Tissue Distribution of Kir7.1 Inwardly Rectifying K+ Channel Probed in a Knock-in Mouse Expressing a Haemagglutinin-Tagged Protein.

PubMed

Cornejo, Isabel; Villanueva, Sandra; Burgos, Johanna; López-Cayuqueo, Karen I; Chambrey, Régine; Julio-Kalajzić, Francisca; Buelvas, Neudo; Niemeyer, María I; Figueiras-Fierro, Dulce; Brown, Peter D; Sepúlveda, Francisco V; Cid, L P

2018-01-01

Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K + channel present in epithelia where it shares membrane localization with the Na + /K + -pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA) epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb + currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation studies. It

Molecular characterization of partial fusion gene and C-terminus extension length of haemagglutinin-neuraminidase gene of recently isolated Newcastle disease virus isolates in Malaysia

PubMed Central

2010-01-01

Background Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene. Results The coding region of eleven NDV isolates fusion (F) gene and carboxyl terminal region of haemagglutinin-neuraminidase (HN) gene including extensions were amplified by reverse transcriptase PCR and directly sequenced. All the isolates have shown to have non-synonymous to synonymous base substitution rate ranging between 0.081 - 0.264 demonstrating presence of negative selection. Analysis based on F gene showed the characterized isolates possess three different types of protease cleavage site motifs; namely 112RRQKRF117, 112RRRKRF117 and 112GRQGRL117 and appear to show maximum identities with isolates in the region such as cockatoo/14698/90 (Indonesia), Ch/2000 (China), local isolate AF2240 indicating the high similarity of isolates circulating in the South East Asian countries. Meanwhile, one of the isolates resembles commonly used lentogenic vaccine strains. On further characterization of the HN gene, Malaysian isolates had C-terminus extensions of 0, 6 and 11 amino acids. Analysis of the phylogenetic tree revealed that the existence of three genetic groups; namely, genotype II, VII and VIII. Conclusions The study concluded that the occurrence of three types of NDV genotypes and presence of varied carboxyl terminus extension lengths among Malaysian isolates incriminated for sporadic cases. PMID:20691110

Molecular characterization of partial fusion gene and C-terminus extension length of haemagglutinin-neuraminidase gene of recently isolated Newcastle disease virus isolates in Malaysia.

PubMed

Berhanu, Ayalew; Ideris, Aini; Omar, Abdul R; Bejo, Mohd Hair

2010-08-08

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene. The coding region of eleven NDV isolates fusion (F) gene and carboxyl terminal region of haemagglutinin-neuraminidase (HN) gene including extensions were amplified by reverse transcriptase PCR and directly sequenced. All the isolates have shown to have non-synonymous to synonymous base substitution rate ranging between 0.081 - 0.264 demonstrating presence of negative selection. Analysis based on F gene showed the characterized isolates possess three different types of protease cleavage site motifs; namely 112RRQKRF117, 112RRRKRF117 and 112GRQGRL117 and appear to show maximum identities with isolates in the region such as cockatoo/14698/90 (Indonesia), Ch/2000 (China), local isolate AF2240 indicating the high similarity of isolates circulating in the South East Asian countries. Meanwhile, one of the isolates resembles commonly used lentogenic vaccine strains. On further characterization of the HN gene, Malaysian isolates had C-terminus extensions of 0, 6 and 11 amino acids. Analysis of the phylogenetic tree revealed that the existence of three genetic groups; namely, genotype II, VII and VIII. The study concluded that the occurrence of three types of NDV genotypes and presence of varied carboxyl terminus extension lengths among Malaysian isolates incriminated for sporadic cases.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

PubMed

Wong, Jack H; Wan, Chung T; Ng, Tzi B

2010-01-15

A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells. Copyright (c) 2009 Society of Chemical Industry.

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.

PubMed

Weyer, U; Possee, R D

1991-12-01

A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.

Emergence of canine distemper virus strains with two amino acid substitutions in the haemagglutinin protein, detected from vaccinated carnivores in North-Eastern China in 2012-2013.

PubMed

Zhao, Jianjun; Zhang, Hailing; Bai, Xue; Martella, Vito; Hu, Bo; Sun, Yangang; Zhu, Chunsheng; Zhang, Lei; Liu, Hao; Xu, Shujuan; Shao, Xiqun; Wu, Wei; Yan, Xijun

2014-04-01

A total of 16 strains of canine distemper virus (CDV) were detected from vaccinated minks, foxes, and raccoon dogs in four provinces in North-Eastern China between the end of 2011 and 2013. Upon sequence analysis of the haemagglutinin gene and comparison with wild-type CDV from different species in the same geographical areas, two non-synonymous single nucleotide polymorphisms were identified in 10 CDV strains, which led to amino acid changes at positions 542 (isoleucine to asparagine) and 549 (tyrosine to histidine) of the haemagglutinin protein coding sequence. The change at residue 542 generated a potentially novel N-glycosylation site. Masking of antigenic epitopes by sugar moieties might represent a mechanism for evasion of virus neutralising antibodies and reduced protection by vaccination. Copyright © 2014 Elsevier Ltd. All rights reserved.

ALP gene expression in cDNA samples from bone tissue engineering using a HA/TCP/Chitosan scaffold

NASA Astrophysics Data System (ADS)

Stephanie, N.; Katarina, H.; Amir, L. R.; Gunawan, H. A.

2017-08-01

This study examined the potential use of hydroxyapatite (HA)/tricalcium phosphate (TCP)/Chitosan as a bone tissue engineering scaffold. The potential for using HA/TCP/chitosan as a scaffold was analyzed by measuring expression of the ALP osteogenic gene in cDNA from bone biopsies from four Macaque nemestrina. Experimental conditions included control (untreated), treatment with HA/TCP 70:30, HA/TCP 50:50, and HA/TCP/chitosan. cDNA samples were measured quantitively with Real-Time PCR (qPCR) and semi-quantitively by gel electrophoresis. There were no significant differences in ALP gene expression between treatment subjects after two weeks, but the HA/TCP/chitosan treatment gave the highest level of expression after four weeks. The scaffold using the HA/TCP/chitosan combination induced a higher level of expression of the osteogenic gene ALP than did scaffold without chitosan.

Substitutions in position 222 of haemagglutinin of pandemic influenza A (H1N1) 2009 viruses in Spain.

PubMed

Ledesma, Juan; Pozo, Francisco; Pérez Ruiz, Mercedes; Navarro, Jose María; Piñeiro, Luis; Montes, Milagros; Pérez Castro, Sonia; Suárez Fernández, Jonathan; García Costa, Juan; Fernández, Mirian; Galán, Juan Carlos; Cuevas, María Teresa; Casas, Inmaculada; Pérez Breña, Pilar

2011-05-01

A change of aspartic acid (D) to glycine (G) at position 222 in the haemagglutinin (HA) protein of pandemic influenza A (H1N1) 2009 viruses was described in Norway on November 2009 with considerable frequency in fatal and severe cases. This change was detected in other countries and was related only with severe disease. Other substitutions to glutamic acid (E) or asparagine (N) at position 222 were detected among pandemic viruses but it is unclear what implications might have in terms of severity. To analyse the appearance of amino acid substitutions at position 222 in the HA protein of circulating viruses in Spain and to determine their relationships with the disease symptoms observed. Pandemic influenza A (H1N1) 2009 viruses detected in respiratory samples of 273 severe and 533 non-severe cases from different Spanish regions were selected for sequencing of a partial segment of HA1 subunit and studied to monitor substitutions at position 222. D222G substitution was only detected in viruses from 14 severe cases (5.12%). D222E was found in viruses from 47 severe (17.21%) and from 52 non-severe cases (9.75%). D222N occurred in viruses from 3 additional severe cases (0.37%). Appearance of D222G and D222E substitution in HA of pandemic influenza A (H1N1) viruses circulating in Spain might be related with severe respiratory disease. Copyright © 2011 Elsevier B.V. All rights reserved.

Haemagglutinin mutations and glycosylation changes shaped the 2012/13 influenza A(H3N2) epidemic, Houston, Texas

PubMed Central

Stucker, K M; Schobel, S A; Olsen, R J; Hodges, H L; Lin, X; Halpin, R A; Fedorova, N; Stockwell, T B; Tovchigrechko, A; Das, S R; Wentworth, D E; Musser, J M

2017-01-01

While the early start and higher intensity of the 2012/13 influenza A virus (IAV) epidemic was not unprecedented, it was the first IAV epidemic season since the 2009 H1N1 influenza pandemic where the H3N2 subtype predominated. We directly sequenced the genomes of 154 H3N2 clinical specimens collected throughout the epidemic to better understand the evolution of H3N2 strains and to inform the H3N2 vaccine selection process. Phylogenetic analyses indicated that multiple co-circulating clades and continual antigenic drift in the haemagglutinin (HA) of clades 5, 3A, and 3C, with the evolution of a new 3C subgroup (3C-2012/13), were the driving causes of the epidemic. Drift variants contained HA substitutions and alterations in the potential N-linked glycosylation sites of HA. Antigenic analysis demonstrated that viruses in the emerging subclade 3C.3 and subgroup 3C-2012/13 were not well inhibited by antisera generated against the 3C.1 vaccine strains used for the 2012/13 (A/Victoria/361/2011) or 2013/14 (A/Texas/50/2012) seasons. Our data support updating the H3N2 vaccine strain to a clade 3C.2 or 3C.3-like strain or a subclade that has drifted further. They also underscore the challenges in vaccine strain selection, particularly regarding HA and neuraminidase substitutions derived during laboratory passage that may alter antigenic testing accuracy. PMID:25990233

Human relevance of an in vitro gene signature in HaCaT for skin sensitization.

PubMed

van der Veen, Jochem W; Hodemaekers, Henny; Reus, Astrid A; Maas, Wilfred J M; van Loveren, Henk; Ezendam, Janine

2015-02-01

The skin sensitizing potential of chemicals is mainly assessed using animal methods, such as the murine local lymph node assay. Recently, an in vitro assay based on a gene expression signature in the HaCaT keratinocyte cell line was proposed as an alternative to these animal methods. Here, the human relevance of this gene signature is assessed through exposure of freshly isolated human skin to the chemical allergens dinitrochlorobenzene (DNCB) and diphenylcyclopropenone (DCP). In human skin, the gene signature shows similar direction of regulation as was previously observed in vitro, suggesting that the molecular processes that drive expression of these genes are similar between the HaCaT cell line and freshly isolated skin, providing evidence for the human relevance of the gene signature. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from breeding foxes, raccoon dogs and minks in China.

PubMed

Zhao, Jian-Jun; Yan, Xi-Jun; Chai, Xiu-Li; Martella, Vito; Luo, Guo-Liang; Zhang, Hai-Ling; Gao, Han; Liu, Ying-Xue; Bai, Xue; Zhang, Lei; Chen, Tao; Xu, Lei; Zhao, Chun-Fei; Wang, Feng-Xue; Shao, Xi-Qun; Wu, Wei; Cheng, Shi-Peng

2010-01-06

Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. Genetic/antigenic heterogeneity has been observed among the various CDV strains, notably in the haemagglutinin (H) gene, that appears as a good target to gather epidemiological information. Based on sequence analysis of the H gene, wild-type CDV strains cluster into distinct geographic lineages (genotypes), irrespective of the species of isolation. The sequence of the H gene of 28 CDV strains detected from both vaccinated and non-vaccinated breeding foxes, raccoon dogs and minks from different geographical areas of China during the years 2004-2008 was determined. All the CDV strains but two (strains HL and HLJ2) were characterized as Asia-1 genotype and were highly similar to each other (96.2-99.7% at the amino acid [aa] level) and to other Asia-1 strains (96.1-99.5% aa) previously detected in China. The CDV strains HL and HLJ2 were both collected from foxes in Heilongjiang province in 2005. Strain HL resembled CDVs of the Arctic genotype (GR88-like) and displayed high aa identity (98.0%) to the Chinese canine strain Liu. By converse, strain HLJ2 was barely related to CDVs of the Asia-2 genotype (88.7-90.3% aa identity), and could represent a novel CDV genotype, tentatively proposed as Asia-3. These results suggest that at least three different CDV genotypes, distantly related (81.8-91.6% aa identity) to the vaccine strains, Onderstepoort-like (America-1 genotype), are currently circulating in breeding foxes, raccoon dogs and minks in China, and that the genotype Asia-1 is predominant. Whether the diversity between wild-type CDVs and the vaccine strains may affect, to some extent, the efficacy of the vaccines deserves further investigations.

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.

PubMed

Agustiningsih, Agustiningsih; Trimarsanto, Hidayat; Setiawaty, Vivi; Artika, I Made; Muljono, David Handojo

2016-08-30

Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.

Silencing the HaHR3 Gene by Transgenic Plant-mediated RNAi to Disrupt Helicoverpa armigera Development

PubMed Central

Xiong, Yehui; Zeng, Hongmei; Zhang, Yuliang; Xu, Dawei; Qiu, Dewen

2013-01-01

RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests. PMID:23630449

Quantifying selection and diversity in viruses by entropy methods, with application to the haemagglutinin of H3N2 influenza

PubMed Central

Pan, Keyao; Deem, Michael W.

2011-01-01

Many viruses evolve rapidly. For example, haemagglutinin (HA) of the H3N2 influenza A virus evolves to escape antibody binding. This evolution of the H3N2 virus means that people who have previously been exposed to an influenza strain may be infected by a newly emerged virus. In this paper, we use Shannon entropy and relative entropy to measure the diversity and selection pressure by an antibody in each amino acid site of H3 HA between the 1992–1993 season and the 2009–2010 season. Shannon entropy and relative entropy are two independent state variables that we use to characterize H3N2 evolution. The entropy method estimates future H3N2 evolution and migration using currently available H3 HA sequences. First, we show that the rate of evolution increases with the virus diversity in the current season. The Shannon entropy of the sequence in the current season predicts relative entropy between sequences in the current season and those in the next season. Second, a global migration pattern of H3N2 is assembled by comparing the relative entropy flows of sequences sampled in China, Japan, the USA and Europe. We verify this entropy method by describing two aspects of historical H3N2 evolution. First, we identify 54 amino acid sites in HA that have evolved in the past to evade the immune system. Second, the entropy method shows that epitopes A and B on the top of HA evolve most vigorously to escape antibody binding. Our work provides a novel entropy-based method to predict and quantify future H3N2 evolution and to describe the evolutionary history of H3N2. PMID:21543352

Structure of novel rat major histocompatibility complex class II genes RT1.Ha and Hb

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

Arimura, Yutaka; Tang, Wei Ran; Koda, Toshiaki

1995-03-01

We have cloned the novel rat MHC class II genes, RT1.Ha and Hb, which are homologous to human HLA-DPA and DPB. RT1.Hb is a pseudogene, whereas RT1.Ha is apparently intact and may have transcriptional potential. In addition, with an RT1.Ha probe, we detecteda single Southern hybridization band in the genome of the mouse. This finding may aford an opportunity to analyze the HLA-DPA homologue in the mouse genome. 18 refs., 4 figs., 1 tab.

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

PubMed Central

Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J.; Peng, Deliang

2016-01-01

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi.

PubMed

Qiao, Fen; Luo, Lilian; Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J; Peng, Deliang

2016-01-01

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in

Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes

PubMed Central

Graziano, Claudio; Giorgi, Massimo; Malentacchi, Cecilia; Mattiuz, Pier Luigi; Porfirio, Berardino

2005-01-01

Background The minor histocompatibility antigens (mHags) are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD) in the recipients of bone marrow from HLA-identical donors. Two alleles have been identified in the mHag HA-1. The correlation between mismatches of the mHag HA-1 and GvHD has been suggested and methods to facilitate large-scale testing were afterwards developed. Methods We used sequence specific primer (SSP) PCR and direct sequencing to detect HA-1 gene polymorphisms in a sample of 131 unrelated Italian subjects. We then set up a novel melting temperature (Tm) assay that may help identification of HA-1 alleles without oligonucleotide probes. Results We report the frequencies of HA-1 alleles in the Italian population and the presence of an intronic 5 base-pair deletion associated with the immunogeneic allele HA-1H. We also detected novel variable sites with respect to the consensus sequence of HA-1 locus. Even though recombination/gene conversion events are documented, there is considerable linkage disequilibrium in the data. The gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism prompted us to try the Tm analysis with SYBR® Green I. We show that the addition of dimethylsulfoxide (DMSO) during the assay yields distinct patterns when amplicons from HA-1H homozygotes, HA-1R homozygotes, and heterozygotes are analysed. Conclusion The possibility to use SYBR® Green I to detect Tm differences between allelic variants is attractive but requires great caution. We succeeded in allele discrimination of the HA-1 locus using a relatively short (101 bp) amplicon, only in the presence of DMSO. We believe that, at least in certain assets, Tm assays may benefit by the addition of DMSO or other agents affecting DNA strand conformation and stability. PMID:16202172

Molecular aspects of zygotic embryogenesis in sunflower (Helianthus annuus L.): correlation of positive histone marks with HaWUS expression and putative link HaWUS/HaL1L.

PubMed

Salvini, Mariangela; Fambrini, Marco; Giorgetti, Lucia; Pugliesi, Claudio

2016-01-01

The link HaWUS/ HaL1L , the opposite transcriptional behavior, and the decrease/increase in positive histone marks bond to both genes suggest an inhibitory effect of WUS on HaL1L in sunflower zygotic embryos. In Arabidopsis, a group of transcription factors implicated in the earliest events of embryogenesis is the WUSCHEL-RELATED HOMEOBOX (WOX) protein family including WUSCHEL (WUS) and other 14 WOX protein, some of which contain a conserved WUS-box domain in addition to the homeodomain. WUS transcripts appear very early in embryogenesis, at the 16-cell embryo stage, but gradually become restricted to the center of the developing shoot apical meristem (SAM) primordium and continues to be expressed in cells of the niche/organizing center of SAM and floral meristems to maintain stem cell population. Moreover, WUS has decisive roles in the embryonic program presumably promoting the vegetative-to-embryonic transition and/or maintaining the identity of the embryonic stem cells. However, data on the direct interaction between WUS and key genes for seed development (as LEC1 and L1L) are not collected. The novelty of this report consists in the characterization of Helianthus annuus WUS (HaWUS) gene and in its analysis regarding the pattern of the methylated lysine 4 (K4) of the Histone H3 and of the acetylated histone H3 during the zygotic embryo development. Also, a parallel investigation was performed for HaL1L gene since two copies of the WUS-binding site (WUSATA), previously identified on HaL1L nucleotide sequence, were able to be bound by the HaWUS recombinant protein suggesting a not described effect of HaWUS on HaL1L transcription.

Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield

PubMed Central

Han, Qiang; Wang, Zhenzhen; He, Yunxin; Xiong, Yehui; Lv, Shun; Li, Shupeng; Zhang, Zhigang; Qiu, Dewen; Zeng, Hongmei

2017-01-01

RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3, a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA-HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing dsHaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera. Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls. PMID:28867769

Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+-ATPases with different enzymatic properties.

PubMed

Luo, Shuhong; Scott, David A; Docampo, Roberto

2002-11-15

Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T. cruzi life cycle.

Genetic diversity of influenza A(H1N1)2009 virus circulating during the season 2010-2011 in Spain.

PubMed

Ledesma, Juan; Pozo, Francisco; Reina, Gabriel; Blasco, Miriam; Rodríguez, Guadalupe; Montes, Milagrosa; López-Miragaya, Isabel; Salvador, Carmen; Reina, Jordi; Ortíz de Lejarazu, Raúl; Egido, Pilar; López Barba, José; Delgado, Concepción; Cuevas, María Teresa; Casas, Inmaculada

2012-01-01

Genetic diversity of influenza A(H1N1)2009 viruses has been reported since the pandemic virus emerged in April 2009. Different genetic clades have been identified and defined based on amino acid substitutions found in the haemagglutinin (HA) protein sequences. In Spain, circulating influenza viruses are monitored each season by the regional laboratories enrolled in the Spanish Influenza Surveillance System (SISS). The analysis of the HA gene sequence helps to detect the genetic diversity and viral evolution. To perform an analysis of the genetic diversity of influenza A(H1N1)2009 viruses circulating in Spain during the season 2010-2011 based on analysis of the HA sequence gene. Phylogenetic analysis based on the HA1 subunit of the haemagglutinin gene was carried out on 220 influenza A(H1N1)2009 viruses circulating during the season 2010-2011. Six different genetic groups were identified among circulating A(H1N1)2009 viruses, five of them were previously reported during season 2010-2011. A new group, characterized by E172K and K308E changes and a proline at position 83, was observed in 12.27% of the Spanish viruses. Co-circulation of six different genetic groups of influenza A(H1N1)2009 viruses was identified in Spain during the season 2010-2011. Nevertheless, at this stage, none of the groups identified to date have resulted in significant antigenic changes according to data collected by World Health Organization Collaborating Centres for influenza surveillance. Copyright © 2011 Elsevier B.V. All rights reserved.

Silencing the HaAK Gene by Transgenic Plant-Mediated RNAi Impairs Larval Growth of Helicoverpa armigera

PubMed Central

Liu, Feng; Wang, Xiao-Dong; Zhao, Yi-Ying; Li, Yan-Jun; Liu, Yong-Chang; Sun, Jie

2015-01-01

Insect pests have caused noticeable economic losses in agriculture, and the heavy use of insecticide to control pests not only brings the threats of insecticide resistance but also causes the great pollution to foods and the environment. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been is currently developed for protection against insect pests. In this study, we used this technology to silence the arginine kinase (AK) gene of Helicoverpa armigera (HaAK), encoding a phosphotransferase that plays a critical role in cellular energy metabolism in invertebrate. Transgenic Arabidopsis plants producing HaAK dsRNA were generated by Agrobacterium-mediated transformation. The maximal mortality rate of 55% was reached when H. armigera first-instar larvae were fed with transgenic plant leaves for 3 days, which was dramatically higher than the 18% mortality recorded in the control group. Moreover, the ingestion of transgenic plants significantly retarded larval growth, and the transcript levels of HaAK were also knocked down by up to 52%. The feeding bioassays further indicated that the inhibition efficiency was correlated with the integrity and concentration of the produced HaAK dsRNA in transgenic plants. These results strongly show that the resistance to H. armigera was improved in transgenic Arabidopsis plants, suggesting that the RNAi targeting of AK has the potential for the control of insect pests. PMID:25552931

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

PubMed

Saini, Ravi Prakash; Raman, Venkat; Dhandapani, Gurusamy; Malhotra, Era Vaidya; Sreevathsa, Rohini; Kumar, Polumetla Ananda; Sharma, Tilak R; Pattanayak, Debasis

2018-01-01

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70-80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

Phylogenetic and nucleotide sequence analysis of influenza A (H1N1) HA and NA genes of strains isolated from Saudi Arabia.

PubMed

Al-Qahtani, Ahmed Ali; Mubin, Muhammad; Dela Cruz, Damian M; Althawadi, Sahar Isa; Ul Rehman, Muhammad Shah Nawaz; Bohol, Marie Fe F; Al-Ahdal, Mohammed N

2017-01-30

In early 2009, a novel influenza A (H1N1) virus appeared in Mexico and rapidly disseminated worldwide. Little is known about the phylogeny and evolutionary dynamics of the H1N1 strain found in Saudi Arabia. Nucleotide sequencing and bioinformatics analyses were used to study molecular variation between the virus isolates. In this report, 72 hemagglutinin (HA) and 45 neuraminidase (NA) H1N1 virus gene sequences, isolated in 2009 from various regions of Saudi Arabia, were analyzed. Genetic characterization indicated that viruses from two different clades, 6 and 7, were circulating in the region, with clade 7, the most widely circulating H1N1 clade globally in 2009, being predominant. Sequence analysis of the HA and NA genes revealed a high degree of sequence identity with the corresponding genes from viruses circulating in the South East Asia region and with the A/California/7/2009 strain. New mutations in the HA gene of pandemic H1N1 (pH1N1) viruses, that could alter viral fitness, were identified. Relaxed-clock and Bayesian Skyline Plot analyses, based on the isolates used in this study and closely related globally representative strains, indicated marginally higher substitution rates than the type strain (5.14×10-3 and 4.18×10-3 substitutions/nucleotide/year in the HA and NA genes, respectively). The Saudi isolates were antigenically homogeneous and closely related to the prototype vaccine strain A/California/7/2009. The antigenic site of the HA gene had acquired novel mutations in some isolates, making continued monitoring of these viruses vital for the identification of potentially highly virulent and drug resistant variants.

The deafness gene dfna5 is crucial for ugdh expression and HA production in the developing ear in zebrafish.

PubMed

Busch-Nentwich, Elisabeth; Söllner, Christian; Roehl, Henry; Nicolson, Teresa

2004-02-01

Over 30 genes responsible for human hereditary hearing loss have been identified during the last 10 years. The proteins encoded by these genes play roles in a diverse set of cellular functions ranging from transcriptional regulation to K(+) recycling. In a few cases, the genes are novel and do not give much insight into the cellular or molecular cause for the hearing loss. Among these poorly understood deafness genes is DFNA5. How the truncation of the encoded protein DFNA5 leads to an autosomal dominant form of hearing loss is not clear. In order to understand the biological role of Dfna5, we took a reversegenetic approach in zebrafish. Here we show that morpholino antisense nucleotide knock-down of dfna5 function in zebrafish leads to disorganization of the developing semicircular canals and reduction of pharyngeal cartilage. This phenotype closely resembles previously isolated zebrafish craniofacial mutants including the mutant jekyll. jekyll encodes Ugdh [uridine 5'-diphosphate (UDP)-glucose dehydrogenase], an enzyme that is crucial for production of the extracellular matrix component hyaluronic acid (HA). In dfna5 morphants, expression of ugdh is absent in the developing ear and pharyngeal arches, and HA levels are strongly reduced in the outgrowing protrusions of the developing semicircular canals. Previous studies suggest that HA is essential for differentiating cartilage and directed outgrowth of the epithelial protrusions in the developing ear. We hypothesize that the reduction of HA production leads to uncoordinated outgrowth of the canal columns and impaired facial cartilage differentiation.

Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif

PubMed Central

Luczo, Jasmina M.; Stambas, John; Durr, Peter A.; Michalski, Wojtek P.

2015-01-01

Summary The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio‐economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host‐pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd. PMID:26467906

Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation.

PubMed

Raghavendran, Hanumantha Rao Balaji; Mohan, Saktiswaren; Genasan, Krishnamurithy; Murali, Malliga Raman; Naveen, Sangeetha Vasudevaraj; Talebian, Sepehr; McKean, Robert; Kamarul, Tunku

2016-03-01

Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of Water-Glass Coating on HA and HA-TCP Samples for MSCs Adhesion, Proliferation, and Differentiation.

PubMed

Bajpai, Indu; Kim, Duk Yeon; Kyong-Jin, Jung; Song, In-Hwan; Kim, Sukyoung

2016-01-01

Ca-P and silicon based materials have become very popular as bone tissue engineering materials. In this study, water-glass (also known as sodium silicate glass) was coated on sintered hydroxyapatite (HA) and HA-TCP (TCP stands for tricalcium phosphate) samples and subsequently heat-treated at 600°C for 2 hrs. X-rays diffraction showed the presence of β- and α-TCP phases along with HA in the HA-TCP samples. Samples without coating, with water-glass coating, and heat-treated after water-glass coating were used to observe the adhesion and proliferation response of bone marrow derived-mesenchymal stem cells (MSCs). Cell culture was carried out for 4 hrs, 1 day, and 7 days. Interestingly, all samples showed similar response for cell adhesion and proliferation up to 7-day culture but fibronectin, E-cadherin, and osteogenic differentiation related genes (osteocalcin and osteopontin) were significantly induced in heat-treated water-glass coated HA-TCP samples. A water-glass coating on Ca-P samples was not found to influence the cell proliferation response significantly but activated some extracellular matrix genes and induced osteogenic differentiation in the MSCs.

Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents.

PubMed

Fooks, A R; Jeevarajah, D; Lee, J; Warnes, A; Niewiesk, S; ter Meulen, V; Stephenson, J R; Clegg, J C

1998-05-01

The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

Mutation tryptophan to leucine at position 222 of haemagglutinin could facilitate H3N2 influenza A virus infection in dogs

PubMed Central

Yang, Guohua; Li, Shoujun; Blackmon, Sherry; Ye, Jianqiang; Bradley, Konrad C.; Cooley, Jim; Smith, Dave; Hanson, Larry; Cardona, Carol; Steinhauer, David A.; Webby, Richard; Liao, Ming

2013-01-01

An avian-like H3N2 influenza A virus (IAV) has recently caused sporadic canine influenza outbreaks in China and Korea, but the molecular mechanisms involved in the interspecies transmission of H3N2 IAV from avian to canine species are not well understood. Sequence analysis showed that residue 222 in haemagglutinin (HA) is predominantly tryptophan (W) in the closely related avian H3N2 IAV, but was leucine (L) in canine H3N2 IAV. In this study, reassortant viruses rH3N2-222L (canine-like) and rH3N2-222W (avian-like) with HA mutation L222W were generated using reverse genetics to evaluate the significance of the L222W mutation on receptor binding and host tropism of H3N2 IAV. Compared with rH3N2-222W, rH3N2-222L grew more rapidly in MDCK cells and had significantly higher infectivity in primary canine tracheal epithelial cells. Tissue-binding assays demonstrated that rH3N2-222L had a preference for canine tracheal tissues rather avian tracheal tissues, whereas rH3N2-222W favoured slightly avian rather canine tracheal tissues. Glycan microarray analysis suggested both rH3N2-222L and rH3N2-222W bound preferentially to α2,3-linked sialic acids. However, the rH3N2-222W had more than twofold less binding affinity than rH3N2-222L to a set of glycans with Neu5Aca2–3Galb1–4(Fuca-)-like or Neu5Aca2–3Galb1–3(Fuca-)-like structures. These data suggest the W to L mutation at position 222 of the HA could facilitate infection of H3N2 IAV in dogs, possibly by increasing the binding affinities of the HA to specific receptors with Neu5Aca2–3Galb1–4(Fuca-) or Neu5Aca2–3Galb1–3(Fuca-)-like structures that are present in dogs. PMID:23994833

Phylogenetic relationships of the HA and NA genes between vaccine and seasonal influenza A(H3N2) strains in Korea

PubMed Central

Park, Sehee; Bae, Joon-Yong; Yoo, Kirim; Cheong, Hee Jin; Noh, Ji Yun; Hong, Kyung Wook; Lemey, Philippe; Vrancken, Bram; Kim, Juwon; Nam, Misun; Yun, Soo-Hyeon; Cho, Woo In; Song, Joon Young; Kim, Woo Joo; Park, Mee Sook; Song, Jin-Won; Kee, Sun-Ho; Song, Ki-Joon; Park, Man-Seong

2017-01-01

Seasonal influenza is caused by two influenza A subtype (H1N1 and H3N2) and two influenza B lineage (Victoria and Yamagata) viruses. Of these antigenically distinct viruses, the H3N2 virus was consistently detected in substantial proportions in Korea during the 2010/11-2013/14 seasons when compared to the other viruses and appeared responsible for the influenza-like illness rate peak during the first half of the 2011/12 season. To further scrutinize possible causes for this, we investigated the evolutionary and serological relationships between the vaccine and Korean H3N2 strains during the 2011/12 season for the main antigenic determinants of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA) genes. In the 2011/12 season, when the number of H3N2 cases peaked, the majority of the Korean strains did not belong to the HA clade of A/Perth/16/2009 vaccine, and no Korean strains were of this lineage in the NA segment. In a serological assay, post-vaccinated human sera exhibited much reduced hemagglutination inhibition antibody titers against the non-vaccine clade Korean H3N2 strains. Moreover, Korean strains harbored several amino acid differences in the HA antigenic sites and in the NA with respect to vaccine lineages during this season. Of these, the HA antigenic site C residues 45 and 261 and the NA residue 81 appeared to be the signatures of positive selection. In subsequent seasons, when H3N2 cases were lower, the HA and NA genes of vaccine and Korean strains were more phylogenetically related to each other. Combined, our results provide indirect support for using phylogenetic clustering patterns of the HA and possibly also the NA genes in the selection of vaccine viruses and the assessment of vaccine effectiveness. PMID:28257427

In-Depth Analysis of HA and NS1 Genes in A(H1N1)pdm09 Infected Patients.

PubMed

Caglioti, Claudia; Selleri, Marina; Rozera, Gabriella; Giombini, Emanuela; Zaccaro, Paola; Valli, Maria Beatrice; Capobianchi, Maria Rosaria

2016-01-01

In March/April 2009, a new pandemic influenza A virus (A(H1N1)pdm09) emerged and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century. Influenza virus may be present in the infected host as a mixture of variants, referred to as quasi-species, on which natural and immune-driven selection operates. Since hemagglutinin (HA) and non-structural 1 (NS1) proteins are relevant in respect of adaptive and innate immune responses, the present study was aimed at establishing the intra-host genetic heterogeneity of HA and NS1 genes, applying ultra-deep pyrosequencing (UDPS) to nasopharyngeal swabs (NPS) from patients with confirmed influenza A(H1N1)pdm09 infection. The intra-patient nucleotide diversity of HA was significantly higher than that of NS1 (median (IQR): 37.9 (32.8-42.3) X 10-4 vs 30.6 (27.4-33.6) X 10-4 substitutions/site, p = 0.024); no significant correlation for nucleotide diversity of NS1 and HA was observed (r = 0.319, p = 0.29). Furthermore, a strong inverse correlation between nucleotide diversity of NS1 and viral load was observed (r = - 0.74, p = 0.004). For both HA and NS1, the variants appeared scattered along the genes, thus indicating no privileged mutation site. Known polymorphisms, S203T (HA) and I123V (NS1), were observed as dominant variants (>98%) in almost all patients; three HA and two NS1 further variants were observed at frequency >40%; a number of additional variants were detected at frequency <6% (minority variants), of which three HA and four NS1 variants were novel. In few patients multiple variants were observed at HA residues 203 and 222. According to the FLUSURVER tool, some of these variants may affect immune recognition and host range; however, these inferences are based on H5N1, and their extension to A(H1N1)pdm09 requires caution. More studies are necessary to address the significance of the composite nature of influenza virus quasi-species within infected patients.

Distribution of gene segments of the pandemic A(H1N1) 2009 virus lineage in pig populations.

PubMed

Okuya, K; Matsuu, A; Kawabata, T; Koike, F; Ito, M; Furuya, T; Taneno, A; Akimoto, S; Deguchi, E; Ozawa, M

2018-05-06

Swine influenza viruses (SIVs) are important not only for pig farming, but also for public health. In fact, pandemic A(H1N1) 2009 viruses [A(H1N1)pdm09] were derived from SIVs. Therefore, timely characterization of locally circulating SIVs is necessary for understanding the global status of SIVs. To genetically characterize SIVs circulating in Japanese pig populations, we isolated 24 SIVs of three subtypes (17 H1N1, four H1N2 and three H3N2 strains) from 14 pig farms in Japan from 2013 to 2016. Genetic analyses revealed that the haemagglutinin (HA) and neuraminidase (NA) genes of the 17 H1N1 and the HA gene of one H1N2, A/swine/Aichi/02/2016 (H1N2), SIVs belonged to the A(H1N1)pdm09 lineage. More importantly, all of the remaining six gene segments (i.e., PB1, PB1, PA, NP, M and NS) of the 24 SIVs, regardless of the HA and NA subtype, were also classified as belonging to the A(H1N1)pdm09 lineage. These results indicate that gene segments of A(H1N1)pdm09 lineage are widely distributed in SIVs circulating in Japanese pig populations In addition, the NA gene of A/swine/Aichi/02/2016 (H1N2) shared less than 88.5% nucleotide identity with that of the closest relative A/swine/Miyagi/5/2003 (H1N2), which was isolated in Japan in 2003. These results indicate the sustained circulation of classical H1N2-derived SIVs with remarkable diversity in the NA genes in Japanese pig populations. These findings highlight the necessity of both intensive biosecurity systems and active SIV surveillance in pig populations worldwide for both animal and public health. © 2018 Blackwell Verlag GmbH.

Topical stabilized retinol treatment induces the expression of HAS genes and HA production in human skin in vitro and in vivo.

PubMed

Li, Wen-Hwa; Wong, Heng-Kuan; Serrano, José; Randhawa, Manpreet; Kaur, Simarna; Southall, Michael D; Parsa, Ramine

2017-05-01

Skin Aging manifests primarily with wrinkles, dyspigmentations, texture changes, and loss of elasticity. During the skin aging process, there is a loss of moisture and elasticity in skin resulting in loss of firmness finally leading to skin sagging. The key molecule involved in skin moisture is hyaluronic acid (HA), which has a significant water-binding capacity. HA levels in skin decline with age resulting in decrease in skin moisture, which may contribute to loss of firmness. Clinical trials have shown that topically applied ROL effectively reduces wrinkles and helps retain youthful appearance. In the current study, ROL was shown to induce HA production and stimulates the gene expression of all three forms of hyaluronic acid synthases (HAS) in normal human epidermal keratinocytes monolayer cultures. Moreover, in human skin equivalent tissues and in human skin explants, topical treatment of tissues with a stabilized-ROL formulation significantly induced the gene expression of HAS mRNA concomitant with an increased HA production. Finally, in a vehicle-controlled human clinical study, histochemical analysis confirmed increased HA accumulation in the epidermis in ROL-treated human skin as compared to vehicle. These results show that ROL increases skin expression of HA, a significant contributing factor responsible for wrinkle formation and skin moisture, which decrease during aging. Taken together with the activity to increase collagen, elastin, and cell proliferation, these studies establish that retinol provides multi-functional activity for photodamaged skin.

A genotype network reveals homoplastic cycles of convergent evolution in influenza A (H3N2) haemagglutinin.

PubMed

Wagner, Andreas

2014-07-07

Networks of evolving genotypes can be constructed from the worldwide time-resolved genotyping of pathogens like influenza viruses. Such genotype networks are graphs where neighbouring vertices (viral strains) differ in a single nucleotide or amino acid. A rich trove of network analysis methods can help understand the evolutionary dynamics reflected in the structure of these networks. Here, I analyse a genotype network comprising hundreds of influenza A (H3N2) haemagglutinin genes. The network is rife with cycles that reflect non-random parallel or convergent (homoplastic) evolution. These cycles also show patterns of sequence change characteristic for strong and local evolutionary constraints, positive selection and mutation-limited evolution. Such cycles would not be visible on a phylogenetic tree, illustrating that genotype network analysis can complement phylogenetic analyses. The network also shows a distinct modular or community structure that reflects temporal more than spatial proximity of viral strains, where lowly connected bridge strains connect different modules. These and other organizational patterns illustrate that genotype networks can help us study evolution in action at an unprecedented level of resolution. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from giant panda and raccoon dogs in China

PubMed Central

2013-01-01

Background Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. In this study, we sequenced and phylogenetic analyses of the hemagglutinin (H) genes from eight canine distemper virus (CDV) isolates obtained from seven raccoon dogs (Nyctereutes procyonoides) and a giant panda (Ailuropoda melanoleuca) in China. Results Phylogenetic analysis of the partial hemagglutinin gene sequences showed close clustering for geographic lineages, clearly distinct from vaccine strains and other wild-type foreign CDV strains, all the CDV strains were characterized as Asia-1 genotype and were highly similar to each other (91.5-99.8% nt and 94.4-99.8% aa). The giant panda and raccoon dogs all were 549Y on the HA protein in this study, irrespective of the host species. Conclusions These findings enhance our knowledge of the genetic characteristics of Chinese CDV isolates, and may facilitate the development of effective strategies for monitoring and controlling CDV for wild canids and non-cainds in China. PMID:23566727

Single gene reassortants identify a critical role for PB1, HA, and NA in the high virulence of the 1918 pandemic influenza virus

PubMed Central

Pappas, Claudia; Aguilar, Patricia V.; Basler, Christopher F.; Solórzano, Alicia; Zeng, Hui; Perrone, Lucy A.; Palese, Peter; García-Sastre, Adolfo; Katz, Jacqueline M.; Tumpey, Terrence M.

2008-01-01

The 1918 influenza pandemic was exceptionally severe, resulting in the death of up to 50 million people worldwide. Here, we show which virus genes contributed to the replication and virulence of the 1918 influenza virus. Recombinant viruses, in which genes of the 1918 virus were replaced with genes from a contemporary human H1N1 influenza virus, A/Texas/36/91 (Tx/91), were generated. The exchange of most 1918 influenza virus genes with seasonal influenza H1N1 virus genes did not alter the virulence of the 1918 virus; however, substitution of the hemagglutinin (HA), neuraminidase (NA), or polymerase subunit PB1 genes significantly affected the ability of this virus to cause severe disease in mice. The 1918 virus virulence observed in mice correlated with the ability of 1918 recombinant viruses to replicate efficiently in human airway cells. In a second series of experiments, eight 1918 1:7 recombinants were generated, in which each Tx/91 virus gene was individually replaced by a corresponding gene from 1918 virus. Replication capacity of the individual 1:7 reassortant viruses was assessed in mouse lungs and human airway cells. Increased virus titers were observed among 1:7 viruses containing individual 1918 HA, NA, and PB1 genes. In addition, the 1918 PB1:Tx/91 (1:7) virus showed a distinctly larger plaque size phenotype than the small plaque phenotype of the 1918 PA:Tx/91 and 1918 PB2:Tx/91 1:7 reassortants. These results highlight the importance of the 1918 HA, NA, and PB1 genes for optimal virus replication and virulence of this pandemic strain. PMID:18287069

Androgen regulation of the human hair follicle: the type I hair keratin hHa7 is a direct target gene in trichocytes.

PubMed

Jave-Suarez, Luis F; Langbein, Lutz; Winter, Hermelita; Praetzel, Silke; Rogers, Michael A; Schweizer, Juergen

2004-03-01

Previous work had shown that most members of the complex human hair keratin family were expressed in terminal scalp hairs. An exception to this rule was the type I hair keratin hHa7, which was only detected in some but not all vellus hairs of the human scalp (Langbein et al, 1999). Here we show that hHa7 exhibits constitutive expression in medullary cells of all types of male and female sexual hairs. Medullated beard, axillary, and pubic hairs arise during puberty from small, unmedullated vellus hairs under the influence of circulating androgens. This suggested an androgen-controlled expression of the hHa7 gene. Further evidence for this assumption was provided by the demonstration of androgen receptor (AR) expression in the nuclei of medullary cells of beard hairs. Moreover, homology search for the semipalindromic androgen receptor-binding element (ARE) consensus sequence GG(A)/(T)ACAnnnTGTTCT in the proximal hHa7 promoter revealed three putative ARE motifs. Electrophoretic mobility shift assays demonstrated the specific binding of AR to all three hHa7 AREs. Their function as AR-responsive elements, either individually or in concert within the hHa7 promoter, could be further confirmed by transfection studies with or without an AR expression vector in PtK2 and prostate PC3-Arwt cells, respectively in the presence or absence of a synthetic androgen. Our study detected hHa7 as the first gene in hair follicle trichocytes whose expression appears to be directly regulated by androgens. As such, hHa7 represents a marker for androgen action on hair follicles and might be a suitable tool for investigations of androgen-dependent hair disorders.

Isolation and phylogenetic characterization of haemagglutinin and neuraminidase genes of H9N2 low pathogenicity avian influenza virus isolated from commercial layers in India.

PubMed

Gowthaman, Vasudevan; Singh, Shambu Dayal; Dhama, Kuldeep; Srinivasan, Palani; Saravanan, Sellappan; Murthy, Thippichettypalayam Ramasamy Gopala Krishna; Sukumar, Kuppanan; Mathapati, Basavaraj; Lebarbenchon, Camille; Malik, Yashpal Singh; Ramakrishnan, Muthannan Andavar

2016-12-01

Avian influenza is a highly infectious and dynamically evolving disease of birds causing high morbidity and mortality. It is caused by avian influenza virus (AIV) that belongs to the family Orthomyxoviridae. Two types of AIV have been described based on their pathogenicity viz. highly pathogenic avian influenza virus that causes severe disease with high mortality and low pathogenic avian influenza virus (LPAI) that generally causes asymptomatic infection or a mild disease. The H9N2 subtype is the widely circulated LPAI type in the world. The H9N2 subtype of was first reported from northern India in March 2003. However, systematical surveillance information for the evolution of H9N2 viruses in poultry flocks of Southern India is lacking. The present study reports the isolation and characterization of H9N2 isolates from the southern parts of the country during the period between May 2010 and September 2011. Out of the 30 poultry flocks investigated, six were found to be positive for HA activity. Further, all the six samples conformed as AIV. Partial nucleotide sequencing of the HA and NA genes revealed that all were belonging to the H9N2 subtype. Phylogenetically, the HA and NA genes of the H9N2 viruses from India clustered with those isolated from Bangladesh, Pakistan and the Middle East, although we were not able to conclude on their exact geographic origin.

Influenza A virus subtypes in wild birds in North-Eastern Spain (Catalonia).

PubMed

Busquets, Núria; Alba, Anna; Napp, Sebastián; Sánchez, Azucena; Serrano, Erika; Rivas, Raquel; Núñez, José I; Majó, Natàlia

2010-04-01

Since the spread of H5N1 highly pathogenic avian influenza virus in 2005, many surveillance programmes have been initiated in poultry and wild birds worldwide. This study describes for the first time the detection of different subtypes of avian influenza viruses (AIV) in wild birds in the West Mediterranean area (Catalonia, North-Eastern Spain). During a 3-year period (from mid-2006 to mid-2009), 1374 birds from 16 different families were examined, and a total of 62 AIV were detected by means of a real-time reverse transcriptase PCR assay. AIV were more frequently detected in Anatidae, Phoenicopteridae, Rallidae and Laridae families. Of the 62 positive samples, 28 AIV could be isolated in embryonated eggs. All isolates were subtyped by haemagglutinin and neuraminidase inhibition techniques and 10 different haemagglutinins (HA) and 7 neuraminidases (NA) were found in 13 different subtype combinations. The most common combinations were H4N6 (22.2%) and H1N1 (18.5%). The HA and NA gene sequences of different AIV subtypes were compared and aligned with those available AIV strains from genome databases. Our studies on AIV phylogenetic analysis revealed that all AIV genes sequenced from wild birds in North-Eastern Spain clustered within Eurasian avian clades, including the sequences of H8, N4 and N5 genes analyzed for the first time in Europe. The results contribute to the understanding of AIV in the Mediterranean area and in Europe. Copyright 2009 Elsevier B.V. All rights reserved.

Activity-guided separation and characterization of new halocin HA3 from fermented broth of Haloferax larsenii HA3.

PubMed

Kumar, Vijay; Tiwari, Santosh Kumar

2017-05-01

Haloferax larsenii HA3 was able to grow optimally in HS medium containing 15% NaCl, at pH 7.2 and 42 °C in aerobic conditions. Strain HA3 was found to be round shape, Gram-negative, catalase-positive, sensitive to bile acid, and resistant to chloramphenicol, and could not utilize arginine. The lipid profile revealed the presence of glycerol diether moiety (GDEM) suggesting Haloarchaea characteristics. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that it was closely related to H. larsenii ZJ206. Interestingly, strain HA3 was found to produce halocin HA3 which was purified using ultrafiltration and chromatography. It was found to be stable up to 80 °C, pH 2.0-10.0, organic solvents, surfactants, and detergents tested. However, the activity of halocin HA3 was completely reduced in the presence of proteinase K and trypsin. It was found to be halocidal against H. larsenii HA10, rupturing cell boundary and leading to cell death. The molecular weight of halocin HA3 was found to be ~13 kDa and MALDI-TOF MS/MS analysis suggested no homology with known halocins. The N-terminal ten amino-acid residues, NH 2 MNLGIILETN-COOH, suggested a new/novel halocin. These properties of halocin HA3 may be applicable for control of Haloarchaea in environments and salted foods.

[Construction of a recombinant HVT virus expressing the HA gene of avian influenza virus H5N1 via Rde/ET recombination system].

PubMed

Lan, Desong; Shi, Xingming; Wang, Yunfeng; Liu, Changjun; Wang, Mei; Cui, Hongyu; Tian, Guobin; Li, Jisong; Tong, Guangzhi

2009-01-01

In recent years,manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). With these BAC clones in hand,we manipulated the genome of HVT by utilizing Red/ET recombination system, and developed a biologically safe live vaccine based on the HVT BACs. In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs,and added inducer L-arabinose into the cells. We prepared the cells into competent cells and electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms into the cells. So the functional cassette was inserted into the U(S)2 locus. Only colonies carrying the modified BAC would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin (HA) gene of (HPAIV) A/Goose/ Guangdong/1/96(H5N1) flanked by the same homology arms. Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates. Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the U(S)2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts (CEFs). We achieved one rescued recombinant virus which designated as rHVT-HA3. The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.

Black rice (Oryza sativa L. var. japonica) hydrolyzed peptides induce expression of hyaluronan synthase 2 gene in HaCaT keratinocytes.

