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Sample records for live cold-adapted flumist

  1. Demystifying FluMist, a new intranasal, live influenza vaccine.

    PubMed

    Mossad, Sherif B

    2003-09-01

    FluMist--a cold-adapted, live-attenuated, trivalent, intranasal influenza virus vaccine approved by the US Food and Drug Administration on June 17, 2003--has been shown to be safe and effective, but its role in the general prevention of influenza is yet to be defined. Intranasal administration is expected to be more acceptable than parenteral, particularly in children, but the potential for the shedding of live virus may pose a risk to anyone with a compromised immune system.

  2. Reversion of Cold-Adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus.

    PubMed

    Zhou, Bin; Meliopoulos, Victoria A; Wang, Wei; Lin, Xudong; Stucker, Karla M; Halpin, Rebecca A; Stockwell, Timothy B; Schultz-Cherry, Stacey; Wentworth, David E

    2016-10-01

    The only licensed live attenuated influenza A virus vaccines (LAIVs) in the United States (FluMist) are created using internal protein-coding gene segments from the cold-adapted temperature-sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature-sensitive phenotype can revert and, if so, the types of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperatures in vitro generated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations, PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect the temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the roles of individual mutations were characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild-type 2009 H1N1 pandemic virus. The live attenuated influenza vaccine FluMist has been proven safe and effective and is widely used in the United States. The phenotype and genotype of the vaccine virus are believed to be very stable, and mutants that cause disease in animals or humans have never been reported. By propagating the virus under

  3. Reversion of Cold-Adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus

    PubMed Central

    Meliopoulos, Victoria A.; Wang, Wei; Lin, Xudong; Stucker, Karla M.; Halpin, Rebecca A.; Stockwell, Timothy B.; Schultz-Cherry, Stacey

    2016-01-01

    ABSTRACT The only licensed live attenuated influenza A virus vaccines (LAIVs) in the United States (FluMist) are created using internal protein-coding gene segments from the cold-adapted temperature-sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature-sensitive phenotype can revert and, if so, the types of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperatures in vitro generated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations, PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect the temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the roles of individual mutations were characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild-type 2009 H1N1 pandemic virus. IMPORTANCE The live attenuated influenza vaccine FluMist has been proven safe and effective and is widely used in the United States. The phenotype and genotype of the vaccine virus are believed to be very stable, and mutants that cause disease in animals or humans have never been reported. By

  4. [New cold-adapted donor strains for live influenza vaccine].

    PubMed

    Gendon, Iu Z; Markushin, S G; Tsfasman, T M; Akopova, I I; Akhmatova, N K; Koptiaeva, I B

    2013-01-01

    Cold-adapted (CA) strains A/Krasnodar/35 and B/Victoria/63 were isolated using passages of A/Krasnodar/101/59 and B/Victoria/2/87 wild type strains at low temperatures. The resulting CA strains possessed TS and CA phenotypes and had a reduced ability to reproduce in mouse lungs and nasal turbinates. They displayed a high protective efficacy in experiments on mice. The two CA strains reproduced well in chick embryos and MDCK cell line without change of TS and CA markers. The CA A/Krasnodar/35 strain during passages at low temperature acquired 13 mutations in the 6 internal genes, 8 of those mutations led to amino acid changes. The CA B/Victoria/63 strain acquired 8 mutations in the internal genes, 6 of which led to amino acid changes. The intranasal vaccination of mice with the CA A/Krasnodar/35 strain led to a transitory suppression of various lymphocyte subpopulations, as well as to an increase in the number of some other cell types. The CA strains in question may be used in the future as attenuation donors for live influenza vaccines.

  5. [Live cold-adapted influenza vaccine: state-of-the-art].

    PubMed

    Gendon, Iu Z

    2011-01-01

    The review characterizes the currently used cold-adapted donor strains of influenza virus attenuation to prepare cold-adapted reassortants with actual epidemic influenza virus strains. It considers new procedures for preparing attenuated influenza virus strains for live influenza vaccines, as well as analytical methods and the genome composition of reassortants. Recent data on the safety of live cold-adapted influenza vaccines (LCAIVs), including those on the genetic stability of vaccine reassortants and the immunogenicity and efficacy of these vaccines for different age groups, are discussed. There is evidence for the design of live human vaccines against avian influenza. It is concluded that LCAIVs are highly effective for immunization of children.

  6. Genetic stability of live, cold-adapted influenza virus components of the FluMist/CAIV-T vaccine throughout the manufacturing process.

    PubMed

    Buonagurio, Deborah A; Bechert, Thomas M; Yang, Chin-Fen; Shutyak, Leonid; D'Arco, Gail A; Kazachkov, Yuriy; Wang, Hai-Ping; Rojas, Eduardo A; O'Neill, Robert E; Spaete, Richard R; Coelingh, Kathleen L; Zamb, Timothy J; Sidhu, Mohinder S; Udem, Stephen A

    2006-03-15

    FluMist is a live-attenuated, trivalent influenza vaccine (LAIV) recently approved for intranasal administration. To demonstrate genetic stability during manufacture of the vaccine viruses in LAIV and a similar vaccine in development (CAIV-T), full genome consensus sequences were determined at multiple manufacturing stages for four influenza type A and five type B strains. The critical cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) mutations were preserved in the virus manufacturing intermediates. Moreover, sequence identity was observed for all vaccine intermediates of the same strain. Minor sequence differences were noted in the shared gene segments of the vaccine viruses and their common progenitor master donor virus (MDV) and several of the hemagglutinin (HA) and neuraminidase (NA) genes contained nucleotide differences when compared to the wild-type parent. Nonetheless, all vaccine viruses retained the ca, ts, and att phenotypes. Thus, genetic and phenotypic stability of the vaccine viruses is maintained during the manufacture of LAIV/CAIV-T vaccines.

  7. Genetic analysis of attenuation markers of cold-adapted X-31 influenza live vaccine donor strain.

    PubMed

    Jang, Yo Han; Jung, Eun-Ju; Lee, Kwang-Hee; Byun, Young Ho; Yang, Seung Won; Seong, Baik Lin

    2016-03-08

    Cold-adapted live attenuated influenza vaccines (CAIVs) have been considered as a safe prophylactic measure to prevent influenza virus infections. The safety of a CAIV depends largely on genetic markers that confer specific attenuation phenotypes. Previous studies with other CAIVs reported that polymerase genes were primarily responsible for the attenuation. Here, we analyzed the genetic mutations and their phenotypic contribution in the X-31 ca strain, a recently developed alternative CAIV donor strain. During the cold-adaptation of its parental X-31 virus, various numbers of sequence changes were accumulated in all six internal genes. Phenotypic analysis with single-gene and multiple-gene reassortant viruses suggests that NP gene makes the largest contribution to the cold-adapted (ca) and temperature-sensitive (ts) characters, while the remaining other internal genes also impart attenuation characters with varying degrees. A balanced contribution of all internal genes to the attenuation suggests that X-31 ca could serve as an ideal master donor strain for CAIVs preventing influenza epidemics and pandemics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Safety and immunogenicity of a novel cold-adapted modified-live equine influenza virus vaccine.

    PubMed

    Tabynov, K; Kydyrbayev, Z; Ryskeldinova, S; Assanzhanova, N; Kozhamkulov, Y; Inkarbekov, D; Sansyzbay, A

    2014-11-01

    To design and evaluate the safety and immunogenicity of a modified-live vaccine to prevent equine influenza virus (EIV) infection based on the novel reassortant cold-adapted strain A/HK/Otar/6:2/2010. Surface proteins (HA, NA) from the wild-type strain A/equine/Otar/764/2007 (H3N8) and internal proteins (PB2, PB1, PA, NP, M, NS) from the attenuated cold-adapted donor strain A/Hong Kong/1/68/162/35CA (H3N2) were included in the vaccine. Horses were administered 10(9.2) EID50 /mL of the modified-live vaccine or saline solution using a nasal spray. The clinical condition of the animals was assessed throughout the study and nasopharyngeal swabs were collected for virus titration. Two yearlings in each group were euthanased on day 5 post vaccination (PV) for histological examination and measurement of viral titres in the organs. Serum samples and nasal secretions were collected to evaluate serological response. Lymphoproliferation after restimulation in vitro was determined to evaluate cell-mediated immunity. To evaluate the protective capacity of the vaccine, the yearlings in both groups were challenged with the wild-type virus at 28 days PV and their clinical condition and serological response was evaluated. Nasal swabs were collected to assess viral shedding from the upper respiratory tract. Single intranasal administration of a modified-live EIV vaccine caused no adverse effects and vaccinated yearlings and pregnant mares did not form detectable levels of antibodies by days 7, 14 and 28 PV, as indicated by the HI reaction and ELISA. Secretory antibodies could be detected on day 7 and reached maximal levels on day 14 PV. In vitro studies showed that the yearlings and pregnant mares both formed a cell-mediated immune response by day 14 PV. The vaccine protected yearlings against challenge with wild-type virus. We conclude that single intranasal administration of the modified-live EIV vaccine was safe in the yearlings and pregnant mares that we treated, and was

  9. Cold adaptations.

    PubMed

    Launay, Jean-Claude; Savourey, Gustave

    2009-07-01

    Nowdays, occupational and recreational activities in cold environments are common. Exposure to cold induces thermoregulatory responses like changes of behaviour and physiological adjustments to maintain thermal balance either by increasing metabolic heat production by shivering and/or by decreasing heat losses consecutive to peripheral cutaneous vasoconstriction. Those physiological responses present a great variability among individuals and depend mainly on biometrical characteristics, age, and general cold adaptation. During severe cold exposure, medical disorders may occur such as accidental hypothermia and/or freezing or non-freezing cold injuries. General cold adaptations have been qualitatively classified by Hammel and quantitatively by Savourey. This last classification takes into account the quantitative changes of the main cold reactions: higher or lower metabolic heat production, higher or lesser heat losses and finally the level of the core temperature observed at the end of a standardized exposure to cold. General cold adaptations observed previously in natives could also be developed in laboratory conditions by continuous or intermittent cold exposures. Beside general cold adaptation, local cold adaptation exists and is characterized by a lesser decrease of skin temperature, a more pronounced cold induced vasodilation, less pain and a higher manual dexterity. Adaptations to cold may reduce the occurrence of accidents and improve human performance as surviving in the cold. The present review describes both general and local cold adaptations in humans and how they are of interest for cold workers.

  10. Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

    SciTech Connect

    Lee, Yoon Jae; Jang, Yo Han; Kim, Paul; Lee, Yun Ha; Lee, Young Jae; Byun, Young Ho; Lee, Kwang-Hee; Kim, Kyusik; Seong, Baik Lin

    2016-04-15

    In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. - Highlights: • Cold-adaptation process induced four amino acid mutations in the HA of X-31 virus. • The four mutations in the HA also contributed to attenuation of the X-31ca virus • N81K mutation was the most significant marker for the attenuation of X-31ca virus. • Introduction of N81K mutation into H3N2 LAIV further attenuated the vaccine. • This approach provides a useful guideline for enhancing the safety of the LAIVs.

  11. Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin.

    PubMed

    Lee, Yoon Jae; Jang, Yo Han; Kim, Paul; Lee, Yun Ha; Lee, Young Jae; Byun, Young Ho; Lee, Kwang-Hee; Kim, Kyusik; Seong, Baik Lin

    2016-04-01

    In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. The cold adapted and temperature sensitive influenza A/Ann Arbor/6/60 virus, the master donor virus for live attenuated influenza vaccines, has multiple defects in replication at the restrictive temperature

    SciTech Connect

    Chan, Winnie; Zhou, Helen; Kemble, George; Jin Hong

    2008-10-25

    We have previously determined that the temperature sensitive (ts) and attenuated (att) phenotypes of the cold adapted influenza A/Ann Arbor/6/60 strain (MDV-A), the master donor virus for the live attenuated influenza A vaccines (FluMist), are specified by the five amino acids in the PB1, PB2 and NP gene segments. To understand how these loci control the ts phenotype of MDV-A, replication of MDV-A at the non-permissive temperature (39 deg. C) was compared with recombinant wild-type A/Ann Arbor/6/60 (rWt). The mRNA and protein synthesis of MDV-A in the infected MDCK cells were not significantly reduced at 39 deg. C during a single-step replication, however, vRNA synthesis was reduced and the nuclear-cytoplasmic export of viral RNP (vRNP) was blocked. In addition, the virions released from MDV-A infected cells at 39 deg. C exhibited irregular morphology and had a greatly reduced amount of the M1 protein incorporated. The reduced M1 protein incorporation and vRNP export blockage correlated well with the virus ts phenotype because these defects could be partially alleviated by removing the three ts loci from the PB1 gene. The virions and vRNPs isolated from the MDV-A infected cells contained a higher level of heat shock protein 70 (Hsp70) than those of rWt, however, whether Hsp70 is involved in thermal inhibition of MDV-A replication remains to be determined. Our studies demonstrate that restrictive replication of MDV-A at the non-permissive temperature occurs in multiple steps of the virus replication cycle.

  13. [Further development (MDCK) of live cold-adapted influenza vaccine: cultivation of vaccine strains in production fermenters].

    PubMed

    Gendon, Iu Z; Markushin, S G; Akopova, I I; Koptiaeva, I B; Nechaeva, E A; Mazurkova, I A; Radaeva, I F; Kolokol'tsova, T D

    2005-01-01

    Optimal conditions were developed for cultivating the cold-adapted reassortant live influenza vaccine (CARLIV) in MDCK cells, which were in their turn cultivated in fermenters with serum-free medium and microcarrier. The use of MDCK cells meets all national and WHO requirements to continuous cells used in the production of biological preparations. CARLIV cultivated under such conditions well preserve their ts-mutations and mutation, which entail substitutions of amino acids, in all CARLIV genome segments. Provided the cultivation conditions are optimal, the output of multivalent CARLIV in a 101 fermenter can reach 100000 doses.

  14. Efficacy and effectiveness of live attenuated influenza vaccine in school-age children.

    PubMed

    Coelingh, Kathleen; Olajide, Ifedapo Rosemary; MacDonald, Peter; Yogev, Ram

    2015-01-01

    Evidence of high efficacy of live attenuated influenza vaccine (LAIV) from randomized controlled trials is strong for children 2-6 years of age, but fewer data exist for older school-age children. We reviewed the published data on efficacy and effectiveness of LAIV in children ≥5 years. QUOSA (Elsevier database) was searched for articles published from January 1990 to June 2014 that included 'FluMist', 'LAIV', 'CAIV', 'cold adapted influenza vaccine', 'live attenuated influenza vaccine', 'live attenuated cold adapted' or 'flu mist'. Studies evaluated included randomized controlled trials, effectiveness and indirect protection studies. This review demonstrates that LAIV has considerable efficacy and effectiveness in school-age children.

  15. Influenza virus vaccine live intranasal--MedImmune vaccines: CAIV-T, influenza vaccine live intranasal.

    PubMed

    2003-01-01

    MedImmune Vaccines (formerly Aviron) has developed a cold-adapted live influenza virus vaccine [FluMist] that can be administered by nasal spray. FluMist is the first live virus influenza vaccine and also the first nasally administered vaccine to be marketed in the US. The vaccine will be formulated to contain live attenuated (att) influenza virus reassortants of the strains recommended by the US Public Health Service for each 'flu season. The vaccine is termed cold-adapted (ca) because the virus has been adapted to replicate efficiently at 25 degrees C in the nasal passages, which are below normal body temperature. The strains used in the seasonal vaccine will also be made temperature sensitive (ts) so that their replication is restricted at 37 degrees C (Type B strains) and 39 degrees C (Type A strains). The combined effect of the antigenic properties and the att, ca and ts phenotypes of the influenza strains contained in the vaccine enables the viruses to replicate in the nasopharynx to produce protective immunity. The original formulation of FluMist requires freezer storage throughout distribution. Because many international markets do not have distribution channels well suited to the sale of frozen vaccines, Wyeth and MedImmune are collaborating to develop a second generation, refrigerator-stable, liquid trivalent cold-adapted influenza vaccine (CAIV-T), which is in phase III trials. Initially, the frozen formulation will only be available in the US. For the 2003-2004 season, FluMist will contain A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2) (A/Moscow/10/99-like) and B/Hong Kong/330/2001. Aviron was acquired by MedImmune on 15 January 2002. Aviron is now a wholly-owned subsidiary of MedImmune and is called MedImmune Vaccines. Aviron acquired FluMist in March 1995 through a Co-operative Research and Development Agreement (CRADA) with the US NIAID, and a licensing agreement with the University of Michigan, Ann Arbor, USA. In June 2000, the CRADA was

  16. Elucidation of the molecular basis for the attenuation of a live, attenuated influenza A H5N1 cold-adapted vaccine virus

    USDA-ARS?s Scientific Manuscript database

    A recombinant, live influenza A H5N1 vaccine candidate with the hemagglutinin (HA) and neuraminidase (NA) genes derived from A/VietNam/1203/04 (H5N1) (H5N1 2004 wt) and the internal protein genes from A/Ann Arbor/6/60 (AA) (H2N2) cold-adapted (ca) virus has been previously shown to be attenuated in ...

  17. Evaluation of two live attenuated cold-adapted H5N1 influenza virus vaccines in healthy adults

    PubMed Central

    Karron, Ruth A.; Talaat, Kawsar; Luke, Catherine; Callahan, Karen; Thumar, Bhagvanji; DiLorenzo, Susan; McAuliffe, Josephine; Schappell, Elizabeth; Suguitan, Amorsolo; Mills, Kimberly; Chen, Grace; Lamirande, Elaine; Coelingh, Kathleen; Jin, Hong; Murphy, Brian R.; Kemble, George; Subbarao, Kanta

    2016-01-01

    Background Development of live attenuated influenza vaccines (LAIV) against avian viruses with pandemic potential is an important public health strategy. Methods and Findings We performed open-label trials to evaluate the safety, infectivity, and immunogenicity of H5N1 VN 2004 AA ca and H5N1 HK 2003 AA ca. Each of these vaccines contains a modified H5 hemagglutinin and unmodified N1 neuraminidase from the respective wild-type (wt) parent virus and the six internal protein gene segments of the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. The H5N1 VN 2004 AA ca vaccine virus was evaluated at dosages of 106.7 TCID50 and 107.5 TCID50, and the H5N1 HK 2003 AA ca vaccine was evaluated at a dosage of 107.5 TCID50. Two doses were administered intranasally to healthy adults in isolation at 4 to 8 week intervals. Vaccine safety was assessed through daily examinations and infectivity was assessed by viral culture and by realtime reverse transcription-polymerase chain reaction testing of nasal wash (NW) specimens. Immunogenicity was assessed by measuring hemagglutination-inhibition (HI) antibodies, neutralizing antibodies, and IgG or IgA antibodies to recombinant (r)H5 VN 2004 hemagglutinin (HA) in serum or NW. Fifty-nine participants were enrolled: 21 received 106.7 TCID50 and 21 received 107.5 TCID50 of H5N1 VN 2004 AA ca and 17 received H5N1 HK 2003 AA ca. Shedding of vaccine virus was minimal, as were HI and neutralizing antibody responses. Fifty-two percent of recipients of 107.5 TCID50 of H5N1 VN 2004 AA ca developed a serum IgA response to rH5 VN 2004 HA. Conclusions The live attenuated H5N1 VN 2004 and HK 2003 AA ca vaccines bearing avian H5 HA antigens were very restricted in replication and were more attenuated than seasonal LAIV bearing human H1, H3 or B HA antigens. The H5N1 AA ca LAIV elicited serum ELISA antibody but not HI or neutralizing antibody responses in healthy adults. (ClinicalTrials.gov Identifiers: NCT00347672 and NCT00488046). PMID:19540952

  18. Study of live recombinant cold-adapted influenza bivalent vaccine of type A for use in children: an epidemiological control trial.

    PubMed

    Alexandrova, G I; Budilovsky, G N; Koval, T A; Polezhaev, F I; Garmashova, L M; Ghendon YuZ; Romanova, Y R; Smorodintsev, A A

    1986-06-01

    Live cold-adapted recombinant bivalent vaccine of influenza type A was studied in a controlled field trial in 1982-1983 among nearly 30,000 children 3-15 years old. The bivalent vaccine consisted of recombinants 47/25/1 (H1N1) and 47/7/2 (H3N2) of wild-type viruses A/Brazil/11/78 (H1N1) and A/Bangkok/1/79 (H3N2) with cold-adapted donor A/Leningrad/134/47/57 (H2N2). The recombinants which received mutant nonglycoprotein genes from cold-adapted donor did not suppress each other after simultaneous inoculation of children and stimulated antibody response to both strains. The bivalent vaccine was completely attenuated for children. It caused less than 1% transient febrile reactions during five days after the first vaccination, including double seronegative individuals with low antibody titres to both vaccinal strains. The cold-adapted bivalent vaccine tested proved to be safe for children according to the analysis of morbidity studies among vaccines and a control group performed during the five days and the following six months after the first immunization. There is a similar distribution of non-influenza illnesses and a statistically significant decrease in influenza-like diseases among vaccines compared to the control group. In the four months after the immunization programme was completed, epidemics of influenza A H1N1 and H3N2 occurred. The incidence of influenza-like diseases was approximately 50% less in the vaccinated than in the control groups. This is the first evidence of safety and protective efficacy of recombinant live influenza vaccine for children 3-15 years of age.

  19. Replication of live attenuated cold-adapted H2N2 influenza virus vaccine candidates in non human primates.

    PubMed

    Broadbent, Andrew J; Santos, Celia P; Paskel, Myeisha; Matsuoka, Yumiko; Lu, Janine; Chen, Zhongying; Jin, Hong; Subbarao, Kanta

    2015-01-01

    The development of an H2N2 vaccine is a priority in pandemic preparedness planning. We previously showed that a single dose of a cold-adapted (ca) H2N2 live attenuated influenza vaccine (LAIV) based on the influenza A/Ann Arbor/6/60 (AA ca) virus was immunogenic and efficacious in mice and ferrets. However, in a Phase I clinical trial, viral replication was restricted and immunogenicity was poor. In this study, we compared the replication of four H2N2 LAIV candidate viruses, AA ca, A/Tecumseh/3/67 (TEC67 ca), and two variants of A/Japan/305/57 (JAP57 ca) in three non-human primate (NHP) species: African green monkeys (AGM), cynomolgus macaques (CM) and rhesus macaques (RM). One JAP57 ca virus had glutamine and glycine at HA amino acid positions 226 and 228 (Q-G) that binds to α2-3 linked sialic acids, and one had leucine and serine that binds to α2-3 and α2-6 linked residues (L-S). The replication of all ca viruses was restricted, with low titers detected in the upper respiratory tract of all NHP species, however replication was detected in significantly more CMs than AGMs. The JAP57 ca Q-G and TEC67 ca viruses replicated in a significantly higher percentage of NHPs than the AA ca virus, with the TEC67 ca virus recovered from the greatest percentage of animals. Altering the receptor specificity of the JAP57 ca virus from α2-3 to both α2-3 and α2-6 linked sialic acid residues did not significantly increase the number of animals infected or the titer to which the virus replicated. Taken together, our data show that in NHPs the AA ca virus more closely reflects the human experience than mice or ferret studies. We suggest that CMs and RMs may be the preferred species for evaluating H2N2 LAIV viruses, and the TEC67 ca virus may be the most promising H2N2 LAIV candidate for further evaluation.

  20. Replication of live attenuated cold-adapted H2N2 influenza virus vaccine candidates in non human primates

    PubMed Central

    Broadbent, Andrew J.; Santos, Celia P.; Paskel, Myeisha; Matsuoka, Yumiko; Lu, Janine; Chen, Zhongying; Jin, Hong; Subbarao, Kanta

    2014-01-01

    The development of an H2N2 vaccine is a priority in pandemic preparedness planning. We previously showed that a single dose of a cold-adapted (ca) H2N2 live attenuated influenza vaccine (LAIV) based on the influenza A/Ann Arbor/6/60 (AA ca) virus was immunogenic and efficacious in mice and ferrets. However, in a Phase I clinical trial, viral replication was restricted and immunogenicity was poor. In this study, we compared the replication of four H2N2 LAIV candidate viruses, AA ca, A/Tecumseh/3/67 (TEC67 ca), and two variants of A/Japan/305/57 (JAP57 ca) in three non-human primate (NHP) species: African green monkeys (AGM), cynomolgus macaques (CM) and rhesus macaques (RM). One JAP57 ca virus had glutamine and glycine at HA amino acid positions 226 and 228 (Q-G) that binds to α2-3 linked sialic acids, and one had leucine and serine that binds to α2-3 and α2-6 linked residues (L-S). The replication of all ca viruses was restricted, with low titers detected in the upper respiratory tract of all NHP species, however replication was detected in significantly more CMs than AGMs. The JAP57 ca Q-G and TEC67 ca viruses replicated in a significantly higher percentage of NHPs than the AA ca virus, with the TEC67 ca virus recovered from the greatest percentage of animals. Altering the receptor specificity of the JAP57 ca virus from α2-3 to both α2-3 and α2-6 linked sialic acid residues did not significantly increase the number of animals infected or the titer to which the virus replicated. Taken together, our data show that in NHPs the AA ca virus more closely reflects the human experience than mice or ferret studies. We suggest that CMs and RMs may be the preferred species for evaluating H2N2 LAIV viruses, and the TEC67 ca virus may be the most promising H2N2 LAIV candidate for further evaluation. PMID:25444799

  1. Advantage of live attenuated cold-adapted influenza A virus over inactivated vaccine for A/Washington/80 (H3N2) wild-type virus infection.

    PubMed

    Clements, M L; Betts, R F; Murphy, B R

    1984-03-31

    The efficacy of live attenuated cold-adapted (ca) reassortant influenza virus vaccine against experimental challenge with homologous wild-type virus 5 to 8 weeks after vaccination was compared with that of licensed inactivated vaccine in 81 seronegative (haemagglutination-inhibition antibody titre less than or equal to 1:8) college students. At a dose of 10(7.5) 50% tissue culture infectious dose (TCID50) (70 HID50, human 50% infectious doses) the live virus vaccine, given intranasally, completely protected against illness caused by wild-type virus, whereas the inactivated vaccine, administered intramuscularly, provided 72% protection. Wild-type virus was recovered from only 13% of live virus vaccinees (10(7.5) TCID50 dose of ca virus) compared with 63% of inactivated virus vaccinees and the few infected live virus vaccinees shed 1000 times less wild-type virus than did infected inactivated virus vaccinees or unvaccinated controls. This striking reduction in virus shedding suggests that influenza transmission may be more efficiently interrupted with live than with inactivated virus vaccination.

  2. Cold-adapted pandemic 2009 H1N1 influenza virus live vaccine elicits cross-reactive immune responses against seasonal and H5 influenza A viruses.

    PubMed

    Jang, Yo Han; Byun, Young Ho; Lee, Yoon Jae; Lee, Yun Ha; Lee, Kwang-Hee; Seong, Baik Lin

    2012-05-01

    The rapid transmission of the pandemic 2009 H1N1 influenza virus (pH1N1) among humans has raised the concern of a potential emergence of reassortment between pH1N1 and highly pathogenic influenza strains, especially the avian H5N1 influenza virus. Here, we report that the cold-adapted pH1N1 live attenuated vaccine (CApH1N1) elicits cross-reactive immunity to seasonal and H5 influenza A viruses in the mouse model. Immunization with CApH1N1 induced both systemic and mucosal antibodies with broad reactivity to seasonal and H5 strains, including HAPI H5N1 and the avian H5N2 virus, providing complete protection against heterologous and heterosubtypic lethal challenges. Our results not only accentuate the merit of using live attenuated influenza virus vaccines in view of cross-reactivity but also represent the potential of CApH1N1 live vaccine for mitigating the clinical severity of infections that arise from reassortments between pH1N1 and highly pathogenic H5 subtype viruses.

  3. Perfluoroalkyl substance serum concentrations and immune response to FluMist vaccination among healthy adults.

    PubMed

    Stein, Cheryl R; Ge, Yongchao; Wolff, Mary S; Ye, Xiaoyun; Calafat, Antonia M; Kraus, Thomas; Moran, Thomas M

    2016-08-01

    Perfluoroalkyl substances (PFAS) were shown to be immunotoxic in laboratory animals. There is some epidemiological evidence that PFAS exposure is inversely associated with vaccine-induced antibody concentration. We examined immune response to vaccination with FluMist intranasal live attenuated influenza vaccine in relation to four PFAS (perfluorooctanoate, perfluorononanoate, perfluorooctane sulfonate, perfluorohexane sulfonate) serum concentrations among 78 healthy adults vaccinated during the 2010-2011 influenza season. We measured anti-A H1N1 antibody response and cytokine and chemokine concentrations in serum pre-vaccination, 3 days post-vaccination, and 30 days post-vaccination. We measured cytokine, chemokine, and mucosal IgA concentration in nasal secretions 3 days post-vaccination and 30 days post-vaccination. Adults with higher PFAS concentrations were more likely to seroconvert after FluMist vaccination as compared to adults with lower PFAS concentrations. The associations, however, were imprecise and few participants seroconverted as measured either by hemagglutination inhibition (9%) or immunohistochemical staining (25%). We observed no readily discernable or consistent pattern between PFAS concentration and baseline cytokine, chemokine, or mucosal IgA concentration, or between PFAS concentration and change in these immune markers between baseline and FluMist-response states. The results of this study do not support a reduced immune response to FluMist vaccination among healthy adults in relation to serum PFAS concentration. Given the study's many limitations, however, it does not rule out impaired vaccine response to other vaccines or vaccine components in either children or adults. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Perfluoroalkyl Substance Serum Concentrations and Immune Response to FluMist Vaccination among Healthy Adults

    PubMed Central

    Stein, Cheryl R; Ge, Yongchao; Wolff, Mary S; Ye, Xiaoyun; Calafat, Antonia M; Kraus, Thomas; Moran, Thomas M

    2016-01-01

    Perfluoroalkyl substances (PFAS) were shown to be immunotoxic in laboratory animals. There is some epidemiological evidence that PFAS exposure is inversely associated with vaccine-induced antibody concentration. We examined immune response to vaccination with FluMist intranasal live attenuated influenza vaccine in relation to four PFAS (perfluorooctanoate, perfluorononanoate, perfluorooctane sulfonate, perfluorohexane sulfonate) serum concentrations among 78 healthy adults vaccinated during the 2010 – 2011 influenza season. We measured anti-A H1N1 antibody response and cytokine and chemokine concentrations in serum pre-vaccination, 3 days post-vaccination, and 30 days post-vaccination. We measured cytokine, chemokine, and mucosal IgA concentration in nasal secretions 3 days post-vaccination and 30 days post-vaccination. Adults with higher PFAS concentrations were more likely to seroconvert after FluMist vaccination as compared to adults with lower PFAS concentrations. The associations, however, were imprecise and few participants seroconverted as measured either by hemagglutination inhibition (9%) or immunohistochemical staining (25%). We observed no readily discernable or consistent pattern between PFAS concentration and baseline cytokine, chemokine, or mucosal IgA concentration, or between PFAS concentration and change in these immune markers between baseline and FluMist-response states. The resuts of this study do not support a reduced immune response to FluMist vaccination among healthy adults in relation to serum PFAS concentration. Given the study’s many limitations, however, it does not rule out impaired vaccine response to other vaccines or vaccine components in either children or adults. PMID:27208468

  5. Human whole body cold adaptation

    PubMed Central

    Daanen, Hein A.M.; Van Marken Lichtenbelt, Wouter D.

    2016-01-01

    ABSTRACT Reviews on whole body human cold adaptation generally do not distinguish between population studies and dedicated acclimation studies, leading to confusing results. Population studies show that indigenous black Africans have reduced shivering thermogenesis in the cold and poor cold induced vasodilation in fingers and toes compared to Caucasians and Inuit. About 40,000 y after humans left Africa, natives in cold terrestrial areas seems to have developed not only behavioral adaptations, but also physiological adaptations to cold. Dedicated studies show that repeated whole body exposure of individual volunteers, mainly Caucasians, to severe cold results in reduced cold sensation but no major physiological changes. Repeated cold water immersion seems to slightly reduce metabolic heat production, while repeated exposure to milder cold conditions shows some increase in metabolic heat production, in particular non-shivering thermogenesis. In conclusion, human cold adaptation in the form of increased metabolism and insulation seems to have occurred during recent evolution in populations, but cannot be developed during a lifetime in cold conditions as encountered in temperate and arctic regions. Therefore, we mainly depend on our behavioral skills to live in and survive the cold. PMID:27227100

  6. Evaluation of replication, immunogenicity and protective efficacy of a live attenuated cold-adapted pandemic H1N1 influenza virus vaccine in non-human primates.

    PubMed

    Boonnak, Kobporn; Paskel, Myeisha; Matsuoka, Yumiko; Vogel, Leatrice; Subbarao, Kanta

    2012-08-17

    We studied the replication of influenza A/California/07/09 (H1N1) wild type (CA09wt) virus in two non-human primate species and used one of these models to evaluate the immunogenicity and protective efficacy of a live attenuated cold-adapted vaccine, which contains the hemagglutinin and neuraminidase from the H1N1 wild type (wt) virus and six internal protein gene segments of the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. We infected African green monkeys (AGMs) and rhesus macaques with 2×10(6) TCID(50) of CA09wt and CA09ca influenza viruses. The virus CA09wt replicated in the upper respiratory tract of all animals but the titers in upper respiratory tract tissues of rhesus macaques were significant higher than in AGMs (mean peak titers 10(4.5) TCID(50)/g and 10(2.0) TCID(50)/g on days 4 and 2 post-infection, respectively; p<0.01). Virus replication was observed in the lungs of all rhesus macaques (10(2.0)-10(5.4) TCID(50)/g) whereas only 2 out of 4 AGMs had virus recovered from the lungs (10(2.5)-10(3.5) TCID(50)/g). The CA09ca vaccine virus was attenuated and highly restricted in replication in both AGMs and rhesus macaques. We evaluated the immunogenicity and protective efficacy of the CA09ca vaccine in rhesus macaques because CA09wt virus replicated more efficiently in this species. One or two doses of vaccine were administered intranasally and intratracheally to rhesus macaques. For the two-dose group, the vaccine was administered 4-weeks apart. Immunogenicity was assessed by measuring hemagglutination-inhibiting (HAI) antibodies in the serum and specific IgA antibodies to CA09wt virus in the nasal wash. One or two doses of the vaccine elicited a significant rise in HAI titers (range 40-320). Two doses of CA09ca elicited higher pH1N1-specific IgA titers than in the mock-immunized group (p<0.01). Vaccine efficacy was assessed by comparing titers of CA09wt challenge virus in the respiratory tract of mock-immunized and CA09ca vaccinated monkeys

  7. A live attenuated cold-adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets

    SciTech Connect

    Joseph, Tomy; McAuliffe, Josephine; Lu, Bin; Vogel, Leatrice; Swayne, David; Jin, Hong; Kemble, George; Subbarao, Kanta

    2008-08-15

    The appearance of human infections caused by avian influenza A H7 subtype viruses underscores their pandemic potential and the need to develop vaccines to protect humans from viruses of this subtype. A live attenuated H7N3 virus vaccine was generated by reverse genetics using the HA and NA genes of a low pathogenicity A/chicken/BC/CN-6/04 (H7N3) virus and the six internal protein genes of the cold-adapted A/Ann Arbor/6/60 ca (H2N2) virus. The reassortant H7N3 BC 04 ca vaccine virus was temperature sensitive and showed attenuation in mice and ferrets. Intranasal immunization with one dose of the vaccine protected mice and ferrets when challenged with homologous and heterologous H7 viruses. The reassortant H7N3 BC 04 ca vaccine virus showed comparable levels of attenuation, immunogenicity and efficacy in mice and ferret models. The safety, immunogenicity, and efficacy of this vaccine in mice and ferrets support the evaluation of this vaccine in clinical trials.

  8. A live attenuated cold-adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets.

    PubMed

    Joseph, Tomy; McAuliffe, Josephine; Lu, Bin; Vogel, Leatrice; Swayne, David; Jin, Hong; Kemble, George; Subbarao, Kanta

    2008-08-15

    The appearance of human infections caused by avian influenza A H7 subtype viruses underscores their pandemic potential and the need to develop vaccines to protect humans from viruses of this subtype. A live attenuated H7N3 virus vaccine was generated by reverse genetics using the HA and NA genes of a low pathogenicity A/chicken/BC/CN-6/04 (H7N3) virus and the six internal protein genes of the cold-adapted A/Ann Arbor/6/60 ca (H2N2) virus. The reassortant H7N3 BC 04 ca vaccine virus was temperature sensitive and showed attenuation in mice and ferrets. Intranasal immunization with one dose of the vaccine protected mice and ferrets when challenged with homologous and heterologous H7 viruses. The reassortant H7N3 BC 04 ca vaccine virus showed comparable levels of attenuation, immunogenicity and efficacy in mice and ferret models. The safety, immunogenicity, and efficacy of this vaccine in mice and ferrets support the evaluation of this vaccine in clinical trials.

  9. [Molecular mechanisms of reversions to the ts+ -phenotype of cold-adapted influenza virus A strains--attenuation donors for live influenza reassortant vaccines].

    PubMed

    Markushin, S G; Akopova, I I; Koptiaeva, I B; Tsfasman, T M; Gendon, Iu Z

    2006-01-01

    A ts+ revertant of cold-adapted (ca) strain A/Leningrad/134/47/57--the attenuation donor for live influenza reassortant vaccines--was obtained by passages of the ca strain in chick embryos at nonpermissive temperatures. The ts+ revertant acquired the ability to grow in chick embryos at 40 degrees C and lost the capacity to reproduce there at 25 degrees C. A complementation-recombination test using the fowl plague virus (FPV0 ts-mutants showed the loss of the ts-phenotype in the RNA-segments of ts+ revertants' genome coding for PB2, NP, and NS (NS2) proteins. However, PCR-restriction analysis revealed a true reversion in RNA-segment coding for PB2 protein only. All the investigated mutations in the ts+ revertant genome were preserved. This phenomenon could be explained by the appearance of intragenic and extragenic suppression mutations in the ts+ revertant genome. The data of the complementation-recombination test suggest that reversion of ts-phenotype occurs more frequently due to extra- or intragenic suppression rather than as a result of a true mutation loss. Estimation of the genetic stability of vaccine ca strains of influenza virus should be based on the combined use of PCR-restriction and complementation tests.

  10. Duration of the protective immune response after prime and booster vaccination of yearlings with a live modified cold-adapted viral vaccine against equine influenza.

    PubMed

    Tabynov, K; Kydyrbayev, Zh; Ryskeldinova, Sh; Assanzhanova, N; Sansyzbay, A

    2014-05-23

    We previously created a live vaccine against equine influenza based the new reassortant cold-adapted (Ca) strain A/HK/Otar/6:2/2010. The live vaccine contains surface proteins (HA, NA) from the wild-type virus A/equine/Otar/764/2007 (Н3N8; American Lineage Florida Clade 2), and internal proteins (PB2, PB1, PA, NP, M, NS) from the attenuated Ca donor virus A/Hong Kong/1/68/162/35CA (H3N2). To determine the safety and duration of the protective immune responses, 90 yearlings were intranasally vaccinated in single mode, double mode at an interval of 42 days (10(7.0) EID50/animal for both vaccinations), or with PBS (control group). Ten animals from each group were challenged with the homologous wild-type virus A/equine/Otar/764/07 (Н3N8) at 1, 2, 3, 4, 5, 6, 9 and 12 months after vaccination. Similarly, 10 animals from each group were challenged with the heterologous wild-type virus A/equine/Sydney/2888-8/07 (Н3N8; American Lineage Florida Clade 1) 12 months after vaccination. The vaccine was completely safe, and single intranasal vaccination of yearlings was capable of inducing statistically significant (from P=0.03 to P<0.0001) clinical and virological protection against the homologous virus; however, only double mode vaccination generated significant (from P=0.02 to P<0.0001) protection against the heterologous virus at 12 months (observation period). Interestingly, this vaccine enables the differentiation of infected and vaccinated animals. On this basis of this study, we recommend double intranasal administration of this vaccine at an interval of 42 days in veterinary practice.

  11. Immunogenicity and protective efficacy of cold-adapted X-31 live attenuated pre-pandemic H5N1 influenza vaccines.

    PubMed

    Jang, Yo Han; Jung, Eun-Ju; Byun, Young Ho; Lee, Kwang-Hee; Lee, Eun-Young; Lee, Yoon Jae; Seong, Baik Lin

    2013-07-18

    Despite global efforts to control influenza viruses, they have taken a heavy toll on human public health worldwide. Among particular threats is highly pathogenic avian H5N1 influenza virus (HPAI) due to not only its high mortality in humans but also possible human-to-human transmission either through reassortment with other human influenza viruses such as 2009 pandemic H1N1 influenza virus, or by genetic mutations. With the aim of developing effective vaccines against the H5N1 viruses, we generated two live attenuated H5N1 vaccine candidates against A/Indonesia/05/2005 (clade 2.1) and A/chicken/Korea/ES/2003 (clade 2.5) strains, in the genetic background of the cold-adapted donor strain of X-31. In mice, a single dose of immunization with each of the two vaccines was highly immunogenic inducing high titers of serum viral-neutralizing and hemagglutinin-inhibiting antibodies against the homologous H5N1 strain. Furthermore, significant levels of cross-clade antibody responses were induced by the vaccines, suggesting a broad-spectrum cross-reactivity against the heterologous H5N1 strains. The immunizations provided solid protections against heterologous lethal challenges with H5N2 virus, significantly reducing the morbidity and challenge virus replications in the respiratory tracts. The robustness of the antibody responses against both the homologous and heterologous strains, together with efficient protection against the lethal H5N2 challenge, strongly support the protection against wild type H5N1 infections. These results could serve as an experimental basis for the development of safe and effective H5N1 pre-pandemic vaccines while further addressing the biosecurity concerns associated with H5N1 HPAI.

  12. The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity and efficacy of a live attenuated influenza A h5N1 cold-adapted vaccine virus

    USDA-ARS?s Scientific Manuscript database

    A recombinant live attenuated influenza virus (LAIV) deltaH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and the six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was attenuated in chickens, mice and fe...

  13. Cold-Adapted Enzymes

    NASA Astrophysics Data System (ADS)

    Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

    In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

  14. Safety of the trivalent, cold-adapted influenza vaccine (CAIV-T) in children.

    PubMed

    Piedra, Pedro A

    2002-04-01

    The trivalent, cold-adapted influenza vaccine (CAIV-T, FluMist, Aviron, Mountain View, CA) is a live attenuated influenza virus vaccine that is administered by nasal spray. CAIV-T is efficacious in preventing influenza virus infection. The vaccine was submitted to the Food and Drug Administration for licensure in healthy children and adults. Universal immunization is being considered in children, and an effective vaccine with minimal adverse reactions is thus required. The published studies on the safety of CAIV-T in children reviewed in this article were clinical trials sponsored by the National Institutes of Health (NIH) conducted in children from 1975 to 1991, clinical trials from 1991 to 1993 sponsored by a cooperative agreement between NIH and Wyeth-Ayerst Research, and clinical trials from 1995 to the present sponsored by a cooperative agreement between NIH and Aviron. Safety assessments included the occurrence of: 1) specific influenza-like symptoms, unexpected symptoms, and use of medications within the first 10 days after vaccination; 2) acute illness and use of medication within 11 to 42 days postvaccination; 3) serious adverse events and rare events within 42 days after vaccination; 4) healthcare utilization within 14 days after vaccination; and 5) acute respiratory symptoms with annual sequential vaccine doses. CAIV-T was safe and well-tolerated. Transient, mild respiratory symptoms were observed in a minority (10%-15%) of children and primarily with the first CAIV-T dose. Vomiting and abdominal pain occurred in fewer than 2 percent of CAIV-T recipients. The gastrointestinal symptoms were mild and of short duration. An excess of illness or use of medication was not observed after the 10th day of vaccination. Sequential annual doses of CAIV-T were well-tolerated and not associated with increased reactogenicity. CAIV-T did not cause an increase in healthcare utilization. Thus CAIV-T is safe in healthy children and should complement the use of inactivated

  15. A live attenuated cold adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets

    USDA-ARS?s Scientific Manuscript database

    The appearance of human infections caused by avian influenza A H7 subtype viruses underscore their pandemic potential and the need to develop vaccines to protect humans from viruses of this subtype. A live attenuated H7N3 virus vaccine was generated by reverse genetics using the HA and NA genes of ...

  16. Identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated cold-passage 45 (cp45) human parainfluenza virus 3 candidate vaccine.

    PubMed

    Skiadopoulos, M H; Surman, S; Tatem, J M; Paschalis, M; Wu, S L; Udem, S A; Durbin, A P; Collins, P L; Murphy, B R

    1999-02-01

    The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp45 independently confers the temperature-sensitive (ts) and attenuation (att) phenotypes but not the cold-adaptation (ca) phenotype (29). cp45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts, ca, and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, and att phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3' N region (i.e., including the leader, N GS, and N coding changes) contributes to the ts phenotype. A recombinant virus containing all the cp45 mutations except those in L was more ts than cp45, illustrating the complex nature of this phenotype. The ca phenotype of cp45 also is a complex composite phenotype

  17. Identification of Mutations Contributing to the Temperature-Sensitive, Cold-Adapted, and Attenuation Phenotypes of the Live-Attenuated Cold-Passage 45 (cp45) Human Parainfluenza Virus 3 Candidate Vaccine

    PubMed Central

    Skiadopoulos, Mario H.; Surman, Sonja; Tatem, Joanne M.; Paschalis, Maribel; Wu, Shin-Lu; Udem, Stephen A.; Durbin, Anna P.; Collins, Peter L.; Murphy, Brian R.

    1999-01-01

    The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp45 independently confers the temperature-sensitive (ts) and attenuation (att) phenotypes but not the cold-adaptation (ca) phenotype (29). cp45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts, ca, and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, and att phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3′ N region (i.e., including the leader, N GS, and N coding changes) contributes to the ts phenotype. A recombinant virus containing all the cp45 mutations except those in L was more ts than cp45, illustrating the complex nature of this phenotype. The ca phenotype of cp45 also is a complex composite phenotype

  18. Comparison of live, attenuated H1N1 and H3N2 cold-adapted and avian-human influenza A reassortant viruses and inactivated virus vaccine in adults.

    PubMed

    Sears, S D; Clements, M L; Betts, R F; Maassab, H F; Murphy, B R; Snyder, M H

    1988-12-01

    The infectivity, immunogenicity, and efficacy of live, attenuated influenza A/Texas/1/85 (H1N1) and A/Bethesda/1/85 (H3N2) avian-human (ah) and cold-adapted (ca) reassortant vaccines were compared in 252 seronegative adult volunteers. The immunogenicity and efficacy of the H1N1 reassortant vaccine were also compared with those of the trivalent inactivated virus vaccine. Each reassortant vaccine was satisfactorily attenuated. The 50% human infectious dose was 10(4.9) for ca H1N1, 10(5.4) for ah H1N1, 10(6.4) for ca H3N2, and 10(6.5) TCID50 for ah H3N2 reassortant virus. Within a subtype, the immunogenicities of ah and ca vaccines were comparable. Five to seven weeks after vaccination, volunteers were challenged with homologous wild-type influenza A virus. The magnitude of shedding of virus after challenge was greater than 100-fold less in H1N1 vaccinees and greater than 10-fold less in H3N2 vaccinees compared with unimmunized controls. The vaccines were equally efficacious, as indicated by an 86%-100% reduction in illness. Thus, the ah A/Mallard/New York/6750/78 and the ca A/Ann Arbor/6/60 reassortant viruses are comparable.

  19. Imparting temperature sensitivity and attenuation in ferrets to A/Puerto Rico/8/34 influenza virus by transferring the genetic signature for temperature sensitivity from cold-adapted A/Ann Arbor/6/60.

    PubMed

    Jin, Hong; Zhou, Helen; Lu, Bin; Kemble, George

    2004-01-01

    The four temperature-sensitive (ts) loci identified in the PB1 and PB2 gene segments of cold-adapted A/Ann Arbor/6/60 influenza virus, the master donor virus for influenza A virus (MDV-A) FluMist vaccines, were introduced into a divergent A/Puerto Rico/8/34 influenza virus strain. Recombinant A/Puerto Rico/8/34 virus with these four introduced ts loci exhibited both ts and att phenotypes similar to those of MDV-A, which could be used as a donor virus for manufacturing large quantities of inactivated influenza virus vaccine against potential pandemic strains.

  20. [Selection and Identification of the Biological Characteristics of a Cold-adapted Genotype G1P[8] ZTR-68 Rotavirus by Serial Cold-adapted Passaging].

    PubMed

    Xie, Li; Mi, Kai; Ye, Jing; Niu, Xianglian; Sun, Xiaoqin; Yi, Shan; Li, Hongjun; Sun, Maosheng

    2015-09-01

    We wished to select a cold-adapted genotype G1P[8] ZTR-68 rotavirus (China southwest strain) in MA104 cells for possible use as a live vaccine. ZTR-68 was recovered originally from children with diarrhea. The virus was cultivated at 37 degrees C at the first passage. Then, the cultivation temperature was decreased stepwise by 3 degrees C per eight passages. In total, the virus was passaged 32 times, and cultivation was terminated at 28 degrees C. Biological characteristics of the virus were analyzed during serial passages. There was no difference between the migration patterns of genomic dsRNA segments according to polyacrylamide gel electrophoresis of original and cold-adapted viruses. Infectious and red cell-agglutination titers of cold-adapted virus were lower than those of the parent virus. Also, the virus formed small-size plaques with irregular shapes at 31 degrees C and 28 degrees C. These results suggested that a genetically stable attenuated virus can be obtained through serial cold-adapted passages. Thus, an alternative strategy is provided by cold-adaption for development of attenuated live rotavirus vaccines.

  1. Different types of cold adaptation in humans.

    PubMed

    Makinen, Tiina Maria

    2010-06-01

    Human adaptation to cold may occur through acclimatization or acclimation and includes genetic, physiologic, morphological or behavioural responses. It has been studied in indigenous populations, during polar or ski expeditions, sporting activities, military training, in urban people, or under controlled conditions involving exposures to cold air or water. Although divergent results exist between the studies, the main cold adaptation responses are either insulative (circulatory adjustments, increase of fat layer) or metabolic (shivering or nonshivering thermogenesis) and may be positive (enhanced) or negative (blunted). The pattern of cold adaptation is dependent on the type (air, water) and intensity (continuous, intermittent) of the cold exposure. In addition, several individual factors like age, sex, body composition, exercise, diet, fitness and health modify the responses to cold. Habituation of thermal sensations to cold develops first, followed by cardiovascular, metabolic and endocrinological responses. If the repeated cold stimulus is discontinued, adaptation will gradually disappear. The functional significance of physiological cold adaptation is unclear, and some of the responses can even be harmful and predispose to cold injuries. The article summarises recent research information concerning with the thermoregulatory responses related to repeated exposures to cold (air or water), and also discusses the determinants of cold adaptation, as well as its functional significance.

  2. Proteomic analysis of endothelial cold-adaptation

    PubMed Central

    2011-01-01

    Background Understanding how human cells in tissue culture adapt to hypothermia may aid in developing new clinical procedures for improved ischemic and hypothermic protection. Human coronary artery endothelial cells grown to confluence at 37°C and then transferred to 25°C become resistant over time to oxidative stress and injury induced by 0°C storage and rewarming. This protection correlates with an increase in intracellular glutathione at 25°C. To help understand the molecular basis of endothelial cold-adaptation, isolated proteins from cold-adapted (25°C/72 h) and pre-adapted cells were analyzed by quantitative proteomic methods and differentially expressed proteins were categorized using the DAVID Bioinformatics Resource. Results Cells adapted to 25°C expressed changes in the abundance of 219 unique proteins representing a broad range of categories such as translation, glycolysis, biosynthetic (anabolic) processes, NAD, cytoskeletal organization, RNA processing, oxidoreductase activity, response-to-stress and cell redox homeostasis. The number of proteins that decreased significantly with cold-adaptation exceeded the number that increased by 2:1. Almost half of the decreases were associated with protein metabolic processes and a third were related to anabolic processes including protein, DNA and fatty acid synthesis. Changes consistent with the suppression of cytoskeletal dynamics provided further evidence that cold-adapted cells are in an energy conserving state. Among the specific changes were increases in the abundance and activity of redox proteins glutathione S-transferase, thioredoxin and thioredoxin reductase, which correlated with a decrease in oxidative stress, an increase in protein glutathionylation, and a recovery of reduced protein thiols during rewarming from 0°C. Increases in S-adenosylhomocysteine hydrolase and nicotinamide phosphoribosyltransferase implicate a central role for the methionine-cysteine transulfuration pathway in increasing

  3. Could human cold adaptation decrease the risk of cardiovascular disease?

    PubMed

    Kralova Lesna, I; Rychlikova, J; Vavrova, L; Vybiral, S

    2015-08-01

    The impact of repeated exposure to cold and cold adaptation on human cardiovascular health is not fully understood. The aim of the study was to determine the effect of cold adaptation on cardiovascular risk factors, thyroid hormones and the capacity of humans to reset the damaging effect of oxidative stress. Ten well cold-adapted winter swimmers (CA) and 16 non-adapted controls (CON) were enroled in this experiment to test whether cold adaptation could influence the parameters of lipoprotein metabolism, cholesterol efflux capacity (CEC), homocysteine, thyroid hormones, antioxidant defence markers (reduced glutathione (GSH), glutathione peroxidase 1 (GPX1), glutathione reductase (GR), catalase (CAT) and paraoxonase 1 (PON1)) and oxidative stress markers (concentration of conjugated dienes (CD)). A decreased apolipoprotein B/apolipoprotein A1 (ApoB/ApoA1) ratio was found in the CA group (p<0.05), but other lipoprotein parameters, including CEC, did not differ significantly. Plasma homocysteine was lower in CA subjects in comparison with controls (p<0.05). Higher triiodothyronine (T3) values were observed in the CA compared to the CON (p<0.05) group, but TSH and other thyroid hormones did not differ between both groups. CA subjects had lower activity of GPX1 (p<0.05), lower concentrations of CD (p<0.05) and increased activities of PON1 (p<0.001) compared to CON subjects. A trend for decreased activity of CAT (p=0.06) in CA compared to CON groups was also observed, but GSH levels did not differ significantly. Zn concentration was higher in the CA group than in the CON group (p<0.001). Human cold adaptation can influence oxidative stress markers. Trends towards the improvement of cardiovascular risk factors in cold-adapted subjects also indicate the positive effect of cold adaptation on cardio-protective mechanisms. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Comparative studies of infectivity, immunogenicity and cross-protective efficacy of live attenuated influenza vaccines containing nucleoprotein from cold-adapted or wild-type influenza virus in a mouse model.

    PubMed

    Isakova-Sivak, Irina; Korenkov, Daniil; Smolonogina, Tatiana; Tretiak, Tatiana; Donina, Svetlana; Rekstin, Andrey; Naykhin, Anatoly; Shcherbik, Svetlana; Pearce, Nicholas; Chen, Li-Mei; Bousse, Tatiana; Rudenko, Larisa

    2017-01-01

    This study sought to improve an existing live attenuated influenza vaccine (LAIV) by including nucleoprotein (NP) from wild-type virus rather than master donor virus (MDV). H7N9 LAIV reassortants with 6:2 (NP from MDV) and 5:3 (NP from wild-type virus) genome compositions were compared with regard to their growth characteristics, induction of humoral and cellular immune responses in mice, and ability to protect mice against homologous and heterologous challenge viruses. Although, in general, the 6:2 reassortant induced greater cell-mediated immunity in C57BL6 mice than the 5:3 vaccine, mice immunized with the 5:3 LAIV were better protected against heterologous challenge. The 5:3 LAIV-induced CTLs also had better in vivo killing activity against target cells loaded with the NP366 epitope of recent influenza viruses. Modification of the genome of reassortant vaccine viruses by incorporating the NP gene from wild-type viruses represents a simple strategy to improve the immunogenicity and cross-protection of influenza vaccines. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Comparative studies of infectivity, immunogenicity and cross-protective efficacy of live attenuated influenza vaccines containing nucleoprotein from cold-adapted or wild-type influenza virus in a mouse model

    PubMed Central

    Isakova-Sivak, Irina; Korenkov, Daniil; Smolonogina, Tatiana; Tretiak, Tatiana; Donina, Svetlana; Rekstin, Andrey; Naykhin, Anatoly; Shcherbik, Svetlana; Pearce, Nicholas; Chen, Li-Mei; Bousse, Tatiana; Rudenko, Larisa

    2016-01-01

    This study sought to improve an existing live attenuated influenza vaccine (LAIV) by including nucleoprotein (NP) from wild-type virus rather than master donor virus (MDV). H7N9 LAIV reassortants with 6:2 (NP from MDV) and 5:3 (NP from wild-type virus) genome compositions were compared with regard to their growth characteristics, induction of humoral and cellular immune responses in mice, and ability to protect mice against homologous and heterologous challenge viruses. Although, in general, the 6:2 reassortant induced greater cell-mediated immunity in C57BL6 mice than the 5:3 vaccine, mice immunized with the 5:3 LAIV were better protected against heterologous challenge. The 5:3 LAIV-induced CTLs also had better in vivo killing activity against target cells loaded with the NP366 epitope of recent influenza viruses. Modification of the genome of reassortant vaccine viruses by incorporating the NP gene from wild-type viruses represents a simple strategy to improve the immunogenicity and cross-protection of influenza vaccines. PMID:27829176

  6. Cold adaptation of parainfluenza virus type 3: induction of three phenotypic markers.

    PubMed

    Belshe, R B; Hissom, F K

    1982-01-01

    In order to attenuate parainfluenza type 3 virus, a wild type strain that was isolated from a child with respiratory disease was adapted to replicate in African green monkey kidney cells at 20 degrees C. Replication at 20 degrees C was not a property of the wild type virus. The virus was serially passaged 45 times in the cold, and clones were selected following passage levels 7, 12, 18, and 45. The population of cold-adapted virus was found to be progressively enriched with temperature sensitive (ts) mutants. After 7 passages in the cold, 1 of 9, and after 12 passages in the cold, 3 of 12 clones were temperature sensitive. Following 18 passages in the cold, 80% of the clones were temperature sensitive and after 45 passages in the cold, all clones were temperature sensitive. In addition to being temperature sensitive each ts clone manifested the tiny plaque morphology. Each temperature-sensitive clone was also cold adapted. Some clones were cold adapted but were not temperature sensitive. The mutants were found to be genetically stable when serially passaged at 32 degrees, 35 degrees, or 39 degrees C. The mutants may possess the necessary degree of attenuation for use as live attenuated intranasal vaccines.

