactivity (P=0.017) and infrequent PIK3CA mutation (P=0.025). In ovarian clear cell carcinomas, TERT promoter mutations were correlated with patient age >45 (P=0.045) and preserved ARID1A expression (P=0.003). In cases of endometrial clear cell carcinoma, TERT promoter mutations were not statistically associated with any other clinicopathological factors. In ovarian clear cell carcinoma patients with early FIGO stage (stages I and II), TERT promoter mutation was an independent prognostic factor and correlated with a shorter disease-free survival and overall survival (P=0.015 and 0.009, respectively). In recurrent ovarian clear cell carcinoma patients with early FIGO stage, TERT promoter mutations were associated with early relapse within 6 months (P=0.018). We concluded that TERT promoter mutations were present in endometrial and ovarian clear cell carcinomas. Distinct molecular alteration patterns in endometrial and ovarian clear cell carcinomas implied different processes of tumorigenesis in these morphologically similar tumors. In ovarian clear cell carcinoma of early FIGO stage, patients with TERT promoter mutation require close follow-up during the initial 6 months following chemotherapy.

Rare beneficial mutations can halt Muller's ratchet

NASA Astrophysics Data System (ADS)

Balick, Daniel; Goyal, Sidhartha; Jerison, Elizabeth; Neher, Richard; Shraiman, Boris; Desai, Michael

2012-02-01

In viral, bacterial, and other asexual populations, the vast majority of non-neutral mutations are deleterious. This motivates the application of models without beneficial mutations. Here we show that the presence of surprisingly few compensatory mutations halts fitness decay in these models. Production of deleterious mutations is balanced by purifying selection, stabilizing the fitness distribution. However, stochastic vanishing of fitness classes can lead to slow fitness decay (i.e. Muller's ratchet). For weakly deleterious mutations, production overwhelms purification, rapidly decreasing population fitness. We show that when beneficial mutations are introduced, a stable steady state emerges in the form of a dynamic mutation-selection balance. We argue this state is generic for all mutation rates and population sizes, and is reached as an end state as genomes become saturated by either beneficial or deleterious mutations. Assuming all mutations have the same magnitude selective effect, we calculate the fraction of beneficial mutations necessary to maintain the dynamic balance. This may explain the unexpected maintenance of asexual genomes, as in mitochondria, in the presence of selection. This will affect in the statistics of genetic diversity in these populations.

Characterization of potential antiviral resistance mutations in hepatitis B virus reverse transcriptase sequences in treatment-naïve Chinese patients.

PubMed

Liu, Bao-Ming; Li, Tong; Xu, Jie; Li, Xiao-Guang; Dong, Jian-Ping; Yan, Ping; Yang, Jing-Xian; Yan, Ling; Gao, Zhi-Yong; Li, Wen-Peng; Sun, Xie-Wen; Wang, Yu-Hua; Jiao, Xiu-Juan; Hou, Chun-Sheng; Zhuang, Hui

2010-03-01

Full-length hepatitis B virus (HBV) reverse transcriptase (RT) sequences were amplified and sequenced among 192 nucleos(t)ide analogue (NA)-naïve Chinese patients with chronic hepatitis B. Deduced amino acids (AAs) at 42 previously reported potential NA resistance (NAr) mutation positions in RT region were analyzed. Patients were found with either B-genotype (28.65%) or C-genotype (71.35%) infections. Rt53, rt91, rt124, rt134, rt221, rt224, rt238 and rt256 were identified as B- and C-genotype-dependent polymorphic AA positions. AA substitutions at 11 classical NAr mutation positions, i.e. rt80, rt169, rt173, rt180, rt181, rt184, rt194, rt202, rt204, rt236 and rt250, were not detected. However, potential NAr mutations were found in 30.73% (59/192) isolates, which involved 18 positions including rt53, rt207, rt229, rt238 and rt256, etc. The concomitant AA changes of HBsAg occurred in 16.67% (32/192) isolates including sG145R mutation. One-third of mutation positions were located in functional RT domains (e.g. rt207 and rt233), A-B interdomains (overlapping HBsAg 'a' determinant and showing most concomitant immune-associated mutations) and non-A-B interdomains (e.g. rt191 and rt213), respectively. Genotypes B and C each showed several preferred positions to mutate. These results might provide insights into understanding the evolution and selection basis of NAr HBV strains under antiviral therapy.

Clinical Outcomes of Lung Transplantation in Patients with Telomerase Mutations

PubMed Central

Tokman, Sofya; Singer, Jonathan P.; Devine, Megan S.; Westall, Glen P.; Aubert, John-David; Tamm, Michael; Snell, Gregory I.; Lee, Joyce S.; Goldberg, Hilary J.; Kukreja, Jasleen; Golden, Jeffrey A.; Leard, Lorriana E.; Garcia, Christine K.; Hays, Steven R.

2017-01-01

Background Successful lung transplantation (LT) for patients with pulmonary fibrosis from telomerase mutations is limited by systemic complications of telomerase dysfunction including myelosuppression, cirrhosis, and malignancy. We describe clinical outcomes among 14 LT recipients with telomerase mutations. Methods Subjects underwent LT between February 2005 and April 2014 at 5 LT centers. We abstracted data from medical records, focusing on outcomes reflecting post-LT treatment effects likely to be complicated by telomerase mutations. Results The median age of subjects was 60.5 years (IQR 52.0–62.0), 64.3% were male, and the mean post-LT observation time was 3.2 years (SD ±2.9). Eleven subjects had a mutation in telomerase reverse transcriptase, 2 in telomerase RNA component, and 1 had an uncharacterized mutation. Ten subjects were leukopenic post-LT; leukopenia prompted cessation of mycophenolate mofetil in 5 and treatment with filgrastim in 4. Six subjects had recurrent lower respiratory tract infections (LRTI), 7 had acute cellular rejection (ACR) (A1), and 4 developed chronic lung allograft dysfunction (CLAD). Ten LT recipients developed chronic renal insufficiency and 8 experienced acute, reversible renal failure. Three developed cancer, none had cirrhosis. Thirteen subjects were alive at data censorship. Conclusions The clinical course for LT recipients with telomerase mutations is complicated by renal disease, leukopenia prompting a change in the immunosuppressive regimen, and recurrent LTRI. In contrast, cirrhosis was absent, ACR was mild, and development of CLAD was comparable to other LT populations. While posing challenges, lung transplantation may be feasible for patients with pulmonary fibrosis due to telomerase mutations. PMID:26169663

Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea.

PubMed

Triques, Karine; Sturbois, Bénédicte; Gallais, Stéphane; Dalmais, Marion; Chauvin, Stéphanie; Clepet, Christian; Aubourg, Sébastien; Rameau, Catherine; Caboche, Michel; Bendahmane, Abdelhafid

2007-09-01

Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3beta-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5-21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project.

Molecular Characterization of the Human Immunodeficiency Virus Type 1 in Women and Their Vertically Infected Children.

PubMed

Vaz, Sara Nunes; Giovanetti, Marta; Rego, Filipe Ferreira de Almeida; Oliveira, Tulio de; Danaviah, Siva; Gonçalves, Maria Luiza Freire; Alcantara, Luiz Carlos Junior; Brites, Carlos

2015-10-01

Approximately 35 million people worldwide are infected with human immunodeficiency virus (HIV) around 3.2 million of whom are children under 15 years. Mother-to-child-transmission (MTCT) of HIV-1 accounts for 90% of all infections in children. Despite great advances in the prevention of MTCT in Brazil, children are still becoming infected. Samples from 19 HIV-1-infected families were collected. DNA was extracted and fragments from gag, pol, and env were amplified and sequenced directly. Phylogenetic reconstruction was performed. Drug resistance analyses were performed in pol and env sequences. We found 82.1% of subtype B and 17.9% of BF recombinants. A prevalence of 43.9% drug resistance-associated mutations in pol sequences was identified. Of the drug-naive children 33.3% presented at least one mutation related to protease inhibitor/nucleoside reverse transcriptase inhibitor/nonnucleoside reverse transcriptase inhibitor (PI/NRTI/NNRTI) resistance. The prevalence of transmitted drug resistance mutations was 4.9%. On env we found a low prevalence of HR1 (4.9%) and HR2 (14.6%) mutations.

The usability of allele-specific PCR and reverse-hybridization assays for KRAS genotyping in Serbian colorectal cancer patients.

PubMed

Brotto, Ksenija; Malisic, Emina; Cavic, Milena; Krivokuca, Ana; Jankovic, Radmila

2013-04-01

Colorectal cancers (CRCs) with wild-type KRAS respond to EGFR-targeted antibody treatment. Analysis of the hotspot clustered mutations in codons 12 and 13 is compulsory before therapy and no standardized methodology for that purpose has been established so far. Since these mutations may have different biological effects and clinical outcome, reliable frequency and types of KRAS mutations need to be determined for individual therapy. The purpose of this study was to describe the KRAS mutation spectrum in a group of 481 Serbian mCRC patients and to compare the general performances of allele-specific PCR and reverse-hybridization assays. KRAS testing was performed with two diagnostic analyses, DxS TheraScreen K-RAS PCR Kit and KRAS StripAssay™. KRAS mutations in codons 12 and 13 were present in 37.6 % of analyzed formalin-fixed paraffin-embedded (FFPE) DNA samples. The seven most frequent mutation types were observed with both assays: p.G12D 34.6 %, p.G12V 24.9 %, p.G12A 10.3 %, p.G12C 8.1 %, p.G12S 5.4 %, p.G12R 1.6 %, and p.G13D 15.1 %. Regarding double mutants, 0.8 % of them were present among all tested samples and 2.2 % among KRAS mutated ones. Two screening approaches that were used in this study have been shown as suitable tests for detecting KRAS mutations in diagnostic settings. In addition, they appear to be good alternatives to methods presently in use. In our experience, both methods showed capacity to detect and identify double mutations which may be important for potential further subgrouping of CRC patients.

Selection Pressure in CD8+ T-cell Epitopes in the pol Gene of HIV-1 Infected Individuals in Colombia. A Bioinformatic Approach

PubMed Central

Acevedo-Sáenz, Liliana; Ochoa, Rodrigo; Rugeles, Maria Teresa; Olaya-García, Patricia; Velilla-Hernández, Paula Andrea; Diaz, Francisco J.

2015-01-01

One of the main characteristics of the human immunodeficiency virus is its genetic variability and rapid adaptation to changing environmental conditions. This variability, resulting from the lack of proofreading activity of the viral reverse transcriptase, generates mutations that could be fixed either by random genetic drift or by positive selection. Among the forces driving positive selection are antiretroviral therapy and CD8+ T-cells, the most important immune mechanism involved in viral control. Here, we describe mutations induced by these selective forces acting on the pol gene of HIV in a group of infected individuals. We used Maximum Likelihood analyses of the ratio of non-synonymous to synonymous mutations per site (dN/dS) to study the extent of positive selection in the protease and the reverse transcriptase, using 614 viral sequences from Colombian patients. We also performed computational approaches, docking and algorithmic analyses, to assess whether the positively selected mutations affected binding to the HLA molecules. We found 19 positively-selected codons in drug resistance-associated sites and 22 located within CD8+ T-cell epitopes. A high percentage of mutations in these epitopes has not been previously reported. According to the docking analyses only one of those mutations affected HLA binding. However, algorithmic methods predicted a decrease in the affinity for the HLA molecule in seven mutated peptides. The bioinformatics strategies described here are useful to identify putative positively selected mutations associated with immune escape but should be complemented with an experimental approach to define the impact of these mutations on the functional profile of the CD8+ T-cells. PMID:25803098

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation.

PubMed

Ferrari, Eleonora; Monzani, Romina; Villella, Valeria R; Esposito, Speranza; Saluzzo, Francesca; Rossin, Federica; D'Eletto, Manuela; Tosco, Antonella; De Gregorio, Fabiola; Izzo, Valentina; Maiuri, Maria C; Kroemer, Guido; Raia, Valeria; Maiuri, Luigi

2017-01-12

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation

PubMed Central

Ferrari, Eleonora; Monzani, Romina; Villella, Valeria R; Esposito, Speranza; Saluzzo, Francesca; Rossin, Federica; D'Eletto, Manuela; Tosco, Antonella; De Gregorio, Fabiola; Izzo, Valentina; Maiuri, Maria C; Kroemer, Guido; Raia, Valeria; Maiuri, Luigi

2017-01-01

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF. PMID:28079883

Recommendations to address the difficulties encountered when determining linezolid resistance from whole genome sequencing data.

PubMed

Beukers, Alicia G; Hasman, Henrik; Hegstad, Kristin; van Hal, Sebastiaan J

2018-05-29

Mutations associated with linezolid resistance within the V domain of 23S rRNA are annotated using an Escherichia coli numbering system. The 23S rRNA gene varies in length, nucleotide sequence and copy number between bacterial species. Consequently, this numbering system is not intuitive and can lead to confusion when locating mutation sites using whole genome sequencing data. Using the mutation G2576T as an example, we demonstrate the difficulties associated with using the E. coli numbering system. © Crown copyright 2018.

The Role of Drosophila Merlin in the Control of Mitosis Exit and Development

DTIC Science & Technology

2006-07-01

schwannomas and is associated with mutations in the tumor suppressor gene called the neurofibromatosis type 2 (NF2) gene (Chang et al., 2005; Neff...been shown to associate with endocytic compartments and because mutations in the genes , such as clathrin and ff16, that are known to be important... mutations in the Drosophila homologues of the human Neurofibromatosis 2 and yeast CDC42 genes using a simple and efficient reverse-genetic method. Genetics

A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus.

PubMed

Qi, Xiaole; Gao, Xiang; Lu, Zhen; Zhang, Lizhou; Wang, Yongqiang; Gao, Li; Gao, Yulong; Li, Kai; Gao, Honglei; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Wang, Xiaomei

2016-07-01

To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

Auto-production of biosurfactants reverses the coffee ring effect in a bacterial system

NASA Astrophysics Data System (ADS)

Sempels, Wouter; de Dier, Raf; Mizuno, Hideaki; Hofkens, Johan; Vermant, Jan

2013-04-01

The deposition of material at the edge of evaporating droplets, known as the ‘coffee ring effect’, is caused by a radially outward capillary flow. This phenomenon is common to a wide array of systems including colloidal and bacterial systems. The role of surfactants in counteracting these coffee ring depositions is related to the occurrence of local vortices known as Marangoni eddies. Here we show that these swirling flows are universal, and not only lead to a uniform deposition of colloids but also occur in living bacterial systems. Experiments on Pseudomonas aeruginosa suggest that the auto-production of biosurfactants has an essential role in creating a homogeneous deposition of the bacteria upon drying. Moreover, at biologically relevant conditions, intricate time-dependent flows are observed in addition to the vortex regime, which are also effective in reversing the coffee ring effect at even lower surfactant concentrations.

Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli.

PubMed

Carter, Michelle Qiu; Brandl, Maria T; Kudva, Indira T; Katani, Robab; Moreau, Matthew R; Kapur, Vivek

2018-01-01

Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro : it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the autotransporter protein Cah confers on E. coli DH5α cells a strong autoaggregative phenotype that is inversely correlated with its ability to form biofilms and plays a strain-specific role in plant and animal colonization by STEC. Although cah is widespread in the STEC population, we detected a mutation rate of 31.3% in cah , which is similar to that reported for rpoS and fimH The formation of cell aggregates due to increased bacterium-to-bacterium interactions may be disadvantageous to bacterial populations under conditions that favor a planktonic state in STEC. Therefore, a loss-of-function mutation in cah is likely a selective trait in STEC when autoaggregative properties become detrimental to bacterial cells and may contribute to the adaptability of STEC to fluctuating environments. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Suppressor Mutation Analysis of the Sensory Rhodopsin I-Transducer Complex: Insights into the Color-Sensing Mechanism

PubMed Central

Jung, Kwang-Hwan; Spudich, John L.

1998-01-01

The molecular complex containing the phototaxis receptor sensory rhodopsin I (SRI) and transducer protein HtrI (halobacterial transducer for SRI) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. One-photon excitation of the complex by orange light elicits attractant responses, while two-photon excitation (orange followed by near-UV light) elicits repellent responses in swimming cells. Several mutations in SRI and HtrI cause an unusual mutant phenotype, called orange-light-inverted signaling, in which the cell produces a repellent response to normally attractant light. We applied a selection procedure for intragenic and extragenic suppressors of orange-light-inverted mutants and identified 15 distinct second-site mutations that restore the attractant response. Two of the 3 suppressor mutations in SRI are positioned at the cytoplasmic ends of helices F and G, and 12 suppressor mutations in HtrI cluster at the cytoplasmic end of the second HtrI transmembrane helix (TM2). Nearly all suppressors invert the normally repellent response to two-photon stimulation to an attractant response when they are expressed with their suppressible mutant alleles or in an otherwise wild-type strain. The results lead to a model for control of flagellar reversal by the SRI-HtrI complex. The model invokes an equilibrium between the A (reversal-inhibiting) and R (reversal-stimulating) conformers of the signaling complex. Attractant light and repellent light shift the equilibrium toward the A and R conformers, respectively, and mutations are proposed to cause intrinsic shifts in the equilibrium in the dark form of the complex. Differences in the strength of the two-photon signal inversion and in the allele specificity of suppression are correlated, and this correlation can be explained in terms of different values of the equilibrium constant (Keq) for the conformational transition in different mutants and mutant-suppressor pairs. PMID:9555883

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

PubMed

Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

2016-11-01

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Characterization of Bacterial Cellulose by Gluconacetobacter hansenii CGMCC 3917.

PubMed

Feng, Xianchao; Ullah, Niamat; Wang, Xuejiao; Sun, Xuchun; Li, Chenyi; Bai, Yun; Chen, Lin; Li, Zhixi

2015-10-01

In this study, comprehensive characterization and drying methods on properties of bacterial cellulose were analyzed. Bacterial cellulose was prepared by Gluconacetobacter hansenii CGMCC 3917, which was mutated by high hydrostatic pressure (HHP) treatment. Bacterial cellulose is mainly comprised of cellulose Iα with high crystallinity and purity. High-water holding and absorption capacity were examined by reticulated structure. Thermogravimetric analysis showed high thermal stability. High tensile strength and Young's modulus indicated its mechanical properties. The rheological analysis showed that bacterial cellulose had good consistency and viscosity. These results indicated that bacterial cellulose is a potential food additive and also could be used for a food packaging material. The high textural stability during freeze-thaw cycles makes bacterial cellulose an effective additive for frozen food products. In addition, the properties of bacterial cellulose can be affected by drying methods. Our results suggest that the bacterial cellulose produced from HHP-mutant strain has an effective characterization, which can be used for a wide range of applications in food industry. © 2015 Institute of Food Technologists®

Spontaneous Mutation Rate in the Smallest Photosynthetic Eukaryotes

PubMed Central

Krasovec, Marc; Eyre-Walker, Adam; Sanchez-Ferandin, Sophie

2017-01-01

Abstract Mutation is the ultimate source of genetic variation, and knowledge of mutation rates is fundamental for our understanding of all evolutionary processes. High throughput sequencing of mutation accumulation lines has provided genome wide spontaneous mutation rates in a dozen model species, but estimates from nonmodel organisms from much of the diversity of life are very limited. Here, we report mutation rates in four haploid marine bacterial-sized photosynthetic eukaryotic algae; Bathycoccus prasinos, Ostreococcus tauri, Ostreococcus mediterraneus, and Micromonas pusilla. The spontaneous mutation rate between species varies from μ = 4.4 × 10−10 to 9.8 × 10−10 mutations per nucleotide per generation. Within genomes, there is a two-fold increase of the mutation rate in intergenic regions, consistent with an optimization of mismatch and transcription-coupled DNA repair in coding sequences. Additionally, we show that deviation from the equilibrium GC content increases the mutation rate by ∼2% to ∼12% because of a GC bias in coding sequences. More generally, the difference between the observed and equilibrium GC content of genomes explains some of the inter-specific variation in mutation rates. PMID:28379581

Chemical and UV Mutagenesis.

PubMed

Bose, Jeffrey L

2016-01-01

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

2004: which HIV-1 drug resistance mutations are common in clinical practice?

PubMed

Cheung, Peter K; Wynhoven, Brian; Harrigan, P Richard

2004-01-01

The emergence of drug resistance remains a major problem for the treatment of HIV-infected patients. However, the variety of mutational patterns that evolve in clinical practice have made the application of resistance data to clinical decision-making challenging. Despite (or because of) an abundance of drug-resistance data from disparate sources, there is only limited information available describing the patterns of drug resistance which usually appear in the clinic. Here we attempt to address this issue by reviewing HIV drug resistance in the population of patients failing antiretroviral therapy in British Columbia, Canada from June 1996 to December 2003 as an example. Our findings suggest that, although hundreds of mutations have been associated with resistance, relatively few key mutations occur at a high frequency. For example, only the nucleoside reverse transcriptase inhibitor (NRTI) mutations M184V, M41L T215Y, D67N, K70R and L210W, non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations K103N and Y181C, and protease inhibitor (PI) mutation L90M, occur in more than 10% of samples tested for resistance in this population. The introduction of new drugs allows for the selection of new mutations. Trends in the prevalence of resistance-associated mutations have generally followed trends in drug usage, but have not always mirrored them. The phenomenon of cross-resistance can play an important role in the efficacy of new antiretroviral agents, even before they become available. The extent of this cross-resistance depends in part on the prevalence of specific mutations in the population of individuals who have previously received antiretroviral therapy. Hence there is a need to determine which mutations are prevalent in the treated population. The tremendous capacity of HIV to adapt means that common resistance pathways are likely to change over time, and new pathways to resistance are likely to continue to be discovered in the future.

Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine.

PubMed Central

Medlin, H K; Zhu, Y Q; Remington, K M; Phillips, T R; North, T W

1996-01-01

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis. PMID:8849258

Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation

PubMed Central

Saito, Samuel Takashi; Trentin, Danielle da Silva; Macedo, Alexandre José; Pungartnik, Cristina; Gosmann, Grace; Silveira, Jaqueline de Deos; Guecheva, Temenouga Nikolova; Henriques, João Antonio Pêgas; Brendel, Martin

2012-01-01

Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI < 3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications. PMID:22548121

Acute toxicity and genotoxicity of fermented traditional medicine oyaksungi-san.

PubMed

Park, Hwayong; Hwang, Youn-Hwan; Ma, Jin Yeul

2017-06-01

The traditional medicine oyaksungi-san (OY) has been prescribed in East Asia for hundreds of years for the treatment of stroke, paralysis, and ataxia. OY also has therapeutic effects on arthralgia, myalgia, and rheumatoid arthritis, and recent studies have shown its protective effects against apoptosis of hippocampal cells and its anti-inflammatory effects on the peripheral blood cells of patient with cerebral infarction. Many studies have explored the use of traditional medicine and herb materials in the development of safe, novel, and effective pharmaceuticals with fewer side effects. These efforts commonly adopt a bioconversion tool for fermentation with beneficial microbes. However, only pharmaceuticals with high levels of safety and low levels of toxicity can be used in healthcare system. OY water extract was fermented with Lactobacillus and assayed for acute toxicity and genotoxicity. Single dose acute toxicity, bacterial reverse mutation, chromosome aberrations, and micronucleus were observed and assayed in rats, histidine/tryptophan auxotrophic bacteria, Chinese hamster ovary fibroblast cells, and mice bone marrow cells, respectively. All the experimental animals showed no abnormal behavior, clinical signs, body weight increases, or mortality. In the bacterial cultures, no revertant colonies were observed. Morphological and numerical chromosomal aberrations were not found in all metaphases examined. Frequency of induced micronuclei was not significantly increased in all doses applied. As a whole, no acute toxicity or genotoxicity were observed in all the assays examined. Therefore, fermented OY is considered to be a safe material that can be used for development of complementary and alternative medicine using bioconversion.

Acute toxicity and genotoxicity study of fermented traditional herb formula Guibi-tang.

PubMed

Park, Hwayong; Hwang, Youn-Hwan; Yang, Hye Jin; Kim, Hyun-Kyu; Song, Kyung Seuk; Ma, Jin Yeul

2014-10-28

Guibi-tang (Guipi-tang in Chinese and Kihi-to in Japanese) is a multi-herb traditional medicine commonly prescribed to treat psychoneurosis in East Asia. Although this medicine has been widely used, there is little available information on the safety and toxicity of Guibi-tang, especially on the fermented one. Guibi-tang, composed of 12 herbs, was fermented with bacteria and lyophilized. Single dose acute toxicity in rats was observed for 14 days after administration. Genetic toxicity of fermented Guibi-tang was evaluated on bacterial reverse mutation in Salmonella and Escherichia spp., chromosome aberrations in Chinese hamster ovary cells, and micronucleus formation in mice. Ingredients in FGBT were identified and quantified by high performance liquid chromatography-mass spectrometry. In acute oral toxicity study, behavior, clinical signs and body weight changes were normal observing in all experimental animals. No revertant colonies were found in any bacterial cultures examined. Morphological or numerical anomalies and significant increased number of aberrant metaphases were not observed. Micronucleus assay showed no significant increases in the frequency of inducing micronuclei in any dose examined. Decursinol, decursin, glycyrrhizin, and 6-gingerol in fermented Guibi-tang were identified and quantitated. As a whole, no acute and genotoxic effects were found in all the assays and parameters analyzed. Fermented Guibi-tang was recognized as safe and non-toxic, and therefore can be used for applications of traditional medicine in modern complementary and alternative therapeutics and health care. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation.

PubMed

Saito, Samuel Takashi; Trentin, Danielle da Silva; Macedo, Alexandre José; Pungartnik, Cristina; Gosmann, Grace; Silveira, Jaqueline de Deos; Guecheva, Temenouga Nikolova; Henriques, João Antonio Pêgas; Brendel, Martin

2012-01-01

Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI < 3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications.

Experimental evolution and the dynamics of genomic mutation rate modifiers.

PubMed

Raynes, Y; Sniegowski, P D

2014-11-01

Because genes that affect mutation rates are themselves subject to mutation, mutation rates can be influenced by natural selection and other evolutionary forces. The population genetics of mutation rate modifier alleles has been a subject of theoretical interest for many decades. Here, we review experimental contributions to our understanding of mutation rate modifier dynamics. Numerous evolution experiments have shown that mutator alleles (modifiers that elevate the genomic mutation rate) can readily rise to high frequencies via genetic hitchhiking in non-recombining microbial populations. Whereas these results certainly provide an explanatory framework for observations of sporadically high mutation rates in pathogenic microbes and in cancer lineages, it is nonetheless true that most natural populations have very low mutation rates. This raises the interesting question of how mutator hitchhiking is suppressed or its phenotypic effect reversed in natural populations. Very little experimental work has addressed this question; with this in mind, we identify some promising areas for future experimental investigation.

Differences in resistance mutations among HIV-1 non-subtype B infections: a systematic review of evidence (1996–2008)

PubMed Central

2009-01-01

Ninety percent of HIV-1-infected people worldwide harbour non-subtype B variants of HIV-1. Yet knowledge of resistance mutations in non-B HIV-1 and their clinical relevance is limited. Although a few reviews, editorials and perspectives have been published alluding to this lack of data among non-B subtypes, no systematic review has been performed to date. With this in mind, we conducted a systematic review (1996–2008) of all published studies performed on the basis of non-subtype B HIV-1 infections treated with antiretroviral drugs that reported genotype resistance tests. Using an established search string, 50 studies were deemed relevant for this review. These studies reported genotyping data from non-B HIV-1 infections that had been treated with either reverse transcriptase inhibitors or protease inhibitors. While most major resistance mutations in subtype B were also found in non-B subtypes, a few novel mutations in non-B subtypes were recognized. The main differences are reflected in the discoveries that: (i) the non-nucleoside reverse transcriptase inhibitor resistance mutation, V106M, has been seen in subtype C and CRF01_AE, but not in subtype B, (ii) the protease inhibitor mutations L89I/V have been reported in C, F and G subtypes, but not in B, (iii) a nelfinavir selected non-D30N containing pathway predominated in CRF01_AE and CRF02_AG, while the emergence of D30N is favoured in subtypes B and D, (iv) studies on thymidine analog-treated subtype C infections from South Africa, Botswana and Malawi have reported a higher frequency of the K65R resistance mutation than that typically seen with subtype B. Additionally, some substitutions that seem to impact non-B viruses differentially are: reverse transcriptase mutations G196E, A98G/S, and V75M; and protease mutations M89I/V and I93L. Polymorphisms that were common in non-B subtypes and that may contribute to resistance tended to persist or become more frequent after drug exposure. Some, but not all, are recognized as minor resistance mutations in B subtypes. These observed differences in resistance pathways may impact cross-resistance and the selection of second-line regimens with protease inhibitors. Attention to newer drug combinations, as well as baseline genotyping of non-B isolates, in well-designed longitudinal studies with long duration of follow up are needed. PMID:19566959

Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics*

PubMed Central

Lukacs, Peter; Gawali, Vaibhavkumar S.; Cervenka, Rene; Ke, Song; Koenig, Xaver; Rubi, Lena; Zarrabi, Touran; Hilber, Karlheinz; Stary-Weinzinger, Anna; Todt, Hannes

2014-01-01

Despite the availability of several crystal structures of bacterial voltage-gated Na+ channels, the structure of eukaryotic Na+ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na+ channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na+ channels. PMID:24947510

Quorum quenching quandary: resistance to antivirulence compounds

PubMed Central

Maeda, Toshinari; García-Contreras, Rodolfo; Pu, Mingming; Sheng, Lili; Garcia, Luis Rene; Tomás, Maria; Wood, Thomas K

2012-01-01

Quorum sensing (QS) is the regulation of gene expression in response to the concentration of small signal molecules, and its inactivation has been suggested to have great potential to attenuate microbial virulence. It is assumed that unlike antimicrobials, inhibition of QS should cause less Darwinian selection pressure for bacterial resistance. Using the opportunistic pathogen Pseudomonas aeruginosa, we demonstrate here that bacterial resistance arises rapidly to the best-characterized compound that inhibits QS (brominated furanone C-30) due to mutations that increase the efflux of C-30. Critically, the C-30-resistant mutant mexR was more pathogenic to Caenorhabditis elegans in the presence of C-30, and the same mutation arises in bacteria responsible for chronic cystic fibrosis infections. Therefore, bacteria may evolve resistance to many new pharmaceuticals thought impervious to resistance. PMID:21918575

LytN, a Murein Hydrolase in the Cross-wall Compartment of Staphylococcus aureus, Is Involved in Proper Bacterial Growth and Envelope Assembly*

PubMed Central

Frankel, Matthew B.; Hendrickx, Antoni P. A.; Missiakas, Dominique M.; Schneewind, Olaf

2011-01-01

Cell cycle progression for the spherical microbe Staphylococcus aureus requires the coordinated synthesis and remodeling of peptidoglycan. The majority of these rearrangements takes place at the mid-cell, in a compartment designated the cross-wall. Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. One such YSIRK-containing protein is the murein hydrolase LytN. lytN mutations precipitate structural damage to the cross-wall and interfere with staphylococcal growth. Overexpression of lytN also affects growth and triggers rupture of the cross-wall. The lytN phenotype can be reversed by the controlled expression of lytN but not by adding purified LytN to staphylococcal cultures. LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-l-alanine amidase and d-alanyl-glycine endopeptidase. Thus, LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the staphylococcal cell cycle. PMID:21784864

Safety evaluation of a proprietary food-grade, dried fermentate preparation of Saccharomyces cerevisiae.

PubMed

Schauss, Alexander G; Glavits, R; Endres, John; Jensen, Gitte S; Clewell, Amy

2012-01-01

A safety evaluation was performed for EpiCor, a product produced by a proprietary fermentation process using Saccharomyces cerevisiae. Studies included the following assays: bacterial reverse mutation, mouse lymphoma cell mutagenicity, mitogenicity assay in human peripheral lymphocytes, and a cytochrome P450 ([CYP] CYP1A2 and CYP3A4) induction assessment as well as 14-day acute, 90-day subchronic, and 1-year chronic oral toxicity studies in rats. No evidence of genotoxicity or mitogenicity was seen in any of the in vitro or in vivo studies. The CYP assessment showed no interactions or inductions. No toxic clinical symptoms or histopathological lesions were observed in the acute, subchronic, or chronic oral toxicity studies in the rat. Results of the studies performed indicate that EpiCor does not possess genotoxic activity and has a low order of toxicity that is well tolerated when administered orally. The no observable adverse effect level (NOAEL) was 1500 mg/kg body weight (bw)/d for the 90-day study and 800 mg/kg bw/d for the 1 year study, for the highest doses tested.

Mutagenicity and Acute Oral Toxicity Test for Herbal Poultry Feed Supplements.

PubMed

Srinivasa Rao, Boddapati; Chandrasekaran, C V; Srikanth, H S; Sasikumar, Murugan; Edwin Jothie, R; Haseena, Begum; Bharathi, Bethapudi; Selvam, Ramasamy; Prashanth, D'Souza

2018-01-01

Herbal products are being used and trusted globally for thousands of years for their health benefits and limited side effects. Globally, a general belief amongst the consumers is that herbal supplements are always safe because they are "natural." But later, research reveals that they may not be safe. This raises concern on their safety and implications for their use as feed supplement or medicine. Toxicity testing can reveal some of the risks that may be associated with use of herbs, therefore avoiding potential harmful effects. The present study was designed to investigate five poultry feed supplements (PFS), EGMAX® (to revitalize ovarian activity), FEED-X ™ (feed efficiency enhancer), KOLIN PLUS ™ (natural replacer of synthetic choline chloride), PHYTOCEE® (natural defence enhancer), and STODI® (to prevent and control loose droppings), for their possible mutagenicity and toxicity. Bacterial reverse mutation (BRMT) and acute oral toxicity tests were employed to assess the PFS for their possible mutagenicity and toxicity. Results indicated that the PFS were devoid of mutagenic effects in BRMT and showed higher safety profile in rodent acute oral toxicity test.