PubMed

Sim, Gwan Sub; Lee, Dong-Hwan; Kim, Jin-Hwa; An, Sung-Kwan; Choe, Tae-Boo; Kwon, Tae-Jong; Pyo, Hyeong-Bae; Lee, Bum-Chun

2007-02-01

Black rice (Oryza sativa L. var. japonica) has been used in folk medicine in Asia. To understand the effects of black rice hydrolyzed peptides (BRP) from germinated black rice, we assessed the expression levels of about 20,000 transcripts in BRP-treated HaCaT keratinocytes using human 1A oligo microarray analysis. As a result, the BRP treatment showed a differential expression ratio of more than 2-fold: 745 were activated and 1,011 were repressed. One of the most interesting findings was a 2-fold increase in hyaluronan synthase 2 (HAS2) gene expression by BRP. Semiquantitative RT-PCR showed that BRP increased HAS2 mRNA in dose-dependent manners. ELISA showed that BRP effectively increased hyaluronan (HA) production in HaCaT keratinocytes.

Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion.

PubMed

Peacock, Thomas P; Benton, Donald J; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; McCauley, John W; Barclay, Wendy S; Iqbal, Munir

2017-03-22

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the 'Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages.

Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion

PubMed Central

Peacock, Thomas P; Benton, Donald J; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; McCauley, John W; Barclay, Wendy S; Iqbal, Munir

2017-01-01

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the ‘Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages. PMID:28325922

HA222 polymorphism in Influenza A(H1N1) 2009 isolates from Intensive Care Units and ambulatory patients during three influenza seasons.

PubMed

Corcioli, F; Arvia, R; Pierucci, F; Clausi, V; Bonizzoli, M; Peris, A; Azzi, A

2014-02-13

Amino acid substitutions which can affect the receptor binding specificity of the influenza virus, like the substitution of aspartic acid with glycine in position 222 of the haemagglutinin (HA) of influenza virus A(H1N1) 2009, have been associated with increased viral pathogenicity and increased tropism for the lower respiratory tract. In this paper, the polymorphic site 222 and the site 223 of the HA1 polypeptide of H1N1 2009 viruses were analyzed in order to better clarify the role of these substitutions in H1N1 2009 virus virulence. Viral strains included in this study were collected in Tuscany during 3 different influenza seasons from patients with severe as well as with mild forms of influenza caused by A(H1N1) 2009 virus. In addition, the oseltamivir resistance of the H1N1 2009 strains circulating during the same seasons was monitored with the aim to evaluate whether these changes in the HA and in neuraminidase (NA) tend to be linked and to influence each other. Altogether, the results indicate that in severe forms of influenza viral population is more variable than in mild influenza, as regards the site 222. The frequency of such substitutions varied among the three seasons, it was highest in the season 2010-2011 and very low in the season 2012-2013. However these differences were not significant. Copyright © 2013 Elsevier B.V. All rights reserved.

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

PubMed

Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

2015-01-02

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhanced humoural and cellular immune responses to influenza H7N9 antigen HA1-2 fused with flagellin in chickens.

PubMed

Song, Li; Xiong, Dan; Hu, Maozhi; Kang, Xilong; Pan, Zhiming; Jiao, Xinan

2017-06-21

Sudden increases in the number of human A (H7N9) cases reported during December and January have been observed in previous years. Most reported infection cases are due to prior exposure to live poultry or potentially contaminated environments. Low pathogenicity of influenza A (H7N9) virus in avian species complicates timely discovery of infected birds. Therefore, there is a pressing need to develop safe and effective anti-H7N9 vaccines for poultry to reduce the risk of human infection and prevent the emergence of novel mutated strains. In addition to a good antigen, an effective vaccine also requires an appropriate adjuvant to enhance its immunogenicity. Previously, we generated an H7N9 influenza recombinant subunit vaccine (HA1-2-fliC), in which haemagglutinin globular head domain (HA1-2) was fused with flagellin (fliC), a potent TLR5 ligand, and demonstrated that HA1-2-fliC elicited effective HA1-2-specific immune responses in mice. In this study, we determined flagellin-induced expression profiles of cytokines and chemokines in different types of avian immune cells in vitro and ex vivo. We found that flagellin significantly increased the expression levels of CXCL inflammatory chemokines (CXCLi1 and CXCLi2) and CCL chemokines (MIP-1β and MCP-3) in avian macrophage HD11 cells. In addition, HA1-2-fliC induced significant upregulation of cytokines (IL-1β, IL-6, IL-18 and IFN-γ) and chemokines (CXCLi1, CXCLi2 and MIP-1β) in ex vivo splenic lymphocytes and peripheral blood mononuclear cells (PBMCs), suggesting that flagellin promoted immune responses of avian cells in vitro. We also evaluated specific humoural and cellular immune responses induced by HA1-2-fliC and found that chickens immunised intramuscularly with HA1-2-fliC showed significantly higher HA1-2-specific immunoglobulin (Ig)G titers in serum. Furthermore, HA1-2-fliC potentiated cellular immune responses, as reflected by an increase in CD4 + and CD8 + T cells and proliferation of PBMCs. Significantly

Reassortment between Influenza B Lineages and the Emergence of a Coadapted PB1–PB2–HA Gene Complex

PubMed Central

Dudas, Gytis; Bedford, Trevor; Lycett, Samantha; Rambaut, Andrew

2015-01-01

Influenza B viruses make a considerable contribution to morbidity attributed to seasonal influenza. Currently circulating influenza B isolates are known to belong to two antigenically distinct lineages referred to as B/Victoria and B/Yamagata. Frequent exchange of genomic segments of these two lineages has been noted in the past, but the observed patterns of reassortment have not been formalized in detail. We investigate interlineage reassortments by comparing phylogenetic trees across genomic segments. Our analyses indicate that of the eight segments of influenza B viruses only segments coding for polymerase basic 1 and 2 (PB1 and PB2) and hemagglutinin (HA) proteins have maintained separate Victoria and Yamagata lineages and that currently circulating strains possess PB1, PB2, and HA segments derived entirely from one or the other lineage; other segments have repeatedly reassorted between lineages thereby reducing genetic diversity. We argue that this difference between segments is due to selection against reassortant viruses with mixed-lineage PB1, PB2, and HA segments. Given sufficient time and continued recruitment to the reassortment-isolated PB1–PB2–HA gene complex, we expect influenza B viruses to eventually undergo sympatric speciation. PMID:25323575

Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression.

PubMed Central

Brown, K; Buchmann, A; Balmain, A

1990-01-01

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz[a]anthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G ---- A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G ---- T and codon 61 A ---- T transversions in papillomas; only the G ---- T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy. Images PMID:2105486

Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells.

PubMed

Stanhill, A; Levin, V; Hendel, A; Shachar, I; Kazanov, D; Arber, N; Kaminski, N; Engelberg, D

2006-03-09

Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

PubMed

Hidema, Shizu; Fukuda, Tomokazu; Hiraoka, Yuichi; Mizukami, Hiroaki; Hayashi, Ryotaro; Otsuka, Ayano; Suzuki, Shingo; Miyazaki, Shinji; Nishimori, Katsuhiko

2016-05-01

The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR. © 2015 Wiley Periodicals, Inc.

Mutations in the haemagglutinin protein and their effect in transmission of highly pathogenic avian influenza (HPAI) H5N1 virus in sub-optimally vaccinated chickens.

PubMed

Sitaras, Ioannis; Rousou, Xanthoula; Peeters, Ben; de Jong, Mart C M

2016-11-04

Transmission of highly pathogenic avian influenza (HPAI) viruses in poultry flocks is associated with huge economic losses, culling of millions of birds, as well as human infections and deaths. In the cases where vaccination against avian influenza is used as a control measure, it has been found to be ineffective in preventing transmission of field strains. Reports suggest that one of the reasons for this is the use of vaccine doses much lower than the ones recommended by the manufacturer, resulting in very low levels of immunity. In a previous study, we selected for immune escape mutants using homologous polyclonal sera and used them as vaccines in transmission experiments. We concluded that provided a threshold of immunity is reached, antigenic distance between vaccine and challenge strains due to selection need not result in vaccine escape. Here, we evaluate the effect that the mutations in the haemagglutinin protein of our most antigenically-distant mutant may have in the transmission efficiency of this mutant to chickens vaccinated against the parent strain, under sub-optimal vaccination conditions resembling those often found in the field. In this study we employed reverse genetics techniques and transmission experiments to examine if the HA mutations of our most antigenically-distant mutant affect its efficiency to transmit to vaccinated chickens. In addition, we simulated sub-optimal vaccination conditions in the field, by using a very low vaccine dose. We find that the mutations in the HA protein of our most antigenically-distant mutant are not enough to allow it to evade even low levels of vaccination-induced immunity. Our results suggest that - for the antigenic distances we investigated - vaccination can reduce transmission of an antigenically-distant strain compared to the unvaccinated groups, even when low vaccine doses are used, resulting in low levels of immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Blinded study determination of high sensitivity and specificity microchip electrophoresis-SSCP/HA to detect mutations in the p53 gene.

PubMed

Hestekin, Christa N; Lin, Jennifer S; Senderowicz, Lionel; Jakupciak, John P; O'Connell, Catherine; Rademaker, Alfred; Barron, Annelise E

2011-11-01

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)-SSCP/HA. We demonstrate the ability of ME-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5-9 in a blinded study in an analysis time of <10 min. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Blinded study determination of high sensitivity and specificity microchip electrophoresis–SSCP/HA to detect mutations in the p53 gene

PubMed Central

Hestekin, Christa N.; Lin, Jennifer S.; Senderowicz, Lionel; Jakupciak, John P.; O’Connell, Catherine; Rademaker, Alfred; Barron, Annelise E.

2012-01-01

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here we demonstrate the first blinded study using microchip electrophoresis-SSCP/HA. We demonstrate the ability of microchip electrophoresis-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5–9 in a blinded study in an analysis time of less than 10 minutes. PMID:22002021

Nanodiamond enhances immune responses in mice against recombinant HA/H7N9 protein.

PubMed

Pham, Ngoc Bich; Ho, Thuong Thi; Nguyen, Giang Thu; Le, Thuy Thi; Le, Ngoc Thu; Chang, Huan-Cheng; Pham, Minh Dinh; Conrad, Udo; Chu, Ha Hoang

2017-10-05

The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). These results

Mumps virus F gene and HN gene sequencing as a molecular tool to study mumps virus transmission.

PubMed

Gouma, Sigrid; Cremer, Jeroen; Parkkali, Saara; Veldhuijzen, Irene; van Binnendijk, Rob S; Koopmans, Marion P G

2016-11-01

Various mumps outbreaks have occurred in the Netherlands since 2004, particularly among persons who had received 2 doses of measles, mumps, and rubella (MMR) vaccination. Genomic typing of pathogens can be used to track outbreaks, but the established genotyping of mumps virus based on the small hydrophobic (SH) gene sequences did not provide sufficient resolution. Therefore, we expanded the sequencing to include fusion (F) gene and haemagglutinin-neuraminidase (HN) gene sequences in addition to the SH gene sequences from 109 mumps virus genotype G strains obtained between 2004 and mid 2015 in the Netherlands. When the molecular information from these 3 genes was combined, we were able to identify separate mumps virus clusters and track mumps virus transmission. The analyses suggested that multiple mumps virus introductions occurred in the Netherlands between 2004 and 2015 resulting in several mumps outbreaks throughout this period, whereas during some local outbreaks the molecular data pointed towards endemic circulation. Combined analysis of epidemiological data and sequence data collected in 2015 showed good support for the phylogenetic clustering. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of the interaction between subunits of the botulinum toxin complex produced by serotype D through tryptic susceptibility of the isolated components and complex forms.

PubMed

Suzuki, Tomonori; Watanabe, Toshihiro; Mutoh, Shingo; Hasegawa, Kimiko; Kouguchi, Hirokazu; Sagane, Yoshimasa; Fujinaga, Yukako; Oguma, Keiji; Ohyama, Tohru

2005-05-01

The 650 kDa large toxin complex (L-TC) produced by Clostridium botulinum serotype D strain 4947 (D-4947) has a subunit structure composed of unnicked components, i.e. neurotoxin (NT), non-toxic non-haemagglutinin (NTNHA) and three haemagglutinin subcomponents (HA-70, HA-33 and HA-17). In this study, subunit interactions were investigated through the susceptibilities of the toxin components to limited trypsin proteolysis. Additionally, complex forms were reconstituted in vitro by various combinations of individual components. Trypsin treatment of intact D-4947 L-TC led to the formation of mature L-TC with nicks at specific sites of each component, which is usually observed in other strains of serotype D. NT, NTNHA and HA-17 were cleaved at their specific sites in either the single or complex forms, but HA-33 showed no sign of proteolysis. Unlike the other components, HA-70 was digested into random fragments as a single form, but it was cleaved into two fragments in the complex form. Based on the relative position of exposed or hidden regions of the individual components in the complex derived from their tryptic susceptibilities, an assembly model is proposed for the arrangement of individual subunits in the botulinum L-TC.

Phylogenetic Analysis of H6 Influenza Viruses Isolated from Rosy-Billed Pochards (Netta peposaca) in Argentina Reveals the Presence of Different HA Gene Clusters ▿

PubMed Central

Rimondi, Agustina; Xu, Kemin; Craig, Maria Isabel; Shao, Hongxia; Ferreyra, Hebe; Rago, Maria Virginia; Romano, Marcelo; Uhart, Marcela; Sutton, Troy; Ferrero, Andrea; Perez, Daniel R.; Pereda, Ariel

2011-01-01

Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. We report the characterization of 5 avian influenza viruses of the H6 hemagglutinin (HA) subtype isolated from rosy-billed pochards (Netta peposaca). Three of these viruses were paired to an N2 NA subtype, while the other two were of the N8 subtype. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The presence of NS alleles A and B was also identified. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. Animal studies suggested that these Argentine H6 viruses could replicate and transmit inefficiently in chickens, indicating limited adaptation to poultry. Our results highlight the importance of continued influenza virus surveillance in wild birds of South America, especially considering the unique evolution of these viruses. PMID:21976652

Osteoinductive-nanoscaled silk/HA composite scaffolds for bone tissue engineering application.

PubMed

Huang, Xiaowei; Bai, Shumeng; Lu, Qiang; Liu, Xi; Liu, Shanshan; Zhu, Hesun

2015-10-01

Osteoinductive silk/hydroxyapatite (HA) composite scaffolds for bone regeneration were prepared by combining silk with HA/silk core-shell nanoparticles. The HA/silk nanoparticles were directly dispersed in silk solution to form uniform silk/HA blend and then composite scaffolds after a freeze-drying process. The HA/silk nanoparticles uniformly distributed in silk scaffolds at nanometer scale at varying HA content up to 40%, and substantially improved the compressive strength of the scaffolds produced. Rat bone mesenchymal stem cells (rBMSCs) were cultured in these scaffolds and cell proliferation was analyzed by confocal microscopy and DNA assay. Gene expression and biochemical assays were employed to study the influence of increasing HA/silk nanoparticles on in vitro osteogenic differentiation of rBMSCs. Increasing HA/silk nanoparticles inside silk scaffolds improved the growth and osteogenic capability of rBMSCs in the absence of osteogenic growth factors, and also significantly increased the calcium and collagen I deposition. In addition, compared to silk/HA composite scaffolds containing HA aggregates, the scaffolds loaded with HA/silk nanoparticles showed remarkably higher stiffness and better osteogenic property at same HA content, implying a preferable microenvironment for rBMSCs. These results suggest that the osteogenic property as well as mechanical property of silk/HA scaffolds could be further improved through fabricating their structure and topography at nanometer scale, providing more suitable systems for bone regeneration. © 2014 Wiley Periodicals, Inc.

The PA and HA Gene-Mediated High Viral Load and Intense Innate Immune Response in the Brain Contribute to the High Pathogenicity of H5N1 Avian Influenza Virus in Mallard Ducks

PubMed Central

Hu, Jiao; Hu, Zenglei; Mo, Yiqun; Wu, Qiwen; Cui, Zhu; Duan, Zhiqiang; Huang, Junqing; Chen, Hongzhi; Chen, Yuxin; Gu, Min; Wang, Xiaoquan; Hu, Shunlin; Liu, Huimou; Liu, Wenbo; Liu, Xiaowen

2013-01-01

Most highly pathogenic avian influenza A viruses cause only mild clinical signs in ducks, serving as an important natural reservoir of influenza A viruses. However, we isolated two H5N1 viruses that are genetically similar but differ greatly in virulence in ducks. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is low pathogenic. To determine the genetic basis for the high virulence of CK10 in ducks, we generated a series of single-gene reassortants between CK10 and GS10 and tested their virulence in ducks. Expression of the CK10 PA or hemagglutinin (HA) gene in the GS10 context resulted in increased virulence and virus replication. Conversely, inclusion of the GS10 PA or HA gene in the CK10 background attenuated the virulence and virus replication. Moreover, the PA gene had a greater contribution. We further determined that residues 101G and 237E in the PA gene contribute to the high virulence of CK10. Mutations at these two positions produced changes in virulence, virus replication, and polymerase activity of CK10 or GS10. Position 237 plays a greater role in determining these phenotypes. Moreover, the K237E mutation in the GS10 PA gene increased PA nuclear accumulation. Mutant GS10 viruses carrying the CK10 HA gene or the PA101G or PA237E mutation induced an enhanced innate immune response. A sustained innate response was detected in the brain rather than in the lung and spleen. Our results suggest that the PA and HA gene-mediated high virus replication and the intense innate immune response in the brain contribute to the high virulence of H5N1 virus in ducks. PMID:23926340

Characterization and ecology of a type A influenzavirus isolated from a shearwater

PubMed Central

Downie, Jean C.; Webster, R. G.; Schild, G. C.; Dowdle, Walter R.; Laver, W. G.

1973-01-01

An influenzavirus isolated from a shearwater bird nesting on Tryon Island on the Australian Great Barrier Reef in 1971 has been more extensively characterized. Haemagglutinin subunits were isolated from the shearwater virus and from the antigenically related avian influenzaviruses A/turkey/Mass./65 (Hav6N2) and A/duck/Penn./69 (Hav6N1). Maps of the tryptic peptides from the heavy polypeptides (HA1) of the haemagglutinin subunits of the three viruses showed a number of differences, but peptide maps of the light polypeptides (HA2) were almost identical, suggesting that these had almost the same amino acid sequence. Extensive tests confirmed that the neuraminidase of the shearwater virus was not related antigenically to any known neuraminidase. The sera collected from pelagic birds nesting on islands in the Capricorn—Bunker group in 1970 were devoid of any antibodies to the shearwater virus, while a high proportion of the sera collected from birds on the same islands in 1972 (one year after the isolation of the shearwater virus) had antibodies to the haemagglutinin and neuraminidase of the shearwater virus, some to a high titre. Thus, the shearwater virus appeared to have only recently been introduced into the area from where it was isolated. ImagesFig. 1Fig. 2Fig. 3 PMID:4548383

Gene fusions AHRR-NCOA2, NCOA2-ETV4, ETV4-AHRR, P4HA2-TBCK, and TBCK-P4HA2 resulting from the translocations t(5;8;17)(p15;q13;q21) and t(4;5)(q24;q31) in a soft tissue angiofibroma

PubMed Central

Panagopoulos, Ioannis; Gorunova, Ludmila; Viset, Trond; Heim, Sverre

2016-01-01

We present an angiofibroma of soft tissue with the karyotype 46,XY,t(4;5)(q24;q31),t(5;8;17)(p15;q13;q21) [8]/46,XY,t(1;14)(p31;q32)[2]/46,XY[3]. RNA-sequencing showed that the t(4;5)(q24;q31) resulted in recombination of the genes TBCK on 4q24 and P4HA2 on 5q31.1 with generation of an in-frame TBCK-P4HA2 and the reciprocal but out-of-frame P4HA2-TBCK fusion transcripts. The putative TBCK-P4HA2 protein would contain the kinase, the rhodanese-like domain, and the Tre-2/Bub2/Cdc16 (TBC) domains of TBCK together with the P4HA2 protein which is a component of the prolyl 4-hydroxylase. The t(5;8;17)(p15;q13;q21) three-way chromosomal translocation targeted AHRR (on 5p15), NCOA2 (on 8q13), and ETV4 (on 17q21) generating the in-frame fusions AHRR-NCOA2 and NCOA2-ETV4 as well as an out-of-frame ETV4-AHRR transcript. In the AHRR-NCOA2 protein, the C-terminal part of AHRR is replaced by the C-terminal part of NCOA2 which contains two activation domains. The NCOA2-ETV4 protein would contain the helix-loop-helix, PAS_9 and PAS_11, CITED domains, the SRC-1 domain of NCOA2 and the ETS DNA-binding domain of ETV4. No fusion gene corresponding to t(1;14)(p31;q32) was found. Our findings indicate that, in spite of the recurrence of AHRR-NCOA2 in angiofibroma of soft tissue, additional genetic events (or fusion genes) might be required for the development of this tumor. PMID:27633981

Gene fusions AHRR-NCOA2, NCOA2-ETV4, ETV4-AHRR, P4HA2-TBCK, and TBCK-P4HA2 resulting from the translocations t(5;8;17)(p15;q13;q21) and t(4;5)(q24;q31) in a soft tissue angiofibroma.

PubMed

Panagopoulos, Ioannis; Gorunova, Ludmila; Viset, Trond; Heim, Sverre

2016-11-01

We present an angiofibroma of soft tissue with the karyotype 46,XY,t(4;5)(q24;q31),t(5;8;17)(p15;q13;q21)[8]/46,XY,t(1;14)(p31;q32)[2]/46,XY[3]. RNA‑sequencing showed that the t(4;5)(q24;q31) resulted in recombination of the genes TBCK on 4q24 and P4HA2 on 5q31.1 with generation of an in‑frame TBCK‑P4HA2 and the reciprocal but out‑of‑frame P4HA2‑TBCK fusion transcripts. The putative TBCK‑P4HA2 protein would contain the kinase, the rhodanese‑like domain, and the Tre‑2/Bub2/Cdc16 (TBC) domains of TBCK together with the P4HA2 protein which is a component of the prolyl 4‑hydroxylase. The t(5;8;17)(p15;q13;q21) three‑way chromosomal translocation targeted AHRR (on 5p15), NCOA2 (on 8q13), and ETV4 (on 17q21) generating the in‑frame fusions AHRR‑NCOA2 and NCOA2‑ETV4 as well as an out‑of‑frame ETV4‑AHRR transcript. In the AHRR‑NCOA2 protein, the C‑terminal part of AHRR is replaced by the C‑terminal part of NCOA2 which contains two activation domains. The NCOA2‑ETV4 protein would contain the helix‑loop‑helix, PAS_9 and PAS_11, CITED domains, the SRC‑1 domain of NCOA2 and the ETS DNA‑binding domain of ETV4. No fusion gene corresponding to t(1;14)(p31;q32) was found. Our findings indicate that, in spite of the recurrence of AHRR‑NCOA2 in angiofibroma of soft tissue, additional genetic events (or fusion genes) might be required for the development of this tumor.

Reversible Association of the Hemagglutinin Subcomplex, HA-33/HA-17 Trimer, with the Botulinum Toxin Complex.

PubMed

Sagane, Yoshimasa; Mutoh, Shingo; Koizumi, Ryosuke; Suzuki, Tomonori; Miyashita, Shin-Ichiro; Miyata, Keita; Ohyama, Tohru; Niwa, Koichi; Watanabe, Toshihiro

2017-10-01

Botulinum neurotoxin (BoNT) associates with nontoxic proteins, either a nontoxic nonhemagglutinin (NTNHA) or the complex of NTNHA and hemagglutinin (HA), to form M- or L-toxin complexes (TCs). Single BoNT and NTNHA molecules are associated and form M-TC. A trimer of the 70-kDa HA protein (HA-70) attaches to the M-TC to form M-TC/HA-70. Further, 1-3 arm-like 33- and 17-kDa HA molecules (HA-33/HA-17 trimer), consisting of 1 HA-17 protein and 2 HA-33 proteins, can attach to the M-TC/HA-70 complex, yielding 1-, 2-, and 3-arm L-TC. In this study, the purified 1- and 2-arm L-TCs spontaneously converted into another L-TC species after acquiring the HA-33/HA-17 trimer from other TCs during long-term storage and freezing/thawing. Transmission electron microscopy analysis provided evidence of the formation of detached HA-33/HA-17 trimers in the purified TC preparation. These findings provide evidence of reversible association/dissociation of the M-TC/HA-70 complex with the HA-33/HA-17 trimers, as well as dynamic conversion of the quaternary structure of botulinum TC in culture.

Surveillance of human influenza A(H3N2) virus from 1999 to 2009 in southern Italy.

PubMed

DE Donno, A; Idolo, A; Quattrocchi, M; Zizza, A; Gabutti, G; Romano, A; Grima, P; Donatelli, I; Guido, M

2014-05-01

The aim of this study was to evaluate the presence of influenza virus co-infections in humans and changes in the genetic variability of A(H3N2) virus strains in southern Italy from 1999 to 2009. A partial sequence of the haemagglutinin (HA) gene by human influenza H3N2 strains identified in oropharyngeal swabs from patients with influenza-like illness was analysed by DNA sequencing and a phylogenetic analysis was performed. During the seasons 1999-2000, 2002-2003, 2004-2005 and 2008-2009, the influenza viruses circulating belonged to subtype H3N2. However, A(H1N1) subtype virus and B type were respectively prevalent during the 2000-2001, 2006-2007, 2007-2008 and 2001-2002, 2003-2004, 2005-2006 seasons. The HA sequences appeared to be closely related to the sequence of the influenza A vaccine strain. Only the 2002-2003 season was characterized by co-circulation of two viral lineages: A/New York/55/01(H3N2)-like virus of the previous season and A/Fujian/411/02(H3N2)-like virus, a new H3 variant. In this study, over the decade analysed, no significant change was seen in the sequences of the HA gene of H3 viruses isolated.

Haemagglutinin-neuraminidase from HPIV3 mediates human NK regulation of T cell proliferation via NKp44 and NKp46.

PubMed

McQuaid, Samantha; Loughran, Sinead; Power, Patrick; Maguire, Paula; Walls, Dermot; Grazia Cusi, Maria; Orvell, Claes; Johnson, Patricia

2018-06-01

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

Intense circulation of A/H5N1 and other avian influenza viruses in Cambodian live-bird markets with serological evidence of sub-clinical human infections.

PubMed

Horm, Srey Viseth; Tarantola, Arnaud; Rith, Sareth; Ly, Sowath; Gambaretti, Juliette; Duong, Veasna; Y, Phalla; Sorn, San; Holl, Davun; Allal, Lotfi; Kalpravidh, Wantanee; Dussart, Philippe; Horwood, Paul F; Buchy, Philippe

2016-07-20

Surveillance for avian influenza viruses (AIVs) in poultry and environmental samples was conducted in four live-bird markets in Cambodia from January through November 2013. Through real-time RT-PCR testing, AIVs were detected in 45% of 1048 samples collected throughout the year. Detection rates ranged from 32% and 18% in duck and chicken swabs, respectively, to 75% in carcass wash water samples. Influenza A/H5N1 virus was detected in 79% of samples positive for influenza A virus and 35% of all samples collected. Sequence analysis of full-length haemagglutinin (HA) and neuraminidase (NA) genes from A/H5N1 viruses, and full-genome analysis of six representative isolates, revealed that the clade 1.1.2 reassortant virus associated with Cambodian human cases during 2013 was the only A/H5N1 virus detected during the year. However, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HA and NA genes revealed co-circulation of at least nine low pathogenic AIVs from HA1, HA2, HA3, HA4, HA6, HA7, HA9, HA10 and HA11 subtypes. Four repeated serological surveys were conducted throughout the year in a cohort of 125 poultry workers. Serological testing found an overall prevalence of 4.5% and 1.8% for antibodies to A/H5N1 and A/H9N2, respectively. Seroconversion rates of 3.7 and 0.9 cases per 1000 person-months participation were detected for A/H5N1 and A/H9N2, respectively. Peak AIV circulation was associated with the Lunar New Year festival. Knowledge of periods of increased circulation of avian influenza in markets should inform intervention measures such as market cleaning and closures to reduce risk of human infections and emergence of novel AIVs.

Sugar-induced conformational change found in the HA-33/HA-17 trimer of the botulinum toxin complex.

PubMed

Sagane, Yoshimasa; Hayashi, Shintaro; Matsumoto, Takashi; Miyashita, Shin-Ichiro; Inui, Ken; Miyata, Keita; Yajima, Shunsuke; Suzuki, Tomonori; Hasegawa, Kimiko; Yamano, Akihito; Nishikawa, Atsushi; Ohyama, Tohru; Watanabe, Toshihiro; Niwa, Koichi

2013-08-30

Large-sized botulinum toxin complex (L-TC) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (HA) complex. The HA complex is formed by association of three HA-70 molecules and three HA-33/HA-17 trimers, comprised of a single HA-17 and two HA-33 proteins. The HA-33/HA-17 trimer isolated from serotype D L-TC has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important role in toxin delivery through the intestinal cell wall. In this study, we determined the solution structure of the HA-33/HA-17 trimer by using small-angle X-ray scattering (SAXS). The SAXS image of HA-33/HA-17 exhibited broadly similar appearance to the crystal image of the complex. On the other hand, in the presence of N-acetylneuraminic acid, glucose and galactose, the solution structure of the HA-33/HA-17 trimer was drastically altered compared to the structure in the absence of the sugars. Sugar-induced structural change of the HA-33/HA-17 trimer may contribute to cell binding and subsequent transport across the intestinal cell layer. Copyright © 2013 Elsevier Inc. All rights reserved.

Conformational divergence in the HA-33/HA-17 trimer of serotype C and D botulinum toxin complex.

PubMed

Sagane, Yoshimasa; Hayashi, Shintaro; Akiyama, Tomonori; Matsumoto, Takashi; Hasegawa, Kimiko; Yamano, Akihito; Suzuki, Tomonori; Niwa, Koichi; Watanabe, Toshihiro; Yajima, Shunsuke

2016-08-05

Clostridium botulinum produces a large toxin complex (L-TC) comprising botulinum neurotoxin associated with auxiliary nontoxic proteins. A complex of 33- and 17-kDa hemagglutinins (an HA-33/HA-17 trimer) enhances L-TC transport across the intestinal epithelial cell layer via binding HA-33 to a sugar on the cell surface. At least two subtypes of serotype C/D HA-33 exhibit differing preferences for the sugars sialic acid and galactose. Here, we compared the three-dimensional structures of the galactose-binding HA-33 and HA-33/HA-17 trimers produced by the C-Yoichi strain. Comparisons of serotype C/D HA-33 sequences reveal a variable region with relatively low sequence similarity across the C. botulinum strains; the variability of this region may influence the manner of sugar-recognition by HA-33. Crystal structures of sialic acid- and galactose-binding HA-33 are broadly similar in appearance. However, small-angle X-ray scattering revealed distinct solution structures for HA-33/HA-17 trimers. A structural change in the C-terminal variable region of HA-33 might cause a dramatic shift in the conformation and sugar-recognition mode of HA-33/HA-17 trimer. Copyright © 2016 Elsevier Inc. All rights reserved.

Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.

PubMed

Sequeira, Daniela P; Correia, Ricardo; Carrondo, Manuel J T; Roldão, António; Teixeira, Ana P; Alves, Paula M

2018-05-24

Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×10 6 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×10 6 cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×10 6 cells/mL showed comparable HA titers per cell to those of CCI of 2×10 6 cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts.

PubMed

Nagaoka, Aya; Yoshida, Hiroyuki; Nakamura, Sachiko; Morikawa, Tomohiko; Kawabata, Keigo; Kobayashi, Masaki; Sakai, Shingo; Takahashi, Yoshito; Okada, Yasunori; Inoue, Shintaro

2015-12-25

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Immune Escape Mutants of Highly Pathogenic Avian Influenza H5N1 Selected Using Polyclonal Sera: Identification of Key Amino Acids in the HA Protein

PubMed Central

Sitaras, Ioannis; Kalthoff, Donata; Beer, Martin; Peeters, Ben; de Jong, Mart C. M.

2014-01-01

Evolution of Avian Influenza (AI) viruses – especially of the Highly Pathogenic Avian Influenza (HPAI) H5N1 subtype – is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks) is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1), using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA) protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number. PMID:24586231

Minor histocompatibility antigen HA-1 and HA-2 polymorphisms in Taiwan: frequency and application in hematopoietic stem cell transplantation.

PubMed

Lio, Hoi-Yan; Tang, Jih-Luh; Wu, Jui; Wu, Shang-Ju; Lin, Chun-Ying; Yang, Ya-Chien

2010-09-01

Minor histocompatibility antigens influence the occurrence of graft-vs.-host disease and graft-vs.-leukemia effects after hematopoietic stem cell transplantation (HSCT). We determined the population frequencies of HA-1 and HA-2 alleles in Taiwan and exploited their potential applications in allogeneic HSCT. HA-1 and HA-2 were genotyped using polymerase chain reaction and restriction fragment length polymorphism in healthy controls (221 for HA-1 and 306 for HA-2) and HLA-matched donor-recipient sibling pairs with HSCT (92 for HA-1 and 38 for HA-2). The association of genetic polymorphisms with HSCT outcome was evaluated by univariate and multivariate analyses. The allele frequencies in controls were 35.3% and 64.7% for HA-1(H) and HA-1(R), and 89.0% and 11.0% for HA-2(V) and HA-2(M), respectively. HA-1 disparity was denoted in 16.3% of HLA-matched donor-recipient sibling pairs, while it was not associated with HSCT outcome. HA-2 disparity was not observed in the donor-recipient pairs studied. The possibilities of using HA-1 and HA-2 variabilities as molecular markers for hematopoietic chimerism after HSCT were 39.2% and 18.4%, respectively. Our data provide the information on allele and genotype frequencies of HA-1 and HA-2 in a Taiwanese population, and suggest that prospective genomic typing for HA-1 and HA-2 alleles of the donor and recipient could be a useful approach for molecular identification of hematopoietic chimerism after HSCT, rather than prognosis of clinical outcome.

Challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza H5N1 viruses emerging in Vietnam.

PubMed

Slomka, Marek J; To, Thanh L; Tong, Hien H; Coward, Vivien J; Hanna, Amanda; Shell, Wendy; Pavlidis, Theo; Densham, Anstice L E; Kargiolakis, Georgios; Arnold, Mark E; Banks, Jill; Brown, Ian H

2012-01-01

Forty-six chickens and 48 ducks were sampled from four Vietnamese poultry premises in 2009 infected with H5N1 highly pathogenic avian influenza (HPAI) clade 2.3.2 and 2.3.4 viruses, which also differed by cleavage site (CS) sequences in their haemagglutinin (HA) genes. All clinical specimens (n=282), namely tracheal and cloacal swabs plus feathers, were tested by five Eurasian reverse-transcriptase AI RealTime polymerase chain reaction (RRT-PCR) methods. Bayesian modelling showed similar high sensitivity for the validated H5 HA2 RRT-PCR and a new modified M-gene RRT-PCR that utilizes lyophilized reagents. Both were more sensitive than the validated "wet" M-gene RRT-PCR. Another RRT-PCR, which targeted the H5-gene CS region, was effective for clade 2.3.4 detection, but severely compromised for clade 2.3.2 viruses. Reduced sensitivity of the H5 CS and "wet" M-gene RRT-PCRs correlated with mismatches between the target and the primer and/or probe sequences. However, the H5 HA2 RRT-PCR sensitively detected both clade 2.3.2 and 2.3.4 viruses, and agreed with N1 RRT-PCR results. Feather testing from diseased chicken and duck flocks by AI RRT-PCRs resulted in the most sensitive identification of H5N1 HPAI-infected birds. Evolution of new H5N1 HPAI clades remains a concern for currently affected Asian countries, but also for more distant regions where it is important to be prepared for new incursions of H5N1 HPAI viruses. Genetic evidence for adamantane resistance and sensitivity was also observed in isolates from both clades.

Viral reassortment and transmission after co-infection of pigs with classical H1N1 and triple-reassortant H3N2 swine influenza viruses.

PubMed

Ma, Wenjun; Lager, Kelly M; Lekcharoensuk, Porntippa; Ulery, Eva S; Janke, Bruce H; Solórzano, Alicia; Webby, Richard J; García-Sastre, Adolfo; Richt, Jürgen A

2010-09-01

Triple-reassortant swine influenza viruses circulating in North American pigs contain the internal genes derived from swine (matrix, non-structural and nucleoprotein), human [polymerase basic 1 (PB1)] and avian (polymerase acidic and PB2) influenza viruses forming a constellation of genes that is well conserved and is called the triple-reassortant internal gene (TRIG) cassette. In contrast, the external genes [haemagglutinin (HA) and neuraminidase (NA)] are less conserved, reflecting multiple reassortant events that have produced viruses with different combinations of HA and NA genes. This study hypothesized that maintenance of the TRIG cassette confers a selective advantage to the virus. To test this hypothesis, pigs were co-infected with the triple-reassortant H3N2 A/Swine/Texas/4199-2/98 (Tx/98) and the classical H1N1 A/Swine/Iowa/15/1930 viruses and co-housed with a group of sentinel animals. This direct contact group was subsequently moved into contact with a second group of naïve animals. Four different subtypes (H1N1, H1N2, H3N1 and H3N2) of influenza virus were identified in bronchoalveolar lavage fluid collected from the lungs of the experimentally infected pigs, with most of the viruses containing TRIG from the Tx/98 virus. Interestingly, only the intact H3N2 Tx/98 virus was transmitted from the infected pigs to the direct-contact animals and from them to the second contact group of pigs. These results demonstrated that multiple reassortments can occur within a host; however, only specific gene constellations are readily transmissible. It was concluded that certain HA and NA gene pairs, in conjunction with the TRIG cassette, may have a competitive advantage over other combinations for transmission and maintenance in swine.

Differential osteogenic activity of osteoprogenitor cells on HA and TCP/HA scaffold of tissue engineered bone.

PubMed

Ng, Angela M H; Tan, K K; Phang, M Y; Aziyati, O; Tan, G H; Isa, M R; Aminuddin, B S; Naseem, M; Fauziah, O; Ruszymah, B H I

2008-05-01

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering. Copyright 2007 Wiley Periodicals, Inc.

PCL-HA microscaffolds for in vitro modular bone tissue engineering.

PubMed

Totaro, Alessandra; Salerno, Aurelio; Imparato, Giorgia; Domingo, Concepción; Urciuolo, Francesco; Netti, Paolo Antonio

2017-06-01

The evolution of microscaffolds and bone-bioactive surfaces is a pivotal point in modular bone tissue engineering. In this study, the design and fabrication of porous polycaprolactone (PCL) microscaffolds functionalized with hydroxyapatite (HA) nanoparticles by means of a bio-safe and versatile thermally-induced phase separation process is reported. The ability of the as-prepared nanocomposite microscaffolds to support the adhesion, growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in standard and osteogenic media and using dynamic seeding/culture conditions was investigated. The obtained results demonstrated that the PCL-HA nanocomposite microparticles had an enhanced interaction with hMSCs and induced their osteogenic differentiation, even without the exogenous addition of osteogenic factors. In particular, calcium deposition, alizarin red assay, histological analysis, osteogenic gene expression and collagen I secretion were assessed. The results of these tests demonstrated the formation of bone microtissue precursors after 28 days of dynamic culture. These findings suggest that PCL-HA nanocomposite microparticles represent an excellent platform for in vitro modular bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein.

PubMed

Hu, A; Norrby, E

1994-09-01

The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties. (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface. (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not

Elastin-like protein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration.

PubMed

Zhu, Danqing; Wang, Huiyuan; Trinh, Pavin; Heilshorn, Sarah C; Yang, Fan

2017-05-01

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows variation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair. Copyright © 2017 Elsevier Ltd. All rights reserved.

Elastin-like Protein-Hyaluronic acid (ELP-HA) Hydrogels with Decoupled Mechanical and Biochemical cues for Cartilage Regeneration

PubMed Central

Zhu, Danqing; Wang, Huiyuan; Trinh, Pavin; Heilshorn, Sarah C.; Yang, Fan

2018-01-01

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows varyiation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair. PMID:28268018

In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP.

PubMed

Kang, Kyung-Jung; Lee, Min Suk; Moon, Chan-Woong; Lee, Jae-Hoon; Yang, Hee Seok; Jang, Young-Joo

2017-01-01

Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin.

In vitro testing of calcium phosphate (HA, TCP, and biphasic HA-TCP) whiskers.

PubMed

Jalota, Sahil; Bhaduri, Sarit B; Tas, A Cuneyt

2006-09-01

Calcium phosphate [single-phase hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)), single-phase tricalcium phosphate (beta-TCP, Ca(3)(PO(4))(2)), and biphasic HA-TCP] whiskers were formed by using a novel microwave-assisted molten salt mediated process. Aqueous solutions containing NaNO(3), HNO(3), Ca(NO(3))(2) x 4H(2)O, and KH(2)PO(4) (with or without urea) were used as starting reagents. These solutions were irradiated in a household microwave oven for 5 min. As-recovered precursors were then simply stirred in water at room temperature for 1 h to obtain the whiskers of the desired calcium phosphate (CaP) bioceramics. These whiskers were evaluated, respectively, in vitro by (1) soaking those in synthetic body fluid (SBF) solutions at 37 degrees C for one week, and (2) performing cell attachment and total protein assay tests on the neat whiskers by using a mouse osteoblast cell line (7F2). beta-TCP, HA, and HA-TCP biphasic whiskers were all found to possess apatite-inducing ability when soaked in SBF. SBF-soaked whiskers were found to have BET surface areas ranging from 45 to 112 m(2)/g. Although the osteoblast viability and protein concentrations were found to be the highest on the neat HA whiskers, cells were attached and proliferated on all the whiskers.

In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP

PubMed Central

Kang, Kyung-Jung; Lee, Min Suk; Moon, Chan-Woong; Lee, Jae-Hoon

2017-01-01

Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin. PMID:28761445

Effect of molecular weight of hyaluronic acid (HA) on viscoelasticity and particle texturing feel of HA dermal biphasic fillers.

PubMed

Chun, Cheolbyong; Lee, Deuk Yong; Kim, Jin-Tae; Kwon, Mi-Kyung; Kim, Young-Zu; Kim, Seok-Soon

2016-01-01

Hyaluronic acid (HA) dermal biphasic fillers are synthesized for their efficacy in correcting aesthetic defects such as wrinkles, scars and facial contouring defects. The fillers consist of crosslinked HA microspheres suspended in a noncrosslinked HA. To extend the duration of HAs within the dermis and obtain the particle texturing feel, HAs are crosslinked to obtain the suitable mechanical properties. Hyaluronic acid (HA) dermal biphasic fillers are prepared by mixing the crosslinked HA microspheres and the noncrosslinked HAs. The elastic modulus of the fillers increased with raising the volume fraction of the microspheres. The mechanical properties and the particle texturing feel of the fillers made from crosslinked HA (1058 kDa) microspheres suspended in noncrosslinked HA (1368 kDa) are successfully achieved, which are adequate for the fillers. Dermal biphasic HA fillers made from 1058 kDa exhibit suitable elastic moduli (211 to 420 Pa) and particle texturing feel (scale 7 ~ 9).

The Impact of HA Oligomer Content on Physical, Mechanical, and Biologic Properties of Divinyl Sulfone-Crosslinked HA Hydrogels

PubMed Central

Ibrahim, Samir; Kang, Qian K; Ramamurthi, Anand

2009-01-01

In recent studies, we showed that exogenous hyaluronic acid oligomers (HA-o) stimulate functional endothelialization, though native long-chain HA is more bioinert and possibly more biocompatible. Thus, in this study, hydrogels containing high molecular weight (HMW) HA (1×106 Da) and HA oligomer mixtures (HA-o: 0.75–10 kDa) were created by crosslinking with divinyl sulfone (DVS). The incorporation of HA oligomers was found to compromise the physical and mechanical properties of the gels (rheology, apparent crosslinking density, swelling ratio, degradation) and to very mildly enhance inflammatory cell recruitment in vivo; increasing the DVS crosslinker content within the gels in general, had the opposite effect, though the relatively high concentration of DVS within these gels (necessary to create a solid gel) also stimulated a mild sub-cutaneous inflammatory response in vivo and VCAM-1 expression by ECs cultured atop; ICAM-expression levels remained very low irrespective extent of DVS crosslinking or HA-o content. The greatest EC attachment and proliferation (MTT assay) was observed on gels that contained the highest amount of HA-o. The study shows that the beneficial EC response to HA oligomers and biocompatibility of HA is mostly unaltered by their chemical derivatization and crosslinking into a hydrogel. However, the study also demonstrates that the relatively high concentrations of DVS, necessary to create solid gels, compromises their biocompatibility. Moreover, the poor mechanics of even these heavily crosslinked gels, in the context of vascular implantation, necessitates the investigation of other, more appropriate crosslinking agents. Alternately, the outcomes of this study may be used to guide an approach based on chemical immobilization and controlled surface-presentation of both bioactive HA oligomers and more biocompatible HMW HAon synthetic or tissue engineered grafts already in use, without the use of a crosslinker, so that improved, predictable

In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization

PubMed Central

Deglesne, Pierre-Antoine; Arroyo, Rodrigo; Ranneva, Evgeniya; Deprez, Philippe

2016-01-01

Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS® (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts. PMID:26966384

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

PubMed Central

Nicholls, Sarah; Piper, Karen P.; Mohammed, Fiyaz; Dafforn, Timothy R.; Tenzer, Stefan; Salim, Mahboob; Mahendra, Premini; Craddock, Charles; van Endert, Peter; Schild, Hansjörg; Cobbold, Mark; Engelhard, Victor H.; Moss, Paul A. H.; Willcox, Benjamin E.

2009-01-01

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1H versus HA-1R) in the HMHA1 gene. The HA-1H peptide is restricted by HLA-A2 and is immunogenic in HA-1R/R into HA-1H transplants, while HA-1R has been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1R variant. To understand these findings, we determined the structure of an HLA-A2-HA-1H complex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1H peptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition. PMID:19234124

Characterization of New PEEK/HA Composites with 3D HA Network Fabricated by Extrusion Freeforming.

PubMed

Vaezi, Mohammad; Black, Cameron; Gibbs, David M R; Oreffo, Richard O C; Brady, Mark; Moshrefi-Torbati, Mohamed; Yang, Shoufeng

2016-05-26

Addition of bioactive materials such as calcium phosphates or Bioglass, and incorporation of porosity into polyetheretherketone (PEEK) has been identified as an effective approach to improve bone-implant interfaces and osseointegration of PEEK-based devices. In this paper, a novel production technique based on the extrusion freeforming method is proposed that yields a bioactive PEEK/hydroxyapatite (PEEK/HA) composite with a unique configuration in which the bioactive phase (i.e., HA) distribution is computer-controlled within a PEEK matrix. The 100% interconnectivity of the HA network in the biocomposite confers an advantage over alternative forms of other microstructural configurations. Moreover, the technique can be employed to produce porous PEEK structures with controlled pore size and distribution, facilitating greater cellular infiltration and biological integration of PEEK composites within patient tissue. The results of unconfined, uniaxial compressive tests on these new PEEK/HA biocomposites with 40% HA under both static and cyclic mode were promising, showing the composites possess yield and compressive strength within the range of human cortical bone suitable for load bearing applications. In addition, preliminary evidence supporting initial biological safety of the new technique developed is demonstrated in this paper. Sufficient cell attachment, sustained viability in contact with the sample over a seven-day period, evidence of cell bridging and matrix deposition all confirmed excellent biocompatibility.

Biomedical potential of chitosan/HA and chitosan/β-1,3-glucan/HA biomaterials as scaffolds for bone regeneration--A comparative study.

PubMed

Przekora, Agata; Palka, Krzysztof; Ginalska, Grazyna

2016-01-01

The aim of this work was to compare biomedical potential of chitosan/hydroxyapatite (chit/HA) and novel chitosan/β-1,3-glucan/hydroxyapatite (chit/glu/HA) materials as scaffolds for bone regeneration via characterization of their biocompatibility, porosity, mechanical properties, and water uptake behaviour. Biocompatibility of the scaffolds was assessed in direct-contact with the materials using normal human foetal osteoblast cell line. Cytotoxicity and osteoblast proliferation rate were evaluated. Porosity was assessed using computed microtomography analysis and mechanical properties were determined by compression testing. Obtained results demonstrated that chit/HA scaffold possessed significantly better mechanical properties (compressive strength: 1.23 MPa, Young's modulus: 0.46 MPa) than chit/glu/HA material (compressive strength: 0.26 MPa, Young's modulus: 0.25 MPa). However, addition of bacterial β-1,3-glucan to the chit/HA scaffold improved its flexibility and porosity. Moreover, chit/glu/HA scaffold revealed significantly higher water uptake capability (52.6% after 24h of soaking) compared to the chit/HA (30.7%) and thus can serve as a very good drug delivery carrier. Chit/glu/HA scaffold was also more favourable to osteoblast survival (near 100% viability after 24-h culture), proliferation, and spreading compared to the chit/HA (63% viability). The chit/glu/HA possesses better biomedical potential than chit/HA scaffold. Nevertheless, poor mechanical properties of the chit/glu/HA limit its application to non-load bearing implantation area. Copyright © 2015 Elsevier B.V. All rights reserved.