  7. [Genetic stability of cold-adapted influenza viruses].

    PubMed

    Kiseleva, I V; Larionova, N V; Isakova, I N; Rudenko, L G

    2006-01-01

    The stability of cold adaptation, temperature-sensitivity, and marker mutations that are typical of attenuated influenza A and B viruses--master donor strains and their based reassortant vaccine strains was studied. After 5 sequential passages in chick embryos (CE) at resolving temperatures of 32 and 37 degrees C, the master donor strains and vaccine viruses retained their adaptability and temperature sensitive phenotype. Passage at the temperatures maximally permissible for viral reproduction (39 and 38 degrees C for influenza A and B viruses, respectively, aborted infection just during the second passage. After a series of passages at all study temperatures), there was neither loss or nor substitution of the marker mutations typical of the cold-adapted and temperature-sensitive phenotype of attenuated viruses. The study supports the high genetic stability of attenuated cold-adapted influenza A and B viruses during CE passage not only at the optimum, but also at elevated incubation temperatures.

  8. Anti-Biofilm Activities from Marine Cold Adapted Bacteria Against Staphylococci and Pseudomonas aeruginosa

    PubMed Central

    Papa, Rosanna; Selan, Laura; Parrilli, Ermenegilda; Tilotta, Marco; Sannino, Filomena; Feller, Georges; Tutino, Maria L.; Artini, Marco

    2015-01-01

    Microbial biofilms have great negative impacts on the world’s economy and pose serious problems to industry, public health and medicine. The interest in the development of new approaches for the prevention and treatment of bacterial adhesion and biofilm formation has increased. Since, bacterial pathogens living in biofilm induce persistent chronic infections due to the resistance to antibiotics and host immune system. A viable approach should target adhesive properties without affecting bacterial vitality in order to avoid the appearance of resistant mutants. Many bacteria secrete anti-biofilm molecules that function in regulating biofilm architecture or mediating the release of cells from it during the dispersal stage of biofilm life cycle. Cold-adapted marine bacteria represent an untapped reservoir of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. The anti-biofilm activity of cell-free supernatants derived from sessile and planktonic cultures of cold-adapted bacteria belonging to Pseudoalteromonas, Psychrobacter, and Psychromonas species were tested against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa strains. Reported results demonstrate that we have selected supernatants, from cold-adapted marine bacteria, containing non-biocidal agents able to destabilize biofilm matrix of all tested pathogens without killing cells. A preliminary physico-chemical characterization of supernatants was also performed, and these analyses highlighted the presence of molecules of different nature that act by inhibiting biofilm formation. Some of them are also able to impair the initial attachment of the bacterial cells to the surface, thus likely containing molecules acting as anti-biofilm surfactant molecules. The described ability of cold-adapted bacteria to produce effective anti-biofilm molecules paves the way to further characterization of the most promising molecules and to test their

  9. Anti-Biofilm Activities from Marine Cold Adapted Bacteria Against Staphylococci and Pseudomonas aeruginosa.

    PubMed

    Papa, Rosanna; Selan, Laura; Parrilli, Ermenegilda; Tilotta, Marco; Sannino, Filomena; Feller, Georges; Tutino, Maria L; Artini, Marco

    2015-01-01

    Microbial biofilms have great negative impacts on the world's economy and pose serious problems to industry, public health and medicine. The interest in the development of new approaches for the prevention and treatment of bacterial adhesion and biofilm formation has increased. Since, bacterial pathogens living in biofilm induce persistent chronic infections due to the resistance to antibiotics and host immune system. A viable approach should target adhesive properties without affecting bacterial vitality in order to avoid the appearance of resistant mutants. Many bacteria secrete anti-biofilm molecules that function in regulating biofilm architecture or mediating the release of cells from it during the dispersal stage of biofilm life cycle. Cold-adapted marine bacteria represent an untapped reservoir of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. The anti-biofilm activity of cell-free supernatants derived from sessile and planktonic cultures of cold-adapted bacteria belonging to Pseudoalteromonas, Psychrobacter, and Psychromonas species were tested against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa strains. Reported results demonstrate that we have selected supernatants, from cold-adapted marine bacteria, containing non-biocidal agents able to destabilize biofilm matrix of all tested pathogens without killing cells. A preliminary physico-chemical characterization of supernatants was also performed, and these analyses highlighted the presence of molecules of different nature that act by inhibiting biofilm formation. Some of them are also able to impair the initial attachment of the bacterial cells to the surface, thus likely containing molecules acting as anti-biofilm surfactant molecules. The described ability of cold-adapted bacteria to produce effective anti-biofilm molecules paves the way to further characterization of the most promising molecules and to test their

  10. Genetic characterization and protective immunity of cold-adapted attenuated avian H9N2 influenza vaccine.

    PubMed

    Lee, Joo Sub; Kim, Hyun Soo; Seo, Sang Heui

    2008-12-02

    H9N2 influenza viruses are endemic in many Asian countries including China and Korea, and cause a considerable economic loss to chicken industry by reduction in egg production and about 30% mortality. Here we developed live cold-adapted attenuated H9N2 influenza vaccine by adaptation of viruses in hen's eggs at 25 degrees C. Genetic analysis shows that the cold-adapted H9N2 (A/Chicken/Korea/S1/03) viruses contain a total of 44 amino acid substitutions, of which 7 amino acids are identical to the loci identified in the cold-adapted H2N2 (A/Ann Arbor/6/60) vaccine strain compared to genes in wild-type H9N2 (A/Chicken/Korea/S1/03) influenza viruses. When cold-adapted H9N2 (A/Chicken/Korea/S1/03) influenza viruses were inoculated in layers viruses were detectable in the tracheas, not in the lungs, no reduction of egg production and mortality was observed in contrast to the infection of wild-type H9N2 influenza viruses, and CD8+ T lymphocytes expressing IFN-gamma were induced. When layers vaccinated with cold-adapted attenuated H9N2 (A/Chicken/Korea/S1/03) influenza viruses were challenged with wild-type H9N2 (A/Chicken/Korea/521/04) influenza viruses, they were protected from the loss of egg production and mortality. Our results suggest that cold-adapted attenuated H9N2 vaccine can be used for controlling the infection of H9N2 influenza viruses in chickens.

  11. Cold-adapted proteases as an emerging class of therapeutics.

    PubMed

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications.

  12. Cold-adapted vaccine strains of influenza A virus act as dominant negative mutants in mixed infections with wild-type influenza A virus.

    PubMed

    Whitaker-Dowling, P; Lucas, W; Youngner, J S

    1990-04-01

    The cold-adapted reassortant of influenza A, which is a candidate live virus vaccine, interfered with the replication of parental wild-type virus in mixed infections of either MDCK cells or embryonated eggs. The interference occurred at either the permissive or nonpermissive temperature for the cold-adapted virus. In doubly infected cells, the yield of the wild-type virus was reduced by as much as 3000-fold and the protein synthesis phenotype expressed was that of the cold-adapted virus. The interference was detected even when infection with wild-type virus was carried out at a 9-fold excess or 2 hr before infection with the cold-adapted virus. As well as interfering with its wild-type parental virus, the cold-adapted virus also inhibited the replication of a heterologous influenza A subtype. In addition to its immunogenic potential, the ability to interfere with the replication of wild-type viruses is a desirable trait for any live, attenuated virus vaccine.

  13. Highly phosphorylated core oligosaccharide structures from cold-adapted Psychromonas arctica.

    PubMed

    Corsaro, Maria M; Pieretti, Giuseppina; Lindner, Buko; Lanzetta, Rosa; Parrilli, Ermenegilda; Tutino, Maria L; Parrilli, Michelangelo

    2008-01-01

    Many cold habitats contain plenty of microorganisms that represent the most abundant cold-adapted life forms on earth. These organisms have developed a wide range of adaptations that involve the cell wall of the microorganism. In particular, bacteria enhance the synthesis of unsaturated fatty acids of membrane lipids to maintain the membrane fluidity, but very little is known about the adaptational changes in the structure of the lipopolysaccharides (LPSs), the main constituent of the outer leaflet of the outer membrane of Gram-negative bacteria. The aim of this study was to investigate the chemical structure of these LPSs for insight into the temperature-adaptation mechanism. For this objective, the cold-adapted Psychromonas arctica bacterium, which lives in the arctic sea-water near Spitzbergen (Svalbard islands, Arctic) was cultivated at 4 degrees C. The lipooligosaccharides (LOSs) were isolated and analysed by means of chemical analysis and electrospray ionisation high-resolution Fourier transform mass spectrometry. The LOS was then degraded either by mild hydrazinolysis (O-deacylation) or with hot 4 M KOH (N-deacylation). Both products were investigated in detail by using 1H and 13C NMR spectroscopy and mass spectrometry. The core consists of a mixture of species that differ because of the presence of nonstoichiometric D-fructose and/or D-galacturonic acid units.

  14. Generation and protective efficacy of a cold-adapted attenuated avian H9N2 influenza vaccine

    PubMed Central

    Wei, Yandi; Qi, Lu; Gao, Huijie; Sun, Honglei; Pu, Juan; Sun, Yipeng; Liu, Jinhua

    2016-01-01

    To prevent H9N2 avian influenza virus infection in chickens, a long-term vaccination program using inactivated vaccines has been implemented in China. However, the protective efficacy of inactivated vaccines against antigenic drift variants is limited, and H9N2 influenza virus continues to circulate in vaccinated chicken flocks in China. Therefore, developing a cross-reactive vaccine to control the impact of H9N2 influenza in the poultry industry remains a high priority. In the present study, we developed a live cold-adapted H9N2 influenza vaccine candidate (SD/01/10-ca) by serial passages in embryonated eggs at successively lower temperatures. A total of 13 amino acid mutations occurred during the cold-adaptation of this H9N2 virus. The candidate was safe in chickens and induced robust hemagglutination-inhibition antibody responses and influenza virus–specific CD4+ and CD8+ T cell immune responses in chickens immunized intranasally. Importantly, the candidate could confer protection of chickens from homologous and heterogenous H9N2 viruses. These results demonstrated that the cold-adapted attenuated H9N2 virus would be selected as a vaccine to control the infection of prevalent H9N2 influenza viruses in chickens. PMID:27457755

  15. Selection of cold-adapted mutants of human rotaviruses that exhibit various degrees of growth restriction in vitro.

    PubMed Central

    Hoshino, Y; Kapikian, A Z; Chanock, R M

    1994-01-01

    Group A human rotavirus strains D, Wa, DS-1, and P were originally recovered from children with diarrhea. In an attempt to attenuate virulent, wild-type human rotaviruses of major epidemiological importance for use in a live oral vaccine, two reference rotavirus strains, D and DS-1, and two laboratory-generated reassortants, Wa x DS-1 and Wa x P, were subjected to cold adaptation. Collectively, these viruses provide antigenic coverage for both of the clinically important rotavirus VP4 antigens and three of the four important rotavirus VP7 antigens. Mutants of each of these rotaviruses were selected during successive serial passage in primary African green monkey kidney cells at progressively lower suboptimal temperatures (30, 28, and 26 degrees C). The genotype of each mutant appeared to be indistinguishable from that of its wild-type, parental virus. The mutants recovered after 10 serial passages at 30 degrees C exhibited both temperature sensitivity of plaque formation (i.e., a ts phenotype) and the ability to form plaques efficiently at suboptimal temperature (i.e., a cold adaptation [ca] phenotype), in contrast to parental wild-type rotavirus. The succeeding set of 10 serial passages at 28 degrees C selected mutants that exhibited an increased degree of cold adaptation, and three of the mutants exhibited an associated increase in temperature sensitivity. Finally, in the case of three of the strains, the third successive serial passage series, which was performed at 26 degrees C, selected for mutants with an even greater degree of cold adaptation than the previous series and was associated with greater temperature sensitivity in one instance. It appeared that each of the viruses sustained a minimum of four to five mutations during the total selection procedure. The ultimate identification of candidate vaccine viruses that exhibit the desired level of attenuation, immunogenicity, and protective efficacy needed for immunoprophylaxis will require evaluation of

  16. Selection of cold-adapted mutants of human rotaviruses that exhibit various degrees of growth restriction in vitro.

    PubMed

    Hoshino, Y; Kapikian, A Z; Chanock, R M

    1994-11-01

    Group A human rotavirus strains D, Wa, DS-1, and P were originally recovered from children with diarrhea. In an attempt to attenuate virulent, wild-type human rotaviruses of major epidemiological importance for use in a live oral vaccine, two reference rotavirus strains, D and DS-1, and two laboratory-generated reassortants, Wa x DS-1 and Wa x P, were subjected to cold adaptation. Collectively, these viruses provide antigenic coverage for both of the clinically important rotavirus VP4 antigens and three of the four important rotavirus VP7 antigens. Mutants of each of these rotaviruses were selected during successive serial passage in primary African green monkey kidney cells at progressively lower suboptimal temperatures (30, 28, and 26 degrees C). The genotype of each mutant appeared to be indistinguishable from that of its wild-type, parental virus. The mutants recovered after 10 serial passages at 30 degrees C exhibited both temperature sensitivity of plaque formation (i.e., a ts phenotype) and the ability to form plaques efficiently at suboptimal temperature (i.e., a cold adaptation [ca] phenotype), in contrast to parental wild-type rotavirus. The succeeding set of 10 serial passages at 28 degrees C selected mutants that exhibited an increased degree of cold adaptation, and three of the mutants exhibited an associated increase in temperature sensitivity. Finally, in the case of three of the strains, the third successive serial passage series, which was performed at 26 degrees C, selected for mutants with an even greater degree of cold adaptation than the previous series and was associated with greater temperature sensitivity in one instance. It appeared that each of the viruses sustained a minimum of four to five mutations during the total selection procedure. The ultimate identification of candidate vaccine viruses that exhibit the desired level of attenuation, immunogenicity, and protective efficacy needed for immunoprophylaxis will require evaluation of

  17. Molecular and biological changes in the cold-adapted "master strain" A/AA/6/60 (H2N2) influenza virus.

    PubMed Central

    Herlocher, M L; Maassab, H F; Webster, R G

    1993-01-01

    The live cold-adapted (ca) A/AA/6/60 influenza vaccine is being commercially developed for worldwide use in children and is being used as a model for other live vaccines. Although it has been proven safe and immunogenic, the molecular basis of cold adaptation has never been determined. To identify sequence changes responsible for cold adaptation, we have compared the sequence of the master ca vaccine strain to its progenitor wild-type virus, wt A/AA/6/60 E2 (wt2). Only 4 nt differences encoding 2 aa differences were found in three gene segments. Computer-predicted RNA folds project different secondary structures between the ca and wt2 molecules based on the two silent differences between them. Genes coding for the acidic polymerase, matrix, and nonstructural proteins are identical between the two viruses. The few differences found in the ca A/AA/6/60 virus after its long stepwise passage at 25 degrees C in primary chicken kidney cells suggest that cold adaptation resulted in greater genetic stability for the highly variable RNA genome. PMID:8327480

  18. Molecular and biological changes in the cold-adapted "master strain" A/AA/6/60 (H2N2) influenza virus.

    PubMed

    Herlocher, M L; Maassab, H F; Webster, R G

    1993-07-01

    The live cold-adapted (ca) A/AA/6/60 influenza vaccine is being commercially developed for worldwide use in children and is being used as a model for other live vaccines. Although it has been proven safe and immunogenic, the molecular basis of cold adaptation has never been determined. To identify sequence changes responsible for cold adaptation, we have compared the sequence of the master ca vaccine strain to its progenitor wild-type virus, wt A/AA/6/60 E2 (wt2). Only 4 nt differences encoding 2 aa differences were found in three gene segments. Computer-predicted RNA folds project different secondary structures between the ca and wt2 molecules based on the two silent differences between them. Genes coding for the acidic polymerase, matrix, and nonstructural proteins are identical between the two viruses. The few differences found in the ca A/AA/6/60 virus after its long stepwise passage at 25 degrees C in primary chicken kidney cells suggest that cold adaptation resulted in greater genetic stability for the highly variable RNA genome.

  19. From genotype to phenotype: unraveling the complexities of cold adaptation in forest trees

    Treesearch

    Glenn T. Howe; Sally N. Aitken; David B. Neale; Kathleen D. Jermstad; Nicholas C. Wheeler; Tony H.H Chen

    2003-01-01

    Adaptation to winter cold in temperate and boreal trees involves complex genetic, physiological, and developmental processes. Genecological studies demonstrate the existence of steep genetic clines for cold adaptation traits in relation to environmental (mostly temperature related) gradients. Population differentiation is generally stronger for cold adaptation traits...

  20. Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

    PubMed

    Mock, Thomas; Otillar, Robert P; Strauss, Jan; McMullan, Mark; Paajanen, Pirita; Schmutz, Jeremy; Salamov, Asaf; Sanges, Remo; Toseland, Andrew; Ward, Ben J; Allen, Andrew E; Dupont, Christopher L; Frickenhaus, Stephan; Maumus, Florian; Veluchamy, Alaguraj; Wu, Taoyang; Barry, Kerrie W; Falciatore, Angela; Ferrante, Maria I; Fortunato, Antonio E; Glöckner, Gernot; Gruber, Ansgar; Hipkin, Rachel; Janech, Michael G; Kroth, Peter G; Leese, Florian; Lindquist, Erika A; Lyon, Barbara R; Martin, Joel; Mayer, Christoph; Parker, Micaela; Quesneville, Hadi; Raymond, James A; Uhlig, Christiane; Valas, Ruben E; Valentin, Klaus U; Worden, Alexandra Z; Armbrust, E Virginia; Clark, Matthew D; Bowler, Chris; Green, Beverley R; Moulton, Vincent; van Oosterhout, Cock; Grigoriev, Igor V

    2017-01-26

    The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

  1. Anthranilate degradation by a cold-adapted Pseudomonas sp.

    PubMed

    Kim, Dockyu; Yoo, Miyoun; Kim, Eungbin; Hong, Soon Gyu

    2015-03-01

    An alpine soil bacterium Pseudomonas sp. strain PAMC 25931 was characterized as eurypsychrophilic (both psychrophilic and mesotolerant) with a broad temperature range of 5-30 °C both for anthranilate (2-aminobenzoate) degradation and concomitant cell growth. Two degradative gene clusters (antABC and catBCA) were detected from a fosmid clone in the PAMC 25931 genomic library; each cluster was confirmed to be specifically induced by anthranilate. When expressed in Escherichia coli, the recombinant AntABC (anthranilate 1,2-dioxygenase, AntDO) converted anthranilate into catechol, exhibiting strict specificity toward anthranilate. Recombinant CatA (catechol 1,2-dioxygenase, C12O) from the organism was active over a broad temperature range (5-37 °C). However, CatA rapidly lost the enzyme activity when incubated at above 25 °C. For example, 1 h-preincubation at 37 °C resulted in 100% loss of enzyme activity, while a counterpart from mesophilic Pseudomonas putida mt-2 did not show any negative effect on the initial enzyme activity. These results suggest that CatA is a new cold-adapted thermolabile enzyme, which might be a product through the adaptation process of PAMC 25931 to naturally cold environments and contribute to its ability to grow on anthranilate there.

  2. Cold adaptation regulated by cryptic prophage excision in Shewanella oneidensis

    PubMed Central

    Zeng, Zhenshun; Liu, Xiaoxiao; Yao, Jianyun; Guo, Yunxue; Li, Baiyuan; Li, Yangmei; Jiao, Nianzhi; Wang, Xiaoxue

    2016-01-01

    Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation. PMID:27482926

  3. Cold-adapted poliovirus mutants bypass a postentry replication block.

    PubMed

    Dove, A W; Racaniello, V R

    1997-06-01

    In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.

  4. Cold adaptation regulated by cryptic prophage excision in Shewanella oneidensis.

    PubMed

    Zeng, Zhenshun; Liu, Xiaoxiao; Yao, Jianyun; Guo, Yunxue; Li, Baiyuan; Li, Yangmei; Jiao, Nianzhi; Wang, Xiaoxue

    2016-12-01

    Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation.

  5. Genomic and Expression Analyses of Cold-Adapted Microorganisms.

    SciTech Connect

    Bakermans, Corien; Bergholz, Peter W.; Rodrigues, Debora F.; Vishnivetskaya, T.; Ayala-del-Río, Hector L.; Tiedje, James M.

    2011-01-01

    Contents 7.1 Introduction 7.2 Ecological evidence of bacterial adaptation to cold 7.2.1 Characteristics of cold environments and implications for microbial ecology 7.2.2 Ecological adaptation in Exiguobacterium spp. and Psychrobacter spp. 7.3 Gene Expression Responses to the Cold 7.3.1 Fundamentals of Gene Expression Responses to Cold 7.3.2 Acclimation for Life in Cold Habitats 7.3.2.1 Translation and Chaperone Proteins: Safeguarding the functional units of cellular physiology 7.3.2.2 Carbon and Energy metabolism: resource efficiency over long generation times 7.3.2.3 Amino Acid Biosynthesis: Species-specific responses to species-specific deficiencies 7.3.2.4 Compatible solutes: a concomitant response in cryoenvironments 7.3.2.5 Membrane fluidity: A major role in the overall metabolic rate at temperature 7.3.2.6 The cell wall at low temperature: A poorly understood growth rate determinant 7.3.2.7 Transporters: The balance between local nutrient uptake and depletion 7.3.2.8 Genome plasticity. The potential role of transposases and repeated sequences. 7.4 Protein adaptations to cold 7.4.1 The low temperature challenge 7.4.2 The stability activity relationship 7.4.3 Structural features of cold adapted enzymes. 7.4.4 Hydrophobic interactions 7.4.5 Electrostatic interactions 7.4.5.1 Arginine 7.4.5.2 Acidic residues 7.4.6 Structural elements 7.4.6.1 -helices and -sheets 7.4.6.2 Proline and Glycine 7.4.6.3 Disordered regions 7.5 Comparison of cold- and warm-adapted Exiguobacterium strains 7.5.1 Phylogeny reflects adaptations to environmental conditions 7.5.2 Genomic comparison of two strains 7.6 Summary and future directions

  6. [Bioremediation of petroleum hydrocarbon-contaminated soils by cold-adapted microorganisms: research advance].

    PubMed

    Wang, Shi-jie; Wang, Xiang; Lu, Gui-lan; Wang, Qun-hui; Li, Fa-sheng; Guo, Guan-lin

    2011-04-01

    Cold-adapted microorganisms such as psychrotrophs and psychrophiles widely exist in the soils of sub-Arctic, Arctic, Antarctic, alpine, and high mountains, being the important microbial resources for the biodegradation of petroleum hydrocarbons at low temperature. Using the unique advantage of cold-adapted microorganisms to the bioremediation of petroleum hydrocarbon-contaminated soils in low temperature region has become a research hotspot. This paper summarized the category and cold-adaptation mechanisms of the microorganisms able to degrade petroleum hydrocarbon at low temperature, biodegradation characteristics and mechanisms of different petroleum fractions under the action of cold-adapted microorganisms, bio-stimulation techniques for improving biodegradation efficiency, e. g., inoculating petroleum-degrading microorganisms and adding nutrients or bio-surfactants, and the present status of applying molecular biotechnology in this research field, aimed to provide references to the development of bioremediation techniques for petroleum hydrocarbon-contaminated soils.

  7. Biotechnological Potential of Cold Adapted Pseudoalteromonas spp. Isolated from 'Deep Sea' Sponges.

    PubMed

    Borchert, Erik; Knobloch, Stephen; Dwyer, Emilie; Flynn, Sinéad; Jackson, Stephen A; Jóhannsson, Ragnar; Marteinsson, Viggó T; O'Gara, Fergal; Dobson, Alan D W

    2017-06-19

    The marine genus Pseudoalteromonas is known for its versatile biotechnological potential with respect to the production of antimicrobials and enzymes of industrial interest. We have sequenced the genomes of three Pseudoalteromonas sp. strains isolated from different deep sea sponges on the Illumina MiSeq platform. The isolates have been screened for various industrially important enzymes and comparative genomics has been applied to investigate potential relationships between the isolates and their host organisms, while comparing them to free-living Pseudoalteromonas spp. from shallow and deep sea environments. The genomes of the sponge associated Pseudoalteromonas strains contained much lower levels of potential eukaryotic-like proteins which are known to be enriched in symbiotic sponge associated microorganisms, than might be expected for true sponge symbionts. While all the Pseudoalteromonas shared a large distinct subset of genes, nonetheless the number of unique and accessory genes is quite large and defines the pan-genome as open. Enzymatic screens indicate that a vast array of enzyme activities is expressed by the isolates, including β-galactosidase, β-glucosidase, and protease activities. A β-glucosidase gene from one of the Pseudoalteromonas isolates, strain EB27 was heterologously expressed in Escherichia coli and, following biochemical characterization, the recombinant enzyme was found to be cold-adapted, thermolabile, halotolerant, and alkaline active.

  8. Biotechnological Potential of Cold Adapted Pseudoalteromonas spp. Isolated from ‘Deep Sea’ Sponges

    PubMed Central

    Borchert, Erik; Knobloch, Stephen; Dwyer, Emilie; Flynn, Sinéad; Jackson, Stephen A.; Jóhannsson, Ragnar; Marteinsson, Viggó T.; O’Gara, Fergal; Dobson, Alan D. W.

    2017-01-01

    The marine genus Pseudoalteromonas is known for its versatile biotechnological potential with respect to the production of antimicrobials and enzymes of industrial interest. We have sequenced the genomes of three Pseudoalteromonas sp. strains isolated from different deep sea sponges on the Illumina MiSeq platform. The isolates have been screened for various industrially important enzymes and comparative genomics has been applied to investigate potential relationships between the isolates and their host organisms, while comparing them to free-living Pseudoalteromonas spp. from shallow and deep sea environments. The genomes of the sponge associated Pseudoalteromonas strains contained much lower levels of potential eukaryotic-like proteins which are known to be enriched in symbiotic sponge associated microorganisms, than might be expected for true sponge symbionts. While all the Pseudoalteromonas shared a large distinct subset of genes, nonetheless the number of unique and accessory genes is quite large and defines the pan-genome as open. Enzymatic screens indicate that a vast array of enzyme activities is expressed by the isolates, including β-galactosidase, β-glucosidase, and protease activities. A β-glucosidase gene from one of the Pseudoalteromonas isolates, strain EB27 was heterologously expressed in Escherichia coli and, following biochemical characterization, the recombinant enzyme was found to be cold-adapted, thermolabile, halotolerant, and alkaline active. PMID:28629190

  9. Searching for signatures of cold adaptations in modern and archaic humans: hints from the brown adipose tissue genes.

    PubMed

    Sazzini, M; Schiavo, G; De Fanti, S; Martelli, P L; Casadio, R; Luiselli, D

    2014-09-01

    Adaptation to low temperatures has been reasonably developed in the human species during the colonization of the Eurasian landmass subsequent to Out of Africa migrations of anatomically modern humans. In addition to morphological and cultural changes, also metabolic ones are supposed to have favored human isolation from cold and body heat production and this can be hypothesized also for most Neandertal and at least for some Denisovan populations, which lived in geographical areas that strongly experienced the last glacial period. Modulation of non-shivering thermogenesis, for which adipocytes belonging to the brown adipose tissue are the most specialized cells, might have driven these metabolic adaptations. To perform an exploratory analysis aimed at looking into this hypothesis, variation at 28 genes involved in such functional pathway was investigated in modern populations from different climate zones, as well as in Neandertal and Denisovan genomes. Patterns of variation at the LEPR gene, strongly related to increased heat dissipation by mitochondria, appeared to have been shaped by positive selection in modern East Asians, but not in Europeans. Moreover, a single potentially cold-adapted LEPR allele, different from the supposed adaptive one identified in Homo sapiens, was found also in Neandertal and Denisovan genomes. These findings suggest that independent mechanisms for cold adaptations might have been developed in different non-African human groups, as well as that the evolution of possible enhanced thermal efficiency in Neandertals and in some Denisovan populations has plausibly entailed significant changes also in other functional pathways than in the examined one.

  10. Evaluation of cold-adapted and temperature-sensitive mutants of parainfluenza virus type 3 in weanling hamsters.

    PubMed

    Crookshanks, F K; Belshe, R B

    1984-01-01

    Cold-adapted (ca) and temperature-sensitive (ts) mutants of parainfluenza virus 3 were produced by serially passaging wild type (wt) parainfluenza virus 3 at 20 degrees C 45 times. Previously, plaque-purified viruses (clones) were selected from the wt parent and from cold passage levels 7, 12, 18, and 45 and characterized in vitro. In the present study, we evaluated at least one mutant from each cold passage level for attenuation in hamsters. Four of the five mutants tested were ts and ca (one each from cold passage levels 7, 12, 18, and 45) and one was ca but not ts (from cold passage 18). Groups of hamsters were inoculated intranasally with either parent wt or mutant virus. Four or five hamsters from each group were sacrificed prior to inoculation and daily on the first, second, third, fourth, and sixth or seventh day postinoculation and the amount of virus in nasal turbinates and lungs was quantitated. The quantity of virus recovered from the hamsters and the parainfluenza virus 3 antibody titers were inversely related to the cold passage level. Two of the mutants did not replicate in hamsters. Cold adaptation of parainfluenza virus 3 resulted in progressive attenuation of the virus in weanling hamsters. These highly attenuated mutants are suitable for evaluation in children as live virus vaccines.

  11. Efficacy of a cold-adapted, intranasal, equine influenza vaccine: challenge trials.

    PubMed

    Townsend, H G; Penner, S J; Watts, T C; Cook, A; Bogdan, J; Haines, D M; Griffin, S; Chambers, T; Holland, R E; Whitaker-Dowling, P; Youngner, J S; Sebring, R W

    2001-11-01

    A randomised, controlled, double-blind, influenza virus, aerosol challenge of horses was undertaken to determine the efficacy of a cold-adapted, temperature sensitive, modified-live virus, intranasal, equine influenza vaccine. Ninety 11-month-old influenza-naïve foals were assigned randomly to 3 groups (20 vaccinates and 10 controls per group) and challenged 5 weeks, 6 and 12 months after a single vaccination. Challenges were performed on Day 0 in a plastic-lined chamber. Between Days 1 and 10, animals were examined daily for evidence of clinical signs of influenza. Nasal swabs for virus isolation were obtained on Day 1 and Days 1 to 8 and blood samples for serology were collected on Days 1, 7 and 14. There was no adverse response to vaccination in any animal. Following challenge at 5 weeks and 6 months, vaccinates had significantly lower clinical scores (P = 0.0001 and 0.005, respectively), experienced smaller increases in rectal temperature (P = 0.0008 and 0.0007, respectively) and shed less virus (P<0.0001 and P = 0.03, respectively) over fewer days (P<0.0001 and P = 0.002, respectively) than did the controls. After the 12 month challenge, rectal temperatures (P = 0.006) as well as the duration (P = 0.03) and concentration of virus shed (P = 0.04) were significantly reduced among vaccinated animals. The results of this study showed that 6 months after a single dose of vaccine the duration and severity of clinical signs were markedly reduced amongst vaccinated animals exposed to a severe live-virus challenge. Appropriate use of this vaccine should lead to a marked reduction in the frequency, severity and duration of outbreaks of equine influenza in North America.

  12. Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

    PubMed Central

    Cao, Yi; Li, Jie; Jiang, Na

    2014-01-01

    Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats, Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the pta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5′ untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5′ UTR was present in the pta-ackA transcript. Removal of the 5′ UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5′ UTRs to pta-ackA mRNA increased its in vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5′ UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptive M. mazei zm-15. PMID:24317083

  13. Mechanism for stabilizing mRNAs involved in methanol-dependent methanogenesis of cold-adaptive Methanosarcina mazei zm-15.

    PubMed

    Cao, Yi; Li, Jie; Jiang, Na; Dong, Xiuzhu

    2014-02-01

    Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats, Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the pta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5' untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5' UTR was present in the pta-ackA transcript. Removal of the 5' UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5' UTRs to pta-ackA mRNA increased its in vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5' UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptive M. mazei zm-15.

  14. Specific temperature-induced perturbations of secondary mRNA structures are associated with the cold-adapted temperature-sensitive phenotype of influenza A virus.

    PubMed

    Chursov, Andrey; Kopetzky, Sebastian J; Leshchiner, Ignaty; Kondofersky, Ivan; Theis, Fabian J; Frishman, Dmitrij; Shneider, Alexander

    2012-10-01

    For decades, cold-adapted, temperature-sensitive (ca/ts) strains of influenza A virus have been used as live attenuated vaccines. Due to their great public health importance it is crucial to understand the molecular mechanism(s) of cold adaptation and temperature sensitivity that are currently unknown. For instance, secondary RNA structures play important roles in influenza biology. Thus, we hypothesized that a relatively minor change in temperature (32-39°C) can lead to perturbations in influenza RNA structures and, that these structural perturbations may be different for mRNAs of the wild type (wt) and ca/ts strains. To test this hypothesis, we developed a novel in silico method that enables assessing whether two related RNA molecules would undergo (dis)similar structural perturbations upon temperature change. The proposed method allows identifying those areas within an RNA chain where dissimilarities of RNA secondary structures at two different temperatures are particularly pronounced, without knowing particular RNA shapes at either temperature. We identified such areas in the NS2, PA, PB2 and NP mRNAs. However, these areas are not identical for the wt and ca/ts mutants. Differences in temperature-induced structural changes of wt and ca/ts mRNA structures may constitute a yet unappreciated molecular mechanism of the cold adaptation/temperature sensitivity phenomena.

  15. Behavioral buffering of global warming in a cold-adapted lizard.

    PubMed

    Ortega, Zaida; Mencía, Abraham; Pérez-Mellado, Valentín

    2016-07-01

    Alpine lizards living in restricted areas might be particularly sensitive to climate change. We studied thermal biology of Iberolacerta cyreni in high mountains of central Spain. Our results suggest that I. cyreni is a cold-adapted thermal specialist and an effective thermoregulator. Among ectotherms, thermal specialists are more threatened by global warming than generalists. Alpine lizards have no chance to disperse to new suitable habitats. In addition, physiological plasticity is unlikely to keep pace with the expected rates of environmental warming. Thus, lizards might rely on their behavior in order to deal with ongoing climate warming. Plasticity of thermoregulatory behavior has been proposed to buffer the rise of environmental temperatures. Therefore, we studied the change in body and environmental temperatures, as well as their relationships, for I. cyreni between the 1980s and 2012. Air temperatures have increased more than 3.5°C and substrate temperatures have increased by 6°C in the habitat of I. cyreni over the last 25 years. However, body temperatures of lizards have increased less than 2°C in the same period, and the linear relationship between body and environmental temperatures remains similar. These results show that alpine lizards are buffering the potential impact of the increase in their environmental temperatures, most probably by means of their behavior. Body temperatures of I. cyreni are still cold enough to avoid any drop in fitness. Nonetheless, if warming continues, behavioral buffering might eventually become useless, as it would imply spending too much time in shelter, losing feeding, and mating opportunities. Eventually, if body temperature exceeds the thermal optimum in the near future, fitness would decrease abruptly.

  16. [Cloning and expression of endoglucanase of marine cold-adapted bacteria Pseudoalteromonas sp. MB-1].

    PubMed

    You, Yin-wei; Wang, Tian-hong

    2005-02-01

    The cold-adapted gram-negative rod bacterium MB-1 which could secret cellulase was screened from mud of the bottom of the Huanghai. According to the sequence of 16S rDNA, this bacterium screened was identified as one species of Pseudoalteromonas and was named as Pseudoalteromonas sp. MB-1. The gene celA encoding cold-adapted endogluanase was cloned and then jointed to pGEX-4T-1 to construct expression plasmid pGEX-celA which was expressed in E. coli BL21. Analysis to the supernatant of E. coli sonicate revealed that the concentration of GST-CelA was about 78.5 mg/L. Properties of the fusion enzyme of GST-CelA including the optimum temperature at 35 degrees C and the optimum pH about 7.2, showed that this fusion enzyme still belonged to cold-adapted enzyme and neutral enzyme. The result lays solid base for the fundamental theory and application research on cold-adapted cellulase from Pseudoalteromonas sp. MB-1.

  17. The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

    PubMed Central

    Sotelo-Mundo, Rogerio R.; Lopez-Zavala, Alonso A.; Garcia-Orozco, Karina D.; Arvizu-Flores, Aldo A.; Velazquez-Contreras, Enrique F.; Valenzuela-Soto, Elisa M.; Rojo-Dominguez, Arturo; Kanost, Michael R.

    2008-01-01

    Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of α-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5−30 °C. These results together with measured thermodynamical activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins. PMID:17979817

  18. The Lysozyme from Insect (Manduca sexta) is a Cold-Adapted Enzyme

    SciTech Connect

    Sotelo-Mundo,R.; Lopez-Zavala, A.; Garcia-Orozco, K.; Arvizu-Flores, A.; Velazquez-Contreras, E.; Valenzuela-Soto, E.; Rojo-Dominguez, A.; Kanost, M.

    2007-01-01

    Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of {alpha}-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 {sup o}C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.

  19. [Cold adaptation strategy in insects inhabiting central Yakutia].

    PubMed

    Li, N G; Averenskiĭ, A I

    2007-01-01

    Cold hardiness in 20 insect species living in extremely cold climate of Yakutia has been investigated for the first time. It was shown that the Yakutian insects prefer to use the strategy of freeze tolerance according to which they produce special substances initiating the freezing of hemolymph at high subzero temperatures. The presence of ice-nucleating agents in the haemolymph of insects belonging to the phylogenetic group of Lepidopteran was shown. We postulate that Pieris rapae may shift between the different cold hardiness strategies when they move from moderately cold regions to a more severe environment.

  20. Energy balance and cold adaptation in the octopus Pareledone charcoti.

    PubMed

    Daly; Peck

    2000-03-15

    A complete energy balance equation is calculated for the Antarctic octopus Pareledone charcoti at 0 degrees C. Energy used in respiration, growth, and excretion of nitrogenous and faecal waste, was recorded along with the total consumption of energy through food, for three specimens of P. charcoti (live weights: 73, 51 and 29 g). Growth rates were very slow for cephalopods, with a mean daily increase in body weight of only 0.11%. Assimilation efficiencies were high, between 95.4 and 97.0%, which is consistent with previous work on octopods. The respiration rate in P. charcoti was low, with a mean of 2.45 mg O(2) h(-1) for a standard animal of 150 g wet mass at 0 degrees C. In the North Sea octopus Eledone cirrhosa, respiration rates of 9.79 mg O(2) h(-1) at 11.5 degrees C and 4.47 mg O(2) h(-1) at 4.5 degrees C for a standard animal of 150 g wet mass were recorded. Respiration rates between P. charcoti and E. cirrhosa were compared using a combined Q(10) value between P. charcoti at 0 degrees C and E. cirrhosa at 4.5 degrees C. This suggests that P. charcoti are respiring at a level predicted by E. cirrhosa rates at 4.5 and 11.5 degrees C extrapolated to 0 degrees C along the curve Q(10)=3, with no evidence of metabolic compensation for low temperature.

  1. Protein cold adaptation: Role of physico-chemical parameters in adaptation of proteins to low temperatures.

    PubMed

    Shokrollahzade, Soheila; Sharifi, Fatemeh; Vaseghi, Akbar; Faridounnia, Maryam; Jahandideh, Samad

    2015-10-21

    During years 2007 and 2008, we published three papers (Jahandideh, 2007a, JTB, 246, 159-166; Jahandideh, 2007b, JTB, 248, 721-726; Jahandideh, 2008, JTB, 255, 113-118) investigating sequence and structural parameters in adaptation of proteins to low temperatures. Our studies revealed important features in cold-adaptation of proteins. Here, we calculate values of a new set of physico-chemical parameters and perform a comparative systematic analysis on a more comprehensive database of psychrophilic-mesophilic homologous protein pairs. Our obtained results confirm that psychrophilicity rules are not merely the inverse rules of thermostability; for instance, although contact order is reported as a key feature in thermostability, our results have shown no significant difference between contact orders of psychrophilic proteins compared to mesophilic proteins. We are optimistic that these findings would help future efforts to propose a strategy for designing cold-adapted proteins.

  2. Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification.

    PubMed

    Jónsdóttir, Gudrún; Bjarnason, Jón Bragi; Gudmundsdóttir, Agústa

    2004-01-01

    Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.

  3. Cold-adapted organic solvent tolerant alkalophilic family I.3 lipase from an Antarctic Pseudomonas.

    PubMed

    Ganasen, Menega; Yaacob, Norhayati; Rahman, Raja Noor Zaliha Raja Abd; Leow, Adam Thean Chor; Basri, Mahiran; Salleh, Abu Bakar; Ali, Mohd Shukuri Mohamad

    2016-11-01

    Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li(+), Na(+), K(+), Rb(+) and Cs(+) after 30min treatment. Heavy metal ions such as Cu(2+), Fe(3+) and Zn(2+) inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature.

  4. Attenuated temperature-sensitive respiratory syncytial virus mutants generated by cold adaptation.

    PubMed

    Randolph, V B; Kandis, M; Stemler-Higgins, P; Kennelly, M S; McMullen, Y M; Speelman, D J; Weeks-Levy, C

    1994-09-01

    Two strains of respiratory syncytial virus (RSV), RSV 2B and RSV 3A (representing subgroup B and A virus respectively) were cold-adapted by passaging in Vero cells for up to 42 weeks at successively lower temperatures down to 20 degrees C. Successful cold adaptation of the virus population was dependent on the amount of time the cultures were maintained at the various low temperatures, as well as on the strain of virus used. Temperature-sensitive (TS) mutants appeared in the cold passaged virus populations; however, the majority of the virus variants remained predominantly non-TS. Four RSV 2B and three RSV 3A TS mutants were selected for further characterization. These seven TS mutants retained their fusion phenotype and two major neutralizing antibody epitopes, and displayed varying levels of temperature sensitivity. Six of the seven mutants had a cold-adapted (CA) phenotype. All of the RSV 2B mutants were highly attenuated in cotton rats and two of the mutants elicited relatively high levels of neutralizing antibody and were able to protect rats against virus challenge. The RSV 3A TS mutants grew well in the nose but poorly in the cotton rat lungs, as did the parental 3A virus. All 3A mutants elicited high titers of neutralizing antibody and provided complete protection against virus challenge. These mutants showed varying levels of temperature sensitivity in vitro and attenuation in vivo and represent potential vaccine candidates.

  5. Cold-adapted tubulins in the glacier ice worm, Mesenchytraeus solifugus.

    PubMed

    Tartaglia, Lawrence J; Shain, Daniel H

    2008-11-01

    Glacier ice worms, Mesenchytraeus solifugus and related species, are the only known annelids that survive obligately in glacier ice and snow. One fundamental component of cold temperature adaptation is the ability to polymerize tubulin, which typically depolymerizes at low physiological temperatures (e.g., <10 degrees C) in most temperate species. In this study, we isolated two alpha-tubulin (Msalpha1, Msalpha2) and two beta-tubulin (Msbeta1, Msbeta2) subunits from an ice worm cDNA library, and compared their predicted amino acid sequences with homologues from other cold-adapted organisms (e.g., Antarctic fish, ciliate) in an effort to identify species-specific amino acid substitutions that contribute to cold temperature-dependent tubulin polymerization. Our comparisons and predicted protein structures suggest that ice worm-specific amino acid substitutions stabilize lateral contact associations, particularly between beta-tubulin protofilaments, but these substitutions occur at different positions in comparison with other cold-adapted tubulins. The ice worm tubulin gene family appears relatively small, comprising one primary alpha- and one primary beta-tubulin monomers, though minor isoforms and pseudogenes were identified. Our analyses suggest that variation occurs in the strategies (i.e., species-specific amino acid substitutions, gene number) by which cold-adapted taxa have evolved the ability to polymerize tubulin at low physiological temperatures.

  6. Comparative Immunogenicities of Frozen and Refrigerated Formulations of Live Attenuated Influenza Vaccine in Healthy Subjects▿

    PubMed Central

    Block, Stan L.; Reisinger, Keith S.; Hultquist, Micki; Walker, Robert E.

    2007-01-01

    The frozen version of live attenuated influenza vaccine (LAIV; FluMist) was compared with a newly licensed, refrigerated formulation, the cold-adapted influenza vaccine, trivalent (CAIV-T), for their immunogenicity, safety, and tolerability in healthy subjects 5 to 49 years of age. Eligible subjects were randomized 1:1 to receive CAIV-T or frozen LAIV. Subjects 5 to 8 years of age received two doses of vaccine 46 to 60 days apart; subjects 9 to 49 years of age received one dose of vaccine. Equivalent immunogenicities were defined as serum hemagglutination inhibition (HAI) geometric mean titer (GMT) ratios >0.5 and <2.0 for each of the three vaccine-specific strains. A total of 376 subjects 5 to 8 years of age and 566 subjects 9 to 49 years of age were evaluable. Postvaccination HAI GMT ratios were equivalent for CAIV-T and LAIV. The GMT ratios of CAIV-T/LAIV for the H1N1, H3N2, and B strains were 1.24, 1.02, and 1.00, respectively, for the 5- to 8-year-old age group and 1.14, 1.12, and 0.96, respectively, for the 9- to 49-year-old age group. Seroresponse/seroconversion rates (fourfold or greater rise) were similar in both age groups for each of the three vaccine strains. Within 28 days, the most frequent reactogenicity event in the CAIV-T and LAIV groups was runny nose/nasal congestion, which occurred at higher rates after dose 1 (44% and 42%, respectively) than after dose 2 (41% and 29%, respectively) in the 5- to 8-year-old group. Otherwise, the rates of adverse events (AEs) were similar between the treatment groups and the two age cohorts, with no serious AEs related to the study vaccines. The immunogenicities, reactogenicity events, and AEs were comparable for refrigerated CAIV-T and frozen LAIV. PMID:17724151

  7. Cold Adaptation, Ca2+ Dependency and Autolytic Stability Are Related Features in a Highly Active Cold-Adapted Trypsin Resistant to Autoproteolysis Engineered for Biotechnological Applications

    PubMed Central

    Olivera-Nappa, Alvaro; Reyes, Fernando; Andrews, Barbara A.; Asenjo, Juan A.

    2013-01-01

    Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca2+ dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10° C and at least 50% more active than pig trypsin up to 50° C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca2+ concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca2+ dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done. PMID:23951314

  8. Identification and immobilization of a novel cold-adapted esterase, and its potential for bioremediation of pyrethroid-contaminated vegetables.

    PubMed

    Fan, Xinjiong; Liang, Weiqu; Li, Yanfang; Li, He; Liu, Xiaolong

    2017-09-11

    Pyrethroids are potentially harmful to living organisms and ecosystems. Thus, concerns have been raised about pyrethroid residues and their persistence in agricultural products. To date, although several pyrethroid-hydrolyzing enzymes have been cloned, very few reports are available on pyrethroid-hydrolyzing enzymes with cold adaptation, high hydrolytic activity and good reusability, indispensable properties in practical bioremediation of pyrethroid-contaminated vegetables. Here, a novel gene (est684) encoding pyrethroid-hydrolyzing esterase was isolated from the Mao-tofu metagenome for the first time. Est684 encoded a protein of 227 amino acids and was expressed in Escherichia coli BL21 (DE3) in soluble form. The optimum temperature was 18 °C. It maintained 46.1% of activity at 0 °C and over 50% of its maximal activity at 4-35 °C. With the goal of enhancing stability and recycling biocatalysts, we used mesoporous silica SBA-15 as a nanometer carrier for the efficient immobilization of Est684 by the absorption method. The best conditions were an esterase-to-silica ratio of 0.96 mg/g (w/w) and an adsorption time of 30 min at 10 °C. Under these conditions, the recovery of enzyme activity was 81.3%. A large improvement in the thermostability of Est684 was achieved. The half-life (T1/2) of the immobilized enzyme at 35 °C was 6 h, 4 times longer than the soluble enzyme. Interestingly, the immobilized Est684 had less loss in enzyme activity up to 12 consecutive cycles, and it retained nearly 54% of its activity after 28 cycles, indicating excellent operational stability. Another noteworthy characteristic was its high catalytic activity. It efficiently hydrolyzed cyhalothrin, cypermethrin, and fenvalreate in pyrethroid-contaminated cucumber within 5 min, reaching over 85% degradation efficiency after four cycles. A novel cold-adapted pyrethroid-hydrolyzing esterase was screened from the Mao-tofu metagenome. This report is the first on immobilizing pyrethroid

  9. Phenotypic and genetic analyses of the heterogeneous population present in the cold-adapted master donor strain: A/Leningrad/134/17/57 (H2N2).

    PubMed

    Youil, R; Kiseleva, I; Kwan, W-S; Szymkowiak, C; Toner, T J; Su, Q; Klimov, A; Rudenko, L; Shaw, A R

    2004-06-15

    For the past three decades the cold-adapted (ca) and temperature sensitive (ts) master donor strain, A/Leningrad/134/17/57 (H2N2) has been successfully used as the basis for the live attenuated reassortant influenza A vaccine. This donor strain was developed from A/Leningrad/134/57 (H2N2) wild-type (wt) virus following 17 passages in eggs at 25 degrees C. Our detailed investigation has revealed that the A/Leningrad/134/17/57 (Len/17) master donor stock is a mixed population comprised of numerous variants of the ca/ts Len/17 influenza virus. We have identified these variants to exhibit a broad range in their temperature sensitive phenotype when assayed on Madin-Darby canine kidney (MDCK) cells at 37 degrees C. A selection of these variant clones has been fully characterized by sequencing in order to understand the variability in the ts phenotype. This study has also addressed the feasibility of using cell culture technology for the propagation and subsequent manufacturing of the cold-adapted influenza vaccine (CAIV), particularly with respect to retaining the defined mutations that contribute toward the ca/ts phenotype.

  10. [Analysis of mutations in the genome of cold-adapted strains of influenza A virus using extended modification of polymerase chain restriction method].

    PubMed

    Kiseleva, I V; Klimov, A I

    2002-01-01

    Cold-adapted influenza viruses A/Leningrad/13 4/17/5 7 (H2N2) (Len/17) and A/Leningrad/I 34/4 7/57 (H2N2) (Len/47) are used in Russia to prepare live reassortant cold-adapted influenza vaccines (LIV) for adults and children, respectively. Comparison between the nucleotide sequences of the Len/17 strain and the initial wild-type strain A/Leningrad/13 4/5 7 (H2N2) revealed ten nucleotide substitutions (eight of them encoding). Four additional substitutions (three encoding) were found in the genome of the Len/47 virus. Gene segment restriction site (PCR-restriction) analysis was used for identification of the genotype of reassortant influenza viruses. Conventional methods of PCR-restriction analysis detect only five encoding nucleotides substitutions in the internal genes of the Len/17 and seven substitutions in the internal genes of the Len/47 virus. An extended modification of the PCR-restriction method detect all encoding mutations in the internal genes of the Len/17 and Len/47 viruses (eight and eleven encoding substitutions, respectively). This method is advantageous for genome composition analysis of reassortant influenza vaccine strains and for investigating the genetic stability of LIV during replication in vaccines.

  11. Cold adaptation: structural and functional characterizations of psychrophilic and mesophilic acetate kinase.

    PubMed

    Tang, Md Abul Kashem; Motoshima, Hiroyuki; Watanabe, Keiichi

    2014-08-01

    Acetate kinase catalyzes the reversible magnesium-dependent phosphoryl transfer from ATP to acetate to form acetyl phosphate and ADP. Here, we report functional and some structural properties of cold-adapted psychrotrophic enzyme; acetate kinase with those from mesophilic counterpart in Escherichia coli K-12. Recombinant acetate kinase from Shewanella sp. AS-11 (SAK) and E. coli K-12 (EAK) were purified to homogeneity following affinity chromatography and followed by Super Q column chromatography as reported before [44]. Both purified enzymes are shared some of the common properties such as (similar molecular mass, amino acid sequence and similar optimum pH), but characterized shift in the apparent optimum temperature of specific activity to lower temperature as well as by a lower thermal stability compared with EAK. The functional comparisons reveal that SAK is a cold adapted enzyme, having a higher affinity to acetate than EAK. In the acetyl phosphate and ADP-forming direction, the catalytic efficiency (k(cat)/K(m)) for acetate was 8.0 times higher for SAK than EAK at 10 °C. The activity ratio of SAK to EAK was increased with decreasing temperature in both of the forward and backward reactions. Furthermore, the activation energy, enthalpy and entropy in both reaction directions that catalyzed by SAK were lower than those catalyzed by EAK. The model structure of SAK showed the significantly reduced numbers of salt bridges and cation-pi interactions as compared with EAK. These results suggest that weakening of intramolecular electrostatic interactions of SAK is involved in a more flexible structure which is likely to be responsible for its cold adaptation.

  12. Testing phylogeographic hypotheses in a Euro-Siberian cold-adapted leaf beetle with coalescent simulations.

    PubMed

    Mardulyn, Patrick; Mikhailov, Yuri E; Pasteels, Jacques M

    2009-10-01

    Few studies to date have investigated the impact of Pleistocene climatic oscillations on the genetic diversity of cold-adapted species. We focus on the geographic distribution of genetic diversity in a Euro-Siberian boreo-montane leaf beetle, Gonioctena pallida. We present the molecular variation from three independent gene fragments over the entire geographic range of this insect. The observed sequence variation identifies a genetic diversity hot spot in the Carpathian Mountains, in central Europe, which reveals the presence of (1) an ancestral refuge population or (2) a secondary contact zone in this area. Modeling of population evolution in a coalescent framework allowed us to favor the ancestral refuge hypothesis. These analyses suggest that the Carpathian Mountains served as a refuge for G. pallida, whereas the rest of the species distribution, that spans a large portion of Europe and Asia, experienced a dramatic reduction in genetic variation probably associated to bottlenecks and/or founder events. We estimated the time of isolation of the ancestral refuge population, using an approximate Bayesian method, to be larger than 90,000 years. If true, the current pattern of genetic variation in this cold-adapted organism was shaped by a climatic event predating by far the end of the last ice age.

  13. Diversity and bioprospecting of fungal communities associated with endemic and cold-adapted macroalgae in Antarctica.

    PubMed

    Godinho, Valéria M; Furbino, Laura E; Santiago, Iara F; Pellizzari, Franciane M; Yokoya, Nair S; Pupo, Diclá; Alves, Tânia M A; Junior, Policarpo A S; Romanha, Alvaro J; Zani, Carlos L; Cantrell, Charles L; Rosa, Carlos A; Rosa, Luiz H

    2013-07-01

    We surveyed the distribution and diversity of fungi associated with eight macroalgae from Antarctica and their capability to produce bioactive compounds. The collections yielded 148 fungal isolates, which were identified using molecular methods as belonging to 21 genera and 50 taxa. The most frequent taxa were Geomyces species (sp.), Penicillium sp. and Metschnikowia australis. Seven fungal isolates associated with the endemic Antarctic macroalgae Monostroma hariotii (Chlorophyte) displayed high internal transcribed spacer sequences similarities with the psychrophilic pathogenic fungus Geomyces destructans. Thirty-three fungal singletons (66%) were identified, representing rare components of the fungal communities. The fungal communities displayed high diversity, richness and dominance indices; however, rarefaction curves indicated that not all of the fungal diversity present was recovered. Penicillium sp. UFMGCB 6034 and Penicillium sp. UFMGCB 6120, recovered from the endemic species Palmaria decipiens (Rhodophyte) and M. hariotii, respectively, yielded extracts with high and selective antifungal and/or trypanocidal activities, in which a preliminary spectral analysis using proton nuclear magnetic resonance spectroscopy indicated the presence of highly functionalised aromatic compounds. These results suggest that the endemic and cold-adapted macroalgae of Antarctica shelter a rich, diversity and complex fungal communities consisting of a few dominant indigenous or mesophilic cold-adapted species, and a large number of rare and/or endemic taxa, which may provide an interesting model of algal-fungal interactions under extreme conditions as well as a potential source of bioactive compounds.

  14. Diversity and bioprospecting of fungal communities associated with endemic and cold-adapted macroalgae in Antarctica

    PubMed Central

    Godinho, Valéria M; Furbino, Laura E; Santiago, Iara F; Pellizzari, Franciane M; Yokoya, Nair S; Pupo, Diclá; Alves, Tânia MA; S Junior, Policarpo A; Romanha, Alvaro J; Zani, Carlos L; Cantrell, Charles L; Rosa, Carlos A; Rosa, Luiz H

    2013-01-01

    We surveyed the distribution and diversity of fungi associated with eight macroalgae from Antarctica and their capability to produce bioactive compounds. The collections yielded 148 fungal isolates, which were identified using molecular methods as belonging to 21 genera and 50 taxa. The most frequent taxa were Geomyces species (sp.), Penicillium sp. and Metschnikowia australis. Seven fungal isolates associated with the endemic Antarctic macroalgae Monostroma hariotii (Chlorophyte) displayed high internal transcribed spacer sequences similarities with the psychrophilic pathogenic fungus Geomyces destructans. Thirty-three fungal singletons (66%) were identified, representing rare components of the fungal communities. The fungal communities displayed high diversity, richness and dominance indices; however, rarefaction curves indicated that not all of the fungal diversity present was recovered. Penicillium sp. UFMGCB 6034 and Penicillium sp. UFMGCB 6120, recovered from the endemic species Palmaria decipiens (Rhodophyte) and M. hariotii, respectively, yielded extracts with high and selective antifungal and/or trypanocidal activities, in which a preliminary spectral analysis using proton nuclear magnetic resonance spectroscopy indicated the presence of highly functionalised aromatic compounds. These results suggest that the endemic and cold-adapted macroalgae of Antarctica shelter a rich, diversity and complex fungal communities consisting of a few dominant indigenous or mesophilic cold-adapted species, and a large number of rare and/or endemic taxa, which may provide an interesting model of algal–fungal interactions under extreme conditions as well as a potential source of bioactive compounds. PMID:23702515

  15. The Antarctic Chlamydomonas raudensis: an emerging model for cold adaptation of photosynthesis.

    PubMed

    Dolhi, Jenna M; Maxwell, Denis P; Morgan-Kiss, Rachael M

    2013-09-01

    Permanently cold habitats dominate our planet and psychrophilic microorganisms thrive in cold environments. Environmental adaptations unique to psychrophilic microorganisms have been thoroughly described; however, the vast majority of studies to date have focused on cold-adapted bacteria. The combination of low temperatures in the presence of light is one of the most damaging environmental stresses for a photosynthetic organism: in order to survive, photopsychrophiles (i.e. photosynthetic organisms adapted to low temperatures) balance temperature-independent reactions of light energy capture/transduction with downstream temperature-dependent metabolic processes such as carbon fixation. Here, we review research on photopsychrophiles with a focus on an emerging model organism, Chlamydomonas raudensis UWO241 (UWO241). UWO241 is a psychrophilic green algal species and is a member of the photosynthetic microbial eukaryote community that provides the majority of fixed carbon for ice-covered lake ecosystems located in the McMurdo Dry Valleys, Antarctica. The water column exerts a range of environmental stressors on the phytoplankton community that inhabits this aquatic ecosystem, including low temperatures, extreme shade of an unusual spectral range (blue-green), high salinity, nutrient deprivation and extremes in seasonal photoperiod. More than two decades of work on UWO241 have produced one of our most comprehensive views of environmental adaptation in a cold-adapted, photosynthetic microbial eukaryote.