Toxicological assessment of Ashitaba Chalcone.

PubMed

Maronpot, Robert R

2015-03-01

The plant Angelica keiskei contains two main physiologically active flavonoid chalcones, 4-hydroxyderricin and xanthoangelol. Known as ashitaba in Japan, powder from the sap is widely consumed for its medicinal properties in Asia as a dietary supplement. Limited previously reported mammalian studies were without evidence of toxicity. GLP studies reported here, including a bacterial reverse mutation assay, a chromosome aberration assay, and an in vivo micronucleus assay are negative for genotoxicity. A GLP- compliant 90-day repeated oral gavage study of ashitaba yellow sap powder containing 8.45% chalcones in Sprague Dawley rats resulted in expected known physiological effects on coagulation parameters and plasma lipids at 300 and 1000 mg/kg/day. Ashitaba-related pathology included a dose-related male rat-specific alpha 2-urinary globulin nephropathy at 100, 300, and 1000 mg/kg/day and jejunal lymphangiectasia in both sexes at 1000 mg/kg/day. All other study parameters and histopathological changes were incidental or not of toxicological concern. Based on these studies ashitaba chalcone powder is not genotoxic with a NOAEL of 300 mg/kg in male and female rats. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mutations in the sigma subunit of E. coli RNA polymerase which affect positive control of transcription.

PubMed

Hu, J C; Gross, C A

1985-01-01

The sigma subunits of bacterial RNA polymerases are required for the selective initiation of transcription. We have isolated and characterized mutations in rpoD, the gene which encodes the major form of sigma in E. coli, which affect the selectivity of transcription. These mutations increase the expression of araBAD up to 12-fold in the absence of CAP-cAMP. Expression of lac is unaffected, while expression of malT-activated operons is decreased. We determined the DNA sequence of 17 independently isolated mutations, and found that they consist of three different changes in a single CGC arginine codon at position 596 in the sigma polypeptide.

Engineering microbes for efficient production of chemicals

DOEpatents

Gong, Wei; Dole, Sudhanshu; Grabar, Tammy; Collard, Andrew Christopher; Pero, Janice G; Yocum, R Rogers

2015-04-28

This present invention relates to production of chemicals from microorganisms that have been genetically engineered and metabolically evolved. Improvements in chemical production have been established, and particular mutations that lead to those improvements have been identified. Specific examples are given in the identification of mutations that occurred during the metabolic evolution of a bacterial strain genetically engineered to produce succinic acid. This present invention also provides a method for evaluating the industrial applicability of mutations that were selected during the metabolic evolution for increased succinic acid production. This present invention further provides microorganisms engineered to have mutations that are selected during metabolic evolution and contribute to improved production of succinic acid, other organic acids and other chemicals of commercial interest.

Culturable bacterial diversity from a feed water of a reverse osmosis system, evaluation of biofilm formation and biocontrol using phages.

PubMed

Belgini, D R B; Dias, R S; Siqueira, V M; Valadares, L A B; Albanese, J M; Souza, R S; Torres, A P R; Sousa, M P; Silva, C C; De Paula, S O; Oliveira, V M

2014-10-01

Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.

Newly emerging mutations in the matrix genes of the human influenza A(H1N1)pdm09 and A(H3N2) viruses reduce the detection sensitivity of real-time reverse transcription-PCR.

PubMed

Yang, Ji-Rong; Kuo, Chuan-Yi; Huang, Hsiang-Yi; Wu, Fu-Ting; Huang, Yi-Lung; Cheng, Chieh-Yu; Su, Yu-Ting; Chang, Feng-Yee; Wu, Ho-Sheng; Liu, Ming-Tsan

2014-01-01

New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.

Towards rationally redesigning bacterial signaling systems using information encoded in abundant sequence data

NASA Astrophysics Data System (ADS)

Cheng, Ryan; Morcos, Faruck; Levine, Herbert; Onuchic, Jose

2014-03-01

An important challenge in biology is to distinguish the subset of residues that allow bacterial two-component signaling (TCS) proteins to preferentially interact with their correct TCS partner such that they can bind and transfer signal. Detailed knowledge of this information would allow one to search sequence-space for mutations that can systematically tune the signal transmission between TCS partners as well as re-encode a TCS protein to preferentially transfer signals to a non-partner. Motivated by the notion that this detailed information is found in sequence data, we explore the mutual sequence co-evolution between signaling partners to infer how mutations can positively or negatively alter their interaction. Using Direct Coupling Analysis (DCA) for determining evolutionarily conserved interprotein interactions, we apply a DCA-based metric to quantify mutational changes in the interaction between TCS proteins and demonstrate that it accurately correlates with experimental mutagenesis studies probing the mutational change in the in vitro phosphotransfer. Our methodology serves as a potential framework for the rational design of TCS systems as well as a framework for the system-level study of protein-protein interactions in sequence-rich systems. This research has been supported by the NSF INSPIRE award MCB-1241332 and by the CTBP sponsored by the NSF (Grant PHY-1308264).

Insights in connecting phenotypes in bacteria to coevolutionary information

NASA Astrophysics Data System (ADS)

Cheng, Ryan; Morcos, Faruck; Hayes, Ryan; Helm, Rodney; Levine, Herbert; Onuchic, Jose

It has long been known that protein sequences are far from random. These sequences have been evolutionarily selected to maintain their ability to fold into stable, three-dimensional folded structures as well as their ability to form macromolecular assemblies, perform catalytic functions, etc. For these reasons, there exist quantifiable mutational patterns in the collection of sequence data for a protein family arising from the need to maintain favorable residue-residue interactions to facilitate folding as well as cellular function. Here, we focus on studying the correlated mutational patterns that give rise to interaction specificity in bacterial two-component signaling (TCS) systems. TCS proteins have evolved to be able to preferentially bind and transfer a phosphate group to their signaling partner while avoiding phosphotransfer with non-partners. We infer a Potts model Hamiltonian governing the correlated mutational patterns that are observed in the sequence data of TCS partners and apply this model to recently published in vivo mutational data. Our findings further support the notion that statistical models built from sequence data can be used to predict bacterial phenotypes as well as engineer interaction specificity between non-partner TCS proteins. This research has been supported by the NSF INSPIRE Award (MCB-1241332) and by the CTBP sponsored by the NSF (Grant PHY- 1427654).

Distinct signatures for mutator sensitivity of lacZ reversions and for the spectrum of lacI/lacO forward mutations on the chromosome of nondividing Escherichia coli.

PubMed Central

Bharatan, Shanti M; Reddy, Manjula; Gowrishankar, J

2004-01-01

A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature. Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells. The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5. In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate. Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5. Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants. This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state. PMID:15020459

Quinolone Resistance Reversion by Targeting the SOS Response.

PubMed

Recacha, E; Machuca, J; Díaz de Alba, P; Ramos-Güelfo, M; Docobo-Pérez, F; Rodriguez-Beltrán, J; Blázquez, J; Pascual, A; Rodríguez-Martínez, J M

2017-10-10

Suppression of the SOS response has been postulated as a therapeutic strategy for potentiating antimicrobial agents. We aimed to evaluate the impact of its suppression on reversing resistance using a model of isogenic strains of Escherichia coli representing multiple levels of quinolone resistance. E. coli mutants exhibiting a spectrum of SOS activity were constructed from isogenic strains carrying quinolone resistance mechanisms with susceptible and resistant phenotypes. Changes in susceptibility were evaluated by static (MICs) and dynamic (killing curves or flow cytometry) methodologies. A peritoneal sepsis murine model was used to evaluate in vivo impact. Suppression of the SOS response was capable of resensitizing mutant strains with genes encoding three or four different resistance mechanisms (up to 15-fold reductions in MICs). Killing curve assays showed a clear disadvantage for survival (Δlog 10 CFU per milliliter [CFU/ml] of 8 log units after 24 h), and the in vivo efficacy of ciprofloxacin was significantly enhanced (Δlog 10 CFU/g of 1.76 log units) in resistant strains with a suppressed SOS response. This effect was evident even after short periods (60 min) of exposure. Suppression of the SOS response reverses antimicrobial resistance across a range of E. coli phenotypes from reduced susceptibility to highly resistant, playing a significant role in increasing the in vivo efficacy. IMPORTANCE The rapid rise of antibiotic resistance in bacterial pathogens is now considered a major global health crisis. New strategies are needed to block the development of resistance and to extend the life of antibiotics. The SOS response is a promising target for developing therapeutics to reduce the acquisition of antibiotic resistance and enhance the bactericidal activity of antimicrobial agents such as quinolones. Significant questions remain regarding its impact as a strategy for the reversion or resensitization of antibiotic-resistant bacteria. To address this question, we have generated E. coli mutants that exhibited a spectrum of SOS activity, ranging from a natural SOS response to a hypoinducible or constitutively suppressed response. We tested the effects of these mutations on quinolone resistance reversion under therapeutic concentrations in a set of isogenic strains carrying different combinations of chromosome- and plasmid-mediated quinolone resistance mechanisms with susceptible, low-level quinolone resistant, resistant, and highly resistant phenotypes. Our comprehensive analysis opens up a new strategy for reversing drug resistance by targeting the SOS response. Copyright © 2017 Recacha et al.

GSTA1 Expression Is Correlated With Aldosterone Level in KCNJ5-Mutated Adrenal Aldosterone-Producing Adenoma.

PubMed

Li, Xintao; Wang, Baojun; Tang, Lu; Zhang, Yu; Chen, Luyao; Gu, Liangyou; Zhang, Fan; Ouyang, Jinzhi; Zhang, Xu

2018-03-01

KCNJ5 mutation is a major cause of aldosterone-producing adenomas (APAs). The development of APA apart from KCNJ5 mutation is less investigated. To investigate other mechanisms affecting aldosterone secretion apart from KCNJ5. Six pairs of KCNJ5-mutated, high and low aldosterone-secreting APAs, five non-KCNJ5-mutated APAs, and four normal adrenal glands were assayed by Affymetrix GeneChip Human Transcriptome Array 2.0. A total of 113 APA samples were investigated to explore the expression of glutathione-S-transferase A1 (GSTA1). H295R cells were used to verify the function of GSTA1. GSTA1 was the top gene downregulated in high-aldosterone KCNJ5-mutated APAs. GSTA1 was also downregulated in KCNJ5-mutated APAs compared with wild-type KCNJ5 APAs. Accordingly, mutant KCNJ5 decreased GSTA1 messenger RNA and protein expression levels. GSTA1 overexpression suppressed aldosterone secretion whether in wild-type or mutant KCNJ5 H295R cells. Adding ethacrynic acid or silencing of GSTA1 increased aldosterone secretion by increasing reactive oxygen species (ROS), superoxide, H2O2 levels, and Ca2+ influx. The expression of the transcription factors NR4A1, NR4A2, and CAMK1 and intracellular Ca2+ were significantly upregulated by GSTA1 inhibition. The reduced form of NAD phosphate oxidase inhibitor or H2O2 scavenger or blocking calmodulin or calcium channels could significantly reduce aldosterone secretion in GSTA1-inhibited cells. (1) GSTA1 expression is reversely correlated with aldosterone level in KCNJ5-mutated APAs, (2) GSTA1 regulates aldosterone secretion by ROS and Ca2+ signaling, and (3) KCNJ5 mutation downregulates GSTA1 expression, and overexpression of GSTA1 reverses increased aldosterone in KCNJ5-mutated adrenal cells.

FOXL2 is a female sex-determining gene in the goat.

PubMed

Boulanger, Laurent; Pannetier, Maëlle; Gall, Laurence; Allais-Bonnet, Aurélie; Elzaiat, Maëva; Le Bourhis, Daniel; Daniel, Nathalie; Richard, Christophe; Cotinot, Corinne; Ghyselinck, Norbert B; Pailhoux, Eric

2014-02-17

The origin of sex reversal in XX goats homozygous for the polled intersex syndrome (PIS) mutation was unclear because of the complexity of the mutation that affects the transcription of both FOXL2 and several long noncoding RNAs (lncRNAs). Accumulating evidence suggested that FOXL2 could be the sole gene of the PIS locus responsible for XX sex reversal, the lncRNAs being involved in transcriptional regulation of FOXL2. In this study, using zinc-finger nuclease-directed mutagenesis, we generated several fetuses, of which one XX individual bears biallelic mutations of FOXL2. Our analysis demonstrates that FOXL2 loss of function dissociated from loss of lncRNA expression is sufficient to cause an XX female-to-male sex reversal in the goat model and, as in the mouse model, an agenesis of eyelids. Both developmental defects were reproduced in two newborn animals cloned from the XX FOXL2(-/-) fibroblasts. These results therefore identify FOXL2 as a bona fide female sex-determining gene in the goat. They also highlight a stage-dependent role of FOXL2 in the ovary, different between goats and mice, being important for fetal development in the former but for postnatal maintenance in the latter. Copyright © 2014 Elsevier Ltd. All rights reserved.

Occurrence of transmitted HIV-1 drug resistance among Drug-naïve pregnant women in selected HIV-care centres in Ghana.

PubMed

Martin-Odoom, Alexander; Adiku, Theophilus; Delgado, Elena; Lartey, Margaret; Ampofo, William K

2017-03-01

Access to antiretroviral therapy in Ghana has been scaled up across the country over the last decade. This study sought to determine the occurrence of transmitted HIV-1 drug resistance in pregnant HIV-1 positive women yet to initiate antiretroviral therapy at selected HIV Care Centres in Ghana. Plasma specimens from twenty-six (26) HIV seropositive pregnant women who were less than 28weeks pregnant with their first pregnancy and ART naïve were collected from selected HIV care centres in three (3) regions in Ghana. Genotypic testing was done for the reverse transcriptase gene and the sequences generated were analyzed for HIV-1 drug resistance mutations using the Stanford University HIV Drug Resistance Database. Resistance mutations associated with the reverse transcriptase gene were detected in 4 (15.4%) of the participants. At least one major drug resistance mutation in the reverse transcriptase gene was found in 3 (11.5%) of the women. The detection of transmitted HIV-1 drug resistance in this drug-naïve group in two regional HIV care sites is an indication of the need for renewed action in monitoring the emergence of transmitted HIV-1 drug resistance in Ghana. None declared.

Solid papillary carcinoma with reverse polarity of the breast harbors specific morphologic, immunohistochemical and molecular profile in comparison with other benign or malignant papillary lesions of the breast: a comparative study of 9 additional cases.

PubMed

Alsadoun, Nadjla; MacGrogan, Gaëtan; Truntzer, Caroline; Lacroix-Triki, Magali; Bedgedjian, Isabelle; Koeb, Marie-Hélène; El Alam, Elsy; Medioni, Dan; Parent, Michel; Wuithier, Pascal; Robert, Isabelle; Boidot, Romain; Arnould, Laurent

2018-05-21

Solid papillary carcinoma with reverse polarity is a rare breast cancer of favorable prognosis that can be difficult to diagnose. We report here nine additional cases of this tumor, and we describe its morphologic, immunohistochemical and molecular profile in comparison to other types of papillary and micropapillary lesions of the breast that are intraductal papilloma with usual ductal hyperplasia, encapsulated papillary carcinoma, solid papillary carcinoma and invasive micropapillary carcinoma. We studied nine cases of this special papillary tumor and six of each other types mentioned above. We found that solid papillary carcinoma with reverse polarity harbor specific morphologic features as cuboid or tall cells with abundant eosinophilic cytoplasms located at the basal pole giving the impression of reverse nuclear polarity. Nuclei were sometimes grooved. Immunohistochemistry demonstrated the lack of myoepithelial cells, as in encapsulated papillary carcinoma and solid papillary carcinoma, questioning their invasive nature. Seven of nine solid papillary carcinoma with reverse polarity showed a low Ki67 proliferative index (Ki67 <5%). They showed expression of CK5/6 as in intraductal papilloma with usual ductal hyperplasia. They showed expression of calretinin and a low or lack of hormonal receptor (HR) expression that were not observed in other breast tumors studied. By whole-exome analysis, seven of nine solid papillary carcinomas with reverse polarity (78%) harbored a hotspot mutation in IDH2 (R172) that was totally absent in other groups. Six of nine tumors (67%) also harbored PRUNE2 mutation, including the two IDH2 wild-type cases. We also demonstrated for the first time in this breast tumor, immunostaining with a specific antibody IDH1/2 mutant R132/R172 (7/9) that can highlight IDH2 mutation. Moreover, transcriptomic analysis showed that proteoglycan pathway was significantly enriched. Our findings support the fact that solid papillary carcinoma with reverse polarity is a singular breast neoplasm that can be distinguished from other papillary breast tumors.

Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing.

PubMed

Bierle, Craig J; Anderholm, Kaitlyn M; Wang, Jian Ben; McVoy, Michael A; Schleiss, Mark R

2016-08-01

The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx

PubMed Central

Suárez, Gabriel A.; Renda, Brian A.; Dasgupta, Aurko

2017-01-01

ABSTRACT The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting “clean-genome” ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. PMID:28667117

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

PubMed

Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

2017-09-01

The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting "clean-genome" ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. Copyright © 2017 American Society for Microbiology.

Ela2 mutations and clinical manifestations in familial congenital neutropenia.

PubMed

Shiohara, Masaaki; Shigemura, Tomonari; Saito, Shoji; Tanaka, Miyuki; Yanagisawa, Ryu; Sakashita, Kazuo; Asada, Hiroshi; Ishii, Eizaburo; Koike, Kazutoshi; Chin, Motoaki; Kobayashi, Masao; Koike, Kenichi

2009-05-01

Three familial cases of each of severe congenital neutropenia (SCN) and cyclic neutropenia (CN) in addition to 3 sporadic cases of SCN were analyzed for neutrophil elastase (Ela2) gene mutation. The contents of the neutrophil-specific granule proteins cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were also analyzed in SCN. Genomic DNA was extracted from the patients' peripheral blood or bone marrow, and the coding sequence of the Ela2 gene was amplified by polymerase chain reaction and subjected to direct sequencing. The contents of antimicrobial peptides were analyzed by flow cytometry. Three cases of familial SCN (P13L, R52P, and S97L), 2 of familial CN (W212stop and P110L), and 1 of sporadic SCN (V72M) were shown to have heterozygous mutations in the Ela2 gene. W212stop found in a familial CN case was a novel mutation of Ela2. Prophylactic treatment for growth factors or antibiotic prophylaxis against bacterial infection was useful for lowering the frequency of infectious episodes. Adult patients tended to have less frequent infections compared with minors in the same family. The contents of both cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were significantly reduced in SCN compared with healthy controls. Prophylaxis by growth factor or antibiotics is useful for decreasing risks of bacterial infections in SCN and CN. Adults were likely to have less frequent infections than children in familial cases of SCN and CN with the same mutation of Ela2.

Global Comparison of Drug Resistance Mutations After First-Line Antiretroviral Therapy Across Human Immunodeficiency Virus-1 Subtypes

PubMed Central

Huang, Austin; Hogan, Joseph W.; Luo, Xi; DeLong, Allison; Saravanan, Shanmugam; Wu, Yasong; Sirivichayakul, Sunee; Kumarasamy, Nagalingeswaran; Zhang, Fujie; Phanuphak, Praphan; Diero, Lameck; Buziba, Nathan; Istrail, Sorin; Katzenstein, David A.; Kantor, Rami

2016-01-01

Background. Human immunodeficiency virus (HIV)-1 drug resistance mutations (DRMs) often accompany treatment failure. Although subtype differences are widely studied, DRM comparisons between subtypes either focus on specific geographic regions or include populations with heterogeneous treatments. Methods. We characterized DRM patterns following first-line failure and their impact on future treatment in a global, multi-subtype reverse-transcriptase sequence dataset. We developed a hierarchical modeling approach to address the high-dimensional challenge of modeling and comparing frequencies of multiple DRMs in varying first-line regimens, durations, and subtypes. Drug resistance mutation co-occurrence was characterized using a novel application of a statistical network model. Results. In 1425 sequences, 202 subtype B, 696 C, 44 G, 351 circulating recombinant forms (CRF)01_AE, 58 CRF02_AG, and 74 from other subtypes mutation frequencies were higher in subtypes C and CRF01_AE compared with B overall. Mutation frequency increased by 9%–20% at reverse transcriptase positions 41, 67, 70, 184, 215, and 219 in subtype C and CRF01_AE vs B. Subtype C and CRF01_AE exhibited higher predicted cross-resistance (+12%–18%) to future therapy options compared with subtype B. Topologies of subtype mutation networks were mostly similar. Conclusions. We find clear differences in DRM outcomes following first-line failure, suggesting subtype-specific ecological or biological factors that determine DRM patterns. PMID:27419147

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria

PubMed Central

Bonhoeffer, Sebastian

2018-01-01

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the numbers of cell divisions are crucial but neglected parameters in the evolvability of a population, and we provide experimental and computational tools and methods to study evolvability under stress, leading to a reassessment of the magnitude and significance of the stress-induced mutagenesis paradigm. PMID:29750784

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria.

PubMed

Frenoy, Antoine; Bonhoeffer, Sebastian

2018-05-01

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the numbers of cell divisions are crucial but neglected parameters in the evolvability of a population, and we provide experimental and computational tools and methods to study evolvability under stress, leading to a reassessment of the magnitude and significance of the stress-induced mutagenesis paradigm.

UVR2 ensures transgenerational genome stability under simulated natural UV-B in Arabidopsis thaliana

PubMed Central

Willing, Eva-Maria; Piofczyk, Thomas; Albert, Andreas; Winkler, J. Barbro; Schneeberger, Korbinian; Pecinka, Ales

2016-01-01

Ground levels of solar UV-B radiation induce DNA damage. Sessile phototrophic organisms such as vascular plants are recurrently exposed to sunlight and require UV-B photoreception, flavonols shielding, direct reversal of pyrimidine dimers and nucleotide excision repair for resistance against UV-B radiation. However, the frequency of UV-B-induced mutations is unknown in plants. Here we quantify the amount and types of mutations in the offspring of Arabidopsis thaliana wild-type and UV-B-hypersensitive mutants exposed to simulated natural UV-B over their entire life cycle. We show that reversal of pyrimidine dimers by UVR2 photolyase is the major mechanism required for sustaining plant genome stability across generations under UV-B. In addition to widespread somatic expression, germline-specific UVR2 activity occurs during late flower development, and is important for ensuring low mutation rates in male and female cell lineages. This allows plants to maintain genome integrity in the germline despite exposure to UV-B. PMID:27905394

Long-term persistence of primary genotypic resistance after HIV-1 seroconversion.

PubMed

Pao, David; Andrady, Ushan; Clarke, Janette; Dean, Gillian; Drake, Susan; Fisher, Martin; Green, Tanya; Kumar, Siva; Murphy, Maurice; Tang, Alan; Taylor, Stephen; White, David; Underhill, Gillian; Pillay, Deenan; Cane, Patricia

2004-12-15

Primary infection with drug-resistant HIV-1 is well documented. We have followed up patients infected with such viruses to determine the stability of resistance-associated mutations. Fourteen patients who experienced primary infection with genotypic evidence of resistance were followed for up to 3 years. Drug resistance-associated mutations persisted over time in most patients studied. In particular, M41L, T69N, K103N, and T215 variants within reverse transcriptase (RT) and multidrug resistance demonstrated little reversion to wild-type virus. By contrast, Y181C and K219Q in RT, occurring alone, disappeared within 25 and 9 months, respectively. Multidrug resistance in 2 patients was found to be stable for up to 18 months, the maximum period studied. We conclude that certain resistance-associated mutations are highly stable and these data support the recommendation that all new HIV diagnoses in areas where primary resistance may occur should undergo genotyping irrespective of whether the date of seroconversion is known.

Adaptive changes in HIV-1 subtype C proteins during early infection are driven by changes in HLA-associated immune pressure

PubMed Central

Treurnicht, F.K.; Seoighe, C.; Martin, D.P.; Wood, N.; Abrahams, M-R.; de Assis Rosa, D.; Bredell, H.; Woodman, Z.; Hide, W.; Mlisana, K.; Karim, S Abdool; Gray, C.M.; Williamson, C.

2009-01-01

It is unresolved whether recently transmitted human immunodeficiency viruses (HIV) have genetic features that specifically favour their transmissibility. To identify potential “transmission signatures”, we compared 20 full-length HIV-1 subtype C genomes from primary infections, with 66 sampled from ethnically and geographically matched individuals with chronic infections. Controlling for recombination and phylogenetic relatedness, we identified 39 sites at which amino acid frequency spectra differed significantly between groups. These sites were predominantly located within Env, Pol and Gag (14/39, 9/39 and 6/39 respectively) and were significantly clustered (33/39) within known immunoreactive peptides. Within 6 months of infection we detected reversion-to-consensus mutations at 14 sites and potential CTL escape mutations at seven. Here we provide evidence that frequent reversion mutations probably allows the virus to recover replicative fitness which, together with immune escape driven by the HLA alleles of the new hosts, differentiate sequences from chronic infections from those sampled shortly after transmission. PMID:19913270

RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon.

PubMed

Tang, Hongxing; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Li, Huimin; He, Lifang; Liu, Bin

2008-06-15

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.

Cancer-associated TERT promoter mutations abrogate telomerase silencing

PubMed Central

Chiba, Kunitoshi; Johnson, Joshua Z; Vogan, Jacob M; Wagner, Tina; Boyle, John M; Hockemeyer, Dirk

2015-01-01

Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells. DOI: http://dx.doi.org/10.7554/eLife.07918.001 PMID:26194807

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

PubMed Central

Rodríguez-Marí, Adriana; Cañestro, Cristian; BreMiller, Ruth A.; Nguyen-Johnson, Alexandria; Asakawa, Kazuhide; Kawakami, Koichi; Postlethwait, John H.

2010-01-01

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination. PMID:20661450

Genotoxicity assessment of multispecies probiotics using reverse mutation, mammalian chromosomal aberration, and rodent micronucleus tests.

PubMed

Chiu, Yi-Jen; Nam, Mun-Kit; Tsai, Yueh-Ting; Huang, Chun-Chi; Tsai, Cheng-Chih

2013-01-01

Genotoxicity assessment is carried out on freeze dried powder of cultured probiotics containing Lactobacillus rhamnosus LCR177, Bifidobacterium adolescentis BA286, and Pediococcus acidilactici PA318. Ames tests, in vitro mammalian chromosome aberration assay, and micronucleus tests in mouse peripheral blood are performed. For 5 strains of Salmonella Typhimurium, the Ames tests show no increased reverse mutation upon exposure to the test substance. In CHO cells, the frequency of chromosome aberration does not increase in responding to the treatment of probiotics. Likewise, the frequency of micronucleated reticulocytes in probiotics-fed mice is indistinguishable from that in the negative control group. Taken together, the toxicity assessment studies suggest that the multispecies probiotic mixture does not have mutagenic effects on various organisms.

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

PubMed Central

Fitzgibbon, J E; Howell, R M; Haberzettl, C A; Sperber, S J; Gocke, D J; Dubin, D T

1992-01-01

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient. Images PMID:1317143

A Novel Point Mutation at Position 156 of Reverse Transcriptase from Feline Immunodeficiency Virus Confers Resistance to the Combination of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine and 3′-Azido-3′-Deoxythymidine

PubMed Central

Smith, Robert A.; Remington, Kathryn M.; Preston, Bradley D.; Schinazi, Raymond F.; North, Thomas W.

1998-01-01

Mutants of feline immunodeficiency virus (FIV) resistant to (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3′-azido-3′-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5′-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation. PMID:9499094

Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome–associated PTPN11 mutation

PubMed Central

Marin, Talita M.; Keith, Kimberly; Davies, Benjamin; Conner, David A.; Guha, Prajna; Kalaitzidis, Demetrios; Wu, Xue; Lauriol, Jessica; Wang, Bo; Bauer, Michael; Bronson, Roderick; Franchini, Kleber G.; Neel, Benjamin G.; Kontaridis, Maria I.

2011-01-01

LEOPARD syndrome (LS) is an autosomal dominant “RASopathy” that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11Y279C/+ (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients. PMID:21339643

Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration.

PubMed

Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

2005-04-01

The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

A repetitive mutation and selection system for bacterial evolution to increase the specific affinity to pancreatic cancer cells.

PubMed

Osawa, Masaki

2018-01-01

It is difficult to target and kill cancer cells. One possible approach is to mutate bacteria to enhance their binding to cancer cells. In the present study, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis were randomly mutated, and then were positively and negatively selected for binding cancer vs normal cells. With repetitive mutation and selection both bacteria successfully evolved to increase affinity to the pancreatic cancer cell line (Mia PaCa-2) but not normal cells (HPDE: immortalized human pancreatic ductal epithelial cells). The mutant E. coli and B. subtilis strains bound to Mia PaCa-2 cells about 10 and 25 times more than to HPDE cells. The selected E. coli strain had mutations in biofilm-related genes and the regulatory region for a type I pilus gene. Consistent with type I pili involvement, mannose could inhibit the binding to cells. The results suggest that weak but specific binding is involved in the initial step of adhesion. To test their ability to kill Mia PaCa-2 cells, hemolysin was expressed in the mutant strain. The hemolysin released from the mutant strain was active and could kill Mia PaCa-2 cells. In the case of B. subtilis, the initial binding to the cells was a weak interaction of the leading pole of the motile bacteria. The frequency of this interaction to Mia PaCa-2 cells dramatically increased in the evolved mutant strain. This mutant strain could also specifically invade beneath Mia PaCa-2 cells and settle there. This type of mutation/selection strategy may be applicable to other combinations of cancer cells and bacterial species.

Emergence of resistance mutations in simian immunodeficiency virus (SIV)-infected rhesus macaques receiving non-suppressive antiretroviral therapy (ART)

DOE PAGES

Policicchio, Benjamin Bruno; Sette, Paola; Xu, Cuiling; ...

2018-02-21

Two SIVmac251-infected rhesus macaques received tenofovir/emtricitabine with raltegravir intensification. Viral rebound occurred during treatment and sequencing of reverse transcriptase and integrase genes identified multiple resistance mutations. Similar to HIV infection, antiretroviral-resistance mutations may occur in SIV-infected nonhuman primates receiving nonsuppressive ART. As ART administration to nonhuman primates is currently dramatically expanding, fueled by both cure research and the study of HIV-related comorbidities, viral resistance should be factored in the study design and data interpretation

Emergence of resistance mutations in simian immunodeficiency virus (SIV)-infected rhesus macaques receiving non-suppressive antiretroviral therapy (ART)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Policicchio, Benjamin Bruno; Sette, Paola; Xu, Cuiling

Two SIVmac251-infected rhesus macaques received tenofovir/emtricitabine with raltegravir intensification. Viral rebound occurred during treatment and sequencing of reverse transcriptase and integrase genes identified multiple resistance mutations. Similar to HIV infection, antiretroviral-resistance mutations may occur in SIV-infected nonhuman primates receiving nonsuppressive ART. As ART administration to nonhuman primates is currently dramatically expanding, fueled by both cure research and the study of HIV-related comorbidities, viral resistance should be factored in the study design and data interpretation

Clinical and molecular investigation in Chinese patients with glutaric aciduria type I.

PubMed

Zhang, Yanghui; Li, Haoxian; Ma, Ruiyu; Mei, Libin; Wei, Xianda; Liang, Desheng; Wu, Lingqian

2016-01-30

Glutaric aciduria type I (GA-I) is a rare autosomal recessive metabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase (GCDH), leading to an abnormal metabolism of lysine, hydroxylysine and tryptophan. It results in accumulations of glutaric acid, 3-hydroxyglutaric acid and glutaconic acid. Clinical features include the sudden onset of encephalopathy, hypotonia and macrocephaly usually before age 18months. Here we report five cases of GA-I confirmed with mutation analysis. GCDH gene mutations were identified in all five probands with GA-I. Three of them had compound heterozygous mutations and two had homozygous mutations. Mutations of two alleles (c.334G>T and IVS11-11A>G) were novel and both of them were confirmed to be splice site mutations by reverse transcription PCR. Copyright © 2015 Elsevier B.V. All rights reserved.

Nonnucleoside reverse transcriptase inhibitor phenotypic hypersusceptibility can be demonstrated in different assays.

PubMed

Shulman, Nancy S; Delgado, Jamael; Bosch, Ronald J; Winters, Mark A; Johnston, Elizabeth; Shafer, Robert W; Katzenstein, David A; Merigan, Thomas C

2005-05-01

HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible ("hypersusceptible") to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility. To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays. The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT. Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results. NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.

Probing the dynamic reversibility and generation of dynamic combinatorial libraries in the presence of bacterial model oligopeptides as templating guests of tetra-carbohydrazide macrocycles using electrospray mass spectrometry.