HA metabolism in skin homeostasis and inflammatory disease.

PubMed

Kavasi, Rafaela-Maria; Berdiaki, Aikaterini; Spyridaki, Ioanna; Corsini, Emanuela; Tsatsakis, Aristidis; Tzanakakis, George; Nikitovic, Dragana

2017-03-01

Hyaluronan (HA), an unsulfated glycosaminoglycan, is an important component of the complex extracellular matrix network which surrounds and supports cells in tissues. HA is detected in all vertebrate tissues, but the bulk of HA is produced and deposited in the skin. In this review we focus on the role of HA in skin-associated inflammatory disease and wound healing. Properties of HA are directly dependent on its molecular weight. Thus, high molecular weight HA (HMWHA) is deposited in normal tissues during homeostasis and promotes their stability whereas low molecular weight HA fragments (LMWHA), on the other hand, may arise from enzymatic or chemical activities. The degradation of HMWHA to LMWHA fragments, often leads to the generation of biologically active oligosaccharides with different properties and postulated functions in wound scar formation and inflammation. More detailed studies of HA involvement in skin-associated inflammatory disease may result in novel treatment modalities. Copyright © 2017 Elsevier Ltd. All rights reserved.

Heparin-Binding Haemagglutinin, a New Tool for the Detection of Latent Mycobacterium tuberculosis Infection in Hemodialysis Patients

PubMed Central

Dessein, Rodrigue; Corbière, Véronique; Nortier, Joëlle; Dratwa, Max; Gastaldello, Karine; Pozdzik, Agnieszka; Lecher, Sophie; Grandbastien, Bruno; Locht, Camille; Mascart, Françoise

2013-01-01

Background Patients with end-stage renal disease (ESRD) and latently infected with Mycobacterium tuberculosis (LTBI) are at higher risk to develop tuberculosis (TB) than healthy subjects. Interferon-gamma release assays (IGRAs) were reported to be more sensitive than tuberculin skin tests for the detection of infected individuals in dialysis patients. Methods On 143 dialysis patients prospectively enrolled, we compared the results from the QuantiFERON®-TB Gold assay (QFT), to those of an IGRA in response to in vitro stimulation of circulating mononuclear cells with the mycobacterial latency antigen Heparin-Binding Haemagglutinin purified from Mycobacterium bovis BCG (native HBHA, nHBHA). Results Seven patients had a past history of active TB and 1 had an undetermined result with both IGRAs. Among the other 135 patients, 94 had concordant results with the QFT and nHBHA-IGRA, 40.0% being negative and therefore not latently infected, and 29.6% being positive and thus LTBI. Discrepant results between these tests were found for 36 patients positive only with the nHBHA-IGRA and 5 only with the QFT. Conclusions The nHBHA-IGRA is more sensitive than the QFT for the detection of LTBI dialysis patients, and follow-up of the patients will allow us to define the clinical significance of discrepant results between the nHBHA-IGRA and the QFT. PMID:23940693

The effect of the type of HA on the degradation of PLGA/HA composites.

PubMed

Naik, Ashutosh; Shepherd, David V; Shepherd, Jennifer H; Best, Serena M; Cameron, Ruth E

2017-01-01

The aim of this study is to explore the importance of the potentially competing effects of buffering effects of the calcium phosphate filler and particle-mediated water sorption on the degradation products of poly(d,l lactide-co-glycolide (50:50))(PLGA)/hydroxyapatite(HA) composites. Further the influence of type of HA on the mechanical properties of the composites was investigated. Phase pure HA was synthesised via a reaction between aqueous solutions of calcium hydroxide and orthophosphoric acid. The powder produced was either used as produced (uncalcined) or calcined in air or calcined in a humidified argon atmosphere. An in-vitro degradation study was carried out in phosphate buffered saline (PBS). The results obtained indicated that the degradation rate of the composite might be better understood if both the buffering effects and the rate of water sorption by the composites are considered. Copyright © 2016 Elsevier B.V. All rights reserved.

Integrated HPV genomes tend to integrate in gene desert areas in the CaSki, HeLa, and SiHa cervical cancer cell lines.

PubMed

Diao, Ming-Kun; Liu, Chu-Yi; Liu, Hong-Wei; Li, Jin-Tao; Li, Fan; Mehryar, Mohammadreza Mohammadzad; Wang, Yang-Junqi; Zhan, Shao-Bing; Zhou, Yu-Bai; Zhong, Ru-Gang; Zeng, Yi

2015-04-15

The integration preferences of human papillomavirus (HPV) have been intensively studied and contested over recent years. To disclose the integration preferences of high-risk HPV in cervical cancer, HPV transcriptional sites and features in different cervical cancer cell lines were identified. In this study, three cervical cancer cell lines (CaSki, HeLa, and SiHa) were subjected for HPV genome status determination by amplification of papillomavirus oncogene transcripts (APOT) assay. The numbers of viral copies in human genomes and numbers of viral-human fusion mRNAs in three HPV-integrated cervical cancer cell lines were measured and analysed. The results revealed that the gene desert region 8q24 of the HPV type 18 integrated HeLa cell line and the 13q21-22 region of the HPV type 16 integrated CaSki and SiHa cell lines were hotspots for HPV integration, and the numbers of viral copies in the human genomes of the three cell lines that we detected were not in accordance with those reported in previous studies. Integration of the HPV genome into the host cell chromosome suggests that persistent HPV infection is vital for malignant cell transformation and carcinogenesis. This study provides information to benefit health care professionals seeking more comprehensive and accurate diagnostics for HPV-related disease"? Please check, and amend as necessary. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization, Corrosion Resistance, and Cell Response of High-Velocity Flame-Sprayed HA and HA/TiO2 Coatings on 316L SS

NASA Astrophysics Data System (ADS)

Singh, Tejinder Pal; Singh, Harpreet; Singh, Hazoor

2012-09-01

The main aim of this study is to evaluate corrosion and biocompatibility behavior of thermal spray hydroxyapatite (HA) and hydroxyapatite/titania bond (HA/TiO2)-coated 316L stainless steel (316L SS). In HA/TiO2 coatings, TiO2 was used as a bond coat between HA top coat and 316L SS substrate. The coatings were characterized by x-ray diffraction and scanning electron microscopy/energy dispersive spectroscopy, and corrosion resistance determined for the uncoated substrate and the two coatings. The biological behavior was investigated by the cell culture studies using osteosarcoma cell line KHOS-NP (R-970-5). The corrosion resistance of the steel was found to increase after the deposition of the HA and HA/TiO2 bond coatings. Both HA, as well as, HA/TiO2 coatings exhibit excellent bond strength of 49 and 47 MPa, respectively. The cell culture studies showed that HA-coated 316L SS specimens appeared more biocompatible than the uncoated and HA/TiO2-coated 316L SS specimens.

Selective pre-priming of HA-specific CD4 T cells restores immunological reactivity to HA on heterosubtypic influenza infection.

PubMed

Alam, Shabnam; Chan, Cory; Qiu, Xing; Shannon, Ian; White, Chantelle L; Sant, Andrea J; Nayak, Jennifer L

2017-01-01

A hallmark of the immune response to influenza is repeated encounters with proteins containing both genetically conserved and variable components. Therefore, the B and T cell repertoire is continually being remodeled, with competition between memory and naïve lymphocytes. Our previous work using a mouse model of secondary heterosubtypic influenza infection has shown that this competition results in a focusing of CD4 T cell response specificity towards internal virion proteins with a selective decrease in CD4 T cell reactivity to the novel HA epitopes. Strikingly, this shift in CD4 T cell specificity was associated with a diminished anti-HA antibody response. Here, we sought to determine whether the loss in HA-specific reactivity that occurs as a consequence of immunological memory could be reversed by selectively priming HA-specific CD4 T cells prior to secondary infection. Using a peptide-based priming strategy, we found that selective expansion of the anti-HA CD4 T cell memory repertoire enhanced HA-specific antibody production upon heterosubtypic infection. These results suggest that the potentially deleterious consequences of repeated exposure to conserved influenza internal virion proteins could be reversed by vaccination strategies that selectively arm the HA-specific CD4 T cell compartment. This could be a potentially useful pre-pandemic vaccination strategy to promote accelerated neutralizing antibody production on challenge with a pandemic influenza strain that contains few conserved HA epitopes.

The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein.

PubMed

Spierings, Eric; Brickner, Anthony G; Caldwell, Jennifer A; Zegveld, Suzanne; Tatsis, Nia; Blokland, Els; Pool, Jos; Pierce, Richard A; Mollah, Sahana; Shabanowitz, Jeffrey; Eisenlohr, Laurence C; van Veelen, Peter; Ossendorp, Ferry; Hunt, Donald F; Goulmy, Els; Engelhard, Victor H

2003-07-15

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr-->Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti-HA antibody in single in-capillary assay.

PubMed

Wang, Jianhao; Qin, Yuqin; Qin, Haifang; Liu, Li; Ding, Shumin; Teng, Yiwan; Ji, Junling; Qiu, Lin; Jiang, Pengju

2016-08-01

Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 μM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Association between endothelial nitric oxide synthase (ENOS) G894T polymorphism and high altitude (HA) adaptation: a meta-analysis.

PubMed

Lu, Hong-xiang; Wang, Yu-xiao; Chen, Yu; Luo, Yong-jun

2015-11-01

Highland natives adapt well to the hypoxic environment at high altitude (HA). Several genes have been reported to be linked to HA adaptation. Previous studies showed that the endothelial ni- tric oxide synthase (ENOS) G894T polymorphism contributed to the physiology and pathophysiology of hu- mans at HA by regulating the production of NO. In this meta-analysis, we evaluate the association between the ENOS G894T polymorphism and HA adaptation through analyzing the published data. We searched all relevant literature about the ENOS G894T polymorphism and HA adaptation in PubMed, Med- line, and Embase before Step 2015. A random-effects model was applied (Revman 5.0), and study quality was assessed in duplicate. Six studies with 634 HA native cases and 621 low-altitude controls were included in this meta-analysis. From the results, we observed that the wild-type allele G was significantly overrepresented in the HA groups (OR = 1.85; 95% Cl, 1.47-2.33; P < 0.0001). In addition, the GG genotype was significantly associated with HA adaptation (OR = 1.99; 95% Cl, 1.54-2.57; P < 0.0001). Our results showed that in 894 G allele carriers, the GG genotype might be a beneficial factor for HA adaptation through enhancing the level of NO. However, more studies were needed to confirm our findings due to the limited sample size.

The homologous HD-Zip I transcription factors HaHB1 and AtHB13 confer cold tolerance via the induction of pathogenesis-related and glucanase proteins.

PubMed

Cabello, Julieta V; Arce, Agustín L; Chan, Raquel L

2012-01-01

Plants deal with cold temperatures via different signal transduction pathways. The HD-Zip I homologous transcription factors HaHB1 from sunflower and AtHB13 from Arabidopsis were identified as playing a key role in such cold response. The expression patterns of both genes were analyzed indicating an up-regulation by low temperatures. When these genes were constitutively expressed in Arabidopsis, the transgenic plants showed similar phenotypes including cell membrane stabilization under freezing treatments and cold tolerance. An exploratory transcriptomic analysis of HaHB1 transgenic plants indicated that several transcripts encoding glucanases and chitinases were induced. Moreover, under freezing conditions some proteins accumulated in HaHB1 plants apoplasts and these extracts exerted antifreeze activity in vitro. Three genes encoding two glucanases and a chitinase were overexpressed in Arabidopsis and these plants were able to tolerate freezing temperatures. All the obtained transgenic plants exhibited cell membrane stabilization after a short freezing treatment. Finally, HaHB1 and AtHB13 were used to transiently transform sunflower and soybean leading to the up-regulation of HaHB1/AtHB13-target homologues thus indicating the conservation of cold response pathways. We propose that HaHB1 and AtHB13 are involved in plant cold tolerance via the induction of proteins able to stabilize cell membranes and inhibit ice growth. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

PubMed

Zhang, Zhenyu; Zhao, Wei; Li, Deshan; Yang, Jinlong; Zsak, Laszlo; Yu, Qingzhong

2015-08-01

In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy.

Role of induced glutathione-S-transferase from Helicoverpa armigera (Lepidoptera: Noctuidae) HaGST-8 in detoxification of pesticides.

PubMed

Labade, Chaitali P; Jadhav, Abhilash R; Ahire, Mehul; Zinjarde, Smita S; Tamhane, Vaijayanti A

2018-01-01

The present study deals with glutathione-S-transferase (GST) based detoxification of pesticides in Helicoverpa armigera and its potential application in eliminating pesticides from the environment. Dietary exposure of a pesticide mixture (organophosphates - chlorpyrifos and dichlorvos, pyrethroid - cypermethrin; 2-15ppm each) to H. armigera larvae resulted in a dose dependant up-regulation of GST activity and gene expression. A variant GST from H. armigera (HaGST-8) was isolated from larvae fed with 10ppm pesticide mixture and it was recombinantly expressed in yeast (Pichia pastoris HaGST-8). HaGST-8 had a molecular mass of 29kDa and was most active at pH 9 at 30°C. GC-MS and LC-HRMS analysis validated that HaGST-8 was effective in eliminating organophosphate type of pesticides and partially reduced the cypermethrin content (53%) from aqueous solutions. Unlike the untransformed yeast, P. pastoris HaGST-8 grew efficiently in media supplemented with pesticide mixtures (200 and 400ppm each pesticide) signifying the detoxification ability of HaGST-8. The amino acid sequence of HaGST-8 and the already reported sequence of HaGST-7 had just 2 mismatches. The studies on molecular interaction strengths revealed that HaGST-8 had stronger binding affinities with organophosphate, pyrethroid, organochloride, carbamate and neonicotinoid type of pesticides. The abilities of recombinant HaGST-8 to eliminate pesticides and P. pastoris HaGST-8 to grow profusely in the presence of high level of pesticide content can be applied for removal of such residues from food, water resources and bioremediation. Copyright © 2017 Elsevier Inc. All rights reserved.

Hybrid Macro-Porous Titanium Ornamented by Degradable 3D Gel/nHA Micro-Scaffolds for Bone Tissue Regeneration

PubMed Central

Yin, Bo; Ma, Pei; Chen, Jun; Wang, Hai; Wu, Gui; Li, Bo; Li, Qiang; Huang, Zhifeng; Qiu, Guixing; Wu, Zhihong

2016-01-01

Porous titanium is a kind of promising material for bone substitution, while its bio-inert property results in demand of modifications to improve the osteointegration capacity. In this study, gelatin (Gel) and nano-hydroxyapatite (nHA) were used to construct 3D micro-scaffolds in the pores of porous titanium in the ratios of Gel:nHA = 1:0, Gel:nHA = 1:1, and Gel:nHA = 1:3, respectively. Cell attachment and proliferation, and gene and protein expression levels of osteogenic markers were evaluated in MC3T3-E1 cells, followed by bone regeneration assessment in a rabbit radius defect model. All hybrid scaffolds with different composition ratio were found to have significant promotional effects in cell adhesion, proliferation and differentiation, in which the group with Gel:nHA = 1:1 showed the best performance in vitro, as well as the most bone regeneration volume in vivo. This 3D micro-scaffolds modification may be an innovative method for porous titanium ornamentation and shows potential application values in clinic. PMID:27092492

Hybrid Macro-Porous Titanium Ornamented by Degradable 3D Gel/nHA Micro-Scaffolds for Bone Tissue Regeneration.

PubMed

Yin, Bo; Ma, Pei; Chen, Jun; Wang, Hai; Wu, Gui; Li, Bo; Li, Qiang; Huang, Zhifeng; Qiu, Guixing; Wu, Zhihong

2016-04-15

Porous titanium is a kind of promising material for bone substitution, while its bio-inert property results in demand of modifications to improve the osteointegration capacity. In this study, gelatin (Gel) and nano-hydroxyapatite (nHA) were used to construct 3D micro-scaffolds in the pores of porous titanium in the ratios of Gel:nHA = 1:0, Gel:nHA = 1:1, and Gel:nHA = 1:3, respectively. Cell attachment and proliferation, and gene and protein expression levels of osteogenic markers were evaluated in MC3T3-E1 cells, followed by bone regeneration assessment in a rabbit radius defect model. All hybrid scaffolds with different composition ratio were found to have significant promotional effects in cell adhesion, proliferation and differentiation, in which the group with Gel:nHA = 1:1 showed the best performance in vitro, as well as the most bone regeneration volume in vivo. This 3D micro-scaffolds modification may be an innovative method for porous titanium ornamentation and shows potential application values in clinic.

HaLT2- an enhanced lumber grading trainer

Treesearch

Powsiri Klinkhachorn; Charles Gatchell; Charles McMillin; Ravi Kothari; Dennis Yost

1992-01-01

This paper reports on HaLT2, an improved version of HaLT (Hardwood Lumber Traning Program)- a computer program that provides training in lumber grading. The newly added enhancements In HaLT2 will provide training for both novice and experienced hardwood lumber graders in accordance with National Hardwood Lumber Assodation (NHLA) rules. HaLT2 is more accurate, easier to...

Homology modeling study toward identifying structural properties in the HA2 B-loop that would influence the HA1 receptor-binding site.

PubMed

Cueno, Marni E; Imai, Kenichi; Shimizu, Kazufumi; Ochiai, Kuniyasu

2013-07-01

Influenza hemagglutinin (HA) consists of a fibrous globular stem (HA2) inserted into the viral membrane supporting a globular head (HA1). HA1 receptor-binding has been hypothesized to be structurally correlated to the HA2 B-loop, however, this was never fully understood. Here, we elucidated the structural relationship between the HA2 B-loop and the HA1 receptor-binding site (RBS). Throughout this study, we analyzed 2486 H1N1 HA homology models obtained from human, swine and avian strains during 1976-2012. Quality of all homology models were verified before further analyses. We established that amino acid residue 882 is putatively strain-conserved and differs in the human (K882), swine (H882) and avian (N882) strains. Moreover, we observed that the amino acid at residue 882 and, similarly, its orientation has the potential to influence the HA1 RBS diameter measurements which we hypothesize may consequentially affect influenza H1N1 viral infectivity, immune escape, transmissibility, and evolution. Copyright © 2013 Elsevier Inc. All rights reserved.

Modeling the association of space, time, and host species with variation of the HA, NA, and NS genes of H5N1 highly pathogenic avian influenza viruses isolated from birds in Romania in 2005-2007.

PubMed

Alkhamis, Mohammad; Perez, Andres; Batey, Nicole; Howard, Wendy; Baillie, Greg; Watson, Simon; Franz, Stephanie; Focosi-Snyman, Raffaella; Onita, Iuliana; Cioranu, Raluca; Turcitu, Mihai; Kellam, Paul; Brown, Ian H; Breed, Andrew C

2013-09-01

Molecular characterization studies of a diverse collection of avian influenza viruses (AIVs) have demonstrated that AIVs' greatest genetic variability lies in the HA, NA, and NS genes. The objective here was to quantify the association between geographical locations, periods of time, and host species and pairwise nucleotide variation in the HA, NA, and NS genes of 70 isolates of H5N1 highly pathogenic avian influenza virus (HPAIV) collected from October 2005 to December 2007 from birds in Romania. A mixed-binomial Bayesian regression model was used to quantify the probability of nucleotide variation between isolates and its association with space, time, and host species. As expected for the three target genes, a higher probability of nucleotide differences (odds ratios [ORs] > 1) was found between viruses sampled from places at greater geographical distances from each other, viruses sampled over greater periods of time, and viruses derived from different species. The modeling approach in the present study maybe useful in further understanding the molecular epidemiology of H5N1 HPAI virus in bird populations. The methodology presented here will be useful in predicting the most likely genetic distance for any of the three gene segments of viruses that have not yet been isolated or sequenced based on space, time, and host species during the course of an epidemic.

Registration of two double rust resistant germplasms, HA-R12 and HA-R13 for confection sunflower

USDA-ARS?s Scientific Manuscript database

The confection sunflower (Helianthus annuus L.) germplasms HA-R12 (Reg. No. ______, PI 673104) and HA-R13 (Reg. No. ______, PI 673105) were developed by the USDA-ARS, Sunflower and Plant Biology Research Unit in collaboration with the North Dakota Agricultural Experiment Station, and released in Jul...

Hyaluronic acid doped-poly(3,4-ethylenedioxythiophene)/chitosan/gelatin (PEDOT-HA/Cs/Gel) porous conductive scaffold for nerve regeneration.

PubMed

Wang, Shuping; Guan, Shui; Zhu, Zhibo; Li, Wenfang; Liu, Tianqing; Ma, Xuehu

2017-02-01

Conducting polymer, as a "smart" biomaterial, has been increasingly used to construct tissue engineered scaffold for nerve tissue regeneration. In this study, a novel porous conductive scaffold was prepared by incorporating conductive hyaluronic acid (HA) doped-poly(3,4-ethylenedioxythiophene) (PEDOT-HA) nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. The physicochemical characteristics of Cs/Gel scaffold with 0-10wt% PEDOT-HA were analyzed and the results indicated that the incorporation of PEDOT-HA into scaffold increased the electrical and mechanical properties while decreasing the porosity and water absorption. Moreover, in vitro biodegradation of scaffold displayed a declining trend with the PEDOT-HA content increased. About the biocompatibility of conductive scaffold, neuron-like rat phaeochromocytoma (PC12) cells were cultured in scaffold to evaluate cell adhesion and growth. 8% PEDOT-HA/Cs/Gel scaffold had a higher cell adhesive efficiency and cell viability than the other conductive scaffolds. Furthermore, cells in the scaffold with 8wt% PEDOT-HA expressed higher synapse growth gene of GAP43 and SYP compared with Cs/Gel control group. These results suggest that 8%PEDOT-HA/Cs/Gel scaffold is an attractive cell culture conductive substrate which could support cell adhesion, survival, proliferation, and synapse growth for the application in nerve tissue regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Imaging of Homeostatic, Neoplastic, and Injured Tissues by HA-Based Probes

PubMed Central

Veiseh, Mandana; Breadner, Daniel; Ma, Jenny; Akentieva, Natalia; Savani, Rashmin C; Harrison, Rene; Mikilus, David; Collis, Lisa; Gustafson, Stefan; Lee, Ting-Yim; Koropatnick, James; Luyt, Leonard G.; Bissell, Mina J.; Turley, Eva A.

2013-01-01

An increase in hyaluronan (HA) synthesis, cellular uptake, and metabolism occurs during the remodeling of tissue microenvironments following injury and during disease processes such as cancer. We hypothesized that multimodality HA-based probes selectively target and detectably accumulate at sites of high HA metabolism, thus providing a flexible imaging strategy for monitoring disease and repair processes. Kinetic analyses confirmed favorable available serum levels of the probe following intravenous (i.v.) or subcutaneous (s.c.) injection. Nuclear (technetium-HA, 99mTc-HA, and iodine-HA, 125I-HA), optical (fluorescent Texas Red-HA, TR-HA), and magnetic resonance (gadolinium-HA, Gd-HA) probes imaged liver (99mTc-HA), breast cancer cells/xenografts (TR-HA, Gd-HA), and vascular injury (125I-HA, TR-HA). Targeting of HA probes to these sites appeared to result from selective HA receptor-dependent localization. Our results suggest that HA-based probes, which do not require polysaccharide backbone modification to achieve favorable half-life and distribution, can detect elevated HA metabolism in homeostatic, injured, and diseased tissues. PMID:22066590

Modeling the Association of Space, Time, and Host Species with Variation of the HA, NA, and NS Genes of H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Birds in Romania in 2005–2007

PubMed Central

Alkhamis, Mohammad; Perez, Andres; Batey, Nicole; Howard, Wendy; Baillie, Greg; Watson, Simon; Franz, Stephanie; Focosi-Snyman, Raffaella; Onita, Iuliana; Cioranu, Raluca; Turcitu, Mihai; Kellam, Paul; Brown, Ian H.; Breed, Andrew C.

2014-01-01

SUMMARY Molecular characterization studies of a diverse collection of avian influenza viruses (AIVs) have demonstrated that AIVs’ greatest genetic variability lies in the HA, NA, and NS genes. The objective here was to quantify the association between geographical locations, periods of time, and host species and pairwise nucleotide variation in the HA, NA, and NS genes of 70 isolates of H5N1 highly pathogenic avian influenza virus (HPAIV) collected from October 2005 to December 2007 from birds in Romania. A mixed-binomial Bayesian regression model was used to quantify the probability of nucleotide variation between isolates and its association with space, time, and host species. As expected for the three target genes, a higher probability of nucleotide differences (odds ratios [ORs] > 1) was found between viruses sampled from places at greater geographical distances from each other, viruses sampled over greater periods of time, and viruses derived from different species. The modeling approach in the present study maybe useful in further understanding the molecular epidemiology of H5N1 HPAI virus in bird populations. The methodology presented here will be useful in predicting the most likely genetic distance for any of the three gene segments of viruses that have not yet been isolated or sequenced based on space, time, and host species during the course of an epidemic. PMID:24283126

Antibacterial Property of Cold-Sprayed HA-Ag/PEEK Coating

NASA Astrophysics Data System (ADS)

Sanpo, Noppakun; Tan, Meng Lu; Cheang, Philip; Khor, K. A.

2009-03-01

The antibacterial behavior of HA-Ag (silver-doped hydroxyapatite) nanopowder and their composite coatings were investigated against Escherichia coli (DH5α). HA-Ag nanopowder and PEEK (poly-ether-ether-ketone)-based HA-Ag composite powders were synthesized using in-house powder processing techniques. Bacteria culture assay of HA-Ag nanopowder and their composite powders displayed excellent bacteriostatic activity against E. coli. The antibacterial activity increased with increasing concentration of HA-Ag nanoparticle in these composite powders. These nanocomposite powders were subsequently used as feedstock to generate antibacterial coatings via cold spray technology. The ratios of HA-Ag to PEEK in their composite powders were 80:20, 60:40, 40:60, and 20:80 (wt.%). Microstructural characterization and phase analysis of feedstock powders and as-deposited coatings were carried out using FESEM/EDX and XRD. Antibacterial nanocomposite HA-Ag/PEEK coatings were successfully deposited using cold spraying parameters of 11-12 bars at preheated air temperature between 150 and 160 °C. These as-sprayed coatings of HA-Ag/PEEK composite powders comprising varying HA-Ag and PEEK ratios retained their inherent antibacterial property as verified from bacterial assay. The results indicated that the antibacterial activity increased with increasing HA-Ag nanopowder concentration in the composite powder feedstock and cold-sprayed coating.

Role of the HaHOG1 MAP Kinase in Response of the Conifer Root and But Rot Pathogen (Heterobasidion annosum) to Osmotic and Oxidative Stress

PubMed Central

Raffaello, Tommaso; Keriö, Susanna; Asiegbu, Fred O.

2012-01-01

The basidiomycete Heterobasidion annosum (Fr.) Bref. s.l. is a filamentous white rot fungus, considered to be the most economically important pathogen of conifer trees. Despite the severity of the tree infection, very little is known about the molecular and biochemical aspects related to adaptation to abiotic stresses. In this study, the osmotic and oxidative tolerance as well as the role of the HaHOG1 Mitogen Activated Protein Kinase (MAPK) gene were investigated. The transcript levels of the yeast orthologues GPD1, HSP78, STL1, GRE2 and the ATPase pumps ENA1, PMR1, PMC1 known to have an important role in osmotolerance were also quantified under salt osmotic conditions. The HaHOG1 gene was used for a heterologous expression and functional study in the Saccharomyces cerevisiae Δhog1 strain. Moreover, the phosphorylation level of HaHog1p was studied under salt osmotic and oxidative stress. The result showed that H. annosum displayed a decreased growth when exposed to an increased concentration of osmotic and oxidative stressors. GPD1, HSP78, STL1 and GRE2 showed an induction already at 10 min after exposure to salt stress. Among the ATPase pumps studied, PMC1 was highly induced when the fungus was exposed to 0.2 M CaCl2 for 60 min. The heterologous expression of the HaHOG1 sequence in yeast confirmed that the gene is able to restore the osmotolerance and oxidative tolerance in the S. cerevisiae hog1Δ mutant strain. The HaHog1p was strongly phosphorylated in the presence of NaCl, KCl, H2O2 but not in the presence of CaCl2 and MgCl2. The GFP-HaHog1p fusion protein accumulated in the nuclei of the S. cerevisiae hog1Δ cells when exposed to high osmotic conditions but not under oxidative stress. These results provide the first insights about the response of H. annosum to osmotic and oxidative stress and elucidate the role of the HaHOG1 gene in such conditions. PMID:22319614

[Typing and subtyping avian influenza virus using DNA microarrays].

PubMed

Yang, Zhongping; Wang, Xiurong; Tian, Lina; Wang, Yu; Chen, Hualan

2008-07-01

Outbreaks of highly pathogenic avian influenza (HPAI) virus has caused great economic loss to the poultry industry and resulted in human deaths in Thailand and Vietnam since 2004. Rapid typing and subtyping of viruses, especially HPAI from clinical specimens, are desirable for taking prompt control measures to prevent spreading of the disease. We described a simultaneous approach using microarray to detect and subtype avian influenza virus (AIV). We designed primers of probe genes and used reverse transcriptase PCR to prepare cDNAs of AIV M gene, H5, H7, H9 subtypes haemagglutinin genes and N1, N2 subtypes neuraminidase genes. They were cloned, sequenced, reamplified and spotted to form a glass-bound microarrays. We labeled samples using Cy3-dUTP by RT-PCR, hybridized and scanned the microarrays to typing and subtyping AIV. The hybridization pattern agreed perfectly with the known grid location of each probe, no cross hybridization could be detected. Examinating of HA subtypes 1 through 15, 30 infected samples and 21 field samples revealed the DNA microarray assay was more sensitive and specific than RT-PCR test and chicken embryo inoculation. It can simultaneously detect and differentiate the main epidemic AIV. The results show that DNA microarray technology is a useful diagnostic method.

Highly stretchable HA/SA hydrogels for tissue engineering.

PubMed

Zhu, Chengcheng; Yang, Rui; Hua, Xiaobin; Chen, Hong; Xu, Jumei; Wu, Rile; Cen, Lian

2018-04-01

A highly stretchable hyaluronic acid (HA)/sodium alginate (SA) hydrogel was developed in this study based on an interpenetrating polymer network. HA/SA hydrogels were prepared by mixing two polysaccharides followed by covalent crosslinking via epoxy groups on HA molecules and ionic crosslinking via divalent ions on SA chains sequentially. The effect of HA/SA ratio on the pore size and distribution, swelling ratio, elongation and rheological properties as well as protein loading and release properties of HA/SA hydrogels was explored. Moreover, a surface modification method, layer-by-layer (LBL) assembly technique, was applied to modify the hydrogel to evaluate the hydrogel's tenability in varying biological performance. It was then shown that the hydrogels had the pore sizes ranging from 100 to 50 μm. With the increase in SA content of the resulting hydrogels, the pore size, swelling ratio, and storage modulus (G') and loss modulus (G″) of the hydrogel all decreased, whereas the in vitro bulk weight loss was fastened. Moreover, elongation at break (EB) value increased first, reached a peak value and then decreased, that is HA8/SA1 (HA:SA = 8:1) had the highest EB value of 417%. This hydrogel could retain 33.2% of the pre-loaded protein even after 72 h, which could be further attenuated when LBL was used to shell the hydrogel. The growth of fibroblasts on HA8/SA1 hydrogel gave preliminary assessment on its suitability as a cellular carrier, while the LBL modified HA8/SA1 hydrogel also favored the anchoring of keratinocytes, further enhancing its cell carrier role for tissue regeneration, especially skin engineering.

Glycosylation of Residue 141 of Subtype H7 Influenza A Hemagglutinin (HA) Affects HA-Pseudovirus Infectivity and Sensitivity to Site A Neutralizing Antibodies.

PubMed

Alvarado-Facundo, Esmeralda; Vassell, Russell; Schmeisser, Falko; Weir, Jerry P; Weiss, Carol D; Wang, Wei

2016-01-01

Human infections with H7 subtype influenza virus have been reported, including an H7N7 outbreak in Netherlands in 2003 and H7N9 infections in China in 2013. Previously, we reported murine monoclonal antibodies (mAbs) that recognize the antigenic site A of H7 hemagglutinin (HA). To better understand protective immunity of H7 vaccines and vaccine candidate selection, we used these mAbs to assess the antigenic relatedness among two H7 HA isolated from past human infections and determine residues that affect susceptibility to neutralization. We found that these mAbs neutralize pseudoviruses bearing HA of A/Shanghai/02/2013(H7N9), but not A/Netherlands/219/2003(H7N7). Glycosylation of the asparagine residue at position 141 (N141) (N133, H3 HA numbering) in the HA of A/Netherlands/219/2003 HA is responsible for this resistance, and it affects the infectivity of HA-pseudoviruses. The presence of threonine at position 143 (T135, H3 HA numbering) in the HA of A/Netherlands/219/2003, rather than an alanine found in the HA of A/Shanghai/02/2013(H7N9), accounts for these differences. These results demonstrate a key role for glycosylation of residue N141 in affecting H7 influenza HA-mediated entry and sensitivity to neutralizing antibodies, which have implications for candidate vaccine design.

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

PubMed

Cox, Freek; Kwaks, Ted; Brandenburg, Boerries; Koldijk, Martin H; Klaren, Vincent; Smal, Bastiaan; Korse, Hans J W M; Geelen, Eric; Tettero, Lisanne; Zuijdgeest, David; Stoop, Esther J M; Saeland, Eirikur; Vogels, Ronald; Friesen, Robert H E; Koudstaal, Wouter; Goudsmit, Jaap

2016-01-01

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

Osteogenic properties of PBLG-g-HA/PLLA nanocomposites.

PubMed

Liao, Lan; Yang, Shuang; Miron, Richard J; Wei, Junchao; Zhang, Yufeng; Zhang, Meng

2014-01-01

New development of biomaterial scaffolds remains a prominent issue for the regeneration of lost or fractured bone. Of these scaffolds, a number of bioactive polymers have been synthesized and fabricated for diverse biological roles. Although recent evidence has demonstrated that composite scaffolds such as HA/PLLA have improved properties when compared to either HA or PLLA alone, recent investigations have demonstrated that the phase compatibility between HA and PLLA layers is weak preventing optimal enhancement of the mechanical properties and making the composites prone to breakdown. In the present study, poly (γ-benzyl-L-glutamate) modified hydroxyapatite/(poly (L-lactic acid)) (PBLG-g-HA/PLLA) composite scaffolds were fabricated with improved phase compatibility and tested for their osteogenic properties in 18 Wistar female rats by analyzing new bone formation in 3 mm bilateral femur defects in vivo. At time points, 2, 4 and 8 weeks post surgery, bone formation was evaluated by µ-CT and histological analysis by comparing 4 treatment groups; 1) blank defect, 2) PLLA, 3) HA/PLLA and 4) PBLG-g-HA/PLLA scaffolds. The in vivo analysis demonstrated that new bone formation was much more prominent in HA/PLLA and PBLG-g-HA/PLLA groups as depicted by µ-CT, H&E staining and immunohistochemistry for collagen I. TRAP staining was also utilized to determine the influence of osteoclast cell number and staining intensity to the various scaffolds. No significant differences in either staining intensity or osteoclast numbers between all treatment modalities was observed, however blank defects did contain a higher number of osteoclast-like cells. The results from the present study illustrate the potential of PBLG-g-HA/PLLA scaffolds for bone tissue engineering applications by demonstrating favorable osteogenic properties.

Effect of humic acid (HA) on sulfonamide sorption by biochars.

PubMed

Lian, Fei; Sun, Binbin; Chen, Xi; Zhu, Lingyan; Liu, Zhongqi; Xing, Baoshan

2015-09-01

Effect of quantity and fractionation of loaded humic acid (HA) on biochar sorption for sulfonamides was investigated. The HA was applied in two different modes, i.e. pre-coating and co-introduction with sorbate. In pre-coating mode, the polar fractions of HA tended to interact with low-temperature biochars via H-bonding, while the hydrophobic fractions were likely to be adsorbed by high-temperature biochars through hydrophobic and π-π interactions, leading to different composition and structure of the HA adlayers. The influences of HA fractionation on biochar sorption for sulfonamides varied significantly, depending on the nature of interaction between HA fraction and sorbate. Meanwhile, co-introduction of HA with sulfonamides revealed that the effect of HA on sulfonamide sorption was also dependent on HA concentration. These findings suggest that the amount and fractionation of adsorbed HA are tailored by the surface properties of underlying biochars, which differently affect the sorption for organic contaminants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Changeable HA to improve MIPv6 protocol

NASA Astrophysics Data System (ADS)

Hu, Qing-gui

2015-12-01

For mobile IPv6, home agent (HA) plays an important role. Each mobile node (MN) has a home IP address, it will be not changeable. Also, the home agent (HA) of MN is not changeable. This rule provides the convenient for the ongoing communication without interruption. But it has some obvious drawbacks. Here, the new variable HA scheme is proposed. Every MN has a dynamic cache table, recording the information such as its home address, care-of address, and history address etc. If the accumulated time in one region exceeds that in the hometown, the foreign agent (FA) could become home agent (HA), the home agent could become history agent. Later, the performance of the new protocol is simulated with OPNET software, whose result shows the performance of the new protocol works better than that of the traditional protocol.

[Promoting effect of cyclin D1 overexpression on proliferation and epithelial mesenchymal transition of cervical squamous cell carcinoma SiHa cells].

PubMed

Wang, P; Liu, S; Cheng, B; Wu, X Z; Ding, S S; Xu, L; Liu, Y; Duan, L; Sun, S Z

2017-03-08

Objective: To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Methods: Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. Results: The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Conclusions: Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory

Registration of two confection sunflower germplasm Lines, HA-R10 and HA-R11, Resistant to sunflower rust

USDA-ARS?s Scientific Manuscript database

Two confection sunflower (Helianthus annuus L.) germplasm lines, HA-R10 (Reg. No.xxx, PI670043) and HA-R11 (Reg. No.xxx, PI670044) were developed by the USDA-ARS Sunflower and Plant Biology Research Unit in collaboration with the North Dakota Agricultural Experiment Station and released December, 20...

Application of new vaccine technologies for the control of transboundary diseases.

PubMed

Swayne, D E

2004-01-01

Vaccines have played an important role in the control of diseases of livestock and poultry, including Transboundary Diseases. In the future, vaccines will play a greater role in controlling these diseases. Historically, inactivated whole viruses in various adjuvant systems have been used and will continue to be used in the near future. For the future, emerging technologies will allow targeted use of only the protective antigens of the pathogen and will provide the opportunity for differentiating between vaccinated and field-exposed animals. Furthermore, the expression of cytokines by vaccines will afford earlier or greater enhancement of protection than can be achieved by the protective response elicited by the antigenic epitopes of the pathogen alone. Avian influenza (AI) is a good case for studying future trends in vaccine design and use. Inactivated AI virus (AIV) vaccines will continue as the primary vaccines used over the next 10 years. These vaccines will use homologous haemagglutinin sub-types, either from the use of field strains or the generation of new strains through the use of infectious clones produced in the laboratory. The latter will allow creation of high growth reassortants, which will provide consistent high yields of antigen and result in potent vaccines. New viral and bacterial vectors with inserts of AIV haemagglutinin gene will be developed and potentially used in the field. Such new vectors will include herpesvirus-turkey, infectious laryngotracheitis virus, adenoviruses, various types of paramyxoviruses and Salmonella sp. In addition, there is a theoretical possibility of gene-deleted mutants that would allow the use of live AIV vaccines, but the application of such vaccines has inherent dangers for gene reassortment with field viruses in the generation of disease-causing strains. Subunit haemagglutinin protein and DNA haemagglutinin gene vaccines are possible, but with current technologies, the cost is prohibitive. In the future, effective

Generation of recombinant pandemic H1N1 influenza virus with the HA cleavable by bromelain and identification of the residues influencing HA bromelain cleavage.

PubMed

Wang, Weijia; Suguitan, Amorsolo L; Zengel, James; Chen, Zhongying; Jin, Hong

2012-01-20

The proteolytic enzyme bromelain has been traditionally used to cleave the hemagglutinin (HA) protein at the C-terminus of the HA2 region to release the HA proteins from influenza virions. The bromelain cleaved HA (BHA) has been routinely used as an antigen to generate antiserum that is essential for influenza vaccine product release. The HA of the 2009 pandemic H1N1 influenza A/California/7/2009 (CA09) virus could not be cleaved efficiently by bromelain. To ensure timely delivery of BHA for antiserum production, we generated a chimeric virus that contained the HA1 region from CA09 and the HA2 region from the seasonal H1N1 A/South Dakota/6/2007 (SD07) virus that is cleavable by bromelain. The BHA from this chimeric virus was antigenically identical to CA09 and induced high levels of HA-specific antibodies and protected ferrets from wild-type H1N1 CA09 virus challenge. To determine the molecular basis of inefficient cleavage of CA09 HA by bromelain, the amino acids that differed between the HA2 of CA09 and SD07 were introduced into recombinant CA09 virus to assess their effect on bromelain cleavage. The D373N or E374G substitution in the HA2 stalk region of CA09 HA enabled efficient cleavage of CA09 HA by bromelain. Sequence analysis of the pandemic H1N1-like viruses isolated from 2010 revealed emergence of the E374K change. We found that K374 enabled the HA to be cleaved by bromelain and confirmed that the 374 residue is critical for HA bromelain cleavage. Copyright © 2011 Elsevier Ltd. All rights reserved.

Preparation and characterization of bio-composite PEEK/nHA

NASA Astrophysics Data System (ADS)

Jin, Y. S.; Bian, C. C.; Zhang, Z. Q.; Zhao, Y.; Yang, L.

2017-01-01

PEEK/nHA composite material, with excellent mechanical property as polyetheretherketone (PEEK) and biological activity as hydroxyapatite (HA), has attracted wide attention of medical experts and materials science experts. The addition of hydroxyapatite was the decisive factor for biological activity in PEEK/nHA composite. In this paper, acicular nanohydroxyapatite was prepared by chemical precipitation method with Ca(NO3)2, (NH4)2HPO4 as raw material; PEEK/nHA composite was prepared by solution blending and vacuum sintering method. The composite was characterized with FT-IR, XRD, DSC, TG and mechanical property test. Results showed that the composite has good thermal stability and compressive property when the mass ratio of PEEK to nHA is 10:3; and high nHA content can improve the biological activity of the composite, which can meet the basic requirements for bone tissue engineering scaffold.

Hyaluronan (HA) interacting proteins RHAMM and hyaluronidase impact prostate cancer cell behavior and invadopodia formation in 3D HA-based hydrogels.

PubMed

Gurski, Lisa A; Xu, Xian; Labrada, Lyana N; Nguyen, Ngoc T; Xiao, Longxi; van Golen, Kenneth L; Jia, Xinqiao; Farach-Carson, Mary C

2012-01-01

To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, "invadopodia", consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of

Potential usefulness of D2R reporter gene imaging by IBF as gene therapy monitoring for cerebellar neurodegenerative diseases.

PubMed

Shiba, Kazuhiro; Torashima, Takashi; Hirai, Hirokazu; Ogawa, Kazuma; Akhter, Nasima; Nakajima, Kenichi; Kinuya, Seigo; Mori, Hirofumi

2009-02-01

We investigated a gene expression imaging method to examine the level of therapeutic gene expression in the cerebellum. Using a human immunodeficiency virus derived lentivial vector, we expressed the dopamine D(2) receptor (D(2)R) as a reporter protein to mouse cerebellar Purkinje cells. Biodistribution and ex vivo autoradiography studies were performed by giving [(125)I]5-iodo-7-N-[(1-ethyl-2-pyrrolidinyl)methyl]carboxamide-2,3-dihydrobenzofuran ([(125)I]IBF) (1.85 MBq), as a radioactive D(2)R ligand, to model mice expressing the D(2)R with an HA tag (HA-D(2)R) in the cerebellum. In this study, [(125)I]IBF was bound to the D(2)R expressed in the cerebellum of the model mice selectively. Immunostaining was performed to confirm the HA-D(2)R expression in the cerebellum of the model mice. A significant correlation (r=0.900, P<0.001) between areas that expressed HA-D(2)R by immunostaining and areas in which [(125)I]IBF accumulated by the ex vivo autoradiograms was found. These results indicated that radioiodinated IBF is useful as a reporter probe to detect D(2)R reporter gene expression, which can be used for monitoring therapeutic gene expression in the cerebellum.

Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2.

PubMed

Chiapponi, Chiara; Moreno, Ana; Barbieri, Ilaria; Merenda, Marianna; Foni, Emanuela

2012-09-01

In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies. Copyright © 2012 Elsevier B.V. All rights reserved.

Genesis of the novel human-infecting influenza A(H10N8) virus and potential genetic diversity of the virus in poultry, China.

PubMed

Qi, W; Zhou, X; Shi, W; Huang, L; Xia, W; Liu, D; Li, H; Chen, S; Lei, F; Cao, L; Wu, J; He, F; Song, W; Li, Q; Li, H; Liao, M; Liu, M

2014-06-26

Human infection with a novel influenza A(H10N8) virus was first described in China in December 2013. However, the origin and genetic diversity of this virus is still poorly understood. We performed a phylogenetic analysis and coalescent analysis of two viruses from the first case of influenza A(H10N8) (A/Jiangxi-Donghu/346-1/2013 and A/Jiangxi-Donghu/346-2/2013 and a novel A(H10N8) virus (A/chicken/Jiangxi/102/2013) isolated from a live poultry market that the patient had visited. The haemagglutinin (HA), neuraminidase (NA), PA subunit of the virus polymerase complex, nucleoprotein (NP), M and nonstructural protein (NS) genes of the three virus strains shared the same genetic origins. The origins of their HA and NA genes were similar: originally from wild birds to ducks, and then to chickens. The PA, NP, M, and NS genes were similar to those of chicken influenza A(H9N2) viruses. Coalescent analyses showed that the reassortment of these genes from A(H9N2) to A(H10N8) might have occurred at least twice. However, the PB1 and PB2 genes of the chicken A(H10N8) virus most likely originated from H7-like viruses of ducks, while those of the viruses from the case most likely stemmed from A(H9N2) viruses circulating in chickens. The oseltamivir-resistance mutation, R292K (R291K in A(H10N8) numbering) in the NA protein, occurred after four days of oseltamivir treatment. It seems that A(H10N8) viruses might have become established among poultry and their genetic diversity might be much higher than what we have observed.

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

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

Feng, Guoxing; Shi, Hui; Li, Jiong

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 atmore » the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells

Hyperactivation of Ha-ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis

PubMed Central

Mo, Lan; Zheng, Xiaoyong; Huang, Hong-Ying; Shapiro, Ellen; Lepor, Herbert; Cordon-Cardo, Carlos; Sun, Tung-Tien; Wu, Xue-Ru

2007-01-01

Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%–90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity — a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system. PMID:17256055

The sunflower transcription factor HaHB11 confers tolerance to water deficit and salinity to transgenic Arabidopsis and alfalfa plants.

PubMed

Cabello, Julieta V; Giacomelli, Jorge I; Gómez, María C; Chan, Raquel L

2017-09-10

Homeodomain-leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom; members of subfamily I are known to be involved in abiotic stress responses. HaHB11 belongs to this subfamily and it was previously shown that it is able to confer improved yield and tolerance to flooding via a quiescent strategy. Here we show that HaHB11 expression is induced by ABA, NaCl and water deficit in sunflower seedlings and leaves. Arabidopsis transgenic plants expressing HaHB11, controlled either by its own promoter or by the constitutive 35S CaMV, presented rolled leaves and longer roots than WT when grown under standard conditions. In addition, these plants showed wider stems and more vascular bundles. To deal with drought, HaHB11 transgenic plants closed their stomata faster and lost less water than controls, triggering an enhanced tolerance to such stress condition and also to salinity stress. Concomitantly, ABA-synthesis and sensing related genes were differentially regulated in HaHB11 transgenic plants. Either under long-term salinity stress or mild drought stress, HaHB11 transgenic plants did not exhibit yield penalties. Moreover, alfalfa transgenic plants were generated which also showed enhanced drought tolerance. Altogether, the results indicated that HaHB11 was able to confer drought and salinity tolerance via a complex mechanism which involves morphological, physiological and molecular changes. Copyright © 2016 Elsevier B.V. All rights reserved.

Phylogenetic analysis of the haemagglutinin gene of current wild-type canine distemper viruses from South Africa: lineage Africa.