  16. Cold adaptation improves the growth of seasonal influenza B vaccine viruses.

    PubMed

    Kim, Hyunsuh; Schoofs, Peter; Anderson, David A; Tannock, Gregory A; Rockman, Steven P

    2014-05-01

    Gene reassortment has proved useful in improving yields of influenza A antigens of egg-based inactivated vaccines, but similar approaches have been difficult with influenza B antigens. Current regulations for influenza vaccine seed viruses limit the number of egg passages and as a result resultant yields from influenza B vaccine seed viruses are frequently inconsistent. Therefore, reliable approaches to enhance yields of influenza B vaccine seed viruses are required for efficient vaccine manufacture. In the present study three stable cold-adapted (ca) mutants, caF, caM and caB derived from seasonal epidemic strains, B/Florida/4/2006, B/Malaysia/2506/2004 and B/Brisbane/60/2008 were prepared, which produced high hemagglutinin antigen yields and also increased viral yields of reassortants possessing the desired 6:2 gene constellation. The results demonstrate that consistent improvements in yields of influenza B viruses can be obtained by cold adaptation following extended passage. Taken together, the three ca viruses were shown to have potential as donor viruses for the preparation of high-yielding influenza B vaccine viruses by reassortment.

  17. Endoplasmic Reticulum-Associated Degradation Is Required for Cold Adaptation and Regulation of Sterol Biosynthesis in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Loertscher, Jennifer; Larson, Lynnelle L.; Matson, Clinton K.; Parrish, Mark L.; Felthauser, Alicia; Sturm, Aaron; Tachibana, Christine; Bard, Martin; Wright, Robin

    2006-01-01

    Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10 (Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7, CUE1, or DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity. Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed cold-sensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The sterol metabolite profile of ubc7Δ cells was altered relative to that of wild-type cells. Since sterol levels are known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was examined at low temperatures. Cells lacking UBC7, CUE1, or DOA10 were cold sensitive, suggesting that these ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis. PMID:16607018

  18. Optimization of electrostatics as a strategy for cold-adaptation: a case study of cold- and warm-active elastases.

    PubMed

    Papaleo, Elena; Olufsen, Magne; De Gioia, Luca; Brandsdal, Bjørn O

    2007-07-01

    Adaptation to both high and low temperatures requires proteins with special properties. While organisms living at or close to the boiling point of water need to have proteins with increased stability, other properties are required at temperatures close to the freezing point of water. Indeed, it has been shown that enzymes adapted to cold environments are less resistant to heat with a concomitant increased activity as compared to their warm-active counter-parts. Several recent studies have pointed in the direction that electrostatic interactions play a central role in temperature adaptation, and in this study we investigate the role such interactions have in adaptation of elastase from Atlantic salmon and pig. Molecular dynamics (MD) simulations have been used to generate structural ensembles at 283 and 310 K of the psychrophilic and mesophilic elastase, and a total of eight 12 ns simulations have been carried out. Even though the two homologues have a highly similar three-dimensional structure, the location and number of charged amino acids are very different. Based on the simulated structures we find that very few salt-bridges are stable throughout the simulations, and provide little stabilization/destabilization of the proteins as judged by continuum electrostatic calculations. However, the mesophilic elastase is characterized by a greater number of salt-bridges as well as a putative salt-bridge network close to the catalytic site, indicating a higher rigidity of the components involved in the catalytic cycle. In addition, subtle differences are also found in the electrostatic potentials in the vicinity of the catalytic residues, which may explain the increased catalytic efficiency of the cold-adapted elastase.

  19. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

    SciTech Connect

    Simpson, Philippa J.L.; Codd, Rachel

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Cold-adapted phenotype of NapA from the Antarctic bacterium Shewanella gelidimarina. Black-Right-Pointing-Pointer Protein homology model of NapA from S. gelidimarina and mesophilic homologue. Black-Right-Pointing-Pointer Six amino acid residues identified as lead candidates governing NapA cold adaptation. Black-Right-Pointing-Pointer Molecular-level understanding of designing cool-temperature in situ oxyanion sensors. -- Abstract: The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo-MGD) cofactor and one [4Fe-4S] iron-sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap{sub Sgel}) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap{sub Sput}) was examined at varied temperature. Irreversible deactivation of Nap{sub Sgel} and Nap{sub Sput} occurred at 54.5 and 65 Degree-Sign C, respectively. When Nap{sub Sgel} was preincubated at 21-70 Degree-Sign C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 Degree-Sign C, which suggested that Nap{sub Sgel} was poised for optimal catalysis at modest temperatures and, unlike Nap{sub Sput}, did not benefit from thermally-induced refolding. At 20 Degree-Sign C, Nap{sub Sgel} reduced selenate at 16% of the rate of nitrate reduction. Nap{sub Sput} did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap{sub Sgel} that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap{sub Sgel} cold-adapted phenotype. Protein homology modeling of Nap{sub Sgel} using a

  20. Cold adaptation of a psychrophilic chaperonin from Psychrobacter sp. and its application for heterologous protein expression.

    PubMed

    Kim, Han-Woo; Wi, Ah Ram; Jeon, Byoung Wook; Lee, Jun Hyuck; Shin, Seung Chul; Park, Hyun; Jeon, Sung-Jong

    2015-09-01

    A chaperonin, PsyGroELS, from the Antarctic psychrophilic bacterium Psychrobacter sp. PAMC21119, was examined for its role in cold adaptation when expressed in a mesophilic Escherichia coli strain. Growth of E. coli harboring PsyGroELS at 10 °C was increased compared to the control strain. A co-expression system using PsyGroELS was developed to increase productivity of the psychrophilic enzyme PsyEst9. PsyEst9 was cloned and expressed using three E. coli variants that co-expressed GroELS from PAMC21119, E. coli, or Oleispira antarctica RB8(T). Co-expression with PsyGroELS was more effective for the production of PsyEst9 compared tothe other chaperonins. PsyGroELS confers cold tolerance to E. coli, and shows potential as an effective co-expression system for the stable production of psychrophilic proteins.

  1. Metabolic cold adaptation in fishes occurs at the level of whole animal, mitochondria and enzyme.

    PubMed

    White, Craig R; Alton, Lesley A; Frappell, Peter B

    2012-05-07

    Metabolic cold adaptation (MCA), the hypothesis that species from cold climates have relatively higher metabolic rates than those from warm climates, was first proposed nearly 100 years ago and remains one of the most controversial hypotheses in physiological ecology. In the present study, we test the MCA hypothesis in fishes at the level of whole animal, mitochondria and enzyme. In support of the MCA hypothesis, we find that when normalized to a common temperature, species with ranges that extend to high latitude (cooler climates) have high aerobic enzyme (citrate synthase) activity, high rates of mitochondrial respiration and high standard metabolic rates. Metabolic compensation for the global temperature gradient is not complete however, so when measured at their habitat temperature species from high latitude have lower absolute rates of metabolism than species from low latitudes. Evolutionary adaptation and thermal plasticity are therefore insufficient to completely overcome the acute thermodynamic effects of temperature, at least in fishes.

  2. Factors affecting the yield of cold-adapted influenza virus vaccine.

    PubMed

    Yannarell, D A; Hjorth, R N

    1997-03-01

    Various manipulations to production procedures have been investigated in order to discover methods to attain adequate or augmented titers of cold-adapted influenza virus (CAIV) vaccine. The methods modified include those used for reassortant selection and the determination of virus growth parameters. Increased infectivity titers were achieved through selection of high-yielding mutants by isolating multiple plaques during plaque purification of reassortant clones, as well as through optimization of egg incubation times, age, and lot for individual strains. Up to 6-fold increases in virus yield were obtained by selecting high yielding mutants, up to 9-fold increases were achieved by modifying egg incubation times, and a nearly 1 log increase was realized by determining the ideal egg age for individual strains.

  3. The cold adaptability of microorganisms with different carbon source in activated sludge treating synthetical wastewater.

    PubMed

    Niu, Chuan; Geng, Jinju; Ren, Hongqiang; Ding, Lili; Xu, Ke

    2012-11-01

    The cold adaptability of microorganisms with different carbon source under 5°C was studied in activated sludge for treating synthetical wastewater. Phospholipid fatty acid (PLFA) analysis indicated contents of unsaturated fatty acids in cell membrane at 5°C were 13.66% and 24.96% higher for glucose and sodium acetate source than that at 25°C. PLFA biomarkers showed more Gram-negative bacteria enriched than Gram-positive bacteria in low-temperature activated sludge. The Shannon-Wiener diversity analysis demonstrated glucose fed reactor in low temperature had lower PLFA diversity index (1.21-1.30) than that at 25°C and sodium acetate source was reverse (1.08-0.69). The 16S rRNA analysis manifested certain microbes were considerably suitable for existence under cold environment, most of which belong to Gram-negative bacteria.

  4. [Structural and functional reorganization of photosynthetic apparatus in cold adaptation of wheat plants].

    PubMed

    Venzhik, Ju V; Titov, D F; Talanova, V V; Miroslavov, E D; Koteeva, N K

    2012-01-01

    The structural and functional characteristics of the photosynthetic apparatus (PSA) and the cold resistance of wheat seedlings were studied during low-temperature adaptation. It has been established that large chloroplasts with thylakoid system of "sun type" forme in the mesophyll cells in the early hours of plants hardening. At the same time the functional reorganization of the PSA in the leaves of wheat occurs: content of pigments changes, stabilization of the pigment-protein complexes is observed, non-photochemical quenching of excess energy increases. The stabilization of photosynthesis during cold adaptation occurs due to structural and functional reorganization of the PSA. It is assumed that the reorganization of the PSA is a prerequisite for formation of increased cold resistance of leaf cells, and this, along with other physiological and biochemical changes occurring in cells and tissues of plants, allows the plants to survive in chilling.

  5. Efficacy of trivalent, cold-adapted, influenza virus vaccine against influenza A (Fujian), a drift variant, during 2003-2004.

    PubMed

    Halloran, M Elizabeth; Piedra, Pedro A; Longini, Ira M; Gaglani, Manjusha J; Schmotzer, Brian; Fewlass, Charles; Herschler, Gayla B; Glezen, W Paul

    2007-05-16

    In the 2003-2004 influenza season, the predominant circulating influenza A (H3N2) virus in the United States was similar antigenically to A/Fujian/411/2002 (H3N2), a drift variant of A/Panama/2007/99 (H3N2), the vaccine strain. That year, a field study of trivalent live-attenuated influenza vaccine (LAIV-T) was conducted in Temple-Belton, Texas, as part of a larger community-based, non-randomized, open-label study in three communities that began in August 1998 [Gaglani MJ, Piedra PA, Herschler GB, Griffith ME, Kozinetz CA, Riggs MW, et al. Direct effectiveness of the trivalent, cold-adapted, influenza virus vaccine (CAIV-T) against the 2000-2001 influenza A (H1N1) and B epidemic in healthy children. Arch Pediatr Adolesc Med 2004;158:65-73; Piedra PA, Gaglani MJ, Kozinetz CA, Herschler G, Riggs M, Griffith M, et al. Herd immunity in adults against influenza-related illnesses with use of the trivalent-live attenuated influenza vaccine (CAIV-T) in children. Vaccine 2005;23:1540-8; Piedra PA, Gaglani MJ, Riggs M, Herschler G, Fewlass C, Watts M, et al. Live attenuated influenza vaccine, trivalent, is safe in healthy children 18 months to 4 years, 5 to 9 years, and 10 to 18 years of age in a community-based, nonrandomized, open-label trial. Pediatrics 2005;116:397-407]. Participants were healthy children aged 5-18 years. The analysis here concerns 6403 children in the Scott & White Health Plan (SWHP) database living within zip codes of the Temple-Belton area, of whom 1706 received LAIV-T and 548 received trivalent inactivated vaccine (TIV) in 2003, 983 had been previously vaccinated in 1998-2001, but not in 2002-2003 or 2003, and 3166 had never been vaccinated. The main outcome measure was medically-attended acute respiratory illness (MAARI). Surveillance culture results were incorporated into the analysis to estimate efficacy against culture-confirmed influenza illness. Vaccine effectiveness of LAIV-T against MAARI was 26% (95% confidence interval (CI) 11, 39). Vaccine

  6. Characterisation of a cold adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

    PubMed

    Dong, Juan; Gasmalla, Mohammed A A; Zhao, Wei; Sun, Jingtao; Liu, Wenyu; Wang, Mingming; Han, Liang; Yang, Ruijin

    2016-07-13

    A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No.1, Tianshan, China, and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room-temperature plasma (ARTP) method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30°C, pH 9.0 and 25°C, pH 8.5, respectively. EstTB11 was thermally more stable (50°C for 1 hour) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0°C and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4°C. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry. This article is protected by copyright. All rights reserved.

  7. A bacterial acyl aminoacyl peptidase couples flexibility and stability as a result of cold adaptation.

    PubMed

    Brocca, Stefania; Ferrari, Cristian; Barbiroli, Alberto; Pesce, Alessandra; Lotti, Marina; Nardini, Marco

    2016-12-01

    Life in cold environments requires an overall increase in the flexibility of macromolecular and supramolecular structures to allow biological processes to take place at low temperature. Conformational flexibility supports high catalytic rates of enzymes in the cold but in several cases is also a cause of instability. The three-dimensional structure of the psychrophilic acyl aminoacyl peptidase from Sporosarcina psychrophila (SpAAP) reported in this paper highlights adaptive molecular changes resulting in a fine-tuned trade-off between flexibility and stability. In its functional form SpAAP is a dimer, and an increase in flexibility is achieved through loosening of intersubunit hydrophobic interactions. The release of subunits from the quaternary structure is hindered by an 'arm exchange' mechanism, in which a tiny structural element at the N terminus of one subunit inserts into the other subunit. Mutants lacking the 'arm' are monomeric, inactive and highly prone to aggregation. Another feature of SpAAP cold adaptation is the enlargement of the tunnel connecting the exterior of the protein with the active site. Such a wide channel might compensate for the reduced molecular motions occurring in the cold and allow easy and direct access of substrates to the catalytic site, rendering transient movements between domains unnecessary. Thus, cold-adapted SpAAP has developed a molecular strategy unique within this group of proteins: it is able to enhance the flexibility of each functional unit while still preserving sufficient stability. Structural data are available in the Protein Data Bank under the accession number 5L8S. © 2016 Federation of European Biochemical Societies.

  8. Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

    PubMed

    Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

    2016-06-28

    Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

  9. Cloning, expression and structural stability of a cold-adapted ß-Galactosidase from Rahnella sp.R3

    USDA-ARS?s Scientific Manuscript database

    A novel gene was isolated for the first time from a psychrophilic gram-negative bacterium Rahnella sp.R3. It encoded a cold-adapted ß-galactosidase (R-ß-Gal). Recombinant R-ß-Gal was expressed in Escherichia coli BL21 (DE3), purified, and characterized. R-ß-Gal belongs to the glycosyl hydrolase fami...

  10. PB2 and PA genes control the expression of the temperature-sensitive phenotype of cold-adapted B/USSR/60/69 influenza master donor virus.

    PubMed

    Kiseleva, Irina V; Voeten, J Theo M; Teley, Lisette C P; Larionova, Natalia V; Drieszen-van der Cruijsen, Sandra K M; Basten, Stephanie M C; Heldens, Jacco G M; van den Bosch, Han; Rudenko, Larisa G

    2010-04-01

    The cold-adapted (ca) and temperature-sensitive (ts) influenza master donor virus (MDV) B/USSR/60/69 was derived from its wild-type parental virus after successive passages in eggs at 32 degrees C and 25 degrees C. This strain is currently in use for preparing reassortant influenza B vaccine viruses which are used in the Russian trivalent live attenuated influenza vaccine. Vaccine viruses are obtained by classical reassortment of MDV and a currently circulating wild-type virus. The phenotypic properties cold adaptation and temperature sensitivity are inherited from the six genes encoding the internal proteins of the MDV. However, the role of the individual gene segments in temperature sensitivity and thus attenuation is not known. In this study, 35 reassortant viruses of B/USSR/60/69 MDV with current wild-type non-ts influenza B viruses were generated in eggs or MDCK cells and studied in order to identify the genes responsible for their ts phenotype. For each virus the exact genome composition was determined as well as its ts phenotype. The results demonstrated that the polymerase PB2 and PA gene segments of B/USSR/60/69 MDV independently controlled expression of the ts phenotype of B/USSR/60/69 MDV-based reassortant viruses. The other genes coding for internal proteins played no role in this respect. This suggests that mutations in the polymerase genes PB2 and PA play an essential role in attenuation of B/USSR/60/69 MDV-based reassortant influenza B vaccine viruses.

  11. Structural insights into the cold adaptation of the photosynthetic pigment-protein C-phycocyanin from an Arctic cyanobacterium.

    PubMed

    Su, Hai-Nan; Wang, Qian-Min; Li, Chun-Yang; Li, Kang; Luo, Wei; Chen, Bo; Zhang, Xi-Ying; Qin, Qi-Long; Zhou, Bai-Cheng; Chen, Xiu-Lan; Zhang, Yu-Zhong; Xie, Bin-Bin

    2017-04-01

    The cold adaptation mechanism of phycobiliproteins, the major photosynthetic pigment-proteins in cyanobacteria and red algae, has rarely been studied. Here we reported the biochemical, structural, and molecular dynamics simulation study of the C-phycocyanin from Arctic cyanobacterial strain Pseudanabaena sp. LW0831. We characterized the phycobilisome components of LW0831 and obtained their gene sequences. Compared to the mesophilic counterpart from Arthrospira platensis (Ar-C-PC), LW0831 C-phycocyanin (Ps-C-PC) has a decreased thermostability (∆Tm of -16°C), one of the typical features of cold-adapted enzymes. To uncover its structural basis, we resolved the crystal structure of Ps-C-PC 1 at 2.04Å. Consistent with the decrease in thermostability, comparative structural analyses revealed decreased intra-trimer and inter-trimer interactions in Ps-C-PC 1, compared to Ar-C-PC. However, comparative molecular dynamics simulations indicated that Ps-C-PC 1 shows similar flexibilities to Ar-C-PC for both the (αβ)3 trimer and (αβ)6 hexamer. Therefore, the optimization mode is clearly different from cold-adapted enzymes, which usually have increased flexibilities. Detailed analyses demonstrated different optimization modes for the α and β subunits and it was revealed that hydrophobic interactions are key to this difference, though salt bridges, hydrogen bonds, and surface hydrophobicity are also involved. This study is the first report of the structure of cold-adapted phycobiliproteins and provides insights into the cold-adaptation strategies of non-enzyme proteins.

  12. Hot experience for cold-adapted microorganisms: temperature sensitivity of soil enzymes

    NASA Astrophysics Data System (ADS)

    Liu, Shibin; Razavidezfuly, Baharsadat; Kuzyakov, Yakov

    2016-04-01

    The temperature sensitivity of enzymes responsible for organic matter decomposition in cold environment soil, where warming is expected to be greatest is crucial. Based on Michaelis-Menten kinetics and Arrhenius function, we hypothesized that cold-adapted microorganisms will produce high efficient enzymes at cold temperatures (enzymes with lower apparent activation energy (Ea) at cold temperature ranges). To test our hypothesis, 30 g soil of Tibetan Plateau (4100 m a.s.l., annual temperature 2.4 °C) in 4 replicates were incubated for one month over a temperature range of 0-40 °C (with 5 °C steps) and determined the kinetic parameters of six enzymes involved in decomposing organics: cellobiohydrolase and β-glucosidase, which are commonly measured as enzymes responsible for consecutive stages of cellulose degradation; xylanase, which is responsible for breaking down hemicelluloses; acid phosphatase, which mineralizes organic P to phosphate by hydrolyzing phosphoric (mono) ester bonds under acidic conditions. Activities of leucine aminopeptidase and tyrosine aminopeptidase were analyzed to assess the hydrolysis of L-peptide bonds. The apparent activation energy varied between enzymes from 42 (phosphatase) to 54 (cellobiohydrolase) kJ mol-1 corresponding to the Q10 values of the enzyme reactions of 1.8-2.3. The increase of substrate affinity (Km) with temperature was gradual for most tested enzymes from 0-20 °C (enzymes involved in C cycle), (proteases) and 0-40 °C (phosphatase). However, within a high range of temperatures (25-40 °C) the hydrolytic activity was governed by enzymes with nearly constant substrate affinity. Overall, for enzymes involved in C cycle and proteases, a strong increase (30-40%) in Km at high temperatures (25 °C) reflects an expression of multiple isoenzymes each with different temperature optima and probable shift of microbial community. The general trend of catalytic efficiency (Vmax/Km) demonstrated a gradual increase with

  13. Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain

    PubMed Central

    Li, Jiang; Hu, Qiushi; Li, Yuquan; Xu, Yuan

    2015-01-01

    An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg2+, Mn2+, K+; Ca2+, Na+, Ba2+, Zn2+, Cu2+, Co2+, Fe2+, Sr2+ and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries. PMID:26413048

  14. Expression, purification, and characterization of cold-adapted inorganic pyrophosphatase from psychrophilic Shewanella sp. AS-11.

    PubMed

    Ginting, Elvy Like; Iwasaki, Syouhei; Maeganeku, Chihiro; Motoshima, Hiroyuki; Watanabe, Keiichi

    2014-01-01

    In the presence of divalent cations, inorganic pyrophosphatase is activated to hydrolyze inorganic pyrophosphate to inorganic phosphate. Here, we clone, express, purify, and characterize inorganic pyrophosphatase from the psychrophilic Shewanella sp. AS-11 (Sh-PPase). The recombinant Sh-PPase was expressed in Escherichia coli BL21 (DE3) at 20°C using pET16b as an expression vector and purified from the cell extracts by a combination of ammonium sulfate fractionation and anion-exchange chromatography. Sh-PPase was found to be a family II PPase with a subunit molecular mass of 34 kD that preferentially utilizes Mn²⁺ over Mg²⁺ ions for activity. The functional characteristics of Sh-PPase, such as activity, temperature dependency, and thermal inactivation, were greatly influenced by manganese ions. Manganese ion activation increased the enzyme's activity at low temperatures; therefore, it was required to gain the cold-adapted characteristics of Sh-PPase.

  15. Structural characterization of metal binding to a cold-adapted frataxin.

    PubMed

    Noguera, Martín E; Roman, Ernesto A; Rigal, Juan B; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

    2015-06-01

    Frataxin is an evolutionary conserved protein that participates in iron metabolism. Deficiency of this small protein in humans causes a severe neurodegenerative disease known as Friedreich's ataxia. A number of studies indicate that frataxin binds iron and regulates Fe-S cluster biosynthesis. Previous structural studies showed that metal binding occurs mainly in a region of high density of negative charge. However, a comprehensive characterization of the binding sites is required to gain further insights into the mechanistic details of frataxin function. In this work, we have solved the X-ray crystal structures of a cold-adapted frataxin from a psychrophilic bacterium in the presence of cobalt or europium ions. We have identified a number of metal-binding sites, mainly solvent exposed, several of which had not been observed in previous studies on mesophilic homologues. No major structural changes were detected upon metal binding, although the structures exhibit significant changes in crystallographic B-factors. The analysis of these B-factors, in combination with crystal packing and RMSD among structures, suggests the existence of localized changes in the internal motions. Based on these results, we propose that bacterial frataxins possess binding sites of moderate affinity for a quick capture and transfer of iron to other proteins and for the regulation of Fe-S cluster biosynthesis, modulating interactions with partner proteins.

  16. Unusual Lipid A from a Cold-Adapted Bacterium: Detailed Structural Characterization.

    PubMed

    Casillo, Angela; Ziaco, Marcello; Lindner, Buko; Parrilli, Ermenegilda; Schwudke, Dominik; Holgado, Aurora; Verstrepen, Lynn; Sannino, Filomena; Beyaert, Rudi; Lanzetta, Rosa; Tutino, Maria Luisa; Corsaro, Maria Michela

    2017-06-26

    Colwellia psychrerythraea 34H is a Gram-negative cold-adapted microorganism that adopts many strategies to cope with the limitations associated with the low temperatures of its habitat. In this study, we report the complete characterization of the lipid A moiety from the lipopolysaccharide of Colwellia. Lipid A and its partially deacylated derivative were completely characterized by high-resolution mass spectrometry, NMR spectroscopy, and chemical analysis. An unusual structure with a 3-hydroxy unsaturated tetradecenoic acid as a component of the primary acylation pattern was identified. In addition, the presence of a partially acylated phosphoglycerol moiety on the secondary acylation site at the 3-position of the reducing 2-amino-2-deoxyglucopyranose unit caused tremendous natural heterogeneity in the structure of lipid A. Biological-activity assays indicated that C. psychrerythraea 34H lipid A did not show an agonistic or antagonistic effect upon testing in human macrophages. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from Sporosarcina psychrophila

    PubMed Central

    Parravicini, Federica; Natalello, Antonino; Papaleo, Elena; De Gioia, Luca; Doglia, Silvia Maria; Lotti, Marina; Brocca, Stefania

    2013-01-01

    Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution. PMID:23457536

  18. Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster

    PubMed Central

    Williams, CM; Watanabe, M; Guarracino, MR; Ferraro, MB; Edison, AS; Morgan, TJ; Boroujerdi, AFB; Hahn, DA

    2015-01-01

    When ectotherms are exposed to low temperatures, they enter a cold-induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill-coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using NMR spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold-induced perturbations. The metabolites of cold-hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations. PMID:25308124

  19. Expression and purification of a cold-adapted group III trypsin in Escherichia coli.

    PubMed

    Pálsdóttir, Helga Margrét; Gudmundsdóttir, Agústa

    2007-02-01

    The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and purification of a recombinant HP-thioredoxin-trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its purification by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the first biochemically characterized group III trypsin. It has dual substrate specificity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 degrees C with a complete inactivation at 30 degrees C. The aim of the study was to facilitate further studies of recombinant trypsin Y by finding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an efficient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod.

  20. Cold adaptation of the Antarctic haloarchaea Halohasta litchfieldiae and Halorubrum lacusprofundi.

    PubMed

    Williams, Timothy J; Liao, Yan; Ye, Jun; Kuchel, Rhiannon P; Poljak, Anne; Raftery, Mark J; Cavicchioli, Ricardo

    2017-02-20

    Halohasta litchfieldiae represents ∼ 44% and Halorubrum lacusprofundi ∼ 10% of the hypersaline, perennially cold (≥ -20°C) Deep Lake community in Antarctica. We used proteomics and microscopy to define physiological responses of these haloarchaea to growth at high (30°C) and low (10 and 4°C) temperatures. The proteomic data indicate that both species responded to low temperature by modifying their cell envelope including protein N-glycosylation, maintaining osmotic balance and translation initiation, and modifying RNA turnover and tRNA modification. Distinctions between the two species included DNA protection and repair strategies (e.g. roles of UspA and Rad50), and metabolism of glycerol and pyruvate. For Hrr. lacusprofundi, low temperature led to the formation of polyhydroxyalkanoate (PHA) as a storage compound with the process of PHA granule formation occurring by an unknown mechanism. Hrr. lacusprofundi also formed biofilms and synthesized high levels of Hsp20 chaperones. Hht. litchfieldiae was characterized by an active CRISPR system, and elevated levels of the core gene expression machinery, which contrasted markedly to the decreased levels of Hrr. lacusprofundi. These findings greatly expand the understanding of cellular mechanisms of cold adaptation in psychrophilic archaea, and provide insight into how Hht. litchfieldiae gains dominance in Deep Lake. This article is protected by copyright. All rights reserved.

  1. Cultivation of a novel cold-adapted nitrite oxidizing betaproteobacterium from the Siberian Arctic.

    PubMed

    Alawi, Mashal; Lipski, André; Sanders, Tina; Pfeiffer, Eva Maria; Spieck, Eva

    2007-07-01

    Permafrost-affected soils of the Siberian Arctic were investigated with regard to identification of nitrite oxidizing bacteria active at low temperature. Analysis of the fatty acid profiles of enrichment cultures grown at 4 degrees C, 10 degrees C and 17 degrees C revealed a pattern that was different from that of known nitrite oxidizers but was similar to fatty acid profiles of Betaproteobacteria. Electron microscopy of two enrichment cultures grown at 10 degrees C showed prevalent cells with a conspicuous ultrastructure. Sequence analysis of the 16S rRNA genes allocated the organisms to a so far uncultivated cluster of the Betaproteobacteria, with Gallionella ferruginea as next related taxonomically described organism. The results demonstrate that a novel genus of chemolithoautotrophic nitrite oxidizing bacteria is present in polygonal tundra soils and can be enriched at low temperatures up to 17 degrees C. Cloned sequences with high sequence similarities were previously reported from mesophilic habitats like activated sludge and therefore an involvement of this taxon in nitrite oxidation in nonarctic habitats is suggested. The presented culture will provide an opportunity to correlate nitrification with nonidentified environmental clones in moderate habitats and give insights into mechanisms of cold adaptation. We propose provisional classification of the novel nitrite oxidizing bacterium as 'Candidatus Nitrotoga arctica'.

  2. Are mountain habitats becoming more suitable for generalist than cold-adapted lizards thermoregulation?

    PubMed

    Ortega, Zaida; Mencía, Abraham; Pérez-Mellado, Valentín

    2016-01-01

    Mountain lizards are highly vulnerable to climate change, and the continuous warming of their habitats could be seriously threatening their survival. We aim to compare the thermal ecology and microhabitat selection of a mountain lizard, Iberolacerta galani, and a widely distributed lizard, Podarcis bocagei, in a montane area. Both species are currently in close syntopy in the study area, at 1,400 m above the sea level. We determined the precision, accuracy and effectiveness of thermoregulation, and the thermal quality of habitat for both species. We also compared the selection of thermal microhabitats between both species. Results show that I. galani is a cold-adapted thermal specialist with a preferred temperature range of 27.9-29.7 °C, while P. bocagei would be a thermal generalist, with a broader and higher preferred temperature range (30.1-34.5 °C). In addition, I. galani selects rocky substrates while P. bocagei selects warmer soil and leaf litter substrates. The thermal quality of the habitat is higher for P. bocagei than for I. galani. Finally, P. bocagei achieves a significantly higher effectiveness of thermoregulation (0.87) than I. galani (0.80). Therefore, these mountain habitat conditions seem currently more suitable for performance of thermophilic generalist lizards than for cold-specialist lizards.

  3. Expression of a cold-adapted fish trypsin in Pichia pastoris.

    PubMed

    Macouzet, Martin; Simpson, Benjamin K; Lee, Byong H

    2005-06-01

    Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of the enzyme and for trypsins with special properties has driven the interest to clone and express this protease in microorganisms. Reports about expression of recombinant trypsins show wide differences in the degree of success and are contained mainly in patent applications, which disregard the difficulties associated with the developments. Although the yeast Pichia pastoris appears to be the microbial host with the greatest potential for the production of trypsin, it has shown problems when expressing cold-adapted fish trypsins (CAFTs). CAFTs are considered of immense value for their comparative advantage over other trypsins in a number of food-processing and biotechnological applications. Thus, to investigate potential obstacles related to the production of CAFTs in P. pastoris, the cunner fish trypsin (CFT) was cloned in different Pichia expression vectors. The vectors were constructed targeting both internal and secreted expression and keeping the CFT native signal peptide. Western-blotting analysis confirmed the expression with evident differences for each construct, observing a major effect of the leader peptide sequence on the expression patterns. Immobilized nickel affinity chromatography yielded a partially purified recombinant CFT, which exhibited trypsin-specific activity after activation with bovine enterokinase.

  4. Cold adaptation generates mutations associated with the growth of influenza B vaccine viruses.

    PubMed

    Kim, Hyunsuh; Velkov, Tony; Camuglia, Sarina; Rockman, Steven P; Tannock, Gregory A

    2015-10-26

    Seasonal inactivated influenza vaccines are usually trivalent or quadrivalent and are prepared from accredited seed viruses. Yields of influenza A seed viruses can be enhanced by gene reassortment with high-yielding donor strains, but similar approaches for influenza B seed viruses have been largely unsuccessful. For vaccine manufacture influenza B seed viruses are usually adapted for high-growth by serial passage. Influenza B antigen yields so obtained are often unpredictable and selection of influenza B seed viruses by this method can be a rate-limiting step in seasonal influenza vaccine manufacture. We recently have shown that selection of stable cold-adapted mutants from seasonal epidemic influenza B viruses is associated with improved growth. In this study, specific mutations were identified that were responsible for growth enhancement as a consequence of adaptation to growth at lower temperatures. Molecular analysis revealed that the following mutations in the HA, NP and NA genes are required for enhanced viral growth: G156/N160 in the HA, E253, G375 in the NP and T146 in the NA genes. These results demonstrate that the growth of seasonal influenza B viruses can be optimized or improved significantly by specific gene modifications.

  5. Are mountain habitats becoming more suitable for generalist than cold-adapted lizards thermoregulation?

    PubMed Central

    Mencía, Abraham; Pérez-Mellado, Valentín

    2016-01-01

    Mountain lizards are highly vulnerable to climate change, and the continuous warming of their habitats could be seriously threatening their survival. We aim to compare the thermal ecology and microhabitat selection of a mountain lizard, Iberolacerta galani, and a widely distributed lizard, Podarcis bocagei, in a montane area. Both species are currently in close syntopy in the study area, at 1,400 m above the sea level. We determined the precision, accuracy and effectiveness of thermoregulation, and the thermal quality of habitat for both species. We also compared the selection of thermal microhabitats between both species. Results show that I. galani is a cold-adapted thermal specialist with a preferred temperature range of 27.9–29.7 °C, while P. bocagei would be a thermal generalist, with a broader and higher preferred temperature range (30.1–34.5 °C). In addition, I. galani selects rocky substrates while P. bocagei selects warmer soil and leaf litter substrates. The thermal quality of the habitat is higher for P. bocagei than for I. galani. Finally, P. bocagei achieves a significantly higher effectiveness of thermoregulation (0.87) than I. galani (0.80). Therefore, these mountain habitat conditions seem currently more suitable for performance of thermophilic generalist lizards than for cold-specialist lizards. PMID:27280076

  6. Purification and Characterization of a Cold-Adapted Lipase from Oceanobacillus Strain PT-11

    PubMed Central

    Jiewei, Tian; Zuchao, Lei; Peng, Qiu; Lei, Wang; Yongqiang, Tian

    2014-01-01

    We isolated a moderately halophilic lipase-producing bacterium from the saline soil. Based on the morphological, physiological, chemotaxonomic and phylogenetic analysis, the isolate PT-11 was postulated to be a novel species identified as Oceanobacillus rekensis PT-11. The lipase was purified 2.50-fold by Q-Sepharose FF and SP-Sepharose FF chromatography and its molecular mass was estimated to be 23.5 kDa by SDS-PAGE. It was highly active over the broad temperature ranging from 10 to 35°C and showed up to 80% of the maximum activity at 10°C indicating the lipase to be a typical cold-adapted enzyme. The enzyme activity was slightly enhanced by Na+, Li+ and K+. Incubation with detergents, such as Tween-20 and Tween-80, slightly inhibited the enzyme activity; while Triton X-100decreased the enzyme activity. The enzyme was fairly stable in the presence of long-chain alcohols but was highly denatured in hydrophilic solvents such as acetone or short-chain alcohols (C1–C3). PMID:24984141

  7. Metabolic cold adaptation contributes little to the interspecific variation in metabolic rates of 65 species of Drosophilidae.

    PubMed

    Messamah, Branwen; Kellermann, Vanessa; Malte, Hans; Loeschcke, Volker; Overgaard, Johannes

    2017-02-11

    Metabolic cold adaptation (MCA) is a controversial hypothesis suggesting that cold adapted species display an elevated metabolic rate (MR) compared to their warm climate relatives. Here we test for the presence of MCA in 65 species of drosophilid flies reared under common garden conditions. MR was measured at both 10 and 20°C for both sexes and data were analyzed in relation to the natural thermal environment of these species. We found considerable interspecific variation in MR ranging from 1.34 to 8.99µWmg(-1) at 10°C. As predicted by Bergmann's rule body mass of fly species correlated negatively with annual mean temperature (AMT), such that larger species were found in colder environments. Because larger flies have a higher total MR we found MR to vary with AMT, however, after inclusion of mass as a co-variate we found no significant effect of AMT. Furthermore, we did not find that thermal sensitivity of MR (Q10) varied with AMT. Based on this broad collection of species we therefore conclude that there is no adaptive pattern of metabolic cold adaptation within drosophilid species ranging from sub-arctic to tropical environments.

  8. Optimization of cold-adapted lysozyme production from the psychrophilic yeast Debaryomyces hansenii using statistical experimental methods.

    PubMed

    Wang, Quanfu; Hou, Yanhua; Yan, Peisheng

    2012-06-01

    Statistical experimental designs were employed to optimize culture conditions for cold-adapted lysozyme production of a psychrophilic yeast Debaryomyces hansenii. In the first step of optimization using Plackett-Burman design (PBD), peptone, glucose, temperature, and NaCl were identified as significant variables that affected lysozyme production, the formula was further optimized using a four factor central composite design (CCD) to understand their interaction and to determine their optimal levels. A quadratic model was developed and validated. Compared to the initial level (18.8 U/mL), the maximum lysozyme production (65.8 U/mL) observed was approximately increased by 3.5-fold under the optimized conditions. Cold-adapted lysozymes production was first optimized using statistical experimental methods. A 3.5-fold enhancement of microbial lysozyme was gained after optimization. Such an improved production will facilitate the application of microbial lysozyme. Thus, D. hansenii lysozyme may be a good and new resource for the industrial production of cold-adapted lysozymes. © 2012 Institute of Food Technologists®

  9. Novel cold-adaptive Penicillium strain FS010 secreting thermo-labile xylanase isolated from Yellow Sea.

    PubMed

    Hou, Yun-Hua; Wang, Tian-Hong; Long, Hao; Zhu, Hui-Yuan

    2006-02-01

    A novel cold-adaptive xylanolytic Penicillium strain FS010 was isolated from Yellow sea sediments. The marine fungus grew well from 4 to 20 degrees; a lower (0 degrees) or higher (37 degrees) temperature limits its growth. The strain was identified as Penicillium chrysogenum. Compared with mesophilic P. chrysogenum, the cold-adaptive fungus secreted the cold-active xylanase (XYL) showing high hydrolytic activities at low temperature (2-15 degrees) and high sensitivity to high temperature (>50 degrees). The XYL gene was isolated from the cold-adaptive P. chrysogenum FS010 and designated as xyl. The deduced amino acid sequence of the protein encoded by xyl showed high homology with the sequence of glycoside hydrolase family 10. The gene was subcloned into an expression vector pGEX-4T-1 and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase in Escherichia coli BL21. The expression product was purified and subjected to enzymatic characterization. The optimal temperature and pH for recombinant XYL was 25 degrees and 5.5, respectively. Recombinant XYL showed nearly 80% of its maximal activity at 4 degrees and was active in the pH range 3.0-9.5.

  10. Molecular determinants of enzyme cold adaptation: comparative structural and computational studies of cold- and warm-adapted enzymes.

    PubMed

    Papaleo, Elena; Tiberti, Matteo; Invernizzi, Gaetano; Pasi, Marco; Ranzani, Valeria

    2011-11-01

    The identification of molecular mechanisms underlying enzyme cold adaptation is a hot-topic both for fundamental research and industrial applications. In the present contribution, we review the last decades of structural computational investigations on cold-adapted enzymes in comparison to their warm-adapted counterparts. Comparative sequence and structural studies allow the definition of a multitude of adaptation strategies. Different enzymes carried out diverse mechanisms to adapt to low temperatures, so that a general theory for enzyme cold adaptation cannot be formulated. However, some common features can be traced in dynamic and flexibility properties of these enzymes, as well as in their intra- and inter-molecular interaction networks. Interestingly, the current data suggest that a family-centered point of view is necessary in the comparative analyses of cold- and warm-adapted enzymes. In fact, enzymes belonging to the same family or superfamily, thus sharing at least the three-dimensional fold and common features of the functional sites, have evolved similar structural and dynamic patterns to overcome the detrimental effects of low temperatures.

  11. Promoters from a cold-adapted bacterium: definition of a consensus motif and molecular characterization of UP regulative elements.

    PubMed

    Duilio, Angela; Madonna, Stefania; Tutino, Maria Luisa; Pirozzi, Marinella; Sannia, Giovanni; Marino, Gennaro

    2004-04-01

    Although low-temperature tolerant micro-organisms were discovered long ago, limited information is still available on the transcription machinery in cold-adapted bacteria. This knowledge represents a necessary background for the investigation of the adaptation of gene-expression mechanisms at low temperatures. The recent development of a shuttle genetic system for the transformation of the cold-adapted Gram-negative bacterium Pseudoalteromonas haloplanktis strain TAC125 has made possible the isolation of the psychrophilic promoters described in this paper. TAC125 genomic DNA fragments were cloned in the shuttle vector and the promoter-containing recombinant clones were selected for their ability to express a promoter-less lacZ gene. The nucleotide sequence of several selected inserts and the transcription start points of the transcribed m-RNAs were determined. A promoter consensus sequence for Pseudoalteromonas haloplanktis TAC125 was proposed on the basis of a sequence comparison between the various active promoters. Furthermore, the identification and the functional characterization of two UP elements from this cold-adapted bacterium are also reported.

  12. Biogenesis of thermogenic mitochondria in brown adipose tissue of Djungarian hamsters during cold adaptation.

    PubMed Central

    Klingenspor, M; Ivemeyer, M; Wiesinger, H; Haas, K; Heldmaier, G; Wiesner, R J

    1996-01-01

    After cold exposure, cytochrome c oxidase (COX) activity increased about 2.5-fold within 2 weeks in the brown adipose tissue (BAT) of Djungarian hamsters. The mRNAs for COX subunits I and III and the 12 S rRNA, encoded on mitochondrial DNA (mtDNA), as well as mRNAs for COX subunits IV, Va and mitochondrial transcription factor A, encoded in the nucleus, were unchanged when expressed per unit of total tissue RNA. However, since total tissue RNA doubled per BAT depot, while total DNA remained unchanged, the actual levels of these transcripts were increased within BAT cells. In contrast, the abundance of mRNA for uncoupling protein was increased 10-fold, indicating specific activation of this gene. In addition, the maximal rate of protein synthesis analysed in a faithful in organello system was increased 2.5-fold in mitochondria isolated from BAT after 7 days of cold exposure. We conclude from these data that the biogenesis of thermogenic mitochondria in BAT following cold adaptation is achieved by increasing the overall capacity for synthesis of mitochondrial proteins in both compartments, by increasing their mRNAs as well as the ribosomes needed for their translation. In addition, the translational rate for COX subunits as well as all other proteins encoded on mtDNA is increased. Thus the pool of subunits encoded on mtDNA required for assembly of respiratory chain complexes is provided. By comparison with other models of increased mitochondrial biogenesis, we propose that thyroid hormone (generated within BAT cells by 5'-deiodinase, and induced upon sympathetic stimulation), which is a well known regulator of the biogenesis of mitochondria in many tissues, is also the major effector of these adaptive changes in BAT. PMID:8687407

  13. Role of fatty acids in cold adaptation of Antarctic psychrophilic Flavobacterium spp.

    PubMed

    Králová, Stanislava

    2017-09-01

    Cold-loving microorganisms developed numerous adaptation mechanisms allowing them to survive in extremely cold habitats, such as adaptation of the cell membrane. The focus of this study was on the membrane fatty acids of Antarctic Flavobacterium spp., and their adaptation response to cold-stress. Fatty acids and cold-response of Antarctic flavobacteria was also compared to mesophilic and thermophilic members of the genus Flavobacterium. The results showed that the psychrophiles produced more types of major fatty acids than meso- and thermophilic members of this genus, namely C15:1 iso G, C15:0 iso, C15:0 anteiso, C15:1ω6c, C15:0 iso 3OH, C17:1ω6c, C16:0 iso 3OH and C17:0 iso 3OH, summed features 3 (C16:1ω7cand/or C16:1ω6c) and 9 (C16:0 10-methyl and/or C17:1 iso ω9c). It was shown that the cell membrane of psychrophiles was composed mainly of branched and unsaturated fatty acids. The results also implied that Antarctic flavobacteria mainly used two mechanisms of membrane fluidity alteration in their cold-adaptive response. The first mechanism was based on unsaturation of fatty acids, and the second mechanism on de novo synthesis of branched fatty acids. The alteration of the cell membrane was shown to be similar for all thermotypes of members of the genus Flavobacterium. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Expression and biochemical characterization of cold-adapted lipases from Antarctic Bacillus pumilus strains.

    PubMed

    Litantra, Ribka; Lobionda, Stefani; Yim, Joung Han; Kim, Hyung Kwoun

    2013-09-28

    Two lipase genes (bpl1 and bpl3) from Antarctic Bacillus pumilus strains were expressed in Bacillus subtilis. Both recombinant lipases BPL1 and BPL2 were secreted to the culture medium and their activities reached 3.5 U/ml and 5.0 U/ml, respectively. Their molecular masses apparent using SDS-PAGE were 23 kDa for BPL1 and 19 kDa for BPL3. Both lipases were purified to homogeneity using ammonium sulfate precipitation and HiTrap SP FF column and Superose 12 column chromatographies. The final specific activities were estimated to be 328 U/mg for BPL1 and 310 U/mg for BPL3. Both lipases displayed an optimum temperature of 35°C, similar to other mesophilic enzymes. However, they maintained as much as 70% and 80% of the maximum activities at 10°C. Accordingly, their calculated activation energy at a temperature range of 10-35°C was 5.32 kcal/mol for BPL1 and 4.26 kcal/mol for BPL3, typical of cold-adapted enzymes. The optimum pH of BPL1 and BPL3 was 8.5 and 8.0, respectively, and they were quite stable at pH 7.0-11.0, showing their strong alkaline tolerance. Both lipases had a preference toward medium chain length (C6-C10) fatty acid substrates. These results indicate the potential for the two Antarctic B. pumilus lipases as catalysts in bioorganic synthesis, food, and detergent industries.

  15. Genome-wide analysis of cold adaptation in indigenous Siberian populations.

    PubMed

    Cardona, Alexia; Pagani, Luca; Antao, Tiago; Lawson, Daniel J; Eichstaedt, Christina A; Yngvadottir, Bryndis; Shwe, Ma Than Than; Wee, Joseph; Romero, Irene Gallego; Raj, Srilakshmi; Metspalu, Mait; Villems, Richard; Willerslev, Eske; Tyler-Smith, Chris; Malyarchuk, Boris A; Derenko, Miroslava V; Kivisild, Toomas

    2014-01-01

    Following the dispersal out of Africa, where hominins evolved in warm environments for millions of years, our species has colonised different climate zones of the world, including high latitudes and cold environments. The extent to which human habitation in (sub-)Arctic regions has been enabled by cultural buffering, short-term acclimatization and genetic adaptations is not clearly understood. Present day indigenous populations of Siberia show a number of phenotypic features, such as increased basal metabolic rate, low serum lipid levels and increased blood pressure that have been attributed to adaptation to the extreme cold climate. In this study we introduce a dataset of 200 individuals from ten indigenous Siberian populations that were genotyped for 730,525 SNPs across the genome to identify genes and non-coding regions that have undergone unusually rapid allele frequency and long-range haplotype homozygosity change in the recent past. At least three distinct population clusters could be identified among the Siberians, each of which showed a number of unique signals of selection. A region on chromosome 11 (chr11:66-69 Mb) contained the largest amount of clustering of significant signals and also the strongest signals in all the different selection tests performed. We present a list of candidate cold adaption genes that showed significant signals of positive selection with our strongest signals associated with genes involved in energy regulation and metabolism (CPT1A, LRP5, THADA) and vascular smooth muscle contraction (PRKG1). By employing a new method that paints phased chromosome chunks by their ancestry we distinguish local Siberian-specific long-range haplotype signals from those introduced by admixture.

  16. Diversity and cold adaptation of microorganisms isolated from the Schirmacher Oasis, Antarctica

    NASA Astrophysics Data System (ADS)

    Mojib, Nazia; Bej, Asim K.; Hoover, Richard

    2008-08-01

    We have investigated the feasibility of the PCR amplification of the 16S rRNA genes from eubacteria and Archea on samples collected on Whatman FTA filters from Schirmacher Oasis for the study of culture-independent analysis of the microbial diversity. Both conventional PCR and real-time TaqmaTM PCR successfully amplified the targeted genes. A number of diverse groups of psychrotolerant microorganisms with various pigments have been isolated when cultured on agar medium. 16S rRNA gene analysis of these isolates helped us to identify closest taxonomic genus Pseudomonas, Frigoribacterium, Arthrobacter, Flavobacterium, and Janthinobacterium. It is possible that the pigments play protective role from solar UV radiation, which is prevalent in Antarctic continent especially during Austral summer months. Study of the expression of cold adaptive protein CapB and ice-binding protein IBP using western blots showed positive detection of both or either of these proteins in 6 out of 8 isolates. Since the CapB and IBP protein structure greatly varies in microorganisms, it is possible that the 2 isolates with negative results could have a different class of these proteins. The expression of the CapB and the IBP in these isolates suggest that these proteins are essential for the survival in the Antarctic cold and subzero temperatures and protect themselves from freeze-damage. The current study provided sufficient data to further investigate the rich and diverse biota of psychrotolerant extremophiles in the Antarctic Schirmacher Oasis using both culture-independent and culture-based approaches; and understand the mechanisms of cold tolerance.

  17. Aerobic and Anaerobic Thiosulfate Oxidation by a Cold-Adapted, Subglacial Chemoautotroph

    PubMed Central

    Harrold, Zoë R.; Skidmore, Mark L.; Hamilton, Trinity L.; Desch, Libby; Amada, Kirina; van Gelder, Will; Glover, Kevin; Roden, Eric E.

    2015-01-01

    Geochemical data indicate that protons released during pyrite (FeS2) oxidation are important drivers of mineral weathering in oxic and anoxic zones of many aquatic environments, including those beneath glaciers. Oxidation of FeS2 under oxic, circumneutral conditions proceeds through the metastable intermediate thiosulfate (S2O32−), which represents an electron donor capable of supporting microbial metabolism. Subglacial meltwaters sampled from Robertson Glacier (RG), Canada, over a seasonal melt cycle revealed concentrations of S2O32− that were typically below the limit of detection, despite the presence of available pyrite and concentrations of the FeS2 oxidation product sulfate (SO42−) several orders of magnitude higher than those of S2O32−. Here we report on the physiological and genomic characterization of the chemolithoautotrophic facultative anaerobe Thiobacillus sp. strain RG5 isolated from the subglacial environment at RG. The RG5 genome encodes genes involved with pathways for the complete oxidation of S2O32−, CO2 fixation, and aerobic and anaerobic respiration with nitrite or nitrate. Growth experiments indicated that the energy required to synthesize a cell under oxygen- or nitrate-reducing conditions with S2O32− as the electron donor was lower at 5.1°C than 14.4°C, indicating that this organism is cold adapted. RG sediment-associated transcripts of soxB, which encodes a component of the S2O32−-oxidizing complex, were closely affiliated with soxB from RG5. Collectively, these results suggest an active sulfur cycle in the subglacial environment at RG mediated in part by populations closely affiliated with RG5. The consumption of S2O32− by RG5-like populations may accelerate abiotic FeS2 oxidation, thereby enhancing mineral weathering in the subglacial environment. PMID:26712544

  18. Elucidation of different cold-adapted Atlantic cod (Gadus morhua) trypsin X isoenzymes.

    PubMed

    Stefansson, Bjarki; Sandholt, Gunnar B; Gudmundsdottir, Ágústa

    2017-01-01

    Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist. One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (kcat/Km) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use.

  19. Structural and functional characterization of a cold adapted TPM-domain with ATPase/ADPase activity.

    PubMed

    Cerutti, María L; Otero, Lisandro H; Smal, Clara; Pellizza, Leonardo; Goldbaum, Fernando A; Klinke, Sebastián; Aran, Martín

    2016-11-01

    The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.

  20. Genome-Wide Analysis of Cold Adaptation in Indigenous Siberian Populations

    PubMed Central

    Cardona, Alexia; Pagani, Luca; Antao, Tiago; Lawson, Daniel J.; Eichstaedt, Christina A.; Yngvadottir, Bryndis; Shwe, Ma Than Than; Wee, Joseph; Romero, Irene Gallego; Raj, Srilakshmi; Metspalu, Mait; Villems, Richard; Willerslev, Eske; Tyler-Smith, Chris; Malyarchuk, Boris A.; Derenko, Miroslava V.; Kivisild, Toomas

    2014-01-01

    Following the dispersal out of Africa, where hominins evolved in warm environments for millions of years, our species has colonised different climate zones of the world, including high latitudes and cold environments. The extent to which human habitation in (sub-)Arctic regions has been enabled by cultural buffering, short-term acclimatization and genetic adaptations is not clearly understood. Present day indigenous populations of Siberia show a number of phenotypic features, such as increased basal metabolic rate, low serum lipid levels and increased blood pressure that have been attributed to adaptation to the extreme cold climate. In this study we introduce a dataset of 200 individuals from ten indigenous Siberian populations that were genotyped for 730,525 SNPs across the genome to identify genes and non-coding regions that have undergone unusually rapid allele frequency and long-range haplotype homozygosity change in the recent past. At least three distinct population clusters could be identified among the Siberians, each of which showed a number of unique signals of selection. A region on chromosome 11 (chr11:66–69 Mb) contained the largest amount of clustering of significant signals and also the strongest signals in all the different selection tests performed. We present a list of candidate cold adaption genes that showed significant signals of positive selection with our strongest signals associated with genes involved in energy regulation and metabolism (CPT1A, LRP5, THADA) and vascular smooth muscle contraction (PRKG1). By employing a new method that paints phased chromosome chunks by their ancestry we distinguish local Siberian-specific long-range haplotype signals from those introduced by admixture. PMID:24847810

  1. Aerobic and Anaerobic Thiosulfate Oxidation by a Cold-Adapted, Subglacial Chemoautotroph.

    PubMed

    Harrold, Zoë R; Skidmore, Mark L; Hamilton, Trinity L; Desch, Libby; Amada, Kirina; van Gelder, Will; Glover, Kevin; Roden, Eric E; Boyd, Eric S

    2015-12-28

    Geochemical data indicate that protons released during pyrite (FeS2) oxidation are important drivers of mineral weathering in oxic and anoxic zones of many aquatic environments, including those beneath glaciers. Oxidation of FeS2 under oxic, circumneutral conditions proceeds through the metastable intermediate thiosulfate (S2O3 (2-)), which represents an electron donor capable of supporting microbial metabolism. Subglacial meltwaters sampled from Robertson Glacier (RG), Canada, over a seasonal melt cycle revealed concentrations of S2O3 (2-) that were typically below the limit of detection, despite the presence of available pyrite and concentrations of the FeS2 oxidation product sulfate (SO4 (2-)) several orders of magnitude higher than those of S2O3 (2-). Here we report on the physiological and genomic characterization of the chemolithoautotrophic facultative anaerobe Thiobacillus sp. strain RG5 isolated from the subglacial environment at RG. The RG5 genome encodes genes involved with pathways for the complete oxidation of S2O3 (2-), CO2 fixation, and aerobic and anaerobic respiration with nitrite or nitrate. Growth experiments indicated that the energy required to synthesize a cell under oxygen- or nitrate-reducing conditions with S2O3 (2-) as the electron donor was lower at 5.1°C than 14.4°C, indicating that this organism is cold adapted. RG sediment-associated transcripts of soxB, which encodes a component of the S2O3 (2-)-oxidizing complex, were closely affiliated with soxB from RG5. Collectively, these results suggest an active sulfur cycle in the subglacial environment at RG mediated in part by populations closely affiliated with RG5. The consumption of S2O3 (2-) by RG5-like populations may accelerate abiotic FeS2 oxidation, thereby enhancing mineral weathering in the subglacial environment. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Cold adaptation mechanisms in the ghost moth Hepialus xiaojinensis: Metabolic regulation and thermal compensation.

    PubMed

    Zhu, Wei; Zhang, Huan; Li, Xuan; Meng, Qian; Shu, Ruihao; Wang, Menglong; Zhou, Guiling; Wang, Hongtuo; Miao, Lin; Zhang, Jihong; Qin, Qilian

    2016-02-01

    Ghost moths (Lepidoptera: Hepialidae) are cold-adapted stenothermal species inhabiting alpine meadows on the Tibetan Plateau. They have an optimal developmental temperature of 12-16 °C but can maintain feeding and growth at 0 °C. Their survival strategies have received little attention, but these insects are a promising model for environmental adaptation. Here, biochemical adaptations and energy metabolism in response to cold were investigated in larvae of the ghost moth Hepialus xiaojinensis. Metabolic rate and respiratory quotient decreased dramatically with decreasing temperature (15-4 °C), suggesting that the energy metabolism of ghost moths, especially glycometabolism, was sensitive to cold. However, the metabolic rate at 4 °C increased with the duration of cold exposure, indicating thermal compensation to sustain energy budgets under cold conditions. Underlying regulation strategies were studied by analyzing metabolic differences between cold-acclimated (4 °C for 48 h) and control larvae (15 °C). In cold-acclimated larvae, the energy generating pathways of carbohydrates, instead of the overall consumption of carbohydrates, was compensated in the fat body by improving the transcription of related enzymes. The mobilization of lipids was also promoted, with higher diacylglycerol, monoacylglycerol and free fatty acid content in hemolymph. These results indicated that cold acclimation induced a reorganization on metabolic structure to prioritise energy metabolism. Within the aerobic process, flux throughout the tricarboxylic acid (TCA) cycle was encouraged in the fat body, and the activity of α-ketoglutarate dehydrogenase was the likely compensation target. Increased mitochondrial cristae density was observed in the midgut of cold-acclimated larvae. The thermal compensation strategies in this ghost moth span the entire process of energy metabolism, including degration of metabolic substrate, TCA cycle and oxidative phosphorylation, and from an energy budget

  3. Cold adaptive thermogenesis in small mammals from different geographical zones of China.

    PubMed

    Li, Q; Sun, R; Huang, C; Wang, Z; Liu, X; Hou, J; Liu, J; Cai, L; Li, N; Zhang, S; Wang, Y

    2001-07-01

    in the north; (4) a low RMR in warm environments and peak RMR and NST in cold environments enabled M. unguiculatus to tolerate a semi-desert climate; (5) O. curzoniae has unusually high RMR and high NST, acting mainly via increasing NST to adapt to extreme cold of the Qinghai-Tibet Plateau; (6) the adaptation of euthermic S. dauricus to cold is due to an increase in NST and a relaxed homeothermia; and lastly (7) the thyroid hormone is involved in the regulation of cold adaptive thermogenesis in all the species studied.