PubMed

Nour, Hany F; Islam, Tuhidul; Fernández-Lahore, Marcelo; Kuhnert, Nikolai

2012-12-30

Over the past few decades, bacterial resistance to antibiotics has emerged as a real threat to human health. Accordingly, there is an urgent demand for the development of innovative strategies for discovering new antibiotics. We present the first use of tetra-carbohydrazide cyclophane macrocycles in dynamic combinatorial chemistry (DCC) and molecular recognition as chiral hosts binding oligopeptides, which mimic bacterial cell wall. This study introduces an innovative application of electrospray ionisation time-of-flight mass spectrometry (ESI-TOF MS) to oligopeptides recognition using DCC. A small dynamic library composed of eight functionalised macrocycles has been generated in solution and all members were characterised by ESI-TOF MS. We also probed the dynamic reversibility and mechanism of formation of tetra-carbohydrazide cyclophanes in real-time using ESI-TOF MS. Dynamic reversibility of tetra-carbohydrazide cyclophanes is favored under thermodynamic control. The mechanism of formation of tetra-carbohydrazide cyclophanes involves key dialdehyde intermediates, which have been detected and assigned according to their high-resolution m/z values. Three members of the dynamic library bind efficiently in the gas phase to a selection of oligopeptides, unique to bacteria, allowing observation of host/guest complex ions in the gas phase. We probed the mechanism of the [2+2]-cyclocondensation reaction forming library members, proved dynamic reversibility of tetra-carbohydrazide cyclophanes and showed that complex ions formed between library members and hosts can be observed in the gas phase, allowing the solution of an important problem of biological interest. Copyright © 2012 John Wiley & Sons, Ltd.

Ancient genes establish stress-induced mutation as a hallmark of cancer.

PubMed

Cisneros, Luis; Bussey, Kimberly J; Orr, Adam J; Miočević, Milica; Lineweaver, Charles H; Davies, Paul

2017-01-01

Cancer is sometimes depicted as a reversion to single cell behavior in cells adapted to live in a multicellular assembly. If this is the case, one would expect that mutation in cancer disrupts functional mechanisms that suppress cell-level traits detrimental to multicellularity. Such mechanisms should have evolved with or after the emergence of multicellularity. This leads to two related, but distinct hypotheses: 1) Somatic mutations in cancer will occur in genes that are younger than the emergence of multicellularity (1000 million years [MY]); and 2) genes that are frequently mutated in cancer and whose mutations are functionally important for the emergence of the cancer phenotype evolved within the past 1000 million years, and thus would exhibit an age distribution that is skewed to younger genes. In order to investigate these hypotheses we estimated the evolutionary ages of all human genes and then studied the probability of mutation and their biological function in relation to their age and genomic location for both normal germline and cancer contexts. We observed that under a model of uniform random mutation across the genome, controlled for gene size, genes less than 500 MY were more frequently mutated in both cases. Paradoxically, causal genes, defined in the COSMIC Cancer Gene Census, were depleted in this age group. When we used functional enrichment analysis to explain this unexpected result we discovered that COSMIC genes with recessive disease phenotypes were enriched for DNA repair and cell cycle control. The non-mutated genes in these pathways are orthologous to those underlying stress-induced mutation in bacteria, which results in the clustering of single nucleotide variations. COSMIC genes were less common in regions where the probability of observing mutational clusters is high, although they are approximately 2-fold more likely to harbor mutational clusters compared to other human genes. Our results suggest this ancient mutational response to stress that evolved among prokaryotes was co-opted to maintain diversity in the germline and immune system, while the original phenotype is restored in cancer. Reversion to a stress-induced mutational response is a hallmark of cancer that allows for effectively searching "protected" genome space where genes causally implicated in cancer are located and underlies the high adaptive potential and concomitant therapeutic resistance that is characteristic of cancer.

Ancient genes establish stress-induced mutation as a hallmark of cancer

PubMed Central

Orr, Adam J.; Miočević, Milica; Lineweaver, Charles H.; Davies, Paul

2017-01-01

Cancer is sometimes depicted as a reversion to single cell behavior in cells adapted to live in a multicellular assembly. If this is the case, one would expect that mutation in cancer disrupts functional mechanisms that suppress cell-level traits detrimental to multicellularity. Such mechanisms should have evolved with or after the emergence of multicellularity. This leads to two related, but distinct hypotheses: 1) Somatic mutations in cancer will occur in genes that are younger than the emergence of multicellularity (1000 million years [MY]); and 2) genes that are frequently mutated in cancer and whose mutations are functionally important for the emergence of the cancer phenotype evolved within the past 1000 million years, and thus would exhibit an age distribution that is skewed to younger genes. In order to investigate these hypotheses we estimated the evolutionary ages of all human genes and then studied the probability of mutation and their biological function in relation to their age and genomic location for both normal germline and cancer contexts. We observed that under a model of uniform random mutation across the genome, controlled for gene size, genes less than 500 MY were more frequently mutated in both cases. Paradoxically, causal genes, defined in the COSMIC Cancer Gene Census, were depleted in this age group. When we used functional enrichment analysis to explain this unexpected result we discovered that COSMIC genes with recessive disease phenotypes were enriched for DNA repair and cell cycle control. The non-mutated genes in these pathways are orthologous to those underlying stress-induced mutation in bacteria, which results in the clustering of single nucleotide variations. COSMIC genes were less common in regions where the probability of observing mutational clusters is high, although they are approximately 2-fold more likely to harbor mutational clusters compared to other human genes. Our results suggest this ancient mutational response to stress that evolved among prokaryotes was co-opted to maintain diversity in the germline and immune system, while the original phenotype is restored in cancer. Reversion to a stress-induced mutational response is a hallmark of cancer that allows for effectively searching “protected” genome space where genes causally implicated in cancer are located and underlies the high adaptive potential and concomitant therapeutic resistance that is characteristic of cancer. PMID:28441401

Familial knockin mutation of LRRK2 causes lysosomal dysfunction and accumulation of endogenous insoluble α-synuclein in neurons.

PubMed

Schapansky, Jason; Khasnavis, Saurabh; DeAndrade, Mark P; Nardozzi, Jonathan D; Falkson, Samuel R; Boyd, Justin D; Sanderson, John B; Bartels, Tim; Melrose, Heather L; LaVoie, Matthew J

2018-03-01

Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD. Copyright © 2017 Elsevier Inc. All rights reserved.

A diploid wheat TILLING resource for wheat functional genomics

PubMed Central

2012-01-01

Background Triticum monococcum L., an A genome diploid einkorn wheat, was the first domesticated crop. As a diploid, it is attractive genetic model for the study of gene structure and function of wheat-specific traits. Diploid wheat is currently not amenable to reverse genetics approaches such as insertion mutagenesis and post-transcriptional gene silencing strategies. However, TILLING offers a powerful functional genetics approach for wheat gene analysis. Results We developed a TILLING population of 1,532 M2 families using EMS as a mutagen. A total of 67 mutants were obtained for the four genes studied. Waxy gene mutation frequencies are known to be 1/17.6 - 34.4 kb DNA in polyploid wheat TILLING populations. The T. monococcum diploid wheat TILLING population had a mutation frequency of 1/90 kb for the same gene. Lignin biosynthesis pathway genes- COMT1, HCT2, and 4CL1 had mutation frequencies of 1/86 kb, 1/92 kb and 1/100 kb, respectively. The overall mutation frequency of the diploid wheat TILLING population was 1/92 kb. Conclusion The mutation frequency of a diploid wheat TILLING population was found to be higher than that reported for other diploid grasses. The rate, however, is lower than tetraploid and hexaploid wheat TILLING populations because of the higher tolerance of polyploids to mutations. Unlike polyploid wheat, most mutants in diploid wheat have a phenotype amenable to forward and reverse genetic analysis and establish diploid wheat as an attractive model to study gene function in wheat. We estimate that a TILLING population of 5, 520 will be needed to get a non-sense mutation for every wheat gene of interest with 95% probability. PMID:23134614

Surface contact stimulates the just-in-time deployment of bacterial adhesins.

PubMed

Li, Guanglai; Brown, Pamela J B; Tang, Jay X; Xu, Jing; Quardokus, Ellen M; Fuqua, Clay; Brun, Yves V

2012-01-01

The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive. © 2011 Blackwell Publishing Ltd.

A BAC transgenic mouse model reveals neuron subtype-specific effects of a Generalized Epilepsy with Febrile Seizures Plus (GEFS+) mutation

PubMed Central

Tang, Bin; Dutt, Karoni; Papale, Ligia; Rusconi, Raffaella; Shankar, Anupama; Hunter, Jessica; Tufik, Sergio; Yu, Frank H.; Catterall, William A.; Mantegazza, Massimo; Goldin, Alan L.; Escayg, Andrew

2009-01-01

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability. PMID:19409490

77 FR 65824 - Calcium Gluconate; Exemption From the Requirement of a Tolerance

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-31

... the basis of available data (chemical, biochemical, toxicological, and other) and the total dietary... gluconate was not mutagenic in an in vitro chromosome aberration test, bacterial gene mutation test. In...

HIV-1 transmitted drug resistance and genetic diversity among patients from Piauí State, Northeast Brazil.

PubMed

Moura, Maria Edileuza Soares; da Guarda Reis, Mônica Nogueira; Lima, Yanna Andressa Ramos; Eulálio, Kelsen Dantas; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

2015-05-01

HIV-1 transmitted-drug-resistance and genetic diversity are dynamic and may differ in distinct locations/risk groups. In Brazil, increased AIDS incidence and related mortality have been detected in the Northeast region, differently from the epicenter in the Southeast. This cross-sectional study describes transmitted-dru- resistance and HIV-1 subtypes in protease/PR and reverse transcriptase/RT regions among antiretroviral naïve patients from Piauí State, Northeast Brazil. Among 96 patients recruited 89 (92.7%) had HIV-1 PR/RT regions sequenced: 44 females and 45 males, 22 self-declared as men who have sex with men. Transmitted-drug-resistance was investigated by CPR tool (Stanford HIV-1 Drug Resistance/SDRM). HIV-1 subtypes were assigned by REGA and phylogenetic inference. Overall, transmitted-drug-resistance rate was 11.2% (10/89; CI 95%: 5.8-19.1%); 22.7% among men who have sex with men (5/22; CI 95%: 8.8-43.4%), 10% in heterosexual men (2/20; CI 95%: 1.7-29.3%) and 6.8% in women (3/44; CI 95%: 1.8-17.4%). Singleton mutations to protease-inhibitor/PI, nucleoside-reverse-transcriptase-inhibitor/NRTI or non-nucleoside-reverse-transcriptase-inhibitor/NNRTI predominated (8/10): PI mutations (M46L, V82F, L90M); NRTI mutations (M41L, D67N) and NNRTI mutations (K103N/S). Dual class resistance mutations to NRTI and NNRTI were observed: T215L (NRTI), Y188L (NNRTI) and T215N (NRTI), F227L (NNRTI). Subtype B prevailed (86.6%; 77/89), followed by subtype F1 (1.1%, 1/89) and subtype C (1.1%, 1/89). B/F1 and B/C intersubtype recombinants represented 11.2% (10/89). In Piauí State extensive testing of incidence and transmitted-drug-resistance in all populations with risk behaviors may help control AIDS epidemic locally. © 2015 Wiley Periodicals, Inc.

Chromosomal islands of Streptococcus pyogenes and related streptococci: molecular switches for survival and virulence

PubMed Central

Nguyen, Scott V.; McShan, William M.

2014-01-01

Streptococcus pyogenes is a significant pathogen of humans, annually causing over 700,000,000 infections and 500,000 deaths. Virulence in S. pyogenes is closely linked to mobile genetic elements like phages and chromosomal islands (CI). S. pyogenes phage-like chromosomal islands (SpyCI) confer a complex mutator phenotype on their host. SpyCI integrate into the 5′ end of DNA mismatch repair (MMR) gene mutL, which also disrupts downstream operon genes lmrP, ruvA, and tag. During early logarithmic growth, SpyCI excise from the bacterial chromosome and replicate as episomes, relieving the mutator phenotype. As growth slows and the cells enter stationary phase, SpyCI reintegrate into the chromosome, again silencing the MMR operon. This system creates a unique growth-dependent and reversible mutator phenotype. Additional CI using the identical attachment site in mutL have been identified in related species, including Streptococcus dysgalactiae subsp. equisimilis, Streptococcus anginosus, Streptococcus intermedius, Streptococcus parauberis, and Streptococcus canis. These CI have small genomes, which range from 13 to 20 kB, conserved integrase and DNA replication genes, and no identifiable genes encoding capsid proteins. SpyCI may employ a helper phage for packaging and dissemination in a fashion similar to the Staphylococcus aureus pathogenicity islands (SaPI). Outside of the core replication and integration genes, SpyCI and related CI show considerable diversity with the presence of many indels that may contribute to the host cell phenotype or fitness. SpyCI are a subset of a larger family of streptococcal CI who potentially regulate the expression of other host genes. The biological and phylogenetic analysis of streptococcal chromosomal islands provides important clues as to how these chromosomal islands help S. pyogenes and other streptococcal species persist in human populations in spite of antibiotic therapy and immune challenges. PMID:25161960

Chromosomal islands of Streptococcus pyogenes and related streptococci: molecular switches for survival and virulence.

PubMed

Nguyen, Scott V; McShan, William M

2014-01-01

Streptococcus pyogenes is a significant pathogen of humans, annually causing over 700,000,000 infections and 500,000 deaths. Virulence in S. pyogenes is closely linked to mobile genetic elements like phages and chromosomal islands (CI). S. pyogenes phage-like chromosomal islands (SpyCI) confer a complex mutator phenotype on their host. SpyCI integrate into the 5' end of DNA mismatch repair (MMR) gene mutL, which also disrupts downstream operon genes lmrP, ruvA, and tag. During early logarithmic growth, SpyCI excise from the bacterial chromosome and replicate as episomes, relieving the mutator phenotype. As growth slows and the cells enter stationary phase, SpyCI reintegrate into the chromosome, again silencing the MMR operon. This system creates a unique growth-dependent and reversible mutator phenotype. Additional CI using the identical attachment site in mutL have been identified in related species, including Streptococcus dysgalactiae subsp. equisimilis, Streptococcus anginosus, Streptococcus intermedius, Streptococcus parauberis, and Streptococcus canis. These CI have small genomes, which range from 13 to 20 kB, conserved integrase and DNA replication genes, and no identifiable genes encoding capsid proteins. SpyCI may employ a helper phage for packaging and dissemination in a fashion similar to the Staphylococcus aureus pathogenicity islands (SaPI). Outside of the core replication and integration genes, SpyCI and related CI show considerable diversity with the presence of many indels that may contribute to the host cell phenotype or fitness. SpyCI are a subset of a larger family of streptococcal CI who potentially regulate the expression of other host genes. The biological and phylogenetic analysis of streptococcal chromosomal islands provides important clues as to how these chromosomal islands help S. pyogenes and other streptococcal species persist in human populations in spite of antibiotic therapy and immune challenges.

Evolutionary Potential of an RNA Virus

PubMed Central

Makeyev, Eugene V.; Bamford, Dennis H.

2004-01-01

RNA viruses are remarkably adaptable to changing environments. This is medically important because it enables pathogenic viruses to escape the immune response and chemotherapy and is of considerable theoretical interest since it allows the investigation of evolutionary processes within convenient time scales. A number of earlier studies have addressed the dynamics of adapting RNA virus populations. However, it has been difficult to monitor the trajectory of molecular changes in RNA genomes in response to selective pressures. To address the problem, we developed a novel in vitro evolution system based on a recombinant double-stranded RNA bacteriophage, φ6, containing a β-lactamase (bla) gene marker. Carrier-state bacterial cells are resistant to ampicillin, and after several passages, they become resistant to high concentrations of another β-lactam antibiotic, cefotaxime, due to mutations in the virus-borne bla gene. We monitored the changes in bla cDNAs induced by cefotaxime selection and observed an initial explosion in sequence variants with multiple mutations throughout the gene. After four passages, a stable, homogeneous population of bla sequences containing three specific nonsynonymous mutations was established. Of these, two mutations (E104K and G238S) have been previously reported for β-lactamases from cefotaxime-resistant bacterial isolates. These results extend our understanding of the molecular mechanisms of viral adaptation and also demonstrate the possibility of using an RNA virus as a vehicle for directed evolution of heterologous proteins. PMID:14747576

Evolutionary potential of an RNA virus.

PubMed

Makeyev, Eugene V; Bamford, Dennis H

2004-02-01

RNA viruses are remarkably adaptable to changing environments. This is medically important because it enables pathogenic viruses to escape the immune response and chemotherapy and is of considerable theoretical interest since it allows the investigation of evolutionary processes within convenient time scales. A number of earlier studies have addressed the dynamics of adapting RNA virus populations. However, it has been difficult to monitor the trajectory of molecular changes in RNA genomes in response to selective pressures. To address the problem, we developed a novel in vitro evolution system based on a recombinant double-stranded RNA bacteriophage, phi 6, containing a beta-lactamase (bla) gene marker. Carrier-state bacterial cells are resistant to ampicillin, and after several passages, they become resistant to high concentrations of another beta-lactam antibiotic, cefotaxime, due to mutations in the virus-borne bla gene. We monitored the changes in bla cDNAs induced by cefotaxime selection and observed an initial explosion in sequence variants with multiple mutations throughout the gene. After four passages, a stable, homogeneous population of bla sequences containing three specific nonsynonymous mutations was established. Of these, two mutations (E104K and G238S) have been previously reported for beta-lactamases from cefotaxime-resistant bacterial isolates. These results extend our understanding of the molecular mechanisms of viral adaptation and also demonstrate the possibility of using an RNA virus as a vehicle for directed evolution of heterologous proteins.

Mutagenicity of arsenic in mammalian cells: role of reactive oxygen species

NASA Technical Reports Server (NTRS)

Hei, T. K.; Liu, S. X.; Waldren, C.

1998-01-01

Arsenite, the trivalent form of arsenic present in the environment, is a known human carcinogen that lacked mutagenic activity in bacterial and standard mammalian cell mutation assays. We show herein that when evaluated in an assay (AL cell assay), in which both intragenic and multilocus mutations are detectable, that arsenite is in fact a strong dose-dependent mutagen and that it induces mostly large deletion mutations. Cotreatment of cells with the oxygen radical scavenger dimethyl sulfoxide significantly reduces the mutagenicity of arsenite. Thus, the carcinogenicity of arsenite can be explained at least in part by it being a mutagen that depends on reactive oxygen species for its activity.

Phosphatidyl inositol-3 kinase (PIK3CA) E545K mutation confers cisplatin resistance and a migratory phenotype in cervical cancer cells.

PubMed

Arjumand, Wani; Merry, Cole D; Wang, Chen; Saba, Elias; McIntyre, John B; Fang, Shujuan; Kornaga, Elizabeth; Ghatage, Prafull; Doll, Corinne M; Lees-Miller, Susan P

2016-12-13

The phosphatidylinositol-3 kinase (PI3K)/Akt/mTOR signaling pathway is activated in many human cancers. Previously, we reported that patients with early stage cervical cancer whose tumours harbour PIK3CA exon 9 or 20 mutations have worse overall survival in response to treatment with radiation and cisplatin than patients with wild-type PIK3CA. The purpose of this study was to determine whether PIK3CA-E545K mutation renders cervical cancer cells more resistant to cisplatin and/or radiation, and whether PI3K inhibition reverses the phenotype. We found that CaSki cells that are heterozygous for the PIK3CA-E545K mutation are more resistant to cisplatin or cisplatin plus radiation than either HeLa or SiHa cells that express only wild-type PIK3CA. Similarly, HeLa cells engineered to stably express PIK3CA-E545K were more resistant to cisplatin or cisplatin plus radiation than cells expressing only wild-type PIK3CA or with PIK3CA depleted. Cells expressing the PIK3CA-E545K mutation also had constitutive PI3K pathway activation and increased cellular migration and each of these phenotypes was reversed by treatment with the PI3K inhibitor GDC-0941/Pictilisib. Our results suggests that cervical cancer patients whose tumours are positive for the PIK3CA-E545K mutation may benefit from PI3K inhibitor therapy in concert with standard cisplatin and radiation therapy.

Polymorphisms and resistance mutations in the protease and reverse transcriptase genes of HIV-1 F subtype Romanian strains.

PubMed

Paraschiv, Simona; Otelea, Dan; Dinu, Magdalena; Maxim, Daniela; Tinischi, Mihaela

2007-03-01

To evaluate the prevalence of resistance mutations in the genome of HIV-1 F subtype strains isolated from Romanian antiretroviral (ARV) treatment-naïve patients and to assess the phylogenetic relatedness of these strains with other HIV-1 strains. Twenty-nine HIV-1 strains isolated from treatment-naïve adolescents (n=15) and adults (n=14) were included in this study. Resistance genotyping was performed by using Big Dye Terminator chemistry provided by the ViroSeq Genotyping System. The sequences of the protease and reverse transcriptase genes were aligned (ClustalW) and a phylogenetic tree was built (MEGA 3 software). For subtyping purposes, all the nucleotide sequences were submitted to the Stanford database. All the studied strains were found to harbor accessory mutations in the protease gene. The most frequent mutation was M36I (29 of 29 strains), followed by L63T, K20R, and L10V. The number of polymorphisms associated with protease inhibitor resistance was different for the two age groups. Intraphylogenetic divergence was greater for adults than for adolescents infected in childhood. All the strains were found to belong to the F1 subtype. The phylogenetic analysis revealed that Romanian strains clustered together, but distinctly from F1 HIV-1 strains isolated in other parts of the world (Brazil, Finland, and Belgium). Protease secondary mutations are present with high frequency in the HIV-1 F subtype strains isolated from Romanian ARV treatment-naïve patients, but no major resistance mutations were found.

Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability

PubMed Central

Der, Bryan S.; Kluwe, Christien; Miklos, Aleksandr E.; Jacak, Ron; Lyskov, Sergey; Gray, Jeffrey J.; Georgiou, George; Ellington, Andrew D.; Kuhlman, Brian

2013-01-01

Reengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from −11 to −61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and −30 net charge. Mid-charge variants demonstrated ∼3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding. PMID:23741319

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

PubMed

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

PubMed Central

Mizrahi, V; Usdin, M T; Harington, A; Dudding, L R

1990-01-01

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity. Images PMID:1699202

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Studies on Microbial Propagation in the Airborne State

DTIC Science & Technology

1976-11-29

Bacterial growth, Jupiter, Serratia marcescens , Particulate ejection \\ýAirbForne particles, 2Ck\\l ABSTRACT (Continue an reverse side it necessary and Identit...the use of cells in the latter stages of logarithmic growth, the bacterial species Serratia marcescens can sustain more than two cellular...1133 DISTRIBUTION LIST: Dr. Richard S . Young (3 copies, with reprints) Chief, Planetary Biology NASA Headquarters, Code SL Washington, D.C. 20546

Combusting vegetable oils in diesel engines: the impact of unsaturated fatty acids on particle emissions and mutagenic effects of the exhaust.

PubMed

Bünger, Jürgen; Bünger, Jörn F; Krahl, Jürgen; Munack, Axel; Schröder, Olaf; Brüning, Thomas; Hallier, Ernst; Westphal, Götz A

2016-06-01

High particle emissions and strong mutagenic effects were observed after combustion of vegetable oil in diesel engines. This study tested the hypothesis that these results are affected by the amount of unsaturated or polyunsaturated fatty acids of vegetable oils. Four different vegetable oils (coconut oil, CO; linseed oil, LO; palm tree oil, PO; and rapeseed oil, RO) and common diesel fuel (DF) were combusted in a heavy-duty diesel engine. The exhausts were investigated for particle emissions and mutagenic effects in direct comparison with emissions of DF. The engine was operated using the European Stationary Cycle. Particle masses were measured gravimetrically while mutagenicity was determined using the bacterial reverse mutation assay with tester strains TA98 and TA100. Combustion of LO caused the largest amount of total particulate matter (TPM). In comparison with DF, it particularly raised the soluble organic fraction (SOF). RO presented second highest TPM and SOF, followed by CO and PO, which were scarcely above DF. RO revealed the highest number of mutations of the vegetable oils closely followed by LO. PO was less mutagenic, but still induced stronger effects than DF. While TPM and SOF were strongly correlated with the content of polyunsaturated fatty acids in the vegetable oils, mutagenicity had a significant correlation with the amount of total unsaturated fatty acids. This study supports the hypothesis that numbers of double bounds in unsaturated fatty acids of vegetable oils combusted in diesel engines influence the amount of emitted particles and the mutagenicity of the exhaust. Further investigations have to elucidate the causal relationship.

A subchronic toxicity study in rats and genotoxicity tests with an aqueous ethylcellulose dispersion.

PubMed

DeMerlis, C C; Schoneker, D R; Borzelleca, J F

2005-09-01

Surelease Aqueous Ethylcellulose Dispersion is an excipient used as a modified release coating for beads, granules, non-pariels, drug crystals and tablets and for taste masking applications for drug products and dietary supplement products. A study was conducted to assess the toxicity of spray-dried Surelease when administered orally, via dietary admixture, to Sprague-Dawley CD rats (20/sex/group) at dose levels of 0, 2000, 3500, and 5000 mg/kg/day for a period of at least 3 months. After 3 months of treatment, all rats scheduled for terminal sacrifice were killed and selected organs were weighed. Complete macroscopic examinations and histopathological evaluation of selected tissues were conducted on all animals. Neuropathological evaluations were performed on 5 animals/sex/group. No mortality occurred during the study. Clinical observations, ophthalmology, body weight and food consumption, hematology, coagulation, clinical chemistry, urinalysis, functional observational assessments, motor activity, organ weights and ratios and macroscopic and microscopic observations did not reveal any significant, consistent, dose-dependent test article-related adverse effects. The NOAEL (no-observed-adverse-effect-level) is 5000 mg/kg/day, the highest dose tested. A series of genotoxicity tests were conducted with Surelease. Surelease showed no evidence of mutagenic activity in the bacterial reverse mutation test with and without metabolic activation and in the in vitro cell mutation assay under the experimental conditions employed. Surelease did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by gavage in the mouse micronucleus in vivo test procedure. These findings support the safety of Surelease for use as an excipient.

Lower genetic variability of HIV-1 and antiretroviral drug resistance in pregnant women from the state of Pará, Brazil.

PubMed

Machado, Luiz Fernando Almeida; Costa, Iran Barros; Folha, Maria Nazaré; da Luz, Anderson Levy Bessa; Vallinoto, Antonio Carlos Rosário; Ishak, Ricardo; Ishak, Marluisa Oliveira Guimarães

2017-04-12

The present study aimed to describe the genetic diversity of HIV-1, as well as the resistance profile of the viruses identified in HIV-1 infected pregnant women under antiretroviral therapy in the state of Pará, Northern Brazil. Blood samples were collected from 45 HIV-1 infected pregnant to determine the virus subtypes according to the HIV-1 protease (PR) gene and part of the HIV-1 reverse transcriptase (RT) gene by sequencing the nucleotides of these regions. Drug resistance mutations and susceptibility to antiretroviral drugs were analyzed by the Stanford HIV Drug Resistance Database. Out of 45 samples, only 34 could be amplified for PR and 30 for RT. Regarding the PR gene, subtypes B (97.1%) and C (2.9%) were identified; for the RT gene, subtypes B (90.0%), F (6.7%), and C (3.3%) were detected. Resistance to protease inhibitors (PI) was identified in 5.8% of the pregnant, and mutations conferring resistance to nucleoside reverse transcriptase inhibitors were found in 3.3%, while mutations conferring resistance to non-nucleoside reverse transcriptase inhibitors were found in 3.3%. These results showed a low frequency of strains resistant to antiretroviral drugs, the prevalence of subtypes B and F, and the persistent low transmission of subtype C in pregnant of the state of Pará, Brazil.

Virulence of Mycobacterium tuberculosis after Acquisition of Isoniazid Resistance: Individual Nature of katG Mutants and the Possible Role of AhpC.

PubMed

Nieto R, Luisa Maria; Mehaffy, Carolina; Creissen, Elizabeth; Troudt, JoLynn; Troy, Amber; Bielefeldt-Ohmann, Helle; Burgos, Marcos; Izzo, Angelo; Dobos, Karen M

2016-01-01

In the last decade, there were 10 million new tuberculosis cases per year globally. Around 9.5% of these cases were caused by isoniazid resistant (INHr) Mycobacterium tuberculosis (Mtb) strains. Although isoniazid resistance in Mtb is multigenic, mutations in the catalase-peroxidase (katG) gene predominate among the INHr strains. The effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. Here, we assessed differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of the katG gene. We studied one laboratory and one clinically isolated Mtb clonal pair from different genetic lineages. The INHr strain in each pair had one and two katG mutations with significantly reduced levels of the enzyme and peroxidase activity. Both strains share the V1A mutation, while the double mutant clinical INHr had also the novel E3V katG mutation. Four groups of C57BL/6 mice were infected with one of the Mtb strains previously described. We observed a strong reduction in virulence (reduced bacterial growth), lower induction of proinflammatory cytokines and significantly reduced pathology scores in mice infected with the clinical INHr strain compared to the infection caused by its INHs progenitor strain. On the other hand, there was a subtle reduction of bacteria growth without differences in the pathology scores in mice infected with the laboratory INHr strain. Our results also showed distinct alkyl-hydroperoxidase C (AhpC) levels in the katG mutant strains, which could explain the difference in the virulence profile observed. The difference in the AhpC levels between clonal strains was not related to a genetic defect in the gene or its promoter. Cumulatively, our results indicate that the virulence, pathology and fitness of INHr strains could be negatively affected by multiple mutations in katG, lack of the peroxidase activity and reduced AhpC levels.

An assessment of the genetic toxicology of novel boron-containing therapeutic agents.

PubMed

Ciaravino, Vic; Plattner, Jacob; Chanda, Sanjay

2013-06-01

Boron-containing compounds are being studied as potential therapeutic agents. As part of the safety assessment of these therapeutic agents, a battery of genetic toxicology studies was conducted. The battery included a bacterial reverse mutation (Ames) assay, an in vitro chromosome aberration assay in peripheral human lymphocytes, and an in vivo rat micronucleus study. The following compounds represent some of the boron-containing compounds that have been advanced to human clinical trials in various therapeutic areas. The borinic picolinate, AN0128, is an antibacterial compound with anti-inflammatory activity that has been studied in clinical trials for acne and the treatment of mild to moderate atopic dermatitis. AN2690 (tavaborole) is a benzoxaborole in Phase 3 clinical trials for the topical treatment of onychomycosis, a fungal infection of the toenails and fingernails. Another benzoxaborole derivative, AN2728, a phosphodiesterase-4 (PDE4) inhibitor, is in Phase 2 clinical trials for the treatment of atopic dermatitis. AN2898, also a PDE4 inhibitor, has been studied in clinical trials for atopic dermatitis and psoriasis. AN3365 is a leucyl-tRNA synthetase inhibitor that has been in clinical development for the treatment of various Gram-negative bacterial infections. These five representative compounds were negative in the three genotoxicity assays. Furthermore, AN2690 has been studied in mouse and rat 2-year bioassays and was not found to have any carcinogenic potential. These results demonstrate that it is possible to design boron-based therapeutic agents with no genetic toxicology liabilities. Copyright © 2013 Wiley Periodicals, Inc.

Natural mismatch repair mutations mediate phenotypic diversity and drug resistance in Cryptococcus deuterogattii.

PubMed

Billmyre, R Blake; Clancey, Shelly Applen; Heitman, Joseph

2017-09-26

Pathogenic microbes confront an evolutionary conflict between the pressure to maintain genome stability and the need to adapt to mounting external stresses. Bacteria often respond with elevated mutation rates, but little evidence exists of stable eukaryotic hypermutators in nature. Whole genome resequencing of the human fungal pathogen Cryptococcus deuterogattii identified an outbreak lineage characterized by a nonsense mutation in the mismatch repair component MSH2. This defect results in a moderate mutation rate increase in typical genes, and a larger increase in genes containing homopolymer runs. This allows facile inactivation of genes with coding homopolymer runs including FRR1 , which encodes the target of the immunosuppresive antifungal drugs FK506 and rapamycin. Our study identifies a eukaryotic hypermutator lineage spread over two continents and suggests that pathogenic eukaryotic microbes may experience similar selection pressures on mutation rate as bacterial pathogens, particularly during long periods of clonal growth or while expanding into new environments.

Role of reverse phenotyping in interpretation of next generation sequencing data and a review of INPP5E related disorders.