PubMed

Woma, Timothy Y; van Vuuren, Moritz; Bosman, Ana-Mari; Quan, Melvyn; Oosthuizen, Marinda

2010-07-14

There are no reports of CDV isolations in southern Africa, and although CDV is said to have geographically distinct lineages, molecular information of African strains has not yet been documented. Viruses isolated in cell cultures were subjected to reverse transcription-polymerase chain reaction (RT-PCR), and the complete H gene was sequenced and phylogenetically analysed with other strains from GenBank. Phylogenetic comparisons of the complete H gene of CDV isolates from different parts of the world (available in GenBank) with wild-type South African isolates revealed nine clades. All South African isolates form a separate African clade of their own and thus are clearly separated from the American, European, Asian, Arctic and vaccine virus clades. It is likely that only the 'African lineage' of CDV may be circulating in South Africa currently, and the viruses isolated from dogs vaccinated against CDV are not the result of reversion to virulence of vaccine strains, but infection with wild-type strains. (c) 2009 Elsevier B.V. All rights reserved.

Statistical analysis of nucleotide sequences of the hemagglutinin gene of human influenza A viruses.

PubMed Central

Ina, Y; Gojobori, T

1994-01-01

To examine whether positive selection operates on the hemagglutinin 1 (HA1) gene of human influenza A viruses (H1 subtype), 21 nucleotide sequences of the HA1 gene were statistically analyzed. The nucleotide sequences were divided into antigenic and nonantigenic sites. The nucleotide diversities for antigenic and nonantigenic sites of the HA1 gene were computed at synonymous and nonsynonymous sites separately. For nonantigenic sites, the nucleotide diversities were larger at synonymous sites than at nonsynonymous sites. This is consistent with the neutral theory of molecular evolution. For antigenic sites, however, the nucleotide diversities at nonsynonymous sites were larger than those at synonymous sites. These results suggest that positive selection operates on antigenic sites of the HA1 gene of human influenza A viruses (H1 subtype). PMID:8078892

Genome-Wide Analyses Reveal Genes Subject to Positive Selection in Pasteurella multocida

PubMed Central

Cao, Peili; Guo, Dongchun; Liu, Jiasen; Jiang, Qian; Xu, Zhuofei; Qu, Liandong

2017-01-01

Pasteurella multocida, a Gram-negative opportunistic pathogen, has led to a broad range of diseases in mammals and birds, including fowl cholera in poultry, pneumonia and atrophic rhinitis in swine and rabbit, hemorrhagic septicemia in cattle, and bite infections in humans. In order to better interpret the genetic diversity and adaptation evolution of this pathogen, seven genomes of P. multocida strains isolated from fowls, rabbit and pigs were determined by using high-throughput sequencing approach. Together with publicly available P. multocida genomes, evolutionary features were systematically analyzed in this study. Clustering of 70,565 protein-coding genes showed that the pangenome of 33 P. multocida strains was composed of 1,602 core genes, 1,364 dispensable genes, and 1,070 strain-specific genes. Of these, we identified a full spectrum of genes related to virulence factors and revealed genetic diversity of these potential virulence markers across P. multocida strains, e.g., bcbAB, fcbC, lipA, bexDCA, ctrCD, lgtA, lgtC, lic2A involved in biogenesis of surface polysaccharides, hsf encoding autotransporter adhesin, and fhaB encoding filamentous haemagglutinin. Furthermore, based on genome-wide positive selection scanning, a total of 35 genes were subject to strong selection pressure. Extensive analyses of protein subcellular location indicated that membrane-associated genes were highly abundant among all positively selected genes. The detected amino acid sites undergoing adaptive selection were preferably located in extracellular space, perhaps associated with bacterial evasion of host immune responses. Our findings shed more light on conservation and distribution of virulence-associated genes across P. multocida strains. Meanwhile, this study provides a genetic context for future researches on the mechanism of adaptive evolution in P. multocida. PMID:28611758

High strength yttria-reinforced HA scaffolds fabricated via honeycomb ceramic extrusion.

PubMed

Elbadawi, M; Shbeh, M

2018-01-01

The present study investigated the effects of hydroxyapatite (HA) reinforced with yttria on porous scaffolds fabricated via honeycomb ceramic extrusion. Yttria was selected as it has been demonstrated to toughen other ceramics. Moreover, yttria has been surmised to suppress dehydroxylation in HA, a characteristic that prefigures decomposition thereof during sintering into mechanically weaker phases. However, the compressive strength of yttria-reinforced hydroxyapatite (Y-HA) porous scaffolds has hitherto not been reported. Y-HA was synthesised by calcining a commercially available HA with 10wt% yttria at 1000°C. Y-HA was then fabricated into porous scaffolds using an in-house honeycomb extruder, and subsequently sintered at 1200 and 1250°C. The results were compared to the uncalcined as-received commercial powder (AR-HA) and calcined pure HA powder at 1000°C (C-HA). It was discovered that calcination alone caused marked improvements to the stoichiometry, thermal stability, porosity and compressive strength of scaffolds. The improvements were ascribed to the calcined powders with less susceptibility to both agglomeration and enhanced densification. Still, differences were observed between C-HA and Y-HA at 1250°C. The compressive strength increased from 105.9 to 127.3MPa, a larger microporosity was descried and the HA matrix in Y-HA was more stoichiometric. The latter was confirmed by XRD and EDS analyses. Therefore, it was concluded that the reinforcing of hydroxyapatite with yttria improved the compressive strength and suppressed dehydroxylation of porous HA scaffolds. In addition, the compressive strength achieved demonstrated great potential for load-bearing application. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation

PubMed Central

Peralta-Arrieta, Irlanda; Hernández-Sotelo, Daniel; Castro-Coronel, Yaneth; Leyva-Vázquez, Marco Antonio; Illades-Aguiar, Berenice

2017-01-01

Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo. The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer. PMID:28123849

DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation.

PubMed

Peralta-Arrieta, Irlanda; Hernández-Sotelo, Daniel; Castro-Coronel, Yaneth; Leyva-Vázquez, Marco Antonio; Illades-Aguiar, Berenice

2017-01-01

Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo . The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer.

Transport features of nano-hydroxylapatite (n-HA) embedded silicone rubber (SR) systems: influence of SR/n-HA interaction, degree of reinforcement and morphology.

PubMed

M, Bindu; G, Unnikrishnan

2017-09-27

We report the transport characteristics of silicone rubber/nano-hydroxylapatite (SR/n-HA) systems at room temperature with reference to the effects of n-HA loading, morphology and penetrant nature, using toluene, xylene, ethyl acetate and butyl acetate in the liquid phase and methanol, ethanol, 1-propanol, 2-propanol and butanol in the vapour phase as probe molecules. The interaction between the n-HA particles and SR matrix has been confirmed by FTIR analysis. As the n-HA content in the SR matrix increased, the penetrant uptake has been found to decrease. The observations have been correlated with the density and void content of the systems. Scanning electron microscopy images have been found to be complementary to the observed transport features. The reinforcement effect of n-HA particles on the SR matrix has been verified by Kraus equation. Molecular mass between the cross links has been observed to decrease with an increase in n-HA loading. The results have been compared with affine, phantom network, parallel, series and Maxwell models. The transport data have been complemented by observations on biological fluid uptake with urea, d-glucose, KI, saline water, phosphate buffer and artificial urine as the media.

Chromium III histidinate exposure modulates antioxidant gene expression in HaCaT human keratinocytes exposed to oxidative stress

USDA-ARS?s Scientific Manuscript database

While the toxicity of hexavalent chromium is well established, trivalent Cr (Cr(III)) is an essential nutrient involved in insulin and glucose homeostasis. Recently, antioxidant effects of chromium (III) histidinate (Cr(III)His) were reported in HaCaT human keratinocytes exposed to oxidative stress...

Stalking influenza by vaccination with pre-fusion headless HA mini-stem.

PubMed

Valkenburg, Sophie A; Mallajosyula, V Vamsee Aditya; Li, Olive T W; Chin, Alex W H; Carnell, George; Temperton, Nigel; Varadarajan, Raghavan; Poon, Leo L M

2016-03-07

Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies induced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.

Response of human bone marrow-derived MSCs on triphasic Ca-P substrate with various HA/TCP ratio.

PubMed

Bajpai, Indu; Kim, Duk Yeon; Kyong-Jin, Jung; Song, In-Hwan; Kim, Sukyoung

2017-01-01

Calcium phosphates (Ca-P) are used commonly as artificial bone substitutes to control the biodegradation rate of an implant in the body fluid. This study examined the in vitro proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs) on triphasic Ca-P samples. For this aspect, hydroxyapatite (HA), dicalcium phosphate dehydrate (DCPD), and calcium hydroxide (Ca(OH) 2 ) were mixed at various ratios, cold compacted, and sintered at 1250°C in air. X-ray diffraction showed that the β-tricalcium phosphate (TCP) to α-TCP phase transformation increased with increasing DCPD/HA ratio. The micro-hardness deceased with increasing TCP content, whereas the mean grain size and porosity increased with increasing TCP concentration. To evaluate the in vitro degree of adhesion and proliferation on the HA/TCP samples, human BMSCs were incubated on the HA/TCP samples and analyzed by a cells proliferation assay, expression of the extracellular matrix (ECM) genes, such as α-smooth muscle actin (α-SMA) and fibronectin (FN), and FITC-phalloidin fluorescent staining. In terms of the interactions of human BMSCs with the triphasic Ca-P samples, H50T50 (Ca/P = 1.59) markedly enhanced cell spreading, proliferation, FN, and α-SMA compared with H100T0 (Ca/P = 1.67). Interestingly, these results show that among the five HA/TCP samples, H50T50 is the optimal Ca-P composition for in vitro cell proliferation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 72-80, 2017. © 2015 Wiley Periodicals, Inc.

Preparation, optimization and property of PVA-HA/PAA composite hydrogel.

PubMed

Chen, Kai; Liu, Jinlong; Yang, Xuehui; Zhang, Dekun

2017-09-01

PVA-HA/PAA composite hydrogel is prepared by freezing-thawing, PEG dehydration and annealing method. Orthogonal design method is used to choose the optimization combination. Results showed that HA and PVA have the maximum effect on water content. PVA and freezing-thawing cycles have the maximum effect on creep resistance and stress relaxation rate of hydrogel. Annealing temperature and freezing-thawing cycles have the maximum effect on compressive elastic modulus of hydrogel. Comparing with the water content and mechanical properties of 16 kinds of combination, PVA-HA/PAA composite hydrogel with freezing-thawing cycles of 3, annealing temperature of 120°C, PVA of 16%, HA of 2%, PAA of 4% has the optimization comprehensive properties. PVA-HA/PAA composite hydrogel has a porous network structure. There are some interactions between PVA, HA and PAA in hydrogel and the properties of hydrogel are strengthened. The annealing treatment improves the crystalline and crosslinking of hydrogel. Therefore, the annealing PVA-HA/PAA composite hydrogel has good thermostability, strength and mechanical properties. It also has good lubrication property and its friction coefficient is relative low. Copyright © 2017 Elsevier B.V. All rights reserved.

[Construction of porous hydroxyapatite (HA) block loaded with cultured chondrocytes].

PubMed

Yan, M; Dang, G

1999-07-01

To construct a kind of bone healing enhancing implant with cultured chondrocytes bound to hydroxyapatite (HA). Chondrocytes were obtained from the costicartilage of rat and were cultured on the porous HA blocks, 3 mm x 3 mm x 4 mm size, for three and seven days. Scanning electron micrograph was taken to show whether the cells grew outside and inside the pore of HA block. The cells cultured on tiny glass sheet for 2 days were used to prove where the cells come from by in situ hybridization technique with alpha1 (II) cDNA probe. Scanning electron micrographs showed that the pores of the HA surface and inside of the blocks are filled with cultured cells, especially the longer cultured block. The cells were chondrocytes confirmed by in situ hybridization. The porous HA can be used as cell cultured substrate and chondrocyte can adhere and proliferate inside the porous HA block.

New associations: INFG and TGFB1 genes and the inhibitor development in severe haemophilia A.

PubMed

de Alencar, J B; Macedo, L C; de Barros, M F; Rodrigues, C; Shinzato, A H; Pelissari, C B; Machado, J; Sell, A M; Visentainer, J E L

2015-07-01

The development of factor VIII (FVIII) inhibitor is the main complication of replacement therapy in patients with haemophilia A (HA). A ratio of 5-7% of individuals HA develops antibodies (inhibitors) against the FVIII infused during the treatment, thereby reducing their pro-coagulant activity. The immunomodulatory cytokine genes have been related to the risk of development of alloantibodies in several studies, mainly in HA with severe form. We investigated the polymorphisms in regulatory regions of cytokine genes (IL1A, IL1B, IL1R, IL1RA, IL4RA, IL12, INFG, TGFB1, TNF, IL2, IL4, IL6, IL10) that could influence the risk of developing inhibitors in patients with severe HA. The genotyping of cytokine genes of 117 patients with HA was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP) using the protocol recommended by the manufacturer (Invitrogen kit Cytokines(®) , Canoga Park, USA) RESULTS: From the cohort of 117 patients with severe HA, 35 developed inhibitors. There was a higher frequency of +874 T allele in INFG and of +869 TT and TG/TG in TGFB1 genes on patients with inhibitors. This suggests that polymorphisms in INFG and in TGFB1 genes are related to risk of developing inhibitor, and could contribute to a genetic profile of the individual HA for the risk of inhibitors development to FVIII. © 2015 John Wiley & Sons Ltd.

A comparison study on the behavior of human endometrial stem cell-derived osteoblast cells on PLGA/HA nanocomposite scaffolds fabricated by electrospinning and freeze-drying methods.

PubMed

Namini, Mojdeh Salehi; Bayat, Neda; Tajerian, Roxana; Ebrahimi-Barough, Somayeh; Azami, Mahmoud; Irani, Shiva; Jangjoo, Saranaz; Shirian, Sadegh; Ai, Jafar

2018-03-27

An engineered tissue structure is an artificial scaffold combined with cells and signaling factors. Among various polymers, the polylactide-co-glycolide/hydroxyapatite (PLGA/HA) has attracted much attention due to their optimal properties. The aim of this study was to study the behavior of human endometrial stem cell (hEnSC)-derived osteoblast cells cultured on PLGA/HA nanocomposite scaffolds. hEnSCs were isolated and exposed to osteogenic media for 21 days. Differentiated cells were cultured on PLGA/HA synthetic scaffolds. The PLGA/HA-based nanocomposite scaffolds were fabricated using either electrospinning or freeze-drying methods. Behavior of the cells was evaluated a week after seeding hEnSC-derived osteoblast-like cells on these scaffolds. Osteogenesis was investigated in terms of alkaline phosphatase activity, gene expression, immunocytochemistry (ICC), proliferation, and scanning electron microscopy (SEM). Moreover, scaffold properties, such as pore size and morphology of the cells, onto the scaffolds were evaluated using SEM. Furthermore, biocompatibility of these scaffolds was confirmed by 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The matrix mineralization was proved by alizarin red staining, and the osteogenic media-treated cultures positively expressed osteocalcin and osteopontin markers. Moreover, qRT-PCR results confirmed the positive gene expression of osteopontin and osteonectin in the differentiated osteoblast-like cells. The results of behavior assessment of the cultured cells on electrospinning and freeze-dried scaffolds showed that the behavior of the cultured cells on the freeze-dried PLGA/HA scaffolds was significantly better than the electrospinning PLGA/HA scaffolds. It has been shown that the freeze-dried PLGA/HA nanocomposite scaffolds can appropriately support the attachment and proliferation of the differentiated osteoblast cells and are a suitable candidate for bone tissue engineering.

The Efficacy and Safety of HA IDF Plus (with Lidocaine) Versus HA IDF (Without Lidocaine) in Nasolabial Folds Injection: A Randomized, Multicenter, Double-Blind, Split-Face Study.

PubMed

Lee, Jong-Hun; Kim, Seok-Hwan; Park, Eun-Soo

2017-04-01

Injection-related pain of dermal fillers is a consistent and bothersome problem for patients undergoing soft tissue augmentation. Reducing the pain could improve overall patient satisfaction. The purpose of this study was to compare the pain relief, efficacy, and safety of HA IDF plus containing lidocaine with HA IDF without lidocaine during correction of nasolabial folds (NLFs). Sixty-two subjects were enrolled in a randomized, multicenter, double-blind, split-face study of HA IDF plus and HA IDF for NLF correction. For split-face study, HA IDF plus was injected to one side of NLF, and HA IDF was injected to the other side. The first evaluation variable was the injection site pain measured using a 100-mm visual analogue scale (VAS). The second evaluation variables included the global aesthetic improvement scale, wrinkle severity rating scale, and adverse events. Immediately after injection, 91.94% of subjects experienced at least 10 mm decrease in VAS scores at the side injected with HA IDF plus compared with HA IDF, and the rate of subjects is statistically significant. The two fillers were not significantly different in safety profile or wrinkle correction during the follow-up visit. HA IDF plus significantly reduced the injection-related pain during NLFs correction compared with HA IDF without altering clinical outcomes or safety. Both HA IDF plus and HA IDF were considerably tolerated and most adverse reactions were mild and transient. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Neuroprotection and lifespan extension in Ppt1−/− mice by NtBuHA: therapeutic implications for INCL

PubMed Central

Sarkar, Chinmoy; Chandra, Goutam; Peng, Shyiong; Zhang, Zhongjian; Liu, Aiyi; Mukherjee, Anil B.

2013-01-01

Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating childhood neurodegenerative lysosomal storage disease (LSD) that has no effective treatment. It is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1-deficiency impairs the cleavage of thioester linkage in palmitoylated proteins (constituents of ceroid), preventing degradation by lysosomal hydrolases. Consequently, accumulation of lysosomal ceroid leads to INCL. Thioester linkage is cleaved by nucleophilic attack. Hydroxylamine, a potent nucleophilic cellular metabolite, may have therapeutic potential for INCL but its toxicity precludes clinical application. Here we report that a hydroxylamine-derivative, N-(tert-Butyl) hydroxylamine (NtBuHA), is non-toxic, cleaves thioester linkage in palmitoylated proteins and mediates lysosomal ceroid depletion in cultured cells from INCL patients. Importantly, in Ppt1−/− mice, which mimic INCL, NtBuHA crossed the blood-brain-barrier, depleted lysosomal ceroid, suppressed neuronal apoptosis, slowed neurological deterioration and extended lifespan. Our findings provide the proof of concept that thioesterase-mimetic and antioxidant small molecules like NtBuHA are potential drug-targets for thioesterase deficiency diseases like INCL. PMID:24056696

Surface characterization of colloidal-sol gel derived biphasic HA/FA coatings.

PubMed

Cheng, Kui; Zhang, Sam; Weng, Wenjian

2007-10-01

Hydroxyapatite (HA) powders are ultrasonically dispersed in the precursor of fluoridated hydroxyapatite (FHA) or fluorapatite (FA) to form a "colloidal sol". HA/FA biphasic coatings are prepared on Ti6Al4V substrate via dip coating, 150 degrees C drying and 600 degrees C firing. The coatings show homogenous distribution of HA particles in the FA matrix. The relative phase proportion can be tailored by the amount of HA in the colloidal sol. The surfaces of the coatings consist of two kinds of distinct domains: HA and FA, resulting in a compositionally heterogeneous surface. The biphasic coating surface becomes increasingly rougher with HA powders, from around 200 nm of pure FA to 400-600 nm in Ra of biphasic coatings. The rougher biphasic HA/FA surfaces with chemically controllable domains will favor cell attachment, apatite layer deposition and necessary dissolution in clinical applications.

Monitoring Cellular Interactions during T Cell Activation at the Single Molecule Level Using Semiconductor Quantum-Dots

DTIC Science & Technology

2005-05-10

avidin-fusion constructs in mammalian immune cells. To facilitate detection, an epitope tag (HA, derived from the influenza A virus haemagglutinin...Enhanced Green Fluorescent protein (EGFP) cassette. The IRES element (internal ribosome entry site of the encephalomyocarditis virus ) permits both the...AVIDIN CD4 4 AVIDIN tAT .RES’ EGFP TRES’ EGFP IRES’ EGFp 1RES. EGFP g9K LP tar LP gK LP ITTK LIP IE "M33 Ee TM NAMI IAV ~ 𔃺M A ,A,0,, MIMI, A161 TM

Precision Landing and Hazard Avoidance (PL&HA) Domain

NASA Technical Reports Server (NTRS)

Robertson, Edward A.; Carson, John M., III

2016-01-01

The Precision Landing and Hazard Avoidance (PL&HA) domain addresses the development, integration, testing, and spaceflight infusion of sensing, processing, and GN&C (Guidance, Navigation and Control) functions critical to the success and safety of future human and robotic exploration missions. PL&HA sensors also have applications to other mission events, such as rendezvous and docking.

Analysis of tomato plasma membrane H(+)-ATPase gene family suggests a mycorrhiza-mediated regulatory mechanism conserved in diverse plant species.

PubMed

Liu, Junli; Liu, Jianjian; Chen, Aiqun; Ji, Minjie; Chen, Jiadong; Yang, Xiaofeng; Gu, Mian; Qu, Hongye; Xu, Guohua

2016-10-01

In plants, the plasma membrane H(+)-ATPase (HA) is considered to play a crucial role in regulating plant growth and respoding to environment stresses. Multiple paralogous genes encoding different isozymes of HA have been identified and characterized in several model plants, while limited information of the HA gene family is available to date for tomato. Here, we describe the molecular and expression features of eight HA-encoding genes (SlHA1-8) from tomato. All these genes are interrupted by multiple introns with conserved positions. SlHA1, 2, and 4 were widely expressed in all tissues, while SlHA5, 6, and 7 were almost only expressed in flowers. SlHA8, the transcripts of which were barely detectable under normal or nutrient-/salt-stress growth conditions, was strongly activated in arbuscular mycorrhizal (AM) fungal-colonized roots. Extreme lack of SlHA8 expression in M161, a mutant defective to AM fungal colonization, provided genetic evidence towards the dependence of its expression on AM symbiosis. A 1521-bp SlHA8 promoter could direct the GUS reporter expression specifically in colonized cells of transgenic tobacco, soybean, and rice mycorrhizal roots. Promoter deletion assay revealed a 223-bp promoter fragment of SlHA8 containing a variant of AM-specific cis-element MYCS (vMYCS) sufficient to confer the AM-induced activity. Targeted deletion of this motif in the corresponding promoter region causes complete abolishment of GUS staining in mycorrhizal roots. Together, these results lend cogent evidence towards the evolutionary conservation of a potential regulatory mechanism mediating the activation of AM-responsive HA genes in diverse mycorrhizal plant species.

Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of Streptococcus zooepidemicus via the Elimination of the Hyaluronidase-Encoding Gene.

PubMed

Pourzardosht, Navid; Rasaee, Mohammad Javad

2017-06-01

Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kana r -R as a recombinant suicide plasmid. The construct was then transferred into S. zooepidemicus to induce the homologous recombination between the hyaluronidase gene and the kanamycin resistance gene. Gene deletion was confirmed by PCR and enzyme assay. The product was cultured on selectable medium in which the MW of HA was increased from 1.5 to 3.8 MDa. The yield of HA production using the mutant strain was higher in all different concentrations of glucose from 40 to 120 g/l. Moreover, glucose increase results in higher HA production within both wild-type and recombinant strains. However, the growth rate of HA concentration (the slope of the plot), as a consequence of increased glucose concentration, is always higher for the recombinant strain. Unlike the wild-type strain, there was no sharp HA production drop approaching the 6 g/l HA concentration. In conclusion, hyaluronidase activity and HA concentration and MW exhibited a mutual control on each other. Based on our results, deletion of the hyaluronidase gene positively affects the yield and MW of HA.

Promotion of PDT efficacy by HA14-1

NASA Astrophysics Data System (ADS)

Kessel, David; Price, Michael; Haagenson, Kelly

2008-02-01

Photodynamic therapy (PDT) can target the members of the Bcl-2 family that protect cells from the initiation of apoptosis, a well-known death pathway. We examined the ability of HA14-1, a non-peptidic Bcl-2/Bcl-xL antagonist, to promote the efficacy of PDT. The photosensitizer was the porphycene CPO that causes photodamage to Bcl-2 located in the endoplasmic reticulum. Using low PDT doses together with LD5-20 concentrations of HA14-1, we found a marked synergistic effect. These results indicate that such an effect occurs when PDT is coupled with pharmacologic suppression of Bcl-2 function. HA14-1 is an unstable compound that decomposes in aqueous solution. This resulted in a rapid (~60 sec) burst of fluorescence that closely mimicked the properties of many fluorescent probes, but was traced to an effect produced when HA14-1 contacts serum proteins. Other Bcl-2 antagonists that do not produce any intrinsic fluorescence also promoted PDT efficacy. Moreover, briefly storing HA14-1 in aqueous medium until the fluorescent burst is over does not inhibit a subsequent synergistic promotion of PDT efficacy. We conclude that Bcl-2 antagonists can promote the efficacy of low-dose PDT in a manner unrelated to ROS production. The most likely explanation is an enhanced loss of anti-apoptotic Bcl-2 family function such that a threshold for initiation of apoptosis is crossed.

Recombinants of influenza virus type B as potential live vaccine candidates: RNA characterization and evaluation in man.

PubMed Central

Lobmann, M.; Delem, A.; Jovanovic, D.; Peetermans, J.

1981-01-01

Two recombinants (R22 and R75) of the attenuated B/USSR/69 strain Bright and the virulent B/Hong Kong/5/72 and one recombinant (R5) of Bright and the virulent B/Hong Kong /8/73 were selected for genotypic and phenotypic caracterization. All three recombinants had the growth property of the attenuated parent Brigit. Analysis of their RNA's by polyacrylamide gel electrophoresis revealed that, the strains R22 and R75 had derived all their genes from Brigit, those coding for haemagglutinin excepted. These recombinants were clinically evaluated and found to be attenuated and immunogenic. The recombinant R5 which derived, besides the bene coding for the haemagglutinin, several other genes from B/Hong Kong/8/73 was only partly attenuated since it induced influenza-like symptoms in one out of three volunteers. It is concluded that the strain Brigit can be used as a donor of genes for the attenuation of the B/Hong Kong/5/72 virus and that recombinants of influenza type B can be identified, like influenza type A recombinants, by their RNA pattern. Images Plate 1 PMID:7019320

HA-1077 inhibits cell migration/invasion of oral squamous cell carcinoma.

PubMed

Moreira Carboni, Simone de Sales Costa; Rodrigues Lima, Nathália Alves; Pinheiro, Nanci Mendes; Tavares-Murta, Beatriz Martins; Crema, Virgínia Oliveira

2015-10-01

Oral squamous cell carcinoma (OSCC) is the most malignant lesion occurring in the head and neck. The Rho-kinases (ROCKs), effectors of Rho proteins, are involved in actin cytoskeletal organization, cell migration, and maintenance cortex. The HA-1077 inhibits the ROCKs. This study aimed to evaluate the effect of treatment with HA-1077 on cell motility in SCC-4 cells, a cell line originating from human OSCC. F-actin of SCC-4 cells treated or not with HA-1077 (1, 50 and 100 μmol/l), and also HA-1077 50 μmol/l and/or inhibitors Y-27632 30 μmol/l was stained with rhodamine-conjugated phalloidin and analyzed by confocal microscopy. Approximately 1×10 cells/well, control and treated with HA-1077 (25, 50, and 100 μmol/l) were added to the migration plate assay. In addition, 1×10 cells/well, control and treated with HA-1077 50 μmol/l, were tested by invasion assays (plate coated with Matrigel). The inhibition of ROCKs with HA-1077 and/or Y-27632 leads to morphological changes, affecting the organization of the actin. The inhibitory effect of HA-1077 (P<0.0001) was dose dependent as the number of cells migrated at 100 μmol/l was statistically different: 25 μmol/l (P<0.0001) and 50 μmol/l (P<0.01). The number of cells treated with HA-1077 50 μmol/l decreased compared with control cells that invaded through Matrigel (P<0.0001). This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC.

Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site.

PubMed

Mögling, Ramona; Richard, Mathilde J; Vliet, Stefan van der; Beek, Ruud van; Schrauwen, Eefje J A; Spronken, Monique I; Rimmelzwaan, Guus F; Fouchier, Ron A M

2017-06-01

Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.

Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

PubMed

González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

2016-02-01

Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

Epidemiology and Genetic Characterization of H3N8 Equine Influenza Virus Responsible for Clinical Disease in Algeria in 2011.

PubMed

Laabassi, F; Lecouturier, F; Amelot, G; Gaudaire, D; Mamache, B; Laugier, C; Legrand, L; Zientara, S; Hans, A

2015-12-01

An outbreak of equine influenza (EI) was reported in Algeria between May and July, 2011. The outbreak started in Tiaret, in west province of Algeria, and spread to the other parts of the country affecting almost 900 horses in many provinces. The population studied was composed of 325 horses from different groups of age. Clinical sign expression was age dependent. Indeed, a morbidity rate of 14.9% was observed in horses under 15 months old and a rate of 4.95% in horses over 8 years old. Interestingly, the morbidity rate raised sharply to reach 100% in horses aged between 18 months and 7 years. The virus (H3N8) was detected in nasopharyngeal swabs (n = 11) from non-vaccinated horses using a qRT-PCR targeting a portion of the gene encoding the matrix protein (M). The virus isolates were identified as H3N8 by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes and were named from A/equine/Tiaret/1/2011 to A/equine/Tiaret/10/2011. Alignment of HA1 amino acid sequence confirmed that viruses belong to Clade 2 of the Florida sublineage in the American lineage. Moreover, they are closely related to A/equine/Yokohama/aq13/2010, A/equine/Eyragues/1/2010, A/equine/Bokel/2011 and A/equine/Lichtenfeld/2012. Our data indicate that this strain was also circulating in the European horse population in 2010, 2011 and 2012. © 2014 Blackwell Verlag GmbH.

Formation and bioactivity of HA nanorods on micro-arc oxidized zirconium.

PubMed

Zhang, Lan; Zhu, Shaoyu; Han, Yong; Xiao, Chengzhang; Tang, Wu

2014-10-01

A microporous and CaO partially stabilized zirconia (Ca-PSZ) coating covered with hydroxyapatite (HA) nanorods is fabricated on Zr substrate by a hybrid approach of micro-arc oxidation (MAO) and hydrothermal treatment (HT). The effect of P ions in HT solution on the density and morphology of HA was investigated; the hydrophilicity and apatite-forming ability of the Ca-PSZ coating with HA nanorods were also examined. High-density HA nanorods (with a mean diameter of 50 nm and length of 450 nm) grow on the Ca-PSZ coating after HT in a solution containing 0.002 M β-glycerophosphate disodium (β-GP). However, only a few of coarse-grained HA crystallites grow in the MAOed pores after HT in distilled water or in an ammonia aqueous solution with an initial pH value equal to the solution containing 0.002 M β-GP. P ions in the HT solution are thought to significantly promote the formation of HA nanorods. The Ca-PSZ coating covered with HA nanorods displays good hydrophilicity and excellent apatite-inducing ability, and the induced apatite prefers to nucleate on the basal-faceted surfaces of HA nanorods. Copyright © 2014 Elsevier B.V. All rights reserved.

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site

PubMed Central

Diederich, Sandra; Berhane, Yohannes; Embury-Hyatt, Carissa; Hisanaga, Tamiko; Handel, Katherine; Cottam-Birt, Colleen; Ranadheera, Charlene; Kobasa, Darwyn

2015-01-01

ABSTRACT Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin

Influence of carbonate ion in the crystallization medium on the formation and chemical composition of CaHA-SrHA solid solutions

NASA Astrophysics Data System (ADS)

Nikolaev, Anton; Kuz'mina, Maria; Frank-Kamenetskaya, Olga; Zorina, Maina

2015-06-01

The study of the influence of carbonate ions in a solution to Sr-distribution in system «solution-crystal» and to ion substitutions and the non-stoichiometry of formed CaHA-SrHA solid solutions was carried out. The CaHA-SrHA solid solutions were synthesized by precipitation from aqueous solutions with the atomic C/P ratio equal to 0, 0.05 and 0.1 at T = 90 °C. Resulting precipitates were studied using various methods including X-ray powder diffraction, infrared spectroscopy and different chemical analyses. The results of the study have shown that in the range of values of (Ca + Sr)/P in the water solution from 40% to 85%, the presence of carbonate ions (C/P = 0.05-0.1) promotes the incorporation of strontium in the apatite. Crystalline apatite solid solutions formed from water solutions of such composition are more defective compared to apatites that are mainly calcium or strontium. They are characterized by a smaller size coherence scattering domain length along [0 0 1] direction and a greater number of carbonate ions, water molecules and vacancies at the Ca-sites.

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs.

PubMed

Park, Sehee; Lee, Ilseob; Kim, Jin Il; Bae, Joon-Yong; Yoo, Kirim; Kim, Juwon; Nam, Misun; Park, Miso; Yun, Soo-Hyeon; Cho, Woo In; Kim, Yeong-Su; Ko, Yun Young; Park, Man-Seong

2016-10-14

Avian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Crystal structure of the HA3 subcomponent of Clostridium botulinum type C progenitor toxin.

PubMed

Nakamura, Toshio; Kotani, Mao; Tonozuka, Takashi; Ide, Azusa; Oguma, Keiji; Nishikawa, Atsushi

2009-01-30

The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1 (HA-33), HA2 (HA-17), HA3a (HA-22-23), and HA3b (HA-53). The HA3b subcomponent seems to play an important role cooperatively with HA1 in the internalization of the toxin by gastrointestinal epithelial cells via binding of these subcomponents to specific oligosaccharides. In this study, we investigated the sugar-binding specificity of the HA3b subcomponent using recombinant protein fused to glutathione S-transferase and determined the three-dimensional structure of the HA3a-HA3b complex based on X-ray crystallography. The crystal structure was determined at a resolution of 2.6 A. HA3b contains three domains, domains I to III, and the structure of domain I resembles HA3a. In crystal packing, three HA3a-HA3b molecules are assembled to form a three-leaved propeller-like structure. The three HA3b domain I and three HA3a alternate, forming a trimer of dimers. In a database search, no proteins with high structural homology to any of the domains (Z score >10) were found. Especially, HA3a and HA3b domain I, mainly composed of beta-sheets, reveal a unique fold. In binding assays, HA3b bound sialic acid with high affinity, but did not bind galactose, N-acetylgalactosamine, or N-acetylglucosamine. The electron density of liganded N-acetylneuraminic acid was determined by crystal soaking. In the sugar-complex structure, the N-acetylneuraminic acid-binding site was located in the cleft formed between domains II and III of HA3b. This report provides the first determination of the three-dimensional structure of the HA3a-HA3b complex and its sialic acid binding site. Our results will provide useful information for elucidating the mechanism of assembly of the C16S toxin and for understanding the interactions with oligosaccharides on epithelial cells and internalization of the botulinum toxin complex.

Nanocrystallized SrHA/SrHA SrTiO3/SrTiO3 TiO2 multilayer coatings formed by micro-arc oxidation for photocatalytic application

NASA Astrophysics Data System (ADS)

Han, Y.; Chen, D. H.; Zhang, L.

2008-08-01

Novel photocatalytic coatings containing strontium hydroxyapatite (SrHA), strontium titanate (SrTiO3), and TiO2 were formed by micro-arc oxidation (MAO) in an aqueous electrolyte containing strontium acetate and β-glycerophosphate disodium at 530 V for 0.1-5 min. The structure evolution of the coatings was investigated as a function of processing time, and the photocatalytic activity of the coatings was evaluated by measuring the decomposition rate of methyl orange under ultraviolet irradiation. During the MAO processing of the coatings, it was observed that some granules appeared in the electrolyte adjacent to the anode and they increased in amount as the processing time was prolonged. The obtained results show that the granules are amorphous and poorly crystallized SrHA with negative charges. The coating prepared for 5 min presents a microporous structure of SrHA/SrHA-SrTiO3/SrTiO3-TiO2 multilayers, in which the SrHA outermost layer and the SrHA-SrTiO3 intermediate layer are nanocrystallized. It is suggested that formation of the granules, electro-migration of the granules onto the pre-formed layer, and crystallization of the adhered granules are possible mechanisms for the formation of a SrHA/SrHA-SrTiO3/SrTiO3-TiO2 multilayer coating. This coating shows much higher photocatalytic decomposition efficiency relative to the MAO-formed TiO2 coating, and is expected to have an important photocatalytic application.

Crystalline ha coating on peek via chemical deposition

NASA Astrophysics Data System (ADS)

Almasi, D.; Izman, S.; Assadian, M.; Ghanbari, M.; Abdul Kadir, M. R.

2014-09-01

Polyether ether ketone (PEEK) has a similar elastic modulus to bone and can be a suitable alternative to metallic implants. However, PEEK is bioinert and does not integrate well with the surrounding tissues. The current commercial method for solving this problem is by coating PEEK substrates with calcium phosphates via plasma spraying. However, this method produces a low bonding strength between the substrate and the coating layer, as well as non-uniform density of the coating. In this study, chemical deposition was used to deposit HA crystalline particles on PEEK substrate without any subsequent crystallisation process therefore producing crystalline treated layer. EDX results confirmed the deposition of HA, and the XRD results confirmed that the treated layer was crystalline HA. FT-IR analysis confirmed the chemical bonding between HA and the substrate. Surface roughness increased from 24.27 nm to 34.08 nm for 3 min immersion time. The water contact angle showed an increase in wettability of the treated sample from 71.6 to 36.4 degrees, which in turn increased its bioactivity. The proposed method is a suitable alternative to other conventional methods as high temperature was not involved in the process which could damage the surface of the substrate.

HVOF-Sprayed Nano TiO2-HA Coatings Exhibiting Enhanced Biocompatibility

NASA Astrophysics Data System (ADS)

Lima, R. S.; Dimitrievska, S.; Bureau, M. N.; Marple, B. R.; Petit, A.; Mwale, F.; Antoniou, J.

2010-01-01

Biomedical thermal spray coatings produced via high-velocity oxy-fuel (HVOF) from nanostructured titania (n-TiO2) and 10 wt.% hydroxyapatite (HA) (n-TiO2-10wt.%HA) powders have been engineered as possible future alternatives to HA coatings deposited via air plasma spray (APS). This approach was chosen due to (i) the stability of TiO2 in the human body (i.e., no dissolution) and (ii) bond strength values on Ti-6Al-4V substrates more than two times higher than those of APS HA coatings. To explore the bioperformance of these novel materials and coatings, human mesenchymal stem cells (hMSCs) were cultured from 1 to 21 days on the surface of HVOF-sprayed n-TiO2 and n-TiO2-10 wt.%HA coatings. APS HA coatings and uncoated Ti-6Al-4V substrates were employed as controls. The profiles of the hMSCs were evaluated for (i) cellular proliferation, (ii) biochemical analysis of alkaline phosphatase (ALP) activity, (iii) cytoskeleton organization (fluorescent/confocal microscopy), and (iv) cell/substrate interaction via scanning electron microscopy (SEM). The biochemical analysis indicated that the hMSCs cultured on n-TiO2-10 wt.%HA coatings exhibited superior levels of bioactivity than hMSCs cultured on APS HA and pure n-TiO2 coatings. The cytoskeleton organization demonstrated a higher degree of cellular proliferation on the HVOF-sprayed n-TiO2-10wt.%HA coatings when compared to the control coatings. These results are considered promising for engineering improved performance in the next generation of thermally sprayed biomedical coatings.

The Muon Collider as a $H/A$ factory

DOE PAGES

Eichten, Estia; Martin, Adam; Univ. of Notre Dame, IN

2013-11-22

We show that a muon collider is ideally suited for the study of heavy H/A scalars, cousins of the Higgs boson found in two-Higgs doublet models and required in supersymmetric models. The key aspects of H/A are: (1) they are narrow, yet have a width-to-mass ratio far larger than the expected muon collider beam-energy resolution, and (2) the larger muon Yukawa allows efficient s-channel production. We study in detail a representative Natural Supersymmetry model which has a 1.5 Tev H/A withmore » $$m_H$$- $$m_A$$ = 10 Gev. The large event rates at resonant peak allow the determination of the individual H and A resonance parameters (including CP) and the decays into electroweakinos provides a wealth of information unavailable to any other present or planned collider.« less

Possible role of PAPR-1 in protecting human HaCaT cells against cytotoxicity of SiO2 nanoparticles.

PubMed

Gong, Chunmei; Yang, Linqing; Zhou, Jichang; Guo, Xiang; Zhuang, Zhixiong

2017-10-05

Nano-SiO 2 materials play a significant role in the engineered nanomaterials (ENMs) field. The ease of their production as well as their relatively low cost has promoted the wide use of these products in many fields. Nano-SiO 2 exposure is known to cause severe DNA damage; however, the underlying mechanisms remain poorly understood. In a previous study, we found that nano-SiO 2 exposure regulate the expression of the poly(ADP-ribose) polymerases-1 (PARP-1), a pivotal DNA repair gene, in human HaCaT cells. Here, we employed lentivirus-mediated RNA interference (RNAi) to knock down PAPR-1 expression in HaCaT cells and explored the potential role of PARP-1 in nano-SiO 2 induced cytotoxicity. We found that nano-SiO 2 treatment of HaCaT cells causes decreased cell viability, increased apoptosis and DNA damage. Nano-SiO 2 -treated HaCaT cells were also found to have slightly changed cell cycle distribution. Lentivirus-mediated PAPR-1 knockdown partially aggravated cytotoxicity and increased apoptosis induced by nano-SiO 2 treatment. Nano-SiO 2 had significant toxicity to human HaCaT cells and causes DNA damage. PAPR-1 knock-down cell line appears more sensitive to nano-SiO 2 than the control cells in DNA damage. The results suggest that PAPR-1 is involved in protecting cells from damage caused by nano-SiO 2 . Copyright © 2017 Elsevier B.V. All rights reserved.

Hyperinsulinism–hyperammonaemia syndrome: novel mutations in the GLUD1 gene and genotype–phenotype correlations

PubMed Central

Kapoor, Ritika R; Flanagan, Sarah E; Fulton, Piers; Chakrapani, Anupam; Chadefaux, Bernadette; Ben-Omran, Tawfeg; Banerjee, Indraneel; Shield, Julian P; Ellard, Sian; Hussain, Khalid

2009-01-01

Background Activating mutations in the GLUD1 gene (which encodes for the intra-mitochondrial enzyme glutamate dehydrogenase, GDH) cause the hyperinsulinism–hyperammonaemia (HI/HA) syndrome. Patients present with HA and leucine-sensitive hypoglycaemia. GDH is regulated by another intra-mitochondrial enzyme sirtuin 4 (SIRT4). Sirt4 knockout mice demonstrate activation of GDH with increased amino acid-stimulated insulin secretion. Objectives To study the genotype–phenotype correlations in patients with GLUD1 mutations. To report the phenotype and functional analysis of a novel mutation (P436L) in the GLUD1 gene associated with the absence of HA. Patients and methods Twenty patients with HI from 16 families had mutational analysis of the GLUD1 gene in view of HA (n=19) or leucine sensitivity (n=1). Patients negative for a GLUD1 mutation had sequence analysis of the SIRT4 gene. Functional analysis of the novel P436L GLUD1 mutation was performed. Results Heterozygous missense mutations were detected in 15 patients with HI/HA, 2 of which are novel (N410D and D451V). In addition, a patient with a normal serum ammonia concentration (21 μmol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirms that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. No mutations in the SIRT4 gene were identified. Conclusion Patients with HI due to mutations in the GLUD1 gene may have normal serum ammonia concentrations. Hence, GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of HA. A high frequency of epilepsy (43%) was observed in our patients with GLUD1 mutations. PMID:19690084

Identification of Candidate Genes Associated with Leaf Senescence in Cultivated Sunflower (Helianthus annuus L.)

PubMed Central

Moschen, Sebastian; Bengoa Luoni, Sofia; Paniego, Norma B.; Hopp, H. Esteban; Dosio, Guillermo A. A.

2014-01-01

Cultivated sunflower (Helianthus annuus L.), an important source of edible vegetable oil, shows rapid onset of senescence, which limits production by reducing photosynthetic capacity under specific growing conditions. Carbon for grain filling depends strongly on light interception by green leaf area, which diminishes during grain filling due to leaf senescence. Transcription factors (TFs) regulate the progression of leaf senescence in plants and have been well explored in model systems, but information for many agronomic crops remains limited. Here, we characterize the expression profiles of a set of putative senescence associated genes (SAGs) identified by a candidate gene approach and sunflower microarray expression studies. We examined a time course of sunflower leaves undergoing natural senescence and used quantitative PCR (qPCR) to measure the expression of 11 candidate genes representing the NAC, WRKY, MYB and NF-Y TF families. In addition, we measured physiological parameters such as chlorophyll, total soluble sugars and nitrogen content. The expression of Ha-NAC01, Ha-NAC03, Ha-NAC04, Ha-NAC05 and Ha-MYB01 TFs increased before the remobilization rate increased and therefore, before the appearance of the first physiological symptoms of senescence, whereas Ha-NAC02 expression decreased. In addition, we also examined the trifurcate feed-forward pathway (involving ORE1, miR164, and ETHYLENE INSENSITIVE 2) previously reported for Arabidopsis. We measured transcription of Ha-NAC01 (the sunflower homolog of ORE1) and Ha-EIN2, along with the levels of miR164, in two leaves from different stem positions, and identified differences in transcription between basal and upper leaves. Interestingly, Ha-NAC01 and Ha-EIN2 transcription profiles showed an earlier up-regulation in upper leaves of plants close to maturity, compared with basal leaves of plants at pre-anthesis stages. These results suggest that the H. annuus TFs characterized in this work could play important

Identification of candidate genes associated with leaf senescence in cultivated sunflower (Helianthus annuus L.).

PubMed

Moschen, Sebastian; Bengoa Luoni, Sofia; Paniego, Norma B; Hopp, H Esteban; Dosio, Guillermo A A; Fernandez, Paula; Heinz, Ruth A

2014-01-01

Cultivated sunflower (Helianthus annuus L.), an important source of edible vegetable oil, shows rapid onset of senescence, which limits production by reducing photosynthetic capacity under specific growing conditions. Carbon for grain filling depends strongly on light interception by green leaf area, which diminishes during grain filling due to leaf senescence. Transcription factors (TFs) regulate the progression of leaf senescence in plants and have been well explored in model systems, but information for many agronomic crops remains limited. Here, we characterize the expression profiles of a set of putative senescence associated genes (SAGs) identified by a candidate gene approach and sunflower microarray expression studies. We examined a time course of sunflower leaves undergoing natural senescence and used quantitative PCR (qPCR) to measure the expression of 11 candidate genes representing the NAC, WRKY, MYB and NF-Y TF families. In addition, we measured physiological parameters such as chlorophyll, total soluble sugars and nitrogen content. The expression of Ha-NAC01, Ha-NAC03, Ha-NAC04, Ha-NAC05 and Ha-MYB01 TFs increased before the remobilization rate increased and therefore, before the appearance of the first physiological symptoms of senescence, whereas Ha-NAC02 expression decreased. In addition, we also examined the trifurcate feed-forward pathway (involving ORE1, miR164, and ethylene insensitive 2) previously reported for Arabidopsis. We measured transcription of Ha-NAC01 (the sunflower homolog of ORE1) and Ha-EIN2, along with the levels of miR164, in two leaves from different stem positions, and identified differences in transcription between basal and upper leaves. Interestingly, Ha-NAC01 and Ha-EIN2 transcription profiles showed an earlier up-regulation in upper leaves of plants close to maturity, compared with basal leaves of plants at pre-anthesis stages. These results suggest that the H. annuus TFs characterized in this work could play important

H13 influenza viruses in wild birds have undergone genetic and antigenic diversification in nature.

PubMed

Wang, Zu-Jyun; Kikutani, Yuto; Nguyen, Lam Thanh; Hiono, Takahiro; Matsuno, Keita; Okamatsu, Masatoshi; Krauss, Scott; Webby, Richard; Lee, Youn-Jeong; Kida, Hiroshi; Sakoda, Yoshihiro

2018-05-23

Among 16 haemagglutinin (HA) subtypes of avian influenza viruses (AIVs), H13 AIVs have rarely been isolated in wild waterfowl. H13 AIVs cause asymptomatic infection and are maintained mainly in gull and tern populations; however, the recorded antigenic information relating to the viruses has been limited. In this study, 2 H13 AIVs, A/duck/Hokkaido/W345/2012 (H13N2) and A/duck/Hokkaido/WZ68/2012 (H13N2), isolated from the same area in the same year in our surveillance, were genetically and antigenically analyzed with 10 representative H13 strains including a prototype strain, A/gull/Maryland/704/1977 (H13N6). The HA genes of H13 AIVs were phylogenetically divided into 3 groups (I, II, and III). A/duck/Hokkaido/W345/2012 (H13N2) was genetically classified into Group III. This virus was distinct from a prototype strain, A/gull/Maryland/704/1977 (H13N6), and the virus, A/duck/Hokkaido/WZ68/2012 (H13N2), both belonging to Group I. Antigenic analysis indicated that the viruses of Group I were antigenically closely related to those of Group II, but distinct from those of Group III, including A/duck/Hokkaido/W345/2012 (H13N2). In summary, our study indicates that H13 AIVs have undergone antigenic diversification in nature.