  4. [Characterization of cold-adapted influenza strain A/HongKong/1/68/162/35 as a potential donor of attenuation and high reproduction].

    PubMed

    Tsybalova, L M; Gorev, N E; Potapchuk, M V; Repko, I A; Korotkov, A V; Sergeeva, M V; Komissarov, A B; Pisareva, M M; Kuznetsov, V V; Grudinin, M P; Kiselev, O I

    2012-01-01

    Live and inactivated vaccines are currently produced using virus reassortants originating from various gene donors of internal proteins. Based on the pandemic virus A/Hong Kong/1/68 (H3N2), a cold-adapted thermo-sensitive strain A/Hong Kong/1/68/162/35 was generated. It is distinguished for its high reproductive capacity (9-9.5 lg EID50), and hemagglutinating activity (1:1024-1:2048). The strain has ts and ca phenotype: reproductive capacity at t = 39 degrees C is 1.0 lg EID50; at t = 26 degrees C, 8.5 lg EID50. A total of 16 mutations have emerged from comprehensive sequencing of the virus genome. Among them 10 mutations were located in the genes of polymerase complex and NP, with respective amino-acid substitutions. The stability of strain characteristics, such as attenuation to humans and high reproductive capacity, were confirmed by repeated sequencing of the genome after tenfold passing of the virus in chicken embryos. Reassortants of the strain A/Hong Kong/1/68/162/35 with the wild-type viruses have inherited useful features of donor virus.

  5. Effect of simultaneous administration of cold-adapted and wild-type influenza A viruses on experimental wild-type influenza infection in humans.

    PubMed

    Youngner, J S; Treanor, J J; Betts, R F; Whitaker-Dowling, P

    1994-03-01

    On the basis of the ability of the attenuated cold-adapted strain of influenza A virus to suppress disease production in ferrets simultaneously infected with epidemic influenza virus (P. Whitaker-Dowling, H.F. Maassab, and J.S. Youngner, J. Infect. Dis. 164:1200-1202, 1991), an evaluation of the ability of the cold-adapted virus to modify clinical disease in humans was made. Adult volunteers with prechallenge serum hemagglutination-inhibition titers to the influenza A/Kawasaki/86 (H1N1) virus of < or = 1:8 received either 10(7) 50% tissue culture infective doses of the wild-type A/Kawasaki virus or a mixture of 10(7) 50% tissue culture infective doses of each of the wild-type virus and a cold-adapted A/Kawasaki reassortant virus by intranasal drops in a randomized, double-blind fashion. Symptoms and wild-type virus shedding were assessed daily for 6 days following challenge. Results were compared with those derived from another group of volunteers who received only cold-adapted virus. Volunteers who received the mixed inoculum of cold-adapted and wild-type viruses had lower symptom scores than those who received wild-type virus alone, suggesting that coinfection with the cold-adapted virus may modify wild-type virus infection, but the differences were not statistically significant in this small study. The data demonstrate that administration of cold-adapted influenza A virus to humans at the time of wild-type virus infection is a safe procedure.

  6. Genomic, Transcriptomic, and Proteomic Analysis Provide Insights Into the Cold Adaptation Mechanism of the Obligate Psychrophilic Fungus Mrakia psychrophila

    PubMed Central

    Su, Yao; Jiang, Xianzhi; Wu, Wenping; Wang, Manman; Hamid, M. Imran; Xiang, Meichun; Liu, Xingzhong

    2016-01-01

    Mrakia psychrophila is an obligate psychrophilic fungus. The cold adaptation mechanism of psychrophilic fungi remains unknown. Comparative genomics analysis indicated that M. psychrophila had a specific codon usage preference, especially for codons of Gly and Arg and its major facilitator superfamily (MFS) transporter gene family was expanded. Transcriptomic analysis revealed that genes involved in ribosome and energy metabolism were upregulated at 4°, while genes involved in unfolded protein binding, protein processing in the endoplasmic reticulum, proteasome, spliceosome, and mRNA surveillance were upregulated at 20°. In addition, genes related to unfolded protein binding were alternatively spliced. Consistent with other psychrophiles, desaturase and glycerol 3-phosphate dehydrogenase, which are involved in biosynthesis of unsaturated fatty acid and glycerol respectively, were upregulated at 4°. Cold adaptation of M. psychrophila is mediated by synthesizing unsaturated fatty acids to maintain membrane fluidity and accumulating glycerol as a cryoprotectant. The proteomic analysis indicated that the correlations between the dynamic patterns between transcript level changes and protein level changes for some pathways were positive at 4°, but negative at 20°. The death of M. psychrophila above 20° might be caused by an unfolded protein response. PMID:27633791

  7. Biocontrol activity of a cold-adapted yeast from Tibet against gray mold in cherry tomato and its action mechanism.

    PubMed

    Hu, Hao; Wisniewski, Michael E; Abdelfattah, Ahmed; Zheng, Xiaodong

    2017-07-01

    Cold-adapted biocontrol yeast was selected from four yeast isolates from Tibet against gray mold of cherry tomato in cold storage. The strain numbered LB2 showed the best biocontrol activity and identified as Cryptococcus laurentii. Competition for nutrient, space, and induced fruit resistance was also its antagonistic mechanism. Compared with C. laurentii from sea-level place, the reason why LB2 had a better biocontrol activity was studied. More trehalose and proline in cell of LB2 made it exhibit a better cellular activity at low temperature, such as higher population dynamics in the wounds of cherry tomato and more biocontrol-related enzyme secretion, chitinase and β-glucanase. The better oxidative stress tolerance was another characteristic of LB2. Maybe because of the ideal culture condition, there was no obvious difference between these two yeasts in the growth in vitro test at low temperature. Although the same phenomenon existed in the low pH stress test, LB2 still had higher cell concentration under this stress. Comparative transcriptomics method was also applied to analyze the cell activity of LB2 and C. laurentii at different temperatures. The results showed that more active response in the intracellular structure and intracellular metabolic process to cold temperature made LB2 had a better activity. The present study indicated a possibility to select cold-adapted biocontrol yeast from Tibet and also showed its primary action mechanism.

  8. Purification and characterization of an extracellular cold-adapted alkaline lipase produced by psychrotrophic bacterium Yersinia enterocolitica strain KM1.

    PubMed

    Ji, Xiuling; Chen, Guiyuan; Zhang, Qi; Lin, Lianbing; Wei, Yunlin

    2015-06-01

    An extracellular cold-adapted alkaline lipase from the psychrotrophic Yersinia enterocolitica strain KM1 was purified 26-fold to homogeneity. The enzyme was active over a broad range spanning 0-60 °C with an optimum activity at 37 °C, and it was found to be alkaline-preferring with an optimum activity at pH 9.0. The molecular weight was estimated to be 34.3 KDa and monomeric. The lipase could be activated by Ca(2+) and low concentration (10%) of ethanol, dimethyl sulphoxide, methanol, and acetonitrile, whereas it was strongly inhibited by Zn(2+), Cu(2+), SDS, EDTA, and PMSF. Using p-nitrophenyl butyrate as a substrate at 37 °C, the Km and Vmax of the enzyme were found to be 16.58 mM and 5.24 × 10(5)  μM · min(-1), respectively. This extracellular cold-adapted alkaline lipase may be a good candidate for detergents and biocatalysts at low temperature.

  9. A new cold-adapted serine peptidase from Antarctic Lysobacter sp. A03: Insights about enzyme activity at low temperatures.

    PubMed

    Pereira, Jamile Queiroz; Ambrosini, Adriana; Passaglia, Luciane Maria Pereira; Brandelli, Adriano

    2017-10-01

    Currently, there is a great interest for customized biocatalysts that can supply the ongoing demand of industrial processes, but also deal with the growing concern about the environment. In this scenario, cold-adapted enzymes have features that make them very attractive for industrial and biotechnological purposes. Here, we describe A03Pep1, a new cold-adapted serine peptidase isolated from Lysobacter sp. A03 by screening a genomic library. The enzyme is synthesized as a large inactive prepropeptidase that, after intramolecular processing, gives rise to the active form, of 35kDa. The heterologous expression of A03Pep1 was carried out in E. coli cells harboring the vector pGEX-4T-2-a0301. Its activity was optimal at pH 9.0 and 40°C, in the presence of 25mM Ca(2+), which may contribute to the thermal stability of the enzyme. The 3D structure modelling predicted a less deep and more open binding pocket in A03Pep1 than that observed in the crystal structure of its mesophilic homologous AprV2, presumably as a way to enhance the probability of substrate binding at low temperatures. These results provide possible approaches in developing new biotechnologically relevant peptidases active at low to moderate temperatures. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Total lactate dehydrogenase activity of tail muscle is not cold-adapted in nocturnal lizards from cool-temperate habitats.

    PubMed

    Hare, K M; Miller, J H; Clark, A G; Daugherty, C H

    2005-12-01

    The dependence of metabolic processes on temperature constrains the behavior, physiology and ecology of many ectothermic animals. The evolution of nocturnality in lizards, especially in temperate regions, requires adaptations for activity at low temperatures when optimal body temperatures are unlikely to be obtained. We examined whether nocturnal lizards have cold-adapted lactate dehydrogenase (LDH). LDH was chosen as a representative metabolic enzyme. We measured LDH activity of tail muscle in six lizard species (n=123: three nocturnal, two diurnal and one crepuscular) between 5 and 35 degrees C and found no differences in LDH-specific activity or thermal sensitivity among the species. Similarly, the specific activity and thermal sensitivity of LDH were similar between skinks and geckos. Similar enzyme activities among nocturnal and diurnal lizards indicate that there is no selection of temperature specific LDH enzyme activity at any temperature. As many nocturnal lizards actively thermoregulate during the day, LDH may be adapted for a broad range of temperatures rather than adapted specifically for the low temperatures encountered when the animals are active. The total activity of LDH in tropical and temperate lizards is not cold-adapted. More data are required on biochemical adaptations and whole animal thermal preferences before trends can be established.

  11. The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125.

    PubMed

    Howes, Barry D; Giordano, Daniela; Boechi, Leonardo; Russo, Roberta; Mucciacciaro, Simona; Ciaccio, Chiara; Sinibaldi, Federica; Fittipaldi, Maria; Martí, Marcelo A; Estrin, Darío A; di Prisco, Guido; Coletta, Massimo; Verde, Cinzia; Smulevich, Giulietta

    2011-02-01

    The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 α-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.

  12. The ability of a cold-adapted Rhodotorula mucilaginosa strain from Tibet to control blue mold in pear fruit.

    PubMed

    Hu, Hao; Yan, Fujie; Wilson, Charles; Shen, Qing; Zheng, Xiaodong

    2015-12-01

    Cold-adapted yeasts were isolated from soil samples collected in Tibet and evaluated as potential biocontrol agents against blue mold (Penicillium expansum) of pear fruit in cold storage. YC1, an isolate identified as Rhodotorula mucilaginosa, was found to exhibit the greatest biocontrol activity among the different isolates that were screened. A washed cell suspension of YC1 exhibited the best biocontrol activity among three different preparations that were used in the current study. A concentration of 10(8) cells/ml reduced the incidence of decay to 35 %, compared to the control where decay incidence was 100 %. A higher intracellular level of trehalose and a higher proportion of polyunsaturated acids present in YC1, was associated with increased the tolerance of this strain to low temperatures, relative to the other strains that were evaluated. The increased tolerance to low temperature allowed the YC1 strain of yeast to more effectively compete for nutrients and space in wounded pear fruit that had been inoculated with spores of P. expansum and placed in cold storage. The present study demonstrated the ability to select cold-adapted yeasts from cold climates and use them as biocontrol agents of postharvest diseases of fruit placed in cold storage.

  13. Differential cold-adaptation among protein components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125.

    PubMed

    Cotugno, Roberta; Rosaria Ruocco, Maria; Marco, Salvatore; Falasca, Patrizia; Evangelista, Giovanna; Raimo, Gennaro; Chambery, Angela; Di Maro, Antimo; Masullo, Mariorosario; De Vendittis, Emmanuele

    2009-05-01

    Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 degrees C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source.

  14. Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12

    PubMed Central

    2011-01-01

    Background Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food. Results A gene encoding a cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE) and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low temperatures. Conclusion By

  15. Evaluation of bovine, cold-adapted human, and wild-type human parainfluenza type 3 viruses in adult volunteers and in chimpanzees.

    PubMed Central

    Clements, M L; Belshe, R B; King, J; Newman, F; Westblom, T U; Tierney, E L; London, W T; Murphy, B R

    1991-01-01

    In an attempt to evaluate the level of attenuation of live parainfluenza type 3 virus (PIV3) vaccine candidates, we compared the responses of partially immune adult volunteers inoculated intranasally with 10(6) to 10(7) 50% tissue culture infective dose (TCID50) of bovine PIV3 (n = 18) or cold-adapted (ca) PIV3 (n = 37) with those of 28 adults administered 10(6) to 10(7) TCID50 of wild-type PIV3. The candidate vaccine viruses and the wild-type virus were avirulent and poorly infectious for these adults even though all of them had a low level of nasal antibodies to PIV3. To determine whether the ca PIV3 was attenuated, we then administered 10(4) TCID50 of ca PIV3 (cold-passage 12) or wild-type PIV3 intranasally and intratracheally to two fully susceptible chimpanzees, respectively, and challenged the four primates with wild-type virus 1 month later. Compared with wild-type virus, which caused upper respiratory tract illness, the ca PIV3 was highly attenuated and manifested a 500-fold reduction in virus replication in both the upper and lower respiratory tracts of the two immunized animals. Despite restriction of virus replication, infection with ca PIV3 conferred a high level of protective immunity against challenge with wild-type virus. The ca PIV3 which had been passaged 12 times at 20 degrees C did not retain its ts phenotype. These findings indicate that ca PIV3 may be a promising vaccine candidate for human beings if a passage level can be identified that is genetically stable, satisfactorily attenuated, and immunogenic. PMID:1650789

  16. Structural flexibility of the heme cavity in the cold-adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125.

    PubMed

    Giordano, Daniela; Pesce, Alessandra; Boechi, Leonardo; Bustamante, Juan Pablo; Caldelli, Elena; Howes, Barry D; Riccio, Alessia; di Prisco, Guido; Nardini, Marco; Estrin, Dario; Smulevich, Giulietta; Bolognesi, Martino; Verde, Cinzia

    2015-08-01

    Truncated hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two-on-two α-helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group-II truncated hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO), a cold-adapted Antarctic marine bacterium hosting one flavohemoglobin and three distinct truncated hemoglobins. The Ph-2/2HbO aquo-met crystal structure (at 2.21 Å resolution) shows typical features of group-II truncated hemoglobins, namely the two-on-two α-helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal-site hydrogen-bonded network that includes water molecules and several distal-site residues, including His(58)CD1. Analysis of Ph-2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph-2/2HbO can access diverse heme ligation states. Among these, detection of a low-spin heme hexa-coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme-Fe ligand. Altogether, the results show that Ph-2/2HbO maintains the general structural features of group-II truncated hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment.

  17. Purification and characterization of novel raw-starch-digesting and cold-adapted alpha-amylases from Eisenia foetida.

    PubMed

    Ueda, Mitsuhiro; Asano, Tomohiko; Nakazawa, Masami; Miyatake, Kazutaka; Inouye, Kuniyo

    2008-05-01

    Novel raw-starch-digesting and cold-adapted alpha-amylases (Amy I and Amy II) from the earthworm Eisenia foetida were purified to electrophoretically homogeneous states. The molecular weights of both purified enzymes were estimated to be 60,000 by SDS-PAGE. The enzymes were most active at pH 5.5 and 50 degrees C and stable at pH 7.0-9.0 and 50-60 degrees C. Both Amy I and II exhibited activities at 10 degrees C. The enzymes were inhibited by metal ions Cu(2+), Fe(2+), and Hg(2+), and hydrolyzed raw starch into glucose, maltose and maltotriose as end products.

  18. Cold adaptation overrides developmental regulation of sarcolipin expression in mice skeletal muscle: SOS for muscle-based thermogenesis?

    PubMed

    Pant, Meghna; Bal, Naresh C; Periasamy, Muthu

    2015-08-01

    Neonatal mice have a greater thermogenic need than adult mice and may require additional means of heat production, other than the established mechanism of brown adipose tissue (BAT). We and others recently discovered a novel mediator of skeletal muscle-based thermogenesis called sarcolipin (SLN) that acts by uncoupling sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA). In addition, we have shown that SLN expression is downregulated during neonatal development in rats. In this study we probed two questions: (1) is SLN expression developmentally regulated in neonatal mice?; and (2) if so, will cold adaptation override this? Our data show that SLN expression is higher during early neonatal stages and is gradually downregulated in fast twitch skeletal muscles. Interestingly, we demonstrate that cold acclimation of neonatal mice can prevent downregulation of SLN expression. This observation suggests that SLN-mediated thermogenesis can be recruited to a greater extent during extreme physiological need, in addition to BAT.

  19. Crystallization and preliminary X-ray diffraction analysis of a cold-adapted catalase from Vibrio salmonicida

    SciTech Connect

    Riise, Ellen Kristin; Lorentzen, Marit Sjo; Helland, Ronny; Willassen, Nils Peder

    2006-01-01

    Monoclinic (P2{sub 1}) crystals of a His-tagged form of V. salmonicida catalase without cofactor diffract X-rays to 1.96 Å. Catalase (EC 1.11.1.6) catalyses the breakdown of hydrogen peroxide to water and molecular oxygen. Recombinant Vibrio salmonicida catalase (VSC) possesses typical cold-adapted features, with higher catalytic efficiency, lower thermal stability and a lower temperature optimum than its mesophilic counterpart from Proteus mirabilis. Crystals of VSC were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 98.15, b = 217.76, c = 99.28 Å, β = 110.48°. Data were collected to 1.96 Å and a molecular-replacement solution was found with eight molecules in the asymmetric unit.

  20. Use of temperature-sensitive and cold-adapted mutant viruses in immunoprophylaxis of acute respiratory tract disease.

    PubMed

    Chanock, R M; Murphy, B R

    1980-01-01

    Efforts currently are underway to develop mutations in the influenza A viral genome that will bring about a satisfactory level of attenuation and that can be identified by simple in vitro techniques. Two types of donor viruses that bear such mutations are being evaluated. One donor virus possesses temperature-sensitive (ts) mutations on the P1 and P3 genes, while the other donor bears both ts and cold-adaptation (ca) mutations. The mutant genes from these donors were transferred by gene reassortment to recombinant viruses bearing the surface antigens of new epidemic or pandemic viruses, and in every instance a satisfactory level of attenuation was achieved. However, genetic instability remains a formidable problem. Temperature-sensitive mutants of respiratory syncytial virus also have been evaluated for their usefulness in immunoprophylaxis of respiratory tract disease. Although the desired mutant has not been identified, some progress has been made.

  1. The Physarum polycephalum php gene encodes a unique cold-adapted serine-carboxyl peptidase, physarolisin II.

    PubMed

    Nishii, Wataru; Kuriyama, Hiroki; Takahashi, Kenji

    2003-07-10

    The php gene from a true slime mold, Physarum polycephalum, is a late-replicating and transcriptionally active gene. The deduced amino acid sequence of the gene product is homologous to those of the serine-carboxyl peptidase family, including physarolisin I from the same organism, but lacks the propeptide region. In this study, the protein was expressed in Escherichia coli and shown to possess endopeptidase activity with unique substrate specificity. Thus, we named it physarolisin II. The enzyme was revealed to be a kind of cold-adapted enzyme since it was maximally active at 16-22 degrees C. The active enzyme was markedly unstable due to rapid autolysis (t(1/2)= approximately 5 min, at 18 degrees C). At higher temperature, the enzyme was less active but more stable, despite the fact that no gross conformational change was observed by circular dichroism spectroscopy.

  2. Effect of cold-adapted microbial agent inoculation on enzyme activities during composting start-up at low temperature.

    PubMed

    Sun, Qinghong; Wu, Di; Zhang, Zhechao; Zhao, Yue; Xie, Xinyu; Wu, Junqiu; Lu, Qian; Wei, Zimin

    2017-11-01

    In order to put forward a method to promote composting start-up at low ambient temperature, the cold-adapted microbial agent (CAMA) was inoculated in chicken manure (CM), and compared the enzymes activities, including urease, proteases, β-glucosidase and invertase, with no CAMA group (CK). In this study, the temperature of CM reached 50°C in 53h, but it in CK was only around 30°C during the composting process. Moreover, the enzymes exhibited higher activity in CM than CK, indicating the effectiveness of CAMA. Furthermore, redundancy analysis was conducted to study the relationships of CAMA, with enzymes activities and temperature. Results showed that the positive effect of CAMA on the enzyme activities were achieved by affecting the bacterial community structure. Accordingly, we provide a method to guide CAMA inoculation for promoting compost start-up in cold area. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum.

    PubMed

    Zhang, Tao; Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

    2015-08-01

    The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by Pseudogymnoascus destructans (Pd). Recent studies indicated that apart from Pd, there is an abundance of other Pseudogymnoascus species in the hibernacula soil. We developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system for Pd and a related fungus Pseudogymnoascus pannorum (Pp) to advance experimental studies. URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular genetic analyses. A Uracil-Specific Excision Reagent (USER) Friendly pRF-HU2 vector containing Pd or Pp ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by electroporation and the resulting strains were co-cultivated with conidia of Pd or Pp for various durations and temperatures to optimize the ATMT system. Overall, 680 Pd (0.006%) and 1800 Pp (0.018%) transformants were obtained from plating of 10(7) conidia; their recoveries were strongly correlated with the length of the incubation period (96h for Pd; 72h for Pp) and with temperature (15-18°C for Pd; 25°C for Pp). The homologous recombination in transformants was 3.1% for Pd and 16.7% for Pp. The availability of a standardized ATMT system would allow future molecular genetic analyses of Pd and related cold-adapted fungi. Copyright © 2015. Published by Elsevier Inc.

  4. Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil.

    PubMed

    Ko, Kyong-Cheol; Rim, Soon-Ok; Han, Yunjon; Shin, Bong Seok; Kim, Geun-Joong; Choi, Jong Hyun; Song, Jae Jun

    2012-05-01

    A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1-50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1-40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.

  5. Exploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements.

    PubMed

    Berlemont, Renaud; Pipers, Delphine; Delsaute, Maud; Angiono, Federico; Feller, Georges; Galleni, Moreno; Power, Pablo

    2011-01-01

    Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.

  6. Predictive modeling for growth of non- and cold-adapted Listeria Monocytogenes on fresh-cut cantaloupe at different storage temperatures

    USDA-ARS?s Scientific Manuscript database

    The aim of this study was to determine the growth kinetics of Listeria monocytogenes, with and without cold-adaption, on fresh-cut cantaloupe under different storage temperatures. Fresh-cut samples, spot inoculated with a four-strain cocktail of L. monocytogenes (about 3.2 log CFU/g), were exposed t...

  7. Physical and molecular bases of protein thermal stability and cold adaptation.

    PubMed

    Pucci, Fabrizio; Rooman, Marianne

    2017-02-01

    The molecular bases of thermal and cold stability and adaptation, which allow proteins to remain folded and functional in the temperature ranges in which their host organisms live and grow, are still only partially elucidated. Indeed, both experimental and computational studies fail to yield a fully precise and global physical picture, essentially because all effects are context-dependent and thus quite intricate to unravel. We present a snapshot of the current state of knowledge of this highly complex and challenging issue, whose resolution would enable large-scale rational protein design.

  8. Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase.

    PubMed

    Asgeirsson, Bjarni; Adalbjörnsson, Björn Vidar; Gylfason, Gudjón Andri

    2007-06-01

    Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.

  9. Growth promotory potential of the cold adapted diazotroph Pseudomonas migulae S10724 against native green gram (Vigna radiata (L.) Wilczek).

    PubMed

    Suyal, Deep Chandra; Shukla, Anjana; Goel, Reeta

    2014-12-01

    It is being confirmed previously the atmospheric nitrogen fixing ability of the cold adapted Pseudomonas migulae S10724 strain at the fluctuating temperatures. Therefore, net house bioinoculation experiment was performed to determine the effectiveness of inoculation of strain S10724 on the growth enhancement of native green gram (Vigna radiata L. Wilczek). The strain significantly (p < 0.05) stimulated the growth of roots (45.3 %) and shoots (45.6 %) of green gram plants. Furthermore, other growth related parameters viz. fresh and dry weight was also found to be increased significantly. Treated plants typically showed more obvious modifications in their biochemical status also. The total chlorophyll and nitrate reductase activity was increased in S10724 inoculated plant as compared to the control one. Moreover, in vitro seed germination assay revealed that the germination was increased in S10724 strain treated seeds by 22 % at 25 °C while 25 % at 12 °C unlikely to respective controls. The results suggest that P.migulae S10724 strain is a potential plant growth promoting bacterium for legume under fluctuating temperature ranges and therefore, could be used effectively as a low cost bioinoculant in Himalayan agricultural belt successfully.

  10. New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases.

    PubMed

    Ryu, H S; Kim, H K; Choi, W C; Kim, M H; Park, S Y; Han, N S; Oh, T K; Lee, J K

    2006-04-01

    A Photobacterium strain, M37, showing lipolytic activity, was previously isolated from an intertidal flat of the Yellow Sea in Korea and identified as Photobacterium lipolyticum sp. nov. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,023 bp) corresponded to a protein of 340 amino acid residues with a molecular weight of 38,026. No sequence similarity was found with any known bacterial lipases/esterases; instead, the most similar enzymes were several filamentous fungal lipases. Although the similarity was very low (less than 16%), there were many conserved regions over the entire sequence and N-terminal oxyanion hole (RG) region, a signature sequence of filamentous fungal lipases. The novel protein M37 was produced in both a soluble and insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The soluble protein exhibited lipase activity in a pH-stat assay using an olive oil emulsion. The M37 lipase also displayed a maximum activity at 25 degrees C and maintained its activity at a low temperature range (5-25 degrees C) with an activation energy (E(a)) of 2.07 kcal/mol. Accordingly, these results indicate that the M37 lipase from P. lipolyticum sp. nov. is a new cold-adapted enzyme.

  11. A novel cold-adapted and glucose-tolerant GH1 β-glucosidase from Exiguobacterium antarcticum B7.

    PubMed

    Crespim, Elaine; Zanphorlin, Letícia M; de Souza, Flavio H M; Diogo, José A; Gazolla, Alex C; Machado, Carla B; Figueiredo, Fernanda; Sousa, Amanda S; Nóbrega, Felipe; Pellizari, Vivian H; Murakami, Mário T; Ruller, Roberto

    2016-01-01

    A novel GH1 β-glucosidase (EaBgl1A) from a bacterium isolated from Antarctica soil samples was recombinantly overexpressed in Escherichia coli cells and characterized. The enzyme showed unusual pH dependence with maximum activity at neutral pH and retention of high catalytic activity in the pH range 6 to 9, indicating a catalytic machinery compatible with alkaline conditions. EaBgl1A is also a cold-adapted enzyme, exhibiting activity in the temperature range from 10 to 40°C with optimal activity at 30°C, which allows its application in industrial processes using low temperatures. Kinetic characterization revealed an enzymatic turnover (Kcat) of 6.92s(-1) (cellobiose) and 32.98s(-1) (pNPG) and a high tolerance for product inhibition, which is an extremely desirable feature for biotechnological purposes. Interestingly, the enzyme was stimulated by up to 200 mM glucose, whereas the commercial cocktails tested were found fully inhibited at this concentration. These properties indicate EaBgl1A as a promising biocatalyst for biotechnological applications where low temperatures are required.

  12. Cold-adapted and rhizosphere-competent strain of Rahnella sp. with broad-spectrum plant growth-promotion potential.

    PubMed

    Vyas, Pratibha; Joshi, Robin; Sharma, K C; Rahi, Praveen; Gulati, Ashu; Gulati, Arvind

    2010-12-01

    A phosphate-solubilizing bacterial strain isolated from Hippophae rhamnoides rhizosphere was identified as Rahnella sp. based on its phenotypic features and 16S rRNA gene sequence. The bacterial strain showed the growth characteristics of a cold-adapted psychrotroph, with the multiple plant growth-promoting traits of inorganic and organic phosphate solubilization, 1-aminocyclopropane-1- carboxylate-deaminase activity, ammonia generation, and siderophore production. The strain also produced indole- 3-acetic acid, indole-3-acetaldehyde, indole-3-acetamide, indole-3-acetonitrile, indole-3-lactic acid, and indole-3- pyruvic acid in tryptophan-supplemented nutrient broth. Gluconic, citric and isocitric acids were the major organic acids detected during tricalcium phosphate solubilization. A rifampicin-resistant mutant of the strain exhibited high rhizosphere competence without disturbance to the resident microbial populations in pea rhizosphere. Seed bacterization with a charcoal-based inoculum significantly increased growth in barley, chickpea, pea, and maize under the controlled environment. Microplot testing of the inoculum at two different locations in pea also showed significant increase in growth and yield. The attributes of coldtolerance, high rhizosphere competence, and broad-spectrum plant growth-promoting activity exhibited the potential of Rahnella sp. BIHB 783 for increasing agriculture productivity.

  13. Serine hydroxymethyltransferase from the cold adapted microorganism Psychromonas ingrahamii: a low temperature active enzyme with broad substrate specificity.

    PubMed

    Angelaccio, Sebastiana; Florio, Rita; Consalvi, Valerio; Festa, Guido; Pascarella, Stefano

    2012-01-01

    Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  14. Cloning, expression, purification, and characterization of cold-adapted α-amylase from Pseudoalteromonas arctica GS230.

    PubMed

    Lu, Mingsheng; Wang, Shujun; Fang, Yaowei; Li, Huangzhong; Liu, Shu; Liu, Hongfei

    2010-11-01

    A cold-adapted α-amylase (ParAmy) gene from Pseudoalteromonas arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography. The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With an optimum temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7-8.5 at 30 °C for 1 h. ParAmy was activated by Mn(2+), K(+) and Na(+), and inhibited by Hg(2+), Cu(2+), and Fe(3+). N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K (m) and V (max) values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry.

  15. Cold adapted features of Vibrio salmonicida catalase: characterisation and comparison to the mesophilic counterpart from Proteus mirabilis.

    PubMed

    Lorentzen, Marit Sjo; Moe, Elin; Jouve, Hélène Marie; Willassen, Nils Peder

    2006-10-01

    The gene encoding catalase from the psychrophilic marine bacterium Vibrio salmonicida LFI1238 was identified, cloned and expressed in the catalase-deficient Escherichia coli UM2. Recombinant catalase from V. salmonicida (VSC) was purified to apparent homogeneity as a tetramer with a molecular mass of 235 kDa. VSC contained 67% heme b and 25% protoporphyrin IX. VSC was able to bind NADPH, react with cyanide and form compounds I and II as other monofunctional small subunit heme catalases. Amino acid sequence alignment of VSC and catalase from the mesophilic Proteus mirabilis (PMC) revealed 71% identity. As for cold adapted enzymes in general, VSC possessed a lower temperature optimum and higher catalytic efficiency (k (cat)/K (m)) compared to PMC. VSC have higher affinity for hydrogen peroxide (apparent K (m)) at all temperatures. For VSC the turnover rate (k (cat)) is slightly lower while the catalytic efficiency is slightly higher compared to PMC over the temperature range measured, except at 4 degrees C. Moreover, the catalytic efficiency of VSC and PMC is almost temperature independent, except at 4 degrees C where PMC has a twofold lower efficiency compared to VSC. This may indicate that VSC has evolved to maintain a high efficiency at low temperatures.

  16. Is there metabolic cold adaptation in terrestrial ectotherms? Exploring latitudinal compensation in the invasive snail Cornu aspersum.

    PubMed

    Gaitán-Espitia, Juan Diego; Nespolo, Roberto

    2014-07-01

    Lower temperatures, extreme seasonality and shorter growing seasons at higher latitudes are expected to cause a decline in metabolic rates and annual growth rates of ectotherms. If a reduction in the rates of these biological processes involves a reduction in fitness, then organisms may evolve compensatory responses for the constraints imposed by high-latitude habitats. To test the existence of a latitudinal compensation in ectotherms, we used a common-garden experiment to investigate the extent to which the level of energy turnover (measured as standard metabolic rate, SMR) and the energy budget (energy allocation to growth) are affected by climatic constraints in three populations of the land snail Cornu aspersum, distributed across a latitudinal gradient of 1300 km in Chile. Our results did not support the existence of a latitudinal compensation in metabolic rates (metabolic cold adaptation). However, there was a countergradient variation (CnGV) for growth rate in which the highest latitudinal population exhibited greater growth rates than their counterparts from lower latitudes. Surprisingly, this CnGV pattern was accompanied by a lower apparent dry-matter digestibility, which could highlight a differential assimilation of ingested nutrients into somatic tissue, revealing enhanced growth efficiency in snails from the highest latitudinal habitat. Our evidence highlights that adjustments in energy allocation to the digestive machinery and to protein storage could act as a latitudinal compensation for enhanced growth efficiency in snails from the highest latitudinal population. © 2014. Published by The Company of Biologists Ltd.

  17. Distribution of cold adaptation proteins in microbial mats in Lake Joyce, Antarctica: Analysis of metagenomic data by using two bioinformatics tools.

    PubMed

    Koo, Hyunmin; Hakim, Joseph A; Fisher, Phillip R E; Grueneberg, Alexander; Andersen, Dale T; Bej, Asim K

    2016-01-01

    In this study, we report the distribution and abundance of cold-adaptation proteins in microbial mat communities in the perennially ice-covered Lake Joyce, located in the McMurdo Dry Valleys, Antarctica. We have used MG-RAST and R code bioinformatics tools on Illumina HiSeq2000 shotgun metagenomic data and compared the filtering efficacy of these two methods on cold-adaptation proteins. Overall, the abundance of cold-shock DEAD-box protein A (CSDA), antifreeze proteins (AFPs), fatty acid desaturase (FAD), trehalose synthase (TS), and cold-shock family of proteins (CSPs) were present in all mat samples at high, moderate, or low levels, whereas the ice nucleation protein (INP) was present only in the ice and bulbous mat samples at insignificant levels. Considering the near homogeneous temperature profile of Lake Joyce (0.08-0.29 °C), the distribution and abundance of these proteins across various mat samples predictively correlated with known functional attributes necessary for microbial communities to thrive in this ecosystem. The comparison of the MG-RAST and the R code methods showed dissimilar occurrences of the cold-adaptation protein sequences, though with insignificant ANOSIM (R = 0.357; p-value = 0.012), ADONIS (R(2) = 0.274; p-value = 0.03) and STAMP (p-values = 0.521-0.984) statistical analyses. Furthermore, filtering targeted sequences using the R code accounted for taxonomic groups by avoiding sequence redundancies, whereas the MG-RAST provided total counts resulting in a higher sequence output. The results from this study revealed for the first time the distribution of cold-adaptation proteins in six different types of microbial mats in Lake Joyce, while suggesting a simpler and more manageable user-defined method of R code, as compared to a web-based MG-RAST pipeline.

  18. Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 β-glucosidase from Exiguobacterium antarcticum B7.

    PubMed

    Zanphorlin, Leticia Maria; de Giuseppe, Priscila Oliveira; Honorato, Rodrigo Vargas; Tonoli, Celisa Caldana Costa; Fattori, Juliana; Crespim, Elaine; de Oliveira, Paulo Sergio Lopes; Ruller, Roberto; Murakami, Mario Tyago

    2016-03-31

    Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways. Elucidating their adaptive strategies is instrumental to understand how life can thrive in cold ecosystems and to tailor enzymes for biotechnological applications at low temperatures. In this work, we used X-ray crystallography, in solution studies and molecular dynamics simulations to reveal the structural basis for cold adaptation of the GH1 β-glucosidase from Exiguobacterium antarcticum B7. We discovered that the selective pressure of low temperatures favored mutations that redesigned the protein surface, reduced the number of salt bridges, exposed more hydrophobic regions to the solvent and gave rise to a tetrameric arrangement not found in mesophilic and thermophilic homologues. As a result, some solvent-exposed regions became more flexible in the cold-adapted tetramer, likely contributing to enhance enzymatic activity at cold environments. The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers. According to phylogenetic analysis, diverse adaptive strategies to cold environments emerged in the GH1 family, being tetramerization an alternative, not a rule. These findings reveal a novel strategy for enzyme cold adaptation and provide a framework for the semi-rational engineering of β-glucosidases aiming at cold industrial processes.

  19. Comparative genomics of the marine bacterial genus Glaciecola reveals the high degree of genomic diversity and genomic characteristic for cold adaptation.

    PubMed

    Qin, Qi-Long; Xie, Bin-Bin; Yu, Yong; Shu, Yan-Li; Rong, Jin-Cheng; Zhang, Yan-Jiao; Zhao, Dian-Li; Chen, Xiu-Lan; Zhang, Xi-Ying; Chen, Bo; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2014-06-01

    To what extent the genomes of different species belonging to one genus can be diverse and the relationship between genomic differentiation and environmental factor remain unclear for oceanic bacteria. With many new bacterial genera and species being isolated from marine environments, this question warrants attention. In this study, we sequenced all the type strains of the published species of Glaciecola, a recently defined cold-adapted genus with species from diverse marine locations, to study the genomic diversity and cold-adaptation strategy in this genus.The genome size diverged widely from 3.08 to 5.96 Mb, which can be explained by massive gene gain and loss events. Horizontal gene transfer and new gene emergence contributed substantially to the genome size expansion. The genus Glaciecola had an open pan-genome. Comparative genomic research indicated that species of the genus Glaciecola had high diversity in genome size, gene content and genetic relatedness. This may be prevalent in marine bacterial genera considering the dynamic and complex environments of the ocean. Species of Glaciecola had some common genomic features related to cold adaptation, which enable them to thrive and play a role in biogeochemical cycle in the cold marine environments.

  20. Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 β-glucosidase from Exiguobacterium antarcticum B7

    NASA Astrophysics Data System (ADS)

    Zanphorlin, Leticia Maria; de Giuseppe, Priscila Oliveira; Honorato, Rodrigo Vargas; Tonoli, Celisa Caldana Costa; Fattori, Juliana; Crespim, Elaine; de Oliveira, Paulo Sergio Lopes; Ruller, Roberto; Murakami, Mario Tyago

    2016-03-01

    Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways. Elucidating their adaptive strategies is instrumental to understand how life can thrive in cold ecosystems and to tailor enzymes for biotechnological applications at low temperatures. In this work, we used X-ray crystallography, in solution studies and molecular dynamics simulations to reveal the structural basis for cold adaptation of the GH1 β-glucosidase from Exiguobacterium antarcticum B7. We discovered that the selective pressure of low temperatures favored mutations that redesigned the protein surface, reduced the number of salt bridges, exposed more hydrophobic regions to the solvent and gave rise to a tetrameric arrangement not found in mesophilic and thermophilic homologues. As a result, some solvent-exposed regions became more flexible in the cold-adapted tetramer, likely contributing to enhance enzymatic activity at cold environments. The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers. According to phylogenetic analysis, diverse adaptive strategies to cold environments emerged in the GH1 family, being tetramerization an alternative, not a rule. These findings reveal a novel strategy for enzyme cold adaptation and provide a framework for the semi-rational engineering of β-glucosidases aiming at cold industrial processes.

  1. Global mapping transcriptional start sites revealed both transcriptional and post-transcriptional regulation of cold adaptation in the methanogenic archaeon Methanolobus psychrophilus.

    PubMed

    Li, Jie; Qi, Lei; Guo, Yang; Yue, Lei; Li, Yanping; Ge, Weizhen; Wu, Jun; Shi, Wenyuan; Dong, Xiuzhu

    2015-03-18

    Psychrophilic methanogenic Archaea contribute significantly to global methane emissions, but archaeal cold adaptation mechanisms remain poorly understood. Hinted by that mRNA architecture determined secondary structure respond to cold more promptly than proteins, differential RNA-seq was used in this work to examine the genome-wide transcription start sites (TSSs) of the psychrophilic methanogen Methanolobus psychrophilus R15 and its response to cold. Unlike most prokaryotic mRNAs with short 5' untranslated regions (5' UTR, median lengths of 20-40 nt), 51% mRNAs of this methanogen have large 5' UTR (>50 nt). For 24% of the mRNAs, the 5' UTR is >150 nt. This implies that post-transcriptional regulation may be significance in the psychrophile. Remarkably, 219 (14%) genes possessed multiple gene TSSs (gTSSs), and 84 genes exhibited temperature-regulated gTSS selection to express alternative 5' UTR. Primer extension studies confirmed the temperature-dependent TSS selection and a stem-loop masking of ribosome binding sites was predicted from the longer 5' UTRs, suggesting alternative 5' UTRs-mediated translation regulation in the cold adaptation as well. In addition, 195 small RNAs (sRNAs) were detected, and Northern blots confirmed that many sRNAs were induced by cold. Thus, this study revealed an integrated transcriptional and post-transcriptional regulation for cold adaptation in a psychrophilic methanogen.

  2. Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 β-glucosidase from Exiguobacterium antarcticum B7

    PubMed Central

    Zanphorlin, Leticia Maria; de Giuseppe, Priscila Oliveira; Honorato, Rodrigo Vargas; Tonoli, Celisa Caldana Costa; Fattori, Juliana; Crespim, Elaine; de Oliveira, Paulo Sergio Lopes; Ruller, Roberto; Murakami, Mario Tyago

    2016-01-01

    Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways. Elucidating their adaptive strategies is instrumental to understand how life can thrive in cold ecosystems and to tailor enzymes for biotechnological applications at low temperatures. In this work, we used X-ray crystallography, in solution studies and molecular dynamics simulations to reveal the structural basis for cold adaptation of the GH1 β-glucosidase from Exiguobacterium antarcticum B7. We discovered that the selective pressure of low temperatures favored mutations that redesigned the protein surface, reduced the number of salt bridges, exposed more hydrophobic regions to the solvent and gave rise to a tetrameric arrangement not found in mesophilic and thermophilic homologues. As a result, some solvent-exposed regions became more flexible in the cold-adapted tetramer, likely contributing to enhance enzymatic activity at cold environments. The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers. According to phylogenetic analysis, diverse adaptive strategies to cold environments emerged in the GH1 family, being tetramerization an alternative, not a rule. These findings reveal a novel strategy for enzyme cold adaptation and provide a framework for the semi-rational engineering of β-glucosidases aiming at cold industrial processes. PMID:27029646

  3. The Genome Sequence of the psychrophilic archaeon, Methanococcoides burtonii: the Role of Genome Evolution in Cold-adaptation

    SciTech Connect

    Allen, Michelle A.; Lauro, Federico M.; Williams, Timothy J.; Burg, Dominic; Siddiqui, Khawar S.; De Francisci, David; Chong, Kevin W.Y.; Pilak, Oliver; Chew, Hwee H.; De Maere, Matthew Z.; Ting, Lily; Katrib, Marilyn; Ng, Charmaine; Sowers, Kevin R.; Galperin, Michael Y.; Anderson, Iain J.; Ivanova, Natalia; Dalin, Eileen; Martinez, Michelle; Lapidus, Alla; Hauser, Loren; Land, Miriam; Thomas, Torsten; Cavicchioli, Ricardo

    2009-04-01

    Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five tiered Evidence Rating system that ranked annotations from Evidence Rating (ER) 1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall/membrane/envelope biogenesis COG genes are over-represented. Likewise, signal transduction (COG category T) genes are over-represented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two over-represented COG categories appear to have been acquired from {var_epsilon}- and {delta}-proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they play an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have occurred over the

  4. Purification and characterization of a novel cold-adapted phytase from Rhodotorula mucilaginosa strain JMUY14 isolated from Antarctic.

    PubMed

    Yu, Peng; Wang, Xue-Ting; Liu, Jing-Wen

    2015-08-01

    A yeast producing a cold-adapted phytase was isolated from Antarctic deep-sea sediment and identified as a Rhodotorula mucilaginosa strain JMUY14 of basidiomycetous yeasts. It was cultured in fermentation optimized by a response surface methodology based on the Box-Behnken design. The maximum activity of phytase reached 205.447 U ml(-1), which was close to the predicted value of 201.948 U ml(-1) and approximately 3.4 times higher than its initial activity. The extracellular phytase was purified by 15.2-fold to homogeneity with a specific activity of 31,635 U mg(-1) by (NH4 )2 SO4 precipitation, and a combination of DEAE Sepharose Fast Flow, SP Sepharose Fast Flow, and Sephadex G-100. The molecular weight of the purified enzyme was estimated to be 63 kDa and its pI was 4.33. Its optimal temperature and pH were 50 °C and 5.0, respectively. Its activity was 85% at 37 °C, and showed good stability at pH 3.0 ∼ 7.0. When compared with mesophilic counterparts, the phytase not only exhibited a higher activity during 20 ∼ 30 °C but also had a low Km (247 µM) and high kcat (1394 s(-1)). The phytase activity was slightly stimulated in the presence of Mg(2+), Fe(2+), Fe(3+), K(+), Na(+), Ca(2+), EDTA, and EGTA and moderately inhibited by Cu(2+), Zn(2+), Mn(2+), Ag(+), PMSF, SDS, and phenylgloxal hydrate. It was resistant to both pepsin and trypsin. Since the phytase produced by the R. mucilaginosa JMUY14 showed a high specific activity, good pH stability, strong protease resistance, and high activity at low temperature, it has great potential for feed applications, especially in aquaculture.

  5. The genome sequence of the psychrophilic archaeon, Methanococcoides burtonii: the role of genome evolution in cold adaptation

    SciTech Connect

    Allen, Michele A; Lauro, Federico M; Williams, Timothy J; Burg, Dominic; Siddiqui, Khawar S; DeFrancisci, Davide; Chong, Kevin WY; Pilak, Oliver; Chew, Hwee H; DeMaere, Matthew Z; Ting, Lily; Katrib, Marilyn; Ng, Charmaine; Sowers, Kevin R; Galperin, Michael Y.; Anderson, Iain; Ivanova, N; Dalin, Eileen; Martinez, Michele; Lapidus, Alla L.; Hauser, Loren John; Land, Miriam L; Thomas, Torsten; Cavicchioli, Ricardo

    2009-01-01

    Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five-tiered evidence rating (ER) system that ranked annotations from ER1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea, which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino-acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall, membrane, envelope biogenesis COG genes are overrepresented. Likewise, signal transduction (COG category T) genes are overrepresented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two overrepresented COG categories appear to have been acquired from - and -Proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they have an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have occurred over the last several thousand years as it

  6. Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes

    PubMed Central

    Fields, Peter A.; Somero, George N.

    1998-01-01

    To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4 (A4-LDH) from nine Antarctic (−1.86 to 1°C) and three South American (4 to 10°C) notothenioid teleosts. Higher Michaelis–Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in kcat and Km are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A4-LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting kcat, we conclude that notothenioid A4-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation in Km and kcat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase kcat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high Km values relative to warm-adapted orthologs. PMID:9736762

  7. [MECHANISMS OF ATTENUATION OF COLD-ADAPTED STRAIN A/KRASNO- DAR/101/35/59 (H2N2)].

    PubMed

    Markushin, S G; Svitich, O A; Kinkulkina, A R; Koptyaeva, I B; Lisovskaya, K V

    2016-01-01

    Study of mechanisms of attenuation of cold-adapted (ca) influenza virus strain A/ Krasnodar/101/35/59 (H2N2), associated with disruption of NS1 protein functions. Study of interferonogenic activity of ca strain A/Krasnodar/101/35/59 (H2N2), its parent variant A/Krasnodar/101/59 (H2N2), virulent strain A/WSN/33 (H1N1) and a number of single gene and multiple gene reassortants between these strains, obtained using reverse genetics, was carried out. Study of dynamics of IFNβ gene expression was carried out by using a methodical approach of RT-PCR in real time mode. Inclusion of PB-1 gene of ca strain A/ Krasnodar/101/35/59 (H2N2) with reversion to wild type into genome composition of virulent strain A/WSN/33 (H1N1) does not result in a sharp change of interferonogenic activity of the reassortant. At the same time, similar inclusion of PB-1 gene of ca strain resulted in an incredible growth of interferonogenic activity of the reassortant. On the other hand, inclusion of NP-gene of wild type strain A/Krasnodar/101/59 (H2N2) into genome composition of the wild type strain A/WSN/33 did not differ by effect on interferonogenicity of the reassortant from inclusion of NP-gene of ca strain. Both constellations of genes of parent variants and mutations localized in these genes could affect formation of attenuation phenotype of reassortants. The data obtained allow to assume possible mechanisms of attenuation of ca strains, associated with disruption.of NS gene function.

  8. A novel cold-adapted lipase from Sorangium cellulosum strain So0157-2: gene cloning, expression, and enzymatic characterization.

    PubMed

    Cheng, Yuan-Yuan; Qian, Yun-Kai; Li, Zhi-Feng; Wu, Zhi-Hong; Liu, Hong; Li, Yue-Zhong

    2011-01-01

    Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C(10)), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the K(m) values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.

  9. [Genetic and phenotypic analysis of heterogeneous population of a cold-adapted donor of the A/Leningrad/134/17/57 (H2N2) attenuation and of the donor-based reassortant influenza vaccine strains].

    PubMed

    Kiseleva, I V; Klimov, A I; Grigor'eva, E P; Larionova, N V; Aleksandrova, G I; Rudenko, L G

    2005-01-01

    Cold-adapted (CA) temperature sensitive and attenuated virus A/Leningrad/134/17/57 (H2N2) (Len/17) has been recently used in Russia as a donor of internal genes in the preparation of reassortant vaccine strains of CA live influenza vaccine (LIV) for all age groups. The Len/17 population was found to be heterogeneous and to be made up of clones, which differ by combinations of mutations in internal genes. Around 50% of the Len/17 population had clones with all 8 coding mutations in internal genes. The others were made up of clones with mutation combinations, which were different from the original Len/17. The PCR restriction method was used to analyze 5 clones of Len/17 and 8 LIV vaccine strains. There were no Ala-86-Thr mutation in the M2 protein in 4 clones and 3 vaccine strains. The PB-1 gene of 4 clones and 3 vaccine strains had a mutation encoding Met-317-IIe more typical of a more attenuated virus A/Leningrad/134/47/57 (H2N2) (Len/47). The NP protein of a clone had a mutation Leu-341-IIe also typical of Len/47. However, neither the absence of mutation in the M2 gene nor an extra mutation in the PB1 gene affected the attenuation extent of reassortant CALIV.

  10. Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis

    PubMed Central

    Bai, Xi; Yuan, Xianjun; Wen, Aiyou; Li, Junfeng; Bai, Yunfeng

    2016-01-01

    Background Many biotechnological and industrial applications can benefit from cold-adapted EglCs through increased efficiency of catalytic processes at low temperature. In our previous study, Citrobacter farmeri A1 which was isolated from a wood-inhabiting termite Reticulitermes labralis could secrete a cold-adapted EglC. However, its EglC was difficult to purify for enzymatic properties detection because of its low activity (0.8 U/ml). The objective of the present study was to clone and express the C. farmeri EglC gene in Escherichia coli to improve production level and determine the enzymatic properties of the recombinant enzyme. Methods The EglC gene was cloned from C. farmeri A1 by thermal asymmetric interlaced PCR. EglC was transformed into vector pET22b and functionally expressed in E. coli. The recombination protein EglC22b was purified for properties detection. Results SDS-PAGE revealed that the molecular mass of the recombinant endoglucanase was approximately 42 kDa. The activity of the E. coli pET22b-EglC crude extract was 9.5 U/ml. Additionally, it was active at pH 6.5–8.0 with an optimum pH of 7.0. The recombinant enzyme had an optimal temperature of 30–40 °C and exhibited >50% relative activity even at 5 °C, whereas it lost approximately 90% of its activity after incubation at 60 °C for 30 min. Its activity was enhanced by Co2+ and Fe3+, but inhibited by Cd2+, Zn2+, Li+, Triton X-100, DMSO, acetonitrile, Tween 80, SDS, and EDTA. Conclusion These biochemical properties indicate that the recombinant enzyme is a cold-adapted endoglucanase that can be used for various industrial applications. PMID:27843715

  11. Predictive modeling for growth of non- and cold-adapted Listeria monocytogenes on fresh-cut cantaloupe at different storage temperatures.

    PubMed

    Hong, Yoon-Ki; Yoon, Won Byong; Huang, Lihan; Yuk, Hyun-Gyun

    2014-06-01

    The aim of this study was to determine the growth kinetics of Listeria monocytogenes, with and without cold-adaption, on fresh-cut cantaloupe under different storage temperatures. Fresh-cut samples, spot inoculated with a 4-strain cocktail of L. monocytogenes (∼3.2 log CFU/g), were exposed to constant storage temperatures held at 10, 15, 20, 25, or 30 °C. All growth curves of L. monocytogenes were fitted to the Baranyi, modified Gompertz, and Huang models. Regardless of conditions under which cells grew, the time needed to reach 5 log CFU/g decreased with the elevated storage temperature. Experimental results showed that there were no significant differences (P > 0.05) in the maximum growth rate k (log CFU/g h(-1) ) and lag phase duration λ (h) between the cultures of L. monocytogenes with or without previous cold-adaption treatments. No distinct difference was observed in the growth pattern among 3 primary models at various storage temperatures. The growth curves of secondary modeling were fitted on an Arrhenius-type model for describing the relationship between k and temperature of the L. monocytogenes on fresh-cut cantaloupe from 10 to 30 °C. The root mean square error values of secondary models for non- and cold-adapted cells were 0.018, 0.021, and 0.024, and 0.039, 0.026, and 0.017 at the modified Gompertz, Baranyi, and Huang model, respectively, indicating that these 3 models presented the good statistical fit. This study may provide valuable information to predict the growth of L. monocytogenes on fresh-cut cantaloupes at different storage conditions. Listeriosis has occurred and increased along with the increased demand of fresh and fresh-cut fruits and vegetables. This study was conducted to predict the growth of non- and cold-adapted L. monocytogenes on fresh-cut cantaloupe at different temperature using mathematical model. These results can be helpful for risk assessments of L. monocytogenes in fresh-cut cantaloupe. This study provides valuable

  12. [Identification of psychrotrophs SYP-A2-3 producing cold-adapted protease from the No. 1 Glacier of China and study on its fermentation conditions].

    PubMed

    Shi, Jin-song; Wu, Qi-fan; Xu, Zheng-hong; Tao, Wen-yi

    2005-04-01

    The psychrotrophs SYP-A2-3 producing the cold-adapted protease has been isolated from the bacterial samples collected from the No. 1 Glacier of China and identified as Bacillus cereus according to its morphological and physiochemical characteristics and 16s rDNA gene sequence analysis. It could grow between 0 degree C and 38 degrees C while its optimal growth temperature was 25 degrees C and the optimal temperature for its protease production was 15 degrees C. The cold-adapted protease was identified as neutral metallo-protease, the molecular weight was 34.2 kD shown by SDS-PAGE, the optimal pH and temperature for activity was 7.0-8.5 and 42 degrees C, respectively. Various fermentation conditions of its protease production were also investigated. The results showed that casein was the best nitrogen source while glucose and starch were suitable carbon source for its protease production. The initial pH of fermentation broth ranged from 6.5 to 7.0 was optimal. Under optimized conditions, the protease activity produced by SYP-A2-3 could reach 3800 U/mL and 4800 U/mL conducted in shaking flask and 5 L stirred jar experiment, respectively.

  13. Citric acid production from partly deproteinized whey under non-sterile culture conditions using immobilized cells of lactose-positive and cold-adapted Yarrowia lipolytica B9.

    PubMed

    Arslan, Nazli Pinar; Aydogan, Mehmet Nuri; Taskin, Mesut

    2016-08-10

    The present study was performed to produce citric acid (CA) from partly deproteinized cheese whey (DPCW) under non-sterile culture conditions using immobilized cells of the cold-adapted and lactose-positive yeast Yarrowia lipolytica B9. DPCW was prepared using the temperature treatment of 90°C for 15min. Sodium alginate was used as entrapping agent for cell immobilization. Optimum conditions for the maximum CA production (33.3g/L) in non-sterile DPCW medium were the temperature of 20°C, pH 5.5, additional lactose concentration of 20g/L, sodium alginate concentration of 2%, number of 150 beads/100mL and incubation time of 120h. Similarly, maximum citric acid/isocitric acid (CA/ICA) ratio (6.79) could be reached under these optimal conditions. Additional nitrogen and phosphorus sources decreased CA concentration and CA/ICA ratio. Immobilized cells were reused in three continuous reaction cycles without any loss in the maximum CA concentration. The unique combination of low pH and temperature values as well as cell immobilization procedure could prevent undesired microbial contaminants during CA production. This is the first work on CA production by cold-adapted microorganisms under non-sterile culture conditions. Besides, CA production using a lactose-positive strain of the yeast Y. lipolytica was investigated for the first time in the present study.

  14. Functional annotation of the mesophilic-like character of mutants in a cold-adapted enzyme by self-organising map analysis of their molecular dynamics.

    PubMed

    Fraccalvieri, Domenico; Tiberti, Matteo; Pandini, Alessandro; Bonati, Laura; Papaleo, Elena

    2012-10-01

    Multiple comparison of the Molecular Dynamics (MD) trajectories of mutants in a cold-adapted α-amylase (AHA) could be used to elucidate functional features required to restore mesophilic-like activity. Unfortunately it is challenging to identify the different dynamic behaviors and correctly relate them to functional activity by routine analysis. We here employed a previously developed and robust two-stage approach that combines Self-Organising Maps (SOMs) and hierarchical clustering to compare conformational ensembles of proteins. Moreover, we designed a novel strategy to identify the specific mutations that more efficiently convert the dynamic signature of the psychrophilic enzyme (AHA) to that of the mesophilic counterpart (PPA). The SOM trained on AHA and its variants was used to classify a PPA MD ensemble and successfully highlighted the relationships between the flexibilities of the target enzyme and of the different mutants. Moreover the local features of the mutants that mostly influence their global flexibility in a mesophilic-like direction were detected. It turns out that mutations of the cold-adapted enzyme to hydrophobic and aromatic residues are the most effective in restoring the PPA dynamic features and could guide the design of more mesophilic-like mutants. In conclusion, our strategy can efficiently extract specific dynamic signatures related to function from multiple comparisons of MD conformational ensembles. Therefore, it can be a promising tool for protein engineering.