PubMed

de Goede, Christian; Yue, Wyatt W; Yan, Guanhua; Ariyaratnam, Shyamala; Chandler, Kate E; Downes, Laura; Khan, Nasaim; Mohan, Meyyammai; Lowe, Martin; Banka, Siddharth

2016-03-01

Next Generation Sequencing (NGS) is a useful tool in diagnosis of rare disorders but the interpretation of data can be challenging in clinical settings. We present results of extended studies on a family of multiple members with global developmental delay and learning disability, where another research group postulated the underlying cause to be a homozygous RABL6 missense variant. Using data from the Exome Variant Server, we show that missense RABL6 variants are unlikely to cause early onset rare developmental disorder. Protein structural analysis, cellular functional studies and reverse phenotyping proved that the condition in this family is due to a homozygous INPP5E mutation. An in-depth review of mutational and phenotypic spectrum associated with INPP5E demonstrated that mutations in this gene lead to a range of cilliopathy-phenotypes. We use this study as an example to demonstrate the importance of careful clinical evaluation of multiple family members, reverse phenotyping, considering the unknown phenotypic variability of rare diseases, utilizing publically available genomic databases and conducting appropriate bioinformatics and functional studies while interpreting results from NGS in uncertain cases. We emphasize that interpretation of NGS data is an iterative process and its dynamic nature should be explained to patients and families. Our study shows that developmental delay, intellectual disability, hypotonia and ocular motor apraxia are common in INPP5E-related disorders and considerable intra-familial phenotypic variability is possible. We have compiled the INPP5E mutational spectrum and provided novel insights into their molecular mechanisms. Copyright © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Drug Susceptibility and Resistance Mutations After First-Line Failure in Resource Limited Settings

PubMed Central

Wallis, Carole L.; Aga, Evgenia; Ribaudo, Heather; Saravanan, Shanmugam; Norton, Michael; Stevens, Wendy; Kumarasamy, Nagalingeswaran; Bartlett, John; Katzenstein, David

2014-01-01

Background. The development of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been associated with baseline human immunodeficiency virus (HIV)-1 RNA level (VL), CD4 cell counts (CD4), subtype, and treatment failure duration. This study describes drug resistance and levels of susceptibility after first-line virologic failure in individuals from Thailand, South Africa, India, Malawi, Tanzania. Methods. CD4 and VL were captured at AIDs Clinical Trial Group (ACTG) A5230 study entry, a study of lopinavir/ritonavir (LPV/r) monotherapy after first-line virologic failure on an NNRTI regimen. HIV drug-resistance mutation associations with subtype, site, study entry VL, and CD4 were evaluated using Fisher exact and Kruskall–Wallis tests. Results. Of the 207 individuals who were screened for A5230, sequence data were available for 148 individuals. Subtypes observed: subtype C (n = 97, 66%) AE (n = 27, 18%), A1 (n = 12, 8%), and D (n = 10, 7%). Of the 148 individuals, 93% (n = 138) and 96% (n = 142) had at least 1 reverse transcriptase (RT) mutation associated with NRTI and NNRTI resistance, respectively. The number of NRTI mutations was significantly associated with a higher study screening VL and lower study screening CD4 (P < .001). Differences in drug-resistance patterns in both NRTI and NNRTI were observed by site. Conclusions. The degree of NNRTI and NRTI resistance after first-line virologic failure was associated with higher VL at study entry. Thirty-two percent of individuals remained fully susceptible to etravirine and rilpivirine, protease inhibitor resistance was rare. Some level of susceptibility to NRTI remained; however, VL monitoring and earlier virologic failure detection may result in lower NRTI resistance. PMID:24795328

Major groove binding track residues of the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase enhance cDNA synthesis at high temperatures.

PubMed

Matamoros, Tania; Barrioluengo, Verónica; Abia, David; Menéndez-Arias, Luis

2013-12-23

At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.

Interplay Between Antibiotic Resistance and Virulence During Disease Promoted by Multidrug-Resistant Bacteria

PubMed Central

Geisinger, Edward

2017-01-01

Abstract Diseases caused by antibiotic-resistant bacteria in hospitals are the outcome of complex relationships between several dynamic factors, including bacterial pathogenicity, the fitness costs of resistance in the human host, and selective forces resulting from interventions such as antibiotic therapy. The emergence and fate of mutations that drive antibiotic resistance are governed by these interactions. In this review, we will examine how different forms of antibiotic resistance modulate bacterial fitness and virulence potential, thus influencing the ability of pathogens to evolve in the context of nosocomial infections. We will focus on 3 important multidrug-resistant pathogens that are notoriously problematic in hospitals: Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus. An understanding of how antibiotic resistance mutations shape the pathobiology of multidrug-resistant infections has the potential to drive novel strategies that can control the development and spread of drug resistance. PMID:28375515

Mechanisms of quinolone action and microbial response.

PubMed

Hawkey, Peter M

2003-05-01

Over the years, chromosomal mapping of the bacterial genome of Escherichia coli has demonstrated that many loci are associated with quinolone resistance, which is mainly a result of chromosomal mutation or alteration of the quantity or type of porins in the outer membrane of Gram-negative bacteria. There has been one report of a small and confined episode of plasmid-mediated resistance to fluoroquinolones, which did not appear to persist. With the increasingly widespread use of an expanding range of fluoroquinolone antibiotics, a range and mix in individual bacterial isolates of the different mechanisms of resistance to fluoroquinolones will undoubtedly be encountered amongst clinically significant bacteria. Currently, transferable resistance is extremely rare and most resistant bacteria arise from clonal expansion of mutated strains. However, it is conceivable that in the future, horizontal gene transfer may become a more important means of conferring resistance to fluoroquinolones.

Large-Scale Discovery of Induced Point Mutations With High-Throughput TILLING

PubMed Central

Till, Bradley J.; Reynolds, Steven H.; Greene, Elizabeth A.; Codomo, Christine A.; Enns, Linda C.; Johnson, Jessica E.; Burtner, Chris; Odden, Anthony R.; Young, Kim; Taylor, Nicholas E.; Henikoff, Jorja G.; Comai, Luca; Henikoff, Steven

2003-01-01

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms. PMID:12618384

The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions.

PubMed Central

Nghiem, Y; Cabrera, M; Cupples, C G; Miller, J H

1988-01-01

We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions. Images PMID:3128795

The antimicrobial activity of nanoparticles: present situation and prospects for the future

PubMed Central

Wang, Linlin; Hu, Chen; Shao, Longquan

2017-01-01

Nanoparticles (NPs) are increasingly used to target bacteria as an alternative to antibiotics. Nanotechnology may be particularly advantageous in treating bacterial infections. Examples include the utilization of NPs in antibacterial coatings for implantable devices and medicinal materials to prevent infection and promote wound healing, in antibiotic delivery systems to treat disease, in bacterial detection systems to generate microbial diagnostics, and in antibacterial vaccines to control bacterial infections. The antibacterial mechanisms of NPs are poorly understood, but the currently accepted mechanisms include oxidative stress induction, metal ion release, and non-oxidative mechanisms. The multiple simultaneous mechanisms of action against microbes would require multiple simultaneous gene mutations in the same bacterial cell for antibacterial resistance to develop; therefore, it is difficult for bacterial cells to become resistant to NPs. In this review, we discuss the antibacterial mechanisms of NPs against bacteria and the factors that are involved. The limitations of current research are also discussed. PMID:28243086

Severe congenital neutropenia in a multigenerational family with a novel neutrophil elastase (ELANE) mutation.

PubMed

van de Vosse, Esther; Verhard, Els M; Tool, Anton J T; de Visser, Adriëtte W; Kuijpers, Taco W; Hiemstra, Pieter S; van Dissel, Jaap T

2011-02-01

We have analysed a family with nine congenital neutropenia patients in four generations, several of which we have studied in a long-term follow-up of over 25 years. The patients were mild to severe neutropenic and suffered from various recurrent bacterial infections. Mutations in the genes ELANE, CSF3R and GFI1 have been reported in patients with autosomal dominant congenital neutropenias. Using a small-scale linkage analysis with markers around the ELANE, CSF3R, CSF3 and GFI1 genes, we were able to determine that the disease segregated with markers around the ELANE gene. We identified a novel mutation in the ELANE gene in all of the affected family members that was not present in any of the healthy family members. The mutation leads to an A28S missense mutation in the mature protein. None of these patients developed leukaemia. This is the first truly multigenerational family with mutations in ELANE as unambiguous cause of severe congenital neutropenia SCN.

Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans.

PubMed

Shadoud, Lubana; Almahmoud, Iyad; Jarraud, Sophie; Etienne, Jérôme; Larrat, Sylvie; Schwebel, Carole; Timsit, Jean-François; Schneider, Dominique; Maurin, Max

2015-09-01

Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila. We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples. We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment. In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans

PubMed Central

Shadoud, Lubana; Almahmoud, Iyad; Jarraud, Sophie; Etienne, Jérôme; Larrat, Sylvie; Schwebel, Carole; Timsit, Jean-François; Schneider, Dominique; Maurin, Max

2015-01-01

Background Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila. Methods We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples. Findings We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment. Interpretation In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics. PMID:26501115

Within Host Evolution Selects for a Dominant Genotype of Mycobacterium tuberculosis while T Cells Increase Pathogen Genetic Diversity.

PubMed

Copin, Richard; Wang, Xueying; Louie, Eddie; Escuyer, Vincent; Coscolla, Mireia; Gagneux, Sebastien; Palmer, Guy H; Ernst, Joel D

2016-12-01

Molecular epidemiological assessments, drug treatment optimization, and development of immunological interventions all depend on understanding pathogen adaptation and genetic variation, which differ for specific pathogens. Mycobacterium tuberculosis is an exceptionally successful human pathogen, yet beyond knowledge that this bacterium has low overall genomic variation but acquires drug resistance mutations, little is known of the factors that drive its population genomic characteristics. Here, we compared the genetic diversity of the bacteria that established infection to the bacterial populations obtained from infected tissues during murine M. tuberculosis pulmonary infection and human disseminated M. bovis BCG infection. We found that new mutations accumulate during in vitro culture, but that in vivo, purifying selection against new mutations dominates, indicating that M. tuberculosis follows a dominant lineage model of evolution. Comparing bacterial populations passaged in T cell-deficient and immunocompetent mice, we found that the presence of T cells is associated with an increase in the diversity of the M. tuberculosis genome. Together, our findings put M. tuberculosis genetic evolution in a new perspective and clarify the impact of T cells on sequence diversity of M. tuberculosis.

Drug resistance mutations in HIV type 1 isolates from naive patients eligible for first line antiretroviral therapy in JJ Hospital, Mumbai, India.

PubMed

Deshpande, Alake; Karki, Surendra; Recordon-Pinson, Patricia; Fleury, Herve J

2011-12-01

More than 50 HIV-1-infected patients, naive of antiretroviral therapy (ART) but eligible for first line ART in JJ Hospital, Mumbai, India were investigated for surveillance drug resistance mutations (SDRMs); all but one virus belonged to subtype C; we could observe SDRMs to nonnucleoside reverse transcriptase inhibitors and protease inhibitors in 9.6% of the patients.

Directed evolution of cell size in Escherichia coli.

PubMed

Yoshida, Mari; Tsuru, Saburo; Hirata, Naoko; Seno, Shigeto; Matsuda, Hideo; Ying, Bei-Wen; Yomo, Tetsuya

2014-12-17

In bacteria, cell size affects chromosome replication, the assembly of division machinery, cell wall synthesis, membrane synthesis and ultimately growth rate. In addition, cell size can also be a target for Darwinian evolution for protection from predators. This strong coupling of cell size and growth, however, could lead to the introduction of growth defects after size evolution. An important question remains: can bacterial cell size change and/or evolve without imposing a growth burden? The directed evolution of particular cell sizes, without a growth burden, was tested with a laboratory Escherichia coli strain. Cells of defined size ranges were collected by a cell sorter and were subsequently cultured. This selection-propagation cycle was repeated, and significant changes in cell size were detected within 400 generations. In addition, the width of the size distribution was altered. The changes in cell size were unaccompanied by a growth burden. Whole genome sequencing revealed that only a few mutations in genes related to membrane synthesis conferred the size evolution. In conclusion, bacterial cell size could evolve, through a few mutations, without growth reduction. The size evolution without growth reduction suggests a rapid evolutionary change to diverse cell sizes in bacterial survival strategies.

Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Argaw, Takele; Wilson, Carolyn A., E-mail: carolyn.wilson@fda.hhs.gov

Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectorsmore » with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription. - Highlights: • Env cleavage signal impacts infectivity of gammaretroviruses. • Non-infectious mutants have hyper-glycosylated envelope that bind target cells. • Non-infectious mutants have defects in the formation of the double-stranded DNA. • Env cleavage motif has functions beyond cleavage of the env precursor.« less

Lack of chemically induced mutation in repair-deficient mutants of yeast.

PubMed

Prakash, L

1974-12-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.

Lateral Gene Transfer Dynamics in the Ancient Bacterial Genus Streptomyces

PubMed Central

McDonald, Bradon R.

2017-01-01

ABSTRACT Lateral gene transfer (LGT) profoundly shapes the evolution of bacterial lineages. LGT across disparate phylogenetic groups and genome content diversity between related organisms suggest a model of bacterial evolution that views LGT as rampant and promiscuous. It has even driven the argument that species concepts and tree-based phylogenetics cannot be applied to bacteria. Here, we show that acquisition and retention of genes through LGT are surprisingly rare in the ubiquitous and biomedically important bacterial genus Streptomyces. Using a molecular clock, we estimate that the Streptomyces bacteria are ~380 million years old, indicating that this bacterial genus is as ancient as land vertebrates. Calibrating LGT rate to this geologic time span, we find that on average only 10 genes per million years were acquired and subsequently maintained. Over that same time span, Streptomyces accumulated thousands of point mutations. By explicitly incorporating evolutionary timescale into our analyses, we provide a dramatically different view on the dynamics of LGT and its impact on bacterial evolution. PMID:28588130

The absence of N-acetylglucosamine in wall teichoic acids of Listeria monocytogenes modifies biofilm architecture and tolerance to rinsing and cleaning procedures

PubMed Central

Faille, Christine; Sadovskaya, Irina; Charbit, Alain; Benezech, Thierry; Shen, Yang; Loessner, Martin J.; Bautista, Jean Romain; Midelet-Bourdin, Graziella

2018-01-01

The wall teichoic acid (WTA) is the major carbohydrate found within the extracellular matrix of the Listeria monocytogenes biofilm. We first addressed the frequency of spontaneous mutations in two genes (lmo2549 and lmo2550) responsible for the GlcNAcylation in 93 serotype 1/2a strains that were mainly isolated from seafood industries. We studied the impact of mutations in lmo2549 or lmo2550 genes on biofilm formation by using one mutant carrying a natural mutation inactivating the lmo2550 gene (DSS 1130 BFA2 strain) and two EGD-e mutants that lack respective genes by in-frame deletion of lmo2549 or lmo2550 using splicing-by-overlap-extension PCR, followed by allelic exchange mutagenesis. The lmo2550 gene mutation, occurring in around 50% isolates, caused a decrease in bacterial adhesion to stainless steel compared to wild-type EGD-e strain during the adhesion step. On the other hand, bacterial population weren’t significantly different after 24h-biofilm formation. The biofilm architecture was different between the wild-type strain and the two mutants inactivated for lmo2549 or lmo2550 genes respectively with the presence of bacterial micro-colonies for mutants which were not observed in the wild-type EGD-e strain biofilm. These differences might account for the stronger hydrophilic surface exhibited by the mutant cells. Upon a water flow or to a cleaning procedure at a shear stress of 0.16 Pa, the mutant biofilms showed the higher detachment rate compared to wild-type strain. Meanwhile, an increase in the amount of residual viable but non-culturable population on stainless steel was recorded in two mutants. Our data suggests that the GlcNAc residue of WTA played a role in adhesion and biofilm formation of Listeria monocyctogenes. PMID:29320565

The absence of N-acetylglucosamine in wall teichoic acids of Listeria monocytogenes modifies biofilm architecture and tolerance to rinsing and cleaning procedures.

PubMed

Brauge, Thomas; Faille, Christine; Sadovskaya, Irina; Charbit, Alain; Benezech, Thierry; Shen, Yang; Loessner, Martin J; Bautista, Jean Romain; Midelet-Bourdin, Graziella

2018-01-01

The wall teichoic acid (WTA) is the major carbohydrate found within the extracellular matrix of the Listeria monocytogenes biofilm. We first addressed the frequency of spontaneous mutations in two genes (lmo2549 and lmo2550) responsible for the GlcNAcylation in 93 serotype 1/2a strains that were mainly isolated from seafood industries. We studied the impact of mutations in lmo2549 or lmo2550 genes on biofilm formation by using one mutant carrying a natural mutation inactivating the lmo2550 gene (DSS 1130 BFA2 strain) and two EGD-e mutants that lack respective genes by in-frame deletion of lmo2549 or lmo2550 using splicing-by-overlap-extension PCR, followed by allelic exchange mutagenesis. The lmo2550 gene mutation, occurring in around 50% isolates, caused a decrease in bacterial adhesion to stainless steel compared to wild-type EGD-e strain during the adhesion step. On the other hand, bacterial population weren't significantly different after 24h-biofilm formation. The biofilm architecture was different between the wild-type strain and the two mutants inactivated for lmo2549 or lmo2550 genes respectively with the presence of bacterial micro-colonies for mutants which were not observed in the wild-type EGD-e strain biofilm. These differences might account for the stronger hydrophilic surface exhibited by the mutant cells. Upon a water flow or to a cleaning procedure at a shear stress of 0.16 Pa, the mutant biofilms showed the higher detachment rate compared to wild-type strain. Meanwhile, an increase in the amount of residual viable but non-culturable population on stainless steel was recorded in two mutants. Our data suggests that the GlcNAc residue of WTA played a role in adhesion and biofilm formation of Listeria monocyctogenes.

Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

PubMed

Shaw, Wen Hui; Lin, Qianqian; Muhammad, Zikry Zhiwei Bin Roslee; Lee, Jia Jun; Khong, Wei Xin; Ng, Oon Tek; Tan, Eng Lee; Li, Peng

2016-07-01

Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being from Singapore.

Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development.

PubMed

Mozer, B A

2001-05-15

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins. Copyright 2001 Academic Press.

Two new mutations in the MTATP6 gene associated with Leigh syndrome.

PubMed

Moslemi, A-R; Darin, N; Tulinius, M; Oldfors, A; Holme, E

2005-10-01

In this study we have analyzed the mtDNA encoded ATPase 6 and 8 genes ( MTATP6 and MTATP8) in two children with Leigh syndrome (LS) and reduced Mg (2+) ATPase activity in muscle mitochondria. In patient 1, with a mild and reversible phenotype, mutational analysis revealed a heteroplasmic T --> C mutation at nt position 9185 (T9185C) in the MTATP6. The mutation resulted in substitution of a highly conserved leucine to proline at codon 220. The proportion of the mutation was > 97 % in the patient's blood and muscle and 85 % in blood of his asymptomatic mother. Patient 2, with severe clinical phenotype and death at 2 years of age, exhibited a novel heteroplasmic T9191C missense mutation in the MTATP6, which converted a highly conserved leucine to a proline at position 222 of the polypeptide. The proportion of the mutation was 90 % in fibroblasts and 94 % muscle tissue. This mutation was absent in the patient's parents and sister suggesting that the mutation was de novo. Our findings expand the spectrum of mutations causing LS and emphasize the role of MTATP6 gene mutations in pathogenesis of LS.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

PubMed

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

Identification of New Factors Modulating Adhesion Abilities of the Pioneer Commensal Bacterium Streptococcus salivarius

PubMed Central

Couvigny, Benoit; Kulakauskas, Saulius; Pons, Nicolas; Quinquis, Benoit; Abraham, Anne-Laure; Meylheuc, Thierry; Delorme, Christine; Renault, Pierre; Briandet, Romain; Lapaque, Nicolas; Guédon, Eric

2018-01-01

Biofilm formation is crucial for bacterial community development and host colonization by Streptococcus salivarius, a pioneer colonizer and commensal bacterium of the human gastrointestinal tract. This ability to form biofilms depends on bacterial adhesion to host surfaces, and on the intercellular aggregation contributing to biofilm cohesiveness. Many S. salivarius isolates auto-aggregate, an adhesion process mediated by cell surface proteins. To gain an insight into the genetic factors of S. salivarius that dictate host adhesion and biofilm formation, we developed a screening method, based on the differential sedimentation of bacteria in semi-liquid conditions according to their auto-aggregation capacity, which allowed us to identify twelve mutations affecting this auto-aggregation phenotype. Mutations targeted genes encoding (i) extracellular components, including the CshA surface-exposed protein, the extracellular BglB glucan-binding protein, the GtfE, GtfG and GtfH glycosyltransferases and enzymes responsible for synthesis of cell wall polysaccharides (CwpB, CwpK), (ii) proteins responsible for the extracellular localization of proteins, such as structural components of the accessory SecA2Y2 system (Asp1, Asp2, SecA2) and the SrtA sortase, and (iii) the LiaR transcriptional response regulator. These mutations also influenced biofilm architecture, revealing that similar cell-to-cell interactions govern assembly of auto-aggregates and biofilm formation. We found that BglB, CshA, GtfH and LiaR were specifically associated with bacterial auto-aggregation, whereas Asp1, Asp2, CwpB, CwpK, GtfE, GtfG, SecA2 and SrtA also contributed to adhesion to host cells and host-derived components, or to interactions with the human pathogen Fusobacterium nucleatum. Our study demonstrates that our screening method could also be used to identify genes implicated in the bacterial interactions of pathogens or probiotics, for which aggregation is either a virulence trait or an advantageous feature, respectively. PMID:29515553

Short communication: high prevalence of drug resistance in HIV type 1-infected children born in Honduras and Belize 2001 to 2004.

PubMed

Parham, Leda; de Rivera, Ivette Lorenzana; Murillo, Wendy; Naver, Lars; Largaespada, Natalia; Albert, Jan; Karlsson, Annika C

2011-10-01

Antiretroviral therapy has had a great impact on the prevention of mother-to-child transmission (MTCT) of HIV-1. However, development of drug resistance, which could be subsequently transmitted to the child, is a major concern. In Honduras and Belize the prevalence of drug resistance among HIV-1-infected children remains unknown. A total of 95 dried blood spot samples was obtained from HIV-1-infected, untreated children in Honduras and Belize born during 2001 to 2004, when preventive antiretroviral therapy was often suboptimal and consisted of monotherapy with nevirapine or zidovudine. Partial HIV-1 pol gene sequences were successfully obtained from 66 children (Honduras n=55; Belize n=11). Mutations associated with drug resistance were detected in 13% of the Honduran and 27% of the Belizean children. Most of the mutations detected in Honduras (43%) and all mutations detected in Belize were associated with resistance to nonnucleoside reverse transcriptase inhibitors, which was expected from the wide use of nevirapine to prevent MTCT during the study period. In addition, although several mothers reported that they had not received antiretroviral therapy, mutations associated with resistance to nucleoside reverse transcriptase inhibitors and protease inhibitors were found in Honduras. This suggests prior and unreported use of these drugs, or that these women had been infected with resistant virus. The present study demonstrates, for the first time, the presence of drug resistance-associated mutations in HIV-1-infected Honduran and Belizean children.

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension.

PubMed

Johnson, Jennifer A; Hemnes, Anna R; Perrien, Daniel S; Schuster, Manfred; Robinson, Linda J; Gladson, Santhi; Loibner, Hans; Bai, Susan; Blackwell, Tom R; Tada, Yuji; Harral, Julie W; Talati, Megha; Lane, Kirk B; Fagan, Karen A; West, James

2012-03-01

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.

Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration

PubMed Central

Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P. Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

2005-01-01

Background The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. Methods and Findings To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Conclusion Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations. PMID:15839752

PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae.

PubMed

Henriques, J A; Vicente, E J; Leandro da Silva, K V; Schenberg, A C

1989-09-01

The haploid xs9 mutant, originally selected for on the basis of a slight sensitivity to the lethal effect of X-rays, was found to be extremely sensitive to inactivation by 8-methoxypsoralen (8MOP) photoaddition, especially when cells are treated in the G2 phase of the cell cycle. As the xs9 mutation showed no allelism with any of the 3 known pso mutations, it was now given the name of pso4-1. Regarding inactivation, the pso4-1 mutant is also sensitive to mono- (HN1) or bi-functional (HN2) nitrogen mustards, it is slightly sensitive to 254 nm UV radiation (UV), and shows nearly normal sensitivity to 3-carbethoxypsoralen (3-CPs) photoaddition or methyl methanesulfonate (MMS). Regarding mutagenesis, the pso4-1 mutation completely blocks reverse and forward mutations induced by either 8MOP or 3CPs photoaddition, or by gamma-rays. In the cases of UV, HN1, HN2 or MMS treatments, while reversion induction is still completely abolished, forward mutagenesis is only partially inhibited for UV, HN1, or MMS, and it is unaffected for HN2. Besides severely inhibiting induced mutagenesis, the pso4-1 mutation was found to be semi-dominant, to block sporulation, to abolish the diploid resistance effect, and to block induced mitotic recombination, which indicates that the PSO4 gene is involved in a recombinational pathway of error-prone repair, comparable to the E. coli SOS repair pathway.

Risk factors and outcomes for the Q151M and T69 insertion HIV-1 resistance mutations in historic UK data.

PubMed

Stirrup, Oliver T; Dunn, David T; Tostevin, Anna; Sabin, Caroline A; Pozniak, Anton; Asboe, David; Cox, Alison; Orkin, Chloe; Martin, Fabiola; Cane, Patricia

2018-04-16

The prevalence of HIV-1 resistance to antiretroviral therapies (ART) has declined in high-income countries over recent years, but drug resistance remains a substantial concern in many low and middle-income countries. The Q151M and T69 insertion (T69i) resistance mutations in the viral reverse transcriptase gene can reduce susceptibility to all nucleoside/tide analogue reverse transcriptase inhibitors, motivating the present study to investigate the risk factors and outcomes associated with these mutations. We considered all data in the UK HIV Drug Resistance Database for blood samples obtained in the period 1997-2014. Where available, treatment history and patient outcomes were obtained through linkage to the UK Collaborative HIV Cohort study. A matched case-control approach was used to assess risk factors associated with the appearance of each of the mutations in ART-experienced patients, and survival analysis was used to investigate factors associated with viral suppression. A further analysis using matched controls was performed to investigate the impact of each mutation on survival. A total of 180 patients with Q151M mutation and 85 with T69i mutation were identified, almost entirely from before 2006. Occurrence of both the Q151M and T69i mutations was strongly associated with cumulative period of virological failure while on ART, and for Q151M there was a particular positive association with use of stavudine and negative association with use of boosted-protease inhibitors. Subsequent viral suppression was negatively associated with viral load at sequencing for both mutations, and for Q151M we found a negative association with didanosine use but a positive association with boosted-protease inhibitor use. The results obtained in these analyses were also consistent with potentially large associations with other drugs. Analyses were inconclusive regarding associations between the mutations and mortality, but mortality was high for patients with low CD4 at detection. The Q151M and T69i resistance mutations are now very rare in the UK. Our results suggest that good outcomes are possible for people with these mutations. However, in this historic sample, viral load and CD4 at detection were important factors in determining prognosis.

[Analysis on mutation of S gene and P gene of hepatitis B virus in two counties of Sichuan Province].

PubMed

Tong, Wen-Bin; He, Ji-Lan; Sun, Li

2009-02-01

To analyze HBV S gene/P gene mutation in 2 counties (districts) of Sichuan province. HBV DNA were extracted from sera positive both for HBsAg and HBeAg. After PCR and nucleotide sequencing, nucleotide/amino acid mutation in S and P gene were compared and analyzed. Of 47 serum samples from patients with chronic HBV infection, amino acid mutation in 'a' determinant occurred in 12 samples (25.53%,12/47), correlating with T126A, I126T/S, P127T, T131N, M133L, M133T and T140I; high proportion of mutation clustered in first loop of 'a' determinant (92.86%,13/14), rtV207I mutation in C domain of reverse transcription occured in one sample. Naturally occurring mutation in 'a' determinant clustered predominantly in the first loop and usually associated with altered antigenicity, posing a potential threat to successfully vaccinated individuals; Lamivudine-resistant mutant might occur in patient even without nucleotide analogue treatment.

Benzo(a)pyrene and X-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice.

PubMed

Bishop, A J; Kosaras, B; Sidman, R L; Schiestl, R H

2000-12-20

The pink-eyed unstable (p(un)) mutation is the result of a 70kb tandem duplication within the murine p gene. Homologous deletion/recombination of the locus to wild-type occurs spontaneously in embryos and results in pigmented spots in the fur and eye that persist for life. Such deletion events are also inducible by a variety of DNA damaging agents, as we have observed previously with the fur spot assay. Here, we describe the use of the retinal pigment epithelium (RPE) of the eye to detect reversion events induced with two differently acting agents. Benzo(a)pyrene (B(a)P) induces a high frequency, and X-ray exposure a more modest increase, of p(un) reversion in both the fur and the eye. The eye-spot assay requires fewer mice for significant results than the fur spot assay. Previous work had elucidated the cell proliferation pattern in the RPE and a position effect variegation phenotype in the pattern of p(un) reversions, which we have confirmed. Acute exposure to B(a)P or X-rays resulted in an increased frequency of reversion events. The majority of the spontaneous reversions lie toward the periphery of the RPE whereas induced events are found more centrally, closer to the optic nerve head. The induced distribution corresponds to the major sites of cell proliferation in the RPE at the time of exposure, and further advocates the proposal that dividing cells are at highest risk to develop deletions.

Analysis of 16 cystic fibrosis mutations in Mexican patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villalobos-Torres, C.; Rojas-Martinez, A.; Barrera-Saldana, H.A.

1997-04-14

We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation AF508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for AF508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3849 + 10 kb C{r_arrow}T, N1303K, S549N, and 621 + 1 G{r_arrow}T) were detected, andmore » these accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used. 20 refs., 2 tabs.« less

Optic atrophy and a Leigh-like syndrome due to mutations in the c12orf65 gene: report of a novel mutation and review of the literature.

PubMed

Heidary, Gena; Calderwood, Laurel; Cox, Gerald F; Robson, Caroline D; Teot, Lisa A; Mullon, Jennifer; Anselm, Irina

2014-03-01

Combined oxidative phosphorylation deficiency type 7 (COXPD7) is a rare disorder of mitochondrial metabolism that results in optic atrophy and Leigh syndrome-like disease. We describe 2 siblings with compound heterozygous mutations in the recently identified C12orf65 gene who presented with optic atrophy and mild developmental delays and subsequently developed bilateral, symmetric lesions in the brainstem reminiscent of Leigh syndrome. Repeat neuroimaging demonstrated reversibility of the findings in 1 sibling and persistent metabolic stroke in the other. This article highlights the phenotypic manifestations from a novel mutation in the C12orf65 gene and reviews the clinical presentation of the 5 other individuals reported to date who carry mutations in this gene.

Long-range activation of Sox9 in Odd Sex (Ods) mice.

PubMed

Qin, Yangjun; Kong, Ling-kun; Poirier, Christophe; Truong, Cavatina; Overbeek, Paul A; Bishop, Colin E

2004-06-15

The Odd Sex mouse mutation arose in a transgenic line of mice carrying a tyrosinase minigene driven by the dopachrome tautomerase (Dct) promoter region. The minigene integrated 0.98 Mb upstream of Sox9 and was accompanied by a deletion of 134 kb. This mutation causes female to male sex reversal in XX Ods/+ mice, and a characteristic eye phenotype of microphthalmia with cataracts in all mice carrying the transgene. Ods causes sex reversal in the absence of Sry by upregulating Sox9 expression and maintaining a male pattern of Sox9 expression in XX Ods/+ embryonic gonads. This expression, which begins at E11.5, triggers downstream events leading to the formation of a testis. We report here that the 134 kb deletion, in itself, is insufficient to cause sex reversal. We demonstrate that in Ods, the Dct promoter is capable of acting over a distance of 1 Mb to induce inappropriate expression of Sox9 in the retinal pigmented epithelium of the eye, causing the observed microphthalmia. In addition, it induces Sox9 expression in the melanocytes where it causes pigmentation defects. We propose that Ods sex reversal is due to the Dct promoter element interacting with gonad-specific enhancer elements to produce the observed male pattern expression of Sox9 in the embryonic gonads.

Yersinia pestis IS1541 transposition provides for escape from plague immunity.

PubMed

Cornelius, Claire A; Quenee, Lauriane E; Elli, Derek; Ciletti, Nancy A; Schneewind, Olaf

2009-05-01

Yersinia pestis is perhaps the most feared infectious agent due to its ability to cause epidemic outbreaks of plague disease in animals and humans with high mortality. Plague infections elicit strong humoral immune responses against the capsular antigen (fraction 1 [F1]) of Y. pestis, and F1-specific antibodies provide protective immunity. Here we asked whether Y. pestis generates mutations that enable bacterial escape from protective immunity and isolated a variant with an IS1541 insertion in caf1A encoding the F1 outer membrane usher. The caf1A::IS1541 insertion prevented assembly of F1 pili and provided escape from plague immunity via F1-specific antibodies without a reduction in virulence in mouse models of bubonic or pneumonic plague. F1-specific antibodies interfere with Y. pestis type III transport of effector proteins into host cells, an inhibitory effect that was overcome by the caf1A::IS1541 insertion. These findings suggest a model in which IS1541 insertion into caf1A provides for reversible changes in envelope structure, enabling Y. pestis to escape from adaptive immune responses and plague immunity.

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli.

PubMed

Venkat, Sumana; Chen, Hao; Stahman, Alleigh; Hudson, Denver; McGuire, Paige; Gan, Qinglei; Fan, Chenguang

2018-06-22

The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Toxicological evaluation of Euterpe edulis: a potential superfruit to be considered.