HC-HA/PTX3 Purified From Amniotic Membrane as Novel Regenerative Matrix: Insight Into Relationship Between Inflammation and Regeneration.

PubMed

Tseng, Scheffer C G

2016-04-01

Human limbal palisade of Vogt is an ideal model for studying and practicing regenerative medicine due to their accessibility. Nonresolving inflammation is a common manifestation of limbal stem cell deficiency, which is the major cause of corneal blindness, and presents as a threat to the success of transplanted limbal epithelial stem cells. Clinical studies have shown that the efficacy of transplantation of limbal epithelial stem cells can be augmented by transplantation of cryopreserved human amniotic membrane (AM), which exerts anti-inflammatory, antiscarring, and antiangiogenic action to promote wound healing. Review of published data to determine the molecular action mechanism explaining how AM exerts the aforementioned therapeutic actions. From the water-soluble extract of cryopreserved AM, we have biochemically purified one novel matrix component termed heavy chain (HC)-hyaluronan (HA)/pentraxin 3 (PTX3) as the key relevant tissue characteristic responsible for the aforementioned AM's efficacy. Heavy chain-HA is a complex formed by a covalent linkage between HA and HC1 of inter-α-trypsin inhibitor (IαI) by tumor necrosis factor-stimulated gene-6 (TSG-6). This complex may then be tightly associated with PTX3 to form HC-HA/PTX3 complex. Besides exerting an anti-inflammatory, antiscarring, and antiangiogenic effects, HC-HA/PTX3 complex also uniquely maintains limbal niche cells to support the quiescence of limbal epithelial stem cells. We envision that HC-HA/PTX3 purified from AM can be used as a unique substrate to refine ex vivo expansion of limbal epithelial stem cells by maintaining stem cell quiescence, self-renewal and fate decision. Furthermore, it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche.

[Genetic evolution and substitution frequency of avian influenza virus HA gene in chicken H9N2 subtype in China in the last 20 years].

PubMed

Meng, Fang; Xu, Huaiying; Zhang, Wei; Huang, Dihai; Zhang, Zaihui; Liu, Xia; Chang, Weishan; Qin, Zhuoming

2016-01-04

Low pathogenic avian influenza (LPAI) H9N2 subtype virus has been prevalent in domestic poultry in China over two decades. This study was to determine the genetic evolution trend of H9N2 avian influenza virus (AIV) under immune pressure of vaccine. H9 HA sequences of 40 isolates from the present study and 136 pandemic strains and 7 classical strains from China downloaded from GenBank, were genetically analyzed to determine evolution, molecular characteristic, and mutation frequency. Phylogenetic trees analysis suggested that H9N2 subtypes AIV could be clustered into 5 distinct lineages: G1-like, BJ94-like, Y280-like, S2-like and Americans lineages. Most H9N2 isolates in 2005-2014 belonged to S2-like sub-genotype, suggesting that this genotype was the dominate isolates in China. Further more, comparison based on the amino acid sequence showed that different lineages have their distinct characteristics, and significant accumulations of amino acid variation were also found. In addition, in comparison with reference Ck/BJ/1/1994 HA gene, average annual substitution rates of H9N2 pandemic strain nucleotide and amino acid were 5.73 x 10⁻³ and 4.25 x 10⁻³ from 1994 to 2014, respectively. Substitution rate during 2011-2014 were 6.35 x 10⁻³ and 5.32 x 10⁻³, higher than that during the period of 2006-2010 (5.22 x 10⁻³ and 3.70 x 10⁻³) and even much higher than that during the 1999-2005 (0.74 x 10⁻³ and 0.50 x 10⁻³), when the vaccines were initially applied in the field. Overall, these data indicate that the mismatch between H9N2 vaccine strains and pandemic strains drives the virus to quickly mutate.

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

PubMed Central

Beltzer, J P; Spiess, M

1991-01-01

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

The PB2, PA, HA, NP, and NS genes of a highly pathogenic avian influenza virus A/whooper swan/Mongolia/3/2005 (H5N1) are responsible for pathogenicity in ducks

PubMed Central

2013-01-01

Background Wild ducks are the natural hosts of influenza A viruses. Duck influenza, therefore, has been believed inapparent infection with influenza A viruses, including highly pathogenic avian influenza viruses (HPAIVs) in chickens. In fact, ducks experimentally infected with an HPAIV strain, A/Hong Kong/483/1997 (H5N1) (HK483), did not show any clinical signs. Another HPAIV strain, A/whooper swan/Mongolia/3/2005 (H5N1) (MON3) isolated from a dead swan, however, caused neurological dysfunction and death in ducks. Method To understand the mechanism whereby MON3 shows high pathogenicity in ducks, HK483, MON3, and twenty-four reassortants generated between these two H5N1 viruses were compared for their pathogenicity in domestic ducks. Results None of the ducks infected with MON3-based single-gene reassortants bearing the PB2, NP, or NS gene segment of HK483 died, and HK483-based single-gene reassortants bearing PB2, NP, or NS genes of MON3 were not pathogenic in ducks, suggesting that multiple gene segments contribute to the pathogenicity of MON3 in ducks. All the ducks infected with the reassortant bearing PB2, PA, HA, NP, and NS gene segments of MON3 died within five days post-inoculation, as did those infected with MON3. Each of the viruses was assessed for replication in ducks three days post-inoculation. MON3 and multi-gene reassortants pathogenic in ducks were recovered from all of the tissues examined and replicated with high titers in the brains and lungs. Conclusion The present results indicate that multigenic factors are responsible for efficient replication of MON3 in ducks. In particular, virus growth in the brain might correlate with neurological dysfunction and the disease severity. PMID:23374292

New Coll-HA/BT composite materials for hard tissue engineering.

PubMed

Zanfir, Andrei Vlad; Voicu, Georgeta; Busuioc, Cristina; Jinga, Sorin Ion; Albu, Madalina Georgiana; Iordache, Florin

2016-05-01

The integration of ceramic powders in composite materials for bone scaffolds can improve the osseointegration process. This work was aimed to the synthesis and characterization of new collagen-hydroxyapatite/barium titanate (Coll-HA/BT) composite materials starting from barium titanate (BT) nanopowder, hydroxyapatite (HA) nanopowder and collagen (Coll) gel. BT nanopowder was produced by combining two wet-chemical approaches, sol-gel and hydrothermal methods. The resulting materials were characterized in terms of phase composition and microstructure by X-ray diffraction, Raman spectroscopy, scanning electron microscopy and transmission electron microscopy. Moreover, the biocompatibility and bioactivity of the composite materials were assessed by in vitro tests. The synthesized BT particles exhibit an average size of around 35 nm and a spherical morphology, with a pseudo-cubic or tetragonal symmetry. The diffraction spectra of Coll-HA and Coll-HA/BT composite materials indicate a pronounced interaction between Col and the mineral phases, meaning a good mineralization of Col fibres. As well, the in vitro tests highlight excellent osteoinductive properties for all biological samples, especially for Coll-HA/BT composite materials, fact that can be attributed to the ferromagnetic properties of BT. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of incorporation of HA/ZrO(2) into glass ionomer cement (GIC).

PubMed

Gu, Y W; Yap, A U J; Cheang, P; Khor, K A

2005-03-01

Glass ionomer cements (GICs) are a class of bioactive cements that bond directly to bone. In this paper, a new bioactive hydroxyapatite (HA)/zirconia (ZrO(2))-filled GIC composite was developed to improve the biocompatibility and bioactivity of the GICs with the surrounding bone and connective tissues. Nano-sized HA/30 wt% ZrO(2) powders were heat treated at 700 degrees Celsius and 800 degrees Celsius for 3 h to elucidate the influence of the crystallinity of composite powders on the performance of HA/ZrO(2)-GICs. The effects of different volume percentages of HA/ZrO(2) powders (4, 12, 28 and 40 vol%) substituted within GICs were investigated based on their microhardness, compressive strength and diametral tensile strength. The HA/ZrO(2)-GICs composite was soaked in distilled water for 1 day and 1 week before subjecting the samples to mechanical testing. Results showed that the glass and HA/ZrO(2) particles were distributed uniformly in the GIC matrix. The substitution of highly crystalline HA/ZrO(2) improved the mechanical properties of the HA/ZrO(2)-GICs due to the slow resorption rate for highly crystalline powders in distilled water. The mechanical properties of HA/ZrO(2)-GICs increased with increasing soak time due to the continuous formation of aluminium salt bridges, which improved the final strength of the cements. The compositions 4 and 12 vol% HA/ZrO(2)-GICs exhibited superior mechanical properties than the original GICs. The mechanical properties of HA/ZrO(2)-GICs were found to be much better than those of HA-GICs because ZrO(2) has the attributes of high strength, high modulus, and is significantly harder than glass and HA particles. Furthermore, ZrO(2) does not dissolve with increasing soaking time.

Comparative Analysis of Evolutionary Mechanisms of the Hemagglutinin and Three Internal Protein Genes of Influenza B Virus: Multiple Cocirculating Lineages and Frequent Reassortment of the NP, M, and NS Genes

PubMed Central

Lindstrom, Stephen E.; Hiromoto, Yasuaki; Nishimura, Hidekazu; Saito, Takehiko; Nerome, Reiko; Nerome, Kuniaki

1999-01-01

Phylogenetic profiles of the genes coding for the hemagglutinin (HA) protein, nucleoprotein (NP), matrix (M) protein, and nonstructural (NS) proteins of influenza B viruses isolated from 1940 to 1998 were analyzed in a parallel manner in order to understand the evolutionary mechanisms of these viruses. Unlike human influenza A (H3N2) viruses, the evolutionary pathways of all four genes of recent influenza B viruses revealed similar patterns of genetic divergence into two major lineages. Although evolutionary rates of the HA, NP, M, and NS genes of influenza B viruses were estimated to be generally lower than those of human influenza A viruses, genes of influenza B viruses demonstrated complex phylogenetic patterns, indicating alternative mechanisms for generation of virus variability. Topologies of the evolutionary trees of each gene were determined to be quite distinct from one another, showing that these genes were evolving in an independent manner. Furthermore, variable topologies were apparently the result of frequent genetic exchange among cocirculating epidemic viruses. Evolutionary analysis done in the present study provided further evidence for cocirculation of multiple lineages as well as sequestering and reemergence of phylogenetic lineages of the internal genes. In addition, comparison of deduced amino acid sequences revealed a novel amino acid deletion in the HA1 domain of the HA protein of recent isolates from 1998 belonging to the B/Yamagata/16/88-like lineage. It thus became apparent that, despite lower evolutionary rates, influenza B viruses were able to generate genetic diversity among circulating viruses through a combination of evolutionary mechanisms involving cocirculating lineages and genetic reassortment by which new variants with distinct gene constellations emerged. PMID:10196339

Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells.

PubMed

Aina, Valentina; Bergandi, Loredana; Lusvardi, Gigliola; Malavasi, Gianluca; Imrie, Flora E; Gibson, Iain R; Cerrato, Giuseppina; Ghigo, Dario

2013-04-01

A series of Sr-substituted hydroxyapatites (HA), of general formula Ca(10-x)Srx(PO4)6(OH)2, where x=2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials. A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition. To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl2, in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in HA has a positive effect on MG-63 cells

Investigation of boundary conditions for biomimetic HA deposition on titanium oxide surfaces.

PubMed

Lindgren, M; Astrand, M; Wiklund, U; Engqvist, H

2009-07-01

To improve the clinical outcome of metal implants, i.e. earlier loading and reduction of the incidence of revision surgery, better bone bonding ability is wanted. One method to achieve this is to change the surface chemistry to give a surface that facilitates bone bonding in vivo, i.e. a bioactive surface. Crystalline titanium oxide has recently been proven to be bioactive in vitro and is an interesting option to the more common hydroxylapatite (HA) coatings on implants. A materials possible in vitro bioactivity is tested through soaking in simulated body fluid and studies of possible HA formation on the surface. For bioactive materials, the formed HA layer can also be used as a coating. The aim of the current paper is to investigate some boundary conditions for HA formation on crystalline titanium oxide surfaces regarding influence from coating thickness, soaking time and soaking temperature. The influence from soaking time and temperature on the HA growth were investigated on oxidised Ti samples, (24 h at 800 degrees C) resulting in a rutile surface structure. The oxidised samples were tested at three temperatures (4, 37 and 65 degrees C) and four times (1 h, 1 day, 1 week and 4 weeks). The influence from titanium coating thickness on the HA growth was investigated via depositing thin films of crystalline titanium dioxide on Ti plates using a reactive magnetron sputtering process. Four different PVD runs with coating thicknesses between 19 and 74 nm were tested. The soaking temperature had an effect on the HA formation and growth on both rutile surfaces and native oxide on Ti substrates. Higher temperatures lead to easier formation of HA. It was even possible, at 65 degrees C, to grow HA on native titanium oxide from soaking in PBS. The coating quality was better for HA formed at 65 degrees C compared to 37 degrees C. All PVD-coatings showed HA growth after 1 week in PBS at 37 degrees C, thus even very thin coatings of crystalline titanium oxide coatings are

Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

PubMed

Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

2017-01-01

A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

Interface activation and surface characteristics of Ti/TiN/HA coated sintered stainless steels

NASA Astrophysics Data System (ADS)

Choe, Han-Cheol; Ko, Yeong-Mu

2006-02-01

Interface activation and surface characteristics of Ti/TiN/HA film coated sintered stainless steels (SSS) have been investigated by electrochemical and biocompatibility tests. HA (hydroxyapatite), Ti, and Ti/TiN film coatings were applied using electron-beam deposition method (EB-PVD). Ti, Ti/TiN, and Ti/TiN/HA film coated surfaces and layers were investigated by SEM and XPS. The coated films showed micro-columnar structure, and Ti/TiN/HA films were denser than Ti or HA-only film. The corrosion resistance of the HA coating was similar to that of Ti/TiN/HA film coating when Cu content reached 4 wt.%, but the corrosion resistance of the HA coating decreased when Cu content increased from 4 wt.% in 0.9% NaCl solution. Therefore, HA-only coating could ensure corrosion resistance when Cu content does not exceed 4 wt.%. The results of biocompatibility tests of SSS on dogs showed that bone formation and biocompatibility were favorable when Cu content did not exceed 4 wt.%. The biocompatibility with bone was generally favorable in Ti/TiN/HA film coating and HA-only coating, while bone formation was somewhat faster for the HA film coated surface than for the Ti/TiN/HA film coating. Also, good cell growth and osseointegration without toxicity were observed.

HA and double-layer HA-P2O5/CaO glass coatings: influence of chemical composition on human bone marrow cells osteoblastic behavior.

PubMed

Ferraz, M P; Fernandes, M H; Santos, J D; Monteiro, F J

2001-07-01

Human osteoblastic bone marrow derived cells were cultured for 28 days onto the surface of a glass reinforced hydroxyapatite (HA) composite and a commercial type HA plasma sprayed coatings, both in the "as-received" condition and after an immersion treatment with culture medium during 21 days. Cell proliferation and differentiation were analyzed as a function of the chemical composition of the coatings and the immersion treatment. Cell attachment, growth and differentiation of osteoblastic bone marrow cells seeded onto "as-received" plasma sprayed coatings were strongly affected by the time-dependent variation of the surface structure occurring during the first hours of culture. Initial interactions leading to higher amounts of adsorbed protein and zeta potential shifts towards negative charges appeared to result in surface structures with better biological performance. Cultures grown onto the pretreated coatings showed higher rate of cell proliferation and increased functional activity, as compared to those grown onto the corresponding "as-received" materials. However, the cell behavior was similar in the glass composite and HA coatings. The results showed that the glass composites present better characteristics for bone cell growth and function than HA. In addition, this work also provide evidence that the biological performance of the glass composites can be modulated and improved by manipulations in the chemical composition, namely in the content of glass added to HA. Copyright 2001 Kluwer Academic Publishers

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

Bond Dissociation Free Energies (BDFEs) of the Acidic H-A Bonds in HA(*)(-) Radical Anions by Three Different Pathways.

PubMed

Zhao, Yongyu; Bordwell, Frederick G.

1996-09-20

Cleavage of radical anions, HA(*)(-), have been considered to give either H(*) + A(-) (path a) or H(-) + A(*) (path b), and factors determining the preferred mode of cleavage have been discussed. It is conceivable that cleavage to give a proton and a radical dianion, HA(*)(-) right harpoon over left harpoon H(+) + A(*)(2)(-) (path c), might also be feasible. A method, based on a thermodynamic cycle, to estimate the bond dissociation free energy (BDFE) by path c has been devised. Comparison of the BDFEs for cleavage of the radical anions derived from 24 nitroaromatic OH, SH, NH, and CH acids by paths a, b, c has shown that path c is favored thermodynamically.

The Mechanical Properties and Modeling of Creep Behavior of UHMWPE/Nano-HA Composites

NASA Astrophysics Data System (ADS)

Li, Fan; Gao, Lilan; Gao, Hong; Cui, Yun

2017-09-01

Composites with different levels of hydroxyapatite (HA) content and ultra-high molecular weight polyethylene (UHMWPE) were prepared in this work. Mechanical properties of the composites were examined here, and to evaluate the effect of HA particles on the time-dependent behavior of the pure matrix, the creep and recovery performance of composites at various stress levels were also researched. As expected, the addition of HA influenced the time-dependent response of the UHMWPE and the effect had a strong dependence on the HA content. The creep and recovery strain of the composites significantly decreased with increasing HA content, and tensile properties were also impaired, which was due to the concentration of HA fillers. The mechanism and effect of HA dispersed into the UHMWPE matrix were examined by scanning electron microscopy. Additionally, since variations in the adjusted parameters revealed the impact of HA on the creep behavior of the UHMWPE matrix, Findley's model was employed. The results indicated that the analytical model was accurate for the prediction of creep of the pure matrix and its composites.

24 CFR 964.18 - HA role in activities under subparts B & C.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., including the management of specific functions of a public housing development that may be mutually agreeable to the HA and the resident council/resident management corporation. (4) A HA shall provide the... participation in management. (5) If requested, a HA should provide a duly recognized resident council office...

Wear characterization of nano-hydroxyapatite with addition of titanium (HA-Ti)

NASA Astrophysics Data System (ADS)

Rosmamuhamadani, R.; Arawi, A. Z. O.; Talari, M. K.; Mahat, M. M.; Bonnia, N. N.; Sabrina; Yahaya, M.; Sulaiman, S.; Ismail, M. I. S.

2018-04-01

Hydroxyapatite (Ca10 (PO4)6(OH)2, HA), is an attractive material of an inorganic compound whose chemical composition and crystallographic structures are similar to the composition of the bone. A natural source such as egg shells is composed of 94 wt. % of calcium carbonate (CaCO3), which can be calcined as calcium oxide (CaO) by the calcinations process. The efficient temperature to produce CaO is 900 °C for 2 hours. The synthesis of nano-HA was done by the mixing the diammonium phosphate (DAP) and calcium hydroxide (Ca(OH)2) and subjected into a microwave for 30 minutes at 1100 W irradiation power. Ball milling process was used for 30 minutes to mix the nano-HA with different compositions of titanium. These were pressed to form pallets by hand hydraulic pump (force=2300 psi). The pallets then were sintered at 1200 °C with the heating rate of 3 °C/min for 2 hours. The pallets were tested by several mechanical testing including hardness, compression strength and wear. From the results, HA-25wt. %Ti composite gave the highest hardness, compression and coefficient of friction for wear test values which were 89.6 Hv, 82.5MPa and 0.76μ respectively. It showed that by adding Ti to nano-HA, the mechanical properties of nano-HA could be enhanced. The microstructure analyses by optical micrograph showed that nano-HA-Ti particles displayed shape likes needle morphology. The particles showed the high tendency to form the agglomerations.

Cellular proliferation, cellular viability, and biocompatibility of HA-ZnO composites.

PubMed

Saha, Naresh; Dubey, Ashutosh K; Basu, Bikramjit

2012-01-01

One of the important issues in the development of hydroxyapatite (HA)-based biomaterials is the prosthetic infection, which limits wider use of monolithic HA despite superior cellular response. Recently, we reported that ZnO addition to HA can induce bactericidal property. It is therefore important to assess how ZnO addition influences the cytotoxicity property and cell adhesion/proliferation on HA-ZnO composite surfaces in vitro. In the above perspective, the objective of this study is to investigate the cell type and material composition dependent cellular proliferation and viability of pressureless sintered HA-ZnO composites. The combination of cell viability data as well as morphological observations of cultured human osteoblast-like SaOS2 cells and mouse fibroblast L929 cells suggests that HA-ZnO composites containing 10 Wt % or lower ZnO exhibit the ability to support cell adhesion and proliferation. Both SaOS2 and L929 cells exhibit extensive multidirectional network of actin cytoskeleton and cell flattening on the lower ZnO containing (≤10 Wt %) HA-ZnO composites. The in vitro results illustrate how variation in ZnO content can influence significantly the cell vitality, as evaluated using MTT biochemical assay. Also, the critical statistical analysis reveals that ZnO addition needs to be carefully tailored to ensure good in vitro cytocompatibility. The underlying reasons for difference in biological properties are analyzed. It is suggested that surface wettability as well as dissolution of ZnO, both contribute to the observed differences in cellular viability and proliferation. Copyright © 2011 Wiley Periodicals, Inc.

Genome-wide profiling of microRNAs reveals novel insights into the interactions between H9N2 avian influenza virus and avian dendritic cells.

PubMed

Lin, Jian; Xia, Jing; Zhang, Tian; Zhang, Keyun; Yang, Qian

2018-05-10

The antigen-presenting ability of dendritic cells (DCs) plays an important and irreplaceable role in recognising and clearing viruses. Antiviral responses must rapidly defend against infection while minimising inflammatory damage, but the mechanisms that regulate the magnitude of response within an infected cell are not well understood. MicroRNAs (microRNAs), small non-coding RNAs, can regulate mouse or avian DCs to inhibit the infection and replication of avian influenza virus (AIV). Here, we performed a global analysis to understand how avian DCs respond to H9N2 AIV and provide a potential mechanism to explain how avian microRNAs can defend against H9N2 AIV replication. First, we found that both active and inactive H9N2 AIV enhanced the ability of DCs to present antigens and activate T lymphocytes. Next, total microarray analyses suggested that H9N2 AIV stimulation involved protein localisation, nucleotide binding, leucocyte transendothelial migration and MAPK signalling. Moreover, we constructed 551 transcription factor (TF)-miRNA-mRNA loops based on the above analyses. Furthermore, we found that the haemagglutinin (HA) fragment, neither H5N1-HA or H9N2-HA, could not activate DCs, while truncated HA greatly increased the immune function of DCs by activating ERK and STAT3 signalling pathways. Lastly, our results not only suggested that gga-miR1644 targets muscleblind-like protein 2 (MBNL2) to enhance the ability of avian DCs to inhibit virus replication, but also suggested that gga-miR6675 targets the nuclear localisation sequence of polymerase basic protein 1 (PB1) to trigger the silencing of PB1 genes, resulting in the inhibition of H9N2 AIV replication. Altogether, our innovative study will shed new light on the role of avian microRNAs in evoking avian DCs and inhibiting virus replication.

Intra-articular Hyaluronic Acid (HA) and Platelet Rich Plasma (PRP) injection versus Hyaluronic acid (HA) injection alone in Patients with Grade III and IV Knee Osteoarthritis (OA): A Retrospective Study on Functional Outcome.

PubMed

Saturveithan, C; Premganesh, G; Fakhrizzaki, S; Mahathir, M; Karuna, K; Rauf, K; William, H; Akmal, H; Sivapathasundaram, N; Jaspreet, K

2016-07-01

Introduction: Intra-articular hyaluronic acid (HA) is widely utilized in the treatment of knee osteoarthritis whereas platelet rich plasma (PRP) enhances the regeneration of articular cartilage. This study analyses the efficacy of HA and PRP in grade III and IV knee osteoarthritis. Methodology: This is a cross sectional study with retrospective review of 64 patients (101 knees) which includes 56 knees injected with HA+ PRP, and 45 knees with HA only. Results: During the post six months International Knee Documentation Committee (IKDC) evaluation, HA+PRP group showed marked improvement of 24.33 compared to 12.15 in HA group. Decrement in visual analogue score (VAS) in HA+PRP was 1.9 compared to 0.8 in HA group. Conclusion: We propose intra-articular HA and PRP injections as an optional treatment modality in Grade III and IV knee osteoarthritis in terms of functional outcome and pain control for up to six months when arthroplasty is not an option.

Intra-articular Hyaluronic Acid (HA) and Platelet Rich Plasma (PRP) injection versus Hyaluronic acid (HA) injection alone in Patients with Grade III and IV Knee Osteoarthritis (OA): A Retrospective Study on Functional Outcome

PubMed Central

Premganesh, G; Fakhrizzaki, S; Mahathir, M; Karuna, K; Rauf, K; William, H; Akmal, H; Sivapathasundaram, N; Jaspreet, K

2016-01-01

Introduction: Intra-articular hyaluronic acid (HA) is widely utilized in the treatment of knee osteoarthritis whereas platelet rich plasma (PRP) enhances the regeneration of articular cartilage. This study analyses the efficacy of HA and PRP in grade III and IV knee osteoarthritis. Methodology: This is a cross sectional study with retrospective review of 64 patients (101 knees) which includes 56 knees injected with HA+ PRP, and 45 knees with HA only. Results: During the post six months International Knee Documentation Committee (IKDC) evaluation, HA+PRP group showed marked improvement of 24.33 compared to 12.15 in HA group. Decrement in visual analogue score (VAS) in HA+PRP was 1.9 compared to 0.8 in HA group. Conclusion: We propose intra-articular HA and PRP injections as an optional treatment modality in Grade III and IV knee osteoarthritis in terms of functional outcome and pain control for up to six months when arthroplasty is not an option. PMID:28435559

Quantification of hyaluronan (HA) using a simplified fluorophore-assisted carbohydrate electrophoresis (FACE) procedure.

PubMed

Midura, Ronald J; Cali, Valbona; Lauer, Mark E; Calabro, Anthony; Hascall, Vincent C

2018-01-01

Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types. While this previous FACE procedure is relatively straightforward to implement by carbohydrate research investigators, many nonglycoscience laboratories now studying HA biology might have difficulties establishing this prior FACE procedure as a routine assay for HA. To address this need, we have greatly simplified our prior FACE procedure for accurate and reproducible assessment of HA in tissues and cell cultures. This chapter describes in detail this simplified FACE procedure and, because it uses an enzyme that degrades both HA and CS, investigators will also gain additional insight into the quantities of CS in the same samples dedicated for HA analysis. © 2018 Elsevier Inc. All rights reserved.

Mechanical strength of [HA/Bioplastic/Sericin] composite part printed by bioprinter

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

Tontowi, Alva Edy, E-mail: alvaedytontowi@ugm.ac.id; Setiawan, Agris

The aim of this research was to determine the effect of hydroxyapatite (HA) content in printed biocomposite to its mechanical strength. The biocomposite paste was prepared by composing HA, bioplastic and sericin with various ratios of [HA/Bioplastic]: 40/60, 50/50, 60,40 and 70/30. Sericin of 0.3% weight was added to the biocomposite. Mechanical test was conducted to observe tensile (ASTM D 638 type 4) and flexural strength (ASTM D 790). Both type of specimens were fabricated using 3D Printer. Printing process parameter (infill speed, print speed and layer height) were set up as 60 mm/s, 10 mm/s, 0.35 mm, respectively. Resultsmore » showed that biocomposite with [HA/Biplastic]. weight ratio of 60/40(w/w) has an optimum tensile (3.89 ± 1.26 MPa) and flexural strength (2.51 ± 0.45 MPa). Scanning electron microscope observation indicated that microstructure of specimen was influenced by the percentage of the hydroxyapatite. There was no agglomeration of HA particle within the composite.« less

CILogon-HA. Higher Assurance Federated Identities for DOE Science

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

Basney, James

The CILogon-HA project extended the existing open source CILogon service (initially developed with funding from the National Science Foundation) to provide credentials at multiple levels of assurance to users of DOE facilities for collaborative science. CILogon translates mechanism and policy across higher education and grid trust federations, bridging from the InCommon identity federation (which federates university and DOE lab identities) to the Interoperable Global Trust Federation (which defines standards across the Worldwide LHC Computing Grid, the Open Science Grid, and other cyberinfrastructure). The CILogon-HA project expanded the CILogon service to support over 160 identity providers (including 6 DOE facilities) andmore » 3 internationally accredited certification authorities. To provide continuity of operations upon the end of the CILogon-HA project period, project staff transitioned the CILogon service to operation by XSEDE.« less

Design of peptide mimetics to block pro-inflammatory functions of HA fragments.

PubMed

Hauser-Kawaguchi, Alexandra; Luyt, Leonard G; Turley, Eva

2018-01-31

Hyaluronan is a simple extracellular matrix polysaccharide that actively regulates inflammation in tissue repair and disease processes. The native HA polymer, which is large (>500 kDa), contributes to the maintenance of homeostasis. In remodeling and diseased tissues, polymer size is strikingly polydisperse, ranging from <10 kDa to >500 kDa. In a diseased or stressed tissue context, both smaller HA fragments and high molecular weight HA polymers can acquire pro-inflammatory functions, which result in the activation of multiple receptors, triggering pro-inflammatory signaling to diverse stimuli. Peptide mimics that bind and scavenge HA fragments have been developed, which show efficacy in animal models of inflammation. These studies indicate both that HA fragments are key to driving inflammation and that scavenging these is a viable therapeutic approach to blunting inflammation in disease processes. This mini-review summarizes the peptide-based methods that have been reported to date for blocking HA signaling events as an anti-inflammatory therapeutic approach. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Antibacterial activation of hydroxyapatite (HA) with controlled porosity by different antibiotics.

PubMed

Chai, F; Hornez, J-C; Blanchemain, N; Neut, C; Descamps, M; Hildebrand, H F

2007-11-01

In order to prevent the increasing frequency of per-operative infections, bioceramics can be loaded with anti-bacterial agents, which will release with respect to their chemical characteristics. A novel hydroxyapatite (HA) was elaborated with specific internal porosities for using as a bone-bioactive antibiotic (ATB) carrier material. UV spectrophotometry and bacteria inhibition tests were performed for testing the ATB adsorption and the microbiological effectiveness after loading with different antibiotics. The impregnation time, ATB impregnating concentration, impregnation condition and other factors, which might influence the ATB loading effect, were studied by exposure to different releasing solvents and different pathogenic bacteria: Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. It clearly showed that the facility of ATB loading on this porous HA is even possible just under simple non-vacuum impregnation conditions in a not-so-long impregnating interval. The results also showed that, for all three types of ATB (vancomycin, ciprofloxacin and gentamicin), adsorbed amount on the micro-porous HA were hugely higher than that on dense HA. The micro-porosity of test HA had also significantly prolonged the release time of antibiotics even under mimic physiological conditions. Furthermore, it also has primarily proved by a pilot test that the antibacterial efficiency of crude micro-porous HA could be further significantly improved by other methods of functionalization such as cold plasma technique.

Epigenetic control of skin differentiation genes by phytocannabinoids

PubMed Central

Pucci, Mariangela; Rapino, Cinzia; Di Francesco, Andrea; Dainese, Enrico; D'Addario, Claudio; Maccarrone, Mauro

2013-01-01

BACKGROUND AND PURPOSE Endocannabinoid signalling has been shown to have a role in the control of epidermal physiology, whereby anandamide is able to regulate the expression of skin differentiation genes through DNA methylation. Here, we investigated the possible epigenetic regulation of these genes by several phytocannabinoids, plant-derived cannabinoids that have the potential to be novel therapeutics for various human diseases. EXPERIMENTAL APPROACH The effects of cannabidiol, cannabigerol and cannabidivarin on the expression of skin differentiation genes keratins 1 and 10, involucrin and transglutaminase 5, as well as on DNA methylation of keratin 10 gene, were investigated in human keratinocytes (HaCaT cells). The effects of these phytocannabinoids on global DNA methylation and the activity and expression of four major DNA methyltransferases (DNMT1, 3a, 3b and 3L) were also examined. KEY RESULTS Cannabidiol and cannabigerol significantly reduced the expression of all the genes tested in differentiated HaCaT cells, by increasing DNA methylation of keratin 10 gene, but cannabidivarin was ineffective. Remarkably, cannabidiol reduced keratin 10 mRNA through a type-1 cannabinoid (CB1) receptor-dependent mechanism, whereas cannabigerol did not affect either CB1 or CB2 receptors of HaCaT cells. In addition, cannabidiol, but not cannabigerol, increased global DNA methylation levels by selectively enhancing DNMT1 expression, without affecting DNMT 3a, 3b or 3L. CONCLUSIONS AND IMPLICATIONS These findings show that the phytocannabinoids cannabidiol and cannabigerol are transcriptional repressors that can control cell proliferation and differentiation. This indicates that they (especially cannabidiol) have the potential to be lead compounds for the development of novel therapeutics for skin diseases. PMID:23869687

Rapidly sintering of interconnected porous Ti-HA biocomposite with high strength and enhanced bioactivity.

PubMed

Zhang, L; He, Z Y; Zhang, Y Q; Jiang, Y H; Zhou, R

2016-10-01

In this work, interconnected porous Ti-HA biocomposites with enhanced bioactivity, high porosity and compressive strength were prepared by spark plasma sintering (SPS) and space holder method. Pore characteristics, mechanical properties, corrosion behaviors and in vitro bioactivity of the porous Ti-HA were investigated. Results showed that porous Ti-HA with 5-30wt% HA contents possessed not only low elastic modulus of 8.2-15.8GPa (close to that of human bone) but also high compressive strength (86-388MPa). Although the HA partially decomposed and formed secondary phases, the sintered porous Ti-HA can still be good bioactivity. The homogeneity and the thickness of apatite layer increased significantly with the increase of HA. But with the thickness of apatite layer increased, micro-cracks appeared on the surface of porous Ti-30%HA. A model was built to discuss the current distribution and sintering mechanism of HA on Ti matrix during SPS process. It indicated that the excessive addition of HA would deteriorate the sintering quality, thus decreasing the mechanical properties and corrosion resistance. However, the combination of interconnected pore characteristics, low elastic modulus, high compressive strength and enhanced bioactivity might make porous Ti-HA biocomposites prepared by SPS a promising candidate for hard tissue implants. Copyright © 2016 Elsevier B.V. All rights reserved.

Ligand activation of peroxisome proliferator-activated receptor-beta/delta inhibits cell proliferation in human HaCaT keratinocytes.

PubMed

Borland, Michael G; Foreman, Jennifer E; Girroir, Elizabeth E; Zolfaghari, Reza; Sharma, Arun K; Amin, Shantu; Gonzalez, Frank J; Ross, A Catharine; Peters, Jeffrey M

2008-11-01

Although there is strong evidence that ligand activation of peroxisome proliferator-activated receptor (PPAR)-beta/delta induces terminal differentiation and attenuates cell growth, some studies suggest that PPARbeta/delta actually enhances cell proliferation. For example, it was suggested recently that retinoic acid (RA) is a ligand for PPARbeta/delta and potentiates cell proliferation by activating PPARbeta/delta. The present study examined the effect of ligand activation of PPARbeta/delta on cell proliferation, cell cycle kinetics, and target gene expression in human HaCaT keratinocytes using two highly specific PPARbeta/delta ligands [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy acetic acid (GW0742) and 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (GW501516)] and RA. Both PPARbeta/delta ligands and RA inhibited cell proliferation of HaCaT keratinocytes. GW0742 and GW501516 increased expression of known PPARbeta/delta target genes, whereas RA did not; RA increased the expression of known retinoic acid receptor/retinoid X receptor target genes, whereas GW0742 did not affect these genes. GW0742, GW501516, and RA did not modulate the expression of 3-phosphoinositide-dependent protein kinase or alter protein kinase B phosphorylation. GW0742 and RA increased annexin V staining as quantitatively determined by flow cytometry. The effects of GW0742 and RA were also examined in wild-type and PPARbeta/delta-null primary mouse keratinocytes to determine the specific role of PPARbeta/delta in modulating cell growth. Although inhibition of keratinocyte proliferation by GW0742 was PPARbeta/delta-dependent, inhibition of cell proliferation by RA occurred in both genotypes. Results from these studies demonstrate that ligand activation of PPARbeta/delta inhibits keratinocyte proliferation through PPARbeta/delta-dependent mechanisms. In contrast, the observed inhibition of

HDPE-HA composites synthetized by in situ polymerization with different filler content

NASA Astrophysics Data System (ADS)

Hermán, V.; Karam, A.; Albano, C.; Romero, K.; González, G.

2012-07-01

In Situ ethylene polymerization was used to synthesize high density polyethylene - hydroxyapatite (HDPE-HA) composites, employing Cp2ZrCl2/MAO as catalytic system. A good dispersion of HA into the HDPE matrix was obtained when the following synthesis conditions were combined: high stirring velocities (2000 rpm), low quantities of solvent (100 mL), and 10 °C. Under these conditions different filler content was used to synthetized HDPE-HA composites. An interaction between HA and HDPE was obtained by FTIR. On the other hand, thermal analysis indicated that no significant differences were observed between HDPE and the composites.

[Biocompatibility of HA/TCP biphasic ceramics with co-cultured human osteoblasts in vitro].

PubMed

Lu, X; Li, S; Zhang, J; Zhang, Z; Lu, B; Bu, H; Li, Y; Cheng, J

2001-12-01

The biocompatibility of HA/TCP ceramic was evaluated by investigation of attachment and growth of osteoblasts on biomaterial, as well as monitoring the effects of biomaterial on expression of functional phenotypes of co-cultured osteoblasts in vitro. When co-cultured with HA/TCP ceramics, osteoblasts firstly attached to the surface of HA/TCP disk, then attached to notches and grew into the micropores of biomaterial during further culture period. At last, the ceramics were almost packed with osteoblasts. Additionally, osteoblasts co-cultured with HA/TCP were similar to osteoblasts cultured under normal condition in osteoblastic phenotypes; the secreted lots of collagen type I, possess strong activity of Alkaline Phosphatase and mineralized extracellular matrix. The fact that osteoblasts could grow well on HA/TCP ceramics and the biomaterial did not affect their physiological function suggest that HA/TCP ceramic is biocompatible with human osteoblasts.

Structural fatigue in the 34-meter HA-dec antennas

NASA Technical Reports Server (NTRS)

Van Hek, Ronald A.; Saldua, Benjamin P.

1991-01-01

Three 26-m hour-angle/declination (HA-dec) antennas, designed for a life span of 20 years, were built in the early 1960s for the NASA Deep Space Network. After 16 years the antennas were upgraded. The design required a structural weight increase of about 50 percent in both the HA and dec structures to achieve the desired improvements. The fatigue caused by the resulting stress-reversal conditions is discussed. The structural failures and their analyses are described.

Molecular mapping of three nuclear male sterility mutant genes in cultivated sunflower (Helianthus annuus L.)

USDA-ARS?s Scientific Manuscript database

The nuclear male sterility (NMS) trait is a useful tool for sunflower (Helianthus annuus L.) breeding and genetic programs. Previously, we induced NMS mutants in cultivated line HA 89. The mutants possessed single recessive genes, ms6, ms7, and ms8, respectively, in NMS HA 89-872, NMS HA 89-552, and...

The matrix gene segment destabilizes the acid and thermal stability of the hemagglutinin of pandemic live attenuated influenza virus vaccines.

PubMed

O'Donnell, Christopher D; Vogel, Leatrice; Matsuoka, Yumiko; Jin, Hong; Subbarao, Kanta

2014-11-01

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. 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 investigated the HA stability

Prediction of IOI-HA scores using speech reception thresholds and speech discrimination scores in quiet.

PubMed

Brännström, K Jonas; Lantz, Johannes; Nielsen, Lars Holme; Olsen, Steen Østergaard

2014-02-01

Outcome measures can be used to improve the quality of the rehabilitation by identifying and understanding which variables influence the outcome. This information can be used to improve outcomes for clients. In clinical practice, pure-tone audiometry, speech reception thresholds (SRTs), and speech discrimination scores (SDSs) in quiet or in noise are common assessments made prior to hearing aid (HA) fittings. It is not known whether SRT and SDS in quiet relate to HA outcome measured with the International Outcome Inventory for Hearing Aids (IOI-HA). The aim of the present study was to investigate the relationship between pure-tone average (PTA), SRT, and SDS in quiet and IOI-HA in both first-time and experienced HA users. SRT and SDS were measured in a sample of HA users who also responded to the IOI-HA. Fifty-eight Danish-speaking adult HA users. The psychometric properties were evaluated and compared to previous studies using the IOI-HA. The associations and differences between the outcome scores and a number of descriptive variables (age, gender, fitted monaurally/binaurally with HA, first-time/experienced HA users, years of HA use, time since last HA fitting, best ear PTA, best ear SRT, or best ear SDS) were examined. A multiple forward stepwise regression analysis was conducted using scores on the separate IOI-HA items, the global score, and scores on the introspection and interaction subscales as dependent variables to examine whether the descriptive variables could predict these outcome measures. Scores on single IOI-HA items, the global score, and scores on the introspection (items 1, 2, 4, and 7) and interaction (items 3, 5, and 6) subscales closely resemble those previously reported. Multiple regression analysis showed that the best ear SDS predicts about 18-19% of the outcome on items 3 and 5 separately, and about 16% on the interaction subscale (sum of items 3, 5, and 6) CONCLUSIONS: The best ears SDS explains some of the variance displayed in the IOI-HA

HaCaT anchorage blockade leads to oxidative stress, DNA damage and DNA methylation changes.

PubMed

da Silva, Rodrigo A; Sammartino Mariano, Flavia; Planello, Aline C; Line, Sergio R P; de Souza, Ana Paula

2015-07-01

Cell adhesion plays an important role in neoplastic transformation. Thus, anchorage-independent growth and epithelial-mesenchymal transition, which are features associated to anoikis-resistance, are vital steps in cancer progression and metastatic colonization. Cell attachment loss may induce intracellular oxidative stress, which triggers DNA damage as methylation changes. HaCaT lineage cells were submitted to periods of 1, 3, 5 and 24 h of anchorage blockage with the purpose of study of oxidative stress effect on changes in the DNA methylation pattern, derived from attachment blockade. Through this study, HaCaT anchorage blockage-induced oxidative stress was reported to mediate alterations in global DNA methylation changes and into TP53 gene promoter pattern during anoikis-resistance acquisition. Furthermore, at the first experimental time-periods (1, 3 and 5 h), genome hypermethylation was found; however, genome hypomethylation was observed in later time-periods (24 h) of attachment impediment. The TP 53 methylation analyses were performed after 24 h of replated anoikis-resistance cells and same methylation pattern was observed, occurring an early (1 and 3 h) hypermethylation that was followed by late (5 and 24 h) hypomethylation. However, LINE-1, a marker of genomic instability, was perceived in time-dependent hypomethylation. The mRNA levels of the DNMTs enzymes were influenced by cell attachment blockage, but non-conclusive results were obtained in order to match DNMTs transcription to pattern methylation results. In conclusion, DNA damage was found, leaded by oxidative stress that has come up from HaCaT anchorage blockade, which rises a global genome hypomethylation tendency as consequence, which might denote genomic instability.

Molecular Epidemiology of a novel re-assorted epidemic strain of equine influenza virus in Pakistan in 2015-16.

PubMed

Khan, Amjad; Mushtaq, Muhammad Hassan; Ahmad, Mansur Ud Din; Nazir, Jawad; Farooqi, Shahid Hussain; Khan, Asghar

2017-08-15

A widespread epidemic of equine influenza (EI) occurred in nonvaccinated equine population across multiple districts in Khyber Pakhtunkhwa Province of Pakistan during 2015-2016. An epidemiological surveillance study was conducted from Oct 2015 to April 2016 to investigate the outbreak. EI virus strains were isolated in embryonated eggs from suspected equines swab samples and were subjected to genome sequencing using M13 tagged segment specific primers. Phylogenetic analyses of the nucleotide sequences were concluded using Geneious. Haemagglutinin (HA), Neuraminidase (NA), Matrix (M) and nucleoprotein (NP) genes nucleotide and amino acid sequences of the isolated viruses were aligned with those of OIE recommended, FC-1, FC-2, and contemporary isolates of influenza A viruses from other species. HA and NA genes amino acid sequences were very similar to Tennessee/14 and Malaysia/15 of FC-1 and clustered with the contemporary isolates recently reported in the USA. Phylogenetic analysis showed that these viruses were mostly identical (with 99.6% and 97.4% nucleotide homology) to, and were reassortants containing chicken/Pakistan/14 (H7N3) and Canine/Beijing/10 (H3N2) like M and NP genes. Genetic analysis indicated that A/equine/Pakistan/16 viruses were most probably the result of several re-assortments between the co-circulating avian and equine viruses, and were genetically unlike the other equine viruses due to the presence of H7N3 or H3N2 like M and NP genes. Epidemiological data analysis indicated the potential chance of mixed, and management such as mixed farming system by keeping equine, canine and backyard poultry together in confined premises as the greater risk factors responsible for the re-assortments. Other factors might have contributed to the spread of the epidemic, including low awareness level, poor control of equine movements, and absence of border control disease strategies. Copyright © 2017 Elsevier B.V. All rights reserved.

Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12 -induced cell transformation.

PubMed

Lin, Hsiu-Kuan; Lin, Hsi-Hui; Chiou, Yu-Wei; Wu, Ching-Lung; Chiu, Wen-Tai; Tang, Ming-Jer

2018-05-01

Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-Ras V12 gene) transformation by Ha-Ras V12 . Cav1 overexpression abrogates the Ha-Ras V12 -driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-Ras V12 -inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-Ras V12 , was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-Ras V12 - and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-Ras V12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-Ras V12 -Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-Ras V12 -driven cell transformation. © 2018 The Authors

A GRAS-like gene of sunflower (Helianthus annuus L.) alters the gibberellin content and axillary meristem outgrowth in transgenic Arabidopsis plants.

PubMed

Fambrini, M; Mariotti, L; Parlanti, S; Salvini, M; Pugliesi, C

2015-11-01

The GRAS proteins belong to a plant transcriptional regulator family that function in the regulation of plant growth and development. Despite their important roles, in sunflower only one GRAS gene (HaDella1) with the DELLA domain has been reported. Here, we provide a functional characterisation of a GRAS-like gene from Helianthus annuus (Ha-GRASL) lacking the DELLA motif. The Ha-GRASL gene contains an intronless open reading frame of 1,743 bp encoding 580 amino acids. Conserved motifs in the GRAS domain are detected, including VHIID, PFYRE, SAW and two LHR motifs. Within the VHII motif, the P-H-N-D-Q-L residues are entirely maintained. Phylogenetic analysis reveals that Ha-GRASL belongs to the SCARECROW LIKE4/7 (SCL4/7) subfamily of the GRAS consensus tree. Accumulation of Ha-GRASL mRNA at the adaxial boundaries from P6/P7 leaf primordia suggests a role of Ha-GRASL in the initiation of median and basal axillary meristems (AMs) of sunflower. When Ha-GRASL is over-expressed in Arabidopsis wild-type plants, the number of lateral bolts increases differently from untransformed plants. However, Ha-GRASL slightly affects the lateral suppressor (las-4-) mutation. Therefore, we hypothesise that Ha-GRASL and LAS are not functionally equivalent. The over-expression of Ha-GRASL reduces metabolic flow of gibberellins (GAs) in Arabidopsis and this modification could be relevant in AM development. Phylogenetic analysis includes LAS and SCL4/7 in the same major clade, suggesting a more recent separation of these genes with respect to other GRAS members. We propose that some features of their ancestor, as well as AM initiation and outgrowth, are partially retained in both LAS and SCL4/7. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

v-myb transformation of Xeroderma pigmentosum human fibroblasts: Overexpression of the c-Ha-ras oncogene in the transformed cells

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

Michelin, S.; Varlet, I.; Sarasin, A.

1991-10-01

Human Xeroderma pigmentosum normal' fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental normal' AS16 cells and a revertant clone (ASKXAmore » Cl 1.1 G). The results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.« less

A simplified Sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses.

PubMed

Deng, Yi-Mo; Spirason, Natalie; Iannello, Pina; Jelley, Lauren; Lau, Hilda; Barr, Ian G

2015-07-01

Full genome sequencing of influenza A viruses (IAV), including those that arise from annual influenza epidemics, is undertaken to determine if reassorting has occurred or if other pathogenic traits are present. Traditionally IAV sequencing has been biased toward the major surface glycoproteins haemagglutinin and neuraminidase, while the internal genes are often ignored. Despite the development of next generation sequencing (NGS), many laboratories are still reliant on conventional Sanger sequencing to sequence IAV. To develop a minimal and robust set of primers for Sanger sequencing of the full genome of IAV currently circulating in humans. A set of 13 primer pairs was designed that enabled amplification of the six internal genes of multiple human IAV subtypes including the recent avian influenza A(H7N9) virus from China. Specific primers were designed to amplify the HA and NA genes of each IAV subtype of interest. Each of the primers also incorporated a binding site at its 5'-end for either a forward or reverse M13 primer, such that only two M13 primers were required for all subsequent sequencing reactions. This minimal set of primers was suitable for sequencing the six internal genes of all currently circulating human seasonal influenza A subtypes as well as the avian A(H7N9) viruses that have infected humans in China. This streamlined Sanger sequencing protocol could be used to generate full genome sequence data more rapidly and easily than existing influenza genome sequencing protocols. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Serological Evidence of Pandemic H1N1 Influenza Virus Infections in Greek Swine.