  15. Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

    PubMed Central

    Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra

    2017-01-01

    ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the

  16. Characterization of Reverse Genetics-Derived Cold-Adapted Master Donor Virus A/Leningrad/134/17/57 (H2N2) and Reassortants with H5N1 Surface Genes in a Mouse Model

    PubMed Central

    Isakova-Sivak, Irina; Chen, Li-Mei; Bourgeois, Melissa; Matsuoka, Yumiko; Voeten, J. Theo M.; Heldens, Jacco G. M.; van den Bosch, Han; Klimov, Alexander; Rudenko, Larisa; Cox, Nancy J.

    2014-01-01

    Live attenuated influenza vaccines (LAIV) offer significant advantages over subunit or split inactivated vaccines to mitigate an eventual influenza pandemic, including simpler manufacturing processes and more cross-protective immune responses. Using an established reverse genetics (rg) system for wild-type (wt) A/Leningrad/134/1957 and cold-adapted (ca) A/Leningrad/134/17/1957 (Len17) master donor virus (MDV), we produced and characterized three rg H5N1 reassortant viruses carrying modified HA and intact NA genes from either A/Vietnam/1203/2004 (H5N1, VN1203, clade 1) or A/Egypt/321/2007 (H5N1, EG321, clade 2) virus. A mouse model of infection was used to determine the infectivity and tissue tropism of the parental wt viruses compared to the ca master donor viruses as well as the H5N1 reassortants. All ca viruses showed reduced replication in lungs and enhanced replication in nasal epithelium. In addition, the H5N1 HA and NA enhanced replication in lungs unless it was restricted by the internal genes of the ca MDV. Mice inoculated twice 4 weeks apart with the H5N1 reassortant LAIV candidate viruses developed serum hemagglutination inhibition HI and IgA antibody titers to the homologous and heterologous viruses consistent with protective immunity. These animals remained healthy after challenge inoculation with a lethal dose with homologous or heterologous wt H5N1 highly pathogenic avian influenza (HPAI) viruses. The profiles of viral replication in respiratory tissues and the immunogenicity and protective efficacy characteristics of the two ca H5N1 candidate LAIV viruses warrant further development into a vaccine for human use. PMID:24648485

  17. Molecular mechanisms of reversion to the ts+ (non-temperature-sensitive) phenotype of influenza A cold-adapted (ca) virus strains.

    PubMed

    Tsfasman, T M; Markushin, S G; Akopova, I I; Ghendon, Y Z

    2007-10-01

    A ts+ ca- (non-temperature-sensitive, non-cold-adapted) revertant of the A/Leningrad/134/47/57 ca strain influenza virus [A/Leningrad/134/47/ts+18/1957(H2N2)], obtained in our previous study, lost phenotypic manifestation of ts mutations by the PB2, NP and NS genes, although, according to sequencing data, it acquired only two true reversions of a mutation in the PB2 and PB1 genes. Direct sequencing showed the appearance of 27 additional mutations (13 coding) in the genes encoding the PB2, PB1, PA, NP, M and NS proteins of the revertant, along with the above-mentioned two true reversions. We conjecture that some of these mutations suppressed phenotypic manifestation of ts mutations in the NS and NP genes.

  18. Contributions of two-component regulatory systems, alternative sigma factors, and negative regulators to Listeria monocytogenes cold adaptation and cold growth.

    PubMed

    Chan, Yvonne C; Hu, Yuewei; Chaturongakul, Soraya; Files, Kali D; Bowen, Barbara M; Boor, Kathryn J; Wiedmann, Martin

    2008-02-01

    The ability of Listeria monocytogenes to grow at refrigeration temperatures is critical for transmission of this foodborne pathogen. We evaluated the contributions of different transcriptional regulators and two-component regulatory systems to L. monocytogenes cold adaptation and cold growth. L. monocytogenes parent strain 10403S and selected isogenic null mutants in genes encoding four alternative sigma factors (sigB, sigH, sigC, and sigL), two regulators of sigmaB (rsbT and rsbV), two negative regulators (ctsR and hrcA), and 15 two-component response regulators were grown in brain heart infusion broth at 4 degrees C with (i) a high-concentration starting inoculum (10(8) CFU/ml), (ii) a low-concentration starting inoculum (102 CFU/ml), and (iii) a high-concentration starting inoculum of cold-adapted cells. With a starting inoculum of 10(8) CFU/ml, null mutants in genes encoding selected alternative sigma factors (DeltasigH, DeltasigC, and DeltasigL), a negative regulator (DeltactsR), regulators of sigmaB (DeltarsbT and DeltarsbV), and selected two-component response regulators (DeltalisR, Deltalmo1172, and Deltalmo1060) had significantly reduced growth (P < 0.05) compared with the parent strain after 12 days at 4 degrees C. The growth defect for DeltasigL was limited and was not confirmed by optical density (OD600) measurement data. With a starting inoculum of 102 CFU/ml and after monitoring growth at 4 degrees C over 84 days, only the DeltactsR strain had a consistent but limited growth defect; the other mutant strains had either no growth defects or limited growth defects apparent at only one or two of the nine sampling points evaluated during the 84-day growth period (DeltasigB, DeltasigC, and Deltalmo1172). With a 10(8) CFU/ml starting inoculum of cold-adapted cells, none of the mutant strains that had a growth defect when inoculation was performed with cells pregrown at 37 degrees C had reduced growth as compared with the parent strain after 12 days at 4

  19. A cold-adapted carbohydrate esterase from the oil-degrading marine Bacterium Microbulbifer thermotolerans DAU221: gene cloning, purification, and characterization.

    PubMed

    Lee, Yong-Suk; Heo, Jae Bok; Lee, Je-Hoon; Choi, Yong-Lark

    2014-07-01

    A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the α/β-class of protein consisted of a central six-stranded β-sheet flanked by eight α-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were 15°C and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50°C or 60°C for 6 h, and 89% of its enzyme activity when preincubated at 70°C for 1h . The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by Na(+) and Mg(2+) ions but was strongly inhibited by Cu(+) and Hg(2+) ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a Km of 0.278 mM and a kcat of 1.9 s(-1). Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

  20. Structure Prediction of a Novel Exo-β-1,3-Glucanase: Insights into the Cold Adaptation of Psychrophilic Yeast Glaciozyma antarctica PI12.

    PubMed

    Mohammadi, Salimeh; Parvizpour, Sepideh; Razmara, Jafar; Abu Bakar, Farah Diba; Illias, Rosli Md; Mahadi, Nor Muhammad; Murad, Abdul MunirAbdul

    2016-07-30

    We report a detailed structural analysis of the psychrophilic exo-β-1,3-glucanase (GaExg55) from Glaciozyma antarctica PI12. This study elucidates the structural basis of exo-1,3-β-1,3-glucanase from this psychrophilic yeast. The structural prediction of GaExg55 remains a challenge because of its low sequence identity (37 %). A 3D model was constructed for GaExg55. Threading approach was employed to determine a suitable template and generate optimal target-template alignment for establishing the model using MODELLER9v15. The primary sequence analysis of GaExg55 with other mesophilic exo-1,3-β-glucanases indicated that an increased flexibility conferred to the enzyme by a set of amino acids substitutions in the surface and loop regions of GaExg55, thereby facilitating its structure to cold adaptation. A comparison of GaExg55 with other mesophilic exo-β-1,3-glucanases proposed that the catalytic activity and structural flexibility at cold environment were attained through a reduced amount of hydrogen bonds and salt bridges, as well as an increased exposure of the hydrophobic side chains to the solvent. A molecular dynamics simulation was also performed using GROMACS software to evaluate the stability of the GaExg55 structure at varying low temperatures. The simulation result confirmed the above findings for cold adaptation of the psychrophilic GaExg55. Furthermore, the structural analysis of GaExg55 with large catalytic cleft and wide active site pocket confirmed the high activity of GaExg55 to hydrolyze polysaccharide substrates.

  1. Protection of weanling hamsters from experimental infection with wild-type parainfluenza virus type 3 (para 3) by cold-adapted mutants of para 3.

    PubMed

    Crookshanks-Newman, F K; Belshe, R B

    1986-02-01

    Parainfluenza virus type 3 (para 3) was adapted to replicate at 20 degrees C, a nonpermissive temperature for wild-type (wt) para 3. Serial passage at 20 degrees C resulted in the generation of cold-adapted (ca) and temperature-sensitive (ts) mutants. These mutant viruses have been characterized both in vitro and in vivo [Belshe and Hissom (1982): Journal of Medical Virology 10:235-242; Crookshanks and Belshe (1984): Journal of Medical Virology 13:243-249]. We now report the evaluation of three mutants (clone 1150, passaged 12 times in the cold [cp12], clone 1146, passaged 18 times in the cold [cp18], and clone 1328, passaged 45 times in the cold [cp45]) for their ability to protect hamsters from infection by wild-type para 3. Ether-anesthetized male syrian hamsters were intranasally vaccinated with either wt para 3 (clone 127) or one of the ca para 3 mutants and on day 28 post-vaccination; each animal was intranasally challenged with 10(5.0) pfu of wt para 3. On days 1, 2, 3, and 4 post-challenge, 4 to 13 hamsters from each group were sacrificed, and the quantity of para 3 in the nasal turbinates and lungs was determined. Wt virus induced protection from challenge. cp12, cp18, and cp45 reduced the peak titer of wt replication in the lungs by greater than 100-fold, tenfold, and tenfold, respectively. The duration of virus replication was shortened also by intranasal vaccination with the mutants. These data give evidence of an inverse relationship between the degree of protection induced by vaccination with cold-adapted mutants and the number of passages of the virus in the cold.

  2. Cold shock genes cspA and cspB from Caulobacter crescentus are posttranscriptionally regulated and important for cold adaptation.

    PubMed

    Mazzon, Ricardo R; Lang, Elza A S; Silva, Carolina A P T; Marques, Marilis V

    2012-12-01

    Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.

  3. A live attenuated cold adapted influenza A H7N7 candidate vaccine virus confers protection against homologous and heterologous H7 viruses in mice and ferrets

    USDA-ARS?s Scientific Manuscript database

    Highly pathogenic avian influenza (HPAI) H7 subtype viruses pose a pandemic threat to humans because of their ability to transmit directly from domestic poultry to humans. The recent cases of HPAI H7 virus infection in humans underscore their pandemic potential and a need to develop a vaccine to pr...

  4. Cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics: new insights into relationship between conformational flexibility and hydrogen bonding.

    PubMed

    Xie, Bin-Bin; Bian, Fei; Chen, Xiu-Lan; He, Hai-Lun; Guo, Jun; Gao, Xiang; Zeng, Yin-Xin; Chen, Bo; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2009-04-03

    Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.

  5. A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purification and characterization.

    PubMed

    Choo, D W; Kurihara, T; Suzuki, T; Soda, K; Esaki, N

    1998-02-01

    A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G. Feller, M. Thirty, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381-388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897-4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C4 and C6) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35 degrees C. The enzyme was unstable at temperatures higher than 45 degrees C. The Km of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn2+, Cu2+, Fe3+, and Hg2+ but was not affected by phenylmethylsulfonyl fluoride and bisnitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.

  6. Biochemical characterization of a novel cold-adapted GH39 β-agarase, AgaJ9, from an agar-degrading marine bacterium Gayadomonas joobiniege G7.

    PubMed

    Jung, Subin; Lee, Chang-Ro; Chi, Won-Jae; Bae, Chang-Hwan; Hong, Soon-Kwang

    2017-03-01

    Gayadomonas joobiniege G7 is an agar-degrading marine bacterium belonging to a novel genus. Genomic sequencing of G. joobiniege revealed that AgaJ9 (formerly YjdB) belonging to the glycoside hydrolase (GH) 39 family. It showed the highest similarity (47% identity) to a putative β-agarase from Catenovulum agarivorans DS-2, an agar-degrading marine bacterium sharing the highest similarity in the nucleotide sequence of 16s rRNA gene with G. joobiniege G7. The agaJ9 gene encodes a protein (134 kDa) of 1205 amino acids, including a 23-amino acid signal peptide. The agarase activity of purified AgaJ9 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ9 activity were determined as 5 and 25 °C, respectively. Notably, AgaJ9 is a cold-adapted β-agarase retaining more than 80% of its activity even at a temperature of 5 °C. In addition, gel filtration chromatography revealed that AgaJ9 exists as two forms, dimer and monomer. Although the two forms had similar enzymatic properties, their kinetic parameters were different. The K m and V max of dimeric AgaJ9 for agarose was 0.68 mg/ml (5.7 × 10(-6) M) and 17.2 U/mg, respectively, whereas the monomeric form had a K m of 1.43 mg/ml (1.2 × 10(-5) M) and V max of 10.7 U/mg. Thin-layer chromatography and agarose-liquefying analyses revealed that AgaJ9 is an endo-type β-agarase that hydrolyzes agarose into neoagarotetraose and neoagarobiose. This study is the first report of a GH39 β-agarase with a cold-adapted enzymatic feature, a unique attribute, which may be useful for industrial applications.

  7. Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

    PubMed

    Sanders, Barbara P; de Los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G; Song, Yutong; Cooper, Gillian; Crawt, Laura E; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2016-03-01

    The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive

  8. Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine

    PubMed Central

    Sanders, Barbara P.; de los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G.; Song, Yutong; Cooper, Gillian; Crawt, Laura E.; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H. H. V.; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2016-01-01

    The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive

  9. A novel cold-adapted and highly salt-tolerant esterase from Alkalibacterium sp. SL3 from the sediment of a soda lake.

    PubMed

    Wang, Guozeng; Wang, Qiaohuang; Lin, Xianju; Ng, Tzi Bun; Yan, Renxiang; Lin, Juan; Ye, Xiuyun

    2016-02-26

    A novel esterase gene (estSL3) was cloned from the Alkalibacterium sp. SL3, which was isolated from the sediment of soda lake Dabusu. The 636-bp full-length gene encodes a polypeptide of 211 amino acid residues that is closely related with putative GDSL family lipases from Alkalibacterium and Enterococcus. The gene was successfully expressed in E. coli, and the recombinant protein (rEstSL3) was purified to electrophoretic homogeneity and characterized. rEstSL3 exhibited the highest activity towards pNP-acetate and had no activity towards pNP-esters with acyl chains longer than C8. The enzyme was highly cold-adapted, showing an apparent temperature optimum of 30 °C and remaining approximately 70% of the activity at 0 °C. It was active and stable over the pH range from 7 to 10, and highly salt-tolerant up to 5 M NaCl. Moreover, rEstSL3 was strongly resistant to most tested metal ions, chemical reagents, detergents and organic solvents. Amino acid composition analysis indicated that EstSL3 had fewer proline residues, hydrogen bonds and salt bridges than mesophilic and thermophilic counterparts, but more acidic amino acids and less hydrophobic amino acids when compared with other salt-tolerant esterases. The cold active, salt-tolerant and chemical-resistant properties make it a promising enzyme for basic research and industrial applications.

  10. Gene cloning and catalytic characterization of cold-adapted lipase of Photobacterium sp. MA1-3 isolated from blood clam.

    PubMed

    Kim, Young Ok; Khosasih, Vivia; Nam, Bo-Hye; Lee, Sang-Jun; Suwanto, Antonius; Kim, Hyung Kwoun

    2012-12-01

    A lipase-producing Photobacterium strain (MA1-3) was isolated from the intestine of a blood clam caught at Namhae, Korea. The lipase gene was cloned by shotgun cloning and encoded 340 amino acids with a molecular mass of 38,015 Da. It had a very low sequence identity with other bacterial lipases, with the exception of that of Photobacterium lipolyticum M37 (83.2%). The MA1-3 lipase was produced in soluble form when Escherichia coli cells harboring the gene were cultured at 18°C. Its optimum temperature and pH were 45°C and pH 8.5, respectively. Its activation energy was calculated to be 2.69 kcal/mol, suggesting it to be a cold-adapted lipase. Its optimum temperature, temperature stability, and substrate specificity were quite different from those of M37 lipase, despite the considerable sequence similarities. Meanwhile, MA1-3 lipase performed a transesterification reaction using olive oil and various alcohols including methanol, ethanol, 1-propanol, and 1-butanol. In the presence of t-butanol as a co-solvent, this lipase produced biodiesel using methanol and plant or waste oils. The highest biodiesel conversion yield (73%) was achieved using waste soybean oil and methanol at a molar ratio of 1:5 after 12 h using 5 units of lipase.

  11. Sequence and structural investigation of a novel psychrophilic α-amylase from Glaciozyma antarctica PI12 for cold-adaptation analysis.

    PubMed

    Ramli, Aizi Nor Mazila; Azhar, Mohd Akmal; Shamsir, Mohd Shahir; Rabu, Amir; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad; Illias, Rosli Md

    2013-08-01

    A novel α-amylase was isolated successfully from Glaciozyma antarctica PI12 using DNA walking and reverse transcription-polymerase chain reaction (RT-PCR) methods. The structure of this psychrophilic α-amylase (AmyPI12) from G. antarctica PI12 has yet to be studied in detail. A 3D model of AmyPI12 was built using a homology modelling approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9.9. Analysis of the AmyPI12 model revealed the presence of binding sites for a conserved calcium ion (CaI), non-conserved calcium ions (CaII and CaIII) and a sodium ion (Na). Compared with its template-the thermostable α-amylase from Bacillus stearothermophilus (BSTA)-the binding of CaII, CaIII and Na ions in AmyPI12 was observed to be looser, which suggests that the low stability of AmyPI12 allows the protein to work at different temperature scales. The AmyPI12 amino acid sequence and model were compared with thermophilic α-amylases from Bacillus species that provided the highest structural similarities with AmyPI12. These comparative studies will enable identification of possible determinants of cold adaptation.

  12. Range shifts or extinction? Ancient DNA and distribution modelling reveal past and future responses to climate warming in cold-adapted birds.

    PubMed

    Lagerholm, Vendela K; Sandoval-Castellanos, Edson; Vaniscotte, Amélie; Potapova, Olga R; Tomek, Teresa; Bochenski, Zbigniew M; Shepherd, Paul; Barton, Nick; Van Dyck, Marie-Claire; Miller, Rebecca; Höglund, Jacob; Yoccoz, Nigel G; Dalén, Love; Stewart, John R

    2017-04-01

    Global warming is predicted to cause substantial habitat rearrangements, with the most severe effects expected to occur in high-latitude biomes. However, one major uncertainty is whether species will be able to shift their ranges to keep pace with climate-driven environmental changes. Many recent studies on mammals have shown that past range contractions have been associated with local extinctions rather than survival by habitat tracking. Here, we have used an interdisciplinary approach that combines ancient DNA techniques, coalescent simulations and species distribution modelling, to investigate how two common cold-adapted bird species, willow and rock ptarmigan (Lagopus lagopus and Lagopus muta), respond to long-term climate warming. Contrary to previous findings in mammals, we demonstrate a genetic continuity in Europe over the last 20 millennia. Results from back-casted species distribution models suggest that this continuity may have been facilitated by uninterrupted habitat availability and potentially also the greater dispersal ability of birds. However, our predictions show that in the near future, some isolated regions will have little suitable habitat left, implying a future decrease in local populations at a scale unprecedented since the last glacial maximum.

  13. Safety, immunogencity, and efficacy of a cold-adapted A/Ann Arbor/6/60 (H2N2) vaccine in mice and ferrets

    SciTech Connect

    Chen, Grace L.; Lamirande, Elaine W.; Jin Hong; Kemble, George; Subbarao, Kanta

    2010-03-01

    We studied the attenuation, immunogenicity and efficacy of the cold-adapted A/Ann Arbor/6/60 (AA ca) (H2N2) virus in mice and ferrets to evaluate its use in the event of an H2 influenza pandemic. The AA ca virus was restricted in replication in the respiratory tract of mice and ferrets. In mice, 2 doses of vaccine elicited a > 4-fold rise in hemagglutination-inhibition (HAI) titer and resulted in complete inhibition of viral replication following lethal homologous wild-type virus challenge. In ferrets, a single dose of the vaccine elicited a > 4-fold rise in HAI titer and conferred complete protection against homologous wild-type virus challenge in the upper respiratory tract. In both mice and ferrets, the AA ca virus provided significant protection from challenge with heterologous H2 virus challenge in the respiratory tract. The AA ca vaccine is safe, immunogenic, and efficacious against homologous and heterologous challenge in mice and ferrets, supporting the evaluation of this vaccine in clinical trials.

  14. Safety, immunogencity, and efficacy of a cold-adapted A/Ann Arbor/6/60 (H2N2) vaccine in mice and ferrets.

    PubMed

    Chen, Grace L; Lamirande, Elaine W; Jin, Hong; Kemble, George; Subbarao, Kanta

    2010-03-01

    We studied the attenuation, immunogenicity and efficacy of the cold-adapted A/Ann Arbor/6/60 (AA ca) (H2N2) virus in mice and ferrets to evaluate its use in the event of an H2 influenza pandemic. The AA ca virus was restricted in replication in the respiratory tract of mice and ferrets. In mice, 2 doses of vaccine elicited a >4-fold rise in hemagglutination-inhibition (HAI) titer and resulted in complete inhibition of viral replication following lethal homologous wild-type virus challenge. In ferrets, a single dose of the vaccine elicited a >4-fold rise in HAI titer and conferred complete protection against homologous wild-type virus challenge in the upper respiratory tract. In both mice and ferrets, the AA ca virus provided significant protection from challenge with heterologous H2 virus challenge in the respiratory tract. The AA ca vaccine is safe, immunogenic, and efficacious against homologous and heterologous challenge in mice and ferrets, supporting the evaluation of this vaccine in clinical trials. Published by Elsevier Inc.

  15. Proteomic analysis of cold adaptation in a Siberian permafrost bacterium--Exiguobacterium sibiricum 255-15 by two-dimensional liquid separation coupled with mass spectrometry.

    PubMed

    Qiu, Yinghua; Kathariou, Sophia; Lubman, David M

    2006-10-01

    Bacterial cold adaptation in Exiguobacterium sibiricum 255-15 was studied on a proteomic scale using a 2-D liquid phase separation coupled with MS technology. Whole-cell lysates of E. sibiricum 255-15 grown at 4 degrees C and 25 degrees C were first fractionated according to pI by chromatofocusing (CF), and further separated based on hydrophobicity by nonporous silica RP HPLC (NPS-RP-HPLC) which was on-line coupled with an ESI-TOF MS for intact protein M(r) measurement and quantitative interlysate comparison. Mass maps were created to visualize the differences in protein expression between different growth temperatures. The differentially expressed proteins were then identified by PMF using a MALDI-TOF MS and peptide sequencing by MS/MS with a MALDI quadrupole IT TOF mass spectrometer (MALDI-QIT-TOF MS). A total of over 500 proteins were detected in this study, of which 256 were identified. Among these proteins 39 were cold acclimation proteins (Caps) that were preferentially or uniquely expressed at 4 degrees C and three were homologous cold shock proteins (Csps). The homologous Csps were found to be similarly expressed at 4 degrees C and 25 degrees C, where these three homologous Csps represent about 10% of the total soluble proteins at both 4 degrees C and 25 degrees C.

  16. Cold adaptation of eicosapentaenoic acid-less mutant of Shewanella livingstonensis Ac10 involving uptake and remodeling of synthetic phospholipids containing various polyunsaturated fatty acids.

    PubMed

    Sato, Sho; Kurihara, Tatsuo; Kawamoto, Jun; Hosokawa, Masashi; Sato, Satoshi B; Esaki, Nobuyoshi

    2008-11-01

    An Antarctic psychrotrophic bacterium, Shewanella livingstonensis Ac10, produces cis-5,8,11,14,17-eicosapentaenoic acid (EPA), a long-chain polyunsaturated fatty acid (LPUFA), as a component of membrane phospholipids at low temperatures. The EPA-less mutant generated by disruption of the EPA synthesis gene becomes cold-sensitive. We studied whether the cold sensitivity could be suppressed by supplementation of various LPUFAs. The EPA-less mutant was cultured at 6 degrees C in the presence of synthetic phosphatidylethanolamines (PEs) that contained oleic acid at the sn-1 position and various C20 fatty acids with different numbers of double bonds from zero to five or cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) at the sn-2 position. Mass spectrometric analyses revealed that all these fatty acids became components of various PE and phosphatidylglycerol species together with shorter partner fatty acids, indicating that large-scale remodeling followed the incorporation of synthetic PEs. As the number of double bonds in the sn-2 acyl chain decreased, the growth rate decreased and the cells became filamentous. The growth was restored to the wild-type level only when the medium was supplemented with phospholipids containing EPA or DHA. We found that about a half of DHA was converted into EPA. The results suggest that intact EPA is best required for cold adaptation of this bacterium.

  17. A novel cold-adapted and highly salt-tolerant esterase from Alkalibacterium sp. SL3 from the sediment of a soda lake

    PubMed Central

    Wang, Guozeng; Wang, Qiaohuang; Lin, Xianju; Bun Ng, Tzi; Yan, Renxiang; Lin, Juan; Ye, Xiuyun

    2016-01-01

    A novel esterase gene (estSL3) was cloned from the Alkalibacterium sp. SL3, which was isolated from the sediment of soda lake Dabusu. The 636-bp full-length gene encodes a polypeptide of 211 amino acid residues that is closely related with putative GDSL family lipases from Alkalibacterium and Enterococcus. The gene was successfully expressed in E. coli, and the recombinant protein (rEstSL3) was purified to electrophoretic homogeneity and characterized. rEstSL3 exhibited the highest activity towards pNP-acetate and had no activity towards pNP-esters with acyl chains longer than C8. The enzyme was highly cold-adapted, showing an apparent temperature optimum of 30 °C and remaining approximately 70% of the activity at 0 °C. It was active and stable over the pH range from 7 to 10, and highly salt-tolerant up to 5 M NaCl. Moreover, rEstSL3 was strongly resistant to most tested metal ions, chemical reagents, detergents and organic solvents. Amino acid composition analysis indicated that EstSL3 had fewer proline residues, hydrogen bonds and salt bridges than mesophilic and thermophilic counterparts, but more acidic amino acids and less hydrophobic amino acids when compared with other salt-tolerant esterases. The cold active, salt-tolerant and chemical-resistant properties make it a promising enzyme for basic research and industrial applications. PMID:26915906

  18. Cloning, sequencing and expression analysis of the first cellulase gene encoding cellobiohydrolase 1 from a cold-adaptive Penicillium chrysogenum FS010.

    PubMed

    Hou, Yunhua; Wang, Tianhong; Long, Hao; Zhu, Huiyuan

    2007-02-01

    A cellobiohydrolase 1 gene (cbh1) was cloned from Penicillium chrysogenum FS010 by a modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). DNA sequencing shows that cbh1 has an open reading frame of 1590 bp, encoding a putative protein of 529 amino acid residues. The deduced amino acid sequence revealed that CBHI has a modular structure with a predicted molecular mass of 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region. The putative gene product is homologous to fungal cellobiohydrolases in Family 7 of the glycosyl hydrolases. A novel cbh1 promoter (1.3 kb) was also cloned and sequenced, which contains seven putative binding sites (5'-SYGGRG-3') for the carbon catabolite repressor CRE1. Effect of various carbon sources to the cbh1 transcription of P. chrysogenum was examined by Northern analysis, suggesting that the expression of cbh1 is regulated at transcriptional level. The cbh1 gene in cold-adaptive fungus P. chysogenum was expressed as an active enzyme in Saccharomyces cerevisiae H158. The recombinant CBHI accumulated intracellularly and could not be secreted into the medium.

  19. Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase Family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524

    PubMed Central

    Chen, Xiu-Lan; Dong, Sheng; Xu, Fei; Dong, Fang; Li, Ping-Yi; Zhang, Xi-Ying; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Xie, Bin-Bin

    2016-01-01

    Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low Tm of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5–1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics. PMID:27486451

  20. Two cold-induced family 19 glycosyl hydrolases from cherimoya (Annona cherimola) fruit: an antifungal chitinase and a cold-adapted chitinase.

    PubMed

    Goñi, Oscar; Sanchez-Ballesta, María T; Merodio, Carmen; Escribano, María I

    2013-11-01

    Two cold-induced chitinases were isolated and purified from the mesocarp cherimoyas (Annona cherimola Mill.) and they were characterised as acidic endochitinases with a Mr of 24.79 and 47.77kDa (AChi24 and AChi48, respectively), both family 19 glycosyl hydrolases. These purified chitinases differed significantly in their biochemical and biophysical properties. While both enzymes had similar optimal acidic pH values, AChi24 was enzymatically active and stable at alkaline pH values, as well as displaying an optimal temperature of 45°C and moderate thermostability. Kinetic studies revealed a great catalytic efficiency of AChi24 for oligomeric and polymeric substrates. Conversely, AChi48 hydrolysis showed positive co-operativity that was associated to a mixture of different functional oligomeric states through weak transient protein interactions. The rise in the AChi48 kcat at increasing enzyme concentrations provided evidence of its oligomerisation. AChi48 chitinase was active and stable in a broad acidic pH range, and while it was relatively labile as temperatures increased, with an optimal temperature of 35°C, it retained about 50% of its maximal activity from 5 to 50°C. Thermodynamic characterisation reflected the high kcat of AChi48 and the remarkably lower ΔH(‡), ΔS(‡) and ΔG(‡) values at 5°C compared to AChi24, indicating that the hydrolytic activity of AChi48 was less thermodependent. In vitro functional studies revealed that AChi24 had a strong antifungal defence potential against Botrytis cinerea, whereas they displayed no cryoprotective or antifreeze activity. Hence, based on biochemical, thermodynamic and functional data, this study demonstrates that two acidic endochitinases are induced at low temperatures in a subtropical fruit, and that one of them acts in an oligomeric cold-adapted manner.

  1. Parallel N- and C-Terminal Truncations Facilitate Purification and Analysis of a 155-kDa Cold-Adapted Type-I Pullulanase.

    PubMed

    Elleuche, Skander; Krull, Alina; Lorenz, Ute; Antranikian, Garabed

    2017-02-01

    The cold-adapted pullulanase Pul13A is an industrial useful amylolytic enzyme, but its low solubility is the major bottleneck to produce the protein in recombinant form. In a previous approach, a complex and time-consuming purification strategy including a step-wise dialysis procedure using decreasing concentrations of urea to renature the insoluble protein from inclusion bodies had been established. In this study, a truncation strategy was developed to facilitate the purification and handling of the type-I pullulanase. Pul13A has a size of 155-kDa with a multidomain architecture that is composed of the following predicted modules: CBM41/E-set/Amy-Pul/DUF3372/E-set/E-set/E-set, with CBM and E-set domains being putative carbohydrate-binding modules, Amy-Pul is the catalytic region and DUF is a domain of unknown function. Consecutive N- and C-terminal deletions of domains were applied to construct minimized enzyme variants retaining pullulanase activity and exhibiting improved renaturation efficiencies. A total of seven truncation constructs were generated and tested, which still led to the production of inclusion bodies. However, the parallel deletion of the exterior CBM41 and E-set domain enabled the direct refolding of active enzymes during one-step dialysis in urea-free buffer. Catalytic properties of truncation construct Pul13A-N1/C1 were not impaired indicating that this enzyme variant may be superior for industrial applications over the full-length pullulanase.

  2. Psychrobacter proteolyticus sp. nov., a psychrotrophic, halotolerant bacterium isolated from the Antarctic krill Euphausia superba Dana, excreting a cold-adapted metalloprotease.

    PubMed

    Denner, E B; Mark, B; Busse, H J; Turkiewicz, M; Lubitz, W

    2001-04-01

    An Antarctic marine bacterium (strain 116) excreting an extracellular cold-adapted metalloprotease was subjected to a detailed polyphasic taxonomic investigation. Strain 116 was previously isolated from the stomach of a specimen of the Antarctic krill Euphasia superba Dana and tentatively characterized as Sphingomonas paucimobilis 116. The 16S rDNA sequence analysis showed that the strain is in fact related to species of the genus Psychrobacter, next to Psychrobacter glacincola (97.4% similarity). Sequence similarities between strain 116 and other Psychrobacter species ranged from 96.9% (with P. urativorans) to 95.4% (with P. immobilis). Key phenotypic characteristics as well as chemotaxonomic features of the bacterium were congruent with the description of the genus Psychrobacter i.e. cells were strictly aerobic, strongly oxidase-positive, psychrotrophic, halotolerant, gram-negative non-motile coccobacilli, with ubiquinone-8 as the main respiratory lipoquinone and 18:1 cis 9, 16:1 cis and 17:1 (omega8c being the predominant cellular fatty acids. The G+C content of the DNA was 43.6 mol%. DNA-DNA hybridization studies showed that the relatedness between strain 116 and Psychrobacter glacinola is only 62.2%. Further differences were apparent in whole-cell SDS-PAGE protein pattern, cellular fatty acid profile and in a number of physiological and biochemical characteristics as well as in enzymatic activities. Tolerance to 5% bile salts, nitrate reduction, citrate utilization, acid production from carbohydrates, alkaline phosphatase, acid phosphatase, C4 esterase, C14 lipase and valine arylamidase were found to differentiate strain 116 from Psychrobacter glacincola. On the basis of this phenotypic and molecular evidences, strain 116, previously known as Sphingomonas paucimobilis 116, was recognized as a new species of the genus Psychrobacter for which the name Psychrobacter proteolyticus is proposed. Strain 116 has been deposited in the Collection de l'Institut Pasteur

  3. Seasonal variation in expression pattern of genes under HSP70 : Seasonal variation in expression pattern of genes under HSP70 family in heat- and cold-adapted goats (Capra hircus).

    PubMed

    Banerjee, Dipak; Upadhyay, Ramesh C; Chaudhary, Umesh B; Kumar, Ravindra; Singh, Sohanvir; Ashutosh; G, Jagan Mohanarao; Polley, Shamik; Mukherjee, Ayan; Das, Tapan K; De, Sachinandan

    2014-05-01

    Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.

  4. [Phylogenetic diversity and cold-adaptive hydrolytic enzymes of culturable psychrophilic bacteria associated with sea ice from high latitude ocean, Artic].

    PubMed

    Yu, Yong; Li, Hui-Rong; Chen, Bo; Zeng, Yin-Xin; He, Jian-Feng

    2006-04-01

    showing 100% similarity each other are retrieved from the database, eleven from Antarctic seawater bacteria, three from Antarctic sea-ice bacteria, one from Spitzbergen sea-ice bacteria, two from Chukchi Sea sea-ice bacteria, two from Canadian Basin sea-ice bacteria (in this study) and one from uncultured bacterium clone PDA-OTU11 associated with the coral Pocillopora damicornis from the Great Barrier Reef. These may indicate that the physiological and geographic barriers appear to be permeable and some bacterial species can survive in different environment. The majority of the bacterial strains are able to secrete diversity cold-adaptive hydrolytic enzymes into the medium at 4 degrees C. The isolates that are able to degrade Tween-80, glutin, and starch account for, respectively, 62.6%, 51.4% and 40.5%.

  5. Comparative studies of wild-type and 'cold-mutant' (temperature sensitive) influenza viruses: geneology of the matrix (M) and non-structural (NS) proteins in recombinant cold-adapted H3N2 viruses.

    PubMed

    Kendal, A P; Cox, N J; Murphy, B R; Spring, S B; Maassab, H F

    1977-10-01

    The matrix (M) protein of the H2N2 virus A/Ann Arbor/6/60 may be distinguished from M protein of several H3N2 viruses and A/New Jersey/76 (HSWINI) by SDS acrylamide gel electrophoresis using a discontinuous buffer system. The smallest RNA (RNA 8) of the A/Ann Arbor/6/60 virus may be distinguished from RNA 8 of several H3N2 viruses by acrylamide gel electrophoresis in 3% or 3-6% gels in the absence of urea, if electrophoresis is done at 30 to 36 degrees C or 20 degrees C respectively. Ten clones of conditionally-lethal temperature-sensitive (ts) mutants were studied, which derived their cold-adaption and ts genes from mutant A/Ann Arbor/6/60, and their haemagglutinin from the H3N2 virus A/Scotland/840/74. Each clone was found to derive its M protein from A/Ann Arbor/6/60 mutant, and its RNA 8 from A/Scotland/840/74. The only assignment of genes 7 and 8 consistent with these findings for the recombinants is that in each parent virus (and in the recombinants) gene 7 codes for M protein, and gene 8 for NS protein. Furthermore, it may be concluded from the results that the biologically important ts lesions in the A/Ann Arbor/6/60 mutant parent are not present in the NS gene. In addition to the recombinants of A/Ann Arbor/6/60 and A/Scotland/840/74, five independent ts/cold-adapted recombinants of A/Ann Arbor/6/60 mutant with H3N2 and HSWINI wild-type viruses were examined, and all were found to contain the M protein of the A/Ann Arbor/6/60 mutant parent. This is suggestive that M protein may be at least partially responsible for the cold-adaptation and/or ts properties of the A/Ann Arbor/6/60 mutant and the recombinants.

  6. Evaluation of the genetic stability of the temperature-sensitive PB2 gene mutation of the influenza A/Ann Arbor/6/60 cold-adapted vaccine virus.

    PubMed

    Treanor, J; Perkins, M; Battaglia, R; Murphy, B R

    1994-12-01

    A single-gene reassortant bearing the PB2 gene of the A/Ann Arbor/6/60 cold-adapted virus in the background of the A/Korea/82 (H3N2) wild-type virus is a temperature-sensitive (ts) virus with an in vitro shutoff temperature of 38 degrees C. A single mutation at amino acid (aa) at 265 (Asp-Ser) of the PB2 protein is responsible for the ts phenotype. This ts single-gene PB2 reassortant virus was serially passaged at elevated temperatures in Madin-Darby canine kidney cells to generate ts+ phenotypic revertant viruses. Four ts+ phenotypically revertant viruses were derived independently, and each possessed a shutoff temperature for replication in vitro of > 40 degrees C. Each of the four phenotypically revertant viruses replicated efficiently in the upper and lower respiratory tracts of mice and hamsters, unlike the PB2 single-gene reassortant virus, confirming that the ts phenotype was responsible for the attenuation of this virus in rodents. Mating the ts+ revertants with wild-type virus yielded ts progeny in high frequency, indicating that the loss of ts phenotype was due to a suppressor mutation which was mapped to the PA gene in each of the four independently derived ts phenotypic revertants. Nucleotide sequence analysis confirmed the absence of new mutations on the PB2 gene and the presence of predicted amino acid changes in the PA proteins of the revertant viruses. These studies suggest that single amino acid changes at aa 245 (Glu-Lys) or 347 (Asp-Asn) of the PA protein can completely suppress the ts and attenuation phenotypes specified by the Asp-Ser mutation at aa 265 of the PB2 protein of the A/Ann Arbor/6/60 cold-adapted virus.

  7. A live attenuated H7N7 candidate vaccine virus induces neutralizing antibody that confers protection from challenge in mice, ferrets and monkeys

    USDA-ARS?s Scientific Manuscript database

    A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics using the modified hemagglutinin (HA) and neuraminidase (NA) genes of HP A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca) (...

  8. [Advantages and disadvantages of inactivated and live influenza vaccine].

    PubMed

    Gendon, Iu Z

    2004-01-01

    Published data related with comparison studies of safety, efficiency and some other properties of cold-adapted live influenza vaccine (LIV) and of inactivated influenza vaccine (IIV) are analyzed. LIV and IIV do not differ by systemic reactions after administration; however, it is not ruled out that there can be unfavorable reactions in vaccination of persons with allergy to the chicken-embryo proteins as well as in cases of persistence/reversion of cold-adapted strain observed in vaccination of persons with primary impairments of the immune system. There are no convincing data, up to now, on that LIV is superior to IIV in coping with influenza pandemics. The efficiency of LIV and IIV for children aged 3 years and more and for healthy adults is virtually identical. Additional controllable field comparative studies of LIV and IIV efficiency in immunization of elderly persons are needed. Limited data on LIV efficiency for children aged 2 months and more were obtained. The need in a 2-stage vaccination of all age group with the aim of ensuring responses to all 3 LIV components is, certainly, a LIV disadvantage. In case of IIV, the 2-stage vaccination is needed only for persons who were not ill with influenza. The intranasal LIV administration has, from the practical and psychological standpoints, an advantage before the IIV administration by syringe. The ability of LIV to protect from the drift influenza-virus variations could be its advantage before IIV; still, more research is needed to verify it. Transplantable cell lines meeting the WHO requirements could be an optimal substrate for the production of LIV and IIV. Children are the optimal age group for influenza prevention by cold-adapted LIV, whereas, IIV fits better for vaccination of adults and elderly persons.

  9. Live Healthy, Live Longer

    MedlinePlus

    ... Human Services. More Health News on: Exercise and Physical Fitness Health Screening Healthy Living Recent Health News Related MedlinePlus Health Topics Exercise and Physical Fitness Health Screening Healthy Living About MedlinePlus Site Map ...

  10. Molecular basis of live-attenuated influenza virus.

    PubMed

    He, Wen; Wang, Wei; Han, Huamin; Wang, Lei; Zhang, Ge; Gao, Bin

    2013-01-01

    Human influenza is a seasonal disease associated with significant morbidity and mortality. The most effective means for controlling infection and thereby reducing morbidity and mortality is vaccination with a three inactivated influenza virus strains mixture, or by intranasal administration of a group of three different live attenuated influenza vaccine strains. Comparing to the inactivated vaccine, the attenuated live viruses allow better elicitation of a long-lasting and broader immune (humoral and cellular) response that represents a naturally occurring transient infection. The cold-adapted (ca) influenza A/AA/6/60 (H2N2) (AA ca) virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. Similarly, the influenza A components of live-attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47) along with virulent epidemic strains. However, the mechanism of temperature-sensitive attenuation is largely elusive. To understand how modification at genetic level of influenza virus would result in attenuation of human influenza virus A/PR/8/34 (H1N1,A/PR8), we investigated the involvement of key mutations in the PB1 and/or PB2 genes in attenuation of influenza virus in vitro and in vivo. We have demonstrated that a few of residues in PB1 and PB2 are critical for the phenotypes of live attenuated, temperature sensitive influenza viruses by minigenome assay and real-time PCR. The information of these mutation loci could be used for elucidation of mechanism of temperature-sensitive attenuation and as a new strategy for influenza vaccine development.

  11. Comparative studies of wild-type and "cold-mutant" (temperature-sensitive) influenza viruses: polypeptide synthesis by an Asian (H2N2) strain and its cold-adapted variant.

    PubMed

    Kendal, A P; Kiley, M P; Maassab, H F

    1973-12-01

    The structure and replication of a cold-adapted, temperature-sensitive (TS) mutant of an Asian (H2N2) influenza virus was compared with that of its wild-type (WT) parent. Viruses were grown in a chicken kidney cell system, and at the nonpermissive temperature of 40 C, production of infectious TS virus was about 100,000-fold less than at 35 C, in contrast to WT virus. Major structural polypeptides of each virus grown at 35 C were similar, except that the hemagglutinin glycopolypeptide (HA) of the TS virions was slightly more heterogenous than that of WT virions. Synthesis of viral polypeptides was examined by sodium dodecyl sulfate acrylamide gel electrophoresis of pulse-labeled infected cells. This revealed a defect in the synthesis of TS viral hemagglutinin that was most pronounced at the nonpermissive temperature. Other TS viral polypeptides appeared to be synthesized normally at 40 C. A defect in the TS virus hemagglutinin was also indicated by serological studies that demonstrated that TS virus hemagglutinin had lost antigenic sites present on the WT virus. Thus, it is concluded that the virus mutant examined contains lesions in the hemagglutinin gene, although the possibility of additional unrecognized lesions is not excluded.

  12. Development of an Alternative Modified Live Influenza B Virus Vaccine

    PubMed Central

    Finch, Courtney; Sutton, Troy; Obadan, Adebimpe; Aguirre, Isabel; Wan, Zhimin; Lopez, Diego; Geiger, Ginger; Gonzalez-Reiche, Ana Silvia; Ferreri, Lucas

    2017-01-01

    ABSTRACT Influenza B virus (IBV) is considered a major human pathogen, responsible for seasonal epidemics of acute respiratory illness. Two antigenically distinct IBV hemagglutinin (HA) lineages cocirculate worldwide with little cross-reactivity. Live attenuated influenza virus (LAIV) vaccines have been shown to provide better cross-protective immune responses than inactivated vaccines by eliciting local mucosal immunity and systemic B cell- and T cell-mediated memory responses. We have shown previously that incorporation of temperature-sensitive (ts) mutations into the PB1 and PB2 subunits along with a modified HA epitope tag in the C terminus of PB1 resulted in influenza A viruses (IAV) that are safe and effective as modified live attenuated (att) virus vaccines (IAV att). We explored whether analogous mutations in the IBV polymerase subunits would result in a stable virus with an att phenotype. The PB1 subunit of the influenza B/Brisbane/60/2008 strain was used to incorporate ts mutations and a C-terminal HA tag. Such modifications resulted in a B/Bris att strain with ts characteristics in vitro and an att phenotype in vivo. Vaccination studies in mice showed that a single dose of the B/Bris att candidate stimulated sterilizing immunity against lethal homologous challenge and complete protection against heterologous challenge. These studies show the potential of an alternative LAIV platform for the development of IBV vaccines. IMPORTANCE A number of issues with regard to the effectiveness of the LAIV vaccine licensed in the United States (FluMist) have arisen over the past three seasons (2013–2014, 2014–2015, and 2015–2016). While the reasons for the limited robustness of the vaccine-elicited immune response remain controversial, this problem highlights the critical importance of continued investment in LAIV development and creates an opportunity to improve current strategies so as to develop more efficacious vaccines. Our laboratory has developed an

  13. [ts-Mutations in the genomes of cold-adapted variants of influenza A/Hong Kong/1/68(H3N2) and A/Victoria/35/72(H3N2) viruses].

    PubMed

    Zhikhareva, I V; Medvedeva, T E; Aleksandrova, G I; Klimov, A I

    1993-05-01

    The phenotype and localization of ts mutations in genomes of the influenza A/Victoria/30-ir (A/Vic/30-ir) and A/Hong Kong/17-ir (A/HK/17-ir) cold-adapted (ca) viruses were studied. Using the recombination analysis in chick embryo fibroblasts (CEF) we determined that influenza A/HK/17-ir ca virus carries ts mutations in three "internal" genes, i.e., PB1, NP and M, and influenza A/Vic/30-ir ca virus carries ones in four genes, i.e., PA, NP, M and NS. We have revealed ts mutations for NA gene in none of these viruses. Prior to the analysis of ts mutations in HA gene of influenza A/HK/17-ir and A/Vic/30-ir ca viruses, three cloning steps were performed in chick embryos (CE) by the method of limiting dilutions at 34 degrees C followed by selection of some strains with the most prominent ts phenotype. The cloned strains with such phenotypes were shown to repeat stable results within the recombination analysis in CE, i.e., none from the cloned strains of A/HK/17-ir ca virus recombined in CE at 40 degrees C with the 46 ts mutant, while recombination of this mutant with the cloned A/Vic/30-ir ca strains led to formation of the ts progeny. Thereafter our data result in conclusion that ts mutations in the PA gene must lead to some insignificant contribution for the expression of general ts phenotype among the ca strains as far as this sign is clearly displayed by both viruses, although only one of them, i.e., A/HK/17-ir carries ts mutation in the HA gene.

  14. The complete nucleotide sequence of two cold-adapted, temperature-sensitive attenuated mutant vaccine viruses (cp12 and cp45) derived from the JS strain of human parainfluenza virus type 3 (PIV3).

    PubMed

    Stokes, A; Tierney, E L; Sarris, C M; Murphy, B R; Hall, S L

    1993-10-01

    Two cold-passaged mutant vaccine viruses (cp12 and cp45) derived from the JS wild-type (wt) strain of human parainfluenza virus type 3 (PIV3) have been sequenced. These mutant viruses display the cold-adapted (ca), temperature-sensitive (ts), and attenuation (att) phenotypes. Sequence data indicate that both cp12 and cp45 sustained nucleotide substitutions during cold passage and subsequent cloning. Fifteen nucleotide changes were present in cp12 and 18 in cp45. Of these changes, some were present in the sequence of the prototype wt strain (Wash/47885/57) or were non-coding changes present in the open reading frames (ORFs). These were considered unlikely to be of significance in contributing to phenotypic differences between the mutants and the JS wt. There were nine remaining changes in cp12 and eight in cp45 that would most likely contribute to their phenotypes. For cp12, two were non-coding changes in regulatory regions, one in the 3' genome leader and one in the NP gene transcription start signal. The remaining seven changes resulted in amino acid substitutions in NP, F, HN, and L. For cp45, two mutations were in a non-coding regulatory region, the 3' genome leader. The remaining six changes resulted in amino acid substitutions in F, HN, and L. Only one amino acid substitution was conserved between cp12 and cp45 (a valine to alanine change at position 384 of the HN gene). These results should prove useful in the future in understanding the genetic basis of attenuation of the cold-passaged PIV3 candidate vaccine viruses.

  15. A live attenuated vaccine prevents replication and transmission of H7N9 virus in mammals

    PubMed Central

    Kong, Huihui; Zhang, Qianyi; Gu, Chunyang; Shi, Jianzhong; Deng, Guohua; Ma, Shujie; Liu, Jinxiong; Chen, Pucheng; Guan, Yuntao; Jiang, Yongping; Chen, Hualan

    2015-01-01

    The continued spread of the newly emerged H7N9 viruses among poultry in China, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. An MF59-adjuvant H7N9 inactivated vaccine is reported to be well-tolerated and immunogenic in humans; however a study in ferrets indicated that while a single dose of the inactivated H7N9 vaccine reduced disease severity, it did not prevent virus replication and transmission. In this study, we used reverse genetics to produce a cold-adapted, live attenuated H7N9 vaccine (H7N9/AAca) that contains wild-type HA and NA genes from AH/1, and the backbone of the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AAca). H7N9/AAca was attenuated in mice and ferrets, and induced robust neutralizing antibody responses in rhesus mice, ferrets, and guinea pigs immunized once or twice intranasally. The animals immunized twice were completely protected from H7N9 virus challenge. Importantly, the animals vaccinated once were fully protected from transmission when exposed to or in contact with the H7N9 virus-inoculated animals. These results demonstrate that a cold-adapted H7N9 vaccine can prevent H7N9 virus transmission; they provide a compelling argument for further testing of this vaccine in human trials. PMID:26058711

  16. Cold-active and NaCl-tolerant exo-inulinase from a cold-adapted Arthrobacter sp. MN8 and its potential for use in the production of fructose at low temperatures.

    PubMed

    Zhou, Junpei; Lu, Qian; Peng, Mozhen; Zhang, Rui; Mo, Minghe; Tang, Xianghua; Li, Junjun; Xu, Bo; Ding, Junmei; Huang, Zunxi

    2015-03-01

    An exo-inulinase gene was cloned from Arthrobacter sp. MN8, a cold-adapted bacterium isolated from lead-zinc-rich soil. The gene was expressed in Escherichia coli BL21(DE3). The resultant 505-residue polypeptide (InuAMN8) showed the highest identity (81.1%) with the putative levanase from Arthrobacter phenanthrenivorans Sphe3 (ADX73279) and shared 57.8% identity with the exo-inulinase from Bacillus sp. snu-7 (AAK00768). The purified recombinant InuAMN8 (rInuAMN8) showed an apparently optimal activity at 35°C, and 75.3%, 39.4%, and 15.8% of its maximum activity at 20°C, 10°C, and 0°C, respectively. After pre-incubation for 60 min at 50°C and 55°C, the rInuAMN8 exhibited 69.8% and 17.7% of its initial activity, respectively. The apparent Km values of rInuAMN8 towards inulin were 2.8, 1.5, 1.2, 5.3, and 8.2 mM at 0°C, 10°C, 20°C, 30°C, and 35°C, respectively. Inulin and Jerusalem artichoke tubers were effectively hydrolyzed to release fructose by rInuAMN8 at 0°C, 10°C, and 35°C. Compared with its hyperthermophilic and thermophilic counterparts, the exo-inulinase had less aromatic amino acid F and more hydrophobic amino acid A. In addition, the purified rInuAMN8 retained 127.9%-88.4% inulinase activity at 3.5%-15.0% (w/v) NaCl concentrations. Zn(2+) and Pb(2+) at 10 mM exhibited little or no effect on the enzyme activity. This paper is the first to report a cold-active and/or NaCl-tolerant exo-inulinase from the genus Arthrobacter. The exo-inulinase rInuAMN8 shows a potential for use in the production of fructose at low temperatures. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

    PubMed Central

    Powers, D C; Fries, L F; Murphy, B R; Thumar, B; Clements, M L

    1991-01-01

    In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group. PMID:2037667

  18. Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells.

    PubMed

    Lohr, Verena; Genzel, Yvonne; Jordan, Ingo; Katinger, Dietmar; Mahr, Stefan; Sandig, Volker; Reichl, Udo

    2012-10-30

    Current influenza vaccines are trivalent or quadrivalent inactivated split or subunit vaccines administered intramuscularly, or live attenuated influenza vaccines (LAIV) adapted to replicate at temperatures below body temperature and administered intranasally. Both vaccines are considered safe and efficient, but due to differences in specific properties may complement each other to ensure reliable vaccine coverage. By now, licensed LAIV are produced in embryonated chicken eggs. In the near future influenza vaccines for human use will also be available from adherent MDCK or Vero cell cultures, but a scalable suspension process may facilitate production and supply with vaccines. We evaluated the production of cold-adapted human influenza virus strains in the duck suspension cell line AGE1.CR.pIX using a chemically-defined medium. One cold-adapted A (H1N1) and one cold-adapted B virus strain was tested, as well as the reference strain A/PR/8/34 (H1N1). It is shown that a medium exchange is not required for infection and that maximum virus titers are obtained for 1 × 10⁻⁶ trypsin units per cell. 1 L bioreactor cultivations showed that 4 × 10⁶ cells/mL can be infected without a cell density effect achieving titers of 1 × 10⁸ virions/mL after 24 h. Overall, this study demonstrates that AGE1.CR.pIX cells support replication of LAIV strains in a chemically-defined medium using a simple process without medium exchanges. Moreover, the process is fast with peak titers obtained 24 h post infection and easily scalable to industrial volumes as neither microcarriers nor medium replacements are required.

  19. Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells

    PubMed Central

    2012-01-01

    Background Current influenza vaccines are trivalent or quadrivalent inactivated split or subunit vaccines administered intramuscularly, or live attenuated influenza vaccines (LAIV) adapted to replicate at temperatures below body temperature and administered intranasally. Both vaccines are considered safe and efficient, but due to differences in specific properties may complement each other to ensure reliable vaccine coverage. By now, licensed LAIV are produced in embryonated chicken eggs. In the near future influenza vaccines for human use will also be available from adherent MDCK or Vero cell cultures, but a scalable suspension process may facilitate production and supply with vaccines. Results We evaluated the production of cold-adapted human influenza virus strains in the duck suspension cell line AGE1.CR.pIX using a chemically-defined medium. One cold-adapted A (H1N1) and one cold-adapted B virus strain was tested, as well as the reference strain A/PR/8/34 (H1N1). It is shown that a medium exchange is not required for infection and that maximum virus titers are obtained for 1 × 10-6 trypsin units per cell. 1 L bioreactor cultivations showed that 4 × 106 cells/mL can be infected without a cell density effect achieving titers of 1 × 108 virions/mL after 24 h. Conclusions Overall, this study demonstrates that AGE1.CR.pIX cells support replication of LAIV strains in a chemically-defined medium using a simple process without medium exchanges. Moreover, the process is fast with peak titers obtained 24 h post infection and easily scalable to industrial volumes as neither microcarriers nor medium replacements are required. PMID:23110398

  20. Assisted Living

    MedlinePlus

    ... but they don't need full-time nursing care. Some assisted living facilities are part of retirement ... change. Assisted living costs less than nursing home care. It is still fairly expensive. Older people or ...

  1. Immunogenicity and protective efficacy of a live attenuated vaccine against the 2009 pandemic A H1N1 in mice and ferrets.

    PubMed

    Yang, PengHui; Duan, YueQiang; Wang, Cheng; Xing, Li; Gao, Xiao; Tang, Chong; Luo, DeYan; Zhao, ZhongPeng; Jia, Weihong; Peng, Daxin; Liu, Xiufan; Wang, Xiliang

    2011-01-17

    A novel 2009 influenza A (H1N1) virus was transmitted from humans to humans worldwide. The live attenuated monovalent A H1N1 vaccine (LAMV) for intranasal administration has shown promising immunogenicity and safety in clinical trials and for human use, but the experimental data based on LAMV is incomplete. In this study, using reverse genetic technology, we produced a cold-adapted (ca), live attenuated BJ/AA ca that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2009 pandemic A H1N1 isolate, A/Beijing/501/2009 virus (BJ501), and the remaining six internal gene segments from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). BJ/AA ca exhibited phenotypes of temperature sensitivity (ts), ca, and attenuation (att). The candidate BJ/AA ca was immunogenic in mice and induced strong mucosal secretory IgA (sIgA) in the respiratory tract. Two dosages of intranasal immunization induced robust HI antibodies and offered efficient protection against challenge by the wild-type (wt) 2009 pandemic A H1N1 (A/Beijing/501/2009 or A/California/07/2009) in mice and ferrets. These results support the evaluation of this vaccine made from a wt strain isolated in China for clinical trials. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  2. Living Laboratories

    ERIC Educational Resources Information Center

    Mules, B. R.

    1976-01-01

    Presented is a review of various methods of keeping live animals, including scorpions, spiders, crabs, crayfish, shrimp, ants, fish, mice, and birds, as well as plants as a school science project/display. (SL)

  3. Assisted Living

    MedlinePlus

    ... Recreational activities Security Transportation How to Choose a Facility A good match between a facility and a resident's needs depends as much on the philosophy and services of the assisted living facility as it does on the quality of care. ...

  4. Greener Living

    EPA Pesticide Factsheets

    Learn about how to live a more environmentally friendly life by reducing your environmental footprint, enhancing sustainability, using clean energy, water efficiency, composting, selecting a fuel efficient vehicle, and reducing waste.