PubMed

Felzenszwalb, Israel; da Costa Marques, Monica Regina; Mazzei, José L; Aiub, Claudia A F

2013-08-01

Superfruits have a high nutritional value due to their richness in nutrients, antioxidants, proven or potential health benefits and taste appeal. However, there are no scientific criteria for defining which fruits are superfruits. In Brazil, several palms have an edible palm heart, the best known and most widely appreciated of which is called Acai (Euterpe oleracea). Euterpe edulis Mart., commonly called jussara, is an evergreen species that grows in the rainforest. Having initially been consumed in the form of juice and pulp, they have since been incorporated as an ingredient in many foods. A risk assessment to identify adverse health effects is a prerequisite for taking forward the development of new drugs, cosmetics and foods. To make a toxicological evaluation of E. edulis, in the present work this prerequisite was met by an interdisciplinary network that performed mass spectroscopy analyses, blood biochemistry, genotoxicity, bacterial reverse mutation and cytotoxicity assays. Positive mutagenicity results were detected for Salmonella typhimurium TA97 at low doses, and positive results were also obtained for the mammalian erythrocyte micronucleus assay, indicating that the pulp of E. edulis contains compounds with the capacity to induce mutagenicity and clastogenic/aneugenic effects. Copyright © 2013 Elsevier Ltd. All rights reserved.

Safety Evaluation of Turmeric Polysaccharide Extract: Assessment of Mutagenicity and Acute Oral Toxicity

PubMed Central

Velusami, Chandrasekaran Chinampudur; Boddapati, Srinivasa Rao; Hongasandra Srinivasa, Srikanth; Richard, Edwin Jothie; Balasubramanian, Murali

2013-01-01

Curcuma longa Linn. (Zingiberaceae) commonly known as turmeric has long been used for centuries as a spice and household remedy. The present study was carried out to assess the possible mutagenic potential and acute oral toxicity of polysaccharide extract of turmeric rhizome (NR-INF-02) using standard tests. The standard battery of in vitro genotoxicity tests, bacterial reverse mutation test (BRMT), chromosome aberration (CA), and micronucleus (MN) tests were employed to assess the possible mutagenic activity of NR-INF-02 (Turmacin). The results showed no mutagenic effect with NR-INF-02 up to a dose of 5000 µg/mL in BRMT. The results on CA and MN tests revealed the non clastogenic activity of NR-INF-02 in a dose range of 250.36 to 2500 µg/mL with and without metabolic activation (S9). In acute oral toxicity study, NR-INF-02 was found to be safe up to 5 g/kg body weight in Wistar rats. Overall, results indicated that polysaccharide extract of C. longa was found to be genotoxically safe and also exhibited maximum tolerable dose of more than 5 g/kg rat body weight. PMID:24455673

Genotoxicity potential of a new natural formicide.

PubMed

Cotelle, Sylvie; Testolin, Renan C; Foltête, Anne-Sophie; Bossardi-Rissardi, Georgiana; Silveira, Rosilene A; Radetski, Claudemir M

2012-03-01

Assessment of environmental impacts from pesticide utilization should include genotoxicity studies, where the possible effects of mutagenic/genotoxic substances on individuals are assessed. In this study, the genotoxicity profile of the new formicide Macex® was evaluated with two genotoxicity tests, namely, the micronucleus test with mouse bone marrow and Vicia faba, and a mutagenicity test using the Ames Salmonella assay. The bacterial reverse mutation test (Salmonella typhimurium strains TA97, TA98, TA100, TA102, and TA1535), the Vicia root tip and mouse micronucleus tests were conducted according to published protocols. In the range of the formicide Macex® concentrations tested from 0.06 to 1.0 g L⁻¹ (or mgkg⁻¹ in the mouse test), no genotoxicity was observed in the prokaryotic or eukaryotic test organisms. However, at Macex® concentrations of 0.5 g L⁻¹ and above a significant decrease in the mitotic index (P ≤ 0.05) in the V. faba was observed. Micronucleus formation was likewise increased in the test organism at concentrations starting at 2.0 g L⁻¹. These data allow us to classify this natural formicide preparation as a product with no geno-environmental-impact when applied at recommended concentrations.

Modeling physiological resistance in bacterial biofilms.

PubMed

Cogan, N G; Cortez, Ricardo; Fauci, Lisa

2005-07-01

A mathematical model of the action of antimicrobial agents on bacterial biofilms is presented. The model includes the fluid dynamics in and around the biofilm, advective and diffusive transport of two chemical constituents and the mechanism of physiological resistance. Although the mathematical model applies in three dimensions, we present two-dimensional simulations for arbitrary biofilm domains and various dosing strategies. The model allows the prediction of the spatial evolution of bacterial population and chemical constituents as well as different dosing strategies based on the fluid motion. We find that the interaction between the nutrient and the antimicrobial agent can reproduce survival curves which are comparable to other model predictions as well as experimental results. The model predicts that exposing the biofilm to low concentration doses of antimicrobial agent for longer time is more effective than short time dosing with high antimicrobial agent concentration. The effects of flow reversal and the roughness of the fluid/biofilm are also investigated. We find that reversing the flow increases the effectiveness of dosing. In addition, we show that overall survival decreases with increasing surface roughness.

Transmitted Antiretroviral Drug Resistance in Newly HIV-Infected and Untreated Patients in Ségou and Bamako, Mali

PubMed Central

Fofana, Djeneba Bocar; Maiga, Aichatou Chehy; Diallo, Fodie; Ait-Arkoub, Zaina; Daou, Fatoumata; Cisse, Mamadou; Sarro, Yaya dit Sadio; Oumar, Aboubacar Alassane; Sylla, Aliou; Katlama, Christine; Taiwo, Babafemi; Murphy, Robert; Tounkara, Anatole; Marcelin, Anne-Genevieve; Calvez, Vincent

2013-01-01

Abstract The WHO recommends regular surveillance for transmitted antiretroviral drug-resistant viruses in HIV antiretroviral treatment (ART)-naive patients in resource-limited settings. This study aimed to assess the prevalence of mutations associated with resistance in ART-naive patients newly diagnosed with HIV in Bamako and Ségou in Mali. HIV-positive patients who never received ART were recruited in Bamako and Ségou, Mali. The reverse transcriptase (RT) and protease (PR) genes of these patients were sequenced by the “ViroSeq” method. Analysis and interpretation of the resistance were made according to the WHO 2009 list of drug resistance mutations. In all, 51/54 (94.4%) sample patients were sequenced. The median age (IQR) of our patients was 24 (22–27) years and the median CD4 count was 380 (340–456) cells/mm3. The predominant subtype was recombinant HIV-1 CRF02_AG (66.7%) followed by CRF06_cpx (12%) and CRF09_cpx (4%). Four patients had mutations associated with resistance, giving an overall prevalence of resistance estimated at 7.9%. There were two (4%) patients with nucleoside reverse transcriptase inhibitor (NRTI) mutations (one M184V and one T215Y), two (4%) with non-NRTI mutations (two K103N), and one (2%) with a protease inhibitor mutation (one I54V). The prevalence of primary resistance in newly infected patients in Mali is moderate (7.9%). This indicates that the standard NNRTI-based first-line regimen used in Mali is suboptimal for some patients. This study should be done regularly to inform clinical practice. PMID:22823755

An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle.

PubMed

Fritz, Sébastien; Hoze, Chris; Rebours, Emmanuelle; Barbat, Anne; Bizard, Méline; Chamberlain, Amanda; Escouflaire, Clémentine; Vander Jagt, Christy; Boussaha, Mekki; Grohs, Cécile; Allais-Bonnet, Aurélie; Philippe, Maëlle; Vallée, Amélie; Amigues, Yves; Hayes, Benjamin J; Boichard, Didier; Capitan, Aurélien

2018-04-18

Researching depletions in homozygous genotypes for specific haplotypes among the large cohorts of animals genotyped for genomic selection is a very efficient strategy to map recessive lethal mutations. In this study, by analyzing real or imputed Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes from more than 250,000 Holstein animals, we identified a new locus called HH6 showing significant negative effects on conception rate and nonreturn rate at 56 d in at-risk versus control mating. We fine-mapped this locus in a 1.1-Mb interval and analyzed genome sequence data from 12 carrier and 284 noncarrier Holstein bulls. We report the identification of a strong candidate mutation in the gene encoding SDE2 telomere maintenance homolog (SDE2), a protein essential for genomic stability in eukaryotes. This A-to-G transition changes the initiator ATG (methionine) codon to ACG because the gene is transcribed on the reverse strand. Using RNA sequencing and quantitative reverse-transcription PCR, we demonstrated that this mutation does not significantly affect SDE2 splicing and expression level in heterozygous carriers compared with control animals. Initiation of translation at the closest in-frame methionine codon would truncate the SDE2 precursor by 83 amino acids, including the cleavage site necessary for its activation. Finally, no homozygote for the G allele was observed in a large population of nearly 29,000 individuals genotyped for the mutation. The low frequency (1.3%) of the derived allele in the French population and the availability of a diagnostic test on the Illumina EuroG10K SNP chip routinely used for genomic evaluation will enable rapid and efficient selection against this deleterious mutation. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

PubMed Central

Le Nouën, Cyril; McCarty, Thomas; Brown, Michael; Smith, Melissa Laird; Lleras, Roberto; Dolan, Michael A.; Mehedi, Masfique; Yang, Lijuan; Luongo, Cindy; Liang, Bo; Munir, Shirin; DiNapoli, Joshua M.; Mueller, Steffen; Wimmer, Eckard; Collins, Peter L.; Buchholz, Ursula J.

2017-01-01

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates. PMID:28049853

Effect of dolutegravir in combination with Nucleoside Reverse Transcriptase Inhibitors (NRTIs) on people living with HIV who have pre-existing NRTI mutations.

PubMed

Sörstedt, Erik; Carlander, Christina; Flamholc, Leo; Hejdeman, Bo; Svedhem, Veronica; Sönnerborg, Anders; Gisslén, Magnus; Yilmaz, Aylin

2018-05-01

Until the introduction of dolutegravir (DTG), people living with HIV (PLWH) who have developed nucleoside reverse transcriptase inhibitor (NRTI) mutations have had few other treatment options outside of regimens based on ritonavir-boosted protease inhibitors (PI/r). Here we report treatment results among PLWH in Sweden with pre-existing NRTI mutations on antiretroviral treatment (ART) with DTG and one to two NRTIs. All PLWH on ART with DTG and one to two NRTIs with pre-existing NRTI mutations were retrospectively identified from the National InfCare HIV database. As controls, PLWH on PI/r and one to two NRTIs, matched according to Genotypic Susceptibility Score and observation time, were included. Data were collected as long as the study population was on treatment with DTG; controls were monitored for the same interval. Outcome was classified as either treatment success or failure. In total, 244 participants (122 individuals treated with DTG and 122 individuals treated with PI/r) were included. Median observation time was 78 weeks (interquartile range 50-98 weeks) for participants on DTG and 75 weeks (50-101 weeks) for individuals on PI/r. Viral failure was detected in four individuals treated with DTG and three individuals treated with PI/r, resulting in similar success rates of 96.7% and 97.5%, respectively. No new mutations were found among participants with treatment failure. DTG in combination with one to two NRTIs was as efficient as PI/r in individuals with pre-existing NRTI mutations in this setting. It may be considered an alternative to PI/r-based ART even in the presence of NRTI resistance. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases.

PubMed

Farhat, M; Poissonnier, A; Hamze, A; Ouk-Martin, C; Brion, J-D; Alami, M; Feuillard, J; Jayat-Vignoles, C

2014-05-01

Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated.

Persistence of transmitted HIV-1 drug resistance mutations associated with fitness costs and viral genetic backgrounds.

PubMed

Yang, Wan-Lin; Kouyos, Roger D; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Scherrer, Alexandra U; Shilaih, Mohaned; Hinkley, Trevor; Petropoulos, Christos; Bonhoeffer, Sebastian; Günthard, Huldrych F

2015-03-01

Transmission of drug-resistant pathogens presents an almost-universal challenge for fighting infectious diseases. Transmitted drug resistance mutations (TDRM) can persist in the absence of drugs for considerable time. It is generally believed that differential TDRM-persistence is caused, at least partially, by variations in TDRM-fitness-costs. However, in vivo epidemiological evidence for the impact of fitness costs on TDRM-persistence is rare. Here, we studied the persistence of TDRM in HIV-1 using longitudinally-sampled nucleotide sequences from the Swiss-HIV-Cohort-Study (SHCS). All treatment-naïve individuals with TDRM at baseline were included. Persistence of TDRM was quantified via reversion rates (RR) determined with interval-censored survival models. Fitness costs of TDRM were estimated in the genetic background in which they occurred using a previously published and validated machine-learning algorithm (based on in vitro replicative capacities) and were included in the survival models as explanatory variables. In 857 sequential samples from 168 treatment-naïve patients, 17 TDRM were analyzed. RR varied substantially and ranged from 174.0/100-person-years;CI=[51.4, 588.8] (for 184V) to 2.7/100-person-years;[0.7, 10.9] (for 215D). RR increased significantly with fitness cost (increase by 1.6[1.3,2.0] per standard deviation of fitness costs). When subdividing fitness costs into the average fitness cost of a given mutation and the deviation from the average fitness cost of a mutation in a given genetic background, we found that both components were significantly associated with reversion-rates. Our results show that the substantial variations of TDRM persistence in the absence of drugs are associated with fitness-cost differences both among mutations and among different genetic backgrounds for the same mutation.

Variations and heredity in bacterial colonies

PubMed Central

Čepl, Jaroslav; Blahůšková, Anna; Neubauer, Zdeněk; Markoš, Anton

2016-01-01

ABSTRACT Spontaneous variation in appearance was studied in bacterial colonies of Serratia marcescens F morphotype1: (i) A defined array of non-heritable phenotype variations does appear repeatedly; (ii) The presence of colonies of different bacterial species will narrow the variability toward the typical F appearance, as if such an added environmental factor curtailed the capacity of colony morphospace; (iii) Similarly the morphospace becomes reduced by random mutations leading to new, heritable morphotypes—at the same time opening a new array of variations typical for the mutant but not accessible directly from the original F morphospace. Results are discussed in context with biphasic model of early morphogenesis applicable to all multicellular bodies. PMID:28042382

Connection Subdomain Mutations in HIV-1 Subtype-C Treatment-Experienced Patients Enhance NRTI and NNRTI Drug Resistance

PubMed Central

Delviks-Frankenberry, Krista A.; Lengruber, Renan B.; Santos, Andre F.; Silveira, Jussara M.; Soares, Marcelo A.; Kearney, Mary F.; Maldarelli, Frank; Pathak, Vinay K.

2012-01-01

Mutations in the connection subdomain (CN) and RNase H domain (RH) of HIV-1 reverse transcriptase (RT) from subtype B-infected patients enhance nucleoside and nonnucleoside RT inhibitor (NRTI and NNRTI) resistance by affecting the balance between polymerization and RNase H activity. To determine whether CN mutations in subtype C influence drug sensitivity, single genome sequencing was performed on Brazilian subtype C-infected patients failing RTI therapy. CN mutations identified were similar to subtype B, including A376S, A400T, Q334D, G335D, N348I, and A371V, and increased AZT resistance in the presence of thymidine analog mutations. CN mutations also enhanced NNRTI resistance in the presence of classical NNRTI mutations: etravirine resistance was enhanced 6- to 11-fold in the presence of L100I/K103N/Y181C. These results indicate that selection of CN mutations in treatment-experienced patients also occurs in subtype-C-infected patients and are likely to provide valuable information in predicting clinical RTI resistance. PMID:23068886

THE GENOTOXICITY OF AMBIENT OUTDOOR AIR, A REVIEW: SALMONELLA MUTAGENICITY

EPA Science Inventory

The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity

Abstract
Mutagens in urban air pollution come from anthropogenic sources (especially combustion sources) and are products of airborne chemical reactions. Bacterial mutation tests have been used ...

Mutagenic effect of accelerated heavy ions on bacterial cells

NASA Astrophysics Data System (ADS)

Boreyko, A. V.; Krasavin, E. A.

2011-11-01

The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac-, ton B-, col B) mutations for Esherichia coli cells and reverse his- → His+ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear "dose-effect" dependences are typical. The quadratic character of mutagenesis dose curves is determined by the "interaction" of two independent "hitting" events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific features of energy transfer of the radiations that affect the character of induced DNA damage, and the efficiency inducible and constitutive cell repair systems. The growth of relative biological efficiency of heavy charged particles is determined by the growth of the damage yield of the DNA participating in the formation of radiation-induced effects, and higher efficiency of inducible repair systems. It was established that the LET value ( L max) for which the maximum (according to the applied irradiation criteria) coefficients of relative biological efficiency are observed varies depending on the character of the registered radiation induced effect. It was demonstrated that for gene mutations and induction of precision excision of mobile elements the values of L max are realized in a LET range of ≈20 keV/μm. For lethal effects of irradiation and induction of deletion mutations the value of L max is ≈ 100 and 50 keV/μm, respectively. The differences in the L max for the studied radiation gene effectis are determined by the different type of DNA damage participating in the mutation process. A molecular model of the formation of gene mutations in Escherichia coli cells under the action of ionizing radiation was proposed. Basic DNA radiation damage and main repair ways were considered in the framework of this model. The basis is the idea of the decisive role of mutagenic, error-prone, branch of SOS repair in fixing premutation DNA damage into point mutations. It was demonstrated that the central mechanism in this process is the formation of an inducible multi-enzymatic complex including the DNA polymerase V (Umu C), RecA-protease, SSB proteins, subunits of DNA polymerase III, performing erroneous DNA synthesis on the damaged matrix. A mathematical model of induction of gene mutations under ultraviolet cell irradiation was developed based on the molecular model.

MANAGEMENT OF ENDOCRINE DISEASE: Reversible hypogonadotropic hypogonadism.

PubMed

Dwyer, Andrew A; Raivio, Taneli; Pitteloud, Nelly

2016-06-01

Congenital hypogonadotropic hypogonadism (CHH) is characterized by lack of puberty and infertility. Traditionally, it has been considered a life-long condition yet cases of reversibility have been described wherein patients spontaneously recover function of the reproductive axis following treatment. Reversibility occurs in both male and female CHH cases and appears to be more common (~10-15%) than previously thought. These reversal patients span a range of GnRH deficiency from mild to severe and many reversal patients harbor mutations in genes underlying CHH. However, to date there are no clear factors for predicting reversible CHH. Importantly, recovery of reproductive axis function may not be permanent. Thus, CHH is not always life-long and the incidence of reversal warrants periodic treatment withdrawal with close monitoring and follow-up. Reversible CHH highlights the importance of environmental (epigenetic) factors such as sex steroid treatment on the reproductive axis in modifying the phenotype. This review provides an overview and an update on what is known about this phenomenon. © 2016 European Society of Endocrinology.

Tertiary model of a plant cellulose synthase

PubMed Central

Sethaphong, Latsavongsakda; Haigler, Candace H.; Kubicki, James D.; Zimmer, Jochen; Bonetta, Dario; DeBolt, Seth; Yingling, Yaroslava G.

2013-01-01

A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded β-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose–synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs. PMID:23592721

The molecular chaperone HSP70 binds to and stabilizes NOD2, an important protein involved in Crohn disease.

PubMed

Mohanan, Vishnu; Grimes, Catherine Leimkuhler

2014-07-04

Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Molecular Chaperone HSP70 Binds to and Stabilizes NOD2, an Important Protein Involved in Crohn Disease*

PubMed Central

Mohanan, Vishnu; Grimes, Catherine Leimkuhler

2014-01-01

Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. PMID:24790089

Cysteine Supplementation May be Beneficial in a Subgroup of Mitochondrial Translation Deficiencies.

PubMed

Bartsakoulia, Marina; Mϋller, Juliane S; Gomez-Duran, Aurora; Yu-Wai-Man, Patrick; Boczonadi, Veronika; Horvath, Rita

2016-08-30

Mitochondrial encephalomyopathies are severe, relentlessly progressive conditions and there are very few effective therapies available to date. We have previously suggested that in two rare forms of reversible mitochondrial disease (reversible infantile respiratory chain deficiency and reversible infantile hepatopathy) supplementation with L-cysteine can improve mitochondrial protein synthesis, since cysteine is required for the 2-thiomodification of mitochondrial tRNAs. We studied whether supplementation with L-cysteine or N-acetyl-cysteine (NAC) results in any improvement of the mitochondrial function in vitro in fibroblasts of patients with different genetic forms of abnormal mitochondrial translation. We studied in vitro in fibroblasts of patients carrying the common m.3243A>G and m.8344A>G mutations or autosomal recessive mutations in genes affecting mitochondrial translation, whether L-cysteine or N-acetyl-cysteine supplementation have an effect on mitochondrial respiratory chain function. Here we show that supplementation with L-cysteine, but not with N-acetyl-cysteine partially rescues the mitochondrial translation defect in vitro in fibroblasts of patients carrying the m.3243A>G and m.8344A>G mutations. In contrast, N-acetyl-cysteine had a beneficial effect on mitochondrial translation in TRMU and MTO1 deficient fibroblasts. Our results suggest that L-cysteine or N-acetyl-cysteine supplementation may be a potential treatment for selected subgroups of patients with mitochondrial translation deficiencies. Further studies are needed to explore the full potential of cysteine supplementation as a treatment for patients with mitochondrial disease.

Evaluation of the genotoxicity of extracts of Houttuynia cordata Thunb.

PubMed

Kang, Chang Keun; Hah, Dae Sik; Kim, Chung Hui; Kim, Euikyung; Kim, Jong Shu

2012-01-01

The present study was conducted to evaluate the activity of methanol extracts from Houttuynia cordata Thunb. (HC) in a reverse mutation assay in Salmonella typhimurium, and a chromosome aberration assay in the Chinese hamster ovary (CHO) cell line and to evaluate its effect on the occurrence of polychromatic erythrocytes in mice. In the reverse mutation assay using Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2urvA(-), methanol extracts of HC (5, 2.5, 1.25, 0.62, or 0.312 mg/plate) did not induce reverse mutations in the presence or absence of an S9 metabolic activation mixture. In the chromosome aberration test using CHO cells, methanol extracts (1.25, 2.5 or 5 μg/ml) caused a few incidences of structural and numerical aberrations, in both of absence or presence of an S9 metabolic activation mixture, but in comparison with the positive control group, these incidences were not significantly increased. In the mouse micronucleus test, no significant increases in the occurrence of micronucleated polychromatic erythrocytes were observed in male ICR mice that were orally administered methanol extracts of HC at doses of 2.0, 1.0, or 0.5 g/kg. From these results, we concluded that the methanol extracts of HC did not induce harmful effects on genes in bacteria, a mammalian cell system or in mouse bone marrow cells. Thus, HC's use for health promotion and/or a sick remedy for humans may be safe.

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Inducible CYP2J2 and Its Product 11,12-EET Promotes Bacterial Phagocytosis: A Role for CYP2J2 Deficiency in the Pathogenesis of Crohn’s Disease?

PubMed Central

Bystrom, Jonas; Thomson, Scott J.; Johansson, Jörgen; Edin, Matthew L.; Zeldin, Darryl C.; Gilroy, Derek W.; Smith, Andrew M.; Bishop-Bailey, David

2013-01-01

The epoxygenase CYP2J2 has an emerging role in inflammation and vascular biology. The role of CYP2J2 in phagocytosis is not known and its regulation in human inflammatory diseases is poorly understood. Here we investigated the role of CYP2J2 in bacterial phagocytosis and its expression in monocytes from healthy controls and Crohns disease patients. CYP2J2 is anti-inflammatory in human peripheral blood monocytes. Bacterial LPS induced CYP2J2 mRNA and protein. The CYP2J2 arachidonic acid products 11,12-EET and 14,15-EET inhibited LPS induced TNFα release. THP-1 monocytes were transformed into macrophages by 48h incubation with phorbol 12-myristate 13-acetate. Epoxygenase inhibition using a non-selective inhibitor SKF525A or a selective CYP2J2 inhibitor Compound 4, inhibited E. coli particle phagocytosis, which could be specifically reversed by 11,12-EET. Moreover, epoxygenase inhibition reduced the expression of phagocytosis receptors CD11b and CD68. CD11b also mediates L. monocytogenes phagocytosis. Similar, to E. coli bioparticle phagocytosis, epoxygenase inhibition also reduced intracellular levels of L. monocytogenes, which could be reversed by co-incubation with 11,12-EET. Disrupted bacterial clearance is a hallmark of Crohn’s disease. Unlike macrophages from control donors, macrophages from Crohn’s disease patients showed no induction of CYP2J2 in response to E. coli. These results demonstrate that CYP2J2 mediates bacterial phagocytosis in macrophages, and implicates a defect in the CYP2J2 pathway may regulate bacterial clearance in Crohn’s disease. PMID:24058654

Inducible CYP2J2 and its product 11,12-EET promotes bacterial phagocytosis: a role for CYP2J2 deficiency in the pathogenesis of Crohn's disease?

PubMed

Bystrom, Jonas; Thomson, Scott J; Johansson, Jörgen; Edin, Matthew L; Zeldin, Darryl C; Gilroy, Derek W; Smith, Andrew M; Bishop-Bailey, David

2013-01-01

The epoxygenase CYP2J2 has an emerging role in inflammation and vascular biology. The role of CYP2J2 in phagocytosis is not known and its regulation in human inflammatory diseases is poorly understood. Here we investigated the role of CYP2J2 in bacterial phagocytosis and its expression in monocytes from healthy controls and Crohns disease patients. CYP2J2 is anti-inflammatory in human peripheral blood monocytes. Bacterial LPS induced CYP2J2 mRNA and protein. The CYP2J2 arachidonic acid products 11,12-EET and 14,15-EET inhibited LPS induced TNFα release. THP-1 monocytes were transformed into macrophages by 48h incubation with phorbol 12-myristate 13-acetate. Epoxygenase inhibition using a non-selective inhibitor SKF525A or a selective CYP2J2 inhibitor Compound 4, inhibited E. coli particle phagocytosis, which could be specifically reversed by 11,12-EET. Moreover, epoxygenase inhibition reduced the expression of phagocytosis receptors CD11b and CD68. CD11b also mediates L. monocytogenes phagocytosis. Similar, to E. coli bioparticle phagocytosis, epoxygenase inhibition also reduced intracellular levels of L. monocytogenes, which could be reversed by co-incubation with 11,12-EET. Disrupted bacterial clearance is a hallmark of Crohn's disease. Unlike macrophages from control donors, macrophages from Crohn's disease patients showed no induction of CYP2J2 in response to E. coli. These results demonstrate that CYP2J2 mediates bacterial phagocytosis in macrophages, and implicates a defect in the CYP2J2 pathway may regulate bacterial clearance in Crohn's disease.

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis.

PubMed

Machado, Diana; Coelho, Tatiane S; Perdigão, João; Pereira, Catarina; Couto, Isabel; Portugal, Isabel; Maschmann, Raquel De Abreu; Ramos, Daniela F; von Groll, Andrea; Rossetti, Maria L R; Silva, Pedro A; Viveiros, Miguel

2017-01-01

Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis . A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55 , and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often disregarded by the tuberculosis specialists in favor of the almost undisputed importance of antibiotic target-gene mutations for the resistance in M. tuberculosis .

REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES1

PubMed Central

Lee, W. H.; Ordal, Z. John

1963-01-01

Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207–217. 1963.—It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates. Images PMID:16561987

Cell culture-adaptive mutations of NS5A affect replication of hepatitis C virus differentially depending on the viral genotypes.

PubMed

Chung, Aeri; Jin, Bora; Han, Kwang-Hyub; Ahn, Sang Hoon; Kim, Seungtaek

2017-01-01

Most of HCV RNAs require cell culture-adaptive mutations for efficient replication in cell culture and a number of such mutations have been described including a well-known S2204I substitution mutation in NS5A protein. In contrast, the replication of genotype 2a JFH1 RNA in cell culture does not require any cell culture-adaptive mutation. Rather, the presence of S2204I mutation impaired the JFH1 RNA replication. In this study, we examined the effect of reversions and substitutions of NS5A cell culture-adaptive mutations on virus replication in different genotypic backgrounds after either placing genotype 1a NS5A in the genotype 2a JFH1 or vice versa. The results from this investigation suggest that the S2204I mutation affects HCV RNA replication differentially depending on the viral genotypes but that the effect was not simply explained by the genotypic background. Perhaps, the effect of the S2204I mutation on HCV replication reflects both intra- and intergenic interactions of NS5A protein. J. Med. Virol. 89:146-152, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

[Identification of a novel splicing mutation of PHEX gene in a pedigree affected with X-linked hypophosphatemia].

PubMed

Li, Jie; Xu, Peiwen; Huang, Sexin; Gao, Ming; Zou, Yang; Kang, Ranran; Gao, Yuan

2017-04-10

To identify potential mutation of PHEX gene in two patients from a family affected with X-linked hypophosphatemia (XLH). PCR and Sanger sequencing were performed on blood samples from the patients and 100 healthy controls. Reverse transcription-PCR (RT-PCR) was used to determine the mRNA expression in patient samples. A splicing site mutation, IVS21+2T>G, was found in the PHEX gene in both patients but not among the 100 healthy controls. RT-PCR confirmed that exon 21 of the PHEX gene was deleted. The novel splicing mutation IVS21+2T>G of the PHEX gene probably underlies the XLH in this pedigree. At the mRNA level, the mutation has led to removal of exon 21 and shift of the open reading frame (p.Val691fsx), resulting in premature termination of protein translation.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. III. Dose-response pattern of mutation induction in UV-irradiated rev2ts cells.

PubMed

Siede, W; Eckardt, F

1986-01-01

Recent studies regarding the influence of cycloheximide on the temperature-dependent increase in survival and mutation frequencies of a thermoconditional rev2 mutant lead to the suggestion that the REV2-coded mutagenic repair function is UV-inducible. In the present study we show that stationary-phase rev2ts cells are characterized by a biphasic linear-quadratic dose-dependence of mutation induction ("mutation kinetics") of ochre alleles at 23 degrees C (permissive temperature) but linear kinetics at the restrictive temperature of 36 degrees C. Mathematical analysis using a model based on Poisson statistics and a further mathematical procedure, the calculation of "apparent survival", support the assumption that the quadratic component of the reverse mutation kinetics investigated can be attributed to a UV-inducible component of mutagenic DNA repair controlled by the REV2 gene.

Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine.

PubMed Central

Gu, Z; Gao, Q; Li, X; Parniak, M A; Wainberg, M A

1992-01-01

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety. Images PMID:1279198

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.

PubMed

Chantreau, Maxime; Grec, Sébastien; Gutierrez, Laurent; Dalmais, Marion; Pineau, Christophe; Demailly, Hervé; Paysant-Leroux, Christine; Tavernier, Reynald; Trouvé, Jean-Paul; Chatterjee, Manash; Guillot, Xavier; Brunaud, Véronique; Chabbert, Brigitte; van Wuytswinkel, Olivier; Bendahmane, Abdelhafid; Thomasset, Brigitte; Hawkins, Simon

2013-10-15

Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics. A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species. We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics

PubMed Central

2013-01-01

Background Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics. Results A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species. Conclusions We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax. PMID:24128060

Dynamic of mutational events in variable number tandem repeats of Escherichia coli O157:H7.

PubMed

Bustamante, A V; Sanso, A M; Segura, D O; Parma, A E; Lucchesi, P M A

2013-01-01

VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs) on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10(-05) to 1.8 × 10(-03) mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10(-03) mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study.

Contrasting Phenotypes in Resistance to Thyroid Hormone Alpha Correlate with Divergent Properties of Thyroid Hormone Receptor α1 Mutant Proteins.

PubMed

Moran, Carla; Agostini, Maura; McGowan, Anne; Schoenmakers, Erik; Fairall, Louise; Lyons, Greta; Rajanayagam, Odelia; Watson, Laura; Offiah, Amaka; Barton, John; Price, Susan; Schwabe, John; Chatterjee, Krishna

2017-07-01

Resistance to thyroid hormone alpha (RTHα), a disorder characterized by tissue-selective hypothyroidism and near-normal thyroid function tests due to thyroid receptor alpha gene mutations, is rare but probably under-recognized. This study sought to correlate the clinical characteristics and response to thyroxine (T4) therapy in two adolescent RTHα patients with the properties of the THRA mutation, affecting both TRα1 and TRα2 proteins, they harbored. Clinical, auxological, biochemical, and physiological parameters were assessed in each patient at baseline and after T4 therapy. Heterozygous THRA mutations occurring de novo were identified in a 17-year-old male (patient P1; c.788C>T, p.A263V mutation) investigated for mild pubertal delay and in a 15-year-old male (patient P2; c.821T>C, p.L274P mutation) with short stature (0.4th centile), skeletal dysplasia, dysmorphic facies, and global developmental delay. Both individuals exhibited macrocephaly, delayed dentition, and constipation, together with a subnormal T4/triiodothyronine (T3) ratio, low reverse T3 levels, and mild anemia. When studied in vitro, A263V mutant TRα1 was transcriptionally impaired and inhibited the function of its wild-type counterpart at low (0.01-10 nM) T3 levels, with higher T3 concentrations (100 nM-1 μM) reversing dysfunction and such dominant negative inhibition. In contrast, L274P mutant TRα1 was transcriptionally inert, exerting significant dominant negative activity, only overcome with 10 μM of T3. Mirroring this, normal expression of KLF9, a TH-responsive target gene, was achieved in A263V mutation-containing peripheral blood mononuclear cells following 1 μM of T3 exposure, but with markedly reduced expression levels in L274P mutation-containing peripheral blood mononuclear cells, even with 10 μM of T3. Following T4 therapy, growth, body composition, dyspraxia, and constipation improved in P1, whereas growth retardation and constipation in P2 were unchanged. Neither A263V nor L274P mutations exhibited gain or loss of function in the TRα2 background, and no additional phenotype attributable to this was discerned. This study correlates a milder clinical phenotype and favorable response to T4 therapy in a RTHα patient (P1) with heterozygosity for mutant TRα1 exhibiting partial, T3-reversible, loss of function. In contrast, a more severe clinical phenotype refractory to hormone therapy was evident in another case (P2) associated with severe, virtually irreversible, dysfunction of mutant TRα1.