PubMed

Kyriakis, C S; Papatsiros, V G; Athanasiou, L V; Valiakos, G; Brown, I H; Simon, G; Van Reeth, K; Tsiodras, S; Spyrou, V; Billinis, C

2016-08-01

The introduction of the 2009 pandemic H1N1 (pH1N1) influenza virus in pigs changed the epidemiology of influenza A viruses (IAVs) in swine in Europe and the rest of the world. Previously, three IAV subtypes were found in the European pig population: an avian-like H1N1 and two reassortant H1N2 and H3N2 viruses with human-origin haemagglutinin (HA) and neuraminidase proteins and internal genes of avian decent. These viruses pose antigenically distinct HAs, which allow the retrospective diagnosis of infection in serological investigations. However, cross-reactions between the HA of pH1N1 and the HAs of the other circulating H1 IAVs complicate serological diagnosis. The prevalence of IAVs in Greek swine has been poorly investigated. In this study, we examined and compared haemagglutination inhibition (HI) antibody titres against previously established IAVs and pH1N1 in 908 swine sera from 88 herds, collected before and after the 2009 pandemic. While we confirmed the historic presence of the three IAVs established in European swine, we also found that 4% of the pig sera examined after 2009 had HI antibodies only against the pH1N1 virus. Our results indicate that pH1N1 is circulating in Greek pigs and stress out the importance of a vigorous virological surveillance programme. © 2015 Blackwell Verlag GmbH.

Hyaluronic Acid Enhances Gene Delivery into the Cochlea

PubMed Central

Shibata, Seiji B.; Cortez, Sarah R.; Wiler, James A.; Swiderski, Donald L.

2012-01-01

Abstract Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application. PMID:22074321

Hyaluronic acid enhances gene delivery into the cochlea.

PubMed

Shibata, Seiji B; Cortez, Sarah R; Wiler, James A; Swiderski, Donald L; Raphael, Yehoash

2012-03-01

Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

Preparation and characterization of NiW-nHA composite catalyst for hydrocracking

NASA Astrophysics Data System (ADS)

Zhou, Gang; Hou, Yongzhao; Liu, Lei; Liu, Hongru; Liu, Can; Liu, Jing; Qiao, Huiting; Liu, Wenyong; Fan, Yubo; Shen, Shituan; Rong, Long

2012-11-01

The synthesis, characterization and catalytic capability of the NiW-nano-hydroxyapatite (NiW-nHA) composite were investigated in this paper. The NiW-nHA catalyst was prepared by a co-precipitation method. Then Fourier transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and energy dispersive spectroscopy (EDX) were used to analyze this material. In addition, the catalytic capacity of the NiW-nHA composite was also examined by FT-IR and gas chromatography (GC). The results of FT-IR analysis indicated that Ni, W and nHA combined closely. TEM observation revealed that this catalyst was needle shaped and the crystal retained a nanometer size. XRD data also suggested that a new phase of CaWO4 appeared and the lattice parameters of nHA changed in this system. nHA was the carrier of metals. The rates of Ni/W-loading were 73.24% and 65.99% according to the EDX data, respectively. Furthermore, the conversion of 91.88% Jatropha oil was achieved at 360 °C and 3 MPa h-1 over NiW-nHA catalyst. The straight chain alkanes ranging from C15 to C18 were the main components in the production. The yield of C15-C18 alkanes was up to 83.56 wt%. The reaction pathway involved hydrocracking of the C&z.dbd;C bonds of these triglycerides from Jatropha oil. This paper developed a novel non-sulfided catalyst to obtain a ``green biofuel'' from vegetable oil.

Ligand Activation of Peroxisome Proliferator-Activated Receptor-β/δ Inhibits Cell Proliferation in Human HaCaT KeratinocytesS

PubMed Central

Borland, Michael G.; Foreman, Jennifer E.; Girroir, Elizabeth E.; Zolfaghari, Reza; Sharma, Arun K.; Amin, Shantu; Gonzalez, Frank J.; Ross, A. Catharine; Peters, Jeffrey M.

2009-01-01

Although there is strong evidence that ligand activation of peroxisome proliferator-activated receptor (PPAR)-β/δ induces terminal differentiation and attenuates cell growth, some studies suggest that PPARβ/δ actually enhances cell proliferation. For example, it was suggested recently that retinoic acid (RA) is a ligand for PPARβ/δ and potentiates cell proliferation by activating PPARβ/δ. The present study examined the effect of ligand activation of PPARβ/δ on cell proliferation, cell cycle kinetics, and target gene expression in human HaCaT keratinocytes using two highly specific PPARβ/δ ligands [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy acetic acid (GW0742) and 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (GW501516)] and RA. Both PPARβ/δ ligands and RA inhibited cell proliferation of HaCaT keratinocytes. GW0742 and GW501516 increased expression of known PPARβ/δ target genes, whereas RA did not; RA increased the expression of known retinoic acid receptor/retinoid X receptor target genes, whereas GW0742 did not affect these genes. GW0742, GW501516, and RA did not modulate the expression of 3-phosphoinositide-dependent protein kinase or alter protein kinase B phosphorylation. GW0742 and RA increased annexin V staining as quantitatively determined by flow cytometry. The effects of GW0742 and RA were also examined in wild-type and PPARβ/δ-null primary mouse keratinocytes to determine the specific role of PPARβ/δ in modulating cell growth. Although inhibition of keratinocyte proliferation by GW0742 was PPARβ/δ-dependent, inhibition of cell proliferation by RA occurred in both genotypes. Results from these studies demonstrate that ligand activation of PPARβ/δ inhibits keratinocyte proliferation through PPARβ/δ-dependent mechanisms. In contrast, the observed inhibition of cell proliferation in mouse and human keratinocytes by RA is

Fabrication and characterization of plasma-sprayed HA/SiO(2) coatings for biomedical application.

PubMed

Morks, M F

2008-01-01

Fused silica powder has been mixed with hydroxyapatite (HA) powder and plasma sprayed by using gas tunnel-type plasma jet. The influence of silica content (10 wt% and 20 wt%) on the microstructure and mechanical properties of HA-silica coatings was investigated. For investigating the microstructure and mechanical properties of HA-silica coatings, SUS 304 stainless steel was used as substrate material. The spraying was carried out on roughened substrate in an atmospheric chamber. Scanning electron microscope micrographs of cross-sectioned HA/SiO(2) coatings showed that the sprayed HA coatings with 10 and 20 wt% SiO(2) have dense structure with low porosity compared to the pure HA coatings. On the other hand, as the amount of silica was increased the coatings became denser, harder and exhibited high abrasive wear resistance. The presence of silica significantly improved the adhesive strength of HA/SiO(2) coatings mainly due to the increase in bonding strength of the coating at the interface.

The optimal SAM surface functional group for producing a biomimetic HA coating on Ti.

PubMed

Liu, D P; Majewski, P; O'Neill, B K; Ngothai, Y; Colby, C B

2006-06-15

Commercial interest is growing in biomimetic methods that employ self assembled mono-layers (SAMs) to produce biocompatible HA coatings on Ti-based orthopedic implants. Recently, separate studies have considered HA formation for various SAM surface functional groups. However, these have often neglected to verify crystallinity of the HA coating, which is essential for optimal bioactivity. Furthermore, differing experimental and analytical methods make performance comparisons difficult. This article investigates and evaluates HA formation for four of the most promising surface functional groups: --OH, --SO(3)H, --PO(4)H(2) and --COOH. All of them successfully formed a HA coating at Ca/P ratios between 1.49 and 1.62. However, only the --SO(3)H and --COOH end groups produced a predominantly crystalline HA. Furthermore, the --COOH end group yielded the thickest layer and possessed crystalline characteristics very similar to that of the human bone. The --COOH end group appears to provide the optimal SAM surface interface for nucleation and growth of biomimetic crystalline HA. Intriguingly, this finding may lend support to explanations elsewhere of why human bone sialoprotein is such a potent nucleator of HA and is attributed to the protein's glutamic acid-rich sequences.

Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers, tolerogens and irritants.

PubMed

Mohamadzadeh, M; Müller, M; Hultsch, T; Enk, A; Saloga, J; Knop, J

1994-12-01

To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensitizer (2,4-dinitrofluorobenzene, 3-n-pentadecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Interleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, interleukin-8 production was not detectable in unstimulated normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immunogenic or inflammatory signals in man.

High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex.

PubMed

Lee, Kwangkook; Lam, Kwok-Ho; Kruel, Anna Magdalena; Perry, Kay; Rummel, Andreas; Jin, Rongsheng

2014-04-04

Botulinum neurotoxins (BoNTs) are produced as progenitor toxin complexes (PTCs) by Clostridium botulinum. The PTCs are composed of BoNT and non-toxic neurotoxin-associated proteins (NAPs), which serve to protect and deliver BoNT through the gastrointestinal tract in food borne botulism. HA33 is a key NAP component that specifically recognizes host carbohydrates and helps enrich PTC on the intestinal lumen preceding its transport across the epithelial barriers. Here, we report the crystal structure of HA33 of type B PTC (HA33/B) in complex with lactose at 1.46Å resolution. The structural comparisons among HA33 of serotypes A-D reveal two different HA33-glycan interaction modes. The glycan-binding pockets on HA33/A and B are more suitable to recognize galactose-containing glycans in comparison to the equivalent sites on HA33/C and D. On the contrary, HA33/C and D could potentially recognize Neu5Ac as an independent receptor, whereas HA33/A and B do not. These findings indicate that the different oral toxicity and host susceptibility observed among different BoNT serotypes could be partly determined by the serotype-specific interaction between HA33 and host carbohydrate receptors. Furthermore, we have identified a key structural water molecule that mediates the HA33/B-lactose interactions. It provides the structural basis for development of new receptor-mimicking compounds, which have enhanced binding affinity with HA33 through their water-displacing moiety. Copyright © 2014 Elsevier Inc. All rights reserved.

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests.

PubMed

Tsukamoto, Kenji; Panei, Carlos Javier; Javier, Panei Carlos; Shishido, Makiko; Noguchi, Daigo; Pearce, John; Kang, Hyun-Mi; Jeong, Ok Mi; Lee, Youn-Jeong; Nakanishi, Koji; Ashizawa, Takayoshi

2012-01-01

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10(1.5), 10(2.3), and 10(3.1) 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.

J-Plus: Measuring Ha Emission Line Flux In The Nearby Universe

NASA Astrophysics Data System (ADS)

Logroño-García, Rafael; Vilella-Rojo, Gonzalo; López-San Juan, Carlos; Varela, Jesús; Viironen, Kerttu

2017-10-01

In the present presentation we aim to validate the methodology designed to extract the Ha emission line flux from J-PLUS data, a twelve optical filter survey carried out with the 2 deg² field of view T80Cam camera, mounted at the JAST/T80 telescope in the OAJ, Teruel, Spain. We use the information of the twelve J-PLUS bands, including the J0660 narrow-band filter located at rest-frame Ha, over 42 deg² to extract de-reddened and [NII] decontaminated Ha emission line fluxes of 46 star-forming regions with previous SDSS and/or CALIFA spectroscopic information. The agreement of the J-PLUS photometric Ha flux and the spectroscopic one is remarkable, with a ratio R = 1,01 +/- 0,27. This demonstrates that we are able to recover reliable Ha fluxes from J-PLUS photometric data. With an expected final area of 8,500 deg2, the large J-PLUS footprint will permit the study of the spatially resolved star formation rate of thousands nearby galaxies at z 0,015, as well as the influence of the close environment. As an illustrative example, we looked to the close pair of interacting galaxies NGC3994 and NGC3995, finding an enhancement of the star formation rate not only in the central part of NGC3994 but also in outer parts of the disc.

Incoming human papillomavirus 16 genome is lost in PML protein-deficient HaCaT keratinocytes.

PubMed

Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Keiffer, Timothy R; Guion, Lucile G M; DiGiuseppe, Stephen; Scott, Rona S; Sapp, Martin

2017-05-01

Human papillomaviruses (HPVs) target promyelocytic leukemia (PML) nuclear bodies (NBs) during infectious entry and PML protein is important for efficient transcription of incoming viral genome. However, the transcriptional down regulation was shown to be promoter-independent in that heterologous promoters delivered by papillomavirus particles were also affected. To further investigate the role of PML protein in HPV entry, we used small hairpin RNA to knockdown PML protein in HaCaT keratinocytes. Confirming previous findings, PML knockdown in HaCaT cells reduced HPV16 transcript levels significantly following infectious entry without impairing binding and trafficking. However, when we quantified steady-state levels of pseudogenomes in interphase cells, we found strongly reduced genome levels compared with parental HaCaT cells. Because nuclear delivery was comparable in both cell lines, we conclude that viral pseudogenome must be removed after successful nuclear delivery. Transcriptome analysis by gene array revealed that PML knockdown in clonal HaCaT cells was associated with a constitutive interferon response. Abrogation of JAK1/2 signaling prevented genome loss, however, did not restore viral transcription. In contrast, knockdown of PML protein in HeLa cells did not affect HPV genome delivery and transcription. HeLa cells are transformed by HPV18 oncogenes E6 and E7, which have been shown to interfere with the JAK/Stat signaling pathway. Our data imply that PML NBs protect incoming HPV genomes. Furthermore, they provide evidence that PML NBs are key regulators of the innate immune response in keratinocytes. Promyelocytic leukemia nuclear bodies (PML NBs) are important for antiviral defense. Many DNA viruses target these subnuclear structures and reorganize them. Reorganization of PML NBs by viral proteins is important for establishment of infection. In contrast, HPVs require the presence of PML protein for efficient transcription of incoming viral genome. Our

Analysis of recombinant H7N9 wild-type and mutant viruses in pigs shows that the Q226L mutation in HA is important for transmission.

PubMed

Liu, Qinfang; Zhou, Bin; Ma, Wenjun; Bawa, Bhupinder; Ma, Jingjiao; Wang, Wei; Lang, Yuekun; Lyoo, Young; Halpin, Rebecca A; Lin, Xudong; Stockwell, Timothy B; Webby, Richard; Wentworth, David E; Richt, Juergen A

2014-07-01

The fact that there have been more than 300 human infections with a novel avian H7N9 virus in China indicates that this emerging strain has pandemic potential. Furthermore, many of the H7N9 viruses circulating in animal reservoirs contain putative mammalian signatures in the HA and PB2 genes that are believed to be important in the adaptation of other avian strains to humans. To date, the definitive roles of these mammalian-signature substitutions in transmission and pathogenesis of H7N9 viruses remain unclear. To address this we analyzed the biological characteristics, pathogenicity, and transmissibility of A/Anhui/1/2013 (H7N9) virus and variants in vitro and in vivo using a synthetically created wild-type virus (rAnhui-WT) and two mutants (rAnhui-HA-226Q and rAnhui-PB2-627E). All three viruses replicated in lungs of intratracheally inoculated pigs, yet nasal shedding was limited. The rAnhui-WT and rAnhui-PB2-627E viruses were transmitted to contact animals. In contrast, the rAnhui-HA-226Q virus was not transmitted to sentinel pigs. Deep sequencing of viruses from the lungs of infected pigs identified substitutions arising in the viral population (e.g., PB2-T271A, PB2-D701N, HA-V195I, and PB2-E627K reversion) that may enhance viral replication in pigs. Collectively, the results demonstrate that critical mutations (i.e., HA-Q226L) enable the H7N9 viruses to be transmitted in a mammalian host and suggest that the myriad H7N9 genotypes circulating in avian species in China and closely related strains (e.g., H7N7) have the potential for further adaptation to human or other mammalian hosts (e.g., pigs), leading to strains capable of sustained human-to-human transmission. Importance: The genomes of the zoonotic avian H7N9 viruses emerging in China have mutations in critical genes (PB2-E627K and HA-Q226L) that may be important in their pandemic potential. This study shows that (i) HA-226L of zoonotic H7N9 strains is critical for binding the α-2,6-linked receptor and

Bone remodelling around HA-coated acetabular cups

PubMed Central

Nielsen, P. T.; Søballe, K.

2006-01-01

This study was designed to investigate bone remodelling around the cup in cementless THA. Previous studies indicate an advantage of better sealing of the bone-prosthesis interface by HA/TCP coating of implants, inhibiting polyethylene-induced osteolysis. One hundred patients gave informed consent to participate in a controlled randomized study between porous coated Trilogy versus Trilogy Calcicoat (HA/TCP coated). The cup was inserted in press-fit fixation. The femoral component was a cementless porous coated titanium alloy stem (Bi-Metric), with a modular 28-mm CrCo head. The Harris Hip Score (HHS) and bone mineral density (BMD) determined by DEXA scanning were used to study the effect. Measurements revealed no difference between the two groups after 3 years either in the clinical outcome or in terms of periprosthetic bone density. Patients with a body mass index above normal regained more bone mineral than patients with normal weight. This finding supports the assumption that load is beneficial to bone remodelling. PMID:16761153

Sulforaphane suppresses ultraviolet B-induced inflammation in HaCaT keratinocytes and HR-1 hairless mice.

PubMed

Shibata, Akira; Nakagawa, Kiyotaka; Yamanoi, Hiroko; Tsuduki, Tsuyoshi; Sookwong, Phumon; Higuchi, Ohki; Kimura, Fumiko; Miyazawa, Teruo

2010-08-01

Ultraviolet B (UVB) irradiation induces skin damage and inflammation. One way to reduce the inflammation is via the use of molecules termed photochemopreventive agents. Sulforaphane (4-methylsulfinylbutyl isothiocyanate, SF), which is found in cruciferous vegetables, is known for its potent physiological properties. This study was designed to evaluate the effect of SF on skin inflammation in vitro and in vivo. In in vitro study using immortalized human keratinocytes (HaCaT), UVB caused marked inflammatory responses [i.e., decrease of HaCaT viability and increase of production of an inflammatory marker interleukin-6 (IL-6)]. SF recovered the cell proliferation and suppressed the IL-6 production. These anti-inflammatory effects of SF were explained by its ability to reduce UVB-induced inflammatory gene expressions [IL-6, IL-1beta and cyclooxgenase-2 (COX-2)]. Because SF seems to have an impact on COX-2 expression, we focused on COX-2 and found that SF reduced UVB-induced COX-2 protein expression. In support of this, PGE(2) released from HaCaT was suppressed by SF. Western blot analysis revealed that SF inhibited p38, ERK and SAPK/JNK activation, indicating that the inhibition of mitogen-activated protein kinases (MAPK) by SF would attenuate the expression of inflammatory mediators (e.g., COX-2), thereby reducing inflammatory responses. Moreover, we conducted skin thickening assay using HR-1 hairless mice and found that UVB-induced skin thickness, COX-2 protein expression and hyperplasia were all suppressed by feeding SF to the mice. These results suggest that SF has a potential use as a compound for protection against UVB-induced skin inflammation. Copyright 2010 Elsevier Inc. All rights reserved.

Detection of the HA-33 protein in botulinum neurotoxin type G complex by mass spectrometry.

PubMed

Kalb, Suzanne R; Baudys, Jakub; Barr, John R

2015-10-23

The disease botulism is caused by intoxication with botulinum neurotoxins (BoNTs), extremely toxic proteins which cause paralysis. This neurotoxin is produced by some members of the Clostridium botulinum and closely related species, and is produced as a protein complex consisting of the neurotoxin and neurotoxin-associated proteins (NAPs). There are seven known serotypes of BoNT, A-G, and the composition of the NAPs can differ between these serotypes. It was previously published that the BoNT/G complex consisted of BoNT/G, nontoxic-nonhemagglutinin (NTNH), Hemagglutinin 70 (HA-70), and HA-17, but that HA-33, a component of the protein complex of other serotypes of BoNT, was not found. Components of the BoNT/G complex were first separated by SDS-PAGE, and bands corresponding to components of the complex were digested and analyzed by LC-MS/MS. Gel bands were identified with sequence coverages of 91% for BoNT/G, 91% for NTNH, 89% for HA-70, and 88% for HA-17. Notably, one gel band was also clearly identified as HA-33 with 93% sequence coverage. The BoNT/G complex consists of BoNT/G, NTNH, HA-70, HA-17, and HA-33. These proteins form the progenitor form of BoNT/G, similar to all other HA positive progenitor toxin complexes.

Influence of Nano-HA Coated Bone Collagen to Acrylic (Polymethylmethacrylate) Bone Cement on Mechanical Properties and Bioactivity

PubMed Central

Li, Tao; Weng, Xisheng; Bian, Yanyan; Zhou, Lei; Cui, Fuzhai; Qiu, Zhiye

2015-01-01

Objective This research investigated the mechanical properties and bioactivity of polymethylmethacrylate (PMMA) bone cement after addition of the nano-hydroxyapatite(HA) coated bone collagen (mineralized collagen, MC). Materials & Methods The MC in different proportions were added to the PMMA bone cement to detect the compressive strength, compression modulus, coagulation properties and biosafety. The MC-PMMA was embedded into rabbits and co-cultured with MG 63 cells to exam bone tissue compatibility and gene expression of osteogenesis. Results 15.0%(wt) impregnated MC-PMMA significantly lowered compressive modulus while little affected compressive strength and solidification. MC-PMMA bone cement was biologically safe and indicated excellent bone tissue compatibility. The bone-cement interface crosslinking was significantly higher in MC-PMMA than control after 6 months implantation in the femur of rabbits. The genes of osteogenesis exhibited significantly higher expression level in MC-PMMA. Conclusions MC-PMMA presented perfect mechanical properties, good biosafety and excellent biocompatibility with bone tissues, which has profoundly clinical values. PMID:26039750

Influence of Nano-HA Coated Bone Collagen to Acrylic (Polymethylmethacrylate) Bone Cement on Mechanical Properties and Bioactivity.

PubMed

Li, Tao; Weng, Xisheng; Bian, Yanyan; Zhou, Lei; Cui, Fuzhai; Qiu, Zhiye

2015-01-01

This research investigated the mechanical properties and bioactivity of polymethylmethacrylate (PMMA) bone cement after addition of the nano-hydroxyapatite(HA) coated bone collagen (mineralized collagen, MC). The MC in different proportions were added to the PMMA bone cement to detect the compressive strength, compression modulus, coagulation properties and biosafety. The MC-PMMA was embedded into rabbits and co-cultured with MG 63 cells to exam bone tissue compatibility and gene expression of osteogenesis. 15.0%(wt) impregnated MC-PMMA significantly lowered compressive modulus while little affected compressive strength and solidification. MC-PMMA bone cement was biologically safe and indicated excellent bone tissue compatibility. The bone-cement interface crosslinking was significantly higher in MC-PMMA than control after 6 months implantation in the femur of rabbits. The genes of osteogenesis exhibited significantly higher expression level in MC-PMMA. MC-PMMA presented perfect mechanical properties, good biosafety and excellent biocompatibility with bone tissues, which has profoundly clinical values.

Mechanical, thermal, rheological and morphological behaviour of irradiated PP/HA composites

NASA Astrophysics Data System (ADS)

Ramírez, C.; Albano, C.; Karam, A.; Domínguez, N.; Sánchez, Y.; González, G.

2005-07-01

Hydroxyapatite (HA) reinforced polypropylene (PP) composites are being developed as bone graft materials. In this research, the effect of γ irradiation on mechanical, rheological, thermal and morphological behaviour of PP-HA composites was studied. The melt flow index of polymer increased markedly when it was exposed to radiation. This is indicative of chain scission reaction as the predominant process. During the tensile testing, the composites exhibited brittle behaviour, showing no fluency point. Elongation at break showed a tendency to decrease with the increase in radiation dose while stress at break did not show significant variation with radiation dose. High HA content (>20%) and radiation dose (25 kGy) had significant influence on thermal stability.

Electrophoretic deposition of double-layer HA/Al composite coating on NiTi.

PubMed

Karimi, Esmaeil; Khalil-Allafi, Jafar; Khalili, Vida

2016-01-01

In order to improve the bioactivity of NiTi alloys, which are being known as the suitable materials for biomedical applications, numerous NiTi disks were electrophoretically coated by hetero-coagulated hydroxyapatite/aluminum composite coatings in three main voltages from suspensions with different Al concentrations. In this paper, the amount of Ni ions release and bioactivity of prepared samples as well as bonding strength of the coating to substrate were investigated. The surface characterization of the coating by XRD, EDX, SEM, and FTIR showed that HA particles bonded by Al particles. It caused the formation of a free crack coating on NiTi disks. Moreover, the bonding strength of HA/Al coatings to NiTi substrate were improved by two times as compared to that of the pure HA coatings. Immersing of coated samples in SBF for 1 week showed that apatite formation ability was improved on HA/Al composite coating and Ni ions release from the surface of composite coating decreased. These results induce the appropriate bioactivity and biocompatibility of the deposited HA/Al composite coatings on NiTi disks. Copyright © 2015 Elsevier B.V. All rights reserved.

3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity.

PubMed

Poldervaart, Michelle T; Goversen, Birgit; de Ruijter, Mylene; Abbadessa, Anna; Melchels, Ferry P W; Öner, F Cumhur; Dhert, Wouter J A; Vermonden, Tina; Alblas, Jacqueline

2017-01-01

In bone regenerative medicine there is a need for suitable bone substitutes. Hydrogels have excellent biocompatible and biodegradable characteristics, but their visco-elastic properties limit their applicability, especially with respect to 3D bioprinting. In this study, we modified the naturally occurring extracellular matrix glycosaminoglycan hyaluronic acid (HA), in order to yield photo-crosslinkable hydrogels with increased mechanical stiffness and long-term stability, and with minimal decrease in cytocompatibility. Application of these tailor-made methacrylated hyaluronic acid (MeHA) gels for bone tissue engineering and 3D bioprinting was the subject of investigation. Visco-elastic properties of MeHA gels, measured by rheology and dynamic mechanical analysis, showed that irradiation of the hydrogels with UV light led to increased storage moduli and elastic moduli, indicating increasing gel rigidity. Subsequently, human bone marrow derived mesenchymal stromal cells (MSCs) were incorporated into MeHA hydrogels, and cell viability remained 64.4% after 21 days of culture. Osteogenic differentiation of MSCs occurred spontaneously in hydrogels with high concentrations of MeHA polymer, in absence of additional osteogenic stimuli. Addition of bone morphogenetic protein-2 (BMP-2) to the culture medium further increased osteogenic differentiation, as evidenced by increased matrix mineralisation. MeHA hydrogels demonstrated to be suitable for 3D bioprinting, and were printed into porous and anatomically shaped scaffolds. Taken together, photosensitive MeHA-based hydrogels fulfilled our criteria for cellular bioprinted bone constructs within a narrow window of concentration.

3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity

PubMed Central

Poldervaart, Michelle T.; Goversen, Birgit; de Ruijter, Mylene; Abbadessa, Anna; Melchels, Ferry P. W.; Öner, F. Cumhur; Dhert, Wouter J. A.; Vermonden, Tina

2017-01-01

In bone regenerative medicine there is a need for suitable bone substitutes. Hydrogels have excellent biocompatible and biodegradable characteristics, but their visco-elastic properties limit their applicability, especially with respect to 3D bioprinting. In this study, we modified the naturally occurring extracellular matrix glycosaminoglycan hyaluronic acid (HA), in order to yield photo-crosslinkable hydrogels with increased mechanical stiffness and long-term stability, and with minimal decrease in cytocompatibility. Application of these tailor-made methacrylated hyaluronic acid (MeHA) gels for bone tissue engineering and 3D bioprinting was the subject of investigation. Visco-elastic properties of MeHA gels, measured by rheology and dynamic mechanical analysis, showed that irradiation of the hydrogels with UV light led to increased storage moduli and elastic moduli, indicating increasing gel rigidity. Subsequently, human bone marrow derived mesenchymal stromal cells (MSCs) were incorporated into MeHA hydrogels, and cell viability remained 64.4% after 21 days of culture. Osteogenic differentiation of MSCs occurred spontaneously in hydrogels with high concentrations of MeHA polymer, in absence of additional osteogenic stimuli. Addition of bone morphogenetic protein-2 (BMP-2) to the culture medium further increased osteogenic differentiation, as evidenced by increased matrix mineralisation. MeHA hydrogels demonstrated to be suitable for 3D bioprinting, and were printed into porous and anatomically shaped scaffolds. Taken together, photosensitive MeHA-based hydrogels fulfilled our criteria for cellular bioprinted bone constructs within a narrow window of concentration. PMID:28586346

A Modified ELISA Accurately Measures Secretion of High Molecular Weight Hyaluronan (HA) by Graves' Disease Orbital Cells

PubMed Central

Krieger, Christine C.

2014-01-01

Excess production of hyaluronan (hyaluronic acid [HA]) in the retro-orbital space is a major component of Graves' ophthalmopathy, and regulation of HA production by orbital cells is a major research area. In most previous studies, HA was measured by ELISAs that used HA-binding proteins for detection and rooster comb HA as standards. We show that the binding efficiency of HA-binding protein in the ELISA is a function of HA polymer size. Using gel electrophoresis, we show that HA secreted from orbital cells is primarily comprised of polymers more than 500 000. We modified a commercially available ELISA by using 1 million molecular weight HA as standard to accurately measure HA of this size. We demonstrated that IL-1β-stimulated HA secretion is at least 2-fold greater than previously reported, and activation of the TSH receptor by an activating antibody M22 from a patient with Graves' disease led to more than 3-fold increase in HA production in both fibroblasts/preadipocytes and adipocytes. These effects were not consistently detected with the commercial ELISA using rooster comb HA as standard and suggest that fibroblasts/preadipocytes may play a more prominent role in HA remodeling in Graves' ophthalmopathy than previously appreciated. PMID:24302624

Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA.

PubMed

Lam, Kwok-Ho; Sikorra, Stefan; Weisemann, Jasmin; Maatsch, Hannah; Perry, Kay; Rummel, Andreas; Binz, Thomas; Jin, Rongsheng

2018-04-23

The extreme toxicity of botulinum neurotoxins (BoNTs) relies on their specific cleavage of SNARE proteins, which eventually leads to muscle paralysis. One newly identified mosaic toxin, BoNT/HA (aka H or FA), cleaves VAMP-2 at a unique position between residues L54 and E55, but the molecular basis underlying VAMP-2-recognition of BoNT/HA remains poorly characterized. Here, we report a ∼2.09 Å resolution crystal structure of the light chain protease domain of BoNT/HA (LC/HA). Structural comparison between LC/HA and LC of BoNT/F1 (LC/F1) reveals distinctive hydrophobic and electrostatic features near the active sites, which may explain their different VAMP-2 cleavage sites. When compared to BoNT/F5 that cleaves VAMP-2 at the same site as BoNT/HA, LC/HA displays higher affinity for VAMP-2, which could be caused by their different surface charge properties surrounding a VAMP-2 exosite-binding cleft. Furthermore, systematic mutagenesis studies on VAMP-2 and structural modeling demonstrate that residues R47 to K59 spanning the cleavage site in VAMP-2 may adopt a novel extended conformation when interacting with LC/HA and LC/F5. Taken together, our structure provides new insights into substrate-recognition of BoNT/HA and paves the way for rational design of small molecule or peptide inhibitors against LC/HA.

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests

USGS Publications Warehouse

Tsukamoto, K.; Javier, P.C.; Shishido, M.; Noguchi, D.; Pearce, J.; Kang, H.-M.; Jeong, O.M.; Lee, Y.-J.; Nakanishi, K.; Ashizawa, T.

2012-01-01

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10 1.5, 10 2.3, and 10 3.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples. Copyright ?? 2012, American Society for Microbiology. All Rights Reserved.

The first Swedish H1N2 swine influenza virus isolate represents an uncommon reassortant.

PubMed

Bálint, Adám; Metreveli, Giorgi; Widén, Frederik; Zohari, Siamak; Berg, Mikael; Isaksson, Mats; Renström, Lena Hm; Wallgren, Per; Belák, Sándor; Segall, Thomas; Kiss, István

2009-10-28

The European swine influenza viruses (SIVs) show considerable diversity comprising different types of H1N1, H3N2, and H1N2 strains. The intensifying full genome sequencing efforts reveal further reassortants within these subtypes. Here we report the identification of an uncommon reassortant variant of H1N2 subtype influenza virus isolated from a pig in a multisite herd where H1N2 swine influenza was diagnosed for the first time in Sweden during the winter of 2008-2009. The majority of the European H1N2 swine influenza viruses described so far possess haemagglutinin (HA) of the human-like H1N2 SIV viruses and the neuraminidase (NA) of either the European H1N2 or H3N2 SIV-like viruses. The Swedish isolate has an avian-like SIV HA and a H3N2 SIV-like NA, which is phylogenetically more closely related to H3N2 SIV NAs from isolates collected in the early '80s than to the NA of H3N2 origin of the H1N2 viruses isolated during the last decade, as depicted by some German strains, indicative of independent acquisition of the NA genes for these two types of reassortants. The internal genes proved to be entirely of avian-like SIV H1N1 origin. The prevalence of this SIV variant in pig populations needs to be determined, as well as the suitability of the routinely used laboratory reagents to analyze this strain.The description of this H1N2 SIV adds further information to influenza epidemiology and supports the necessity of surveillance for influenza viruses in pigs.

Changes in redox properties of Humic Acid (HA) upon sorption to alumina

NASA Astrophysics Data System (ADS)

Orsetti, Silvia; Haderlein, Stefan B.; Visser, Anna-Neva

2014-05-01

The interaction between humic substances and soil minerals may change important properties and reactivity of the organic matter. In particular, we are interested whether changes in the redox properties of a HA (namely total electron exchange capacity and redox state) occur upon sorption to redox inactive minerals. Sorption of Pahokee Peat humic acid to Al2O3 was studied at pH value of 7.0 in batch experiments, at several HA/oxide ratio. All experiments were conducted in anoxic environment. The required equilibration time was determined by taking aliquots of the suspension at several time intervals and registering the UV-vis spectra of the supernatant; apparent sorption equilibrium (no decrease in UV-vis signal) was achieved after 5 days approximately. Both the suspension (mineral+sorbed HA, plus supernatant) and the supernatant after centrifugation were analyzed using mediated electrochemical techniques, and the electron donating and accepting capacities (EDC and EAC, respectively) were determined. In addition, SUVA was calculated for each batch. These preliminary results show a slight increase in the SUVA of the supernatant upon sorption, which would indicate a preferential sorption of more aliphatic fractions. Interestingly, the total electron exchange capacities (EEC) of the supernatants showed no significant differences to that of the stock HA, whereas the EEC of the whole suspension showed values up to twice the one from the stock HA. The EDC/EAC (which can be interpreted as a measure of the redox state of the sample) also showed same values for stock and supernatants, being the values of the whole suspensions towards the reduced side. Therefore, such preliminary results would indicate not a change in the redox properties of the dissolved HA, but only for the sorbed one. The sorbed fraction seems to present higher redox activity (higher EEC) and a more reduced state than the stock HA. Given the absence of redox transfer between the HA and the oxide, it could

Re-establishment of the IMS Hydroacoustic Station HA04, Crozet Islands, France.

NASA Astrophysics Data System (ADS)

Haralabus, Georgios; Stanley, Jerry; Zampolli, Mario; Grenard, Patrick; Nielsen, Peter; Le Bras, Ronan; Brown, David; Bittner, Paulina; Wang, Haijun; Gore, Jane; Amir, Menachem; Bereza, Slava

2017-04-01

The incorporation of the hydroacoustic station HA04, Crozet Islands, France, into the International Monitoring System (IMS) of the Comprehensive Nuclear-Test-Ban Treaty Organisation (CTBTO) is a 17 year saga that had a happy ending on 29 December 2016. On that day, following a major engineering and logistical undertaking, the station was successfully installed. While still in its initial testing phase, HA04 sends continuously quality data at the International Data Centre (IDC), pending official certification and promotion to mainstream operational status. Similarly to most other cabled hydroacoustic stations in the IMS, HA04 is comprised of two triplets of moored hydrophones deployed on both sides of Possession Island (Crozet Islands) sending uninterrupted data to a shore facility via submarine fiber optic cables. The designed frequency pass-band is 1 - 100 Hz. Data are relayed to Vienna via a shore based satellite link in real time. According to CTBTO's standard requirements, the design life of HA04 is at least 20 years, maintenance-free for the underwater system. An outline of the main phases of this project, highlights from the installation operations and examples of received hydroacoustic signals associated with recent underwater seismic activity in the Indian Ocean as well as vocalizations from marine mammals acquired by the new HA04 are presented here. HA04 is scheduled to be fully integrated into the operational platform of IDC in the next six months, which will enable registered researchers to access archived monitoring data and processing software, or via the National Data Centres (NDCs).

Fractal analysis of polyferric chloride-humic acid (PFC-HA) flocs in different topological spaces.

PubMed

Wang, Yili; Lu, Jia; Baiyu, Du; Shi, Baoyou; Wang, Dongsheng

2009-01-01

The fractal dimensions in different topological spaces of polyferric chloride-humic acid (PFC-HA) flocs, formed in flocculating different kinds of humic acids (HA) water at different initial pH (9.0, 7.0, 5.0) and PFC dosages, were calculated by effective density-maximum diameter, image analysis, and N2 absorption-desorption methods, respectively. The mass fractal dimensions (Df) of PFC-HA flocs were calculated by bi-logarithm relation of effective density with maximum diameter and Logan empirical equation. The Df value was more than 2.0 at initial pH of 7.0, which was 11% and 13% higher than those at pH 9.0 and 5.0, respectively, indicating the most compact flocs formed in flocculated HA water at initial pH of 7.0. The image analysis for those flocs indicates that after flocculating the HA water at initial pH greater than 7.0 with PFC flocculant, the fractal dimensions of D2 (logA vs. logdL) and D3 (logVsphere VS. logdL) of PFC-HA flocs decreased with the increase of PFC dosages, and PFC-HA flocs showed a gradually looser structure. At the optimum dosage of PFC, the D2 (logA vs. logdL) values of the flocs show 14%-43% difference with their corresponding Df, and they even had different tendency with the change of initial pH values. However, the D2 values of the flocs formed at three different initial pH in HA solution had a same tendency with the corresponding Dr. Based on fractal Frenkel-Halsey-Hill (FHH) adsorption and desorption equations, the pore surface fractal dimensions (Ds) for dried powders of PFC-HA flocs formed in HA water with initial pH 9.0 and 7.0 were all close to 2.9421, and the Ds values of flocs formed at initial pH 5.0 were less than 2.3746. It indicated that the pore surface fractal dimensions of PFC-HA flocs dried powder mainly show the irregularity from the mesopore-size distribution and marcopore-size distribution.

HA117 endows HL60 cells with a stem-like signature by inhibiting the degradation of DNMT1 via its ability to down-regulate expression of the GGL domain of RGS6

PubMed Central

Li, Shuangshuang; Wu, Huan; Wang, Yi; Li, Xiaoqing; Guo, Yuxia; Liang, Shaoyan

2017-01-01

All-trans retinoic acid (ATRA) induces complete remission in almost all patients with acute promyelocytic leukemia (APL) via its ability to induce the in vivo differentiation of APL blasts. However, prolonged ATRA treatment can result in drug resistance. In previous studies, we generated a multi-drug-resistant HL60/ATRA cell line and found it to contain a new drug resistance-related gene segment, HA117. In this study, we demonstrate that ATRA induces multi-drug-resistant subpopulations of HL60 cells with a putative stem-like signature by up-regulating the expression of the new gene segment HA117. Western blot analysis and quantitative real-time PCR demonstrated that HA117 causes alternative splicing of regulator of G-protein signaling 6 (RGS6) and down-regulation of the expression of the GGL domain of RGS6, which plays an important role in DNA methyltransferase 1 (DNMT1) degradation. Moreover, DNMT1 expression was increased in multi-drug resistance HL60/ATRA cells. Knockdown of HA117 restored expression of the GGL domain and blocked DNMT1 expression. Moreover, resistant cells displayed a putative stem-like signature with increased expression of cancer steam cell markers CD133 and CD123. The stem cell marker, Nanog, was significantly up-regulated. In conclusion, our study shows that HA117 potentially promotes the stem-like signature of the HL60/ATRA cell line by inhibiting by the ubiquitination and degradation of DNMT1 and by down-regulating the expression of the GGL domain of RGS6. These results throw light on the cellular events associated with the ATRA-induced multi-drug resistance phenotype in acute leukemia. PMID:28665981

75 FR 55323 - Ha-Best, Inc.; Notice of Availability of Environmental Assessment

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-10

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Project No. 12492-001] Ha-Best, Inc.; Notice of Availability of Environmental Assessment August 31, 2010. In accordance with the National... Office of Energy Projects has reviewed Ha-Best's application for license for the Miner Shoal Waterpower...

Pathogen evolution and disease emergence in carnivores.

PubMed

McCarthy, Alex J; Shaw, Marie-Anne; Goodman, Simon J

2007-12-22

Emerging infectious diseases constitute some of the most pressing problems for both human and domestic animal health, and biodiversity conservation. Currently it is not clear whether the removal of past constraints on geographical distribution and transmission possibilities for pathogens alone are sufficient to give rise to novel host-pathogen combinations, or whether pathogen evolution is also generally required for establishment in novel hosts. Canine distemper virus (CDV) is a morbillivirus that is prevalent in the world dog population and poses an important conservation threat to a diverse range of carnivores. We performed an extensive phylogenetic and molecular evolution analysis on complete sequences of all CDV genes to assess the role of selection and recombination in shaping viral genetic diversity and driving the emergence of CDV in non-dog hosts. We tested the specific hypothesis that molecular adaptation at known receptor-binding sites of the haemagglutinin gene is associated with independent instances of the spread of CDV to novel non-dog hosts in the wild. This hypothesis was upheld, providing compelling evidence that repeated evolution at known functional sites (in this case residues 530 and 549 of the haemagglutinin molecule) is associated with multiple independent occurrences of disease emergence in a range of novel host species.

Safety and persistence of the humoral and cellular immune responses induced by 2 doses of an AS03-adjuvanted A(H1N1)pdm09 pandemic influenza vaccine administered to infants, children and adolescents: Two open, uncontrolled studies.

PubMed

Garcia-Sicilia, José; Arístegui, Javier; Omeñaca, Félix; Carmona, Alfonso; Tejedor, Juan C; Merino, José M; García-Corbeira, Pilar; Walravens, Karl; Bambure, Vinod; Moris, Philippe; Caplanusi, Adrian; Gillard, Paul; Dieussaert, Ilse

2015-01-01

In children, 2 AS03-adjuvanted A(H1N1)pdm09 vaccine doses given 21 days apart were previously shown to induce a high humoral immune response and to have an acceptable safety profile up to 42 days following the first vaccination. Here, we analyzed the persistence data from 2 open-label studies, which assessed the safety, and humoral and cell-mediated immune responses induced by 2 doses of this vaccine. The first study was a phase II, randomized trial conducted in 104 children aged 6-35 months vaccinated with the A(H1N1)pdm09 vaccine containing 1.9 µg haemagglutinin antigen (HA) and AS03B (5.93 mg tocopherol) and the second study, a phase III, non-randomized trial conducted in 210 children and adolescents aged 3-17 years vaccinated with the A(H1N1)pdm09 vaccine containing 3.75 µg HA and AS03A (11.86 mg tocopherol). Approximately one year after the first dose, all children with available data were seropositive for haemagglutinin inhibition and neutralising antibody titres, but a decline in geometric mean antibody titres was noted. The vaccine induced a cell-mediated immune response in terms of antigen-specific CD4(+) T-cells, which persisted up to one year post-vaccination. The vaccine did not raise any safety concern, though these trials were not designed to detect rare events. In conclusion, 2 doses of the AS03-adjuvanted A(H1N1)pdm09 vaccine at 2 different dosages had a clinically acceptable safety profile, and induced high and persistent humoral and cell-mediated immune responses in children aged 6-35 months and 3-17 years. These studies have been registered at www.clinicaltrials.gov NCT00971321 and NCT00964158.

Influence of the concentrations of hyaluronic acid on the properties and biocompatibility of Cs-Gel-HA membranes.

PubMed

Liu, Haifeng; Yin, Yuji; Yao, Kangde; Ma, Dongrui; Cui, Lei; Cao, Yilin

2004-08-01

The object of this study was to investigate the relationship between the concentrations of HA solutions and the physicochemical properties and the biocompatibility of Cs-Gel-HA membranes. The addition of different concentrations of HA not only improved the wettability significantly and extended the degradation time of Cs-Gel-HA membranes, but also changed their mechanical properties. The concentration of HA had a significant influence on the biocompatibility of Cs-Gel-HA membranes. Results demonstrated that it was only the concentrations of HA in a certain range (0.01-0.1%), that could promote the cell adhesion, migration and proliferation, while the concentration of HA was above 0.1% it would either reduce or even inhibit these behaviors.

Masking of antigenic epitopes by antibodies shapes the humoral immune response to influenza

PubMed Central

Zarnitsyna, Veronika I.; Ellebedy, Ali H.; Davis, Carl; Jacob, Joshy; Ahmed, Rafi; Antia, Rustom

2015-01-01

The immune responses to influenza, a virus that exhibits strain variation, show complex dynamics where prior immunity shapes the response to the subsequent infecting strains. Original antigenic sin (OAS) describes the observation that antibodies to the first encountered influenza strain, specifically antibodies to the epitopes on the head of influenza's main surface glycoprotein, haemagglutinin (HA), dominate following infection with new drifted strains. OAS suggests that responses to the original strain are preferentially boosted. Recent studies also show limited boosting of the antibodies to conserved epitopes on the stem of HA, which are attractive targets for a ‘universal vaccine’. We develop multi-epitope models to explore how pre-existing immunity modulates the immune response to new strains following immunization. Our models suggest that the masking of antigenic epitopes by antibodies may play an important role in describing the complex dynamics of OAS and limited boosting of antibodies to the stem of HA. Analysis of recently published data confirms model predictions for how pre-existing antibodies to an epitope on HA decrease the magnitude of boosting of the antibody response to this epitope following immunization. We explore strategies for boosting of antibodies to conserved epitopes and generating broadly protective immunity to multiple strains. PMID:26194761

The duration of hyaluronidase and optimal timing of hyaluronic acid (HA) filler reinjection after hyaluronidase injection.

PubMed

Kim, H J; Kwon, S B; Whang, K U; Lee, J S; Park, Y L; Lee, S Y

2018-02-01

Hyaluronidase injection is a commonly performed treatment for overcorrection or misplacement of hyaluronic acid (HA) filler. Many patients often wants the HA filler reinjection after the use of hyaluronidase, though the optimal timing of reinjection of HA filler still remains unknown. To provide the optimal time interval between hyaluronidase injections and HA filler reinjections. 6 Sprague-Dawley rats were injected with single monophasic HA filler. 1 week after injection, the injected sites were treated with hyaluronidase. Then, HA fillers were reinjected sequentially with differing time intervals from 30 minutes to 14 days. 1 hour after the reinjection of the last HA filler, all injection sites were excised for histologic evaluation. 3 hours after reinjection of HA filler, the appearance of filler material became evident again, retaining its shape and volume. 6 hours after reinjection, the filler materials restored almost its original volume and there were no significant differences from the positive control. Our data suggest that the hyaluronidase loses its effect in dermis and subcutaneous tissue within 3-6 hours after the injection and successful engraftment of reinjected HA filler can be accomplished 6 hours after the injection.

Long-term variation in influenza A virus prevalence and subtype diversity in migratory mallards in northern Europe

PubMed Central

Latorre-Margalef, Neus; Tolf, Conny; Grosbois, Vladimir; Avril, Alexis; Bengtsson, Daniel; Wille, Michelle; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.; Olsen, Björn; Waldenström, Jonas

2014-01-01

Data on long-term circulation of pathogens in wildlife populations are seldom collected, and hence understanding of spatial–temporal variation in prevalence and genotypes is limited. Here, we analysed a long-term surveillance series on influenza A virus (IAV) in mallards collected at an important migratory stopover site from 2002 to 2010, and characterized seasonal dynamics in virus prevalence and subtype diversity. Prevalence dynamics were influenced by year, but retained a common pattern for all years whereby prevalence was low in spring and summer, but increased in early autumn with a first peak in August, and a second more pronounced peak during October–November. A total of 74 haemagglutinin (HA)/neuraminidase (NA) combinations were isolated, including all NA and most HA (H1–H12) subtypes. The most common subtype combinations were H4N6, H1N1, H2N3, H5N2, H6N2 and H11N9, and showed a clear linkage between specific HA and NA subtypes. Furthermore, there was a temporal structuring of subtypes within seasons based on HA phylogenetic relatedness. Dissimilar HA subtypes tended to have different temporal occurrence within seasons, where the subtypes that dominated in early autumn were rare in late autumn, and vice versa. This suggests that build-up of herd immunity affected IAV dynamics in this system. PMID:24573857

[Long distance-PCR for detection of factor VIII gene inversion in patients with severe hemophilia A].