  5. Living Laboratories

    ERIC Educational Resources Information Center

    Mules, B. R.

    1976-01-01

    Presented is a review of various methods of keeping live animals, including scorpions, spiders, crabs, crayfish, shrimp, ants, fish, mice, and birds, as well as plants as a school science project/display. (SL)

  6. Healthy Living

    MedlinePlus

    ... changes to your lifestyle. By taking steps toward healthy living, you can help reduce your risk of ... Get the screening tests you need Maintain a healthy weight Eat a variety of healthy foods, and ...

  7. Bachelor Living

    ERIC Educational Resources Information Center

    Germer, Sondra

    1974-01-01

    Male high school students in a Bachelor Living Class observed methods of child care including bottle feeding, spoon feeding, changing diapers, and method of holding. The purpose was for the students to grasp a better understanding of child development. (EK)

  8. Countryside Live!

    ERIC Educational Resources Information Center

    Best, Andrew; Richardson, Gary

    2006-01-01

    The "Countryside Live!" events, organised by the Countryside Foundation for Education (CFE), provide a unique opportunity for urban children to explore a whole new area of possibilities and learning, through becoming aware at first-hand of what goes on in the countryside. The event at Staunton Country Park, Havant, Hampshire, which took…

  9. Independent Living.

    ERIC Educational Resources Information Center

    Nathanson, Jeanne H., Ed.

    1994-01-01

    This issue of "OSERS" addresses the subject of independent living of individuals with disabilities. The issue includes a message from Judith E. Heumann, the Assistant Secretary of the Office of Special Education and Rehabilitative Services (OSERS), and 10 papers. Papers have the following titles and authors: "Changes in the…

  10. Outdoor Living.

    ERIC Educational Resources Information Center

    Cotter, Kathy

    Course objectives and learning activities are contained in this curriculum guide for a 16-week home economics course which teaches cooking and sewing skills applicable to outdoor living. The course goals include increasing male enrollment in the home economics program, developing students' self-confidence and ability to work in groups, and…

  11. Retiring Lives

    ERIC Educational Resources Information Center

    Carnell, Eileen, Ed.; Lodge, Caroline, Ed.

    2009-01-01

    "Retiring Lives" presents fourteen personal real life stories from people at various stages of retiring. Each author recounts their own story about retiring, bringing together many aspects of the experiences: the social, psychological and practical. These inspirational and illustrated stories will encourage the reader to hold up these…

  12. Retiring Lives

    ERIC Educational Resources Information Center

    Carnell, Eileen, Ed.; Lodge, Caroline, Ed.

    2009-01-01

    "Retiring Lives" presents fourteen personal real life stories from people at various stages of retiring. Each author recounts their own story about retiring, bringing together many aspects of the experiences: the social, psychological and practical. These inspirational and illustrated stories will encourage the reader to hold up these…

  13. Living History

    ERIC Educational Resources Information Center

    Walsh, Mark

    2005-01-01

    John Tinker and Mary Beth Tinker are back in a classroom in their hometown, once again wearing black armbands and drawing attention to a war. Now in their 50s, the siblings are living symbols of constitutional rights for secondary school students. In 1965, they and a handful of others were suspended for wearing black armbands to their public…

  14. Living Jointness

    DTIC Science & Technology

    1994-01-01

    reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of...COVERED 00-00-1993 to 00-00-1994 4. TITLE AND SUBTITLE Living Jointness 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR...and the peacetime activities of all services other than participation in joint exercises. It challenges the existing joint command structure because

  15. ISS Live!

    NASA Technical Reports Server (NTRS)

    Price, Jennifer; Harris, Philip; Hochstetler, Bruce; Guerra, Mark; Mendez, Israel; Healy, Matthew; Khan, Ahmed

    2013-01-01

    International Space Station Live! (ISSLive!) is a Web application that uses a proprietary commercial technology called Lightstreamer to push data across the Internet using the standard http port (port 80). ISSLive! uses the push technology to display real-time telemetry and mission timeline data from the space station in any common Web browser or Internet- enabled mobile device. ISSLive! is designed to fill a unique niche in the education and outreach areas by providing access to real-time space station data without a physical presence in the mission control center. The technology conforms to Internet standards, supports the throughput needed for real-time space station data, and is flexible enough to work on a large number of Internet-enabled devices. ISSLive! consists of two custom components: (1) a series of data adapters that resides server-side in the mission control center at Johnson Space Center, and (2) a set of public html that renders the data pushed from the data adapters. A third component, the Lightstreamer server, is commercially available from a third party and acts as an intermediary between custom components (1) and (2). Lightstreamer also provides proprietary software libraries that are required to use the custom components. At the time of this reporting, this is the first usage of Web-based, push streaming technology in the aerospace industry.

  16. Biophysics of Cold Adaptation and Acclimatization: Microbial Decomposition.

    DTIC Science & Technology

    1984-03-01

    L - A -I. 3 1983 Field Work Fall (pre abscission) leaf litter of seven vascular plant species native to Alaskan Arctic north slope tundra habitats was...Five hundred prepared litter bags were placed in the field at three sites near Barrow, Atquasuk and Driftwood, all within arctic tundra habitats . One...plateau marked with an expanse of obligotrophic lakes, ponds and aquatic habitats , which cover 50% to 85% of the land’s surface area and polygonally

  17. Engineering a substrate-specific cold-adapted subtilisin.

    PubMed

    Tindbaek, Nikolaj; Svendsen, Allan; Oestergaard, Peter Rahbek; Draborg, Henriette

    2004-02-01

    One region predicted to be highly flexible for a psychrophilic enzyme, TA39 subtilisin (S39), was transferred in silico to the mesophilic subtilisin, savinase (EC 3.4.21.62), from Bacillus lentus (clausii). The engineered hybrid and savinase were initially investigated by molecular dynamic simulations at 300 K to show binding region and global flexibility. The predicted S39 region consists of 12 residues, which due to homology between the subtilisins, results in a total change of eight residues. By site-directed modifications, the region was transferred to the binding region of savinase, thus a savinase-S39 hybrid, named H5, was constructed. The designed hybrid showed the same temperature optimum and pH profile as savinase, but H5 had higher specific activity on the synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) at all temperatures measured and, at the same time, H5 showed a decrease in thermostability. The H5 hybrid showed broader substrate specificity, measured at room temperature, due to an increase in catalytic efficiency on AAPF, AAPA and FAAF compared with savinase (N-succinyl-XXXX-pNA; XXXX = AAPF, AAPA and FAAF). The H5 hybrid showed increased activity at low temperature, increased binding region and global flexibility, as investigated by molecular dynamic simulations, and global destabilization from differential scanning calorimetry measurements. These psychrophilic characteristics indicated an increase in binding site flexibility, probably due to the modifications P129S, S130G, P131E, and thus we show that it is possible to increase low temperature activity and global flexibility by engineered flexibility in the binding region.

  18. A cold-adapted endo-arabinanase from Penicillium chrysogenum.

    PubMed

    Sakamoto, T; Ihara, H; Kozaki, S; Kawasaki, H

    2003-12-05

    Previously, three arabinan-degrading enzymes were isolated from Penicillium chrysogenum 31B. Here we describe another arabinan-degrading enzyme, termed Abnc, from the culture filtrate of the same organism. Analysis of the reaction products of debranched arabinan by high-performance anion-exchange chromatography (HPAEC) revealed that Abnc cleaved the substrate in an endo manner and that the final major product was arabinotriose. The molecular mass of Abnc was estimated to be 35 kDa by SDS-PAGE. Enzyme activity of Abnc was highest at pH 6.0 to 7.0. The enzyme was stable up to 30 degrees C and showed optimum activity at 30 to 40 degrees C. Compared with a mesophilic counterpart from Aspergillus niger, Abnc exhibited a lower thermal stability and optimum enzyme activity at lower temperatures. Production of Abnc in P. chrysogenum was found to be strongly induced by arabinose-containing polymers and required a longer culture time than did other arabinanase isozymes in this strain.

  19. Living Nanomachines

    NASA Astrophysics Data System (ADS)

    Carlier, M.-F.; Helfer, E.; Wade, R.; Haraux, F.

    The living cell is a kind of factory on the microscopic scale, in which an assembly of modular machines carries out, in a spatially and temporally coordinated way, a whole range of activities internal to the cell, including the synthesis of substances essential to its survival, intracellular traffic, waste disposal, and cell division, but also activities related to intercellular communication and exchanges with the outside world, i.e., the ability of the cell to change shape, to move within a tissue, or to organise its own defence against attack by pathogens, injury, and so on. These nanomachines are made up of macromolecular assemblies with varying degrees of complexity, forged by evolution, within which work is done as a result of changes in interactions between proteins, or between proteins and nucleic acids, or between proteins and membrane components. All these cell components measure a few nanometers across, so the mechanical activity of these nanomachines all happens on the nanometric scale. The directional nature of the work carried out by biological nanomachines is associated with a dissipation of energy. As examples of protein assemblies, one could mention the proteasome, which is responsible for the degradation of proteins, and linear molecular motors such as actomyosin, responsible for muscle contraction, the dynein-microtubule system, responsible for flagellar motility, and the kinesin-microtubule system, responsible for transport of vesicles, which transform chemical energy into motion. Nucleic acid-protein assemblies include the ribosome, responsible for synthesising proteins, polymerases, helicases, elongation factors, and the machinery of DNA replication and repair; the mitotic spindle is an integrated system involving several of these activities which drive chromosome segregation. The machinery coupling membranes and proteins includes systems involved in the energy metabolism, such as the ATP synthase rotary motor, signalling cascades, endocytosis

  20. Reverse genetic platform for inactivated and live-attenuated influenza vaccine.

    PubMed

    Jung, Eun Ju; Lee, Kwang Hee; Seong, Baik Lin

    2010-02-28

    Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high- growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6+2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/ MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.

  1. Reverse genetic platform for inactivated and live-attenuated influenza vaccine

    PubMed Central

    Jung, Eun-Ju; Lee, Kwang-Hee

    2010-01-01

    Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high-growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6 + 2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics. PMID:20054235

  2. [Living better or living longer].

    PubMed

    Sauvy, A

    1987-01-01

    It has been just 2 centuries since France began to struggle seriously against mortality and excess fertility. Life expectancy, which for millenia had been under 30 years at birth, began to increase because of the discovery of effective treatments, improved production and standards of living, and access of large numbers of persons to health care. France, in the 2nd half of the 18th century, became the first country in which fertility regulation was achieved on a wide scale. The failure of England, a country of similar culture, to follow suit until a century later remains unexplained. After World War II, simple and fairly inexpensive means of mortality control, such as vaccines and water purifiers, became widely distributed throughout the developing world. These countries, which traditionally had mortality rates of 35 or 40/1000 and fertility of 40-45/1000, experienced rapid declines in mortality rates while their fertility remained constant or even increased. Because antinatal techniques diffused so much more slowly, the equilibrium of births and deaths was disturbed as rates of increase of 2 or 3% per year became common. Although the inhabitants of poor countries were not concerned, perhaps through ignorance of what was occurring, the rich countries were alarmed by the increase. Their principal objective became to spread contraception in the poor countries. The available methods at the time, however, were none too reliable. When oral contraceptive pills became available, fertility dropped to very low levels in Europe but such factors as cost and illiteracy discouraged use in many underdeveloped countries. Fertility declined in a few insular states such as Hong Kong, Taiwan, and Singapore even before the appearance of pills. Life expectancies in developing countries except a few in Africa have increased since World War II and are now higher than in Europe at the turn of the century. "Health for all by the year 2000" is an astonishing slogan for a serious

  3. From Living Carbocationic to Living Radical Polymerization.

    DTIC Science & Technology

    1994-06-30

    C 1 -OFFICE OF NAVAL RESEARCH CONTRACT N00014-94-1-0101 R&T Code 31321075 Technical Report No. 18 FROM " LIVING " CARBOCATIONIC TO " LIVING " RADICAL...NUMBERS "From Living " Carbocationic to " Living " ’jical Polymerization N00014-94-1-01I01 6. AUTHOR(S) Krzysztof Matyjaszewski 7. PERFORMING ORGANIZATION NAME...1994) 1Za. DISTRIBUTION I AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE 13. ABSTRACT (Maximum 200 words) " Living " carbocationic polymerization is

  4. The transcriptome of the bowhead whale Balaena mysticetus reveals adaptations of the longest-lived mammal

    PubMed Central

    Seim, Inge; Ma, Siming; Zhou, Xuming; Gerashchenko, Maxim V.; Lee, Sang-Goo; Suydam, Robert; George, John C.; Bickham, John W.; Gladyshev, Vadim N.

    2014-01-01

    Mammals vary dramatically in lifespan, by at least two-orders of magnitude, but the molecular basis for this difference remains largely unknown. The bowhead whale Balaena mysticetus is the longest-lived mammal known, with an estimated maximal lifespan in excess of two hundred years. It is also one of the two largest animals and the most cold-adapted baleen whale species. Here, we report the first genome-wide gene expression analyses of the bowhead whale, based on the de novo assembly of its transcriptome. Bowhead whale or cetacean-specific changes in gene expression were identified in the liver, kidney and heart, and complemented with analyses of positively selected genes. Changes associated with altered insulin signaling and other gene expression patterns could help explain the remarkable longevity of bowhead whales as well as their adaptation to a lipid-rich diet. The data also reveal parallels in candidate longevity adaptations of the bowhead whale, naked mole rat and Brandt's bat. The bowhead whale transcriptome is a valuable resource for the study of this remarkable animal, including the evolution of longevity and its important correlates such as resistance to cancer and other diseases. PMID:25411232

  5. Possible outcomes of reassortment in vivo between wild type and live attenuated influenza vaccine strains.

    PubMed

    Kiseleva, Irina; Dubrovina, Irina; Bazhenova, Ekaterina; Fedorova, Ekaterina; Larionova, Natalie; Rudenko, Larisa

    2012-12-07

    Reassortment of influenza viruses in nature has been well documented. Genetic reassortment plays a key role in emergence of new influenza A strains, including pandemic viruses. Permissive host can be simultaneously coinfected with multiple influenza viruses. During genetic reassortment gene segments are exchanged between parental viruses that may lead to some enhancement of virulence of reassortant progeny. At present, vaccination with live attenuated cold-adapted (ca) reassortant vaccine (LAIV) is used as an effective public health measure for influenza prophylaxis. However, there are concerns about a potential of simultaneous infection of human host with ca and wild type (wt) influenza viruses which might produce progeny that contain novel, more virulent genotypes. The aim of this study was to investigate potential consequences of reassortment of wt with LAIV strains in vivo. We demonstrated that reassortment of wt viruses with ca strains in guinea pigs have resulted in progeny virus which caused reduced macroscopic lesions of chicken embryos. According to phenotypical data 95% (19 out of 20) isolated reassortants were restricted in replication at elevated temperature of 40°C. None of reassortants were more virulent than wt parents, or revealed significantly higher macroscopic lesions than wt parental viruses. Our results suggest that genetic reassortment between wt and vaccine strain is unlikely to lead to virulent reassortant progeny. These findings provide additional support of LAIV safety data. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Field studies on the annual activity and the metabolic responses of a land snail population living in high altitude.

    PubMed

    Staikou, Alexandra; Tachtatzis, George; Feidantsis, Konstantinos; Michaelidis, Basile

    2016-01-01

    In the context of the metabolic cold adaptation hypothesis (MCA), we investigated a) the life and activity cycle characteristics and b) the metabolic responses of the endemic land snail species Cattania trizona olympica living at 1100m altitude in Olympus mountain (Greece). Field observations on the annual activity cycle of C. trizona olympica revealed that snails' activity was restricted mainly between the end of May and September, when the higher temperatures were recorded, while first matings were recorded in July and the last ones in mid September indicating a restricted favorable time period for reproduction. The activities of enzymes of intermediate metabolism showed a periodic seasonal pattern of change which seems to be closely related to the pattern of annual changes of air temperature and most of them exhibited higher activities during the coldest and warmest periods of the year. Moreover the data indicate a distinct differentiation of fuel oxidation during arousal and reproductive periods with lipid oxidation, apart from carbohydrates, contributing significantly to ATP turnover during reproductive activity. The higher enzymatic activities, determined in the tissues of C. trizona olympica than the corresponding ones determined in the tissues of the land snail species living at low altitudes, might indicate higher sensitivity of the intermediate metabolism and ATP turnover in C. trizona olympica to changes in environmental factors. Although the latter seems to be in line with the MCA hypothesis, it needs further investigation on metabolic rates to support it. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Administration for Community Living

    MedlinePlus

    ... Information for Current Grantees About ACL Organization Why Community Living? Authorizing Statutes Budget Mandatory Grant Allocations Strategic ... Final Rule Get ACL Updates OAA Reauthorization Why Community Living? FEATURES #InclusionWorks IL Final Rule Get ACL ...

  8. Living with VHL

    MedlinePlus

    ... Videos Contact Us Search Patients & Caregivers / Living with VHL VHL disease is a lifelong condition. However, with appropriate measures, people can effectively manage the VHL and lead full and productive lives. Early diagnosis, ...

  9. Living with VHL

    MedlinePlus

    ... News & Events Contact Us Website References Search Patients / Living with VHL VHL disease is a lifelong condition. ... regularly surveillance, appropriate treatment and emotional support, and living a healthy lifestyle are all keys to effectively ...

  10. Living with Heart Block

    MedlinePlus

    ... Share this page from the NHLBI on Twitter. Living With Heart Block First-degree heart block may ... whether you need ongoing care for your condition. Living With a Pacemaker People who have third-degree ...

  11. Living with Pulmonary Hypertension

    MedlinePlus

    ... Share this page from the NHLBI on Twitter. Living With Pulmonary Hypertension Pulmonary hypertension (PH) has no ... seek care right away. Emotional Issues and Support Living with PH may cause fear, anxiety, depression, and ...

  12. Living with Vasculitis

    MedlinePlus

    ... Share this page from the NHLBI on Twitter. Living With Vasculitis The outcome of vasculitis is hard ... effects of your medicines. Emotional Issues and Support Living with a chronic condition may cause fear, anxiety, ...

  13. Living with Sarcoidosis

    MedlinePlus

    ... page from the NHLBI on Twitter. Living With Sarcoidosis Sarcoidosis has no cure, but you can take ... Content: NEXT >> Featured Video Living With and Managing Sarcoidosis 05/18/2011 This video—presented by the ...

  14. Independent Living Course

    ERIC Educational Resources Information Center

    Tipping, Joyce

    1978-01-01

    Designed to help handicapped persons who have been living a sheltered existence develop independent living skills, this course is divided into two parts. The first part consists of a five-day apartment live-in experience, and the second concentrates on developing the learners' awareness of community resources and consumer skills. (BM)

  15. Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus.

    PubMed

    Gambaryan, A S; Boravleva, E Y; Lomakina, N F; Kropotkina, E A; Gordeychuk, I V; Chvala, I A; Drygin, V V; Klenk, H-D; Matrosovich, M N

    Development of an effective, broadly-active and safe vaccine for protection of poultry from H5N1 highly pathogenic avian influenza viruses (HPAIVs) remains an important practical goal. In this study we used a low pathogenic wild aquatic bird virus isolate А/duck/Moscow/4182/2010 (H5N3) (dk/4182) as a live candidate vaccine. We compared this virus with four live 1:7 reassortant anti-H5N1 candidate vaccine viruses with modified hemagglutinin from either A/Vietnam/1203/04 (H5N1) or A/Kurgan/3/05 (H5N1) and the rest of the genes from either H2N2 cold-adapted master strain A/Leningrad/134/17/57 (rVN-Len and rKu-Len) or H6N2 virus A/gull/Moscow/3100/2006 (rVN-gull and rKu-gull). The viruses were tested in parallel for pathogenicity, immunogenicity and protective effectiveness in chickens using aerosol, intranasal and oral routes of immunization. All five viruses showed zero pathogenicity indexes in chickens. Viruses rVN-gull and rKu-gull were immunogenic and protective, but they were insufficiently attenuated and caused significant mortality of 1-day-old chickens. The viruses with cold-adapted backbones (rVN-Len and rKu-Len) were completely nonpathogenic, but they were significantly less immunogenic and provided lower protection against lethal challenge with HPAIV A/Chicken/Kurgan/3/05 (H5N1) as compared with three other vaccine candidates. Unlike other four viruses, dk/4182 was both safe and highly immunogenic in chickens of any age regardless of inoculation route. Single administration of 106 TCID50 of dk/4182 virus via drinking water provided complete protection of 30-days-old chickens from 100 LD50 of the challenge virus. Our results suggest that low pathogenic viruses of wild aquatic birds can be used as safe and effective live poultry vaccines against highly pathogenic avian viruses.

  16. Being a Living Donor: Risks

    MedlinePlus

    ... Living Donation / Being a Living Donor / Risks Facts History Organs Frequently Asked Questions Discussing Living Donation Types Related Non-Related Non-Directed Paired Donation Blood Type Incompatible Positive Crossmatch Being a Living Donor ...

  17. Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses.

    PubMed

    Zhou, Bin; Li, Yan; Speer, Scott D; Subba, Anju; Lin, Xudong; Wentworth, David E

    2012-05-21

    The licensed live attenuated influenza A vaccine (LAIV) in the United States is created by making a reassortant containing six internal genes from a cold-adapted master donor strain (ca A/AA/6/60) and two surface glycoprotein genes from a circulating/emerging strain (e.g., A/CA/7/09 for the 2009/2010 H1N1 pandemic). Technologies to rapidly create recombinant viruses directly from patient specimens were used to engineer alternative LAIV candidates that have genomes composed entirely of vRNAs from pandemic or seasonal strains. Multiple mutations involved in the temperature-sensitive (ts) phenotype of the ca A/AA/6/60 master donor strain were introduced into a 2009 H1N1 pandemic strain rA/New York/1682/2009 (rNY1682-WT) to create rNY1682-TS1, and additional mutations identified in other ts viruses were added to rNY1682-TS1 to create rNY1682-TS2. Both rNY1682-TS1 and rNY1682-TS2 replicated efficiently at 30°C and 33°C. However, rNY1682-TS1 was partially restricted, and rNY1682-TS2 was completely restricted at 39°C. Additionally, engineering the TS1 or TS2 mutations into a distantly related human seasonal H1N1 influenza A virus also resulted pronounced restriction of replication in vitro. Clinical symptoms and virus replication in the lungs of mice showed that although rNY1682-TS2 and the licensed FluMist(®)-H1N1pdm LAIV that was used to combat the 2009/2010 pandemic were similarly attenuated, the rNY1682-TS2 was more protective upon challenge with a virulent mutant of pandemic H1N1 virus or a heterologous H1N1 (A/PR/8/1934) virus. This study demonstrates that engineering key temperature sensitive mutations (PB1-K391E, D581G, A661T; PB2-P112S, N265S, N556D, Y658H) into the genomes of influenza A viruses attenuates divergent human virus lineages and provides an alternative strategy for the generation of LAIVs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Living Willow Huts

    ERIC Educational Resources Information Center

    Keeler, Rusty

    2007-01-01

    Living Willow Huts are inexpensive to make, fun to plant, easy to grow, and make beautiful spaces for children. They involve planting dormant willow shoots in the ground and weaving them into shapes that will sprout and grow over time. People have been creating similar living architecture throughout the world for centuries in the forms of living…

  19. Living with Respiratory Failure

    MedlinePlus

    ... smoking. Emotional Issues and Support Living with respiratory failure may cause fear, anxiety, depression, and stress. Talk about how you feel with your health care team. Talking to a professional counselor also can ... to living with respiratory failure. You can see how other people who have ...

  20. Recognizing Safety and Liveness.

    DTIC Science & Technology

    1986-01-01

    in terms of "bad things" and "good things".* In [ Sistla 85], an attempt is made to give syntactic charcrizations for safety and live- ness properties...properris, and for a subset of the livene= proMe- ties, called absolute liveness propertie Fally, [ Sistla 85] proves that the states of a Buchi

  1. Living Willow Huts

    ERIC Educational Resources Information Center

    Keeler, Rusty

    2007-01-01

    Living Willow Huts are inexpensive to make, fun to plant, easy to grow, and make beautiful spaces for children. They involve planting dormant willow shoots in the ground and weaving them into shapes that will sprout and grow over time. People have been creating similar living architecture throughout the world for centuries in the forms of living…

  2. Short-Lived Antigen Recognition but Not Viral Infection at a Defined Checkpoint Programs Effector CD4 T Cells To Become Protective Memory.

    PubMed

    Bautista, Bianca L; Devarajan, Priyadharshini; McKinstry, K Kai; Strutt, Tara M; Vong, Allen M; Jones, Michael C; Kuang, Yi; Mott, Daniel; Swain, Susan L

    2016-11-15

    Although memory CD4 T cells are critical for effective immunity to pathogens, the mechanisms underlying their generation are still poorly defined. We find that following murine influenza infection, most effector CD4 T cells undergo apoptosis unless they encounter cognate Ag at a defined stage near the peak of effector generation. Ag recognition at this memory checkpoint blocks default apoptosis and programs their transition to long-lived memory. Strikingly, we find that viral infection is not required, because memory formation can be restored by the addition of short-lived, Ag-pulsed APC at this checkpoint. The resulting memory CD4 T cells express an enhanced memory phenotype, have increased cytokine production, and provide protection against lethal influenza infection. Finally, we find that memory CD4 T cell formation following cold-adapted influenza vaccination is boosted when Ag is administered during this checkpoint. These findings imply that persistence of viral Ag presentation into the effector phase is the key factor that determines the efficiency of memory generation. We also suggest that administering Ag at this checkpoint may improve vaccine efficacy. Copyright © 2016 by The American Association of Immunologists, Inc.

  3. Development and identification of a new Vero cell-based live attenuated influenza B vaccine by a modified classical reassortment method.

    PubMed

    Yang, Fan; Ma, Lei; Zhou, Jian; Wu, Yinjie; Gao, Jingxia; Song, Shaohui; Geng, Xingliang; Guo, Qi; Li, Zhuofan; Li, Weidong; Liao, Guoyang; Li, Yufeng

    2017-08-01

    It was to generate a new Vero and cold-adapted live attenuated influenza B vaccine with enough safety and immunogenicity. According to modified classical reassortment method, the donor strain was B/Yunnan/2/2005Vca(B), and the parental virus strain was B/Brisbane/60/2008wt. After co-infection in Vero cells, the prepared antibody serum inhibited the donor strain growth, and screening conditions inhibited the parental virus growth, which induced the growth of the new reassortant virus B/Brisbane/60/2008Vca(B) grow. Through intraperitoneal injection (i.j.) and intranasal injection (n.j.) we evaluated the safety and immunogenicity of the vaccine. A high-yield of the reassortant virus was produced in Vero cells at 25°C, similar to the donor strains. After sequencing, it was found that B/Brisbane/60/2008Vca(B) Hemagglutinin (HA) and Neuraminidase (NA) gene fragments were from B/Brisbane/60/2008wt, while the other 6 gene fragments were from B/Yunnan/2/2005Vca(B). The n.j. immune pathway experiments showed no significant differences between the treatment and the PBS control group with respect to weight changes (P > 0.5). Furthermore, the new strain had a sufficient geometric mean titter (GMT) against B/Brisbane/60/2008wt. The new reassortant live attenuated influenza B vaccine was safe and having enough immune stimulating ability.

  4. Engineering living functional materials.

    PubMed

    Chen, Allen Y; Zhong, Chao; Lu, Timothy K

    2015-01-16

    Natural materials, such as bone, integrate living cells composed of organic molecules together with inorganic components. This enables combinations of functionalities, such as mechanical strength and the ability to regenerate and remodel, which are not present in existing synthetic materials. Taking a cue from nature, we propose that engineered 'living functional materials' and 'living materials synthesis platforms' that incorporate both living systems and inorganic components could transform the performance and the manufacturing of materials. As a proof-of-concept, we recently demonstrated that synthetic gene circuits in Escherichia coli enabled biofilms to be both a functional material in its own right and a materials-synthesis platform. To demonstrate the former, we engineered E. coli biofilms into a chemical-inducer-responsive electrical switch. To demonstrate the latter, we engineered E. coli biofilms to dynamically organize biotic-abiotic materials across multiple length scales, template gold nanorods, gold nanowires, and metal/semiconductor heterostructures, and synthesize semiconductor nanoparticles (Chen, A. Y. et al. (2014) Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 13, 515-523.). Thus, tools from synthetic biology, such as those for artificial gene regulation, can be used to engineer the spatiotemporal characteristics of living systems and to interface living systems with inorganic materials. Such hybrids can possess novel properties enabled by living cells while retaining desirable functionalities of inorganic systems. These systems, as living functional materials and as living materials foundries, would provide a radically different paradigm of materials performance and synthesis-materials possessing multifunctional, self-healing, adaptable, and evolvable properties that are created and organized in a distributed, bottom-up, autonomously assembled, and environmentally sustainable manner.

  5. Engineering Living Functional Materials

    PubMed Central

    2016-01-01

    Natural materials, such as bone, integrate living cells composed of organic molecules together with inorganic components. This enables combinations of functionalities, such as mechanical strength and the ability to regenerate and remodel, which are not present in existing synthetic materials. Taking a cue from nature, we propose that engineered ‘living functional materials’ and ‘living materials synthesis platforms’ that incorporate both living systems and inorganic components could transform the performance and the manufacturing of materials. As a proof-of-concept, we recently demonstrated that synthetic gene circuits in Escherichia coli enabled biofilms to be both a functional material in its own right and a materials-synthesis platform. To demonstrate the former, we engineered E. coli biofilms into a chemical-inducer-responsive electrical switch. To demonstrate the latter, we engineered E. coli biofilms to dynamically organize biotic-abiotic materials across multiple length scales, template gold nanorods, gold nanowires, and metal/semiconductor heterostructures, and synthesize semiconductor nanoparticles (Chen, A. Y. et al. (2014) Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater.13, 515–523.). Thus, tools from synthetic biology, such as those for artificial gene regulation, can be used to engineer the spatiotemporal characteristics of living systems and to interface living systems with inorganic materials. Such hybrids can possess novel properties enabled by living cells while retaining desirable functionalities of inorganic systems. These systems, as living functional materials and as living materials foundries, would provide a radically different paradigm of materials performance and synthesis–materials possessing multifunctional, self-healing, adaptable, and evolvable properties that are created and organized in a distributed, bottom-up, autonomously assembled, and environmentally sustainable manner. PMID

  6. Living with Hearing Loss

    MedlinePlus

    ... Current Issue Past Issues Special Section: Focus on Communication Living with Hearing Loss Past Issues / Fall 2008 ... the United States suffer some form of disordered communication. The National Institute on Deafness and Other Communication ...

  7. Living with Stepparents

    MedlinePlus

    ... get divorced . This is never an easy decision. Divorce can be painful for parents, but eventually, each ... ON THIS TOPIC Kids Talk About: Marriage and Divorce (Video) Living With a Single Parent Foster Families ...

  8. Living Beyond Breast Cancer

    MedlinePlus

    ... Types of Complementary Therapy Acupuncture Art Therapy Expressive Writing Guided Imagery Hypnosis Massage Therapy Mindfulness-Based Stress ... Conference Past Conferences Meet-Ups Twitter Chats Webinars Writing Workshops Young Women's Initiative Fundraising Events Galas Living ...

  9. Living with Bronchopulmonary Dysplasia

    MedlinePlus

    ... Living With Bronchopulmonary Dysplasia Caring for a premature infant can be challenging. You may have: Emotional pain, ... tiredness). Frustration that you can't breastfeed your infant right away. (You can pump and store your ...

  10. Living with COPD: Nutrition

    MedlinePlus

    ... Diseases > Lung Disease Lookup > COPD > Living With COPD Nutrition Most people are surprised to learn that the ... asking your doctor or visiting the Academy of Nutrition and Dietetics at EatRight.org . Be sure to ...

  11. Thalassemia: Healthy Living

    MedlinePlus

    ... severe anemia and possible organ damage from iron overload, respectively. Healthy Choices for People Living with Thalassemia ... Center on Birth Defects and Developmental Disabilities Related Information UDC System File Formats Help: How do I ...

  12. Living with Paralysis

    MedlinePlus

    ... are available to answer your questions. Call toll-free 1-800-539-7309 Mon-Fri, 9am-5pm ... are people living with or impacted by paralysis. Free services and downloads > Paralysis Resource Guide Our free ...

  13. Fluorescence Live Cell Imaging

    PubMed Central

    Ettinger, Andreas

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

  14. Hand Hygiene Saves Lives

    MedlinePlus

    ... at Home Smoke-free Multiunit Housing The Quiet Killer Healthy Living Healthy Eating A Grocery Store’s Healthy ... Things Safe Teen Drivers Life Stages & Populations A Killer in Indian Country Baby Steps: Learn the Signs. ...

  15. Living with Pulmonary Embolism

    MedlinePlus

    ... on Twitter. Living With Pulmonary Embolism Pulmonary embolism (PE) usually is treated in a hospital. After leaving ... you're taking medicine. Medicines used to treat PE can thin your blood too much. This can ...

  16. Living with Fanconi Anemia

    MedlinePlus

    ... from the NHLBI on Twitter. Living With Fanconi Anemia Improvements in blood and marrow stem cell transplants ... November 1, 2011 Twitter Facebook YouTube Google+ SITE INDEX ACCESSIBILITY PRIVACY STATEMENT FOIA NO FEAR ACT OIG ...

  17. Live Virus Smallpox Vaccine

    MedlinePlus

    ... Submit What's this? Submit Button The Live Virus Smallpox Vaccine Language: English Español (Spanish) Recommend on Facebook ... the vaccinia virus. Who should NOT get the smallpox vaccine? People most likely to have side effects ...

  18. Living Day by Day

    PubMed Central

    Kaplan, Rachel L.; Khoury, Cynthia El; Field, Emily R. S.; Mokhbat, Jacques

    2016-01-01

    We examined the meaning of living with HIV/AIDS among women in Lebanon. Ten women living with HIV/AIDS (WLWHA) described their experiences via semistructured in-depth interviews. They navigated a process of HIV diagnosis acceptance that incorporated six overlapping elements: receiving the news, accessing care, starting treatment, navigating disclosure decisions, negotiating stigma, and maintaining stability. Through these elements, we provide a framework for understanding three major themes that were constructed during data analysis: Stand by my side: Decisions of disclosure; Being “sick” and feeling “normal”: Interacting with self, others, and society; and Living day by day: focusing on the present. We contribute to the existing literature by providing a theoretical framework for understanding the process of diagnosis and sero-status acceptance among WLWHA. This was the first study of its kind to examine the meaning of living with HIV/AIDS among women in a Middle Eastern country. PMID:28462340

  19. Influenza Vaccine, Live Intranasal

    MedlinePlus

    ... the recombinant influenza vaccine (RIV). The nasal spray flu vaccine (live attenuated influenza vaccine or LAIV) should NOT ... to your doctor or pharmacist about the best flu vaccine option for you or your family.

  20. Healthy Living after Stroke

    MedlinePlus

    ... Stories Stroke Heroes Among Us Healthy Living After Stroke Nutrition Good nutrition is one way to reduce ... reviewed on 04/30/2014. Register for the Stroke Rehab Webinar Join rehab experts as they discuss ...

  1. ASHRAE's Living Laboratory

    SciTech Connect

    Jarnagin, Ronald E.; Brambley, Michael R.

    2008-10-01

    ASHRAE recently remodeled its headquarters building in Atlanta with the intention of making the building a LEED Gold building. As part of that renovation the building was enhanced with additional sensors and monitoring equipment to allow it to serve as a Living Laboratory for use by members and the general public to study the detailed energy use and performance of buildings. This article provides an overview of the Living Laboratory and its capabilities.

  2. Living-cell microarrays.

    PubMed

    Yarmush, Martin L; King, Kevin R

    2009-01-01

    Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment.

  3. Psychoanalysis and creative living.

    PubMed

    Rubin, Jeffrey B

    2003-01-01

    Psychoanalysis is ambivalent about creativity and its own creative potential. On the one hand, psychoanalysis offers enormous resources for elucidating obstacles to creativity, that way of living, making and relating to self and others that is fresh, vital, unpredictable and open to feedback and evolution. On the other hand, when we analysts know too much beforehand about what a work of art really means or the fundamental and singular motives of creativity, then psychoanalysis unconsciously partakes of a perverse scenario in which the work of art serves as merely a means to the author's ends and is psychologically colonized. When psychoanalysis is The Discipline That Knows, then art has nothing new to teach psychoanalysts and our field is impoverished. "Psychoanalysis and Creative Living" attempts to elucidate how psychoanalysis could work through this tension between its creative and perverse possibilities and foster creative living.

  4. Living My Family's Story

    PubMed Central

    Underhill, Meghan L.; Lally, Robin M.; Kiviniemi, Marc T.; Murekeyisoni, Christine; Dickerson, Suzanne S.

    2013-01-01

    Background Based on known or suggested genetic risk factors, a growing number of women now live with knowledge of a potential cancer diagnosis that may never occur. Given this, it is important to understand the meaning of living with high risk for hereditary breast cancer. Objective The objective of the study was to explore how women at high risk for hereditary breast cancer (1) form self-identity, (2) apply self-care strategies toward risk, and (3) describe the meaning of care through a high-risk breast program. Methods Interpretive hermeneutic phenomenology guided the qualitative research method. Women at high risk for hereditary breast cancer were recruited from a high-risk breast program. Open-ended interview questions focused on experiences living as women managing high risk for breast cancer. Consistent with hermeneutic methodology, the principal investigator led a team to analyze the interview transcripts. Results Twenty women participated in in-depth interviews. Analysis revealed that women describe their own identity based on their family story and grieve over actual and potential familial loss. This experience influences self-care strategies, including seeking care from hereditary breast cancer risk experts for early detection and prevention, as well as maintaining a connection for early treatment “when” diagnosis occurs. Conclusions Healthy women living with high risk for hereditary breast cancer are living within the context of their family cancer story, which influences how they define themselves and engage in self-care. Implications for Practice Findings present important practical, research, and policy information regarding health promotion, psychosocial assessment, and support for women living with this risk. PMID:22544165

  5. Cryopreservation of Living Organs

    NASA Astrophysics Data System (ADS)

    Tanasawa, Ichiro; Nagata, Shinichi; Kimura, Naohiro

    Cryopreservation is considered to be the most promising way of preserving living organs or tissues for a long period of time without casuing any damage to their biological functions. However, cryopreservation has been succeeded only for simple and small-size tissues such as spermatozoon, ovum, erythrocyte, bone marrow and cornea. Cryopreservation of more complex and large-scale organs are not yet succssful. The authors have attempted to establish a technique for cryopreservation of larger living organs. An experiment was carried out using daphnia (water flea). The optimum rates of freezing and thawing were determined together with the optimum selection of cryoprotectant. High recovery rate was achieved under these conditions.

  6. Introduction of Living Polymerization. Living and/or Controlled Polymerization

    DTIC Science & Technology

    1994-06-30

    separated into kinetic and synthetic. The intention of this paper is not to review existing and proclaimed living systems but to discuss the essence of a...o1 OFFICE OF NAVAL RESEARCH S4 CONTRACT N00014-94-1-0101 I R&T Code 31321075 Technical Report No. 10 INTRODUCTION TO LIVING POLYMERIZATION. LIVING ...TITLE AND SUBTITLE S. FUNDING NUMBERS Introduction to Living Polymerization. Living and/or Controlled Polymerization N00014-94-1-0101 6. AUTHOR(S

  7. Design for Living

    ERIC Educational Resources Information Center

    Rosenblum, Todd

    2011-01-01

    Bringing a newborn home from the hospital can come with stress for any parent. Coming home with twins can be double the stress. This article shares the story of a couple faced with this situation 12 years ago with the birth of twins, one was born with complications. They lived in a Colonial until the twins were almost five years old, at which time…

  8. Living with HIV

    MedlinePlus

    ... Abroad Treatment Basic Statistics Get Tested Find an HIV testing site near you. Enter ZIP code or city Follow HIV/AIDS CDC HIV CDC HIV/AIDS See RSS | ... Syndicated Content Website Feedback HIV/AIDS Living With HIV Language: English Spanish Recommend on Facebook Tweet Share ...

  9. Living with Aplastic Anemia

    MedlinePlus

    ... Content: NEXT >> Featured Video Clinical Trials for Rare Blood Diseases (Neal Young, M.D.) 05/17/2012 In ... in the lives of people who have rare blood and bone marrow diseases, such as aplastic anemia. // Non Object? Updated: August ...

  10. Microholography of Living Organisms.

    ERIC Educational Resources Information Center

    Solem, Johndale C.; Baldwin, George C.

    1982-01-01

    By using intense pulsed coherent x-ray sources it will be possible to obtain magnified three-dimensional images of living elementary biological structures at precisely defined instants. Discussed are sources/geometrics for x-ray holography, x-radiation interactions, factors affecting resolution, recording the hologram, high-intensity holography,…

  11. Family Living Supplement.

    ERIC Educational Resources Information Center

    Truitt, Debbie

    This family living supplement contains 125 supplemental ideas and strategies designed to help vocational home economics teachers increase student motivation and enrich the teaching process. Ideas and strategies are organized into seven sections. These are career planning, securing a job, and career success; managing financial resources, buying…

  12. Moab's Living Room

    ERIC Educational Resources Information Center

    Berry, John N., III

    2007-01-01

    This article describes the Grand County Public Library (GCPL) which was awarded the 2007 Best Small Library in America, an award sponsored by "Library Journal" and the Bill & Melinda Gates Foundation. Some 4800 of Grand County, Utah's 8,826 people live in Moab and the rest in the adjacent Spanish Valley and environs. The locals are a…

  13. The Living Periodic Table

    ERIC Educational Resources Information Center

    Nahlik, Mary Schrodt

    2005-01-01

    To help make the abstract world of chemistry more concrete eighth-grade students, the author has them create a living periodic table that can be displayed in the classroom or hallway. This display includes information about the elements arranged in the traditional periodic table format, but also includes visual real-world representations of the…

  14. The Living Periodic Table

    ERIC Educational Resources Information Center

    Nahlik, Mary Schrodt

    2005-01-01

    To help make the abstract world of chemistry more concrete eighth-grade students, the author has them create a living periodic table that can be displayed in the classroom or hallway. This display includes information about the elements arranged in the traditional periodic table format, but also includes visual real-world representations of the…

  15. The living building

    SciTech Connect

    McLennan, J.F.

    1998-07-01

    If one is to increase the energy performance of buildings beyond what is now possible, one can no longer afford to think of a building's systems and components as independent of one another. With emerging trends in building technology, it is becoming possible to design buildings (or groups of buildings) that respond to their environments as naturally as do living organisms. The living building integrates advances in glazing technology, photovoltaics, daylight-integrated lighting controls, HVAC and ecological waste management in conjunction with direct digital controls to respond actively to temperature, humidity, heat gain, cooling, lighting levels, and ventilation. This revolutionary building is the building of the future; it maximizes energy savings due to the inherent efficiency of an intelligent, interconnected system in which the envelope, lighting, and HVAC are always aware of and responding to each other's needs. While some of the technologies for such a system are already in use and resulting energy savings documented, it is not until advances such as electrochromic glazing reach the market that the level of integration necessary to produce the living building will be possible. This paper explores the limits of the living building's capacity to learn from environmental forces and regulate itself; the paper then examines emerging technologies that have demonstrated the potential to make such systemic integration and unprecedented energy savings possible.

  16. Living Systems Energy Module

    SciTech Connect

    1995-09-26

    The Living Systems Energy Module, renamed Voyage from the Sun, is a twenty-lesson curriculum designed to introduce students to the major ways in which energy is important in living systems. Voyage from the Sun tells the story of energy, describing its solar origins, how it is incorporated into living terrestrial systems through photosynthesis, how it flows from plants to herbivorous animals, and from herbivores to carnivores. A significant part of the unit is devoted to examining how humans use energy, and how human impact on natural habitats affects ecosystems. As students proceed through the unit, they read chapters of Voyage from the Sun, a comic book that describes the flow of energy in story form (Appendix A). During the course of the unit, an ``Energy Pyramid`` is erected in the classroom. This three-dimensional structure serves as a classroom exhibit, reminding students daily of the importance of energy and of the fragile nature of our living planet. Interactive activities teach students about adaptations that allow plants and animals to acquire, to use and to conserve energy. A complete list of curricular materials and copies of all activity sheets appear in Appendix B.

  17. Live-cell imaging

    PubMed Central

    Cole, Richard

    2014-01-01

    It would be hard to argue that live-cell imaging has not changed our view of biology. The past 10 years have seen an explosion of interest in imaging cellular processes, down to the molecular level. There are now many advanced techniques being applied to live cell imaging. However, cellular health is often under appreciated. For many researchers, if the cell at the end of the experiment has not gone into apoptosis or is blebbed beyond recognition, than all is well. This is simply incorrect. There are many factors that need to be considered when performing live-cell imaging in order to maintain cellular health such as: imaging modality, media, temperature, humidity, PH, osmolality, and photon dose. The wavelength of illuminating light, and the total photon dose that the cells are exposed to, comprise two of the most important and controllable parameters of live-cell imaging. The lowest photon dose that achieves a measureable metric for the experimental question should be used, not the dose that produces cover photo quality images. This is paramount to ensure that the cellular processes being investigated are in their in vitro state and not shifted to an alternate pathway due to environmental stress. The timing of the mitosis is an ideal canary in the gold mine, in that any stress induced from the imaging will result in the increased length of mitosis, thus providing a control model for the current imagining conditions. PMID:25482523

  18. Learning from Live Theater

    ERIC Educational Resources Information Center

    Greene, Jay P.; Hitt, Collin; Kraybill, Anne; Bogulski, Cari A.

    2015-01-01

    Culturally enriching field trips matter. They produce significant benefits for students on a variety of educational outcomes that schools and communities care about. This experiment on the effects of field trips to see live theater demonstrates that seeing plays is an effective way to teach academic content; increases student tolerance by…

  19. Living with Cystic Fibrosis

    MedlinePlus

    ... page from the NHLBI on Twitter. Living With Cystic Fibrosis If you or your child has cystic fibrosis (CF), you should learn as much as you can ... with your doctors to learn how to manage CF. Ongoing Care Having ongoing medical care by a ...

  20. Learning from Live Theater

    ERIC Educational Resources Information Center

    Greene, Jay P.; Hitt, Collin; Kraybill, Anne; Bogulski, Cari A.

    2015-01-01

    Culturally enriching field trips matter. They produce significant benefits for students on a variety of educational outcomes that schools and communities care about. This experiment on the effects of field trips to see live theater demonstrates that seeing plays is an effective way to teach academic content; increases student tolerance by…

  1. New Lives of Teachers

    ERIC Educational Resources Information Center

    Day, Christopher

    2012-01-01

    The work and lives of teachers have always been subject to external influence as those who are nearing the end of their careers will attest, but it is arguable that what is new over the last two decades is the pace, complexity, and intensity of change as governments have responded to the shrinking world of economic competitiveness and social…

  2. Living or Nonliving?

    ERIC Educational Resources Information Center

    Legaspi, Britt; Straits, William

    2011-01-01

    Categorizing organisms as living or nonliving things may seem to be intuitive by nature. Yet, it is regulated by scientific criteria. Students come to school with rules already in place. Their categorizing criteria have already been influenced by their personal experiences, also known as observations and inferences. They believe that all things…

  3. Living or Nonliving?

    ERIC Educational Resources Information Center

    Legaspi, Britt; Straits, William

    2011-01-01

    Categorizing organisms as living or nonliving things may seem to be intuitive by nature. Yet, it is regulated by scientific criteria. Students come to school with rules already in place. Their categorizing criteria have already been influenced by their personal experiences, also known as observations and inferences. They believe that all things…

  4. Native American Independent Living.

    ERIC Educational Resources Information Center

    Clay, Julie Anna

    1992-01-01

    Examines features of independent living philosophy with regard to compatibility with Native American cultures, including definition or conceptualization of disability; self-advocacy; systems advocacy; peer counseling; and consumer control and involvement. Discusses an actualizing process as one method of resolving cultural conflicts and…

  5. Measuring Retirees' Living Costs.

    ERIC Educational Resources Information Center

    Lamale, Helen H.

    1978-01-01

    The author evaluates the Consumer Price Index (CPI) used by the Bureau of Labor Statistics (BLS) to make cost-of-living adjustments to retirement benefits and considers the need for a separate retiree index. Stating that the CPI has underestimated inflation's impact on retirees, she recommends revised BLS retiree budgets. (MF)

  6. Living the Dream.

    ERIC Educational Resources Information Center

    Rogers, Cynthia; Lemay, Carol

    1991-01-01

    Describes one elementary school's "Living the Dream" award program named in honor of Dr. Martin Luther King, Jr. Students read and reviewed books and presented the award to the author of a recent picture book that focused on multicultural awareness. A list of suggested titles is included. (SM)

  7. You Live, You Learn

    ERIC Educational Resources Information Center

    Biesta, Gert

    2008-01-01

    The Learning Lives project, a four-year study into the learning biographies and trajectories of adults, was conducted by a team of researchers from the universities of Stirling, Exeter, Brighton and Leeds as part of the Teaching and Learning Research Programme (TLRP) of the Economic and Social Research Council, and has just been completed. Whereas…

  8. Design for Living

    ERIC Educational Resources Information Center

    Rosenblum, Todd

    2011-01-01

    Bringing a newborn home from the hospital can come with stress for any parent. Coming home with twins can be double the stress. This article shares the story of a couple faced with this situation 12 years ago with the birth of twins, one was born with complications. They lived in a Colonial until the twins were almost five years old, at which time…

  9. Test Pattern For Living.

    ERIC Educational Resources Information Center

    Johnson, Nicholas

    The lives of Americans today are ruled by interlocking corporations. These corporations together present only one kind of life as viable: the consumer's life. Television is their main means of presenting this view. One cannot choose something he does not know about, and many Americans are not sufficiently informed of the alternatives to make an…

  10. The Living Museum.

    ERIC Educational Resources Information Center

    Merrill, Mary

    1992-01-01

    Presents a lesson plan for a second-grade class project. Suggests that the students will learn to identify the word "famous," complete a timeline for a famous person, learn facts about the person, and express individual thoughts and feelings. Explains the steps involved in the presentation of a living museum where students portray famous…

  11. Generation and Characterization of Live Attenuated Influenza A(H7N9) Candidate Vaccine Virus Based on Russian Donor of Attenuation.

    PubMed

    Shcherbik, Svetlana; Pearce, Nicholas; Balish, Amanda; Jones, Joyce; Thor, Sharmi; Davis, Charles Todd; Pearce, Melissa; Tumpey, Terrence; Cureton, David; Chen, Li-Mei; Villanueva, Julie; Bousse, Tatiana L

    2015-01-01

    Avian influenza A (H7N9) virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV) are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes. LAIV candidate A/Anhui/1/2013(H7N9)-CDC-LV7A (abbreviated as CDC-LV7A), based on the Russian MDV, A/Leningrad/134/17/57 (H2N2), was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering) in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9) virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7. Our data indicate that the A/Anhui/1/2013(H7N9)-CDC-LV7A reassortant virus is a safe and genetically stable candidate vaccine virus that is now available for

  12. Generation and Characterization of Live Attenuated Influenza A(H7N9) Candidate Vaccine Virus Based on Russian Donor of Attenuation

    PubMed Central

    Shcherbik, Svetlana; Pearce, Nicholas; Balish, Amanda; Jones, Joyce; Thor, Sharmi; Davis, Charles Todd; Pearce, Melissa; Tumpey, Terrence; Cureton, David; Chen, Li-Mei; Villanueva, Julie; Bousse, Tatiana L.

    2015-01-01

    Background Avian influenza A (H7N9) virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV) are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes. Methodology/Principal Findings LAIV candidate A/Anhui/1/2013(H7N9)-CDC-LV7A (abbreviated as CDC-LV7A), based on the Russian MDV, A/Leningrad/134/17/57 (H2N2), was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering) in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9) virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7. Conclusions/Significance Our data indicate that the A/Anhui/1/2013(H7N9)-CDC-LV7A reassortant virus is a safe and

  13. Living related liver transplantation.

    PubMed

    Makuuchi, M; Kawarazaki, H; Iwanaka, T; Kamada, N; Takayama, T; Kumon, M

    1992-01-01

    Liver transplantation from a brain death donor has not yet been accepted in Japan. The only alternative method at present is transplantation from a living donor. After the first successful living related liver transplantation was performed by Strong in Brisbane, Australia, Japanese hepatic and transplant surgeons also began to perform such operations. As of February 1991, 16 living related liver transplantations had already been performed in Japan, mainly for children with biliary atresia. Five of these patients subsequently died, however, our patient has survived more than 1 year, and she is presently leading a normal school life. The most important issue regarding living related liver transplantation is to ensure the donor's safety. For this purpose, we conducted a preoperative banking of the donor's own blood and plasma. In addition, a selective vascular occlusion was carried out to reduce blood loss during the resection of the liver. Intraoperative color Doppler ultrasonography was introduced for evaluating the circulation of the graft. By using this modality, the following three points were able to be accurately estimated in order to obtain optimal graft perfusion: 1) The most suitable position for the graft to be fixed to the abdominal wall, 2) whether or not the abdominal wall could be closed and 3) the indication for a ligation of the collateral veins to form a porto-systemic shunt. Thanks to these procedures, living related liver transplantations have now become an acceptable transplant method, however, a transplantation from a cadaver that is brain dead but still has a beating heart is still absolutely necessary for adult recipients. Therefore, in the future, both methods should be performed.

  14. Watersheds: where we live

    USGS Publications Warehouse

    Vandas, Stephen; Farrar, Frank

    1996-01-01

    We all live in a watershed. Animals and plants all live there with us. Everyone affects what happens in a watershed by how we treat the natural resources. So what is a watershed? It is the land area that drains water to a stream, river, lake, or ocean. Water travels over the Earth's surface across forest land, farm fields, pastures, suburban lawns, and city streets, or it seeps into the soil and makes its way to a stream as local ground water. Watersheds come in many different shapes and sizes. Some contain mountains and hills, and others are nearly flat. A watershed can be affected by many different activities and events. Construction of cities and towns, farming, logging, and the application and disposal of many garden and household chemicals can affect the quantity and quality of water flowing from a watershed.

  15. Improving assisted living care.

    PubMed

    Gregory, Nancy; Gesell, Sabina B; Widmer, Tom

    2007-01-01

    In the absence of a national measurement system, private vendors of satisfaction measurement and improvement services have played a crucial role in the quality movement in the assisted living industry. Survey responses from 175 resident-family dyads at 20 facilities were analyzed to identify priorities for service improvement from the customers' perspective. They include improving care provided by aides and management, meal service, and activities. Practical solutions for addressing these issues are presented.

  16. Interior view of former living porch, now living area extension, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior view of former living porch, now living area extension, facing east. - Albrook Air Force Station, Field Officer's Quarters, West side of Dargue Avenue Circle, Balboa, Former Panama Canal Zone, CZ

  17. Living Room Mantel Profile, Door Jamb, Window Sill, Baseboard, Living ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Living Room Mantel Profile, Door Jamb, Window Sill, Baseboard, Living Room Fireplace Details, Door Profile - National Home for Disabled Volunteer Soldiers - Battle Mountain Sanitarium, Governor's Quarters, 500 North Fifth Street, Hot Springs, Fall River County, SD

  18. Interior view of living area and living porch showing structural ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior view of living area and living porch showing structural system, facing north. - Albrook Air Force Station, Company Officer's Quarters, East side of Canfield Avenue, Balboa, Former Panama Canal Zone, CZ

  19. Live from the Arctic

    NASA Astrophysics Data System (ADS)

    Haines-Stiles, G.; Warnick, W. K.; Warburton, J.; Sunwood, K.

    2003-12-01

    For reasons of geography and geophysics, the poles of our planet, the Arctic and Antarctica, are places where climate change appears first: they are global canaries in the mine shaft. But while Antarctica (its penguins and ozone hole, for example) has been relatively well-documented in recent books, TV programs and journalism, the far North has received somewhat less attention. This project builds on and advances what has been done to date to share the people, places, and stories of the North with all Americans through multiple media, over several years. In a collaborative project between the Arctic Research Consortium of the United States (ARCUS) and PASSPORT TO KNOWLEDGE, Live from the Arctic will bring the Arctic environment to the public through a series of primetime broadcasts, live and taped programming, interactive virtual field trips, and webcasts. The five-year project will culminate during the 2007-2008 International Polar Year (IPY). Live from the Arctic will: A. Promote global understanding about the value and world -wide significance of the Arctic, B. Bring cutting-edge research to both non-formal and formal education communities, C. Provide opportunities for collaboration between arctic scientists, arctic communities, and the general public. Content will focus on the following four themes. 1. Pan-Arctic Changes and Impacts on Land (i.e. snow cover; permafrost; glaciers; hydrology; species composition, distribution, and abundance; subsistence harvesting) 2. Pan-Arctic Changes and Impacts in the Sea (i.e. salinity, temperature, currents, nutrients, sea ice, marine ecosystems (including people, marine mammals and fisheries) 3. Pan-Arctic Changes and Impacts in the Atmosphere (i.e. precipitation and evaporation; effects on humans and their communities) 4. Global Perspectives (i.e. effects on humans and communities, impacts to rest of the world) In The Earth is Faster Now, a recent collection of comments by members of indigenous arctic peoples, arctic

  20. Live from the Arctic

    NASA Astrophysics Data System (ADS)

    Warnick, W. K.; Haines-Stiles, G.; Warburton, J.; Sunwood, K.