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization.

PubMed

Guo, Qian; Yu, Yan; Zhu, Yan Ling; Zhao, Xiu Qin; Liu, Zhi Guang; Zhang, Yuan Yuan; Li, Gui Lian; Wei, Jian Hao; Wu, Yi Mou; Wan, Kang Lin

2015-01-01

A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

A Reverse-Genetics Mutational Analysis of the Barley HvDWARF Gene Results in Identification of a Series of Alleles and Mutants with Short Stature of Various Degree and Disturbance in BR Biosynthesis Allowing a New Insight into the Process.

PubMed

Gruszka, Damian; Gorniak, Malgorzata; Glodowska, Ewelina; Wierus, Ewa; Oklestkova, Jana; Janeczko, Anna; Maluszynski, Miroslaw; Szarejko, Iwona

2016-04-22

Brassinosteroids (BRs) are plant steroid hormones, regulating a broad range of physiological processes. The largest amount of data related with BR biosynthesis has been gathered in Arabidopsis thaliana, however understanding of this process is far less elucidated in monocot crops. Up to now, only four barley genes implicated in BR biosynthesis have been identified. Two of them, HvDWARF and HvBRD, encode BR-6-oxidases catalyzing biosynthesis of castasterone, but their relation is not yet understood. In the present study, the identification of the HvDWARF genomic sequence, its mutational and functional analysis and characterization of new mutants are reported. Various types of mutations located in different positions within functional domains were identified and characterized. Analysis of their impact on phenotype of the mutants was performed. The identified homozygous mutants show reduced height of various degree and disrupted skotomorphogenesis. Mutational analysis of the HvDWARF gene with the "reverse genetics" approach allowed for its detailed functional analysis at the level of protein functional domains. The HvDWARF gene function and mutants' phenotypes were also validated by measurement of endogenous BR concentration. These results allowed a new insight into the BR biosynthesis in barley.

Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii.

PubMed

Li, Jianwei; Handler, Alfred M

2017-09-28

Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2), using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) homology-directed repair gene-editing. Ds-tra-2 ts2 mutants developed as normal fertile XX and XY adults at permissive temperatures below 20 °C, but at higher restrictive temperatures (26 to 29 °C) chromosomal XX females developed as sterile intersexuals with a predominant male phenotype, while XY males developed with normal morphology, but were sterile. The temperature-dependent function of the Ds-TRA-2 ts2 protein was also evident by the up- and down-regulation of female-specific Ds-Yolk protein 1 (Ds-Yp1) gene expression by temperature shifts during adulthood. This study confirmed the temperature-dependent function of a gene-edited mutation and provides a new method for the more general creation of conditional mutations for functional genomic analysis in insects, and other organisms. Furthermore, it provides a temperature-dependent system for creating sterile male populations useful for enhancing the efficacy of biologically-based programs, such as the sterile insect technique (SIT), to control D. suzukii and other insect pest species of agricultural and medical importance.

In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor.

PubMed

Javanbakht, H; Ptak, R G; Chow, E; Yan, J M; Russell, J D; Mankowski, M K; Hogan, P A; Hogg, J H; Vora, H; Hang, J Q; Li, Y; Su, G; Paul, A; Cammack, N; Klumpp, K; Heilek, G

2010-05-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

Low prevalence of primary HIV resistance in western Massachusetts.

PubMed

Iarikov, Dmitri E; Irizarry-Acosta, Melina; Martorell, Claudia; Hoffman, Robert P; Skiest, Daniel J

2010-01-01

Most studies of primary antiretroviral (ARV) resistance have been conducted in large metropolitan areas with reported rates of 8% to 25%. We collected data on 99 HIV-1-infected antiretroviral-naive patients from several sites in Springfield, MA, who underwent genotypic resistance assay between 2004 and 2008. Only major resistance mutations per International AIDS Society-USA (IAS-USA) drug resistance mutations list were considered. The prevalence of resistance was 5% (5 of 99). Three patients had one nonnucleoside reverse transcriptase inhibitor (NNRTI) mutation: 103N, 103N, and 190A, 1 patient had a protease inhibitor (PI) mutation: 90M; and 1 patient had 3-class resistance with NNRTI: 181C, 190A, PI: 90M, and nucleoside analogue reverse transcriptase inhibitor (NRTI): 41L, 210W. Mean time from HIV diagnosis to resistance testing was shorter in patients with resistance versus those without: 9 (range 0.3-42 months) versus 27 (range 0.1-418 months), P = .11. There was a trend to lower mean CD4 count in those with resistance, 170 versus 318 cells/mm(3), P = .06. No differences were noted in gender, age, HIV risk category, or HIV RNA level. The low prevalence of primary resistance may be explained by differences in demographic and risk factors or may reflect the time from infection to resistance testing. Our findings emphasize the importance of continued resistance surveillance.

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients

PubMed Central

2012-01-01

Abstract In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. Patients and methods A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Results Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. Conclusion The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973 PMID:23044036

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients.

PubMed

Elmi Abar, Aden; Jlizi, Asma; Darar, Houssein Youssouf; Kacem, Mohamed Ali Ben Hadj; Slim, Amine

2012-10-08

In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973.

Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

PubMed Central

Kriegshäuser, Gernot; Fabjani, Gerhild; Ziegler, Barbara; Zöchbauer-Müller, Sabine; End, Adelheid; Zeillinger, Robert

2011-01-01

This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples. PMID:22272089

Finding Relational Associations in HIV Resistance Mutation Data

NASA Astrophysics Data System (ADS)

Richter, Lothar; Augustin, Regina; Kramer, Stefan

HIV therapy optimization is a hard task due to rapidly evolving mutations leading to drug resistance. Over the past five years, several machine learning approaches have been developed for decision support, mostly to predict therapy failure from the genotypic sequence of viral proteins and additional factors. In this paper, we define a relational representation for an important part of the data, namely the sequences of a viral protein (reverse transcriptase), their mutations, and the drug resistance(s) associated with those mutations. The data were retrieved from the Los Alamos National Laboratories' (LANL) HIV databases. In contrast to existing work in this area, we do not aim directly for predictive modeling, but take one step back and apply descriptive mining methods to develop a better understanding of the correlations and associations between mutations and resistances. In our particular application, we use the Warmr algorithm to detect non-trivial patterns connecting mutations and resistances. Our findings suggest that well-known facts can be rediscovered, but also hint at the potential of discovering yet unknown associations.

High frequency, spontaneous motA mutations in Campylobacter jejuni strain 81-176.

PubMed

Mohawk, Krystle L; Poly, Frédéric; Sahl, Jason W; Rasko, David A; Guerry, Patricia

2014-01-01

Campylobacter jejuni is an important cause of bacterial diarrhea worldwide. The pathogenesis of C. jejuni is poorly understood and complicated by phase variation of multiple surface structures including lipooligosaccharide, capsule, and flagellum. When C. jejuni strain 81-176 was plated on blood agar for single colonies, the presence of translucent, non-motile colonial variants was noted among the majority of opaque, motile colonies. High-throughput genomic sequencing of two flagellated translucent and two opaque variants as well as the parent strain revealed multiple genetic changes compared to the published genome. However, the only mutated open reading frame common between the two translucent variants and absent from the opaque variants and the parent was motA, encoding a flagellar motor protein. A total of 18 spontaneous motA mutations were found that mapped to four distinct sites in the gene, with only one class of mutation present in a phase variable region. This study exemplifies the mutative/adaptive properties of C. jejuni and demonstrates additional variability in C. jejuni beyond phase variation.

Development of potent in vivo mutagenesis plasmids with broad mutational spectra

PubMed Central

Badran, Ahmed H.; Liu, David R.

2015-01-01

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms. PMID:26443021

Development of potent in vivo mutagenesis plasmids with broad mutational spectra.

PubMed

Badran, Ahmed H; Liu, David R

2015-10-07

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms.

Colorectal cancer mutational profiles correlate with defined microbial communities in the tumor microenvironment.

PubMed

Burns, Michael B; Montassier, Emmanuel; Abrahante, Juan; Priya, Sambhawa; Niccum, David E; Khoruts, Alexander; Starr, Timothy K; Knights, Dan; Blekhman, Ran

2018-06-20

Variation in the gut microbiome has been linked to colorectal cancer (CRC), as well as to host genetic variation. However, we do not know whether, in addition to baseline host genetics, somatic mutational profiles in CRC tumors interact with the surrounding tumor microbiome, and if so, whether these changes can be used to understand microbe-host interactions with potential functional biological relevance. Here, we characterized the association between CRC microbial communities and tumor mutations using microbiome profiling and whole-exome sequencing in 44 pairs of tumors and matched normal tissues. We found statistically significant associations between loss-of-function mutations in tumor genes and shifts in the abundances of specific sets of bacterial taxa, suggestive of potential functional interaction. This correlation allows us to statistically predict interactions between loss-of-function tumor mutations in cancer-related genes and pathways, including MAPK and Wnt signaling, solely based on the composition of the microbiome. In conclusion, our study shows that CRC microbiomes are correlated with tumor mutational profiles, pointing towards possible mechanisms of molecular interaction.

Step Inside NIH's Sickle Cell Branch

MedlinePlus

... attack it. This is gene transfer, which requires gene editing. Sickle cell disease is caused by a single ... can reverse that mutation in the DNA using gene editing, we could change that one mutant gene back ...

Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile, and accurate RNA structure analysis

PubMed Central

Smola, Matthew J.; Rice, Greggory M.; Busan, Steven; Siegfried, Nathan A.; Weeks, Kevin M.

2016-01-01

SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures. PMID:26426499

Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART

PubMed Central

Vazquez-Guillen, Jose Manuel; Palacios-Saucedo, Gerardo C.; Rivera-Morales, Lydia G.; Garcia-Campos, Jorge; Ortiz-Lopez, Rocio; Noguera-Julian, Marc; Paredes, Roger; Vielma-Ramirez, Herlinda J.; Ramirez, Teresa J.; Chavez-Garcia, Marcelino; Lopez-Guillen, Paulo; Briones-Lara, Evangelina; Sanchez-Sanchez, Luz M.; Vazquez-Martinez, Carlos A.; Rodriguez-Padilla, Cristina

2016-01-01

Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM’s) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM’s in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM’s during STI. PMID:26807922

DNA Mutations Mediate Microevolution between Host-Adapted Forms of the Pathogenic Fungus Cryptococcus neoformans

PubMed Central

Magditch, Denise A.; Liu, Tong-Bao; Xue, Chaoyang; Idnurm, Alexander

2012-01-01

The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a “switch” from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease. PMID:23055925

A Novel Nonnucleoside Analogue That Inhibits Human Immunodeficiency Virus Type 1 Isolates Resistant to Current Nonnucleoside Reverse Transcriptase Inhibitors▿

PubMed Central

Zhang, Zhijun; Xu, Wen; Koh, Yung-Hyo; Shim, Jae Hoon; Girardet, Jean-Luc; Yeh, Li-Tain; Hamatake, Robert K.; Hong, Zhi

2007-01-01

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance, and serious side effects can compromise the benefits of the two current drugs in this class (efavirenz and nevirapine). In this study, we report a novel and potent NNRTI, VRX-480773, that inhibits viruses from efavirenz-resistant molecular clones and most NNRTI-resistant clinical HIV-1 isolates tested. In vitro mutation selection experiments revealed that longer times were required for viruses to develop resistance to VRX-480773 than to efavirenz. RT mutations selected by VRX-480773 after 3 months of cell culture in the presence of 1 nM VRX-480773 carried the Y181C mutation, resulting in a less-than-twofold increase in resistance to the compound. A virus containing the double mutation V106I-Y181C emerged after 4 months, causing a sixfold increase in resistance. Viruses containing additional mutations of D123G, F227L, and T369I emerged when the cultures were incubated with increasing concentrations of VRX-480773. Most of the resistant viruses selected by VRX-480773 are susceptible to efavirenz. Oral administration of VRX-480773 to dogs resulted in plasma concentrations that were significantly higher than those required for the inhibition of wild-type and mutant viruses. These results warrant further clinical development of VRX-480773 for the treatment of HIV infection in both NNRTI-naive and -experienced patients. PMID:17116677

A novel nonnucleoside analogue that inhibits human immunodeficiency virus type 1 isolates resistant to current nonnucleoside reverse transcriptase inhibitors.

PubMed

Zhang, Zhijun; Xu, Wen; Koh, Yung-Hyo; Shim, Jae Hoon; Girardet, Jean-Luc; Yeh, Li-Tain; Hamatake, Robert K; Hong, Zhi

2007-02-01

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance, and serious side effects can compromise the benefits of the two current drugs in this class (efavirenz and nevirapine). In this study, we report a novel and potent NNRTI, VRX-480773, that inhibits viruses from efavirenz-resistant molecular clones and most NNRTI-resistant clinical HIV-1 isolates tested. In vitro mutation selection experiments revealed that longer times were required for viruses to develop resistance to VRX-480773 than to efavirenz. RT mutations selected by VRX-480773 after 3 months of cell culture in the presence of 1 nM VRX-480773 carried the Y181C mutation, resulting in a less-than-twofold increase in resistance to the compound. A virus containing the double mutation V106I-Y181C emerged after 4 months, causing a sixfold increase in resistance. Viruses containing additional mutations of D123G, F227L, and T369I emerged when the cultures were incubated with increasing concentrations of VRX-480773. Most of the resistant viruses selected by VRX-480773 are susceptible to efavirenz. Oral administration of VRX-480773 to dogs resulted in plasma concentrations that were significantly higher than those required for the inhibition of wild-type and mutant viruses. These results warrant further clinical development of VRX-480773 for the treatment of HIV infection in both NNRTI-naive and -experienced patients.

In vitro and in vivo evaluation of fluoroquinolone resistance associated with DNA gyrase mutations in Francisella tularensis, including in tularaemia patients with treatment failure.

PubMed

Sutera, V; Hoarau, G; Renesto, P; Caspar, Y; Maurin, M

2017-09-01

Fluoroquinolones (FQs) are highly effective for treating tularaemia, a zoonosis caused by Francisella tularensis, but failures and relapses remain common in patients with treatment delay or immunocompromised status. FQ-resistant strains of F. tularensis harboring mutations in the quinolone-resistance determining region (QRDR) of gyrA and gyrB, the genes encoding subunits A and B of DNA gyrase, have been selected in vitro. Such mutants have never been isolated from humans as this microorganism is difficult to culture. In this study, the presence of FQ-resistant mutants of F. tularensis was assessed in tularaemia patients using combined culture- and PCR-based approaches. We analyzed 42 F. tularensis strains and 82 tissue samples collected from 104 tularaemia cases, including 32 (30.7%) with FQ treatment failure or relapse. Forty F. tularensis strains and 55 clinical samples were obtained before any FQ treatment, while 2 strains and 15 tissue samples were collected after treatment. FQ resistance was evaluated by the minimum inhibitory concentration (MIC) for the bacterial strains, and by newly developed PCR-based methods targeting the gyrA and gyrB QRDRs for both the bacterial strains and the clinical samples. None of the F. tularensis strains displayed an increased MIC compared with FQ-susceptible controls. Neither gyrA nor gyrB QRDR mutation was found in bacterial strains and tissue samples tested, including those from patients with FQ treatment failure or relapse. Further phenotypic and genetic resistance traits should be explored to explain the poor clinical response to FQ treatment in such tularaemia patients. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

PubMed

Metts, J; West, J; Doares, S H; Matthysse, A G

1991-02-01

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

Hepatitis B virus genetic mutations and evolution in liver diseases

PubMed Central

Shen, Tao; Yan, Xin-Min

2014-01-01

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus of the Hepadnaviridae family and is approximately 3.2 kb in length. Owing to a lack of proofreading capacity during reverse transcription and a high replication rate, HBV exhibits as quasispecies. To detect the genetic mutations of HBV, many methods with different sensitivities and throughputs were developed. According to documentary records, HBV mutation and evolution were important vial parameters in predicting disease progression and therapeutic outcome. In this review, we separately discussed the correlation between HBV genomic mutations in four open reading frames and liver disease progression. Since some of the results were controversial from different laboratories, it remains to be seen whether functional analyses will confirm their role in modifying the course of infection. PMID:24833874

Pyrosequencing for Microbial Identification and Characterization

PubMed Central

Cummings, Patrick J.; Ahmed, Ray; Durocher, Jeffrey A.; Jessen, Adam; Vardi, Tamar; Obom, Kristina M.

2013-01-01

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns. PMID:23995536

Pyrosequencing for microbial identification and characterization.

PubMed

Cummings, Patrick J; Ahmed, Ray; Durocher, Jeffrey A; Jessen, Adam; Vardi, Tamar; Obom, Kristina M

2013-08-22

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Rapid Evolution and the Importance of Recombination to the Gastroenteric Pathogen Campylobacter jejuni

PubMed Central

Gabriel, Edith; Leatherbarrow, Andrew J.H.; Cheesbrough, John; Gee, Steven; Bolton, Eric; Fox, Andrew; Hart, C. Anthony; Diggle, Peter J.; Fearnhead, Paul

2009-01-01

Responsible for the majority of bacterial gastroenteritis in the developed world, Campylobacter jejuni is a pervasive pathogen of humans and animals, but its evolution is obscure. In this paper, we exploit contemporary genetic diversity and empirical evidence to piece together the evolutionary history of C. jejuni and quantify its evolutionary potential. Our combined population genetics–phylogenetics approach reveals a surprising picture. Campylobacter jejuni is a rapidly evolving species, subject to intense purifying selection that purges 60% of novel variation, but possessing a massive evolutionary potential. The low mutation rate is offset by a large effective population size so that a mutation at any site can occur somewhere in the population within the space of a week. Recombination has a fundamental role, generating diversity at twice the rate of de novo mutation, and facilitating gene flow between C. jejuni and its sister species Campylobacter coli. We attempt to calibrate the rate of molecular evolution in C. jejuni based solely on within-species variation. The rates we obtain are up to 1,000 times faster than conventional estimates, placing the C. jejuni–C. coli split at the time of the Neolithic revolution. We weigh the plausibility of such recent bacterial evolution against alternative explanations and discuss the evidence required to settle the issue. PMID:19008526

Mechanisms of Antibiotic Resistance

PubMed Central

Munita, Jose M.; Arias, Cesar A.

2015-01-01

Emergence of resistance among the most important bacterial pathogens is recognized as a major public health threat affecting humans worldwide. Multidrug-resistant organisms have emerged not only in the hospital environment but are now often identified in community settings, suggesting that reservoirs of antibiotic-resistant bacteria are present outside the hospital. The bacterial response to the antibiotic “attack” is the prime example of bacterial adaptation and the pinnacle of evolution. “Survival of the fittest” is a consequence of an immense genetic plasticity of bacterial pathogens that trigger specific responses that result in mutational adaptations, acquisition of genetic material or alteration of gene expression producing resistance to virtually all antibiotics currently available in clinical practice. Therefore, understanding the biochemical and genetic basis of resistance is of paramount importance to design strategies to curtail the emergence and spread of resistance and devise innovative therapeutic approaches against multidrug-resistant organisms. In this chapter, we will describe in detail the major mechanisms of antibiotic resistance encountered in clinical practice providing specific examples in relevant bacterial pathogens. PMID:27227291

Use of bacteriophage to target bacterial surface structures required for virulence: a systematic search for antibiotic alternatives.

PubMed

Orndorff, Paul E

2016-11-01

Bacteriophages (phage) that infect pathogenic bacteria often attach to surface receptors that are coincidentally required for virulence. Receptor loss or modification through mutation renders mutants both attenuated and phage resistant. Such attenuated mutants frequently have no apparent laboratory growth defects, but in the host, they fail to exhibit properties needed to produce disease such as mucosal colonization or survival within professional phagocytic cells. The connection between attenuation and phage resistance has been exploited in experimental demonstrations of phage therapy. In such experiments, phage resistant mutants that arise naturally during therapy are inconsequential because of their attenuated status. A more contemporary approach to exploiting this connection involves identifying small effector molecules, identified in high-throughput screens, that inhibit one or more of the steps needed to produce a functioning phage receptor. Since such biosynthetic steps are unique to bacteria, inhibitors can be utilized therapeutically, in lieu of antibiotics. Also, since the inhibitor is specific to a particular bacterium or group of bacteria, no off-target resistance is generated in the host's commensal bacterial population. This brief review covers examples of how mutations that confer phage resistance produce attenuation, and how this coincidental relationship can be exploited in the search for the next generation of therapeutic agents for bacterial diseases.

Limitations to estimating bacterial cross-species transmission using genetic and genomic markers: inferences from simulation modeling

PubMed Central

Benavides, Julio A; Cross, Paul C; Luikart, Gordon; Creel, Scott

2014-01-01

Cross-species transmission (CST) of bacterial pathogens has major implications for human health, livestock, and wildlife management because it determines whether control actions in one species may have subsequent effects on other potential host species. The study of bacterial transmission has benefitted from methods measuring two types of genetic variation: variable number of tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs). However, it is unclear whether these data can distinguish between different epidemiological scenarios. We used a simulation model with two host species and known transmission rates (within and between species) to evaluate the utility of these markers for inferring CST. We found that CST estimates are biased for a wide range of parameters when based on VNTRs and a most parsimonious reconstructed phylogeny. However, estimations of CST rates lower than 5% can be achieved with relatively low bias using as low as 250 SNPs. CST estimates are sensitive to several parameters, including the number of mutations accumulated since introduction, stochasticity, the genetic difference of strains introduced, and the sampling effort. Our results suggest that, even with whole-genome sequences, unbiased estimates of CST will be difficult when sampling is limited, mutation rates are low, or for pathogens that were recently introduced. PMID:25469159

Foot-and-Mouth Disease (FMD) Virus 3C Protease Mutant L127P: Implications for FMD Vaccine Development.

PubMed

Puckette, Michael; Clark, Benjamin A; Smith, Justin D; Turecek, Traci; Martel, Erica; Gabbert, Lindsay; Pisano, Melia; Hurtle, William; Pacheco, Juan M; Barrera, José; Neilan, John G; Rasmussen, Max

2017-11-15

The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria. IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production. Copyright © 2017 Puckette et al.

Chance and necessity in the genome evolution of endosymbiotic bacteria of insects.

PubMed

Sabater-Muñoz, Beatriz; Toft, Christina; Alvarez-Ponce, David; Fares, Mario A

2017-06-01

An open question in evolutionary biology is how does the selection-drift balance determine the fates of biological interactions. We searched for signatures of selection and drift in genomes of five endosymbiotic bacterial groups known to evolve under strong genetic drift. Although most genes in endosymbiotic bacteria showed evidence of relaxed purifying selection, many genes in these bacteria exhibited stronger selective constraints than their orthologs in free-living bacterial relatives. Remarkably, most of these highly constrained genes had no role in the host-symbiont interactions but were involved in either buffering the deleterious consequences of drift or other host-unrelated functions, suggesting that they have either acquired new roles or their role became more central in endosymbiotic bacteria. Experimental evolution of Escherichia coli under strong genetic drift revealed remarkable similarities in the mutational spectrum, genome reduction patterns and gene losses to endosymbiotic bacteria of insects. Interestingly, the transcriptome of the experimentally evolved lines showed a generalized deregulation of the genome that affected genes encoding proteins involved in mutational buffering, regulation and amino acid biosynthesis, patterns identical to those found in endosymbiotic bacteria. Our results indicate that drift has shaped endosymbiotic associations through a change in the functional landscape of bacterial genes and that the host had only a small role in such a shift.

Mini-review: Strategies for Variation and Evolution of Bacterial Antigens

PubMed Central

Foley, Janet

2015-01-01

Across the eubacteria, antigenic variation has emerged as a strategy to evade host immunity. However, phenotypic variation in some of these antigens also allows the bacteria to exploit variable host niches as well. The specific mechanisms are not shared-derived characters although there is considerable convergent evolution and numerous commonalities reflecting considerations of natural selection and biochemical restraints. Unlike in viruses, mechanisms of antigenic variation in most bacteria involve larger DNA movement such as gene conversion or DNA rearrangement, although some antigens vary due to point mutations or modified transcriptional regulation. The convergent evolution that promotes antigenic variation integrates various evolutionary forces: these include mutations underlying variant production; drift which could remove alleles especially early in infection or during life history phases in arthropod vectors (when the bacterial population size goes through a bottleneck); selection not only for any particular variant but also for the mechanism for the production of variants (i.e., selection for mutability); and overcoming negative selection against variant production. This review highlights the complexities of drivers of antigenic variation, in particular extending evaluation beyond the commonly cited theory of immune evasion. A deeper understanding of the diversity of purpose and mechanisms of antigenic variation in bacteria will contribute to greater insight into bacterial pathogenesis, ecology and coevolution with hosts. PMID:26288700

Limitations to estimating bacterial cross-speciestransmission using genetic and genomic markers: inferencesfrom simulation modeling

USGS Publications Warehouse

Julio Andre, Benavides; Cross, Paul C.; Luikart, Gordon; Scott, Creel

2014-01-01

Cross-species transmission (CST) of bacterial pathogens has major implications for human health, livestock, and wildlife management because it determines whether control actions in one species may have subsequent effects on other potential host species. The study of bacterial transmission has benefitted from methods measuring two types of genetic variation: variable number of tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs). However, it is unclear whether these data can distinguish between different epidemiological scenarios. We used a simulation model with two host species and known transmission rates (within and between species) to evaluate the utility of these markers for inferring CST. We found that CST estimates are biased for a wide range of parameters when based on VNTRs and a most parsimonious reconstructed phylogeny. However, estimations of CST rates lower than 5% can be achieved with relatively low bias using as low as 250 SNPs. CST estimates are sensitive to several parameters, including the number of mutations accumulated since introduction, stochasticity, the genetic difference of strains introduced, and the sampling effort. Our results suggest that, even with whole-genome sequences, unbiased estimates of CST will be difficult when sampling is limited, mutation rates are low, or for pathogens that were recently introduced.

Crossroads between Bacterial and Mammalian Glycosyltransferases

PubMed Central

Brockhausen, Inka

2014-01-01

Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen–Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures. PMID:25368613

Development of positive control materials for DNA-based detection of cystic fibrosis: Cloning and sequencing of 31 mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iovannisci, D.; Brown, C.; Winn-Deen, E.

1994-09-01

The cloning and sequencing of the gene associated with cystic fibrosis (CF) now provides the opportunity for earlier detection and carrier screening through DNA-based detection schemes. To date, over 300 mutations have been reported to the CF Consortium; however, only 30 mutations have been observed frequently enough world-wide to warrant routine screening. Many of these mutations are not available as cloned material or as established tissue culture cell lines to aid in the development of DNA-based detection assays. We have therefore cloned the 30 most frequently reported mutations, plus the mutation R347H due to its association with male infertility (31more » mutations, total). Two approaches were employed: direct PCR amplification, where mutations were available from patient sources, and site-directed PCR mutagenesis of normal genomic DNA to generate the remaining mutations. After amplification, products were cloned into a sequencing vector, bacterial transformants were screened by a novel method (PCR/oligonucleotide litigation assay/sequence-coded separation), and plamid DNA sequences determined by automated fluorescent methods on the Applied Biosystems 373A. Mixing of the clones allows the construction of artificial genotypes useful as positive control material for assay validation. A second round of mutagenesis, resulting in the construction of plasmids bearing multiple mutations, will be evaluated for their utility as reagent control materials in kit development.« less

Simulation of gene evolution under directional mutational pressure

NASA Astrophysics Data System (ADS)

Dudkiewicz, Małgorzata; Mackiewicz, Paweł; Kowalczuk, Maria; Mackiewicz, Dorota; Nowicka, Aleksandra; Polak, Natalia; Smolarczyk, Kamila; Banaszak, Joanna; R. Dudek, Mirosław; Cebrat, Stanisław

2004-05-01

The two main mechanisms generating the genetic diversity, mutation and recombination, have random character but they are biased which has an effect on the generation of asymmetry in the bacterial chromosome structure and in the protein coding sequences. Thus, like in a case of two chiral molecules-the two possible orientations of a gene in relation to the topology of a chromosome are not equivalent. Assuming that the sequence of a gene may oscillate only between certain limits of its structural composition means that the gene could be forced out of these limits by the directional mutation pressure, in the course of evolution. The probability of the event depends on the time the gene stays under the same mutation pressure. Inversion of the gene changes the directional mutational pressure to the reciprocal one and hence it changes the distance of the gene to its lower and upper bound of the structural tolerance. Using Monte Carlo methods we were able to simulate the evolution of genes under experimentally found mutational pressure, assuming simple mechanisms of selection. We found that the mutation and recombination should work in accordance to lower their negative effects on the function of the products of coding sequences.

HlyU Is a Positive Regulator of Hemolysin Expression in Vibrio anguillarum ▿

PubMed Central

Li, Ling; Mou, Xiangyu; Nelson, David R.

2011-01-01

The two hemolysin gene clusters previously identified in Vibrio anguillarum, the vah1 cluster and the rtxACHBDE cluster, are responsible for the hemolytic and cytotoxic activities of V. anguillarum in fish. In this study, we used degenerate PCR to identify a positive hemolysin regulatory gene, hlyU, from the unsequenced V. anguillarum genome. The hlyU gene of V. anguillarum encodes a 92-amino-acid protein and is highly homologous to other bacterial HlyU proteins. An hlyU mutant was constructed, which exhibited an ∼5-fold decrease in hemolytic activity on sheep blood agar with no statistically significant decrease in cytotoxicity of the wild-type strain. Complementation of the hlyU mutation restored both hemolytic activity and cytotoxic activity. Both semiquantitative reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR) were used to examine expression of the hemolysin genes under exponential and stationary-phase conditions in wild-type, hlyU mutant, and hlyU complemented strains. Compared to the wild-type strain, expression of rtx genes decreased in the hlyU mutant, while expression of vah1 and plp was not affected in the hlyU mutant. Complementation of the hlyU mutation restored expression of the rtx genes and increased vah1 and plp expression to levels higher than those in the wild type. The transcriptional start sites in both the vah1-plp and rtxH-rtxB genes' intergenic regions were determined using 5′ random amplification of cDNA ends (5′-RACE), and the binding sites for purified HlyU were discovered using DNA gel mobility shift experiments and DNase protection assays. PMID:21764937

Folding and Stabilization of Native-Sequence-Reversed Proteins

PubMed Central

Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

2016-01-01

Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

Folding and Stabilization of Native-Sequence-Reversed Proteins

NASA Astrophysics Data System (ADS)

Zhang, Yuanzhao; Weber, Jeffrey K.; Zhou, Ruhong

2016-04-01

Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.

Control of biofouling by xanthine oxidase on seawater reverse osmosis membranes from a desalination plant: enzyme production and screening of bacterial isolates from the full-scale plant.

PubMed

Nagaraj, V; Skillman, L; Li, D; Xie, Z; Ho, G

2017-07-01

Control of biofouling on seawater reverse osmosis (SWRO) membranes is a major challenge as treatments can be expensive, damage the membrane material and often biocides do not remove the polymers in which bacteria are embedded. Biological control has been largely ignored for biofouling control. The objective of this study was to demonstrate the effectiveness of xanthine oxidase enzyme against complex fouling communities and then identify naturally occurring bacterial strains that produce the free radical generating enzyme. Initially, 64 bacterial strains were isolated from different locations of the Perth Seawater Desalination Plant. In our preceding study, 25/64 isolates were selected from the culture collection as models for biofouling studies, based on their prevalence in comparison to the genomic bacterial community. In this study, screening of these model strains was performed using a nitroblue tetrazolium assay in the presence of hypoxanthine as substrate. Enzyme activity was measured by absorbance. Nine of 25 strains tested positive for xanthine oxidase production, of which Exiguobacterium from sand filters and Microbacterium from RO membranes exhibited significant levels of enzyme production. Other genera that produced xanthine oxidase were Marinomonas, Pseudomonas, Bacillus, Pseudoalteromonas and Staphylococcus. Strain variations were observed between members of the genera Microbacterium and Bacillus. Xanthine oxidase, an oxidoreductase enzyme that generates reactive oxygen species, is endogenously produced by many bacterial species. In this study, production of the enzyme by bacterial isolates from a full-scale desalination plant was investigated for potential use as biological control of membrane fouling in seawater desalination. We have previously demonstrated that free radicals generated by a commercially available xanthine oxidase in the presence of a hypoxanthine substrate, effectively dispersed biofilm polysaccharides on industrially fouled membranes. Bacterial xanthine oxidase production in the presence of hypoxanthine may prove to be a cost effective, in situ method for alleviation of fouling. © 2017 The Society for Applied Microbiology.

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan.

PubMed

Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph (+)) CML patients in Pakistan. The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region - Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. It is concluded that the co-existence of Ph (+)CML and JAK2 V617F mutation is possible.

Analysis of the Microbial Diversity in the Fecal Material of Giraffes.

PubMed

Schmidt, Jessica M; Henken, Susan; Dowd, Scot E; McLaughlin, Richard William

2018-03-01

Using bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing, the microbiota of the fecal material of seven giraffes living in captivity at the Jacksonville Zoo and Gardens, Jacksonville, FL was investigated. In all samples, the most predominant bacterial phylum was the Firmicutes followed by Bacteroidetes. The most predominant fungi were members of the phylum Ascomycota followed by Neocallimastigomycota in five of seven samples. The reverse was true in the other two samples.