PubMed

Ding, Pei-Fang; Sun, Wei-Sheng; Wang, Qin-You; Liu, De-Chun; Zhang, Xue-Qin; Teng, Bin; Shen, Fa-Kui

2003-08-01

The aim of current study was to detect intron 22 inversion of factor VIII gene in severe hemophilia A (HA) patients and screen the carriers of the gene inversion. Fifty-five cases of severe HA were involved and factor VIII gene inversion was detected and identified by long distance-PCR (LD-PCR) and 0.6% agarose gel electrophoresis. The 11 kb and 12 kb bands indicate the factor VIII gene inversion and non-inversion, respectively. Occurring of both 11 kb and 12 kb bands indicates a carrier of the inversion. The results showed that factor VIII gene inversion existed in 22 out of 55 cases, which accounted for about 40% of total detected patients. Five carriers of factor VIII gene inversion were diagnosed from the members in 15 families. In conclusion, LD-PCR assay is a simple, rapid and accurate method for detection of factor VIII gene inversion, and this approach is helpful in screening, carrier testing, and prenatal diagnosis of severe hemophilia A.

The influence of silanisation on the mechanical and degradation behaviour of PLGA/HA composites.

PubMed

Naik, Ashutosh; Best, Serena M; Cameron, Ruth E

2015-03-01

This study investigates the influence of silanisation on the mechanical and degradation behaviour of PLGA/HA composites. Three different silanes (mercaptopropyl trimethoxy silane (MPTMS), aminopropyl trimethoxy silane (APTMS) and aminopropyltriethoxy silane (APTES)) were applied to HA substrates in order to study the effect of head group (which binds to the polymer) and tail group (which binds to the surface hydroxyl groups in HA). A composite of hydroxyapatite (HA) and poly(d,l lactide-co-glycolide (50:50)) (PLGA) was investigated. The influence of concentration, the reaction time, drying temperature and substrate surface on silanisation was examined. TGA was used to detect the degree of silanisation. HA with MPTMS (1wt.% MPTMS with reaction time of 1h) was used as filler in PLGA-30wt.% HA composites for an in-vitro degradation study carried out in PBS. In addition, the mechanical properties of the composites were studied. Silanisation affects the properties of the composite by improving the bonding at the interface and hence it was found to influence the plastic mechanical properties rather than the elastic mechanical properties or the degradation profile of the composite. Copyright © 2014. Published by Elsevier B.V.

The enhanced anti-tissue adhesive effect of injectable pluronic-HA hydrogel by poly(γ-glutamic acid).

PubMed

Kim, Manse; Hwang, Youngmin; Tae, Giyoong

2016-12-01

The stability of tissue barrier in physiological condition is a key factor to isolate the damaged site from adjacent tissue for anti-tissue adhesion. Although pluronic or pluronic-hyaluronic acid (HA) hydrogel as an injectable formulation can prevent tissue adhesion at the injection site, the anti-tissue adhesion effect is limited due to its poor stability. Herein, we prepared tissue barrier formulations composed of pluronic F127 (F127) and HA mixture (F127-HA) and the effect of the addition of poly(γ-glutamic acid) (PGA) was characterized. All of F127, HA, and F127-HA mixture showed the poor in vitro residence stability less than 3 days. However, by adding PGA into F127-HA mixture, their stability was significantly enhanced by the control of the molecular weight and concentration of PGA. Thus, F127-HA with 10wt% PGA (2000kDa) showed the long-term stability over 10 days. Similarly, the enhanced stability of F127-HA with PGA resulted in the enhanced and excellent in vivo anti-tissue adhesion effect, evidenced by histological analysis and grading of tissue adhesion. Therefore, F127-HA containing PGA could be applied as an efficient injectable tissue barrier for anti-tissue adhesion. Copyright © 2016 Elsevier B.V. All rights reserved.

Green synthesis of nanosilver particles by Aspergillus terreus HA1N and Penicillium expansum HA2N and its antifungal activity against mycotoxigenic fungi.

PubMed

Ammar, H A M; El-Desouky, T A

2016-07-01

The aim of this study was to synthesize silver nanoparticles (AgNPs) by an eco-friendly and low-cost method using the fungi Aspergillus terreus HA1N and Penicillium expansum HA2N as an alternative to chemical procedures mostly requiring drastic experimental conditions emitting toxic chemical byproducts. Also, this study has been extended to evaluate the effect of AgNPs on the growth of some mycotoxigenic fungi and ochratoxin A (OTA) produced by Aspergillus ochraceus. The AgNPs have been characterized by UV-Visible Spectrophotometer, Dynamic Light Scattering (DLS), Fourier Transform Infrared Spectroscopy (FTIR) and Transmission Electron Microscope (TEM). The TEM analysis has revealed that the size of AgNPs ranged between 14 and 25 nm in the case of P. expansum and 10-18 nm in the case of A. terreus. The antifungal activity of AgNP colloids has indicated that the highest inhibition zone was detected with AgNPs synthesized by A. terreus HA1N against all tested fungi. The highest inhibition zone was detected with Aspergillus niger at concentrations 3 and 6 μg of AgNP solution (7·56 ± 0·38 and 11·3 ± 1·8 mm, respectively) while, A. ochraceus showed the maximum inhibition zone (16·33 ± 0·96 mm) at the concentration 9 μg of AgNPs synthesized by A. terreus. The results have also indicated that the AgNPs synthesized by A. terreus and P. expansum at the concentration 220 μg/100 ml media gave the highest reduction of OTA, where the percentages of reduction were 58·87 and 52·18% respectively. The smallest size AgNPs synthesized by A. terreus HA1N are better in their antifungal activity against all tested mycotoxigenic fungi than the largest one synthesized by P. expansum HA2N. This is the first study focused on using AgNPs in control of OTA production. © 2016 The Society for Applied Microbiology.

A Modified Delphi Study to Define "Ah Ha" Moments in Education Settings

ERIC Educational Resources Information Center

Pilcher, Jobeth

2015-01-01

Ah ha moments are often mentioned in education literature. These moments are suggested to be a powerful aspect of learning, yet limited research is present regarding this topic. Ah ha learning moments have also not been defined in the education literature, resulting in the likelihood that each educator and learner may have differing definitions.…

Pl(17) is a novel gene independent of known downy mildew resistance genes in the cultivated sunflower (Helianthus annuus L.).

PubMed

Qi, L L; Long, Y M; Jan, C C; Ma, G J; Gulya, T J

2015-04-01

Pl 17, a novel downy mildew resistance gene independent of known downy mildew resistance genes in sunflowers, was genetically mapped to linkage group 4 of the sunflower genome. Downy mildew (DM), caused by Plasmopara halstedii (Farl.). Berl. et de Toni, is one of the serious sunflower diseases in the world due to its high virulence and the variability of the pathogen. DM resistance in the USDA inbred line, HA 458, has been shown to be effective against all virulent races of P. halstedii currently identified in the USA. To determine the chromosomal location of this resistance, 186 F 2:3 families derived from a cross of HA 458 with HA 234 were phenotyped for their resistance to race 734 of P. halstedii. The segregation ratio of the population supported that the resistance was controlled by a single dominant gene, Pl 17. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) primers were used to identify molecular markers linked to Pl 17. Bulked segregant analysis using 849 SSR markers located Pl 17 to linkage group (LG) 4, which is the first DM gene discovered in this linkage group. An F2 population of 186 individuals was screened with polymorphic SSR and SNP primers from LG4. Two flanking markers, SNP SFW04052 and SSR ORS963, delineated Pl 17 in an interval of 3.0 cM. The markers linked to Pl 17 were validated in a BC3 population. A search for the physical location of flanking markers in sunflower genome sequences revealed that the Pl 17 region had a recombination frequency of 0.59 Mb/cM, which was a fourfold higher recombination rate relative to the genomic average. This region can be considered amenable to molecular manipulation for further map-based cloning of Pl 17.

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion.

PubMed

López, Jesús Adrián; Alvarez-Salas, Luis Marat

2011-06-10

MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology. Copyright © 2011 Elsevier Inc. All rights reserved.

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion

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

Lopez, Jesus Adrian; Alvarez-Salas, Luis Marat, E-mail: lalvarez@cinvestav.mx

Highlights: {yields} In this study we examine miR-34c-3p and miR-34c-5p functions in SiHa cells. {yields} We study miRNA effect on cell proliferation, anchorage independent growth, apoptosis, cell motility and invasion. {yields} We find that miR-34c-3p and miR-34c-5p inhibition of proliferation and anchorage independent growth are exclusive to SiHa cells. {yields} miR-34c-3p induces apoptosis and inhibits cell motility and invasion in SiHa cells. {yields} In this study we conclude that miR-34c-3p functions as a tumor suppressor differ from miR-34c-5p. -- Abstract: MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effectormore » of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology.« less

In vitro modulation of the interaction between HA95 and LAP2beta by cAMP signaling.

PubMed

Martins, Sandra B; Marstad, Anne; Collas, Philippe

2003-09-09

The nuclear envelope mediates key functions by interacting with chromatin. We recently reported an interaction between the chromatin- and nuclear matrix-associated protein HA95 and the inner nuclear membrane integral protein LAP2beta, implicated in initiation of DNA replication (Martins et al. (2003) J. Cell Biol. 160, 177-188). Here, we show that in vitro, interaction between HA95 and LAP2beta is modulated by cAMP signaling via PKA. Exposure of an anti-HA95 immune precipitate from interphase HeLa cells to a mitotic extract promotes ATP-dependent release of LAP2beta from the HA95 complex. This coincides with Ser and Thr phosphorylation of HA95 and LAP2beta. Inhibition of PKA with PKI abolishes phosphorylation of HA95 and dissociation of LAP2beta from HA95, although LAPbeta remains phosphorylated. Antagonizing cAMP signaling in mitotic extract also abolishes the release of LAP2beta from HA95; however, disrupting PKA anchoring to A-kinase anchoring proteins has no effect. Inhibition of CDK activity in the extract greatly reduces LAP2beta phosphorylation but does not prevent LAP2beta release from HA95. Inhibition of PKC, MAP kinase, or CaM kinase II does not affect mitotic extract-induced dissociation of LAP2beta from HA95. PKA phosphorylates HA95 but not LAP2beta in vitro and elicits a release of LAP2beta from HA95. CDK1 or PKC phosphorylates LAP2beta within the HA95 complex, but neither kinase induces LAP2beta release. Our results indicate that in vitro, the interaction between HA95 and LAP2beta is influenced by a PKA-mediated phosphorylation of HA95 rather than by CDK1- or PKC-mediated phosphorylation of LAP2beta. This suggests an additional level of regulation of a chromatin-nuclear envelope interaction in dividing cells.

High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, R 15 , in sunflower (Helianthus annuus L.).

PubMed

Ma, G J; Song, Q J; Markell, S G; Qi, L L

2018-07-01

A novel rust resistance gene, R 15 , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R 15 were identified, facilitating marker-assisted selection of resistance genes. The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F 2 individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R 15 ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R 15 was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

Magneto-optical and catalytic properties of Fe3O4@HA@Ag magnetic nanocomposite

NASA Astrophysics Data System (ADS)

Amir, Md.; Güner, S.; Yıldız, A.; Baykal, A.

2017-01-01

Fe3O4@HA@Ag magnetic nanocomposites (MNCs) were successfully synthesized by the simple reflux method for the removal of azo dyes from the industrial aqueous media. Fe3O4@HA@AgMNCs exhibited high catalytic activity to reduce MB within 20 min from the waste water. The obtained materials were characterized by the means of different techniques. Powder X-ray diffraction (XRD) analysis confirmed the single-phase of Fe3O4 spinel structure. SEM and TEM analysis indicated that Fe3O4@HA@AgMNCs were nanoparticles like structure with small agglomeration. TG result showed that the products contained 9% of HA. The characteristic peaks of HA at 1601 cm-1 and 1703 cm-1 was observed by the means of FT-IR spectra of Fe3O4@HA@AgMNCs. The hysteresis (σ-H) curves revealed Fe3O4@HA@Ag MNCs exhibit a typical superparamagnetic characteristic with a saturation magnetization of 59.11 emu/g and measured magnetic moment is 2.45 μB. The average magnetic particle dimension (Dmag) is 13.25 nm. In accordance, the average crystallite and particle dimensions were obtained as 11.50 nm and 13.10 nm from XRD and TEM measurements, respectively. Magnetocrystalline anisotropy was offered as uniaxial and calculated effective anisotropy constant (Keff) is 2.96×105 Erg/g. The blocking temperature was estimated as 522 K. The size-dependent saturation magnetization suggests the existence of a magnetically dead layer as 0.793 nm for Fe3O4@HA@Ag MNCs. The UV-vis diffuse reflectance spectroscopy (DRS) and Kubelka-Munk theory were applied to determine the optical properties of powder samples. The direct optical energy band gap (Eg) values were estimated from Tauc plots between 1.62 eV and 2.12 eV.

Evaluation of nanohydroxyapaptite (nano-HA) coated epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes.

PubMed

Chu, Chenyu; Deng, Jia; Man, Yi; Qu, Yili

2017-09-01

Collagen is the main component of extracellular matrix (ECM) with desirable biological activities and low antigenicity. Collagen materials have been widely utilized in guided bone regeneration (GBR) surgery due to its abilities to maintain space for hard tissue growth. However, pure collagen lacks optimal mechanical properties. In our previous study, epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes, with better biological activities and enhanced mechanical properties, may promote osteoblast proliferation, but their effect on osteoblast differentiation is not very significant. Nanohydroxyapatite (nano-HA) is the main component of mineral bone, which possesses exceptional bioactivity properties including good biocompatibility, high osteoconductivity and osteoinductivity, non-immunogenicity and non-inflammatory behavior. Herein, by analyzing the physical and chemical properties as well as the effects on promoting bone regeneration, we have attempted to present a novel EGCG-modified collagen membrane with nano-HA coating, and have found evidence that the novel collagen membrane may promote bone regeneration with a better surface morphology, without destroying collagen backbone. To evaluate the surface morphologies, chemical and mechanical properties of pure collagen membranes, epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes, nano-HA coated collagen membranes, nano-HA coated EGCG-collagen membranes, (ii) to evaluate the bone regeneration promoted by theses membranes. In the present study, collagen membranes were divided into 4 groups: (1) untreated collagen membranes (2) EGCG cross-linked collagen membranes (3) nano-HA modified collagen membranes (4) nano-HA modified EGCG-collagen membranes. Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) were used to evaluate surface morphologies and chemical properties, respectively. Mechanical properties were determined by differential scanning calorimeter (DSC

Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds

PubMed Central

Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

2006-01-01

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2 × 108 pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P > 0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P = 0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting

Adenoviral gene delivery to primary human cutaneous cells and burn wounds.

PubMed

Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

2006-01-01

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has

Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

PubMed

Persson, Maria; Lorite, Gabriela S; Kokkonen, Hanna E; Cho, Sung-Woo; Lehenkari, Petri P; Skrifvars, Mikael; Tuukkanen, Juha

2014-09-01

The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Increasing vitamin A in post-weaning diets reduces food intake and body weight and modifies gene expression in brains of male rats born to dams fed a high multivitamin diet.

PubMed

Sánchez-Hernández, Diana; Cho, Clara E; Kubant, Ruslan; Reza-López, Sandra A; Poon, Abraham N; Wang, Jingzhou; Huot, Pedro S P; Smith, Christopher E; Anderson, G Harvey

2014-10-01

High multivitamin gestational diets (HV, 10-fold AIN-93G levels) increase body weight (BW) and food intake (FI) in rat offspring weaned to a recommended multivitamin (RV), but not to a HV diet. We hypothesized that high vitamin A (HA) alone, similar to HV, in post-weaning diets would prevent these effects of the HV maternal diet consistent with gene expression in FI and reward pathways. Male offspring from dams fed HV diets were weaned to a high vitamin A (HA, 10-fold AIN-93G levels), HV or RV diet for 29 weeks. BW, FI, expression of genes involved in regulation of FI and reward and global and gene-specific DNA methylation of pro-opiomelanocortin (POMC) in the hypothalamus were measured. Both HV and HA diets slowed post-weaning weight gain and modified gene expression in offspring compared to offspring fed an RV post-weaning diet. Hypothalamic POMC expression in HA offspring was not different from either HV or RV, and dopamine receptor 1 was 30% (P<.05) higher in HA vs. HV, but not different from RV group. Hippocampal expression of serotonin receptor 1A (40%, P<.01), dopamine receptor 2 (40%, P<.05) and dopamine receptor 5 (70%, P<.0001) was greater in HA vs. RV fed pups and is 40% (P<.01), 50% (P<.05) and 40% (P<.0001) in HA vs. HV pups, respectively. POMC DNA methylation was lower in HA vs. RV offspring (P<.05). We conclude that high vitamin A in post-weaning diets reduces post-weaning weight gain and FI and modifies gene expression in FI and reward pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

[Risk related to the transmission of H5N1 subtype avian influenza virus in the environment of poultry markets in Changsha, China].

PubMed

Zhang, Ru-sheng; Ou, Xin-hua; Song, Ke-yun; Yuan, Jie; Chen, Tian-mu; Xiao, Shan; Sun, Bian-cheng

2012-08-01

To investigate the risk of H5N1 subtype avian influenza virus (AIV) transmission in the poultry market environment in Changsha city. H5N1 antibody levels among the groups related occupational exposure and AIV nucleic acid in the environment of poultry markets were detected. The characteristics of haemagglutinin (HA) genes of H5N1 AIV in the environment were analyzed. One district and one county from Changsha city were selected randomly and two poultry markets at inner city or township levels were selected in the same district or county respectively. H5N1 antibody of the occupational exposure groups in the poultry market was tested and AIV nucleic acid in the poultry market environment monitored. One hundred and two blood samples of the occupational exposure groups were tested for H5N1 antibody with single radioimmunoassay diffusion hemolysis (SRH) while 160 environment samples (from sewage, birds stools, feathers and smearing samples of poultry cages) in the poultry market were also detected for AIV nucleic acid with real-time PCR method. Four sewage samples of H5N1 subtype AIV were collected from poultry markets in Changsha, and the HA genes of H5N1 subtype AIV amplified by RT-PCR and then sequenced with TA cloning. Amino acid sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega 5.0 software. The results through H5N1 antibody monitoring program showed that H5N1 antibody positive rates from workers were 25.5% (26/102), 50.0% (9/18) and 25.4% (17/67) respectively in the poultry markets of township and inner cities. H5N1 antibody positive rate in the township poultry markets was higher than in the inner cities poultry markets. from the surveillance on AIV nucleic acid showed that the overall H5 subtype positive rate in Changsha poultry markets was 31.3% (50/160), and the positive rate of townships poultry markets was 37.3% (31/83), which were both higher than those from the inner cities poultry markets (24.7%, 19/77). H5 subtype AIV

The use of acetone to enhance the infiltration of HA nanoparticles into a demineralized dentin collagen matrix.

PubMed

Besinis, Alexandros; van Noort, Richard; Martin, Nicolas

2016-03-01

This study investigates the role of acetone, as a carrier for nano-hydroxyapatite (nano-HA) in solution, to enhance the infiltration of fully demineralized dentin with HA nanoparticles (NPs). Dentin specimens were fully demineralized and subsequently infiltrated with two types of water-based nano-HA solutions (one containing acetone and one without). Characterization of the dentin surfaces and nano-HA particles was performed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface wettability and infiltration capacity of the nano-HA solutions were quantified by means of contact angle measurements and energy dispersive X-ray spectroscopy (EDS), respectively. Contact angle measurements were taken at baseline and repeated at regular intervals to assess the effect of acetone. The P and Ca levels of infiltrated dentin specimens were measured and compared to sound dentin and non-infiltrated controls. The presence of acetone resulted in an eight-fold decrease in the contact angles of the nano-HA solutions recorded on the surface of demineralized dentin compared to nano-HA solutions without acetone (one-way ANOVA, p<0.05). Perfect wetting of the demineralized dentin surface was achieved 5min after the application of the nano-HA solution containing acetone. Infiltration of demineralized dentin with the nano-HA solution containing acetone restored the lost mineral content by 50%, whereas the mean mineralization values for P and Ca in dentin treated with the acetone-free nano-HA solution were less than 6%. Acetone was shown to act as a vehicle to enhance the capacity to infiltrate demineralized dentin with HA NPs. The successful infiltration of dentin collagen with HA NPs provides a suitable scaffold, whereby the infiltrated HA NPs have the potential to act as seeds that may initiate heterogenous mineral growth when exposed to an appropriate mineral-rich environment. Copyright © 2015 Academy of Dental Materials. Published by Elsevier

Regulated release of a novel non-viral gene delivery vector from electrospun coaxial fiber mesh scaffolds

NASA Astrophysics Data System (ADS)

Saraf, Anita

The development of novel strategies for tissue engineering entails the evolution of biopolymers into multifunctional constructs that can support the proliferation of cells and stimulate their differentiation into functional tissues. With that in mind, biocompatible polymers were fabricated into a novel gene delivery agent as well as three dimensional scaffolds that act as reservoirs and controlled release constructs. To fabricate a novel gene delivery agent a commercially available cationic polymer, poly(ethylenimine), PEI, was chemically conjugated to a ubiquitous glycosaminoglycan, hyaluronic acid (HA). The novel polymer, PEI-HA, had significantly reduced toxicity and improved transfection efficiency with multipotent human mesenchymal stem cells. This transfection efficiency could further be modulated by changing the concentration of sodium chloride and temperature used to assemble PEI-HA/DNA complexes. To facilitate the regulated delivery of these complexes in the context of tissue engineering, an emerging technology for scaffold fabrication, coaxial electrospinning was adapted to include PEI-HA and plasmid DNA within the scaffold fibers. Initially, a factorial design was employed to assess the influence of processing parameters in the absence of gene delivery vectors and plasmids. The study elucidated the role of sheath polymer concentration and core polymer concentration and molecular weight and the presence of sodium chloride on fiber diameters and morphologies. Subsequently, PEI-HA and plasmid DNA were entrapped within the sheath and core compartments of these fibers and the influence of processing parameters was assessed in the context of fiber diameter, release kinetics and transfection efficiency over a period of 60 days. The release of PEI-HA was found to be dependent upon the loading dose of the vector and plasmid. However, the transfection efficiency correlated to the core polymer properties, concentration and molecular weight. The processing

Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes

PubMed Central

2013-01-01

Background Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes. Results We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs). Conclusions Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3. PMID:24001316

Crystallization and preliminary X-ray analysis of the HA3 component of Clostridium botulinum type C progenitor toxin

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

Nakamura, Toshio; Tonozuka, Takashi; Kotani, Mao

2007-12-01

HA3, a 70 kDa haemagglutinating protein, is a precursor form of HA3a and HA3b, the subcomponents of Clostridium botulinum type C 16S progenitor toxin. In this report, recombinant HA3 protein was overexpressed in Escherichia coli, purified and crystallized. HA3, a 70 kDa haemagglutinating protein, is a precursor form of HA3a and HA3b, the subcomponents of Clostridium botulinum type C 16S progenitor toxin. In this report, recombinant HA3 protein was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.6 Å resolution and the crystal belonged to the hexagonal space group P6{sub 3}. Matthews coefficient and self-rotation functionmore » calculations indicate that there is probably one molecule of HA3 in the asymmetric unit. A search for heavy-atom derivatives has been undertaken.« less

24 CFR 882.805 - HA application process, ACC execution, and pre-rehabilitation activities.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false HA application process, ACC... § 882.805 HA application process, ACC execution, and pre-rehabilitation activities. (a) Review. When... applications in accordance with the guidelines, rating criteria, and procedures published in the NOFA. (b) ACC...

Precision Extruding Deposition for Freeform Fabrication of PCL and PCL-HA Tissue Scaffolds

NASA Astrophysics Data System (ADS)

Shor, L.; Yildirim, E. D.; Güçeri, S.; Sun, W.

Computer-aided tissue engineering approach was used to develop a novel Precision Extrusion Deposition (PED) process to directly fabricate Polycaprolactone (PCL) and composite PCL/Hydroxyapatite (PCL-HA) tissue scaffolds. The process optimization was carried out to fabricate both PCL and PCL-HA (25% concentration by weight of HA) with a controlled pore size and internal pore structure of the 0°/90° pattern. Two groups of scaffolds having 60 and 70% porosity and with pore sizes of 450 and 750 microns, respectively, were evaluated for their morphology and compressive properties using Scanning Electron Microscopy (SEM) and mechanical testing. The surface modification with plasma was conducted on PCL scaffold to increase the cellular attachment and proliferation. Our results suggested that inclusion of HA significantly increased the compressive modulus from 59 to 84 MPa for 60% porous scaffolds and from 30 to 76 MPa for 70% porous scaffolds. In vitro cell-scaffolds interaction study was carried out using primary fetal bovine osteoblasts to assess the feasibility of scaffolds for bone tissue engineering application. In addition, the results in surface hydrophilicity and roughness show that plasma surface modification can increase the hydrophilicity while introducing the nano-scale surface roughness on PCL surface. The cell proliferation and differentiation were calculated by Alamar Blue assay and by determining alkaline phosphatase activity. The osteoblasts were able to migrate and proliferate over the cultured time for both PCL as well as PCL-HA scaffolds. Our study demonstrated the viability of the PED process to the fabricate PCL and PCL-HA composite scaffolds having necessary mechanical property, structural integrity, controlled pore size and pore interconnectivity desired for bone tissue engineering.

Influence of temperature and aging time on HA synthesized by the hydrothermal method.

PubMed

Kothapalli, C R; Wei, M; Legeros, R Z; Shaw, M T

2005-05-01

The influence of temperature and aging time on the morphology and mechanical properties of nano-sized hydroxyapatite (HA) synthesized by a hydrothermal method is reported here. The pre-mixed reactants were poured into a stirred autoclave and reacted at temperatures between 25-250 degrees C for 2-10 h. HA powders thus obtained were examined using X-ray diffraction (XRD), high-resolution field emission scanning electron microscopy (FESEM) and a particle size analyzer. It was found that the aspect ratio of the particles increased with the reaction temperature. The length of the HA particles increased with the reaction temperature below 170 degrees C, but it decreased when the temperature was raised above 170 degrees C. The agglomerates of HA particles were formed during synthesis, and their sizes were strongly dependent on reaction temperatures. As the reaction temperature increased, the agglomerate size decreased (p = 0.008). The density of the discs pressed from these samples reached 85-90% of the theoretical density after sintering at 1200 degrees C for 1 h. No decomposition to other calcium phosphates was detected at this sintering temperature. A correlation existed (p = 0.05) between the agglomerate sizes of HA particles synthesized at various conditions and their sintered densities. With the increase of the agglomerate size, the sintered density of the HA compact decreased. It was found that both the sintered density and flexural strength increased with increasing aging time and reaction temperature. A maximum flexural strength of 78 MPa was observed for the samples synthesized at 170 degrees C for 5 h with the predicted average at these conditions being 65 MPa. These samples attained an average sintered density of 88%.

Microstructure and mechanical properties of plasma sprayed HA/YSZ/Ti-6Al-4V composite coatings.

PubMed

Khor, K A; Gu, Y W; Pan, D; Cheang, P

2004-08-01

Plasma sprayed hydroxyapatite (HA) coatings on titanium alloy substrate have been used extensively due to their excellent biocompatibility and osteoconductivity. However, the erratic bond strength between HA and Ti alloy has raised concern over the long-term reliability of the implant. In this paper, HA/yttria stabilized zirconia (YSZ)/Ti-6Al-4V composite coatings that possess superior mechanical properties to conventional plasma sprayed HA coatings were developed. Ti-6Al-4V powders coated with fine YSZ and HA particles were prepared through a unique ceramic slurry mixing method. The so-formed composite powder was employed as feedstock for plasma spraying of the HA/YSZ/Ti-6Al-4V coatings. The influence of net plasma energy, plasma spray standoff distance, and post-spray heat treatment on microstructure, phase composition and mechanical properties were investigated. Results showed that coatings prepared with the optimum plasma sprayed condition showed a well-defined splat structure. HA/YSZ/Ti-6Al-4V solid solution was formed during plasma spraying which was beneficial for the improvement of mechanical properties. There was no evidence of Ti oxidation from the successful processing of YSZ and HA coated Ti-6Al-4V composite powders. Small amount of CaO apart from HA, ZrO(2) and Ti was present in the composite coatings. The microhardness, Young's modulus, fracture toughness, and bond strength increased significantly with the addition of YSZ. Post-spray heat treatment at 600 degrees C and 700 degrees C for up to 12h was found to further improve the mechanical properties of coatings. After the post-spray heat treatment, 17.6% increment in Young's modulus (E) and 16.3% increment in Vicker's hardness were achieved. The strengthening mechanisms of HA/YSZ/Ti-6Al-4V composite coatings were related to the dispersion strengthening by homogeneous distribution of YSZ particles in the matrix, the good mechanical properties of Ti-6Al-4V and the formation of solid solution among HA

Development of a novel rapid immunochromatographic test specific for the H5 influenza virus.

PubMed

Miyagawa, Eiji; Kogaki, Hiroyuki; Uchida, Yoshiaki; Fujii, Nobuyuki; Shirakawa, Takashi; Sakoda, Yoshihrio; Kida, Hiroshi

2011-05-01

Three anti-H5 influenza virus monoclonal antibody (mAb) clones, IFH5-26, IFH5-115 and IFH5-136, were obtained by immunising a BALB/C mouse with inactivated A/duck/Hokkaido/Vac-1/04 (H5N1). These mAbs were found to recognise specifically the haemagglutinin (HA) epitope of the influenza H5 subtypes by western blotting with recombinant HAs; however, these mAbs have no neutralising activity for A/duck/Hokkaido/84/02 (H5N3) or A/Puerto Ric/8/34 (H1N1). Each epitope of these mAbs was a conformational epitope that was formed from the regions located between 46 to 60 amino acids (aa) and 312 to 322 aa for IFH5-115, from 101 to 113 aa and 268 to 273 aa for IFH5-136 and from 61 to 80 aa and 290 to 300 aa for IFH5-26. The epitopes were located in the loop regions between the receptor region and alpha-helix structure in haemagglutinin 1 (HA1). Influenza A virus H5-specific rapid immunochromatographic test kits were tested as solid phase antibody/alkaline phosphate-conjugated mAb in the following three combinations: IFH5-26/IFH5-115, IFH5-136/IFH5-26 and IFH5-136/IFH5-115. In every combination, only influenza A H5 subtypes were detected. For effective clinical application, rapid dual discrimination immunochromatographic test kits in combination with H5 HA-specific mAb, IFA5-26 and IFA5-115 and the influenza A NP NP-specific mAb, FVA2-11, were developed. The dual discrimination immunochromatographic tests kits detected influenza A virus H5 subtypes as H5 line-positive and all influenza A subtypes as A line-positive simultaneously. The dual discrimination immunochromatographic test kits may be useful for discriminating highly pathogenic avian influenza A H5N1 viruses from seasonal influenza A virus, as well as for confirming influenza infection status in human, avian and mammalian hosts. Copyright © 2011 Elsevier B.V. All rights reserved.

Stimulation of TLRs by LMW-HA induces self-defense mechanisms in vaginal epithelium.

PubMed

Dusio, Giuseppina F; Cardani, Diego; Zanobbio, Laura; Mantovani, Martina; Luchini, Patrizia; Battini, Lorenzo; Galli, Valentina; Diana, Angela; Balsari, Andrea; Rumio, Cristiano

2011-07-01

The innate immune system is present throughout the female reproductive tract and functions in synchrony with the adaptive immune system to provide protection in a way that enhances the chances for fetal survival, while protecting against potential pathogens. Recent data show that activation of Toll-like receptor (TLR)2 and 4 by low-molecular weight hyaluronic acid (LMW-HA) in the epidermis induces secretion of the antimicrobial peptide β-defensin 2. In the present work, we show that LMW-HA induces vaginal epithelial cells to release different antimicrobial peptides, via activation of TLR2 and TLR4. Further, we found that LMW-HA favors repair of vaginal epithelial injury, involving TLR2 and TLR4, and independently from its classical receptor CD44. This wound-healing activity of LMW-HA is dependent from an Akt/phosphatidylinositol 3 kinase pathway. Therefore, these findings suggest that the vaginal epithelium is more than a simple physical barrier to protect against invading pathogens: on the contrary, this surface acts as efficient player of innate host defense, which may modulate its antimicrobial properties and injury restitution activity, following LMW-HA stimulation; this activity may furnish an additional protective activity to this body compartment, highly and constantly exposed to microbiota, ameliorating the self-defense of the vaginal epithelium in both basal and pathological conditions.

Molecular cloning, phylogenetic analysis, and expression patterns of LATERAL SUPPRESSOR-LIKE and REGULATOR OF AXILLARY MERISTEM FORMATION-LIKE genes in sunflower (Helianthus annuus L.).

PubMed

Fambrini, Marco; Salvini, Mariangela; Pugliesi, Claudio

2017-03-01

The wild sunflower (Helianthus annuus) plants develop a highly branched form with numerous small flowering heads. The origin of a no branched sunflower, producing a single large head, has been a key event in the domestication process of this species. The interaction between hormonal factors and several genes organizes the initiation and outgrowth of axillary meristems (AMs). From sunflower, we have isolated two genes putatively involved in this process, LATERAL SUPPRESSOR (LS)-LIKE (Ha-LSL) and REGULATOR OF AXILLARY MERISTEM FORMATION (ROX)-LIKE (Ha-ROXL), encoding for a GRAS and a bHLH transcription factor (TF), respectively. Typical amino acid residues and phylogenetic analyses suggest that Ha-LSL and Ha-ROXL are the orthologs of the branching regulator LS and ROX/LAX1, involved in the growth habit of both dicot and monocot species. qRT-PCR analyses revealed a high accumulation of Ha-LSL transcripts in roots, vegetative shoots, and inflorescence shoots. By contrast, in internodal stems and young leaves, a lower amount of Ha-LSL transcripts was observed. A comparison of transcription patterns between Ha-LSL and Ha-ROXL revealed some analogies but also remarkable differences; in fact, the gene Ha-ROXL displayed a low expression level in all organs analyzed. In situ hybridization (ISH) analysis showed that Ha-ROXL transcription was strongly restricted to a small domain within the boundary zone separating the shoot apical meristem (SAM) and the leaf primordia and in restricted regions of the inflorescence meristem, beforehand the separation of floral bracts from disc flower primordia. These results suggested that Ha-ROXL may be involved to establish a cell niche for the initiation of AMs as well as flower primordia. The accumulation of Ha-LSL transcripts was not restricted to the boundary zones in vegetative and inflorescence shoots, but the mRNA activity was expanded in other cellular domains of primary shoot apical meristem as well as AMs. In addition, Ha

Experimental infection of clade 1.1.2 (H5N1), clade 2.3.2.1c (H5N1) and clade 2.3.4.4 (H5N6) highly pathogenic avian influenza viruses in dogs.

PubMed

Lyoo, K S; Na, W; Phan, L V; Yoon, S W; Yeom, M; Song, D; Jeong, D G

2017-12-01

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in Asia, the haemagglutinin (HA) gene of this virus lineage has continued to evolve in avian populations, and H5N1 lineage viruses now circulate concurrently worldwide. Dogs may act as an intermediate host, increasing the potential for zoonotic transmission of influenza viruses. Virus transmission and pathologic changes in HPAI clade 1.1.2 (H5N1)-, 2.3.2.1c (H5N1)- and 2.3.4.4 (H5N6)-infected dogs were investigated. Mild respiratory signs and antibody response were shown in dogs intranasally infected with the viruses. Lung histopathology showed lesions that were associated with moderate interstitial pneumonia in the infected dogs. In this study, HPAI H5N6 virus replication in dogs was demonstrated for the first time. Dogs have been suspected as a "mixing vessel" for reassortments between avian and human influenza viruses to occur. The replication of these three subtypes of the H5 lineage of HPAI viruses in dogs suggests that dogs could serve as intermediate hosts for avian-human influenza virus reassortment if they are also co-infected with human influenza viruses. © 2017 Blackwell Verlag GmbH.

Trypsin pre-treatment corrects SRID over-estimation of immunologically active, pre-fusion HA caused by mixed immunoprecipitin rings.

PubMed

Wen, Yingxia; Palladino, Giuseppe; Xie, Yuhong; Ferrari, Annette; Ma, Xiuwen; Han, Liqun; Dormitzer, Philip R; Settembre, Ethan C

2016-06-17

Influenza vaccines are the primary intervention to prevent the substantial health burden of seasonal and pandemic influenza. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on their content of immunologically active (capable of eliciting functional antibodies) hemagglutinin (HA). Single-radial immunodiffusion (SRID), the standard in vitro potency assay in the field, is believed to specifically detect immunologically active HA. We confirmed that, with conformationally homogeneous HA preparations, SRID specifically detected native, pre-fusion HA, which elicited influenza neutralizing and hemagglutination inhibiting (HI) antibodies in mice, and it did not detect low-pH stressed, post-fusion HA, which was selectively removed from the SRID gel during a blotting step and was significantly less immunologically active. This selective detection was due to the SRID format, not a conformational specificity of the sheep antiserum used in the SRID, as the same antiserum detected non-stressed and low-pH stressed HA similarly when used in an ELISA format. However, when low-pH stressed HA was mixed with non-stressed HA, SRID detected both forms in mixed immunoprecipitin rings, leading to over-quantification of pre-fusion HA. We previously reported that trypsin digestion of antigen samples selectively degrade stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique, reversed-phase high pressure liquid chromatography (RP-HPLC), can specifically quantify trypsin-resistant, immunologically active, pre-fusion HA. Here, we report that trypsin digestion can also improve the specificity of SRID so that it can quantify immunologically active, pre-fusion HA when it is mixed with less immunologically active, post-fusion HA. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of microRNAs and genes associated with hyperandrogenism in the follicular fluid of women with polycystic ovary syndrome.

PubMed

Xue, Yunping; Lv, Juan; Xu, Pengfei; Gu, Lin; Cao, Jian; Xu, Lingling; Xue, Kai; Li, Qian

2018-05-01

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease, which is characterized by hyperandrogenism (HA), chronic anovulation, polycystic ovaries, insulin resistance, and obesity. At present, the mechanism by which PCOS/HA occurs has not been fully elucidated, thus, the mechanisms behind and interventions for HA in PCOS are current hot topics in research. MiRNAs have recently been shown to serve as diagnostic or prognostic biomarkers in patients with cancer. Thus, we are currently focused on studying the altered expression of miRNAs in follicular fluid and their correlation with HA in PCOS. Illumina deep sequencing technology was used to explore different miRNAs in the follicular fluid of women with PCOS/HA and in the follicular fluid of women in a control group. Target prediction databases were then used to analyse the target genes of different expressed miRNAs, and GO analysis and the KEGG pathway database were used to identify the functions and the main biochemical and signalling pathways of differentially expressed target genes. The expression levels of 263 miRNAs were significantly different (>2-fold up-regulated or <0.5-fold down-regulated, P < 0.05) between the two groups of women. For example, the expression levels of miRNA (200a-3p, 10b-3p, 200b-3p, 29c-3p, 99a-3p, and 125a-5p) were significantly increased, while there was a decreased expression of miR-105-3p in PCOS patients with respect to the control. Literature has shown that the above seven miRNAs were associated with HA in PCOS. Furthermore, 31 770 genes were predicted to be targets of the 263 differentially expressed microRNAs. GO analysis and the KEGG pathway database showed involvement of these target genes in HA in PCOS. These results suggest the presence of differentially expressed miRNAs in the follicular fluid of women with PCOS/HA versus women in the control group. The potential role of these microRNAs was elucidated using bioinformatics tools and was found to be

Optimally designed collagen/polycaprolactone biocomposites supplemented with controlled release of HA/TCP/rhBMP-2 and HA/TCP/PRP for hard tissue regeneration.

PubMed

Kim, WonJin; Jang, Chul Ho; Kim, GeunHyung

2017-09-01

Collagen has been widely used as a very promising material to regenerate various tissues. It is a chief component of the extracellular matrix, and encourages various biological effects conducive to tissue regeneration. However, poor mechanical stability, low processability, and high level of water absorption can lead to impaired control of growth factor release and have impeded the use of collagen as a functional biomedical scaffold. Here, to overcome the shortcomings of collagen scaffolds, we have additively manufactured collagen/polycaprolactone (PCL) biocomposites supplemented with a bioceramic (hydroxyapatite (HA)/β-tricalcium-phosphate (TCP)) and two growth factors (recombinant human bone morphogenetic protein-2 [rhBMP-2] and platelet-rich plasma [PRP]). Various weight fractions of PCL in the collagen/PCL composites were manipulated to select optimal growth factor release and highly active cellular responses. After the optimal concentration of PCL in the collagen/PCL scaffold was determined, biocomposites supplemented with bioceramic/growth-factors were fabricated. Continuously released growth factors were assumed to increase the in vitro cellular activities of the osteoblast-like cells (MG63) cultured on the biocomposites. In vitro cellular responses, including osteogenic activities, were examined, and results showed that compared to the HA/TCP/rhBMP-2 supplemented scaffold the HA/TCP/PRP biocomposites provide significantly high cellular activities (cell proliferation: >1.3-fold) and mineralization (calcium deposition: >1.4-fold, osteocalcin: >2.6-fold) sufficient for regenerating bone tissue. Copyright © 2017. Published by Elsevier B.V.

Vaccine-induced anti-HA2 antibodies promote virus fusion and enhance influenza virus respiratory disease.

PubMed

Khurana, Surender; Loving, Crystal L; Manischewitz, Jody; King, Lisa R; Gauger, Phillip C; Henningson, Jamie; Vincent, Amy L; Golding, Hana

2013-08-28

Vaccine-induced disease enhancement has been described in connection with several viral vaccines in animal models and in humans. We investigated a swine model to evaluate mismatched influenza vaccine-associated enhanced respiratory disease (VAERD) after pH1N1 infection. Vaccinating pigs with whole inactivated H1N2 (human-like) virus vaccine (WIV-H1N2) resulted in enhanced pneumonia and disease after pH1N1 infection. WIV-H1N2 immune sera contained high titers of cross-reactive anti-pH1N1 hemagglutinin (HA) antibodies that bound exclusively to the HA2 domain but not to the HA1 globular head. No hemagglutination inhibition titers against pH1N1 (challenge virus) were measured. Epitope mapping using phage display library identified the immunodominant epitope recognized by WIV-H1N2 immune sera as amino acids 32 to 77 of pH1N1-HA2 domain, close to the fusion peptide. These cross-reactive anti-HA2 antibodies enhanced pH1N1 infection of Madin-Darby canine kidney cells by promoting virus membrane fusion activity. The enhanced fusion activity correlated with lung pathology in pigs. This study suggests a role for fusion-enhancing anti-HA2 antibodies in VAERD, in the absence of receptor-blocking virus-neutralizing antibodies. These findings should be considered during the evaluation of universal influenza vaccines designed to elicit HA2 stem-targeting antibodies.

Full genome comparison and characterization of avian H10 viruses with different pathogenicity in Mink (Mustela vison) reveals genetic and functional differences in the non-structural gene

PubMed Central

2010-01-01

Background The unique property of some avian H10 viruses, particularly the ability to cause severe disease in mink without prior adaptation, enabled our study. Coupled with previous experimental data and genetic characterization here we tried to investigate the possible influence of different genes on the virulence of these H10 avian influenza viruses in mink. Results Phylogenetic analysis revealed a close relationship between the viruses studied. Our study also showed that there are no genetic differences in receptor specificity or the cleavability of the haemagglutinin proteins of these viruses regardless of whether they are of low or high pathogenicity in mink. In poly I:C stimulated mink lung cells the NS1 protein of influenza A virus showing high pathogenicity in mink down regulated the type I interferon promoter activity to a greater extent than the NS1 protein of the virus showing low pathogenicity in mink. Conclusions Differences in pathogenicity and virulence in mink between these strains could be related to clear amino acid differences in the non structural 1 (NS1) protein. The NS gene of mink/84 appears to have contributed to the virulence of the virus in mink by helping the virus evade the innate immune responses. PMID:20591155

Fabrication and characterization of nanoengineered biocompatible n-HA/chitosan-tamarind seed polysaccharide: Bio-inspired nanocomposites for bone tissue engineering.

PubMed

Shakir, Mohammad; Zia, Iram; Rehman, Abdur; Ullah, Rizwan

2018-05-01

In this communication we describe the fabrication of nano-hydroxyapatite/chitosan-tamarind seed polysaccharide (n-HA/CS-TSP) nanocomposite with a weight ratio of 70/20/10, 70/15/15 and 70/10/20, respectively through a co-precipitation method. A comparative assessment of the properties of n-HA/CS-TSP and n-HA/CS nanocomposites was done by FT-IR, SEM-EDX, TEM, TGA/DTA, XRD and mechanical testing. The results suggested strong chemical interactions between the three components, decreased particle size and homogeneous dispersion of n-HA particles in n-HA/CS-TSP as compared to n-HA/CS. The n-HA/CS-TSP (70/10/20) showed the most porous and rough surface, enhanced thermal stability and highest compressive strength (4.0 MPa) and modulus (81 MPa). In addition, n-HA/CS-TSP (70/10/20) exhibited greater swelling character, acceptable degradation and increased biomineralization in simulated body fluid (SBF) as compared to n-HA/CS-TSP (70/20/10, 70/15/15) and n-HA/CS nanocomposites. The superior non-toxic response with MG-63 cells and better haemocompatibility was observed with n-HA/CS-TSP (70/15/15). Thereby, n-HA/CS-TSP nanocomposites could be promising alternative biomaterials in the field of bone tissue engineering compared to the n-HA/CS nanocomposite. Copyright © 2018 Elsevier B.V. All rights reserved.

Does PEEK/HA Enhance Bone Formation Compared With PEEK in a Sheep Cervical Fusion Model?

PubMed

Walsh, William R; Pelletier, Matthew H; Bertollo, Nicky; Christou, Chris; Tan, Chris

2016-11-01

Polyetheretherketone (PEEK) has a wide range of clinical applications but does not directly bond to bone. Bulk incorporation of osteoconductive materials including hydroxyapatite (HA) into the PEEK matrix is a potential solution to address the formation of a fibrous tissue layer between PEEK and bone and has not been tested. Using in vivo ovine animal models, we asked: (1) Does PEEK-HA improve cortical and cancellous bone ongrowth compared with PEEK? (2) Does PEEK-HA improve bone ongrowth and fusion outcome in a more challenging functional ovine cervical fusion model? The in vivo responses of PEEK-HA Enhanced and PEEK-OPTIMA ® Natural were evaluated for bone ongrowth in the form of dowels implanted in the cancellous and cortical bone of adult sheep and examined at 4 and 12 weeks as well as interbody cervical fusion at 6, 12, and 26 weeks. The bone-implant interface was evaluated with radiographic and histologic endpoints for a qualitative assessment of direct bone contact of an intervening fibrous tissue later. Gamma-irradiated cortical allograft cages were evaluated as well. Incorporating HA into the PEEK matrix resulted in more direct bone apposition as opposed to the fibrous tissue interface with PEEK alone in the bone ongrowth as well as interbody cervical fusions. No adverse reactions were found at the implant-bone interface for either material. Radiography and histology revealed resorption and fracture of the allograft devices in vivo. Incorporating HA into PEEK provides a more favorable environment than PEEK alone for bone ongrowth. Cervical fusion was improved with PEEK-HA compared with PEEK alone as well as allograft bone interbody devices. Improving the bone-implant interface with a PEEK device by incorporating HA may improve interbody fusion results and requires further clinical studies.

Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization

PubMed Central

Chetta, M.; Drmanac, A.; Santacroce, R.; Grandone, E.; Surrey, S.; Fortina, P.; Margaglione, M.