    2003-12-01

    For reasons of geography and geophysics, the poles of our planet, the Arctic and Antarctica, are places where climate change appears first: they are global canaries in the mine shaft. But while Antarctica (its penguins and ozone hole, for example) has been relatively well-documented in recent books, TV programs and journalism, the far North has received somewhat less attention. This project builds on and advances what has been done to date to share the people, places, and stories of the North with all Americans through multiple media, over several years. In a collaborative project between the Arctic Research Consortium of the United States (ARCUS) and PASSPORT TO KNOWLEDGE, Live from the Arctic will bring the Arctic environment to the public through a series of primetime broadcasts, live and taped programming, interactive virtual field trips, and webcasts. The five-year project will culminate during the 2007-2008 International Polar Year (IPY). Live from the Arctic will: A. Promote global understanding about the value and world -wide significance of the Arctic, B. Bring cutting-edge research to both non-formal and formal education communities, C. Provide opportunities for collaboration between arctic scientists, arctic communities, and the general public. Content will focus on the following four themes. 1. Pan-Arctic Changes and Impacts on Land (i.e. snow cover; permafrost; glaciers; hydrology; species composition, distribution, and abundance; subsistence harvesting) 2. Pan-Arctic Changes and Impacts in the Sea (i.e. salinity, temperature, currents, nutrients, sea ice, marine ecosystems (including people, marine mammals and fisheries) 3. Pan-Arctic Changes and Impacts in the Atmosphere (i.e. precipitation and evaporation; effects on humans and their communities) 4. Global Perspectives (i.e. effects on humans and communities, impacts to rest of the world) In The Earth is Faster Now, a recent collection of comments by members of indigenous arctic peoples, arctic

  1. Valuing different human lives.

    PubMed

    Goodwin, Geoffrey P; Landy, Justin F

    2014-04-01

    Do people think of the value of all human lives as equivalent irrespective of age? Affirmations of the equal value of all human lives are culturally prominent, yet much evidence points to the fact that the young are often prioritized over the old in life-and-death decision-making contexts. Studies 1-3 aimed to reconcile this tension by showing that although individuals are seen as more equal with respect to negative rights not to be harmed or killed (though not completely equal), they are seen as less equal with respect to positive rights to be aided or saved. Age exerts a large and systematic impact on decisions about who to save and about whose death is more tragic, suggesting that individuals are seen as possessing differing amounts of contingent value. These initial studies also yielded the novel finding that, although children are prioritized over adults, older children are often prioritized over younger children. Study 4 replicated this finding with a think-aloud methodology; the study showed that the preference for older children appears to be driven by their having had more invested in their lives, their better developed social relations, and their greater understanding of death. Studies 5a-5c demonstrated the independent causal effects of each of these variables on judgments of life's value. Finally, in Studies 6 and 7, mediation methods were used to show that older children's more meaningful social relations primarily explain the greater value of older than of younger children. These findings have implications for bioethics and medical policy.

  2. Microencapsulation Of Living Cells

    NASA Technical Reports Server (NTRS)

    Chang, Manchium; Kendall, James M.; Wang, Taylor G.

    1989-01-01

    In experimental technique, living cells and other biological materials encapsulated within submillimeter-diameter liquid-filled spheres. Sphere material biocompatible, tough, and compliant. Semipermeable, permitting relatively small molecules to move into and out of sphere core but preventing passage of large molecules. New technique promises to make such spherical capsules at high rates and in uniform, controllable sizes. Capsules injected into patient through ordinary hypodermic needle. Promising application for technique in treatment of diabetes. Also used to encapsulate pituitary cells and thyroid hormone adrenocortical cells for treatment of other hormonal disorders, to encapsulate other secreting cells for transplantation, and to package variety of pharmaceutical products and agricultural chemicals for controlled release.

  3. Living in Space

    NASA Technical Reports Server (NTRS)

    Brown, Ray (Editor)

    1993-01-01

    In this educational video from the 'Liftoff to Learning' series, astronauts from the STS-56 Mission (Ken Cockrell, Mike Foale, Ellen Ochoa, Steve Oswald, and Ken Cameron) explain and show through demonstrations how microgravity affects the way astronauts live onboard the Space Shuttle, and how these same daily habits or processes differ on Earth. A tour of the Space Shuttle is given, including the sleeping compartments, the kitchen area, the storage compartments, and the Waste Collection System (or WCS, as they call it). Daily habits (brushing teeth, shampooing hair and bathing, eating,...) are explained and actively illustrated, along with reasons of how these applications differ from their employment on Earth.

  4. Living olefin polymerization processes

    DOEpatents

    Schrock, R.R.; Baumann, R.

    1999-03-30

    Processes for the living polymerization of olefin monomers with terminal carbon-carbon double bonds are disclosed. The processes employ initiators that include a metal atom and a ligand having two group 15 atoms and a group 16 atom or three group 15 atoms. The ligand is bonded to the metal atom through two anionic or covalent bonds and a dative bond. The initiators are particularly stable under reaction conditions in the absence of olefin monomer. The processes provide polymers having low polydispersities, especially block copolymers having low polydispersities. It is an additional advantage of these processes that, during block copolymer synthesis, a relatively small amount of homopolymer is formed.

  5. Living olefin polymerization processes

    DOEpatents

    Schrock, Richard R.; Baumann, Robert

    1999-01-01

    Processes for the living polymerization of olefin monomers with terminal carbon-carbon double bonds are disclosed. The processes employ initiators that include a metal atom and a ligand having two group 15 atoms and a group 16 atom or three group 15 atoms. The ligand is bonded to the metal atom through two anionic or covalent bonds and a dative bond. The initiators are particularly stable under reaction conditions in the absence of olefin monomer. The processes provide polymers having low polydispersities, especially block copolymers having low polydispersities. It is an additional advantage of these processes that, during block copolymer synthesis, a relatively small amount of homopolymer is formed.

  6. Living olefin polymerization processes

    DOEpatents

    Schrock, Richard R.; Baumann, Robert

    2003-08-26

    Processes for the living polymerization of olefin monomers with terminal carbon-carbon double bonds are disclosed. The processes employ initiators that include a metal atom and a ligand having two group 15 atoms and a group 16 atom or three group 15 atoms. The ligand is bonded to the metal atom through two anionic or covalent bonds and a dative bond. The initiators are particularly stable under reaction conditions in the absence of olefin monomer. The processes provide polymers having low polydispersities, especially block copolymers having low polydispersities. It is an additional advantage of these processes that, during block copolymer synthesis, a relatively small amount of homopolymer is formed.

  7. Living olefin polymerization processes

    DOEpatents

    Schrock, Richard R.; Bauman, Robert

    2006-11-14

    Processes for the living polymerization of olefin monomers with terminal carbon-carbon double bonds are disclosed. The processes employ initiators that include a metal atom and a ligand having two group 15 atoms and a group 16 atom or three group 15 atoms. The ligand is bonded to the metal atom through two anionic or covalent bonds and a dative bond. The initiators are particularly stable under reaction conditions in the absence of olefin monomer. The processes provide polymers having low polydispersities, especially block copolymers having low polydispersities. It is an additional advantage of these processes that, during block copolymer synthesis, a relatively small amount of homopolymer is formed.

  8. Living in Space

    NASA Technical Reports Server (NTRS)

    Brown, Ray (Editor)

    1993-01-01

    In this educational video from the 'Liftoff to Learning' series, astronauts from the STS-56 Mission (Ken Cockrell, Mike Foale, Ellen Ochoa, Steve Oswald, and Ken Cameron) explain and show through demonstrations how microgravity affects the way astronauts live onboard the Space Shuttle, and how these same daily habits or processes differ on Earth. A tour of the Space Shuttle is given, including the sleeping compartments, the kitchen area, the storage compartments, and the Waste Collection System (or WCS, as they call it). Daily habits (brushing teeth, shampooing hair and bathing, eating,...) are explained and actively illustrated, along with reasons of how these applications differ from their employment on Earth.

  9. "Living versus Dead":

    PubMed Central

    Chakrabarti, Pratik

    2010-01-01

    Summary The Semple antirabies vaccine was developed by David Semple in India in 1911. Semple introduced a peculiarly British approach within the Pasteurian tradition by using carbolized dead virus. This article studies this unique phase of vaccine research between 1910 and 1935 to show that in the debates and laboratory experiments around the potency and safety of vaccines, categories like "living" and "dead" were often used as ideological and moral denominations. These abstract and ideological debates were crucial in defining the final configuration of the Semple vaccine, the most popular antirabies vaccine used globally, and also in shaping international vaccination policies. PMID:21037397

  10. Communication in Assisted Living*

    PubMed Central

    Williams, Kristine N.; Warren, Carol A.B.

    2009-01-01

    This study of communication in an Assisted Living Facility (ALF) focuses on staff’s interpretive frameworks and situational tactics for managing elderly residents. It is based on interviews with staff and residents in an ALF together with ethnographic fieldwork. As in other quasi-total institutions, staff members engage in control as well as care, monitoring residents for compliance with rules and directives. Residents, aware of the threat of being moved to a nursing home, also monitor their own behavior and cognition in comparison to other residents. Other communication issues include the infantilization of the elderly by staff, and the race, class, and ethnic prejudices of residents. PMID:20107612

  11. Genetic stability of live attenuated vaccines against potentially pandemic influenza viruses.

    PubMed

    Kiseleva, Irina; Dubrovina, Irina; Fedorova, Ekaterina; Larionova, Natalie; Isakova-Sivak, Irina; Bazhenova, Ekaterina; Pisareva, Maria; Kuznetsova, Victoria; Flores, Jorge; Rudenko, Larisa

    2015-12-08

    Ensuring genetic stability is a prerequisite for live attenuated influenza vaccine (LAIV). This study describes the results of virus shedding and clinical isolates' testing of Phase I clinical trials of Russian LAIVs against potentially pandemic influenza viruses in healthy adults. Three live attenuated vaccines against potentially pandemic influenza viruses, H2N2 LAIV, H5N2 LAIV and H7N3 LAIV, generated by classical reassortment in eggs, were studied. For each vaccine tested, subjects were randomly distributed into two groups to receive two doses of either LAIV or placebo at a 3:1 vaccine/placebo ratio. Nasal swabs were examined for vaccine virus shedding by culturing in eggs and by PCR. Vaccine isolates were tested for temperature sensitivity and cold-adaptation (ts/ca phenotypes) and for nucleotide sequence. The majority of nasal wash positive specimens were detected on the first day following vaccination. PCR method demonstrated higher sensitivity than routine virus isolation in eggs. None of the placebo recipients had detectable vaccine virus replication. All viruses isolated from the immunized subjects retained the ts/ca phenotypic characteristics of the master donor virus (MDV) and were shown to preserve all attenuating mutations described for the MDV. These data suggest high level of vaccine virus genetic stability after replication in humans. During manufacture process, no additional mutations occurred in the genome of H2N2 LAIV. In contrast, one amino acid change in the HA of H7N3 LAIV and two additional mutations in the HA of H5N2 LAIV manufactured vaccine lot were detected, however, they did not affect their ts/ca phenotypes. Our clinical trials revealed phenotypic and genetic stability of the LAIV viruses recovered from the immunized volunteers. In addition, no vaccine virus was detected in the placebo groups indicating the lack of person-to-person transmission. LAIV TRIAL REGISTRATION at ClinicalTrials.gov: H7N3-NCT01511419; H5N2-NCT01719783; H2N2-NCT

  12. Living with a Single Parent

    MedlinePlus

    ... Video: Getting an X-ray Living With a Single Parent KidsHealth > For Kids > Living With a Single Parent ... single parents can be a great idea, too. Single Parents and Work Single parents are often working parents ...

  13. Living with a Single Parent

    MedlinePlus

    ... Happens in the Operating Room? Living With a Single Parent KidsHealth > For Kids > Living With a Single Parent ... single parents can be a great idea, too. Single Parents and Work Single parents are often working parents ...

  14. Living with Sickle Cell Disease

    MedlinePlus

    ... from the NHLBI on Twitter. Living With Sickle Cell Disease If you or your child has sickle ... NEXT >> Featured Video Living With and Managing Sickle Cell Disease (Nicholas) 09/02/2011 In this video— ...

  15. Active Living: Promoting Healthy Lifestyles.

    ERIC Educational Resources Information Center

    Swedburg, Randy B.; Izso, Bill

    1994-01-01

    Active Living is a unique Canadian approach in which physical activity is valued and integrated into daily life. Active Living will have an increasing impact on the health, physical education, and recreation fields. The article describes the development of Active Living, its effects on Canadian society, and current initiatives. (SM)

  16. RACE AS LIVED EXPERIENCE

    PubMed Central

    Garcia, John A.; Sanchez, Gabriel R.; Sanchez-Youngman, Shannon; Vargas, Edward D.; Ybarra, Vickie D.

    2015-01-01

    A growing body of social science research has sought to conceptualize race as a multidimensional concept in which context, societal relations, and institutional dynamics are key components. Utilizing a specially designed survey, we develop and use multiple measures of race (skin color, ascribed race, and discrimination experiences) to capture race as “lived experience” and assess their impact on Latinos’ self-rated health status. We model these measures of race as a lived experience to test the explanatory power of race, both independently and as an integrated scale with categorical regression, scaling, and dimensional analyses. Our analyses show that our multiple measures of race have significant and negative effects on Latinos’ self-reported health. Skin color is a dominant factor that impacts self-reported health both directly and indirectly. We then advocate for the utilization of multiple measures of race, adding to those used in our analysis, and their application to other health and social outcomes. Our analysis provides important contributions across a wide range of health, illness, social, and political outcomes for communities of color. PMID:26681972

  17. Living Longer in Mexico

    PubMed Central

    Aguila, Emma; Diaz, Claudia; Fu, Mary Manqing; Kapteyn, Arie; Pierson, Ashley

    2012-01-01

    Abstract This analysis of aging and income security in Mexico establishes that the older population in Mexico is increasing quickly and that this population is especially vulnerable to poverty. Mexican citizens are living longer and overall have experienced an improvement in the quality of life compared to that of prior generations. However, this study demonstrates that social improvements are not affecting the daily lives of all persons equally. The authors attempt to uncover and highlight those differences. One of the primary challenges facing Mexico is a growing older population. The demographic transition in Mexico combined with the lack of formal sources of income in retirement place many older persons in a state of financial insecurity. The information contained in this study and the proposed policy research areas are intended to enlarge the portfolio of options for older Mexicans. The authors analyze wealth and sources of income during retirement, the relationship between health and wealth, urban and rural disparities, and the impact of migration spells to the United States on wealth accumulation and health insurance in Mexico. PMID:28083208

  18. Alternative live-attenuated influenza vaccines based on modifications in the polymerase genes protect against epidemic and pandemic flu.

    PubMed

    Solórzano, Alicia; Ye, Jianqiang; Pérez, Daniel R

    2010-05-01

    Human influenza is a seasonal disease associated with significant morbidity and mortality. Influenza vaccination is the most effective means for disease prevention. We have previously shown that mutations in the PB1 and PB2 genes of the live-attenuated influenza vaccine (LAIV) from the cold-adapted (ca) influenza virus A/Ann Arbor/6/60 (H2N2) could be transferred to avian influenza viruses and produce partially attenuated viruses. We also demonstrated that avian influenza viruses carrying the PB1 and PB2 mutations could be further attenuated by stably introducing a hemagglutinin (HA) epitope tag in the PB1 gene. In this work, we wanted to determine whether these modifications would also result in attenuation of a so-called triple reassortant (TR) swine influenza virus (SIV). Thus, the TR influenza A/swine/Wisconsin/14094/99 (H3N2) virus was generated by reverse genetics and subsequently mutated in the PB1 and PB2 genes. Here we show that a combination of mutations in this TR backbone results in an attenuated virus in vitro and in vivo. Furthermore, we show the potential of our TR backbone as a vaccine that provides protection against the 2009 swine-origin pandemic influenza H1N1 virus (S-OIV) when carrying the surface of a classical swine strain. We propose that the availability of alternative backbones to the conventional ca A/Ann Arbor/6/60 LAIV strain could also be useful in epidemic and pandemic influenza and should be considered for influenza vaccine development. In addition, our data provide evidence that the use of these alternative backbones could potentially circumvent the effects of original antigenic sin (OAS) in certain circumstances.

  19. Live From the Poles

    NASA Astrophysics Data System (ADS)

    Linder, C. A.; Kent, J.; Lippsett, L.

    2006-12-01

    International Polar Year presents an extraordinary opportunity to educate students and the public about science at the icy ends of the Earth. The goal of our proposal is to apply collaborative multimedia approaches to bring the story of four polar research expeditions to the general public and the classroom. The four expeditions (measurement of ice sheet dynamics in Greenland, a study of the McMurdo ecosystem over austral winter, installation of a buoy array in the Beaufort Gyre, and exploration of the Gakkel Ridge) were chosen based on their broad range of disciplines and relevance to the three primary IPY research emphasis areas defined by NSF. A science writer and a professional photographer will join each expedition and file dispatches for a daily Webcast. The posting will feature science updates, logistical challenges, team member profiles, and life at sea (or on the ice). The writer will also coordinate real-time phone patches from PIs in the field to audiences at the Museum of Science, Boston, the Smithsonian National Museum of Natural History, The Field Museum, Chicago, the Houston Museum of Natural Science, the Birch Aquarium, San Diego, the Pacific Science Center, Seattle, National Public Radio "Talk of the Nation: Science Friday," CBS News, and to student "reporters" writing for Scholastic Online. At the museums, the "Live from the Ice" interactive phone calls will be preceded by a background presentation by a scientist, who will also moderate the live discussion between the public and researchers in the field. A 20-30 minute satellite phone call will allow the public to ask the researchers questions about their research while it's happening. In addition to building and promoting an online experience, a museum exhibit featuring models of Arctic instruments and informative kiosks will be developed at the Woods Hole Oceanographic Institution Exhibit Center. Each of our partner museums will also provide a "leave-behind" component to continue to educate

  20. Women's lives, mothers' health.

    PubMed

    Chauliac, M; Masse-raimbault, A M

    1985-01-01

    This document dealing with women's lives and the health of mothers identifies factors conditioning the health and nutritional status of women and girls (life expectancy at birth, maternal mortality rate, and the birthrate); considers nutritional requirements of pregnant and lactating women, weight gain during preganncy, mothers' age and number of children and interbirth interval, maternal nutritional status and breastfeeding, anemia, work and women's health, pregnancy in adolescents, abortion, the growth of small girls and its effect on future pregnancies, and sexual mutilations; and reports on actions aimed at improving the health of women as well as health problems facing rural women. The 3 key concepts of this reflection on women's lives are: women's health should be taken into account as well as children's health; the development of the whole human being should be respected, implying ongoing surveillance of the health status of women and of their children; and the overall living conditions of women within the family and society must be analyzed at the different phases of their life, so as to encourage integrated actions rather than various uncoordinated efforts. Women's health status, like the health status of everyone, depends on a multitude of socioeconomic and sanitational factors. A figure illustrates several of the many interrelations between the various factors which influence the nutritional status of all individuals. Women of childbearing age are at greater risk than other population groups, due to their reproductive function and their ability to nurse children: pregnancy, like lactation, generates metabolic changes and increases nutritional needs. Delivery itself presents a series of risks for the woman's health, and only regular surveillance of pregnancy may prevent many of these. A woman's health status and, most of all her nutritional status during pregnancy and delivery, condition her future health and ability to assume her many tasks as well as

  1. Living with hypertrophic cardiomyopathy.

    PubMed

    Subasic, Kim

    2013-12-01

    The purpose of this study is to provide an insider's account of what it is like to live with hypertrophic cardiomyopathy (HCM), a genetic cardiovascular illness that carries the risk for sudden cardiac death. This study aims to reveal how HCM impacts the family and guides the decision whether or not to pursue genetic testing, how the physical limitations associated with HCM alter being-in-the-world, and how HCM alters social relationships. Fifteen adults with HCM were recruited for a longitudinal, phenomenological, qualitative study through purposive sampling and word of mouth. A total of 45 interviews were conducted by the researcher at a time and place designated by the participant between August 2011 and January 2012. The first interview with each participant was conducted in person. While efforts were made to conduct all interviews in person, a total of three interviews were conducted by telephone as requested by three participants due to scheduling conflicts. Through methods of interpretive phenomenology, three audio-recorded, semistructured interviews occurred over the course of 3 months. Detailed narratives were solicited and transcribed verbatim. Methodological and analytical documentation was supported with the identification of key phrases, similar experiences, themes, and documentation of the rationale for decisions throughout the research process. Participation in genetic testing carries a multitude of personal, familial, financial, and emotional implications. The results of a genetic test elicited an emotional response regardless of whether the results were negative, positive, or inconclusive. Living with a potentially life-threatening illness altered identity, disrupted social relationships, and generated chronic fear and uncertainty. A new normal was re-ordered or transformed by the demands and limitations posed by HCM, and by the person's concerns, priorities, and the meaning of the illness. Results from this study underscore the need for healthcare

  2. Freezing of living cells

    SciTech Connect

    Mazur, P.

    1985-01-01

    It can be calculated that a living cell will survive more than 5000 years at -196/sup 0/C. This ability to essentially stop biological time has important implications in medicine and agriculture, and in biological research. In medicine the chief implications are in the banking of transplantable tissues and organs and in in vitro fertilization. In agriculture the applications stem in part from the role of frozen embryos in amplifying the number of calves produced by high quanlity cows. The problem is how can cells survive both the cooling to such very low temperatures and the return to normal temperatures. The answers involve fundamental characteristics of cells such as the permeability of their surface membranes to water and solutes. These characteristics determine whether or not cells undergo lethal internal ice formation and other response during freezing and thawing. 27 refs., 12 figs.

  3. Living on the edge.

    PubMed

    Hinrichsen, D

    1989-01-01

    A brief update on the destruction of the environment is given. The concern is for the coastal waters and rivers which are polluted daily by raw sewage, industrial waste, and sedimentation, e.g., the Juru in Malaysia, the Pasig in the Philippines, and the Chao Phraya in Thailand are open sewers by the time the rivers reach the sea or bay. Metropolitan Manila's river is said to be biologically dead from pollution, and the bays of Manila and Jakarta suffer from oxygen depletion. Unfortunately, the coastal area maintains population as well as the wealth of marine life. In the US in 1990, 75% of the population will live within 50 miles of a shore including the Great Lakes. 30 southeast Asia's 50 largest cities are located on or near a coast. Over fishing, over population, over developing, and over exploitation are unacceptable; the alternative is for man to correct his mistakes.

  4. Gaia Live in School

    NASA Astrophysics Data System (ADS)

    Walton, N. A.; Barnes, R.; Soubiran, C.; Vogt, S.

    2014-07-01

    Gaia is the European Space Agency's (ESA) next major astronomy telescope mission that was launched December 19, 2013. Gaia will measure accurate distances to about one billion stars across our Milky Way, allowing us to better understand how our galaxy formed and evolved. Gaia will have a profound impact on our understand ing of the Universe and the nature of dark matter, and provide a deeper understanding of how planets form around stars in our local neighbourhood. Gaia scientists and science education advisors are organising a Gaia post-launch event to link approximately forty schools across Europe. The event will include a live stream connection to ESA Gaia Mission Control and local Gaia research students to act as “explainers” and give practical demonstrations in each school. This paper describes the challenges in conducting this Europe-wide event.

  5. Can lean save lives?

    PubMed

    Fillingham, David

    2007-01-01

    The purpose of this paper is to show how over the last 18 months Bolton Hospitals NHS Trust have been exploring whether or not lean methodologies, often known as the Toyota Production System, can indeed be applied to healthcare. This paper is a viewpoint. One's early experience is that lean really can save lives. The Toyota Production System is an amazingly successful way of manufacturing cars. It cannot be simply translated unthinkingly into a hospital but lessons can be learned from it and the method can be adapted and developed so that it becomes owned by healthcare staff and focused towards the goal of improved patient care. Working in healthcare is a stressful and difficult thing. Everyone needs a touch of inspiration and encouragement. Applying lean to healthcare in Bolton seems to be achieving just that for those who work there.

  6. Incentivizing living organ donation.

    PubMed

    Cynowiec, Jessica; Kim, Jennifer; Qazi, Yasir A

    2009-04-01

    The number of organs available for the patients on the transplant waitlist remains at a disproportionate low. All possible methods to curtail this shortage, including providing donors with incentives, have been proposed. This article reviews recent publications addressing the benefits and risks involved in incentivizing living donation. The debate about the ethics, feasibility, and possible models for compensating organ donors has been prominent in recent literature. As certain countries take lead on this initiative, others are cautiously weighing in on the impact implementations of such policies may have on the society, especially on the underprivileged. The shortage of organs has resulted in proposal of strategies that encroach on certain moral and ethical principles. Providing incentives to donors is one such strategy that is likely to receive a lot of attention in the next few years.

  7. Living Liquid Crystals.

    SciTech Connect

    Zhou, Shuang; Sokolov, Andrey; Lavrentovich, Oleg D.; Aranson, Igor S.

    2014-01-28

    Collective motion of self-propelled organisms or synthetic par­ticles, often termed •active fluid,• has attracted enormous atten­tion in the broad scientific community because of its fundamentally nonequilibrium nature. Energy input and interactions among the moving units and the medium lead to complex dynamics. Here,we introduce a class of active matter-living liquid crystals (UCs}­ that combines living swimming bacteria with a lyotropic liquid crystal. The physical properties of LLCs can be controlled by the amount of oxygen available to bacteria, by concentration of ingre­dients, or by temperature. Our studies reveal a wealth of intriguing dynamic phenomena. caused by the coupling between the activity-triggered flow and long-range orientational order of the medium. Among these are (i) nonlinear trajectories of bacterial motion guided by nonuniform director, (ii) local melting of the liquid crystal caused by the bacteria-produced shear flows, (iii) activity-triggered transition from a nonflowing uniform state into a flowing one-dimensional periodic pattern and its evolution into a turbulent array of topological defects, and (iv) birefringence­ enabled visualization of microflow generated by the nanometers­ thick bacterial flagella. Unlike their isotropic counterpart, the LLCs show collective dynamic effects at very low volume fraction of bacteria, on the order of 0.2%. Our work suggests an unorthodox design concept to control and manipulate the dynamic behavior of soft active matter and opens the door for potential biosensing and biomedical applications.

  8. Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production.

    PubMed

    Shcherbik, Svetlana; Sergent, Sheila B; Davis, William G; Shu, Bo; Barnes, John; Kiseleva, Irina; Larionova, Natalie; Klimov, Alexander; Bousse, Tatiana

    2014-01-01

    Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012-2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate. Published by Elsevier B.V.

  9. Creating living machines

    PubMed Central

    Kamm, Roger D.; Bashir, Rashid

    2015-01-01

    Development of increasingly complex integrated cellular systems will be a major challenge for the next decade and beyond, as we apply the knowledge gained from the sub-disciplines of tissue engineering, synthetic biology, micro-fabrication and nanotechnology, systems biology, and developmental biology. In this prospective, we describe the current state-of-the-art in the context of differentiating source cells from more primitive, pluripotent cells, and organizing these cells into populations of a single cell type to produce the components or building blocks of higher order systems and finally, combining multiple cell types, possibly in combination with scaffolds possessing specific physical or chemical properties, to produce greater functionality. As these “living machines” increase in capabilities, exhibit emergent behavior and potentially reveal the ability for self-assembly, self-repair, and even self-replication, questions arise regarding the ethical implications of this work. Future prospects as well as ways of addressing these complex ethical questions will be addressed. PMID:24006130

  10. Live Information Objects

    DTIC Science & Technology

    2011-06-01

    prevent  it.    Users  of  the  system  pulled  information  of  various  kinds  into  the  shared  scenario:  city   maps,  images,  data  from  databases...videos  from  real‐time  feeds  in  the  city   itself.  Working  together,  they  rapidly  solved  their  problem.    The  live  application  could...hold groups of other objects: a  city  object might contain building objects; a  helicopter squadron multiple helicopters, and so forth.   What about data

  11. Exact Law of Live Nature

    NASA Astrophysics Data System (ADS)

    Azbel', Mark Ya.

    Exact law of mortality dynamics in changing populations and environment is derived. The law is universal for all species, from single cell yeast to humans. It includes no characteristics of animal-environment interactions (metabolism etc.) which are a must for life. Such law is unique for live systems with their homeostatic self-adjustment to environment. Its universal dynamics for all animals, with their drastically different biology, evolutionary history, and complexity, is also unique for live systems — cf. different thermodynamics of liquids and glasses. The law which is valid for all live, and only live, systems is a life specific law of nature.

  12. A live attenuated equine H3N8 influenza vaccine is highly immunogenic and efficacious in mice and ferrets.

    PubMed

    Baz, Mariana; Paskel, Myeisha; Matsuoka, Yumiko; Zengel, James; Cheng, Xing; Treanor, John J; Jin, Hong; Subbarao, Kanta

    2015-02-01

    Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of respiratory disease in horses. Although natural infections of humans with EIV have not been reported, experimental inoculation of humans with these viruses can lead to a productive infection and elicit a neutralizing antibody response. Moreover, EIV have crossed the species barrier to infect dogs, pigs, and camels and therefore may also pose a threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different lineages and sublineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a live attenuated candidate vaccine by reverse genetics with the hemagglutinin and neuraminidase genes of the eq/GA/81 wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 (H2N2) vaccine donor virus, which is the backbone of the licensed seasonal live attenuated influenza vaccine. In both mice and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus induced neutralizing antibodies and conferred complete protection from homologous wt virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also induced neutralizing antibodies and conferred complete protection in mice and nearly complete protection in ferrets upon heterologous challenge with the H3N8 (eq/Newmarket/03) wt virus. These data support further evaluation of the eq/GA/81 ca vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus to humans. Equine influenza viruses have crossed the species barrier to infect other mammals such as dogs, pigs, and camels and therefore may also pose a threat to humans. We believe that it is important to develop vaccines against equine influenza viruses in the event that an EIV evolves, adapts, and spreads in humans, causing disease. We generated a live attenuated H3N8 vaccine candidate and demonstrated that the vaccine was immunogenic and protected mice and

  13. A Live Attenuated Equine H3N8 Influenza Vaccine Is Highly Immunogenic and Efficacious in Mice and Ferrets

    PubMed Central

    Baz, Mariana; Paskel, Myeisha; Matsuoka, Yumiko; Zengel, James; Cheng, Xing; Treanor, John J.; Jin, Hong

    2014-01-01

    ABSTRACT Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of respiratory disease in horses. Although natural infections of humans with EIV have not been reported, experimental inoculation of humans with these viruses can lead to a productive infection and elicit a neutralizing antibody response. Moreover, EIV have crossed the species barrier to infect dogs, pigs, and camels and therefore may also pose a threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different lineages and sublineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a live attenuated candidate vaccine by reverse genetics with the hemagglutinin and neuraminidase genes of the eq/GA/81 wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 (H2N2) vaccine donor virus, which is the backbone of the licensed seasonal live attenuated influenza vaccine. In both mice and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus induced neutralizing antibodies and conferred complete protection from homologous wt virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also induced neutralizing antibodies and conferred complete protection in mice and nearly complete protection in ferrets upon heterologous challenge with the H3N8 (eq/Newmarket/03) wt virus. These data support further evaluation of the eq/GA/81 ca vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus to humans. IMPORTANCE Equine influenza viruses have crossed the species barrier to infect other mammals such as dogs, pigs, and camels and therefore may also pose a threat to humans. We believe that it is important to develop vaccines against equine influenza viruses in the event that an EIV evolves, adapts, and spreads in humans, causing disease. We generated a live attenuated H3N8 vaccine candidate and demonstrated that the vaccine was immunogenic and

  14. African Green Monkeys Recapitulate the Clinical Experience with Replication of Live Attenuated Pandemic Influenza Virus Vaccine Candidates

    PubMed Central

    Matsuoka, Yumiko; Suguitan, Amorsolo; Orandle, Marlene; Paskel, Myeisha; Boonnak, Kobporn; Gardner, Donald J.; Feldmann, Friederike; Feldmann, Heinz; Marino, Michael; Jin, Hong; Kemble, George

    2014-01-01

    ABSTRACT Live attenuated cold-adapted (ca) H5N1, H7N3, H6N1, and H9N2 influenza vaccine viruses replicated in the respiratory tract of mice and ferrets, and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild-type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in phase I clinical trials, there were inconsistencies between the observations in animal models and in humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative with respect to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a nonhuman primate model. The distribution of sialic acid (SA) receptors in the respiratory tract of AGMs was similar to that in humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates. IMPORTANCE Ferrets and mice are commonly used for preclinical evaluation of influenza

  15. Lives Worth Living: Religious Education and Social Movements

    ERIC Educational Resources Information Center

    Ayres, Jennifer R.

    2013-01-01

    When people of faith participate in movements for social change, how are their religious and moral identities formed, challenged, and transformed? Although they have explicit and tangible goals as they participate in advocacy, protest, and boycotts, religious social activists also, James Jasper argues, craft "lives worth living" (1997).…

  16. The power of living things: Living memorials as therapeutic landscapes

    Treesearch

    Heather L. McMillen; Lindsay K. Campbell; Erika S. Svendsen

    2017-01-01

    In response to the events of 11 September 2001 (9/11), many communities came together to create living memorials. Many living memorials were established near the crash sites, but others were created across the United States from urban to rural areas, with designs ranging from entire forests to single trees. They were created by surviving family members, supporters of...

  17. Lives Worth Living: Religious Education and Social Movements

    ERIC Educational Resources Information Center

    Ayres, Jennifer R.

    2013-01-01

    When people of faith participate in movements for social change, how are their religious and moral identities formed, challenged, and transformed? Although they have explicit and tangible goals as they participate in advocacy, protest, and boycotts, religious social activists also, James Jasper argues, craft "lives worth living" (1997).…

  18. Probiotics: "living drugs".

    PubMed

    Elmer, G W

    2001-06-15

    The uses, mechanisms of action, and safety of probiotics are discussed. Probiotics are live microorganisms or microbial mixtures administered to improve the patient's microbial balance, particularly the environment of the gastrointestinal tract and the vagina. The yeast Saccharomyces boulardii and the bacterium Lactobacillus rhamnosus, strain GG, have shown efficacy in clinical trials for the prevention of antimicrobial-associated diarrhea. Other probiotics that have demonstrated at least some promise as prophylaxis for this type of diarrhea are Lactobacillus acidophilus, Bifidobacterium longum, and Enterococcus faecium. The use of S. boulardii as an adjunctive treatment to therapy with metronidazole or vancomycin has been found in controlled studies to decrease further recurrences of Clostridium difficile-associated disease. Other gastrointestinal disorders for which probiotics have been studied include traveler's diarrhea, acute infantile diarrhea, and acute diarrhea in adults. Several Lactobacillus species given in yogurt or in tablet or suppository form have shown clinical efficacy as a treatment for vaginal infections. Lactobacillus strains have also been examined as a treatment for urinary-tract infections. Putative mechanisms of action of probiotics include production of pathogen-inhibitory substances, inhibition of pathogen attachment, inhibition of the action of microbial toxins, stimulation of immunoglobulin A, and trophic effects on intestinal mucosa. The available probiotics are considered nonpathogenic, but even benign microorganisms can be infective when a patient is severely debilitated or immunosuppressed. Probiotics have demonstrated an ability to prevent and treat some infections. Effective use of probiotics could decrease patients' exposure to antimicrobials. Additional controlled studies are needed to clearly define the safety and efficacy of these agents.

  19. Viruses as living processes.

    PubMed

    Dupré, John; Guttinger, Stephan

    2016-10-01

    The view that life is composed of distinct entities with well-defined boundaries has been undermined in recent years by the realisation of the near omnipresence of symbiosis. What had seemed to be intrinsically stable entities have turned out to be systems stabilised only by the interactions between a complex set of underlying processes (Dupré, 2012). This has not only presented severe problems for our traditional understanding of biological individuality but has also led some to claim that we need to switch to a process ontology to be able adequately to understand biological systems. A large group of biological entities, however, has been excluded from these discussions, namely viruses. Viruses are usually portrayed as stable and distinct individuals that do not fit the more integrated and collaborative picture of nature implied by symbiosis. In this paper we will contest this view. We will first discuss recent findings in virology that show that viruses can be 'nice' and collaborate with their hosts, meaning that they form part of integrated biological systems and processes. We further offer various reasons why viruses should be seen as processes rather than things, or substances. Based on these two claims we will argue that, far from serving as a counterexample to it, viruses actually enable a deeper understanding of the fundamentally interconnected and collaborative nature of nature. We conclude with some reflections on the debate as to whether viruses should be seen as living, and argue that there are good reasons for an affirmative answer to this question. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. The living publication

    SciTech Connect

    Terwilliger, Thomas C.

    2012-06-04

    Within the ICSTI Insights Series we offer three articles on the 'living publication' that is already available to practitioners in the important field of crystal structure determination and analysis. While the specific examples are drawn from this particular field, we invite readers to draw parallels in their own fields of interest. The first article describes the present state of the crystallographic living publication, already recognized by an ALPSP (Association of Learned and Professional Society Publishers) Award for Publishing Innovation in 2006. The second article describes the potential impact on the record of science as greater post-publication analysis becomes more common within currently accepted data deposition practices, using processed diffraction data as the starting point. The third article outlines a vision for the further improvement of crystallographic structure reports within potentially achievable enhanced data deposition practices, based upon raw (unprocessed) diffraction data. The IUCr in its Commissions and Journals has for many years emphasized the importance of publications being accompanied by data and the interpretation of the data in terms of atomic models. This has been followed as policy by numerous other journals in the field and its cognate disciplines. This practice has been well served by databases and archiving institutions such as the Protein Data Bank (PDB), the Cambridge Crystallographic Data Centre (CCDC), and the Inorganic Crystal Structure Database (ICSD). Normally the models that are archived are interpretations of the data, consisting of atomic coordinates with their displacement parameters, along with processed diffraction data from X-ray, neutron or electron diffraction studies. In our current online age, a reader can not only consult the printed word, but can display and explore the results with molecular graphics software of exceptional quality. Furthermore, the routine availability of processed diffraction data allows

  1. Master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortants used in live attenuated influenza vaccine (LAIV) do not display neurovirulent properties in a mouse model.

    PubMed

    Voeten, J Theo M; Kiseleva, Irina V; Glansbeek, Harrie L; Basten, Stephanie M C; Drieszen-van der Cruijsen, Sandra K M; Rudenko, Larisa G; van den Bosch, Han; Heldens, Jacco G M

    2010-09-01

    Demonstration of the absence of neurovirulent properties of reassortant viruses contained in live attenuated influenza vaccine (LAIV) is a regulatory requirement. A mouse model was used to detect neurovirulent properties of the cold-adapted, temperature-sensitive and attenuated influenza master donor viruses (MDVs) A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortant influenza viruses. A/NWS/33 (H1N1), which is known to be neurovirulent in mice, was used as a positive control. Under conditions where the positive control virus induced symptoms of disease and showed viral replication in the upper respiratory tract as well as in the brain, replication of the influenza master donor viruses and reassortant influenza A and B viruses was limited to the upper respiratory tract where they were administered. None of the mice inoculated with MDVs or reassortant influenza viruses suffered from disease, and no virus or viral replication was observed in the brains of these mice. The results demonstrate the absence of neurovirulent properties of the MDVs and reassortant influenza viruses derived therefrom used in LAIV.

  2. Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2).

    PubMed

    Isakova-Sivak, Irina; Chen, Li-Mei; Matsuoka, Yumiko; Voeten, J Theo M; Kiseleva, Irina; Heldens, Jacco G M; den Bosch, Han van; Klimov, Alexander; Rudenko, Larisa; Cox, Nancy J; Donis, Ruben O

    2011-04-10

    Trivalent live attenuated influenza vaccines whose type A components are based on cold-adapted A/Leningrad/134/17/57 (H2N2) (caLen17) master donor virus (MDV) have been successfully used in Russia for decades to control influenza. The vaccine virus comprises hemagglutinin and neuraminidase genes from the circulating viruses and the remaining six genes from the MDV. The latter confer temperature-sensitive (ts) and attenuated (att) phenotypes. The ts phenotype of the vaccine virus is a critical biological determinant of attenuation of virulence. We developed a plasmid-based reverse genetics system for MDV caLen17 to study the genetic basis of its ts phenotype. Mutations in the polymerase proteins PB1 and PB2 played a crucial role in the ts phenotype of MDV caLen17. In addition, we show that caLen17-specific ts mutations could impart the ts phenotype to the divergent PR8 virus, suggesting the feasibility of transferring the ts phenotype to new viruses of interest for vaccine development. Published by Elsevier Inc.

  3. Community Living Skills: Nutrition I.

    ERIC Educational Resources Information Center

    Kreps, Alice Roelofs; Dreith, Rita Vallero

    One of twenty course guides in the Community Living Skills Guide for the College for Living series, this document provides guidelines and workbook activities for the course, Nutrition I. The series of courses for developmentally disabled adults is intended to supplement residential programs and to aid in orienting institutionalized persons to…

  4. Framework for Healthful Living Education.

    ERIC Educational Resources Information Center

    North Carolina State Dept. of Public Instruction, Raleigh.

    The Healthful Living Education program promotes behaviors that contribute to a healthful lifestyle and improved quality of life for all students. The Framework for Healthy Living Education supports and reinforces the goals and objectives of its three major components: health education, physical education, and alcohol and other drugs. When the…

  5. Connecting with assisted living consumers.

    PubMed

    Cameron, Kathleen A; Pinkowitz, Jackie

    2009-01-01

    Connecting with residents and their family members should be considered an integral part of medication therapy management services that pharmacists provide to assisted living communities. This article provides suggestions on how pharmacists can better connect and communicate with current and future assisted-living consumers and staff to optimize medication use, maintain resident function, and help residents age in place.

  6. SOLO: Self Organizing Live Objects

    DTIC Science & Technology

    2008-12-01

    mance needs can vary in use-dependent ways. For example in a content sharing system, the lag-tolerance for a live - streaming use may depend on the kind...USA, Octo- ber 2005. [15] Qi Huang, Hai Jin, and Xiaofei Liao, “P2P Live Streaming with Tree-Mesh based Hybrid Overlay”, In Pro- ceedings of ICPP

  7. Community Living Skills: Nutrition I.

    ERIC Educational Resources Information Center

    Kreps, Alice Roelofs; Dreith, Rita Vallero

    One of twenty course guides in the Community Living Skills Guide for the College for Living series, this document provides guidelines and workbook activities for the course, Nutrition I. The series of courses for developmentally disabled adults is intended to supplement residential programs and to aid in orienting institutionalized persons to…

  8. Community Living Skills Guide: Sexuality.

    ERIC Educational Resources Information Center

    Breen, Kathy

    One of twenty course guides in the Community Living Skills Guide for the College for Living series, this document provides guidelines and workbook activities for the course, Sexuality. The series of courses for developmentally disabled adults is intended to supplement residential programs and to aid in orienting institutionalized persons to…

  9. Provider lived experience and stigma.

    PubMed

    Harris, J Irene; Leskela, Jennie; Hoffman-Konn, Lisa

    2016-01-01

    Despite professional values about advocacy for people managing mental health challenges, research on mental health providers indicates that this group expresses as much or more stigma than laypeople. This article reports on a continuing education needs assessment of 101 mental health providers, including evaluation of (a) knowledge about recovery-oriented care, (b) work engagement, (c) provider lived experience with mental health challenges, and (d) stigma, measured as disidentification. In this group of providers, recovery knowledge, lived experience, and work engagement were associated with less stigma toward clients. Recovery knowledge and work engagement were associated with less stigma toward other providers with lived experience, but having lived experience was not associated with stigma toward other providers with lived experience. Findings suggest that the professional culture of nondisclosure may be a factor that increases provider stigma and should be a topic for further research. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  10. Where Plumes Live

    NASA Astrophysics Data System (ADS)

    King, S. D.

    2004-12-01

    From the perspective of fluid dynamics, `Plumes or not?' might be the wrong question. Let me begin by defining a few terms. Plume with a `P' is the well-known thermal structure with thin (order 100 km) tail and large, bulbous head that originates at the core-mantle boundary. The thin tail/large, bulbous-head morphology has been generated in a number of laboratory and numerical experiments. It can be seen, for example, on the cover of the famous fluid dynamics text by Batchelor. There is a clearly-defined range of parameters for which this structure is the preferred solution for instabilities arising from a bottom boundary layer in a convecting fluid. For example, a strong temperature-dependent rheology is needed. By contrast, plume with a `p' is any cylindrical or quasi-cylindrical instability originating from a thermal (or thermo-chemical) boundary layer. In fluid dynamics plume is sometimes used interchangeable with jet. Unless there is a very small temperature drop across the core-mantle boundary or a rather remarkable balance between temperature and composition at the base of the mantle, there are almost certainly plumes. (Note the little p.) Are these plumes the thermal structures with thin (order 100 km) tails and large bulbous heads or could they be broad, hot regions such as the degree 2 pattern seen in global seismic tomography images of the lower mantle, or the disconnected droplets seen in chaotic convection? To study this question, I will present a sequence of numerical `experiments' that illustrate the morphology of instabilities from a basal thermal boundary layer, i.e., plumes. Some of the aspects I will present include: spherical geometry, temperature-and pressure-dependence of rheology, internal heating, pressure-dependent coefficient of thermal expansion, variable coefficient of thermal diffusivity, phase transformations, and compositional layering at the base of the mantle. The goal is to map out the parameters and conditions where Plumes live

  11. Molecular dynamics of the salt dependence of a cold-adapted enzyme: endonuclease I.

    PubMed

    Benrezkallah, D; Dauchez, M; Krallafa, A M

    2015-01-01

    The effects of salt on the stability of globular proteins have been known for a long time. In the present investigations, we shall focus on the effect of the salt ions upon the structure and the activity of the endonuclease I enzyme. In the present work, we shall focus on the relationship between ion position and the structural features of the Vibrio salmonicida (VsEndA) enzyme. We will concentrate on major questions such as: how can salt ions affect the molecular structure? What is the activity of the enzyme and which specific regions are directly involved? For that purpose, we will study the behaviour of the VsEndA over different salt concentrations using molecular dynamics (MD) simulations. We report the results of MD simulations of the endonuclease I enzyme at five different salt concentrations. Analysis of trajectories in terms of the root mean square fluctuation (RMSF), radial distribution function, contact numbers and hydrogen bonding lifetimes, indicate distinct differences when changing the concentration of NaCl. Results are found to be in good agreement with experimental data, where we have noted an optimum salt concentration for activity equal to 425 mM. Under this salt concentration, the VsEndA exhibits two more flexible loop regions, compared to the other salt concentrations. When analysing the RMSF of these two specific regions, three residues were selected for their higher mobility. We find a correlation between the structural properties studied here such as the radial distribution function, the contact numbers and the hydrogen bonding lifetimes, and the structural flexibility of only two polar residues. Finally, in the light of the present work, the molecular basis of the salt adaptation of VsEndA enzyme has been explored by mean of explicit solvent and salt treatment. Our results reveal that modulation of the sodium/chloride ions interaction with some specific loop regions of the protein is the strategy followed by this type of psychrophilic enzyme to enhance catalytic activity at the physiological conditions.

  12. Rheological changes in mammalian egg cytoskeleton in response to cold adaptation

    NASA Astrophysics Data System (ADS)

    Prulière, Gérard; Nguyen, Eric; Nguyen Bui Xuan, Renard, Jean-Paul

    1991-05-01

    The ability of mammalian eggs to support freezing is shown to be related to a reversible reorganization of their cytoskeleton induced by organic compounds used as protectants. 1,2 propanediol which is very effective for the successful freezing of early embryonic stages induces the formation of a fairly homogeneous gel within the cytoplasm in which the cortical layer of highly entangled polymers is no longer apparent. In vitro, propanediol influences specifically interactions between actin and binding proteins and induces the formation of microporous and homogeneous gels able to retain a significant fraction of water within their pores. When eggs are first submitted to a partial dehydration at room temperature they become able to support rapid freezing and thawing in the presence of propanediol probably because the remaining cellular water becomes osmotically inactive. This indicates that the rheological changes of the cytoplasm that occur in the presence of propanediol make the cell able to support the physical and chemical modifications that affect their microenvironment at low temperature.

  13. Cloning and Characterization of Cold-Adapted α-Amylase from Antarctic Arthrobacter agilis.

    PubMed

    Kim, Su-Mi; Park, Hyun; Choi, Jong-Il

    2017-03-01

    In this study, the gene encoding an α-amylase from a psychrophilic Arthrobacter agilis PAMC 27388 strain was cloned into a pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3). The recombinant α-amylase with a molecular mass of about 80 kDa was purified by using Ni(2+)-NTA affinity chromatography. This recombinant α-amylase exhibited optimal activity at pH 3.0 and 30 °C and was highly stable at varying temperatures (30-60 °C) and within the pH range of 4.0-8.0. Furthermore, α-amylase activity was enhanced in the presence of FeCl3 (1 mM) and β-mercaptoethanol (5 mM), while CoCl2 (1 mM), ammonium persulfate (5 mM), SDS (10 %), Triton X-100 (10 %), and urea (1 %) inhibited the enzymatic activity. Importantly, the presence of Ca(2+) ions and phenylmethylsulfonyl fluoride (PMSF) did not affect enzymatic activity. Thin layer chromatography (TLC) analysis showed that recombinant A. agilis α-amylase hydrolyzed starch, maltotetraose, and maltotriose, producing maltose as the major end product. These results make recombinant A. agilis α-amylase an attractive potential candidate for industrial applications in the textile, paper, detergent, and pharmaceutical industries.

  14. The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation

    SciTech Connect

    Blanc, Guillaume; Agarkova, Irina; Grimwood, Jane; Kuo, Alan; Brueggeman, Andrew; Dunigan, David D.; Gurnon, James; Ladunga, Istvan; Lindquist, Erika; Lucas, Susan; Pangilinan, Jasmyn; Proschold, Thomas; Salamov, Asaf; Schmutz, Jeremy; Weeks, Donald; Tamada, Takashi; Lomsadze, Alexandre; Borodovsky, Mark; Claverie, Jean-Michel; Grigoriev, Igor V.; Van Etten, James L.

    2012-02-13

    Background Little is known about the mechanisms of adaptation of life to the extreme environmental conditions encountered in polar regions. Here we present the genome sequence of a unicellular green alga from the division chlorophyta, Coccomyxa subellipsoidea C-169, which we will hereafter refer to as C-169. This is the first eukaryotic microorganism from a polar environment to have its genome sequenced. Results The 48.8 Mb genome contained in 20 chromosomes exhibits significant synteny conservation with the chromosomes of its relatives Chlorella variabilis and Chlamydomonas reinhardtii. The order of the genes is highly reshuffled within synteny blocks, suggesting that intra-chromosomal rearrangements were more prevalent than inter-chromosomal rearrangements. Remarkably, Zepp retrotransposons occur in clusters of nested elements with strictly one cluster per chromosome probably residing at the centromere. Several protein families overrepresented in C. subellipsoidae include proteins involved in lipid metabolism, transporters, cellulose synthases and short alcohol dehydrogenases. Conversely, C-169 lacks proteins that exist in all other sequenced chlorophytes, including components of the glycosyl phosphatidyl inositol anchoring system, pyruvate phosphate dikinase and the photosystem 1 reaction center subunit N (PsaN). Conclusions We suggest that some of these gene losses and gains could have contributed to adaptation to low temperatures. Comparison of these genomic features with the adaptive strategies of psychrophilic microbes suggests that prokaryotes and eukaryotes followed comparable evolutionary routes to adapt to cold environments.

  15. Cold adaptive thermogenesis following consumption of certain pungent spice principles: A validation study.

    PubMed

    Pandit, Chaitanya; Anilakumar, K R

    2017-02-01

    Identifying a means to activate or potentiate thermogenic mechanisms through ingestion of dietary compounds have important implications in cold endurance and survival. Although many reports discuss the thermogenic potential of spices, it is surprising that none of the studies verify whether consumption of spices can improve cold endurance. In this study, we have attempted to evaluate if ingestion of certain spices can activate heat-generating mechanisms in the body such that a fall in. core body temperature (CBT) can be delayed or prevented when faced with a cold challenge. Ten commonly used spices in the Indian cuisine were chosen and 70% ethanol extract of the spices were fed orally to male Wistar rats at a dose of 250mg/kg for a period of 7 days. A change in CBT during cold exposure was recorded before and after treatment. At the end of the experiment, plasma norepinephrine and serum free fatty acid levels were estimated. We observed that among the ten spices, treatment with cinnamon and pepper extracts showed significant improvement in comparison to the control group. Based on evidence in literature and the HPLC-MS analysis from our lab, we hypothesized that the effects of the pepper and cinnamon extracts might be due to their piperine and cinnamaldehyde content respectively. However, no improved endurance was observed when they were administered alone. Poor endurance following depletion of endogenous norepinephrine levels using reserpine indicated its involvement in mediating the heat generating processes. However, it is noteworthy that green tea and spice treated animals exhibited a fall in CBT which was lower than their initial fall. In conclusion, our findings provide experimental evidence that ingestion of spices, viz., pepper and cinnamon, might elicit thermogenic responses such that hypothermia can be delayed or prevented upon cold exposure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Protein surface softness is the origin of enzyme cold-adaptation of trypsin.

    PubMed

    Isaksen, Geir Villy; Åqvist, Johan; Brandsdal, Bjørn Olav

    2014-08-01

    Life has effectively colonized most of our planet and extremophilic organisms require specialized enzymes to survive under harsh conditions. Cold-loving organisms (psychrophiles) express heat-labile enzymes that possess a high specific activity and catalytic efficiency at low temperatures. A remarkable universal characteristic of cold-active enzymes is that they show a reduction both in activation enthalpy and entropy, compared to mesophilic orthologs, which makes their reaction rates less sensitive to falling temperature. Despite significant efforts since the early 1970s, the important question of the origin of this effect still largely remains unanswered. Here we use cold- and warm-active trypsins as model systems to investigate the temperature dependence of the reaction rates with extensive molecular dynamics free energy simulations. The calculations quantitatively reproduce the catalytic rates of the two enzymes and further yield high-precision Arrhenius plots, which show the characteristic trends in activation enthalpy and entropy. Detailed structural analysis indicates that the relationship between these parameters and the 3D structure is reflected by significantly different internal protein energy changes during the reaction. The origin of this effect is not localized to the active site, but is found in the outer regions of the protein, where the cold-active enzyme has a higher degree of softness. Several structural mechanisms for softening the protein surface are identified, together with key mutations responsible for this effect. Our simulations further show that single point-mutations can significantly affect the thermodynamic activation parameters, indicating how these can be optimized by evolution.

  17. Dynamics of some parameters of the endocrine and lymphatic systems in rats during cold adaptation

    SciTech Connect

    Borodin, Yu.I.; Sedova, L.A.; Selyatitskaya, V.G.; Shorin, Yu.P.

    1986-02-01

    This paper examines the combined behavior of the endocrine and lymphatic systems in rats at stages of long-term adaptation of the animals to moderate cold. After decapitation of male Wister rats, the corticosterone concentration in the blood plasma was determined by saturation analysis and serum levels of thyroxine (T/sub 4/) and triiodothyronine (T/sub 3/) were determined by radioimmunoassay. The thymus was weighed and the structure of the popliteal lymph nodes (LN) was studied in histological sections stained with hematoxylin and eosin and with azure II-eosin. Morphometry of the structural components of LN was undertaken and the numbers of the various cell forms per 1000 cells were counted in different zones of LN. The increase in activity of the lymphoid tissue in the phase of adaptation may be connected with intensification of the peripheral action of thyroid hormones. During long-term adaptation, in the phase of consistently increased specific resistance, a new type of endocrine-lymphoid relation is formed, and it differs significantly both in the original state and in the acute phase of stress.

  18. Legionella species diversity and dynamics from surface reservoir to tap water: from cold adaptation to thermophily

    PubMed Central

    Lesnik, René; Brettar, Ingrid; Höfle, Manfred G

    2016-01-01

    Water samples of the Drinking Water Supply System (DWSS) of the city of Braunschweig were analysed for its Legionella species composition using genus-specific PCR amplicons and single-strand conformation polymorphism (SSCP) fingerprint analyses based on 16S rRNA genes. These analyses comprised the whole supply chain including raw water, treatment process and large-scale storage, and a seasonal study of finished drinking water sampled monthly from cold and hot tap water. Treatment of raw water had a major impact on Legionella species by reducing their diversity and abundances. The Legionella species composition of the tap water was highly distinct from that of both source waters. In cold water, 8–14 different phylotypes of Legionella (PTLs) were observed per sample with relative abundances ranging from >1% to 53%. In hot water, L. pneumophila was present during all seasons at high relative abundances (8–40%) accompanied by 5–14 other PTLs of which 6 PTLs were in common with cold water. This thermophilic Legionella community, including L. pneumophila, was able to grow in the hot water above 50 °C. Such thermophilic Legionella populations are of general relevance for drinking water management and public health, but also for the ecology and evolution of the genus Legionella. PMID:26528838

  19. Bioprospection of cold-adapted yeasts with biotechnological potential from Antarctica.

    PubMed

    Martorell, María Martha; Ruberto, Lucas Adolfo Mauro; Fernández, Pablo Marcelo; Castellanos de Figueroa, Lucía Inés; Mac Cormack, Walter Patricio

    2017-06-01

    The aim of this study was to investigate the ability to produce extracellular hydrolytic enzymes at low temperature of yeasts isolated from 25 de Mayo island, Antarctica, and to identify those exhibiting one or more of the evaluated enzymatic activities. A total of 105 yeast isolates were obtained from different samples and 66 were identified. They belonged to 12 basidiomycetous and four ascomycetous genera. Most of the isolates were ascribed to the genera Cryptococcus, Mrakia, Cystobasidium, Rhodotorula, Gueomyces, Phenoliferia, Leucosporidium, and Pichia. Results from enzymes production at low temperatures revealed that the Antarctic environment contains metabolically diverse cultivable yeasts, which represent potential tools for biotechnological applications. While most the isolates proved to produce 2-4 of the investigated exoenzymes, two of them evidenced the six evaluated enzymatic activities: Pichia caribbica and Guehomyces pullulans, which were characterized as psycrotolerant and psycrophilic, respectively. In addition, P. caribbica could assimilate several n-alkanes and diesel fuel. The enzyme production profile and hydrocarbons assimilation capacity, combined with its high level of biomass production and the extended exponential growth phase make P. caribbica a promising tool for cold environments biotechnological purposes in the field of cold-enzymes production and oil spills bioremediation as well. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Parallel molecular routes to cold adaptation in eight genera of New Zealand stick insects

    PubMed Central

    Dennis, Alice B.; Dunning, Luke T.; Sinclair, Brent J.; Buckley, Thomas R.

    2015-01-01

    The acquisition of physiological strategies to tolerate novel thermal conditions allows organisms to exploit new environments. As a result, thermal tolerance is a key determinant of the global distribution of biodiversity, yet the constraints on its evolution are not well understood. Here we investigate parallel evolution of cold tolerance in New Zealand stick insects, an endemic radiation containing three montane-occurring species. Using a phylogeny constructed from 274 orthologous genes, we show that stick insects have independently colonized montane environments at least twice. We compare supercooling point and survival of internal ice formation among ten species from eight genera, and identify both freeze tolerance and freeze avoidance in separate montane lineages. Freeze tolerance is also verified in both lowland and montane populations of a single, geographically widespread, species. Transcriptome sequencing following cold shock identifies a set of structural cuticular genes that are both differentially regulated and under positive sequence selection in each species. However, while cuticular proteins in general are associated with cold shock across the phylogeny, the specific genes at play differ among species. Thus, while processes related to cuticular structure are consistently associated with adaptation for cold, this may not be the consequence of shared ancestral genetic constraints. PMID:26355841

  1. Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli.