Kinetics of bacterial potentiometric titrations: the effect of equilibration time on buffering capacity of Pantoea agglomerans suspensions.

PubMed

Kapetas, Leon; Ngwenya, Bryne T; Macdonald, Alan M; Elphick, Stephen C

2011-07-15

Several recent studies have made use of continuous acid-base titration data to describe the surface chemistry of bacterial cells as a basis for accurately modelling metal adsorption to bacteria and other biomaterials of potential industrial importance. These studies do not share a common protocol; rather they titrate in different pH ranges and they use different stability criteria to define equilibration time during titration. In the present study we investigate the kinetics of bacterial titrations and test the effect they have on the derivation of functional group concentrations and acidity constants. We titrated suspensions of Pantoea agglomerans by varying the equilibration time between successive titrant additions until stability of 0.1 or 0.001 mV s(-1) was attained. We show that under longer equilibration times, titration results are less reproducible and suspensions exhibit marginally higher buffering. Fluorescence images suggest that cell lysis is not responsible for these effects. Rather, high DOC values and titration reversibility hysterisis after long equilibration times suggest that variability in buffering is due to the presence of bacterial exudates, as demonstrated by titrating supernatants separated from suspensions of different equilibration times. It is recommended that an optimal equilibration time is always determined with variable stability control and preliminary reversibility titration experiments. Copyright © 2011 Elsevier Inc. All rights reserved.

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

PubMed

Paar, Jack; Doolittle, Mark M; Varma, Manju; Siefring, Shawn; Oshima, Kevin; Haugland, Richard A

2015-05-01

A method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for detecting interferences in RNA recovery and analysis, was developed for the direct, culture-independent detection of genetic markers from FRNA coliphage genogroups I, II & IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods. Strong associations were observed between the occurrence of the putatively human related FRNA genogroup II marker and the densities of the bacterial markers in the stormwater drain and outfall samples. However fewer samples were positive for FRNA coliphage compared to either the general bacterial fecal indicator or the human-related bacterial fecal indicator markers particularly for ambient water samples. Together, these methods show promise as complementary tools for the identification of contaminated storm water drainage systems as well as the determination of human and non-human sources of contamination. Published by Elsevier B.V.

Molecular Characterization of the Bacterial Communities in the Different Compartments of a Full-Scale Reverse-Osmosis Water Purification Plant ▿

PubMed Central

Bereschenko, L. A.; Heilig, G. H. J.; Nederlof, M. M.; van Loosdrecht, M. C. M.; Stams, A. J. M.; Euverink, G. J. W.

2008-01-01

The origin, structure, and composition of biofilms in various compartments of an industrial full-scale reverse-osmosis (RO) membrane water purification plant were analyzed by molecular biological methods. Samples were taken when the RO installation suffered from a substantial pressure drop and decreased production. The bacterial community of the RO membrane biofilm was clearly different from the bacterial community present at other locations in the RO plant, indicating the development of a specialized bacterial community on the RO membranes. The typical freshwater phylotypes in the RO membrane biofilm (i.e., Proteobacteria, Cytophaga-Flexibacter-Bacteroides group, and Firmicutes) were also present in the water sample fed to the plant, suggesting a feed water origin. However, the relative abundances of the different species in the mature biofilm were different from those in the feed water, indicating that the biofilm was actively formed on the RO membrane sheets and was not the result of a concentration of bacteria present in the feed water. The majority of the microorganisms (59% of the total number of clones) in the biofilm were related to the class Proteobacteria, with a dominance of Sphingomonas spp. (27% of all clones). Members of the genus Sphingomonas seem to be responsible for the biofouling of the membranes in the RO installation. PMID:18621875

Interference with the Mannose Binding and Epithelial Cell Adherence of Escherichia coli by Sublethal Concentrations of Streptomycin

PubMed Central

Eisenstein, Barry I.; Ofek, Itzhak; Beachey, Edwin H.

1979-01-01

When Escherichia coli was grown in sublethal concentrations of streptomycin, mannose binding activity and epithelial cell adherence of the E. coli cultures at stationary phase were significantly reduced in the drug-grown organisms. In a strain whose minimal inhibitory concentrations was 30 μg/ml, the percentage of reduction in mannose binding activity was dose related over a range of concentrations between 0.5 and 10 μg/ml streptomycin. Concomitant with the drug-induced suppression of mannose binding activity, antigenic and ultrastructural alterations on the surface of the drug-grown organisms were observed by agglutination tests and electron microscopy, respectively. The streptomycin effect was reversible, required actively growing organisms, and was most apparent in the early log-phase of growth. High doses of antibiotic were ineffective when added to cultures which had acquired mannose binding activity. An isogenic derivative with high-level resistance to streptomycin was obtained as a single-step mutation from the test E. coli strain. Whereas the isogenic mutant possessed mannose binding activity and adhering ability similar to the parent strain, it was resistant to the streptomycin-induced suppression of the two activities at enormous concentrations (up to 10,000 μg/ml) of streptomycin. Taken together the results suggest that the suppression of epithelial cell adherence and mannose binding activity of E. coli grown in sublethal concentrations of streptomycin is a result of classic mechanisms of drug action upon the bacterial ribosome. The results support the possibility that antibiotics may act through mechanisms other than inhibition of growth and bacterial killing to eradicate bacteria from mucosal surfaces. Images PMID:376556

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates

PubMed Central

Govindarajan, Dhanasekaran; Guan, Liming; Meschino, Steven; Fridman, Arthur; Bagchi, Ansu; Pak, Irene; ter Meulen, Jan; Casimiro, Danilo R.; Bett, Andrew J.

2016-01-01

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses. PMID:27008550

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

PubMed

Govindarajan, Dhanasekaran; Guan, Liming; Meschino, Steven; Fridman, Arthur; Bagchi, Ansu; Pak, Irene; ter Meulen, Jan; Casimiro, Danilo R; Bett, Andrew J

2016-01-01

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

The tyrosine-sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner.

PubMed

Mosher, Stephen; Seybold, Heike; Rodriguez, Patricia; Stahl, Mark; Davies, Kelli A; Dayaratne, Sajeewani; Morillo, Santiago A; Wierzba, Michael; Favery, Bruno; Keller, Harald; Tax, Frans E; Kemmerling, Birgit

2013-02-01

The tyrosine-sulfated peptides PSKα and PSY1 bind to specific leucine-rich repeat surface receptor kinases and control cell proliferation in plants. In a reverse genetic screen, we identified the phytosulfokine (PSK) receptor PSKR1 as an important component of plant defense. Multiple independent loss-of-function mutants in PSKR1 are more resistant to biotrophic bacteria, show enhanced pathogen-associated molecular pattern responses and less lesion formation after infection with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. By contrast, pskr1 mutants are more susceptible to necrotrophic fungal infection with Alternaria brassicicola, show more lesion formation and fungal growth which is not observed on wild-type plants. The antagonistic effect on biotrophic and necrotrophic pathogen resistance is reflected by enhanced salicylate and reduced jasmonate responses in the mutants, suggesting that PSKR1 suppresses salicylate-dependent defense responses. Detailed analysis of single and multiple mutations in the three paralogous genes PSKR1, -2 and PSY1-receptor (PSY1R) determined that PSKR1 and PSY1R, but not PSKR2, have a partially redundant effect on plant immunity. In animals and plants, peptide sulfation is catalyzed by a tyrosylprotein sulfotransferase (TPST). Mutants lacking TPST show increased resistance to bacterial infection and increased susceptibility to fungal infection, mimicking the triple receptor mutant phenotypes. Feeding experiments with PSKα in tpst-1 mutants partially restore the defense-related phenotypes, indicating that perception of the PSKα peptide has a direct effect on plant defense. These results suggest that the PSKR subfamily integrates growth-promoting and defense signals mediated by sulfated peptides and modulates cellular plasticity to allow flexible adjustment to environmental changes. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

Generation of gene-corrected iPSC line from Parkinson's disease patient iPSC line with alpha-SNCA A53T mutation.

PubMed

Lee, Seo-Young; Jeong, SangKyun; Kim, Janghwan; Chung, Sun-Ku

2018-06-09

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. PD can result from a mutation of alpha-synuclein (α-SNCA), such as α-SNCA A53T. Using episomal vectors, induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts with the α-SNCA A53T mutation. A huge bacterial artificial chromosome (BAC) harboring the normal α-SNCA gene successfully corrected the α-SNCA A53T-mutant iPSCs. Melting curve analysis for allelic composition indicated that the BAC DNA was precisely targeted to the α-SNCA A53T mutation allele, without random integration. The corrected PD-iPSCs displayed the normal karyotype and pluripotency, with the capability to differentiate to any cell type. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

PubMed

Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

2016-01-29

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

The Nature and Evolution of Genomic Diversity in the Mycobacterium tuberculosis Complex.

PubMed

Brites, Daniela; Gagneux, Sebastien

2017-01-01

The Mycobacterium tuberculosis Complex (MTBC) consists of a clonal group of several mycobacterial lineages pathogenic to a range of different mammalian hosts. In this chapter, we discuss the origins and the evolutionary forces shaping the genomic diversity of the human-adapted MTBC. Advances in whole-genome sequencing have brought invaluable insights into the macro-evolution of the MTBC, and the biogeographical distribution of the different MTBC lineages, the phylogenetic relationships between these lineages. Moreover, micro-evolutionary processes start to be better understood, including those influencing bacterial mutation rates and those governing the fate of new mutations emerging within patients during treatment. Current genomic and epidemiological evidence reflect the fact that, through ecological specialization, the MTBC affecting humans became an obligate and extremely well-adapted human pathogen. Identifying the adaptive traits of human-adapted MTBC and unraveling the bacterial loci that interact with human genomic variation might help identify new targets for developing better vaccines and designing more effective treatments.

Adaptability of non-genetic diversity in bacterial chemotaxis

PubMed Central

Frankel, Nicholas W; Pontius, William; Dufour, Yann S; Long, Junjiajia; Hernandez-Nunez, Luis; Emonet, Thierry

2014-01-01

Bacterial chemotaxis systems are as diverse as the environments that bacteria inhabit, but how much environmental variation can cells tolerate with a single system? Diversification of a single chemotaxis system could serve as an alternative, or even evolutionary stepping-stone, to switching between multiple systems. We hypothesized that mutations in gene regulation could lead to heritable control of chemotactic diversity. By simulating foraging and colonization of E. coli using a single-cell chemotaxis model, we found that different environments selected for different behaviors. The resulting trade-offs show that populations facing diverse environments would ideally diversify behaviors when time for navigation is limited. We show that advantageous diversity can arise from changes in the distribution of protein levels among individuals, which could occur through mutations in gene regulation. We propose experiments to test our prediction that chemotactic diversity in a clonal population could be a selectable trait that enables adaptation to environmental variability. DOI: http://dx.doi.org/10.7554/eLife.03526.001 PMID:25279698

Structural, Functional, and Genetic Analysis of Sorangicin Inhibition of Bacterial RNA Polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campbell,E.; Pavlova, O.; Zenkin, N.

2005-01-01

A combined structural, functional, and genetic approach was used to investigate inhibition of bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP {beta} subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2-3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive tomore » mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.« less

Cystic fibrosis–adapted Pseudomonas aeruginosa quorum sensing lasR mutants cause hyperinflammatory responses

PubMed Central

LaFayette, Shantelle L.; Houle, Daniel; Beaudoin, Trevor; Wojewodka, Gabriella; Radzioch, Danuta; Hoffman, Lucas R.; Burns, Jane L.; Dandekar, Ajai A.; Smalley, Nicole E.; Chandler, Josephine R.; Zlosnik, James E.; Speert, David P.; Bernier, Joanie; Matouk, Elias; Brochiero, Emmanuelle; Rousseau, Simon; Nguyen, Dao

2015-01-01

Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease–dependent cytokine degradation. In subacute pulmonary infections, lasR mutant–infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients. PMID:26457326

Phenotypic convergence in bacterial adaptive evolution to ethanol stress.

PubMed

Horinouchi, Takaaki; Suzuki, Shingo; Hirasawa, Takashi; Ono, Naoaki; Yomo, Tetsuya; Shimizu, Hiroshi; Furusawa, Chikara

2015-09-03

Bacterial cells have a remarkable ability to adapt to environmental changes, a phenomenon known as adaptive evolution. During adaptive evolution, phenotype and genotype dynamically changes; however, the relationship between these changes and associated constraints is yet to be fully elucidated. In this study, we analyzed phenotypic and genotypic changes in Escherichia coli cells during adaptive evolution to ethanol stress. Phenotypic changes were quantified by transcriptome and metabolome analyses and were similar among independently evolved ethanol tolerant populations, which indicate the existence of evolutionary constraints in the dynamics of adaptive evolution. Furthermore, the contribution of identified mutations in one of the tolerant strains was evaluated using site-directed mutagenesis. The result demonstrated that the introduction of all identified mutations cannot fully explain the observed tolerance in the tolerant strain. The results demonstrated that the convergence of adaptive phenotypic changes and diverse genotypic changes, which suggested that the phenotype-genotype mapping is complex. The integration of transcriptome and genome data provides a quantitative understanding of evolutionary constraints.

Minimizing potential resistance: the molecular view--a comment on Courvalin and Trieu-Cuot.

PubMed

Hooper, D C

2001-09-15

The complexity of bacterial resistance to antimicrobial agents is driven by the interplay of many mechanistic and epidemiologic factors. Mechanistically, resistance by target alteration, reduced permeation, and drug inactivation can occur by both chromosomal mutation and acquisition of new genetic elements. Epidemiologically, exposure to antimicrobial agents provides a growth or persistence advantage for any existing resistant bacteria, generally irrespective of the mechanism. When a single chromosomal mutation is sufficient to cause resistance, any such exposure provides a risk of selection, as long as a sufficiently large bacterial population is exposed. Transmission of resistant bacteria can also amplify resistance of any type, but it is particularly important for complex resistance mechanisms that have evolved over time and for mechanisms that depend on infrequent biological events in nature. Because true biological barriers to the development of resistance are likely to be elusive, multiple approaches that address both the use of antimicrobial agents and transmission are necessary to slow the advance of resistance.

Floating and Tether-Coupled Adhesion of Bacteria to Hydrophobic and Hydrophilic Surfaces

PubMed Central

2018-01-01

Models for bacterial adhesion to substratum surfaces all include uncertainty with respect to the (ir)reversibility of adhesion. In a model, based on vibrations exhibited by adhering bacteria parallel to a surface, adhesion was described as a result of reversible binding of multiple bacterial tethers that detach from and successively reattach to a surface, eventually making bacterial adhesion irreversible. Here, we use total internal reflection microscopy to determine whether adhering bacteria also exhibit variations over time in their perpendicular distance above surfaces. Streptococci with fibrillar surface tethers showed perpendicular vibrations with amplitudes of around 5 nm, regardless of surface hydrophobicity. Adhering, nonfibrillated streptococci vibrated with amplitudes around 20 nm above a hydrophobic surface. Amplitudes did not depend on ionic strength for either strain. Calculations of bacterial energies from their distances above the surfaces using the Boltzman equation showed that bacteria with fibrillar tethers vibrated as a harmonic oscillator. The energy of bacteria without fibrillar tethers varied with distance in a comparable fashion as the DLVO (Derjaguin, Landau, Verwey, and Overbeek)-interaction energy. Distance variations above the surface over time of bacteria with fibrillar tethers are suggested to be governed by the harmonic oscillations, allowed by elasticity of the tethers, piercing through the potential energy barrier. Bacteria without fibrillar tethers “float” above a surface in the secondary energy minimum, with their perpendicular displacement restricted by their thermal energy and the width of the secondary minimum. The distinction between “tether-coupled” and “floating” adhesion is new, and may have implications for bacterial detachment strategies. PMID:29649869

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

PubMed

Sorin, Masha; Yung, Eric; Wu, Xuhong; Kalpana, Ganjam V

2006-08-31

INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and reverse transcription complexes may be important for early events of HIV-1 replication.

[Arms racing between human beings and pathogens: NDM-1 and superbugs].

PubMed

Sun, Mingwei; Zheng, Beiwen; Gao, George F; Zhu, Baoli

2010-11-01

Throughout human history, pandemic bacterial diseases such as the plague and tuberculosis have posed an enormous threat to human beings. The discovery of antibiotics has provided us with powerful arsenal for the defense against bacterial infections. However, bacteria are acquiring more and more resistance genes to shield off antibiotics through mutation and horizontal gene transfer. Therefore, novel antibiotics must be produced and the arms race between bacterial pathogens and antibiotics is becoming increasingly intense. Recently, researchers have found that plasmids carrying a new metallo-beta-lactamase gene, blaNDM-1, and many other antibiotics resistance genes can easily spread through bacterial populations and confer recipient stains resistance to nearly all of the current antibiotics. It is a threat to the human health and a great challenge for our medical science, which we are facing. We need to find new ways to fight and win this arms racing.

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation

PubMed Central

2005-01-01

In goats, the PIS (polled intersex syndrome) mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4–5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation. PMID:15601595

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation.

PubMed

Pailhoux, Eric; Vigier, Bernard; Schibler, Laurent; Cribiu, Edmond P; Cotinot, Corinne; Vaiman, Daniel

2005-01-01

In goats, the PIS (polled intersex syndrome) mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4-5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation.

Single Active Site Mutation Causes Serious Resistance of HIV Reverse Transcriptase to Lamivudine: Insight from Multiple Molecular Dynamics Simulations.

PubMed

Moonsamy, Suri; Bhakat, Soumendranath; Walker, Ross C; Soliman, Mahmoud E S

2016-03-01

Molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA), and residue interaction network analysis were employed in order to investigate the molecular mechanism of M184I single mutation which played pivotal role in making the HIV-1 reverse transcriptase (RT) totally resistant to lamivudine. Results showed that single mutations at residue 184 of RT caused (1) distortion of the orientation of lamivudine in the active site due to the steric conflict between the oxathiolane ring of lamivudine and the side chain of beta-branched amino acids Ile at position 184 which, in turn, perturbs inhibitor binding, (2) decrease in the binding affinity by (~8 kcal/mol) when compared to the wild-type, (3) variation in the overall enzyme motion as evident from the PCA for both systems, and (4) distortion of the hydrogen bonding network and atomic interactions with the inhibitor. The comprehensive analysis presented in this report can provide useful information for understanding the drug resistance mechanism against lamivudine. The results can also provide some potential clues for further design of novel inhibitors that are less susceptible to drug resistance.

Forward and reverse mutagenesis in C. elegans

PubMed Central

Kutscher, Lena M.; Shaham, Shai

2014-01-01

Mutagenesis drives natural selection. In the lab, mutations allow gene function to be deciphered. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques. Here we provide an overview of historical and contemporary methods for mutagenesis in C. elegans, and discuss principles and strategies for forward (genome-wide mutagenesis) and reverse (target-selected and gene-specific mutagenesis) genetic studies in this animal. PMID:24449699

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal.

PubMed

Salas-Cortés, L; Jaubert, F; Nihoul-Feketé, C; Brauner, R; Rosemblatt, M; Fellous, M

2000-01-01

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein. Copyright 2001 S. Karger AG, Basel

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Attempt to rescue sex-reversal by transgenic expression of the PISRT1 gene in XX PIS-/- goats.

PubMed

Boulanger, L; Kocer, A; Daniel, N; Pannetier, M; Chesné, P; Heyman, Y; Renault, L; Mandon-Pépin, B; Chavatte-Palmer, P; Vignon, X; Vilotte, J-L; Cotinot, C; Renard, J-P; Pailhoux, E

2008-01-01

The Polled Intersex Syndrome (PIS mutation) in goats leads to an absence of horn and to an early sex-reversal of the XX gonads. This mutation is a deletion of an 11.7-kb DNA fragment showing a tissue-specific regulatory activity. Indeed, in XX PIS(-/-) gonads the deletion of PIS leads to the transcriptional extinction of at least 3 neighboring genes, FOXL2, PFOXic and PISRT1. Among them, only FOXL2 is a 'classical' gene, encoding a highly conserved transcription factor. On the other hand, knock-out of Foxl2 in mice results in an early blocking of follicle formation without sex-reversal. This phenotype discrepancy leads to two hypotheses, either FOXL2 is responsible for XX sex-reversal in goat assuming distinct functions of its protein during ovarian differentiation in different mammals, or other PIS-regulated genes are involved. To assess the second possibility, PISRT1 expression was constitutively restored in XX PIS(-/-) gonads. Six transgenic fetuses were obtained by nuclear transfer and studied at 2 developmental stages, 41 and 46 days post-reconstruction. The gonads of these fetuses appear phenotypically identical to those of cloned non-transgenic controls. Conclusively, this result argues for FOXL2 being responsible for the PIS gonad-associated phenotype. Its invalidation in goat will help to better understand this complex syndrome. Copyright 2008 S. Karger AG, Basel.

Exploring the reversal of enantioselectivity on a zinc-dependent alcohol dehydrogenase† †Electronic supplementary information (ESI) available. See DOI: 10.1039/C7OB00482F Click here for additional data file.

PubMed Central

Maria-Solano, Miguel A.; Romero-Rivera, Adrian

2017-01-01

Alcohol Dehydrogenase (ADH) enzymes catalyse the reversible reduction of prochiral ketones to the corresponding alcohols. These enzymes present two differently shaped active site pockets, which dictate their substrate scope and selectivity. In this study, we computationally evaluate the effect of two commonly reported active site mutations (I86A, and W110T) on a secondary alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) through Molecular Dynamics simulations. Our results indicate that the introduced mutations induce dramatic changes in the shape of the active site, but most importantly they impact the substrate–enzyme interactions. We demonstrate that the combination of Molecular Dynamics simulations with the tools POVME and NCIplot corresponds to a powerful strategy for rationalising and engineering the stereoselectivity of ADH variants. PMID:28436515

[Safety Evaluation of Rare Sugar Syrup: Single-dose Oral Toxicity in Rats, Reverse Mutation Assay, Chromosome Aberration Assay, and Acute Non-Effect Level for Diarrhea of a Single Dose in Humans].

PubMed

Yamada, Takako; Iida, Tetsuo; Takamine, Satoshi; Hayashi, Noriko; Okuma, Kazuhiro

2015-01-01

The safety of rare sugar syrup obtained from high-fructose corn syrup under slightly alkaline conditions was studied. Mutagenicity of rare sugar syrup was assessed by a reverse mutation assay using Salmonella typhimurium and Escherichia coli, and an in vitro chromosomal aberration assay using Chinese hamster lung cell line (CHL/IU). No mutagenicity of rare sugar syrup was detected under these experimental conditions. Oral administration of single dose (15,000 mg/kg) of rare sugar syrup to rats caused no abnormalities, suggesting no adverse effect of rare sugar syrup. In humans, the acute non-effect level of rare sugar syrup for causing diarrhea was estimated as 0.9 g/kg body weight as dry solid base in both males and females.

Reversion of mtDNA depletion in a patient with TK2 deficiency.

PubMed

Vilà, M R; Segovia-Silvestre, T; Gámez, J; Marina, A; Naini, A B; Meseguer, A; Lombès, A; Bonilla, E; DiMauro, S; Hirano, M; Andreu, A L

2003-04-08

Mutations in the thymidine kinase 2 (TK2) gene cause a myopathic form of the mitochondrial DNA depletion syndrome (MDS). Here, the authors report the unusual clinical, biochemical, and molecular findings in a 14-year-old patient in whom pathogenic mutations were identified in the TK2 gene. This report extends the phenotypic expression of primary TK2 deficiency and suggests that factors other than TK2 may modify expression of the clinical phenotype in patients with MDS syndrome.

Intragenic Mapping of Chemically Induced ad-7 Mutants of Schizosaccharomyces pombe

PubMed Central

Loprieno, Nicola

1967-01-01

Thirty adenine-requiring ad-7 mutants of Schizosaccharomyces pombe, induced by ethylmethanesulfonate, methyl-methanesulfonate, and hydroxylamine and exhibiting low spontaneous reversion frequencies, were located by intragenic recombination analysis. Their identification as ad-7 mutants was assessed in relation to two previously mapped ad-7 mutants. Each mutant was found to occupy a distinct mutational site; the smallest recombination fraction observed between the two closest mutational sites was of the order of 0.5 × 10−6. PMID:6051345

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice.

PubMed

Safdar, Adeel; Khrapko, Konstantin; Flynn, James M; Saleem, Ayesha; De Lisio, Michael; Johnston, Adam P W; Kratysberg, Yevgenya; Samjoo, Imtiaz A; Kitaoka, Yu; Ogborn, Daniel I; Little, Jonathan P; Raha, Sandeep; Parise, Gianni; Akhtar, Mahmood; Hettinga, Bart P; Rowe, Glenn C; Arany, Zoltan; Prolla, Tomas A; Tarnopolsky, Mark A

2016-01-01

Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present mitochondrially targeted p53 as a novel therapeutic modality for diseases of mitochondrial etiology.

New mutations affecting induced mutagenesis in yeast.

PubMed

Lawrence, C W; Krauss, B R; Christensen, R B

1985-01-01

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

The effect of transmitted HIV-1 drug resistance on pre-therapy viral load.

PubMed

Harrison, Linda; Castro, Hannah; Cane, Patricia; Pillay, Deenan; Booth, Clare; Phillips, Andrew; Geretti, Anna Maria; Dunn, David

2010-07-31

Reduced replication capacity of viruses expressing drug resistant mutations implies that patients with transmitted drug resistance (TDR) could have lower HIV RNA viral load than those infected with wild-type virus. We performed analysis using data from the UK HIV Drug Resistance Database and the UK CHIC study. Eligible patients had a resistance test performed between 1997 and 2007 while naive to antiretroviral therapy, were 16 years or older, and had a viral load and CD4 cell count measurement within 6 months of this test. Models were adjusted for CD4 cell count, viral subtype, ethnicity, risk group, sex, age, calendar year, clinical centre, and viral load assay. Of a total of 7994 patients included, 709 (9%) had TDR: 604 (85%) had resistance to one drug class only [350 nucleos(t)ide reverse transcriptase inhibitors (NRTIs), 164 non-nucleos(t)ide reverse transcriptase inhibitors (NNRTIs), 90 protease inhibitors (PIs)], 77 (11%) to two classes (42 NRTIs/NNRTIs, 31 NRTIs/PIs, 4 NNRTIs/PIs), and 28 (4%) had resistance to all three classes. The overall mean (SD) viral load at the time of resistance testing was 4.60 (0.82) log(10) copies/ml, and did not differ by class of TDR. However, patients harbouring M184V/I (n = 61) had a significantly lower viral load [adjusted mean difference -0.33 log10 copies/ml (95% CI -0.54 to -0.11), 53% lower (95% CI 22 to 71%), P = 0.002] compared to wild-type virus. Our study provides clear evidence of an in-vivo fitness cost associated with the M184V/I mutation independent of drug effects which select for this mutation. This was not observed for any other mutation, but true effects may have been obscured by reversion of initially resistant viruses to wild-type.

Spore-forming organisms in platelet concentrates: a challenge in transfusion bacterial safety.

PubMed

Störmer, M; Vollmer, T; Kleesiek, K; Dreier, J

2008-12-01

Bacterial detection and pathogen reduction are widely used methods of minimizing the risk of transfusion-transmitted bacterial infection. But, bacterial spores are highly resistant to chemical and physical agents. In this study, we assessed the bacterial proliferation of spore-forming organisms seeded into platelet concentrates (PCs) to demonstrate that spores can enter the vegetative state in PCs during storage. In the in vitro study, PCs were inoculated with 1-10 spores mL(-1)of Bacillus cereus (n = 1), Bacillus subtilis (n = 2) and Clostridium sporogenes (n = 2). Sampling was performed during 6-day aerobic storage at 22 degrees C. The presence of bacteria was assessed by plating culture, automated culture and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Spores of the C. sporogenes do not enter the vegetative phase under PC storage conditions, whereas B. subtilis and B. cereus showed growth in the PC and could be detected using RT-PCR and automated culture. Depending on the species and inoculums, bacterial spores may enter the vegetative phase during PC storage and can be detected by bacterial detection methods.

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input-mutation output relationships.

PubMed

Maharjan, Ram P; Ferenci, Thomas

2017-06-01

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input-mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input-output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection.

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input–mutation output relationships

PubMed Central

Maharjan, Ram P.

2017-01-01

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input–mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input–output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection. PMID:28594817

Predicting human immunodeficiency virus inhibitors using multi-dimensional Bayesian network classifiers.

PubMed

Borchani, Hanen; Bielza, Concha; Toro, Carlos; Larrañaga, Pedro

2013-03-01

Our aim is to use multi-dimensional Bayesian network classifiers in order to predict the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and protease inhibitors given an input set of respective resistance mutations that an HIV patient carries. Multi-dimensional Bayesian network classifiers (MBCs) are probabilistic graphical models especially designed to solve multi-dimensional classification problems, where each input instance in the data set has to be assigned simultaneously to multiple output class variables that are not necessarily binary. In this paper, we introduce a new method, named MB-MBC, for learning MBCs from data by determining the Markov blanket around each class variable using the HITON algorithm. Our method is applied to both reverse transcriptase and protease data sets obtained from the Stanford HIV-1 database. Regarding the prediction of antiretroviral combination therapies, the experimental study shows promising results in terms of classification accuracy compared with state-of-the-art MBC learning algorithms. For reverse transcriptase inhibitors, we get 71% and 11% in mean and global accuracy, respectively; while for protease inhibitors, we get more than 84% and 31% in mean and global accuracy, respectively. In addition, the analysis of MBC graphical structures lets us gain insight into both known and novel interactions between reverse transcriptase and protease inhibitors and their respective resistance mutations. MB-MBC algorithm is a valuable tool to analyze the HIV-1 reverse transcriptase and protease inhibitors prediction problem and to discover interactions within and between these two classes of inhibitors. Copyright © 2012 Elsevier B.V. All rights reserved.

Sanitization of an Automatic Reverse-Osmosis Watering System: Removal of a Clinically Significant Biofilm

PubMed Central

Molk, Denise M; Karr-May, Charlene L; Trang, Elaine D; Sanders, George E

2013-01-01

During environmental monitoring of our institution's rodent watering systems, one vivarium was found to have high bacterial loads in the reverse-osmosis (RO) automatic water system. These findings prompted evaluation of the entire RO water production and distribution system. Investigation revealed insufficient rack and RO system sanitization, leading to heavy biofilm accumulation within the system. Approximately 2 wk after discovery in the water system, one of the bacterial organisms isolated in the water supply, Sphingomonas paucimobilis, was isolated from a peritoneal abscess of a severely immunodeficient B6.Cg-Slc11a1r Rag1tm1Mom/Cwi mouse housed in the same vivarium, suggesting that rodents drinking from this system were being exposed randomly to fragments of biofilm. Plans were developed to sanitize the entire system. Hypercholorination was used first, followed by treatment with a combination of peracetic acid and hydrogen peroxide. Between system sanitizations, a low-level chlorine infusion was added to the system as a biocide. Heterotrophic plate counts and bacterial isolation were performed on water samples obtained before and after sanitization procedures. We here discuss the process of identifying and correcting this important water-quality issue. PMID:23562105

Primer design for a prokaryotic differential display RT-PCR.

PubMed Central

Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-01-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR. PMID:9108168

Primer design for a prokaryotic differential display RT-PCR.

PubMed

Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-05-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.

Methane production in an anaerobic osmotic membrane bioreactor using forward osmosis: Effect of reverse salt flux.

PubMed

Li, Sheng; Kim, Youngjin; Phuntsho, Sherub; Chekli, Laura; Shon, Ho Kyong; Leiknes, TorOve; Ghaffour, Noreddine

2017-09-01

This study investigated the impact of reverse salt flux (RSF) on microbe community and bio-methane production in a simulated fertilizer driven FO-AnMBR system using KCl, KNO 3 and KH 2 PO 4 as draw solutes. Results showed that KH 2 PO 4 exhibited the lowest RSF in terms of molar concentration 19.1mM/(m 2 .h), while for KCl and KNO 3 it was 32.2 and 120.8mM/(m 2 .h), respectively. Interestingly, bio-methane production displayed an opposite order with KH 2 PO 4 , followed by KCl and KNO 3 . Pyrosequencing results revealed the presence of different bacterial communities among the tested fertilizers. Bacterial community of sludge exposed to KH 2 PO 4 was very similar to that of DI-water and KCl. However, results with KNO 3 were different since the denitrifying bacteria were found to have a higher percentage than the sludge with other fertilizers. This study demonstrated that RSF has a negative effect on bio-methane production, probably by influencing the sludge bacterial community via environment modification. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative Genome Analyses of Streptococcus suis Isolates from Endocarditis Demonstrate Persistence of Dual Phenotypic Clones

PubMed Central

Tohya, Mari; Watanabe, Takayasu; Maruyama, Fumito; Arai, Sakura; Ota, Atsushi; Athey, Taryn B. T.; Fittipaldi, Nahuel; Nakagawa, Ichiro; Sekizaki, Tsutomu

2016-01-01

Many bacterial species coexist in the same niche as heterogeneous clones with different phenotypes; however, understanding of infectious diseases by polyphenotypic bacteria is still limited. In the present study, encapsulation in isolates of the porcine pathogen Streptococcus suis from persistent endocarditis lesions was examined. Coexistence of both encapsulated and unencapsulated S. suis isolates was found in 26 out of 59 endocarditis samples. The isolates were serotype 2, and belonged to two different sequence types (STs), ST1 and ST28. The genomes of each of the 26 pairs of encapsulated and unencapsulated isolates from the 26 samples were sequenced. The data showed that each pair of isolates had one or more unique nonsynonymous mutations in the cps gene, and the encapsulated and unencapsulated isolates from the same samples were closest to each other. Pairwise comparisons of the sequences of cps genes in 7 pairs of encapsulated and unencapsulated isolates identified insertion/deletions (indels) ranging from one to 104 bp in different cps genes of unencapsulated isolates. Capsule expression was restored in a subset of unencapsulated isolates by complementation in trans with cps expression vectors. Examination of gene content common to isolates indicated that mutation frequency was higher in ST28 pairs than in ST1 pairs. Genes within mobile genetic elements were mutation hot spots among ST28 isolates. Taken all together, our results demonstrate the coexistence of dual phenotype (encapsulated and unencapsulated) bacterial clones and suggest that the dual phenotypes arose independently in each farm by means of spontaneous mutations in cps genes. PMID:27433935

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

PubMed Central

Geoghegan, James C.; Fleming, Ryan; Damschroder, Melissa; Bishop, Steven M.; Sathish, Hasige A.; Esfandiary, Reza

2016-01-01

ABSTRACT Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies. PMID:27050875

Inability to activate Rac1-dependent forgetting contributes to behavioral inflexibility in mutants of multiple autism-risk genes

PubMed Central

Dong, Tao; He, Jing; Wang, Shiqing; Wang, Lianzhang; Cheng, Yuqi; Zhong, Yi

2016-01-01

The etiology of autism is so complicated because it involves the effects of variants of several hundred risk genes along with the contribution of environmental factors. Therefore, it has been challenging to identify the causal paths that lead to the core autistic symptoms such as social deficit, repetitive behaviors, and behavioral inflexibility. As an alternative approach, extensive efforts have been devoted to identifying the convergence of the targets and functions of the autism-risk genes to facilitate mapping out causal paths. In this study, we used a reversal-learning task to measure behavioral flexibility in Drosophila and determined the effects of loss-of-function mutations in multiple autism-risk gene homologs in flies. Mutations of five autism-risk genes with diversified molecular functions all led to a similar phenotype of behavioral inflexibility indicated by impaired reversal-learning. These reversal-learning defects resulted from the inability to forget or rather, specifically, to activate Rac1 (Ras-related C3 botulinum toxin substrate 1)-dependent forgetting. Thus, behavior-evoked activation of Rac1-dependent forgetting has a converging function for autism-risk genes. PMID:27335463

Biological support media influence the bacterial biofouling community in reverse osmosis water reclamation demonstration plants.

PubMed

Ferrera, Isabel; Mas, Jordi; Taberna, Elisenda; Sanz, Joan; Sánchez, Olga

2015-01-01

The diversity of the bacterial community developed in different stages of two reverse osmosis (RO) water reclamation demonstration plants designed in a wastewater treatment plant (WWTP) in Tarragona (Spain) was characterized by applying 454-pyrosequencing of the 16S rRNA gene. The plants were fed by secondary treated effluent to a conventional pretreatment train prior to the two-pass RO system. Plants differed in the material used in the filtration process, which was sand in one demonstration plant and Scandinavian schists in the second plant. The results showed the presence of a highly diverse and complex community in the biofilms, mainly composed of members of the Betaproteobacteria and Bacteroidetes in all stages, with the presence of some typical wastewater bacteria, suggesting a feed water origin. Community similarities analyses revealed that samples clustered according to filter type, highlighting the critical influence of the biological supporting medium in biofilm community structure.

Dynamic of Mutational Events in Variable Number Tandem Repeats of Escherichia coli O157:H7

PubMed Central

Bustamante, A. V.; Sanso, A. M.; Segura, D. O.; Parma, A. E.; Lucchesi, P. M. A.

2013-01-01

VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs) on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10−05 to 1.8 × 10−03 mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10−03 mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study. PMID:24093095

[Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations].

PubMed

Liu, Yan-chen; Huang, Ai-long; Hu, Yuan; Hu, Jie-li; Lai, Guo-qi; Zhang, Wen-lu

2011-12-01

To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system. 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively. Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test. Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.

Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

PubMed Central

Vergnano, Marta; Wan, Chris

2017-01-01

ABSTRACT We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. PMID:28743817

Stenotrophomonas maltophilia: emergence of multidrug-resistant strains during therapy and in an in vitro pharmacodynamic chamber model.

PubMed Central

Garrison, M W; Anderson, D E; Campbell, D M; Carroll, K C; Malone, C L; Anderson, J D; Hollis, R J; Pfaller, M A

1996-01-01

Emergence of Stenotrophomonas maltophilia as a nosocomial pathogen is becoming increasingly apparent. Pleiotropic resistance characterizes S. maltophilia. Furthermore, a slow growth rate and an increased mutation rate generate discordance between in vitro susceptibility testing and clinical outcome. Despite original susceptibility, drug-resistant strains of S. maltophilia are often recovered from patients receiving beta-lactams, quinolones, or aminoglycosides. Given the disparity among various in vitro susceptibility methods, this study incorporated a unique pharmacodynamic model to more accurately characterize the bacterial time-kill curves and mutation rates of four clinical isolates of S. maltophilia following exposure to simulated multidose regimens of ceftazidime, ciprofloxacin, gentamicin, and ticarcillin-clavulanate. Time-kill data demonstrated regrowth of S. maltophilia with all four agents. With the exception of ticarcillin-clavulanate, viable bacterial counts at the end of 24 h exceeded the starting inoculum. Ciprofloxacin only reduced bacterial counts by less than 1.0 log prior to rapid bacterial regrowth. Resistant mutant strains, identical to their parent strain by pulsed-field gel electrophoresis, were observed following exposure to each class of antibiotic. Mutant strains also had distinct susceptibility patterns. These data are consistent with previous reports which suggest that S. maltophilia, despite susceptibility data that imply that the organism is sensitive, develops multiple forms of resistance quickly and against several classes of antimicrobial agents. Standard in vitro susceptibility methods are not completely reliable for detecting resistant S. maltophilia strains; and therefore, interpretation of these results should be done with caution. In vivo studies are needed to determine optimal therapy against S. maltophilia infections. PMID:9124855

Mechanism of quinolone action and resistance.

PubMed

Aldred, Katie J; Kerns, Robert J; Osheroff, Neil

2014-03-18

Quinolones are one of the most commonly prescribed classes of antibacterials in the world and are used to treat a variety of bacterial infections in humans. Because of the wide use (and overuse) of these drugs, the number of quinolone-resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quinolone action against their enzyme targets. It will then discuss the following three mechanisms that decrease the sensitivity of bacterial cells to quinolones. Target-mediated resistance is the most common and clinically significant form of resistance. It is caused by specific mutations in gyrase and topoisomerase IV that weaken interactions between quinolones and these enzymes. Plasmid-mediated resistance results from extrachromosomal elements that encode proteins that disrupt quinolone-enzyme interactions, alter drug metabolism, or increase quinolone efflux. Chromosome-mediated resistance results from the underexpression of porins or the overexpression of cellular efflux pumps, both of which decrease cellular concentrations of quinolones. Finally, this review will discuss recent advancements in our understanding of how quinolones interact with gyrase and topoisomerase IV and how mutations in these enzymes cause resistance. These last findings suggest approaches to designing new drugs that display improved activity against resistant strains.

Distinct molecular features facilitating ice-binding mechanisms in hyperactive antifreeze proteins closely related to an Antarctic sea ice bacterium.

PubMed

Banerjee, Rachana; Chakraborti, Pratim; Bhowmick, Rupa; Mukhopadhyay, Subhasish

2015-01-01

Antifreeze proteins or ice-binding proteins (IBPs) facilitate the survival of certain cellular organisms in freezing environment by inhibiting the growth of ice crystals in solution. Present study identifies orthologs of the IBP of Colwellia sp. SLW05, which were obtained from a wide range of taxa. Phylogenetic analysis on the basis of conserved regions (predicted as the 'ice-binding domain' [IBD]) present in all the orthologs, separates the bacterial and archaeal orthologs from that of the eukaryotes'. Correspondence analysis pointed out that the bacterial and archaeal IBDs have relatively higher average hydrophobicity than the eukaryotic members. IBDs belonging to bacterial as well as archaeal AFPs contain comparatively more strands, and therefore are revealed to be under higher evolutionary selection pressure. Molecular docking studies prove that the ice crystals form more stable complex with the bacterial as well as archaeal proteins than the eukaryotic orthologs. Analysis of the docked structures have traced out the ice-binding sites (IBSs) in all the orthologs which continue to facilitate ice-binding activity even after getting mutated with respect to the well-studied IBSs of Typhula ishikariensis and notably, all these mutations performing ice-binding using 'anchored clathrate mechanism' have been found to prefer polar and hydrophilic amino acids. Horizontal gene transfer studies point toward a strong selection pressure favoring independent evolution of the IBPs in some polar organisms including prokaryotes as well as eukaryotes because these proteins facilitate the polar organisms to acclimatize to the adversities in their niche, thus safeguarding their existence.

The carotenoid pigments of a marine Bacillus firmus strain.

PubMed

Pane, L; Radin, L; Franconi, G; Carli, A

1996-01-01

As carotenoids have important biological functions, it is important to discover new natural sources of these pigments. The bacterial strains isolated from a sea water rock pool were cultivated on marine agar containing yeast extract and identified by conventional methods. The bacterial pigments were extracted with methanol and analyzed by reversed-phase HPLC with diode array detection. The major pigment of a Bacillus firmus strain was identified as astaxanthin; the results obtained suggest potential use of this bacterium in aquaculture and in pharmaceutical field.

Activating HER2 mutations in HER2 gene amplification negative breast cancer.

PubMed

Bose, Ron; Kavuri, Shyam M; Searleman, Adam C; Shen, Wei; Shen, Dong; Koboldt, Daniel C; Monsey, John; Goel, Nicholas; Aronson, Adam B; Li, Shunqiang; Ma, Cynthia X; Ding, Li; Mardis, Elaine R; Ellis, Matthew J

2013-02-01

Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.

Molecular Diagnostics in Colorectal Carcinoma: Advances and Applications for 2018.

PubMed

Bhalla, Amarpreet; Zulfiqar, Muhammad; Bluth, Martin H

2018-06-01

The molecular pathogenesis and classification of colorectal carcinoma are based on the traditional adenomaecarcinoma sequence, serrated polyp pathway, and microsatellite instability (MSI). The genetic basis for hereditary nonpolyposis colorectal cancer is the detection of mutations in the MLH1, MSH2, MSH6, PMS2, and EPCAM genes. Genetic testing for Lynch syndrome includes MSI testing, methylator phenotype testing, BRAF mutation testing, and molecular testing for germline mutations in MMR genes. Molecular makers with predictive and prognostic implications include quantitative multigene reverse transcriptase polymerase chain reaction assay and KRAS and BRAF mutation analysis. Mismatch repair-deficient tumors have higher rates of programmed death-ligand 1 expression. Cell-free DNA analysis in fluids are proving beneficial for diagnosis and prognosis in these disease states towards effective patient management. Copyright © 2018 Elsevier Inc. All rights reserved.

Proteome analysis of Arabidopsis seedlings exposed to bacterial volatiles.

PubMed

Kwon, Young Sang; Ryu, Choong-Min; Lee, Soohyun; Park, Hyo Bee; Han, Ki Soo; Lee, Jung Han; Lee, Kyunghee; Chung, Woo Sik; Jeong, Mi-Jeong; Kim, Hee Kyu; Bae, Dong-Won

2010-11-01

Plant root-associated bacteria (rhizobacteria) elicit plant basal immunity referred to as induced systemic resistance (ISR) against multiple pathogens. Among multi-bacterial determinants involving such ISR, the induction of ISR and promotion of growth by bacterial volatile compounds was previously reported. To exploit global de novo expression of plant proteins by bacterial volatiles, proteomic analysis was performed after exposure of Arabidopsis plants to the rhizobacterium Bacillus subtilis GB03. Ethylene biosynthesis enzymes were significantly up-regulated. Analysis by quantitative reverse transcriptase polymerase chain reaction confirmed that ethylene biosynthesis-related genes SAM-2, ACS4, ACS12, and ACO2 as well as ethylene response genes, ERF1, GST2, and CHIB were up-regulated by the exposure to bacterial volatiles. More interestingly, the emission of bacterial volatiles significantly up-regulated both key defense mechanisms mediated by jasmonic acid and salicylic acid signaling pathways. In addition, high accumulation of antioxidant proteins also provided evidence of decreased sensitivity to reactive oxygen species during the elicitation of ISR by bacterial volatiles. The present results suggest that the proteomic analysis of plant defense responses in bacterial volatile-mediated ISR can reveal the mechanisms of plant basal defenses orchestrated by endogenous ethylene production pathways and the generation of reactive oxygen species.

Clinicopathological characteristics of TERT promoter mutation and telomere length in hepatocellular carcinoma.

PubMed

Lee, Hye Won; Park, Tae In; Jang, Se Young; Park, Soo Young; Park, Won-Jin; Jung, Soo-Jung; Lee, Jae-Ho

2017-02-01

Promoter mutations in telomerase reverse transcriptase (TERT) and telomere length have been studied in various tumors. In the present study, the frequency and clinical characteristics of TERT promoter mutation and telomere length were studied in hepatocellular carcinoma (HCC). TERT promoter mutation and telomere length were analyzed in 162 tumor samples of the patients with HCC by sequencing and real-time PCR, respectively. The TERT promoter mutation rate was 28.8% (46/160) in HCC and was associated with males (P = 0.027). The telomere length was not significantly different in the presence of a TERT promoter mutation but was shorter in high-grade tumor stages (P = 0.048). Survival analyses showed that poor overall survival was associated with longer telomere length (P = 0.013). However, the TERT promoter mutation did not have a prognostic value for HCC. Multivariate survival analyses demonstrated that the telomere length was an independent prognostic marker for poor overall survival (hazard ratio = 1.75, 95% confidence interval: 1.046-2.913, P = 0.033). These data demonstrated that TERT promoter mutation is a frequent event in HCC; however, telomere length, but not the presence of a TERT promoter mutation, might have potential value as a prognostic indicator of HCC.

Differential Reprogramming of Isogenic Colorectal Cancer Cells by Distinct Activating KRAS Mutations

PubMed Central

2015-01-01

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS. PMID:25599653

A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

PubMed Central

Pauly, Matthew D; Procario, Megan C; Lauring, Adam S

2017-01-01

Influenza virus’ low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7 × 10–6 - 3.0 × 10–5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8 × 10–4 s/n/r for PR8 (H1N1) and 2.5 × 10–4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7–3.6. Our data suggest that each replicated genome will have an average of 2–3 mutations and highlight the importance of mutational load in influenza virus evolution. DOI: http://dx.doi.org/10.7554/eLife.26437.001 PMID:28598328

Surface acidic amino acid of Pseudomonas/Halomonas chimeric nucleoside diphosphate kinase leads effective recovery from heat-denaturation.

PubMed

Tokunaga, Hiroko; Arakawa, Tsutomu; Tokunaga, Masao

2013-07-01

One of the hallmarks of halophilic properties is reversibility of thermal unfolding. A nucleoside diphosphate kinase (NDK) from a moderate halophile Halomonas sp. 593 (HaNDK) follows this behavior. His-tagged chimeric NDK (HisPaHaNDK) consisting of an N-terminal half of a non-halophilic Pseuodomonas aeruginosa NDK (PaNDK) and a Cterminal half of HaNDK loses this reversible property, indicating a critical role of the N-terminal portion of PaNDK in determining the reversibility of the chimeric protein. Various mutations were introduced at Arg45 and Lys61, based on the model NDK structure. It appears that having Glu at position 45 is critical in conferring the thermal reversibility to HisPa- HaNDK chimeric protein.

Genome-Wide Mutation Rate Response to pH Change in the Coral Reef Pathogen Vibrio shilonii AK1.

PubMed

Strauss, Chloe; Long, Hongan; Patterson, Caitlyn E; Te, Ronald; Lynch, Michael

2017-08-22

Recent application of mutation accumulation techniques combined with whole-genome sequencing (MA/WGS) has greatly promoted studies of spontaneous mutation. However, such explorations have rarely been conducted on marine organisms, and it is unclear how marine habitats have influenced genome stability. This report resolves the mutation rate and spectrum of the coral reef pathogen Vibrio shilonii , which causes coral bleaching and endangers the biodiversity maintained by coral reefs. We found that its mutation rate and spectrum are highly similar to those of other studied bacteria from various habitats, despite the saline environment. The mutational properties of this marine bacterium are thus controlled by other general evolutionary forces such as natural selection and genetic drift. We also found that as pH drops, the mutation rate decreases and the mutation spectrum is biased in the direction of generating G/C nucleotides. This implies that evolutionary features of this organism and perhaps other marine microbes might be altered by the increasingly acidic ocean water caused by excess CO 2 emission. Nonetheless, further exploration is needed as the pH range tested in this study was rather narrow and many other possible mutation determinants, such as carbonate increase, are associated with ocean acidification. IMPORTANCE This study explored the pH dependence of a bacterial genome-wide mutation rate. We discovered that the genome-wide rates of appearance of most mutation types decrease linearly and that the mutation spectrum is biased in generating more G/C nucleotides with pH drop in the coral reef pathogen V. shilonii . Copyright © 2017 Strauss et al.

Novel Codon Insert in HIV Type 1 Clade B Reverse Transcriptase Associated with Low-Level Viremia During Antiretroviral Therapy

PubMed Central

Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C.; Richman, Douglas D.; Smith, Davey M.

2014-01-01

Abstract We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach. PMID:24020934

Prevalence and patterns of HIV transmitted drug resistance in Guatemala.

PubMed

Avila-Ríos, Santiago; Mejía-Villatoro, Carlos R; García-Morales, Claudia; Soto-Nava, Maribel; Escobar, Ingrid; Mendizabal, Ricardo; Girón, Amalia; García, Leticia; Reyes-Terán, Gustavo

2011-12-01

To assess human immunodeficiency virus (HIV) diversity and the prevalence of transmitted drug resistance (TDR) in Guatemala. One hundred forty-five antiretroviral treatment-naïve patients referred to the Roosevelt Hospital in Guatemala City were enrolled from October 2010 to March 2011. Plasma HIV pol sequences were obtained and TDR was assessed with the Stanford algorithm and the World Health Organization (WHO) TDR surveillance mutation list. HIV subtype B was highly prevalent in Guatemala (96.6%, 140/145), and a 2.8% (4/145) prevalence of BF1 recombinants and 0.7% (1/145) prevalence of subtype C viruses were found. TDR prevalence for the study period was 8.3% (12/145) with the Stanford database algorithm (score > 15) and the WHO TDR surveillance mutation list. Most TDR cases were associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) (83.3%, 10/12); a low prevalence of nucleoside reverse transcriptase inhibitors and protease inhibitors was observed in the cohort (< 1% for both families). Low selection of antiretroviral drug resistance mutations was found, except for NNRTI-associated mutations. Major NNRTI mutations such as K101E, K103N, and E138K showed higher frequencies than expected in ART-naïve populations. Higher literacy was associated with a greater risk of TDR (odds ratio 4.14, P = 0.0264). This study represents one of the first efforts to describe HIV diversity and TDR prevalence and trends in Guatemala. TDR prevalence in Guatemala was at the intermediate level. Most TDR cases were associated with NNRTIs. Further and continuous TDR surveillance is necessary to gain more indepth knowledge about TDR spread and trends in Guatemala and to optimize treatment outcomes in the country.

Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe.

PubMed

Hofstra, L Marije; Sauvageot, Nicolas; Albert, Jan; Alexiev, Ivailo; Garcia, Federico; Struck, Daniel; Van de Vijver, David A M C; Åsjö, Birgitta; Beshkov, Danail; Coughlan, Suzie; Descamps, Diane; Griskevicius, Algirdas; Hamouda, Osamah; Horban, Andrzej; Van Kasteren, Marjo; Kolupajeva, Tatjana; Kostrikis, Leondios G; Liitsola, Kirsi; Linka, Marek; Mor, Orna; Nielsen, Claus; Otelea, Dan; Paraskevis, Dimitrios; Paredes, Roger; Poljak, Mario; Puchhammer-Stöckl, Elisabeth; Sönnerborg, Anders; Staneková, Danica; Stanojevic, Maja; Van Laethem, Kristel; Zazzi, Maurizio; Zidovec Lepej, Snjezana; Boucher, Charles A B; Schmit, Jean-Claude; Wensing, Annemarie M J; Puchhammer-Stockl, E; Sarcletti, M; Schmied, B; Geit, M; Balluch, G; Vandamme, A-M; Vercauteren, J; Derdelinckx, I; Sasse, A; Bogaert, M; Ceunen, H; De Roo, A; De Wit, S; Echahidi, F; Fransen, K; Goffard, J-C; Goubau, P; Goudeseune, E; Yombi, J-C; Lacor, P; Liesnard, C; Moutschen, M; Pierard, D; Rens, R; Schrooten, Y; Vaira, D; Vandekerckhove, L P R; Van den Heuvel, A; Van Der Gucht, B; Van Ranst, M; Van Wijngaerden, E; Vandercam, B; Vekemans, M; Verhofstede, C; Clumeck, N; Van Laethem, K; Beshkov, D; Alexiev, I; Lepej, S Zidovec; Begovac, J; Kostrikis, L; Demetriades, I; Kousiappa, I; Demetriou, V; Hezka, J; Linka, M; Maly, M; Machala, L; Nielsen, C; Jørgensen, L B; Gerstoft, J; Mathiesen, L; Pedersen, C; Nielsen, H; Laursen, A; Kvinesdal, B; Liitsola, K; Ristola, M; Suni, J; Sutinen, J; Descamps, D; Assoumou, L; Castor, G; Grude, M; Flandre, P; Storto, A; Hamouda, O; Kücherer, C; Berg, T; Braun, P; Poggensee, G; Däumer, M; Eberle, J; Heiken, H; Kaiser, R; Knechten, H; Korn, K; Müller, H; Neifer, S; Schmidt, B; Walter, H; Gunsenheimer-Bartmeyer, B; Harrer, T; Paraskevis, D; Hatzakis, A; Zavitsanou, A; Vassilakis, A; Lazanas, M; Chini, M; Lioni, A; Sakka, V; Kourkounti, S; Paparizos, V; Antoniadou, A; Papadopoulos, A; Poulakou, G; Katsarolis, I; Protopapas, K; Chryssos, G; Drimis, S; Gargalianos, P; Xylomenos, G; Lourida, G; Psichogiou, M; Daikos, G L; Sipsas, N V; Kontos, A; Gamaletsou, M N; Koratzanis, G; Sambatakou, H; Mariolis, H; Skoutelis, A; Papastamopoulos, V; Georgiou, O; Panagopoulos, P; Maltezos, E; Coughlan, S; De Gascun, C; Byrne, C; Duffy, M; Bergin, C; Reidy, D; Farrell, G; Lambert, J; O'Connor, E; Rochford, A; Low, J; Coakely, P; O'Dea, S; Hall, W; Mor, O; Levi, I; Chemtob, D; Grossman, Z; Zazzi, M; de Luca, A; Balotta, C; Riva, C; Mussini, C; Caramma, I; Capetti, A; Colombo, M C; Rossi, C; Prati, F; Tramuto, F; Vitale, F; Ciccozzi, M; Angarano, G; Rezza, G; Kolupajeva, T; Vasins, O; Griskevicius, A; Lipnickiene, V; Schmit, J C; Struck, D; Sauvageot, N; Hemmer, R; Arendt, V; Michaux, C; Staub, T; Sequin-Devaux, C; Wensing, A M J; Boucher, C A B; van de Vijver, D A M C; van Kessel, A; van Bentum, P H M; Brinkman, K; Connell, B J; van der Ende, M E; Hoepelman, I M; van Kasteren, M; Kuipers, M; Langebeek, N; Richter, C; Santegoets, R M W J; Schrijnders-Gudde, L; Schuurman, R; van de Ven, B J M; Åsjö, B; Kran, A-M Bakken; Ormaasen, V; Aavitsland, P; Horban, A; Stanczak, J J; Stanczak, G P; Firlag-Burkacka, E; Wiercinska-Drapalo, A; Jablonowska, E; Maolepsza, E; Leszczyszyn-Pynka, M; Szata, W; Camacho, R; Palma, C; Borges, F; Paixão, T; Duque, V; Araújo, F; Otelea, D; Paraschiv, S; Tudor, A M; Cernat, R; Chiriac, C; Dumitrescu, F; Prisecariu, L J; Stanojevic, M; Jevtovic, Dj; Salemovic, D; Stanekova, D; Habekova, M; Chabadová, Z; Drobkova, T; Bukovinova, P; Shunnar, A; Truska, P; Poljak, M; Lunar, M; Babic, D; Tomazic, J; Vidmar, L; Vovko, T; Karner, P; Garcia, F; Paredes, R; Monge, S; Moreno, S; Del Amo, J; Asensi, V; Sirvent, J L; de Mendoza, C; Delgado, R; Gutiérrez, F; Berenguer, J; Garcia-Bujalance, S; Stella, N; de Los Santos, I; Blanco, J R; Dalmau, D; Rivero, M; Segura, F; Elías, M J Pérez; Alvarez, M; Chueca, N; Rodríguez-Martín, C; Vidal, C; Palomares, J C; Viciana, I; Viciana, P; Cordoba, J; Aguilera, A; Domingo, P; Galindo, M J; Miralles, C; Del Pozo, M A; Ribera, E; Iribarren, J A; Ruiz, L; de la Torre, J; Vidal, F; Clotet, B; Albert, J; Heidarian, A; Aperia-Peipke, K; Axelsson, M; Mild, M; Karlsson, A; Sönnerborg, A; Thalme, A; Navér, L; Bratt, G; Karlsson, A; Blaxhult, A; Gisslén, M; Svennerholm, B; Bergbrant, I; Björkman, P; Säll, C; Mellgren, Å; Lindholm, A; Kuylenstierna, N; Montelius, R; Azimi, F; Johansson, B; Carlsson, M; Johansson, E; Ljungberg, B; Ekvall, H; Strand, A; Mäkitalo, S; Öberg, S; Holmblad, P; Höfer, M; Holmberg, H; Josefson, P; Ryding, U

2016-03-01

Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001. Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0. The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones. Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.

Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations▿ †

PubMed Central

Mitsuya, Yumi; Varghese, Vici; Wang, Chunlin; Liu, Tommy F.; Holmes, Susan P.; Jayakumar, Prerana; Gharizadeh, Baback; Ronaghi, Mostafa; Klein, Daniel; Fessel, W. Jeffrey; Shafer, Robert W.

2008-01-01

T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method—ultradeep pyrosequencing (UDPS; 454 Life Sciences)—to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants (“revertant” samples) compared with samples from untreated persons that lack such revertants (“control” samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants. PMID:18715933

An overview of the molecular and epidemiological features of HIV-1 infection in two major cities of Bahia state, Brazil.

PubMed

Amaral, Amanda Gm; Oliveira, Isabele B; Carneiro, Diego C; Alcantara, Luiz Cj; Monteiro-Cunha, Joana P

2017-06-01

The high mutation rate of the human immunodeficiency virus (HIV) has created a public health challenge because the use of antiretroviral drugs can generate selective pressure that drives resistance in these viruses. The aim of this work was to characterise the molecular and epidemiological profile of HIV in Bahia, Brazil. DNA sequences from regions of HIV gag, pol, and env genes were obtained from previous studies performed in this area between 2002 and 2012. Their genotype and drug-resistance mutations were identified using bioinformatics tools. Clinical and epidemiological data were analysed. Among 263 individuals (46.4% male), 97.5% were asymptomatic and 49.1% were receiving treatment. Most of the individuals were 31 to 40 years old (36.9%) and infected through heterosexual contact (40.7%). The predominant genotype was B (68.1%) followed by BF recombinants (18.6%). Among the individuals infected with either F or BF genotypes, 68.4% were women and 76.8% were infected through heterosexual transmission. The prevalence of associated mutations conferring antiretroviral resistance was 14.2%, with 3.8% of all mutations conferring resistance to protease inhibitors, 9.43% to nucleoside reverse transcriptase inhibitors, and 8.5% to non-nucleoside reverse transcriptase inhibitors. Drug resistance was higher in individuals receiving treatment (26.1%) than in the drug-naïve (4.3%) individuals. This study will contribute to the understanding and monitoring of HIV epidemic in this Brazilian region.

Towards a systemic paradigm in carcinogenesis: linking epigenetics and genetics.

PubMed

Burgio, Ernesto; Migliore, Lucia

2015-04-01

For at least 30 years cancer has been defined as a genetic disease and explained by the so-called somatic mutation theory (SMT), which has dominated the carcinogenesis field. Criticism of the SMT has recently greatly increased, although still not enough to force all SMT supporters to recognize its limits. Various researchers point out that cancer appears to be a complex process concerning a whole tissue; and that genomic mutations, although variably deleterious and unpredictably important in determining the establishment of the neoplastic phenotype, are not the primary origin for a malignant neoplasia. We attempt to describe the inadequacies of the SMT and demonstrate that epigenetics is a more logical cause of carcinogenesis. Many previous models of carcinogenesis fall into two classes: (i) in which some biological changes inside cells alone lead to malignancy; and (ii) requiring changes in stroma/extracellular matrix. We try to make clear that in the (ii) model genomic instability is induced by persistent signals coming from the microenvironment, provoking epigenetic and genetic modifications in tissue stem cells that can lead to cancer. In this perspective, stochastic mutations of DNA are a critical by-product rather then the primary cause of cancer. Indirect support for such model of carcinogenesis comes from the in vitro and vivo experiments showing apparent 'reversion' of cancer phenotypes obtained via physiological factors of cellular differentiation (cytokines and other signaling molecules) or drugs, even if the key mutations are not 'reversed'.

Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene.

PubMed

Poussier, Stéphane; Thoquet, Philippe; Trigalet-Demery, Danièle; Barthet, Séverine; Meyer, Damien; Arlat, Matthieu; Trigalet, André

2003-08-01

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild-type pathogenic to a non-pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild-type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.

Role for Telomerase in Listeria monocytogenes Infection

PubMed Central

Samba-Louaka, Ascel; Stavru, Fabrizia

2012-01-01

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the human telomerase complex. Growing evidence suggests that hTERT also contributes to the cell physiology independently of telomere elongation. However, its role in bacterial infection is unknown. Here we show that hTERT is critical for Listeria monocytogenes infection, as the depletion of hTERT impaired bacterial intracellular replication. In addition, we observed that L. monocytogenes caused a decrease in hTERT levels at early time points of the infectious process. This effect was mediated by the pore-forming toxin listeriolysin O (LLO) and did not require bacterial entry into host cells. Calcium influx through the LLO pores contributed to a proteasome-independent decrease in hTERT protein levels. Together, our data provide evidence that these bacteria trigger hTERT degradation, an event that is detrimental to bacterial replication. PMID:23006849

Reversible synthesis of colanic acid and O-antigen polysaccharides in Salmonella Typhimurium enhances induction of cross-immune responses and provides protection against heterologous Salmonella challenge.

PubMed

Li, Pei; Liu, Qing; Huang, Chun; Zhao, Xinxin; Roland, Kenneth L; Kong, Qingke

2017-05-15

Colanic Acid (CA) and lipopolysaccharide (LPS) are two major mannose-containing extracellular polysaccharides of Salmonella. Their presence on the bacterial surface can mask conserved protective outer membrane proteins (OMPs) from the host immune system. The mannose moiety in these molecules is derived from GDP-mannose, which is synthesized in several steps. The first two steps require the action of phosphomannose isomerase, encoded by pmi (manA), followed by phosphomannomutase, encoded by manB. There are two copies of manB present in the Salmonella chromosome, one located in the cps gene cluster (cpsG) responsible for CA synthesis, and the other in the rfb gene cluster (rfbK) involved in LPS O-antigen synthesis. In this study, it was demonstrated that the products of cpsG and rfbK are isozymes. To evaluate the impact of these genes on O-antigen synthesis, virulence and immunogenicity, single mutations (Δpmi, ΔrfbK or ΔcpsG) and a double mutation (ΔrfbK ΔcpsG) were introduced into both wild-type Salmonella enterica and an attenuated Δcya Δcrp vaccine strain. The Δpmi, ΔrfbK and ΔcpsG ΔrfbK mutants were defective in LPS synthesis and attenuated for virulence. In orally inoculated mice, strain S122 (Δcrp Δcya ΔcpsG ΔrfbK) and its parent S738 (Δcrp Δcya) were both avirulent and colonized internal tissues. Strain S122 elicited higher levels of anti-S. Typhimurium OMP serum IgG than its parent strain. Mice immunized with S122 were completely protected against challenge with wild-type virulent S. Typhimurium and partially protected against challenge with either wild-type virulent S. Choleraesuis or S. Enteritidis. These data indicate that deletions in rfbK and cpsG are useful mutations for inclusion in future attenuated Salmonella vaccine strains to induce cross-protective immunity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

PubMed Central

Siegel, Eli C.

1973-01-01

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage λ. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut+ strains. UV irradiation induced mutations in a mutU4 strain, and phage λ was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4. PMID:4345920