    PubMed

    Niiranen, Laila; Espelid, Sigrun; Karlsen, Christian R; Mustonen, Milla; Paulsen, Steinar M; Heikinheimo, Pirkko; Willassen, Nils P

    2007-03-01

    Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.

  2. The hemoglobins of the cold-adapted Antarctic teleost Cygnodraco mawsoni.

    PubMed

    Caruso, C; Rutigliano, B; Romano, M; di Prisco, G

    1991-06-24

    The blood of the teleost Cygnodraco mawsoni, of the endemic Antarctic family Bathydraconidae, contains a major hemoglobin (Hb 1), accompanied by a minor component (Hb 2, about 5% of total). The two hemoglobins have identical alpha chains and differ by the beta chain. The complete amino acid sequence of the three chains has been elucidated, thus establishing the primary structure of both hemoglobins. The sequences show a 53-65% identity with non-Antarctic poikilotherm fish species; on the other hand, a very high degree of similarity (83-88%) has been found between Hb 1 and the major component of another Antarctic species of a different family. The hemoglobin functional properties relative to oxygen binding have been investigated in intact erythrocytes, 'stripped' hemolysate and purified components of C. mawsoni. The hemoglobins display the Bohr and Root effects, indicating fine regulation of oxygen binding by pH and by the physiological effectors organic phosphates.

  3. Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

    PubMed

    Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

    2016-01-01

    Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

  4. A novel cold-adapted lipase, LP28, from a mesophilic Streptomyces strain.

    PubMed

    Simkhada, Jaya Ram; Yoo, Hah Young; Cho, Seung Sik; Choi, Yun Hee; Kim, Seung Wook; Park, Don Hee; Yoo, Jin Cheol

    2012-01-01

    Fossil fuel is limited but its usage has been growing rapidly, thus the fuel is predicted to be completely running out and causing an unbearable global energy crisis in the near future. To solve this potential crisis, incorporating with increasing environmental concerns, significant attentions have been given to biofuel production in the recent years. With the aim of isolating a microbial biocatalyst with potential application in the field of biofuel, a lipase from Streptomyces sp. CS628, LP28, was purified using hydroxyapatite column chromatography followed by a gel filtration. Molecular weight of LP28 was estimated to be 32,400 Da by SDS-PAGE. The activity was the highest at 30 °C and pH 8.0 and was stable at pH 6.0-8.0 and below 25 °C. The enzyme preferentially hydrolyzed p-nitrophenyl decanoate (C10), a medium chain substrate. Furthermore, LP28 non-specifically hydrolyzed triolein releasing both 1,2- and 1,3-diolein. More importantly, LP28 manifestly catalyzed biodiesel production using palm oil and methanol; therefore, it can be a potential candidate in the field of biofuel.

  5. Extreme preconditioning: cold adaptation through sea swimming as a means to improving surgical outcomes.

    PubMed

    Harper, C Mark

    2012-04-01

    The practice of sea bathing for its health benefits was popularised by Richard Russell in Regency Brighton during the 18th Century. Although the cures he claimed it could effect seem a little far-fetched today, as with many historical remedies, there is much to be gained from revisiting such theories in the light of modern medical research. In this paper I will draw parallels between the surgical stress response and the response to cold exposure and hypothesise how a programme of sea bathing may be used to enhance postoperative recovery and reduce preoperative complications.

  6. Uncovering Mechanisms for Repair and Protection in Cold Environments Through Studies of Cold Adapted Archaea

    DTIC Science & Technology

    2009-12-18

    a role in M. burtonii. Glycine betaine solute-binding protein (Mbur_0503): Given the ability of M. burtonii to grow is dependent upon the...stability of enzymes at low temperatures, this may be facilitated by the intracellular accumulation of osmolytes such as glycine betaine , which serve as

  7. The skin on the move but cold adapted: Fundamental misconceptions in the laboratory and clinic

    PubMed Central

    Ryan, Terence J.

    2010-01-01

    The skin is constantly on the move and at a temperature below 37ºC. The epidermis is a factory, and its blood supply and lymphatic drainage, as well as adipose tissue, are much dependent on movement and influenced by cooling. Neither histopathology (still pictures) nor in vitro studies at 37ºC reflect the true picture. Recent publications neglect older literature exploring these issues. PMID:23130182

  8. Biodegradation of methyl tert-butyl ether by cold-adapted mixed and pure bacterial cultures.

    PubMed

    Zaitsev, G M; Uotila, J S; Häggblom, M M

    2007-04-01

    An aerobic mixed bacterial culture (CL-EMC-1) capable of utilizing methyl tert-butyl ether (MTBE) as the sole source of carbon and energy with a growth temperature range of 3 to 30 degrees C and optimum of 18 to 22 degrees C was enriched from activated sludge. Transient accumulation of tert-butanol (TBA) occurred during utilization of MTBE at temperatures from 3 degrees C to 14 degrees C, but TBA did not accumulate above 18 degrees C. The culture utilized MTBE at a concentration of up to 1.5 g l(-1) and TBA of up to 7 g l(-1). The culture grew on MTBE at a pH range of 5 to 9, with an optimum pH of 6.5 to 7.1. The specific growth rate of the CL-EMC-1 culture on 0.1 g l(-1) of MTBE at 22 degrees C and pH 7.1 was 0.012 h(-1), and the growth yield was 0.64 g (dry weight) g(-1). A new MTBE-utilizing bacterium, Variovorax paradoxus strain CL-8, isolated from the mixed culture utilized MTBE, TBA, 2-hydroxy isobutyrate, lactate, methacrylate, and acetate as sole sources of carbon and energy but not 2-propanol, acetone, methanol, formaldehyde, or formate. Two other isolates, Hyphomicrobium facilis strain CL-2 and Methylobacterium extorquens strain CL-4, isolated from the mixed culture were able to grow on C(1) compounds. The combined consortium could thus utilize all of the carbon of MTBE.

  9. Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes

    PubMed Central

    Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

    2016-01-01

    ABSTRACT Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications. PMID:26710170

  10. Legionella species diversity and dynamics from surface reservoir to tap water: from cold adaptation to thermophily.

    PubMed

    Lesnik, René; Brettar, Ingrid; Höfle, Manfred G

    2016-05-01

    Water samples of the Drinking Water Supply System (DWSS) of the city of Braunschweig were analysed for its Legionella species composition using genus-specific PCR amplicons and single-strand conformation polymorphism (SSCP) fingerprint analyses based on 16S rRNA genes. These analyses comprised the whole supply chain including raw water, treatment process and large-scale storage, and a seasonal study of finished drinking water sampled monthly from cold and hot tap water. Treatment of raw water had a major impact on Legionella species by reducing their diversity and abundances. The Legionella species composition of the tap water was highly distinct from that of both source waters. In cold water, 8-14 different phylotypes of Legionella (PTLs) were observed per sample with relative abundances ranging from >1% to 53%. In hot water, L. pneumophila was present during all seasons at high relative abundances (8-40%) accompanied by 5-14 other PTLs of which 6 PTLs were in common with cold water. This thermophilic Legionella community, including L. pneumophila, was able to grow in the hot water above 50 °C. Such thermophilic Legionella populations are of general relevance for drinking water management and public health, but also for the ecology and evolution of the genus Legionella.

  11. A Live Attenuated H7N3 Influenza Virus Vaccine is Well-tolerated and Immunogenic in a Phase I Trial in Healthy Adults

    PubMed Central

    Talaat, Kawsar R.; Karron, Ruth A.; Callahan, Karen A.; Luke, Catherine J.; DiLorenzo, Susan C.; Chen, Grace L.; Lamirande, Elaine W.; Jin, Hong; Coelingh, Kathy L.; Murphy, Brian R.; Kemble, George; Subbarao, Kanta

    2009-01-01

    Background Live attenuated influenza vaccines (LAIV) are being developed and tested against a variety of influenza viruses with pandemic potential. We describe the results of an open label Phase I trial of a live attenuated H7N3 virus vaccine. Methods and Findings The H7N3 BC 2004/AA ca virus is a live attenuated, cold-adapted, temperature-sensitive influenza virus derived by reverse genetics from the wild-type low pathogenicity avian influenza virus A/chicken/British Columbia/CN-6/2004 (H7N3) and the A/AA/6/60 ca (H2N2) virus that is the Master Donor Virus of the live, intranasal seasonal influenza vaccine. We evaluated the safety, infectivity, and immunogenicity of two doses of 107.5 TCID50 of the vaccine administered by nasal spray 5 weeks apart to normal healthy seronegative adult volunteers in an inpatient isolation unit. The subjects were followed for 2 months after 1 dose of vaccine or for 4 weeks after the second dose. Twenty-one subjects received the first dose of the vaccine, and 17 subjects received two doses. The vaccine was generally well tolerated. No serious adverse events occurred during the trial. The vaccine was highly restricted in replication: 6 (29%) subjects had virus recoverable by culture or by rRT-PCR after the first dose. Replication of vaccine virus was not detected following the second dose. Despite the restricted replication of the vaccine, 90% of the subjects developed an antibody response as measured by any assay: 62% by hemagglutination inhibition assay, 48% by microneutralization assay, 48% by ELISA for H7 HA-specific serum IgG or 71% by ELISA for H7 HA-specific serum IgA, after either one or two doses. Following the first dose, vaccine-specific IgG secreting cells as measured by ELISPOT increased from a mean of 0.1 to 41.6/106 PBMCs; vaccine specific IgA secreting cells increased from 2 to 16.4/106 PBMCs. The antibody secreting cell response after the second dose was less vigorous, which is consistent with the observed low

  12. The Living Expert System (LEXSYS)

    DTIC Science & Technology

    1988-05-12

    LEXSYS Living ~ ARMY SENIORSy tn LEADER DECISION MAKING TOOL FOR U.S. ARMY WAR COLLEGE CARLISLE BARRACKS, PA 17013-5050 VOL. I DISTRIUIn= STATDIIII...for open publication until It has been cleared by the appropriate mUlitav service or government agency. THE LIVING EXPERT SYSTEM A GROUP STUDY PROJECT...LTC, AR R. A. Pomager, Jr., LTC, MP E. R. Ruff, LTC, EN J. D. Tolleson, LTC, QM TITLE: The Living Expert System FORMAT: Group Study DATE: 1 May 1988

  13. The Matrix Gene Segment Destabilizes the Acid and Thermal Stability of the Hemagglutinin of Pandemic Live Attenuated Influenza Virus Vaccines

    PubMed Central

    O'Donnell, Christopher D.; Vogel, Leatrice; Matsuoka, Yumiko; Jin, Hong

    2014-01-01

    ABSTRACT The threat of future influenza pandemics and their potential for rapid spread, morbidity, and mortality has led to the development of pandemic vaccines. We generated seven reassortant pandemic live attenuated influenza vaccines (pLAIVs) with the hemagglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the six internal protein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIV viruses were moderately to highly restricted in replication in seronegative adults; we sought to determine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human influenza viruses and, although they shared the same backbone, the pLAIV viruses had a lower shutoff temperature than seasonal LAIV viruses, suggesting that the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low-pathogenicity avian influenza viruses, as reported by others. However, pLAIV viruses had a consistently higher pH of HA activation and reduced HA thermostability compared to the corresponding wild-type parental viruses. From studies with single-gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60 M gene. Together, the impaired HA acid and thermal stability and temperature sensitivity likely contributed to the restricted replication of the pLAIV viruses we observed in seronegative adults. IMPORTANCE There is increasing evidence that the HA stability of influenza viruses depends on the virus strain and host species and that HA stability can influence replication, virulence, and transmission of influenza A viruses in different species. We

  14. Living Alone With Cognitive Impairment.

    PubMed

    Gibson, Allison K; Richardson, Virginia E

    2017-02-01

    Although most individuals experiencing cognitive impairment (CI) reside with a caregiver, an estimated 800,000 live alone. Such individuals may have an increased risk for injury to self or others through self-neglect as a result of the CI symptoms. While persons living alone with CI have been identified as an important area for needed research, few studies have been able to examine this population due to the challenges of identifying and recruiting study participants. By using the National Health & Aging Trends Study data set, the researchers explored the characteristics to describe this population. The results of this study indicated that the majority of persons living with CI were older, widowed females who were not diagnosed with Alzheimer's or dementia but tested positive on cognitive screening measures. Further, the majority of persons living alone with CI relied on adult children and paid professionals as the primary care providers.

  15. Technology for Independent Living: Sourcebook.

    ERIC Educational Resources Information Center

    Enders, Alexandra, Ed.

    This sourcebook provides information for the practical implementation of independent living technology in the everyday rehabilitation process. "Information Services and Resources" lists databases, clearinghouses, networks, research and development programs, toll-free telephone numbers, consumer protection caveats, selected publications, and…

  16. Living with Diabetic Heart Disease

    MedlinePlus

    ... from the NHLBI on Twitter. Living With Diabetic Heart Disease Diabetic heart disease (DHD) increases the likelihood of earlier and more ... also tend to have less success from certain heart disease treatments, such as coronary artery bypass grafting and ...

  17. Living with Carotid Artery Disease

    MedlinePlus

    ... from the NHLBI on Twitter. Living With Carotid Artery Disease If you have carotid artery disease, you can take steps to manage the ... treatment plan, and getting ongoing care. Having carotid artery disease raises your risk of having a stroke . ...

  18. Living with von Willebrand Disease

    MedlinePlus

    ... from the NHLBI on Twitter. Living With von Willebrand Disease If you have von Willebrand disease (VWD), you ... that they get tested too. Pregnancy and von Willebrand Disease Pregnancy can be a challenge for women who ...

  19. Advanced Melanoma Facebook Live Event

    Cancer.gov

    In case you missed it, watch this recent Facebook Live event about the current state of research and treatment for advanced stage melanoma. To learn more, see our evidence-based information about skin cancer, including melanoma.

  20. Living with Peripheral Arterial Disease

    MedlinePlus

    ... Living With Clinical Trials Links Related Topics Atherosclerosis Coronary Heart Disease Heart Attack Smoking and Your Heart Stroke Send ... D.), you’re more likely to also have coronary heart disease , heart attack , stroke , and transient ischemic attack ("mini- ...

  1. Improving the health and lives of people living in slums.

    PubMed

    Sheuya, Shaaban A

    2008-01-01

    Urban poverty, ill health, and living in slums are intrinsically interwoven. Poverty is multidimensional and there is no agreement on a universal definition. UN-HABITAT has introduced an operational definition of slums that is restricted to legal aspects and excludes the more difficult social dimensions. The World Health Organization definition is more comprehensive and uses a health and social determinants approach that is strongly based on the social conditions in which people live and work. Health and improving the lives of people living in slums is at the top of international development agenda. Proactive strategies to contain new urban populations and slum upgrading are the two key approaches. Regarding the latter, participatory upgrading that most often involves the provision of basic infrastructure is currently the most acceptable intervention in developing countries. In urbanization of poverty, participatory slum upgrading is a necessary but not sufficient condition to reduce poverty and improve the lives of slum dwellers. Empowering interventions that target capacity development and skill transfer of both individuals and community groups--as well as meaningful negotiations with institutions, such as municipal governments, which can affect slum dwellers' lives--appear to be the most promising strategies to improve the slum dwellers' asset bases and health. Non-governmental organizations, training institutions, and international development partners are best placed to facilitate horizontal relationships between individuals, community groups, and vertical relationships with more powerful institutions that affect the slum dwellers' lives. The main challenge appears to be lack of commitment from the key stakeholders to upgrade interventions citywide.

  2. [Passive euthanasia and living will].

    PubMed

    Julesz, Máté

    2014-07-06

    This article deals with the intentional distinction between murder of first degree and passive euthanasia. In Hungary, active euthanasia is considered to be a murder of first degree, whilst the Netherlands, Belgium, Luxemburg and Switzerland have legalized the active form of mercy killing in Europe. The palliative terminal care, when e.g. giving pain-killer morphine to the patient, might result in decreasing the patient's life-span, and thus causing indirect euthanasia. However, the legal institution of living will exists in several counter-euthanasia countries. The living will allows future patients to express their decision in advance to refuse a life-sustaining treatment, e.g. in case of irreversible coma. The institution of living will exists in Germany and in Hungary too. Nevertheless, the formal criteria of living will make it hardly applicable. The patient ought to express his/her will before a notary public in advance, and he/she should hand it over when being hospitalized. If the patient is not able to present his/her living will to his/her doctor in the hospital, then his/her only hope remains that he/she has given a copy of the living will to the family doctor previously, and the family doctor will notify the hospital.

  3. Heidegger, lived experience and method.

    PubMed

    Paley, John

    2014-07-01

    A discussion of the assumption that Heidegger's philosophy in Being And Time provides a warrant for the study of lived experience. It is generally assumed, in nursing as in other disciplines, that Heidegger's philosophy points, uncontroversially, to the study of lived experience. It is also assumed that studies of this type will take the form of qualitative interviews which seek to explore the respondent's experience of a particular phenomenon and to elicit the meanings which the individual concerned attaches to that experience. Being And Time; the philosophical literature on Heidegger since 1999; the literature of experimental social psychology, 1970-2012. According to Heidegger, there is no such thing as 'lived experience'. The concept is embedded in the subject-object dualism that he is attempting to dismantle. In Heideggerian terms, interviews intended to explore 'lived experience' can only reproduce the voice of das Man, the 'They', not the voice of unique individuals. Methods more in keeping with Heidegger's philosophy include observation, naturalistic experiments, some forms of discourse analysis and conceptually associated lines of enquiry involving vocabularies of motive, scripts and the performative aspects of language use. Nursing researchers who wish to embrace Heidegger's philosophy as a basis for their work should abandon 'lived experience' interviews and adopt one of the alternative methods suggested above. Nursing researchers who wish to continue with 'lived experience' interviews should seek an alternative philosophical or theoretical basis for their work. © 2013 John Wiley & Sons Ltd.

  4. Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures.

    PubMed

    Fischer, William A; Chason, Kelly D; Brighton, Missy; Jaspers, Ilona

    2014-03-26

    Influenza viruses are global pathogens that infect approximately 10-20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent

  5. Learning to Live Well with Celiac Disease

    MedlinePlus

    ... turn JavaScript on. Feature: Celiac Disease Learning to Live Well with Celiac Disease Past Issues / Spring 2015 ... Treatment / Four Inches and Seven Pounds… / Learning to Live Well with Celiac Disease / Living Gluten-Free Spring ...

  6. Living Well with Sickle Cell Disease

    MedlinePlus

    ... Information For... Media Policy Makers Living Well with Sickle Cell Disease Language: English Español (Spanish) Recommend on Facebook Tweet Share Compartir People with sickle cell disease can live full lives and enjoy most ...

  7. Video fingerprinting for live events

    NASA Astrophysics Data System (ADS)

    Celik, Mehmet; Haitsma, Jaap; Barvinko, Pavlo; Langelaar, Gerhard; Maas, Martijn

    2009-02-01

    Multimedia fingerprinting (robust hashing) as a content identification technology is emerging as an effective tool for preventing unauthorized distribution of commercial content through user generated content (UGC) sites. Research in the field has mainly considered content types with slow distribution cycles, e.g. feature films, for which reference fingerprint ingestion and database indexing can be performed offline. As a result, research focus has been on improving the robustness and search speed. Live events, such as live sports broadcasts, impose new challenges on a fingerprinting system. For instance, highlights from a soccer match are often available-and viewed-on UGC sites well before the end of the match. In this scenario, the fingerprinting system should be able to ingest and index live content online and offer continuous search capability, where new material is identifiable within minutes of broadcast. In this paper, we concentrate on algorithmic and architectural challenges we faced when developing a video fingerprinting solution for live events. In particular, we discuss how to effectively utilize fast sorting algorithms and a master-slave architecture for fast and continuous ingestion of live broadcasts.

  8. Electronic Interfacing with Living Cells

    NASA Astrophysics Data System (ADS)

    Fleming, James T.

    The direct interfacing of living cells with inorganic electronic materials, components or systems has led to the development of two broad categories of devices that can (1) transduce biochemical signals generated by biological components into electrical signals and (2) transduce electronically generated signals into biochemical signals. The first category of devices permits the monitoring of living cells, the second, enables control of cellular processes. This review will survey this exciting area with emphasis on the fundamental issues and obstacles faced by researchers. Devices and applications that use both prokaryotic (microbial) and eukaryotic (mammalian) cells will be covered. Individual devices described include microbial biofuel cells that produce electricity, bioelectrical reactors that enable electronic control of cellular metabolism, living cell biosensors for the detection of chemicals and devices that permit monitoring and control of mammalian physiology.

  9. Live Imaging of the Lung

    PubMed Central

    Looney, Mark R.; Bhattacharya, Jahar

    2015-01-01

    Live lung imaging has spanned the discovery of capillaries in the frog lung by Malpighi to the current use of single and multiphoton imaging of intravital and isolated perfused lung preparations incorporating fluorescent molecular probes and transgenic reporter mice. Along the way, much has been learned about the unique microcirculation of the lung, including immune cell migration and the mechanisms by which cells at the alveolar-capillary interface communicate with each other. In this review, we highlight live lung imaging techniques as applied to the role of mitochondria in lung immunity, mechanisms of signal transduction in lung compartments, studies on the composition of alveolar wall liquid, and neutrophil and platelet trafficking in the lung under homeostatic and inflammatory conditions. New applications of live lung imaging and the limitations of current techniques are discussed. PMID:24245941

  10. Consumer discourse in assisted living.

    PubMed

    Carder, Paula C; Hernandez, Mauro

    2004-03-01

    The aim of this article is to discuss the cultural construction of the assisted living consumer. Based on theories of consumer studies, it focuses on organizational strategies employed by assisted living practitioners to promote consumer choice and independence while mediating potential risks. Data include field notes, participation in manager-training programs, and interviews with residents and family members during a 22-month study of three Oregon facilities. Consumer discourse is evident in four primary sources, including the state rules, manager-training programs, organizational practices, and an institutional belief in specific consumer demands like independence and choice. Personal care is a complex consumer "good" further complicated by residents with cognitive impairments, family demands, payment sources, and the very novelty of the assisted living philosophy. We conclude with a discussion of benefits and pitfalls based on the use of consumer discourse that represents older persons as active consumers, rather than recipients, of long-term care services.

  11. Honeybee workers with higher reproductive potential live longer lives.

    PubMed

    Kuszewska, Karolina; Miler, Krzysztof; Rojek, Wiktoria; Woyciechowski, Michal

    2017-11-01

    Social insects, especially honeybees, have received much attention in comparative gerontology because of their peculiar and flexible ageing patterns that differ across genetically similar individuals. The longevity of honeybee individuals varies and depends on patterns of gene expression during development; females developing into reproductive individuals (queens) live longer than facultatively sterile workers. Here, we show that rebel workers, which develop under queenless conditions after swarming and have high reproductive potential, live approximately 4days longer in hives and approximately 3days longer in cages than individuals that develop in queenright colonies and have lower reproductive potential; this difference in longevity occurs in both free-flying and caged workers. Moreover, we show that both rebel and normal workers live longer when their ovaries contain more ovarioles. Longer-living rebel workers can benefit the colony because they can fill the generation gap that emerges between workers after queen exchange during swarming. Our findings provide novel evidence that the fecundity of workers in a social insect colony impacts their intrinsic longevity. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Ceratocystis fagacearum in Living and Dead Texas Live Oaks

    Treesearch

    R. Lewis

    1987-01-01

    Ceratocystis fagacearum colonized Texas live oaks (Quercus virginiana var. fusiformis) to a depth of 10 annual increments in sapwood, either before or shortly after initial symptom expression. The fungus survived in dead wood up to 12 months after oak wilt caused crown mortality. Both moist wood at the root...

  13. [Health effects of living habits].

    PubMed

    Vuori, Ilkka

    2015-01-01

    Single healthy living habits such as non-smoking and regular physical activity decrease the risk of common non-communicable diseases, unsuccessful aging and premature death to a small to moderate degree. Their cumulative effects are, however, large. Only a small minority of people adhere well to all healthy living habits or even the healthiest ones. Consequently, the population attributable fractions of major public health problems due to unhealthy lifestyles are large. Substantial improvement of public health calls for policies and programs to influence the root causes of the lifestyles in the multiple environments and systems where they are developed, maintained, and changed.

  14. Astronomy Cast Live: Live Blogging Today's Science to the World

    NASA Astrophysics Data System (ADS)

    Bemrose-Fetter, Rebecca; Gay, P. L.; Astronomy Cast LIVE Team

    2008-05-01

    In today's digital, on-demand society, consumers of information both want to know exactly what is happening as it is happening, and to be able to subscribe to content of their choosing. Meeting the needs of these tech savvy individuals are bloggers, podcasters and vodcasters. Using text, audio, and video to reach their respective audiences, these communicators are the new face of public outreach and journalism, but even their communications means are starting to become passé in the face of live blogging. The idea behind live blogging is simple: Take any person - even an undergraduate - with an Internet connected device, put them someplace interesting, and have them report on what they are seeing and experiencing online in real-time. This new tool is bringing astronomy enthusiasts around the world the thrill of live astronomy announcements, attending talks in real, and being "in the room" with astronomers via an Internet connection. These audiences can be anyone, from any nation, with any age. Beyond the public communications benefits of this program, it is also a program that allows the participation of early undergraduate students in science conferences. To date, two undergraduate students and five E/PO professionals have live blogged text, audio, and video content from three science conferences and a shuttle launch. Together, they have produced over 200 hundred stories that have reached tens of thousands of people around the world. In this poster we describe how we have made astronomy live blogging a reality from both the technical and personal standpoint. This project is funded through NSF grant # 0744944.

  15. The Living Library of NCTE

    ERIC Educational Resources Information Center

    Appleman, Deborah

    2011-01-01

    In this article, the author offers an appreciation of the writers and their books that have shaped her teaching and thinking. Her face-to-face encounters with these books prompted her to question her own beliefs about literacy, about authority, about language, about writing, about literature. She was surrounded by the living animations of the…

  16. Living in the Gifted Family

    ERIC Educational Resources Information Center

    DeVries, Arlene R.

    2010-01-01

    Raising gifted children is both a joy and a challenge. How does one survive and thrive living in a gifted family? Parents play an essential role in helping children develop appreciation and respect for the world and their place in it. Intellectual development and emotional reactions begin at an early age, and many major behavioral patterns are set…

  17. Family Living Including Sex Education.

    ERIC Educational Resources Information Center

    New York City Board of Education, Brooklyn, NY.

    In 1967, the New York Board of Education added a program on Family Living designed for school students from the pre-kindergarten level to the senior level in high school. Tables are used to establish year-by-year concepts to be explored, learning activities to be followed, and general course content relating to the basic concept. In addition,…

  18. Living with wildfire in Colorado

    Treesearch

    Patricia A. Champ; Nicholas Flores; Hannah Brenkert-Smith

    2010-01-01

    In this presentation, we describe results of a survey to homeowners living in wildfire-prone areas of two counties along the Front Range of the Rocky Mountains in Colorado. The survey was designed to elicit information on homeowners' experience with wildfire, perceptions of wildfire risk on their property and neighboring properties, mitigation efforts undertaken...

  19. Living Assessment Passes the Test

    ERIC Educational Resources Information Center

    Suskind, Dorothy C.

    2015-01-01

    The author, a 5th-grade teacher at an independent boys' school, gives a first-person account of how her constant assessments and requirement that her students be active participants in their own learning gainsays the need for high-stakes, standardized testing. She posits a "living assessment" that is intertwined, interactive and…

  20. Living Assessment Passes the Test

    ERIC Educational Resources Information Center

    Suskind, Dorothy C.

    2015-01-01

    The author, a 5th-grade teacher at an independent boys' school, gives a first-person account of how her constant assessments and requirement that her students be active participants in their own learning gainsays the need for high-stakes, standardized testing. She posits a "living assessment" that is intertwined, interactive and…

  1. Where You Live: Risk Assessment

    EPA Pesticide Factsheets

    Where you live page shows visitors to the risk assessment website how to contact their local regional office by state. Since these link to pages maintained by the local offices they will have the most up-to-date contact information.

  2. Senior to Senior: Living Lessons

    ERIC Educational Resources Information Center

    Goff, Kathy

    2004-01-01

    Senior to Senior: Living Lessons is a program created to provide meaningful horticulture therapy activities for community minority elders (60 years of age and older) and senior college students (20 years of age and older) from an Historically Black University. The program's objectives were to promote positive intergenerational relationships and to…

  3. Investigating Evolution with Living Plants.

    ERIC Educational Resources Information Center

    Schlessman, Mark A.

    1997-01-01

    Describes two investigative labs that use live plants to illustrate important biological principles, include quantitative analysis, and require very little equipment. Each lab is adaptable to a variety of class sizes, course contents, and student backgrounds. Topics include the evolution of flower size in Mimulus and pollination of Brassicas. (DDR)

  4. Living History: Elsworth R. Buskirk

    ERIC Educational Resources Information Center

    Tipton, Charles M.

    2009-01-01

    In 2005, the American Physiological Society (APS) initiated the Living History of Physiology Archival Program to recognize senior members who have made significant contributions during their career to the advancement of the discipline and the profession of physiology. Subsequently, the leadership of the APS Section of Environmental and Exercise…

  5. Living History: Clark M. Blatteis

    ERIC Educational Resources Information Center

    Quan, Ning

    2009-01-01

    In 2005, the American Physiological Society (APS) initiated the Living History Project to recognize senior members who have made extraordinary contributions during their career to the advancement of the discipline and profession of physiology. During 2007, the APS Section of Environmental and Exercise Physiology selected Clark M. Blatteis to be…

  6. Shakespeare Live! and Character Counts.

    ERIC Educational Resources Information Center

    Brookshire, Cathy A.

    This paper discusses a live production of Shakespeare's "Macbeth" (in full costume but with no sets) for all public middle school and high school students in Harrisonburg and Rockingham, Virginia. The paper states that the "Character Counts" issues that are covered in the play are: decision making, responsibility and…

  7. Teen Living. 7015. Curriculum Guide.

    ERIC Educational Resources Information Center

    North Carolina State Dept. of Public Instruction, Raleigh. Div. of Vocational and Technical Education Services.

    This curriculum guide was developed as a resource for teachers to use in planning and implementing a competency-based instructional program on teenage living at the high school level. It contains materials for a 2-semester consumer home economics course, based on the North Carolina Program of Studies (revised 1992); it is designed to help students…

  8. Educating Lives for Christian Wisdom

    ERIC Educational Resources Information Center

    Davis, Darin H.; Wadell, Paul J.

    2016-01-01

    This article explores how educating lives for Christian wisdom might serve as an antidote to the vice of "acedia," a prominent feature of the culture of contemporary higher education. After suggesting that the capital vice of "acedia" seems to capture well various facets of our present age and how the pursuit of wisdom serves…

  9. A President Lives with Activism

    ERIC Educational Resources Information Center

    Gooder, Glen G.

    1970-01-01

    College President offers 10 commandments for living with activism: take work, but not self, seriously; keep objective in sight; do not oversimplify; communicate; be prepared; chose proper reaction level; beware November and April (time for an organized activity); watch student funds; resist forces of division; and advance confidently. (Author/CJ)

  10. Chicanas: Their Voices, Their Lives.

    ERIC Educational Resources Information Center

    Lockert, Lucia Fox, Ed.

    This book is an oral history of ten Chicana women ranging in ages from 84 to 24. The collection of interviews reflects how their hard work and determination have significantly changed their lives for the better. The backgrounds of the women vary; some were born in Mexico and moved to the United States. Others were born in the Southwest and later…

  11. Men's Role and Men's Lives.

    ERIC Educational Resources Information Center

    Harrison, James B.

    1978-01-01

    The growing literature on men is clearly a response to the cultural ferment generated by feminism. However, as in the discussion of women's lives since the first advent of feminism, centuries of assumptions do not give way readily to appropriate scientific skepticism. (Author/MC)

  12. Living History: F. Eugene Yates

    ERIC Educational Resources Information Center

    Urquhart, John

    2009-01-01

    In 2005, the American Physiological Society (APS) initiated the Living History of Physiology Archival Program to recognize senior members who have made significant contributions during their career to the advancement of the discipline and the profession of physiology. During 2008, the APS Cardiovascular Section selected Francis Eugene Yates to be…

  13. Living History: Clark M. Blatteis

    ERIC Educational Resources Information Center

    Quan, Ning

    2009-01-01

    In 2005, the American Physiological Society (APS) initiated the Living History Project to recognize senior members who have made extraordinary contributions during their career to the advancement of the discipline and profession of physiology. During 2007, the APS Section of Environmental and Exercise Physiology selected Clark M. Blatteis to be…

  14. Living History: Elsworth R. Buskirk

    ERIC Educational Resources Information Center

    Tipton, Charles M.

    2009-01-01

    In 2005, the American Physiological Society (APS) initiated the Living History of Physiology Archival Program to recognize senior members who have made significant contributions during their career to the advancement of the discipline and the profession of physiology. Subsequently, the leadership of the APS Section of Environmental and Exercise…

  15. Senior to Senior: Living Lessons

    ERIC Educational Resources Information Center

    Goff, Kathy

    2004-01-01

    Senior to Senior: Living Lessons is a program created to provide meaningful horticulture therapy activities for community minority elders (60 years of age and older) and senior college students (20 years of age and older) from an Historically Black University. The program's objectives were to promote positive intergenerational relationships and to…

  16. Living kidney donors and ESRD.

    PubMed

    Ross, Lainie Friedman

    2015-07-01

    There are more than 325 living kidney donors who have developed end-stage renal disease and have been listed on the Organ Procurement and Transplantation Network (OPTN)/United Network for Organ Sharing (UNOS) deceased donor kidney wait list. The OPTN/UNOS database records where these kidney donors are listed and, if they donated after April 1994, where that donation occurred. These 2 locations are often not the same. In this commentary, I examine whether a national living donor registry should be created and whether transplantation centers should be notified when one of their living kidney donors develops end-stage renal disease. I consider and refute 5 potential objections to center notification. I explain that transplantation centers should look back at these cases and input data into a registry to attempt to identify patterns that could improve donor evaluation protocols. Creating a registry and mining the information it contains is, in my view, our moral and professional responsibility to future patients and the transplantation endeavor. As individuals and as a community, we need to acknowledge the many unknown risks of living kidney donation and take responsibility for identifying these risks. We then must share information about these risks, educate prospective donors about them, and attempt to minimize them.

  17. Effective Communication. Successful Living Skills.

    ERIC Educational Resources Information Center

    Oklahoma State Board of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

    This module on effective communication is one of a series of modules designed to help teach students to become more self-sufficient in their personal and professional lives. This module contains teacher and student materials that are planned to help students become more relaxed, prepared, and confident when using written and verbal communications.…

  18. Finding a Place to Live.

    ERIC Educational Resources Information Center

    NatureScope, 1985

    1985-01-01

    Provides background information and student activities on bird habitats, how birds have adapted to living in these habitats, and bird migration. Each activity includes an objective, recommended age level(s), subject area(s), list of materials needed, and procedures. Ready-to-copy student materials (puzzles and worksheets) are included. (JN)

  19. Living history: Clark M. Blatteis.

    PubMed

    Quan, Ning

    2009-03-01

    In 2005, the American Physiological Society (APS) initiated the Living History Project to recognize senior members who have made extraordinary contributions during their career to the advancement of the discipline and profession of physiology. During 2007, the APS Section of Environmental and Exercise Physiology selected Clark M. Blatteis to be profiled in Advances in Physiology Education.

  20. College for Living Instructor's Manual.

    ERIC Educational Resources Information Center

    Templin, Robert G., Jr.; And Others

    This five-part manual was designed to help volunteer instructors in Northern Virginia Community College's College for Living Program to conduct survival and socialization courses for handicapped adults. After introductory material summarizing general principles and specific suggestions, Robert Templin provides information on the skills and…

  1. Living with High Blood Pressure

    MedlinePlus

    ... page from the NHLBI on Twitter. Living With High Blood Pressure If you have high blood pressure, the best thing to do is to talk ... help you track your blood pressure. Pregnancy Planning High blood pressure can cause problems for mother and baby. High ...

  2. Investigating Evolution with Living Plants.

    ERIC Educational Resources Information Center

    Schlessman, Mark A.

    1997-01-01

    Describes two investigative labs that use live plants to illustrate important biological principles, include quantitative analysis, and require very little equipment. Each lab is adaptable to a variety of class sizes, course contents, and student backgrounds. Topics include the evolution of flower size in Mimulus and pollination of Brassicas. (DDR)

  3. Are trees long-lived?

    Treesearch

    Kevin T. Smith

    2009-01-01

    Trees and tree care can capture the best of people's motivations and intentions. Trees are living memorials that help communities heal at sites of national tragedy, such as Oklahoma City and the World Trade Center. We mark the places of important historical events by the trees that grew nearby even if the original tree, such as the Charter Oak in Connecticut or...

  4. I Know Where They Live.

    ERIC Educational Resources Information Center

    Worsley, Ed, Ed.

    This science-oriented reading readiness text is provided in two versions: English and Navajo. It consists of large black-and-white drawings of animals, birds, and insects familiar to the Southwestern U.S. One picture is provided on each page, labeled with the following simple sentences: "This is a [name of the animal]. He lives in [name of…

  5. Living History: F. Eugene Yates

    ERIC Educational Resources Information Center

    Urquhart, John

    2009-01-01

    In 2005, the American Physiological Society (APS) initiated the Living History of Physiology Archival Program to recognize senior members who have made significant contributions during their career to the advancement of the discipline and the profession of physiology. During 2008, the APS Cardiovascular Section selected Francis Eugene Yates to be…

  6. Functional Literacy in People's Lives

    ERIC Educational Resources Information Center

    Rabušicová, Milada; Oplatková, Pavla

    2010-01-01

    The paper presents the results of a qualitative study into the lives of people with inadequate functional literacy skills. The data were collected through a biographical interview with a respondent whose characteristics correspond to those of a hypothetical person likely to exhibit signs of low functional literacy. The characteristics, such as…

  7. PREP for Effective Family Living.

    ERIC Educational Resources Information Center

    Welsch, Marie

    1987-01-01

    The article favorably reviews "PREP for Effective Family Living," a curriculum designed to help adolescents develop social skills and prepare for adult family life. Curriculum strengths include organization of objectives and format of the student materials. A weakness is lack of clear correlation between the student handbook and the…

  8. Finding a Place to Live.

    ERIC Educational Resources Information Center

    NatureScope, 1985

    1985-01-01

    Provides background information and student activities on bird habitats, how birds have adapted to living in these habitats, and bird migration. Each activity includes an objective, recommended age level(s), subject area(s), list of materials needed, and procedures. Ready-to-copy student materials (puzzles and worksheets) are included. (JN)

  9. Teaching Activities of Daily Living.

    ERIC Educational Resources Information Center

    McCormack, James E.

    Provided are strategies for teaching activities of daily living (ADL), which include dressing, eating, grooming, toileting, and basic homemakine, to severely retarded students. Reviewed are the steps necessary to teach ADL skills: ADL assessment, identification of appropriate strategies and tactics, and task analysis. Explained are four common…

  10. Educating Lives for Christian Wisdom

    ERIC Educational Resources Information Center

    Davis, Darin H.; Wadell, Paul J.

    2016-01-01

    This article explores how educating lives for Christian wisdom might serve as an antidote to the vice of "acedia," a prominent feature of the culture of contemporary higher education. After suggesting that the capital vice of "acedia" seems to capture well various facets of our present age and how the pursuit of wisdom serves…

  11. College for Living Instructor's Manual.

    ERIC Educational Resources Information Center

    Templin, Robert G., Jr.; And Others

    This five-part manual was designed to help volunteer instructors in Northern Virginia Community College's College for Living Program to conduct survival and socialization courses for handicapped adults. After introductory material summarizing general principles and specific suggestions, Robert Templin provides information on the skills and…

  12. Piloted ignition of live forest fuels

    Treesearch

    S. McAllister; I. Grenfell; A. Hadlow; W. M. Jolly; M. Finney; J. Cohen

    2012-01-01

    The most unpredictable and uncontrollable wildfires are those that burn in the crowns of live vegetation. The fuels that feed these crown fires are mostly live, green foliage. Unfortunately, little is known about how live fuels combust. To understand how live fuels burn, piloted ignition experiments were performed with lodgepole pine and Douglas-fir. The thermal...

  13. Evaluation of living liver donors.

    PubMed

    Rudow, Dianne LaPointe; Brown, Robert S

    2003-06-01

    As a result of the cadaveric organ shortage, the number of centers performing living donor liver transplantation has increased. Living donor liver transplantation provides immediate organ availability and avoids the risk of life-threatening complications that occur with long waiting times for cadaveric organs; however, it puts a healthy person at risk for little personal gain. A standardized approach to donor evaluation ensures safety to potential donors. Careful medical (physical examination as well as laboratory and radiological evaluation) and psychological evaluation is imperative to reduce donor complications and ensure good outcomes in recipients. A social worker and psychiatrist assess for mental competency, provide emotional support, and can serve as independent donor advocates. Informed understanding and consent are crucial aspects of the evaluation and include ensuring that the donor understands all potential complications and is free of coercion. Safety of the donor must be the highest priority.

  14. Focusing light through living tissue

    NASA Astrophysics Data System (ADS)

    Vellekoop, I. M.; Aegerter, C. M.

    2010-02-01

    Tissues such as skin, fat or cuticle are non-transparent because inhomogeneities in the tissue scatter light. We demonstrate experimentally that light can be focused through turbid layers of living tissue, in spite of scattering. Our method is based on the fact that coherent light forms an interference pattern, even after hundreds of scattering events. By spatially shaping the wavefront of the incident laser beam, this interference pattern was modified to make the scattered light converge to a focus. In contrast to earlier experiments, where light was focused through solid objects, we focused light through living pupae of Drosophila melanogaster. We discuss a dynamic wavefront shaping algorithm that follows changes due to microscopic movements of scattering particles in real time. We relate the performance of the algorithm to the measured timescale of the changes in the speckle pattern and analyze our experiment in the light of Laser Doppler flowmetry. Applications in particle tracking, imaging, and optical manipulation are discussed.

  15. International Living With a Star

    NASA Astrophysics Data System (ADS)

    Brekke, Pål

    2005-08-01

    The response of our space environment to the constantly changing Sun is known as "Space Weather". Sudden ejections of plasma and magnetic field structures from the Sun's atmosphere called coronal mass ejections (CMEs) together with sudden bursts of radiation termed solar flares all cause space weather effects at the Earth. The International Living With a Star (ILWS) programme is a space weather focused and applications driven research programme. Its goal is to develop the scientific understanding necessary to effectively address those aspects of the connected Sun-Earth system that directly affects life and society. Recent large solar storms caused damages to power systems and satellites and disturbed important navigation and communication systems. Furthermore, accurate monitoring of the energy output from the Sun is important for understanding how the Sun contributes to the observed warming of our planet. The Sun is a variable star we better learn how to live with.

  16. Steps to Independent Living Series.

    ERIC Educational Resources Information Center

    Lobb, Nancy

    This set of six activity books and a teacher's guide is designed to help students from eighth grade to adulthood with special needs to learn independent living skills. The activity books have a reading level of 2.5 and address: (1) "How to Get Well When You're Sick or Hurt," including how to take a temperature, see a doctor, and use medicines…

  17. Crime prevention and active living.

    PubMed

    Loukaitou-Sideris, Anastasia; Eck, John E

    2007-01-01

    This paper addresses the question of whether crime is a barrier to active living and if it is, what can be done about it? The authors introduce a theoretical model that addresses how crime might influence physical activity behavior. The core components of the model are: situational characteristics, crime and disorder, fear of crime or disorder, and physical activity. These variables are thought to be moderated through psychological, demographic, environmental and other factors. Research questions that derive from the model are featured.

  18. Imaging Transcription in Living Cells

    PubMed Central

    Darzacq, Xavier; Yao, Jie; Larson, Daniel R.; Causse, Sebastien Z.; Bosanac, Lana; de Turris, Valeria; Ruda, Vera M.; Lionnet, Timothee; Zenklusen, Daniel; Guglielmi, Benjamin; Tjian, Robert; Singer, Robert H.

    2011-01-01

    The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding. PMID:19416065

  19. Viruses: are they living entities?

    PubMed

    Pennazio, Sergio

    2011-01-01

    The essence (living or nonliving entities) of viruses has today become an aporia, i.e. a difficulty inherent in reasoning because they shared four fundamental characteristics with livings (multiplication, genetic information, mutation and evolution) without having the capacity to have an independent life. For much time, however, they were considered minuscule pathogenetic micro-organisms in observance of Koch and Pasteur's 'germ theory' albeit no microbiologist could show their existence except their filterability and pathogenetic action. Only some voices based on experimental results raised against this dogmatic view, in particular those of Beijerinck, Baur and Mrowka, without dipping effectively into the dominant theory. The discovery relative to their nucleoprotein nature made between 1934 and 1936 (Schlesinger as for the phage, and Bawden and co-operators as for Tobacco mosaic virus; TMV), together with the first demonstrations of their structures thanks to electron microscopy (from 1939 onwards) started on casting a new light on their true identity, which could be more clearly identified when, from 1955 onwards, phage and TMV proved to be decisive factors to understand the strategies of replication of the genetic material. Following the new knowledge, the theoretical view relative to viruses changed rather radically and the current view looks on these pathogenetic agents as nonliving aggregates of macromolecules provided with biological properties. There is, however, a current of thought, made explicitly by Lwoff that places viruses as compromise between living and non living and, perhaps, as primitive forms of life which have had great importance for the evolution of cellular life. At any rate, viruses are peculiar entities whose importance cannot be unacknowledged.

  20. Steps to Independent Living Series.

    ERIC Educational Resources Information Center

    Lobb, Nancy

    This set of six activity books and a teacher's guide is designed to help students from eighth grade to adulthood with special needs to learn independent living skills. The activity books have a reading level of 2.5 and address: (1) "How to Get Well When You're Sick or Hurt," including how to take a temperature, see a doctor, and use medicines…

  1. Exact law of live nature

    NASA Astrophysics Data System (ADS)

    Azbel, Mark Ya.

    2005-07-01

    Exact law of mortality dynamics in changing populations and environment is derived. It includes no explicit characteristics of animal- environment interactions (metabolism etc) which are a must for life; it is universal for all animals, from single cell yeast to humans, with their drastically different biology, evolutionary history, and complexity; it is rapidly (within few percent of life span) reversible. Such law is unique for live systems with their homeostatic self-adjustment to environment (cf. thermodynamics of liquids and glasses). The law which is valid for all live, and only live, systems is their specific natural law. Mortality is an instrument of natural selection and biological diversity. Its law, which is preserved in evolution of all species, is a conservation law of mortality, selection, evolution, biology. The law implies new kind of intrinsic mortality and adaptation which dominate in evolutionary unprecedented protected populations and, in contrast to species specific natural selection, proceed via universal stepwise rungs and reduce to universal cellular mechanism. The law demonstrates that intrinsic mortality and at least certain aspects of aging are disposable evolutionary byproducts, and directed genetic and/or biological changes may yield healthy and vital Methuselah lifespan. This is consistent with experiments. Universality implies that single cell yeast may provide a master key to the cellular mechanism of universal mortality, aging, selection, evolution, and its regulation in all animals. One may look for its manifestations in animal cells also, e.g., in their replicative senescence and cancer. Evolutionary origin and genetic nature of universality are suggested.

  2. Women living with environmental illness.

    PubMed

    Chircop, Andrea; Keddy, Barbara

    2003-01-01

    We used a case study approach to explore the experiences of 4 women who live with environmental illness (EI). From the unstructured interviews we found a variety of themes that pointed to the complexity of EI and its severe impact on the lives of these women, their families, and their significant others. The methodology was guided by an ecofeminist approach, which enabled a critical analysis of the data to move beyond the personal to the broader sociopolitical forces shaping society. We identified the following themes from the women's stories: indirect exposure to incitants through people with whom these women come in close physical contact; the phenomenon of burden of proof, meaning that these women are forced to explain and legitimize their illness on a continuous basis; taking refuge from a hostile environment in social isolation to a more controlled environment, not as a matter of choice, but because of the severity of the illness; and, finally, a change in value system was integral to the entire process of living with EI.

  3. Developing live vaccines against plague.

    PubMed

    Sun, Wei; Roland, Kenneth L; Curtiss, Roy

    2011-09-14

    Three great plague pandemics caused by the gram-negative bacterium Yersinia pestis have killed nearly 200 million people and it has been linked to biowarfare in the past. Plague is endemic in many parts of the world. In addition, the risk of plague as a bioweapon has prompted increased research to develop plague vaccines against this disease. Injectable subunit vaccines are being developed in the United States and United Kingdom.  However, the live attenuated Y. pestis-EV NIIEG strain has been used as a vaccine for more than 70 years in the former Soviet Union and in some parts of Asia and provides a high degree of efficacy against plague.  This vaccine has not gained general acceptance because of safety concerns.  In recent years, modern molecular biological techniques have been applied to Y. pestis to construct strains with specific defined mutations designed to create safe, immunogenic vaccines with potential for use in humans and as bait vaccines to reduce the load of Y. pestis in the environment.  In addition, a number of live, vectored vaccines have been reported using attenuated viral vectors or attenuated Salmonella strains to deliver plague antigens. Here we summarize the progress of live attenuated vaccines against plagu.

  4. Exact law of live nature

    NASA Astrophysics Data System (ADS)

    Azbel‧, Mark Ya.

    2005-08-01

    The exact law of mortality dynamics in changing populations and environment is derived. It includes no explicit characteristics of animal-environment interactions (metabolism, etc.) which are a must for life; it is universal for all animals, from single-cell yeast to humans, with their drastically different biology, evolutionary history, and complexity; it is rapidly (within few percent of life span) reversible. Such a law is unique for live systems with their homeostatic self-adjustment to environment (cf. thermodynamics of liquids and glasses). The law which is valid for all live, and only live, systems is their specific natural law. Mortality is an instrument of natural selection and biological diversity. Its law, which is preserved in evolution of all species, is a conservation law of mortality, selection, evolution, biology. The law implies new kinds of intrinsic mortality and adaptation which dominate in evolutionary unprecedented protected populations and, in contrast to species-specific natural selection, proceed via universal stepwise rungs and reduce to universal cellular mechanism. The law demonstrates that intrinsic mortality and at least certain aspects of aging are disposable evolutionary byproducts, and directed genetic and/or biological changes may yield healthy and vital Methuselah lifespan. This is consistent with experiments. Universality implies that single-cell yeast may provide a master key to the cellular mechanism of universal mortality, aging, selection, evolution, and its regulation in all animals. One may look for its manifestations in animal cells also, e.g., in their replicative senescence and cancer. Evolutionary origin and genetic nature of universality are suggested.

  5. Short-Lived Climate Pollution

    NASA Astrophysics Data System (ADS)

    Pierrehumbert, R. T.

    2014-05-01

    Although carbon dioxide emissions are by far the most important mediator of anthropogenic climate disruption, a number of shorter-lived substances with atmospheric lifetimes of under a few decades also contribute significantly to the radiative forcing that drives climate change. In recent years, the argument that early and aggressive mitigation of the emission of these substances or their precursors forms an essential part of any climate protection strategy has gained a considerable following. There is often an implication that such control can in some way make up for the current inaction on carbon dioxide emissions. The prime targets for mitigation, known collectively as short-lived climate pollution (SLCP), are methane, hydrofluo-rocarbons, black carbon, and ozone. A re-examination of the issues shows that the benefits of early SLCP mitigation have been greatly exaggerated, largely because of inadequacies in the methodologies used to compare the climate effects of short-lived substances with those of CO2, which causes nearly irreversible climate change persisting millennia after emissions cease. Eventual mitigation of SLCP can make a useful contribution to climate protection, but there is little to be gained by implementing SLCP mitigation before stringent carbon dioxide controls are in place and have caused annual emissions to approach zero. Any earlier implementation of SLCP mitigation that substitutes to any significant extent for carbon dioxide mitigation will lead to a climate irreversibly warmer than will a strategy with delayed SLCP mitigation. SLCP mitigation does not buy time for implementation of stringent controls on CO2 emissions.

  6. Live from the Moon - Impact!

    NASA Technical Reports Server (NTRS)

    2002-01-01

    On March 24, 1965, a nationwide TV audience watched live video from Ranger 9 as it purposefully crashed into the Moon within the crater Alphonsus. Ranger's six cameras sent back more than 5800 video images during the last 18 minutes of its 3-day journey, the last of the Ranger Project. The last few images show the lunar surface in detail from a few hundred meters above.

    This sequence of images from Camera A was converted from video to film to laser disc to digital files.

  7. Long-lived laser dye

    SciTech Connect

    Fletcher, A.N.

    1986-07-29

    A method is described of obtaining in a flashlamp pumped laser system, a long-lived flashpumped laser dye having a low threshold of lasing and a moderate output comprising the steps of: placing a dye solution comprising a laser dye, the N-methyl tosylate salt of 2-(4-pyridyl)-5-(4-methoxphenyl)oxazole, and a solvent into a laser dye cavity; screening the dye solution from ultraviolet light with an optical filter; flushing the dye solution with an inert gas; and optically pumping the dye solution with a flashlamp to produce laser emission.

  8. Living and morking on Mars.

    NASA Astrophysics Data System (ADS)

    McKay, C. P.

    When it becomes feasible to live and work on Mars, martian resources will be a critical part of the sustaining process. It will be necessary to drink water extracted from the martian environment, to make breathable air and fuel components from the martian atmosphere, and to shield or construct facilities using martian dirt. The ultimate use of martian resources may be the "terraforming" of that planet's global environment into an Earth-like biosphere for a population whose ancestors were born on a distant blue planet.

  9. [Clinical evaluation of living donor].

    PubMed

    Scolari, Maria Piera; Comai, G; La Manna, G; Liviano D'Arcangelo, G; Monti, M; Feliciangeli, G; Stefoni, S

    2009-01-01

    When possible, living donor transplantation represents the best therapeutic strategy for patients suffering from chronic renal failure. Studying the donor allows a complete and thorough clinical, laboratory and instrumental assessment that guarantees good organ function whilst protecting the health of the donor. The main parameters considered within this framework are age, renal function, nephrological complications, comorbidities (diabetes, hypertension, obesity, etc.), malignancies, and infection. Moreover, particular attention is paid to the sociopsychological aspects of the donation, particularly related to the donor, the recipient, and the entire family situation.

  10. Living and Working in Space

    NASA Technical Reports Server (NTRS)

    Roman, Monserrate C.

    2000-01-01

    This document is a presentation about some of the challenges of living and working in space. The presentation shows slides of the Apollo 11 liftoff, Skylab in orbit, a Space Shuttle launch, and a slide of the International Space Station. It reviews the needs and effluents of the astronauts per day, and the Environmental Control and Life Support (ECLS) systems. It shows a flow diagram of the Space Station Regenerative ECLS, which shows the various systems, and how they interact to control the environment and recycle the air, and water. There are other slides some of which show astronauts eating, brushing teeth, shaving, and sipping from a sip bottle while exercising.

  11. Everything About Living in Space

    NASA Image and Video Library

    2017-02-12

    With only five minutes but an unlimited number of questions, you can find out what it’s really like to live on the International Space Station—if you also have NASA astronaut Reid Wiseman, who spent 165 days in space during Expeditions 40 and 41 in 2014. Listen as Wiseman answers the questions you would ask about real life in zero g: how big is the space station, is it very hot or cold there, was the food any good, did you prank-call anyone from orbit, and many many more.

  12. Senior Living: Staying Positive and Moving Forward

    MedlinePlus

    ... Past Issues Feature: Senior Living Staying Positive and Moving Forward Past Issues / Summer 2009 Table of Contents ... page please turn Javascript on. For Juanita Kuhn, moving to an independent living facility is just the ...

  13. CDC Vital Signs: HIV Care Saves Lives

    MedlinePlus

    ... Error processing SSI file Error processing SSI file HIV Care Saves Lives Viral Suppression is Key Language: ... high risk of getting HIV. People living with HIV can Get into HIV medical care as soon ...

  14. Assisted living captures profitable market niche.

    PubMed

    Pallarito, K

    1995-05-08

    The $15 billion assisted-living industry has captured a profitable market niche and created a star on Wall Street. Sunrise Retirement Home of Falls Church (Va.), right, is a facility of the nation's largest assisted-living provider.

  15. Living with Spina Bifida (at different ages)

    MedlinePlus

    ... Us Information For... Media Policy Makers Living With Spina Bifida Language: English Español (Spanish) Recommend on Facebook Tweet ... of the website provides information about living with spina bifida at different ages. Spina bifida affects the entire ...

  16. Live a Full Life with Fibro

    MedlinePlus

    ... Live a Full Life with Fibro Page Content Fibromyalgia is a chronic pain condition that affects 10 ... family, you can live an active life with fibromyalgia. Talking with Your Physician Take the first step ...

  17. Living Free: A Teacher Information Booklet.

    ERIC Educational Resources Information Center

    Mello, Robin

    This workbook helps adolescents learn how to take charge of their own lives and happiness. The underlying idea is to teach them how to live responsibly. By learning to live responsibly, adolescents have the best chance of avoiding drugs, alcohol, and other addictive behaviors such as overeating and overspending. The workbook explains the steps to…

  18. Use of Live Supervision in Counselor Preparation.

    ERIC Educational Resources Information Center

    Bubenzer, Donald L.; And Others

    1991-01-01

    Investigated live supervision in counselor preparation programs by surveying 307 counselor preparation programs. Live supervision was used at 157 institutions and was used in preparing individual, group, and marriage and family counselors. At least 75 percent of programs provided live supervision weekly. Techniques of cotherapy and remote viewing…

  19. Lived Experience of University Continuing Education Leaders

    ERIC Educational Resources Information Center

    Landry, Janice

    2011-01-01

    This article is based on a study that explored the professional lives of eight leaders of continuing education in Canadian universities, with a focus on their administrative role, to provide a deeper understanding of how they live within their practice (lived experience). A practical listing of 56 horizons of experience was identified, useful as…

  20. Gendered Living Arrangements among Children with Disabilities

    ERIC Educational Resources Information Center

    Cohen, Philip N.; Petrescu-Prahova, Miruna

    2006-01-01

    Using data on disabilities from the 2000 Census, we found a consistent pattern of living arrangements that leaves children (aged 5-15 years) with disabilities living disproportionately with women. Children with disabilities are more likely to live with single parents, and especially their mothers, than are other children. Further, those who do not…