2008-01-01

BACKGROUND: Standard methods of mutation detection are time consuming in Hemophilia A (HA) rendering their application unavailable in some analysis such as prenatal diagnosis. OBJECTIVES: To evaluate the feasibility of combinatorial sequencing-by-hybridization (cSBH) as an alternative and reliable tool for mutation detection in FVIII gene. PATIENTS/METHODS: We have applied a new method of cSBH that uses two different colors for detection of multiple point mutations in the FVIII gene. The 26 exons encompassing the HA gene were analyzed in 7 newly diagnosed Italian patients and in 19 previously characterized individuals with FVIII deficiency. RESULTS: Data show that, when solution-phase TAMRA and QUASAR labeled 5-mer oligonucleotide sets mixed with unlabeled target PCR templates are co-hybridized in the presence of DNA ligase to universal 6-mer oligonucleotide probe-based arrays, a number of mutations can be successfully detected. The technique was reliable also in identifying a mutant FVIII allele in an obligate heterozygote. A novel missense mutation (Leu1843Thr) in exon 16 and three novel neutral polymorphisms are presented with an updated protocol for 2-color cSBH. CONCLUSIONS: cSBH is a reliable tool for mutation detection in FVIII gene and may represent a complementary method for the genetic screening of HA patients. PMID:20300295

High Altitude Affects Nocturnal Non-linear Heart Rate Variability: PATCH-HA Study

PubMed Central

Boos, Christopher J.; Bye, Kyo; Sevier, Luke; Bakker-Dyos, Josh; Woods, David R.; Sullivan, Mark; Quinlan, Tom; Mellor, Adrian

2018-01-01

Background: High altitude (HA) exposure can lead to changes in resting heart rate variability (HRV), which may be linked to acute mountain sickness (AMS) development. Compared with traditional HRV measures, non-linear HRV appears to offer incremental and prognostic data, yet its utility and relationship to AMS have been barely examined at HA. This study sought to examine this relationship at terrestrial HA. Methods: Sixteen healthy British military servicemen were studied at baseline (800 m, first night) and over eight consecutive nights, at a sleeping altitude of up to 3600 m. A disposable cardiac patch monitor was used, to record the nocturnal cardiac inter-beat interval data, over 1 h (0200–0300 h), for offline HRV assessment. Non-linear HRV measures included Sample entropy (SampEn), the short (α1, 4–12 beats) and long-term (α2, 13–64 beats) detrend fluctuation analysis slope and the correlation dimension (D2). The maximal rating of perceived exertion (RPE), during daily exercise, was assessed using the Borg 6–20 RPE scale. Results: All subjects completed the HA exposure. The average age of included subjects was 31.4 ± 8.1 years. HA led to a significant fall in SpO2 and increase in heart rate, LLS and RPE. There were no significant changes in the ECG-derived respiratory rate or in any of the time domain measures of HRV during sleep. The only notable changes in frequency domain measures of HRV were an increase in LF and fall in HFnu power at the highest altitude. Conversely, SampEn, SD1/SD2 and D2 all fell, whereas α1 and α2 increased (p < 0.05). RPE inversely correlated with SD1/SD2 (r = -0.31; p = 0.002), SampEn (r = -0.22; p = 0.03), HFnu (r = -0.27; p = 0.007) and positively correlated with LF (r = 0.24; p = 0.02), LF/HF (r = 0.24; p = 0.02), α1 (r = 0.32; p = 0.002) and α2 (r = 0.21; p = 0.04). AMS occurred in 7/16 subjects (43.8%) and was very mild in 85.7% of cases. HRV failed to predict AMS. Conclusion: Non-linear HRV is more sensitive to

High Altitude Affects Nocturnal Non-linear Heart Rate Variability: PATCH-HA Study.

PubMed

Boos, Christopher J; Bye, Kyo; Sevier, Luke; Bakker-Dyos, Josh; Woods, David R; Sullivan, Mark; Quinlan, Tom; Mellor, Adrian

2018-01-01

Background: High altitude (HA) exposure can lead to changes in resting heart rate variability (HRV), which may be linked to acute mountain sickness (AMS) development. Compared with traditional HRV measures, non-linear HRV appears to offer incremental and prognostic data, yet its utility and relationship to AMS have been barely examined at HA. This study sought to examine this relationship at terrestrial HA. Methods: Sixteen healthy British military servicemen were studied at baseline (800 m, first night) and over eight consecutive nights, at a sleeping altitude of up to 3600 m. A disposable cardiac patch monitor was used, to record the nocturnal cardiac inter-beat interval data, over 1 h (0200-0300 h), for offline HRV assessment. Non-linear HRV measures included Sample entropy (SampEn), the short (α1, 4-12 beats) and long-term (α2, 13-64 beats) detrend fluctuation analysis slope and the correlation dimension (D2). The maximal rating of perceived exertion (RPE), during daily exercise, was assessed using the Borg 6-20 RPE scale. Results: All subjects completed the HA exposure. The average age of included subjects was 31.4 ± 8.1 years. HA led to a significant fall in SpO 2 and increase in heart rate, LLS and RPE. There were no significant changes in the ECG-derived respiratory rate or in any of the time domain measures of HRV during sleep. The only notable changes in frequency domain measures of HRV were an increase in LF and fall in HFnu power at the highest altitude. Conversely, SampEn, SD1/SD2 and D2 all fell, whereas α1 and α2 increased ( p < 0.05). RPE inversely correlated with SD1/SD2 ( r = -0.31; p = 0.002), SampEn ( r = -0.22; p = 0.03), HFnu ( r = -0.27; p = 0.007) and positively correlated with LF ( r = 0.24; p = 0.02), LF/HF ( r = 0.24; p = 0.02), α1 ( r = 0.32; p = 0.002) and α2 ( r = 0.21; p = 0.04). AMS occurred in 7/16 subjects (43.8%) and was very mild in 85.7% of cases. HRV failed to predict AMS. Conclusion: Non-linear HRV is more sensitive to

Association between DRD2, 5-HTTLPR, and ALDH2 genes and specific personality traits in alcohol- and opiate-dependent patients.

PubMed

Wang, Tzu-Yun; Lee, Sheng-Yu; Chen, Shiou-Lan; Huang, San-Yuan; Chang, Yun-Hsuan; Tzeng, Nian-Sheng; Wang, Chen-Lin; Hui Lee, I; Yeh, Tzung Lieh; Yang, Yen Kuang; Lu, Ru-Band

2013-08-01

The vulnerability of developing addictions is associated with genetic factors and personality traits. The predisposing genetic variants and personality traits may be common to all addictions or specific to a particular class of addiction. To investigate the relationship between genetic variances, personality traits, and their interactions in addiction are important. We recruited 175 opiate-dependent patients, 102 alcohol-dependent patients, and 111 healthy controls. All participants were diagnosed using DSM-IV criteria and assessed with Tridimensional Personality Questionnaire (TPQ). The dopamine D2 receptor (DRD2), 5-HTT-linked promoter region (5-HTTLPR), and aldehyde dehydrogenase 2 (ALDH2) genes were genotyped using PCR. The genotype frequency of the 5-HTTLPR and ALDH2 was significantly different between the patients and controls (P=0.013, P<0.001, respectively), and borderline significant (P=0.05) for DRD2 polymorphism. Both Novelty Seeking (NS) and Harm Avoidance (HA) scores were higher for patients (P<0.001). After stratification by candidate genes, addicts with ALDH2 *1/*1 interacting with the low-functional group of DRD2 and 5-HTTLPR genes have higher HA traits, whereas addicts with ALDH2 *1/*2 or *2/*2 and low-functional group of DRD2 and 5-HTTLPR genes have higher NS traits. We concluded that addicts, both alcohol- and opiate-dependent patients, have common genetic variants in DRD2 and 5-HTTLPR but specific for ALDH2. Higher NS and HA traits were found in both patient groups with the interaction with DRD2, 5-HTTLPR, and ALDH2 genes. The ALDH2 gene variants had different effect in the NS and HA dimension while the DRD2 and 5-HTTLPR genes did not. Copyright © 2013 Elsevier B.V. All rights reserved.

Illumina MiSeq sequencing reveals microbial community in HA process for dyeing wastewater treatment fed with different co-substrates.

PubMed

Xie, Xuehui; Liu, Na; Ping, Jing; Zhang, Qingyun; Zheng, Xiulin; Liu, Jianshe

2018-06-01

In present study, a hydrolysis acidification (HA) reactor was used for simulated dyeing wastewater treatment. Co-substrates included starch, glucose, sucrose, yeast extract (YE) and peptone were fed sequentially into the HA reactor to enhance the HA process effects. The performance of the HA reactor and the microbial community structure in HA process were investigated under different co-substrates conditions. Results showed that different co-substrates had different influences on the performance of HA reactor. The highest decolorization (50.64%) and COD removal rate (60.73%) of the HA reactor were obtained when sucrose was as the co-substrate. And it found that carbon co-substrates starch, glucose and sucrose exhibited better decolorization and higher COD removal efficiency of the HA reactor than the nitrogen co-substrates YE and peptone. Microbial community structure in the HA process was analyzed by Illumina MiSeq sequencing. Results revealed different co-substrates had different influences on the community structure and microbial diversity in HA process. It was considered that sucrose could enrich the species such as Raoultella, Desulfovibrio, Tolumonas, Clostridium, which might be capable of degrading the dyes. Sucrose was considered to be the best co-substrate of enhancing the HA reactor's performance in this study. This work would provide deep insight into the influence of many different co-substrates on HA reactor performance and microbial communities in HA process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Understanding the undelaying mechanism of HA-subtyping in the level of physic-chemical characteristics of protein.

PubMed

Ebrahimi, Mansour; Aghagolzadeh, Parisa; Shamabadi, Narges; Tahmasebi, Ahmad; Alsharifi, Mohammed; Adelson, David L; Hemmatzadeh, Farhid; Ebrahimie, Esmaeil

2014-01-01

The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896) of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms), percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms) as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search) showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for understanding and

Understanding the Underlying Mechanism of HA-Subtyping in the Level of Physic-Chemical Characteristics of Protein

PubMed Central

Ebrahimi, Mansour; Aghagolzadeh, Parisa; Shamabadi, Narges; Tahmasebi, Ahmad; Alsharifi, Mohammed; Adelson, David L.

2014-01-01

The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896) of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms), percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms) as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search) showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for understanding and

In vitro and in vivo gene delivery using chitosan/hyaluronic acid nanoparticles: Influences of molecular mass of hyaluronic acid and lyophilization on transfection efficiency.

PubMed

Sato, Toshinori; Nakata, Mitsuhiro; Yang, Zhihong; Torizuka, Yu; Kishimoto, Satoko; Ishihara, Masayuki

2017-08-01

Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at -30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration. Copyright © 2017 John Wiley & Sons, Ltd.

Mechanical properties of titanium-hydroxyapatite (Ti-HA) composite coating on stainless steel prepared by thermal spraying

NASA Astrophysics Data System (ADS)

Rosmamuhamadani, R.; Azhar, N. H.; Talari, M. K.; Yahaya, Sabrina M.; Sulaiman, S.; Ismail, M. I. S.

2017-09-01

Addition of hydroxyapatite (HA) can enhance the bioactivity of the common metallic implant due to its similarity with natural bones and teeth. In this investigation, high velocity oxy-fuel (HVOFT) technique was used to deposit titanium-hydroxyapatite (Ti-HA) composite on stainless steel substrate plate with different percentage of HA for biomedical applications. The aim of this research is to investigate the mechanical properties of Ti-HA coating such as hardness, adhesion strength and wear behaviour. The hardness and strength was determined by using SHIMADZU-microhardness Vickers tester and PosiTest AT portable adhesion tester respectively. The wear test was performed by using pin-on-disk equipment and field emission scanning electron microscope (FESEM) used to determine the extent of surface damage. From the results obtained, mechanical properties such as hardness and adhesion strength of titanium (Ti) coating decreased with the increased of HA contents. Meanwhile, the coefficient of friction of Ti-10% HA coating shows the highest value compare to others as three-body abrasion had occurred during the test.

Structural-based designed modular capsomere comprising HA1 for low-cost poultry influenza vaccination.

PubMed

Waneesorn, Jarurin; Wibowo, Nani; Bingham, John; Middelberg, Anton P J; Lua, Linda H L

2018-05-24

Highly pathogenic avian influenza (HPAI) viruses cause a severe and lethal infection in domestic birds. The increasing number of HPAI outbreaks has demonstrated the lack of capabilities to control the rapid spread of avian influenza. Poultry vaccination has been shown to not only reduce the virus spread in animals but also reduce the virus transmission to humans, preventing potential pandemic development. However, existing vaccine technologies cannot respond to a new virus outbreak rapidly and at a cost and scale that is commercially viable for poultry vaccination. Here, we developed modular capsomere, subunits of virus-like particle, as a low-cost poultry influenza vaccine. Modified murine polyomavirus (MuPyV) VP1 capsomere was used to present structural-based influenza Hemagglutinin (HA1) antigen. Six constructs of modular capsomeres presenting three truncated versions of HA1 and two constructs of modular capsomeres presenting non-modified HA1 have been generated. These modular capsomeres were successfully produced in stable forms using Escherichia coli, without the need for protein refolding. Based on ELISA, this adjuvanted modular capsomere (CaptHA1-3C) induced strong antibody response (almost 10 5 endpoint titre) when administered into chickens, similar to titres obtained in the group administered with insect cell-based HA1 proteins. Chickens that received adjuvanted CaptHA1-3C followed by challenge with HPAI virus were fully protected. The results presented here indicate that this platform for bacterially-produced modular capsomere could potentially translate into a rapid-response and low-cost vaccine manufacturing technology suitable for poultry vaccination. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multistep carcinogenesis of normal human fibroblasts. Human fibroblasts immortalized by repeated treatment with Co-60 gamma rays were transformed into tumorigenic cells with Ha-ras oncogenes.

PubMed

Namba, M; Nishitani, K; Fukushima, F; Kimoto, T

1988-01-01

Two normal mortal human fibroblast cell strains were transformed into immortal cell lines, SUSM-1 and KMST-6, by treatment with 4-nitroquinoline 1-oxide (4NQO) and Co-60 gamma rays, respectively. These immortalized cell lines showed morphological changes of cells and remarkable chromosome aberrations, but neither of them grew in soft agar or formed tumors in nude mice. The immortal cell line, KMST-6, was then converted into neoplastic cells by treatment with Harvey murine sarcoma virus (Ha-MSV) or the c-Ha-ras oncogene derived from a human lung carcinoma. These neoplastically transformed cells acquired anchorage-independent growth potential and developed tumors when transplanted into nude mice. All the tumors grew progressively without regression until the animals died of tumors. In addition, the tumors were transplantable into other nude mice. Normal human fibroblasts, on the other hand, were not transformed into either immortal or tumorigenic cells by treatment with Ha-MSV or c-Ha-ras alone. Our present data indicate that (1) the chemical carcinogen, 4NQO, or gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to immortality, and (2) the ras gene played a role in the progression of the immortally transformed cells to more malignant cells showing anchorage-independent growth and tumorigenicity. In other words, the immortalization process of human cells seems to be a pivotal or rate-limiting step in the carcinogenesis of human cells.

Antibiotic-containing hyaluronic acid gel as an antibacterial carrier: Usefulness of sponge and film-formed HA gel in deep infection.

PubMed

Matsuno, Hiroaki; Yudoh, Kazuo; Hashimoto, Masamichi; Himeda, Yasukazu; Miyoshi, Teruzo; Yoshida, Kaoru; Kano, Syogo

2006-03-01

We have developed a novel bioabsorbable antibacterial carrier using hyaluronic acid (HA) gel for prevention and treatment of orthopedic infections. In this study, we investigated the in vivo antibacterial effects of two forms of this new material, an HA gel sponge and an HA gel film. A titanium cylinder was inserted into the intramedullary cavity of each rabbit femur, along with an HA gel sponge or HA gel film containing antibiotics. The HA gel sponge contained gentamycin, vancomycin, tobramycin, or minomycin. The HA gel film contained gentamycin or vancomycin. After 0, 7, and 14 days, the rabbit bone marrow was collected, and the antibacterial activity of the HA gel was determined by agar diffusion test. As a control, we used Septocoll, a commercially available antibacterial carrier. Both the HA gel sponge and HA gel film exhibited antibacterial activity. The present results indicate that HA gel containing antibiotics is a clinically useful bioabsorbable antibacterial carrier. Copyright 2006 Orthopaedic Research Society.

Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

PubMed

Nakamura, Toshio; Tonozuka, Takashi; Ide, Azusa; Yuzawa, Takayuki; Oguma, Keiji; Nishikawa, Atsushi

2008-02-22

Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.

Gaming Sea-based Multinational HA/DR Operations at PACOM Amphibious Leaders Symposium 2016

DTIC Science & Technology

2016-11-01

DISTRIBUTION STATEMENT A. Approved for public release: distribution unlimited. Gaming Sea-based Multinational HA/DR Operations...Marine Corps photo by Cpl. Wesley Timm. Approved by: November 2016 Dr. E.D. McGrady Director, Integration and Gaming Advanced Technology and...TYPE Final2 3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE Gaming Seabased Multinational HA/DR Operations at PACOM 5a. CONTRACT NUMBER N00014-16

3D-printed scaffolds based on PLA/HA nanocomposites for trabecular bone reconstruction

NASA Astrophysics Data System (ADS)

Niaza, K. V.; Senatov, F. S.; Kaloshkin, S. D.; Maksimkin, A. V.; Chukov, D. I.

2016-08-01

In the present work porous PLA scaffolds filled with micro- and nano- HA were studied. Both composites with micro- and nano-HA were obtained by extrusion in the same conditions. Scaffolds were obtained by 3D-printing by fused filament fabrication method. Structure of porous scaffolds was pre-modeled by computer software. Compression and three - point flexural tests were used to study mechanical properties of the scaffolds.

[Study on biocompatibility of hydroxyapatite/high density polyethylene (HA/HDPE) nano-composites artificial ossicle].

PubMed

Wang, Guohui; Zhu, Shaihong; Tan, Guolin; Zhou, Kechao; Huang, Suping; Zhao, Yanzhong; Li, Zhiyou; Huang, Boyun

2008-06-01

This study was aimed to evaluate the biocompatibility of Hydroxyapatite/High density polyethylene (HA/ HDPE) nano-composites artificial ossicle. The percentage of S-period cells were detected by flow cytometry after L929 cells being incubated with extraction of the HA/HDPE nano-composites; the titanium materials for clinical application served as the contrast. In addition, both materials were implanted in animals and the histopathological evaluations were conducted. There were no statistically significant differences between the two groups (P >0.05). The results demonstrated that the HA/HDPE nano-composite artificial ossicle made by our laboratory is of a good biocompatibility and clinical application outlook.

Evaluation of the biological differences of canine and human factor VIII in gene delivery: Implications in human hemophilia treatment

USDA-ARS?s Scientific Manuscript database

The canine is the most important large animal model for testing novel hemophilia A(HA) treatment. It is often necessary to use canine factor VIII (cFIII) gene or protein for the evaluation of HA treatment in the canine model. However, the different biological properties between cFVIII and human FVII...

Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses

PubMed Central

McCormick, Kara; Jiang, Zhiyong; Zhu, Longchao; Lawson, Steven R.; Langenhorst, Robert; Ransburgh, Russell; Brunick, Colin; Tracy, Miranda C.; Hurtig, Heather R.; Mabee, Leah M.; Mingo, Mark; Li, Yanhua; Webby, Richard J.

2015-01-01

Background and Objectives Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes. Methods and Results Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates. Conclusion This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines. PMID:26061265

Neural stem cell proliferation and differentiation in the conductive PEDOT-HA/Cs/Gel scaffold for neural tissue engineering.

PubMed

Wang, Shuping; Guan, Shui; Xu, Jianqiang; Li, Wenfang; Ge, Dan; Sun, Changkai; Liu, Tianqing; Ma, Xuehu

2017-09-26

Engineering scaffolds with excellent electro-activity is increasingly important in tissue engineering and regenerative medicine. Herein, conductive poly(3,4-ethylenedioxythiophene) doped with hyaluronic acid (PEDOT-HA) nanoparticles were firstly synthesized via chemical oxidant polymerization. A three-dimensional (3D) PEDOT-HA/Cs/Gel scaffold was then developed by introducing PEDOT-HA nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. HA, as a bridge, not only was used as a dopant, but also combined PEDOT into the Cs/Gel via chemical crosslinking. The PEDOT-HA/Cs/Gel scaffold was used as a conductive substrate for neural stem cell (NSC) culture in vitro. The results demonstrated that the PEDOT-HA/Cs/Gel scaffold had excellent biocompatibility for NSC proliferation and differentiation. 3D confocal fluorescence images showed cells attached on the channel surface of Cs/Gel and PEDOT-HA/Cs/Gel scaffolds with a normal neuronal morphology. Compared to the Cs/Gel scaffold, the PEDOT-HA/Cs/Gel scaffold not only promoted NSC proliferation with up-regulated expression of Ki67, but also enhanced NSC differentiation into neurons and astrocytes with up-regulated expression of β tubulin-III and GFAP, respectively. It is expected that this electro-active and bio-active PEDOT-HA/Cs/Gel scaffold will be used as a conductive platform to regulate NSC behavior for neural tissue engineering.

Registration of an oilseed sunflower germplasm HA-DM1 resistant to sunflower downy mildew

USDA-ARS?s Scientific Manuscript database

HA-DM1 (Reg. No.xxx, PI 674793) sunflower (Helianthus annuus L.) germplasm was developed and released cooperatively by the USDA-ARS, Sunflower and Plant Biology Research Unit and the North Dakota Agricultural Experiment Station in 2015. HA-DM1 is a BC2F4 derived oilseed maintainer line from the cros...

PLGA/nHA hybrid nanofiber scaffold as a nanocargo carrier of insulin for accelerating bone tissue regeneration

NASA Astrophysics Data System (ADS)

Haider, Adnan; Gupta, Kailash Chandra; Kang, Inn-Kyu

2014-06-01

The development of tissue engineering in the field of orthopedic surgery is booming. Two fields of research in particular have emerged: approaches for tailoring the surface properties of implantable materials with osteoinductive factors as well as evaluation of the response of osteogenic cells to these fabricated implanted materials (hybrid material). In the present study, we chemically grafted insulin onto the surface of hydroxyapatite nanorods (nHA). The insulin-grafted nHAs (nHA-I) were dispersed into poly(lactide-co-glycolide) (PLGA) polymer solution, which was electrospun to prepare PLGA/nHA-I composite nanofiber scaffolds. The morphology of the electrospun nanofiber scaffolds was assessed by field emission scanning electron microscopy (FESEM). After extensive characterization of the PLGA/nHA-I and PLGA/nHA composite nanofiber scaffolds by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray spectrometry (EDS), and transmission electron microscopy (TEM), the PLGA/nHA-I and PLGA/nHA (used as control) composite nanofiber scaffolds were subjected to cell studies. The results obtained from cell adhesion, alizarin red staining, and Von Kossa assay suggested that the PLGA/nHA-I composite nanofiber scaffold has enhanced osteoblastic cell growth, as more cells were proliferated and differentiated. The fact that insulin enhanced osteoblastic cell proliferation will open new possibilities for the development of artificial scaffolds for bone tissue regeneration.

The Source of the PB1 Gene in Influenza Vaccine Reassortants Selectively Alters the Hemagglutinin Content of the Resulting Seed Virus

PubMed Central

Cobbin, Joanna C. A.; Verity, Erin E.; Gilbertson, Brad P.; Rockman, Steven P.

2013-01-01

The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield. PMID:23468502

Intranasal vaccination with an inactivated whole influenza virus vaccine induces strong antibody responses in serum and nasal mucus of healthy adults

PubMed Central

Ainai, Akira; Tamura, Shin-ichi; Suzuki, Tadaki; van Riet, Elly; Ito, Ryo; Odagiri, Takato; Tashiro, Masato; Kurata, Takeshi; Hasegawa, Hideki

2013-01-01

Haemagglutination inhibition (HI) and neutralization (NT) titers as well as haemagglutinin (HA) specific antibody responses were examined in 50 healthy adults aged between 22 and 69 y old after two intranasal administrations of an inactivated whole virus vaccine derived from A/Victoria/210/2009 virus (45 μg HA per dose) at 3 week intervals. Serum HI titers after two-doses of the nasal vaccine showed >2.5-fold rise in the ratio of geometric mean titer upon vaccination, >40% of subjects with a ≥4-fold increase in titer and >70% of subjects with a titer of ≥1:40, all parameters associated with an effective outcome of vaccination in the criteria defined by the European Medicines Agency. Serum neutralizing antibody responses correlated with HI antibody responses, although NT titers were about 2-fold higher than HI titers. These high levels of serum responses were accompanied by high levels of HI and neutralizing antibody responses in nasal mucus as measured in concentrated nasal wash samples that were about 10 times diluted compared with natural nasal mucus. Serum and nasal HI and neutralizing antibody responses consisted of HA-specific IgG and IgA antibody responses, with IgG and IgA antibodies being dominant in serum and nasal responses, respectively. PMID:23896606

Cultivation, LD(50) determination and experimental model of Streptococcus suis serotype 2 strain HA9801.

PubMed

Zhao, Zhanzhong; Wang, Jian; Liu, Peihong; Zhang, Suhua; Gong, Jianpei; Huang, Xiqin; Li, Bin; Xue, Feiqun

2009-04-01

The effects of nutritional components and submerged culture conditions on colony-forming unit (CFU) counts by Streptococcus suis serotype 2 strain HA9801 in flask culture was investigated, and the optimal medium and cultivation conditions was confirmed by using a 50l bioreactor. The LD(50) values of HA9801 in pigs before and after fermentation were 1.8 x 10(7)CFU, which indicated that the virulence of HA9801 was very stable in the fermentation process. In addition, an experimental model that closely mimics naturally occurring disease in conventional pigs was established.

Synopsis of Precision Landing and Hazard Avoidance (PL&HA) Capabilities for Space Exploration

NASA Technical Reports Server (NTRS)

Robertson, Edward A.

2017-01-01

Until recently, robotic exploration missions to the Moon, Mars, and other solar system bodies relied upon controlled blind landings. Because terrestrial techniques for terrain relative navigation (TRN) had not yet been evolved to support space exploration, landing dispersions were driven by the capabilities of inertial navigation systems combined with surface relative altimetry and velocimetry. Lacking tight control over the actual landing location, mission success depended on the statistical vetting of candidate landing areas within the predicted landing dispersion ellipse based on orbital reconnaissance data, combined with the ability of the spacecraft to execute a controlled landing in terms of touchdown attitude, attitude rates, and velocity. In addition, the sensors, algorithms, and processing technologies required to perform autonomous hazard detection and avoidance in real time during the landing sequence were not yet available. Over the past decade, NASA has invested substantial resources on the development, integration, and testing of autonomous precision landing and hazard avoidance (PL&HA) capabilities. In addition to substantially improving landing accuracy and safety, these autonomous PL&HA functions also offer access to targets of interest located within more rugged and hazardous terrain. Optical TRN systems are baselined on upcoming robotic landing missions to the Moon and Mars, and NASA JPL is investigating the development of a comprehensive PL&HA system for a Europa lander. These robotic missions will demonstrate and mature PL&HA technologies that are considered essential for future human exploration missions. PL&HA technologies also have applications to rendezvous and docking/berthing with other spacecraft, as well as proximity navigation, contact, and retrieval missions to smaller bodies with microgravity environments, such as asteroids.

Fabrication of nano-structured HA/CNT coatings on Ti6Al4V by electrophoretic deposition for biomedical applications.

PubMed

Zhang, Bokai; Kwok, Chi Tat; Cheng, Fai Tsun; Man, Hau Chung

2011-12-01

In order to improve the bone bioactivity and osteointegration of metallic implants, hydroxyapatite (HA) is often coated on their surface so that a real bond with the surrounding bone tissue can be formed. In the present study, cathodic electrophoretic deposition (EPD) has been attempted for depositing nanostructured HA coatings on titanium alloy Ti6Al4V followed by sintering at 800 degrees C. Nano-sized HA powder was used in the EPD process to produce dense coatings. Moreover, multiwalled carbon nanotubes (CNTs) were also used to reinforce the HA coating for enhancing its mechanical strength. The surface morphology, compositions and microstructure of the monolithic coating of HA and nanocomposite coatings of HA with different CNT contents (4 to 25%) on Ti6Al4V were investigated by scanning-electron microscopy, energy-dispersive X-ray spectroscopy and Xray diffractometry, respectively. Electrochemical corrosion behavior of the various coatings in Hanks' solution at 37 degrees C was investigated by means of open-circuit potential measurement and cyclic potentiodynamic polarization tests. Surface hardness, adhesion strength and bone bioactivity of the coatings were also studied. The HA and HA/CNT coatings had a thickness of about 10 microm, with corrosion resistance higher than that of the substrate and adhesion strength higher than that of plasma sprayed HA coating. The properties of the composite coatings were optimized by varying the CNT contents. The enhanced properties could be attributed to the use of nano-sized HA particles and CNTs. Compared with the monolithic HA coating, the CNT-reinforced HA coating markedly increased the coating hardness without deteriorating the corrosion resistance or adhesion strength.

Retention of Human-Induced Pluripotent Stem Cells (hiPS) With Injectable HA Hydrogels for Vocal Fold Engineering.

PubMed

Imaizumi, Mitsuyoshi; Li-Jessen, Nicole Y K; Sato, Yuka; Yang, David T; Thibeault, Susan L

2017-04-01

One prospective treatment option for vocal fold scarring is regeneration with an engineered scaffold containing induced pluripotent stem cells (iPS). In the present study, we investigated the feasibility of utilizing an injectable hyaluronic acid (HA) scaffold encapsulated with human-iPS cell (hiPS) for regeneration of vocal folds. Thirty athymic nude rats underwent unilateral vocal fold injury. Contralateral vocal folds served as uninjured controls. Hyaluronic acid hydrogel scaffold, HA hydrogel scaffold containing hiPS, and HA hydrogel scaffold containing hiPS with epidermal growth factor (EGF) were injected in both vocal folds immediately after surgery. One and 2 weeks after injection, larynges were excised for histology, immunohistochemistry, and fluorescence in situ hybridization (FISH). Presence of HA hydrogel was confirmed in vocal folds 1 and 2 weeks post injection. The FISH analysis confirmed the presence and viability of hiPS in the injected vocal folds. Histological results demonstrated that vocal folds injected with HA hydrogel scaffold containing EGF demonstrated less fibrosis than those with HA hydrogel only. Human-iPS survived in injured rat vocal folds. The HA hydrogel with hiPS and EGF ameliorated the fibrotic response. Additional work is necessary to optimize hiPS differentiation and further confirm the safety of hiPS for clinical applications.

Twins in spirit - episode I: comparative preclinical evaluation of [(68)Ga]DOTATATE and [(68)Ga]HA-DOTATATE.

PubMed

Schottelius, Margret; Šimeček, Jakub; Hoffmann, Frauke; Willibald, Marina; Schwaiger, Markus; Wester, Hans-Jürgen

2015-01-01

Recently, an intra-patient comparison demonstrated that the somatostatin (sst) ligand [(68)Ga]HA-DOTATATE ([(68)Ga]DOTA-3-iodo-Tyr(3)-octreotate) provides PET images comparable to or superior to those obtained with [(68)Ga]DOTATATE. To provide a comprehensive basis for nevertheless observed slight differences in tracer biodistribution and dosimetry, the characteristics of [(68)Ga]HA-DOTATATE were investigated in a detailed preclinical study. Affinities of (nat)Ga-HA-DOTATATE and (nat)Ga-DOTATATE to sst1-5 were determined using membrane preparations and [(125)I]SST-28 as radioligand. Internalization into AR42J cells was studied in dual-tracer studies with [(125)I]TOC as internal reference. Biodistribution was investigated using AR42J tumor-bearing CD1 mice, and specificity of tracer uptake was confirmed in competition studies by coinjection of 0.8 mg TOC/kg. Sst2 affinities (IC50) of [(nat)Ga]HA-DOTATATE (1.4 ± 0.8 nM, logP: -3.16) and [(nat)Ga]DOTATATE (1.2 ± 0.6 nM, logP: -3.69) were nearly identical. Both compounds displayed IC50 > 1 μM for sst1,3,4, while sst5 affinity was markedly increased for (nat)Ga-HA-DOTATATE (102 ± 65 nM vs >1 μM for (nat)Ga-DOTATATE). [(nat)Lu]HA-DOTATATE and [(nat)Lu]DOTATATE showed slightly lower, identical sst2 affinities (2.0 ± 1.6 and 2.0 ± 0.8 nM, respectively) and sst3 affinities of 93 ± 1 and 162 ± 16 nM. Internalization of [(68)Ga]HA-DOTATATE was tenfold higher than that of [(125)I]TOC but only sixfold higher for [(68)Ga]DOTATATE and [(177)Lu]HA-DOTATATE. While [(68)Ga]HA-DOTATATE and [(68)Ga]DOTATATE had shown similar target- and non-target uptake in patients, biodistribution studies in mice at 1 h post injection (n = 5) revealed slightly increased non-specific uptake of [(68)Ga]HA-DOTATATE in the blood, liver, and intestines (0.7 ± 0.3, 1.0 ± 0.2, and 4.0 ± 0.7 %iD/g vs 0.3 ± 0.1, 0.5 ± 0.1, and 2.7 ± 0.8 %iD/g for [(68)Ga]DOTATATE). However, sst

HDPE/HA composites obtained in solution: Effect of the gamma radiation

NASA Astrophysics Data System (ADS)

Carmen, Albano; Arquímedes, Karam; Rosestela, Perera; Gema, González; Nohemy, Domínguez; Jeanette, González; Yanixia, Sánchez

2006-06-01

Radiation is employed to sterilize composite materials used in the biomedical field. Due to the changes induced by radiation onto polymeric materials, it is important to study variations in their melt flow index (MFI), as well as in their mechanical and thermal properties. In this work, those previous parameters were determined in composites obtained via solution of a high-density polyethylene (HDPE) in decalin, with different amounts of hydroxyapatite (HA), varying from 10 to 30 parts per hundred, after being exposed to gamma radiation at absorbed doses between 25 and 100 kGy. After the irradiation, the MFI of HDPE dissolved in decalin and precipitated afterwards and without filler increased from 6 to 24 g/10 min at the highest absorbed doses. This behavior was also observed in composites with 10 pph of HA, being the increase less pronounced, specifically in the range between 50 and 100 kGy. Composites with 20 and 30 pph of HA showed a maximum MFI value at 50 kGy, which decreased at higher doses. This implies that the filler begin to exert an influence because it does not melt at the test temperature and consequently, it does not flow. It was observed that Young's modulus increased with HA addition due to rigidity of the ceramic filler. Radiation did not significantly affect this tensile property. On the other hand, the tensile strength did not show significant variations at the different doses but the filler content did affect this property improving it. Finally, elongation at break showed a drastic decrease with filler addition. When the thermal behavior was studied it was noticed that crystallization and melting temperatures remained unchanged. Instead, crystallinity degree slightly increased in the composites, and a little decrease was obtained when they were irradiated.

Research on torsional friction behavior and fluid load support of PVA/HA composite hydrogel.

PubMed

Chen, Kai; Zhang, Dekun; Yang, Xuehui; Cui, Xiaotong; Zhang, Xin; Wang, Qingliang

2016-09-01

Hydrogels have been extensively studied for use as synthetic articular cartilage. This study aimed to investigate (1) the torsional friction contact state and the transformation mechanism of PVA/HA composite hydrogel against CoCrMo femoral head and (2) effects of load and torsional angle on torsional friction behavior. The finite element method was used to study fluid load support of PVA/HA composite hydrogel. Results show fluid loss increases gradually of PVA/HA composite hydrogel with torsional friction time, leading to fluid load support decreases. The contact state changes from full slip state to stick-slip mixed state. As the load increases, friction coefficient and adhesion zone increase gradually. As the torsional angle increases, friction coefficient and slip trend of the contact interface increase, resulting in the increase of the slip zone and the reduction of the adhesion zone. Fluid loss increases of PVA/HA composite hydrogel as the load and the torsional angle increase, which causes the decrease of fluid load support and the increase of friction coefficient. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of sol-gel processing parameters on the phases and microstructures of HA films.

PubMed

Wang, Diangang; Chen, Chuanzhong; Liu, Xiuna; Lei, Tingquan

2007-06-15

Bioactive hydroxyapatite (HA) films were fabricated by a sol-gel method and triethylphosphate and calcium nitrate were used as the phosphorus and calcium precursors, respectively. The effects of the heat treatment temperature, pH level and substrate materials on the phases and microstructures of HA films were studied by X-ray diffraction (XRD), scanning electronic microscopy (SEM) and electronic probe microanalysis (EPMA) and so on. The results show that all the sol-gel films are composed of the phases of HA, CaO, TiO(2) and CaTiO(3). With increasing the calcining temperature, the crystallinity of the films increases, the structure becomes more compact and changes from granular and lamellar to cellular structure, and the Ca/P ratio increases slightly because of the loss of P in the films. The addition of ammonia (adjusting the pH level to be about 7.5) can increase the HA content in the films, and the difference of substrate materials only has a little influence on the microstructure of the sol-gel films.

A Pilot Investigation regarding Speech-Recognition Performance in Noise for Adults with Hearing Loss in the FM+HA Listening Condition

ERIC Educational Resources Information Center

Lewis, M. Samantha; Gallun, Frederick J.; Gordon, Jane; Lilly, David J.; Crandell, Carl

2010-01-01

While the concurrent use of the hearing aid (HA) microphone with frequency modulation (FM) technology can decrease speech-recognition performance, the FM+HA condition is still an important setting for users of both HA and FM technology. The primary goal of this investigation was to evaluate the effect of attenuating HA gain in the FM+HA listening…

[Detection of intron 22 inversion of factor VIII gene in severe hemophilia A patients].

PubMed

Guo, Zhi-ping; Chen, Jian-fang; Qin, Xiu-yu; Zhang, Yao-fang; Yang, Lin-hua

2013-11-01

To investigate the incidence of intron 22 inversion (INV22) of factor VIII (FVIII) gene in severe hemophilia A (HA) patients, clarify its pathological mechanism, and identify INV22 carrier in the female family members. One-stage method was used to assay the FVIII activity (FVIII:C)in 126 severe HA patients with a median age of 14 years old (range: 4 months-63 years). INV22 was analyzed by long-distance polymerase chain reaction (LD-PCR) and pulsed field gel electrophoresis (PFGE), and pedigree were conducted in 3 involved HA families. Of all the 126 severe HA, 52 (41.3%) cases had the INV22. Four females including 3 mothers and 1 sister of probands were diagnosed as INV22 carriers among 11 suspected carrier mosaicisms from 3 INV22 positive HA families. In 8 females from one family without HA history, the patient's mother was a INV22 carrier, but her maternal grandmother, 2 maternal aunts, 2 female siblings and 1 elder female cousin were negative. LD-PCR and PFGE could be used to diagnose severe HA patients with INV22 and identify the carriers.

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

PubMed

Courtney, David G; Kennedy, Edward M; Dumm, Rebekah E; Bogerd, Hal P; Tsai, Kevin; Heaton, Nicholas S; Cullen, Bryan R

2017-09-13

Many viral RNAs are modified by methylation of the N 6 position of adenosine (m 6 A). m 6 A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m 6 A-modified RNAs, but the effects of m 6 A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m 6 A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m 6 A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m 6 A residues modulate IAV RNA function in cis, we mapped m 6 A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m 6 A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m 6 A residues in IAV transcripts enhance viral gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Chimeric Peptide Tat-HA-NR2B9c Improves Regenerative Repair after Transient Global Ischemia.

PubMed

Zhou, Hai-Hui; Zhang, Li; Zhang, Hai-Xia; Zhang, Jin-Ping; Ge, Wei-Hong

2017-01-01

Transient global ischemia (TGI) is a major public health problem, and it heightens the need of effective treatments. The present study was undertaken to investigate whether recombinant polypeptide Tat-HA-NR2B9c improves spatial learning and memory deficits in rats after TGI. Rats were subjected to 20-min ischemia induced by four-vessel occlusion (4-VO) method and daily injected with Tat-HA-NR2B9c (1.12 mg/kg) for 1 week. Tat-HA-NR2B9c increased CREB activity, upregulated B-cell lymphoma-2 (Bcl-2) expression after treated for 24 h. There was a significant increase in dendrite spine density in hippocampal CA1 region and BrdU-positive cells and BrdU/NeuN-positive cells in the dentate gyrus after Tat-HA-NR2B9c treatment, compared with ischemia group at postischemic day 28. Inhibition of the CREB activation by recombinant lentivirus, LV-CREB133-GFP, abolished the upregulation effects of Tat-HA-NR2B9c on Bcl-2 expression. Moreover, Tat-HA-NR2B9c improved the impaired spatial learning and memory ability in Morris water maze. These results suggest that Tat-HA-NR2B9c substantially ameliorated the TGI-induced loss of dendrite spine in hippocampal CA1, increased neurogenesis in dentate gyrus, and significantly improved cognitive abilities by the CREB pathway in rats after transient global cerebral ischemia. It may be served as a treatment for TGI.

Predicting the emergence of H3N2 influenza viruses reveals contrasted modes of evolution of HA and NA antigens.

PubMed

Sandie, Reatha; Aris-Brosou, Stéphane

2014-01-01

Vaccine design for rapidly changing viruses is based on empirical surveillance of strains circulating in a given season to assess those that will most likely spread during the next season. The choice of which strains to include in the vaccine is critical, as an erroneous decision can lead to a nonimmunized human population that will then be at risk in the face of an epidemic or, worse, a pandemic. Here, we present the first steps toward a very general phylogenetic approach to predict the emergence of novel viruses. Our genomic model builds upon natural features of viral evolution such as selection and recombination / reassortment, and incorporates episodic bursts of evolution and or of recombination. As a proof-of-concept, we assess the performance of this model in a retrospective study, focusing: (i) on the emergence of an unexpected H3N2 influenza strain in 2007, and (ii) on a longitudinal design. Based on the analysis of hemagglutinin (HA) and neuraminidase (NA) genes, our results show a lack of predictive power in both experimental designs, but shed light on the mode of evolution of these two antigens: (i) supporting the lack of significance of recombination in the evolution of this influenza virus, and (ii) showing that HA evolves episodically while NA changes gradually.

Serum IgG titres, but not avidity, correlates with neutralizing antibody response after H5N1 vaccination.

PubMed

Pedersen, Gabriel Kristian; Höschler, Katja; Øie Solbak, Sara Marie; Bredholt, Geir; Pathirana, Rishi Delan; Afsar, Aram; Breakwell, Lucy; Nøstbakken, Jane Kristin; Raae, Arnt Johan; Brokstad, Karl Albert; Sjursen, Haakon; Zambon, Maria; Cox, Rebecca Jane

2014-07-31

Influenza H5N1 virus constitutes a pandemic threat and development of effective H5N1 vaccines is a global priority. Anti-influenza antibodies directed towards the haemagglutinin (HA) define a correlate of protection. Both antibody concentration and avidity may be important for virus neutralization and resolving influenza disease. We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with the immunostimulating complex Matrix M™. Sixty adults were intramuscularly immunized with two vaccine doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M™. Serum H5 HA1-specific antibodies and virus neutralization were determined at days 0, 21, 42, 180 and 360 and long-term memory B cells at day 360 post-vaccination. The binding of the HA specific antibodies was measured by avidity NaSCN-elution ELISA and surface plasmon resonance (SPR). The H5 HA1-specific IgG response peaked after the second dose (day 42), was dominated by IgG1 and IgG3 and was highest in the adjuvanted vaccine groups. IgG titres correlated significantly with virus neutralization at all time points (Spearman r≥0.66, p<0.0001). By elution ELISA, serum antibody avidity was highest at days 180 and 360 post vaccination and did not correlate with virus neutralization. Long-lasting H5 HA1-specific memory B cells produced high IgG antibody avidity similar to serum IgG. Maturation of serum antibody avidity continued up to day 360 after influenza H5N1 vaccination. Virus neutralization correlated with serum H5 HA1-specific IgG antibody concentrations and not antibody avidity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Formation of porous HPCL/LPCL/HA scaffolds with supercritical CO2 gas foaming method.

PubMed

Moghadam, M Zahedi; Hassanajili, Sh; Esmaeilzadeh, F; Ayatollahi, M; Ahmadi, M

2017-05-01

Scaffold is a 3D porous structure that is made of different materials, such as synthetic and natural polymers. It plays the role of a synthetic extracellular matrix and permits adhesion, proliferation and differentiation of the cells. Porosity and pore size are the important factors for any 3D scaffold used in bone tissue engineering. In this study, porous scaffolds were prepared by adding hydroxyapatite (HA) nanoparticles as filler to the polymeric matrix of polycaprolactone (PCL) blends with two different molecular weight by using supercritical CO 2 (ScCO 2 ) foaming method. The effect of different parameters such as CO 2 pressure, ratios of the polymers and amount of the filler on the scaffold properties was investigated. The results showed that porosity increased with increment of pressure and decreased with increasing the ratio of the high molecular weight PCL to the low molecular weight PCL in the scaffolds and also HA content. Optimum condition for obtaining adequate porous scaffold of HPCL/LPCL/HA occurred at 140bar and 45°C. The physical and mechanical properties of the prepared scaffolds were characterized using DSC, XRD, FTIR, SEM, contact angle and compression test. By analyzing the results of these tests, optimum sample for cell culture was selected. The biocompatibility of the selected HPCL/LPCL/HA scaffold (HPCL/LPCL 60/40 containing 2.5% HA) was assessed in vitro by using human mesenchymal stem cells (hMSCs). Copyright © 2016 Elsevier Ltd. All rights reserved.

A Novel Strategy for Preparation of Si-HA Coatings on C/C Composites by Chemical Liquid Vaporization Deposition/Hydrothermal Treatments.

PubMed

Xin-Bo, Xiong; Xin-Ye, Ni; Ya-Yun, Li; Cen-Cen, Chu; Ji-Zhao, Zou; Xie-Rong, Zeng

2016-08-05

A novel strategy for the preparation of Si-doped hydroxyapatite (Si-HA) coatings on H2O2-treated carbon/carbon composites (C/C) was developed. HA coating was prepared on C/C through chemical liquid vaporization deposition (CLVD)/hydrothermal treatment. HA coating was immersed in an H2SiO3 solution at an autoclave at 413 K for transformation into Si-HA coating. The effects of H2SiO3 mass contents on the phase, morphology, and composition of the Si-HA coatings were studied through SEM, EDS,XRD, and FTIR. Their bonding performance to C/C was measured through a scratch test. Under the optimal content condition, the in vitro skull osteoblast response behaviors of the Si-HA coating were evaluated. Results showed that SiO3(2-) could enter into the HA lattice and occupy the PO4(3-) sites. Doped SiO3(2-) significantly improved the bonding performance of the HA coating to C/C in comparison with the untreated HA. The adhesive strength of the coatings initially increased and then decreased with increasing H2SiO3 content. Meanwhile, the cohesive strength of the Si-HA coatings was almost nearly identical. The Si-HA coating achieved at a content of 90% H2SiO3 exhibited the best bonding performance, and its osteoblast compatibility in vitro was superior to that of the untreated HA coating on C/C through CLVD/hydrothermal treatment.

A Novel Strategy for Preparation of Si-HA Coatings on C/C Composites by Chemical Liquid Vaporization Deposition/Hydrothermal Treatments

PubMed Central

Xin-bo, Xiong; Xin-ye, Ni; Ya-yun, Li; Cen-cen, Chu; Ji-zhao, Zou; Xie-rong, Zeng

2016-01-01

A novel strategy for the preparation of Si-doped hydroxyapatite (Si-HA) coatings on H2O2-treated carbon/carbon composites (C/C) was developed. HA coating was prepared on C/C through chemical liquid vaporization deposition (CLVD)/hydrothermal treatment. HA coating was immersed in an H2SiO3 solution at an autoclave at 413 K for transformation into Si-HA coating. The effects of H2SiO3 mass contents on the phase, morphology, and composition of the Si-HA coatings were studied through SEM, EDS,XRD, and FTIR. Their bonding performance to C/C was measured through a scratch test. Under the optimal content condition, the in vitro skull osteoblast response behaviors of the Si-HA coating were evaluated. Results showed that SiO32− could enter into the HA lattice and occupy the PO43− sites. Doped SiO32− significantly improved the bonding performance of the HA coating to C/C in comparison with the untreated HA. The adhesive strength of the coatings initially increased and then decreased with increasing H2SiO3 content. Meanwhile, the cohesive strength of the Si-HA coatings was almost nearly identical. The Si-HA coating achieved at a content of 90% H2SiO3 exhibited the best bonding performance, and its osteoblast compatibility in vitro was superior to that of the untreated HA coating on C/C through CLVD/hydrothermal treatment. PMID:27492664

7 CFR 1948.88 - Direct land acquisition by FmHA or its successor agency under Public Law 103-354.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 13 2011-01-01 2009-01-01 true Direct land acquisition by FmHA or its successor... by FmHA or its successor agency under Public Law 103-354. (a) FmHA or its successor agency under... Governor of the State in which the real property is located. FmHA or its successor agency under Public Law...

7 CFR 1945.27 - Relationship between FCIC and FmHA or its successor agency under Public Law 103-354.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 13 2011-01-01 2009-01-01 true Relationship between FCIC and FmHA or its successor...) EMERGENCY Disaster Assistance-General § 1945.27 Relationship between FCIC and FmHA or its successor agency under Public Law 103-354. (a) General. Exhibit A of FmHA Instruction 2000-N (available in any FmHA or...

7 CFR 1948.88 - Direct land acquisition by FmHA or its successor agency under Public Law 103-354.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 13 2010-01-01 2009-01-01 true Direct land acquisition by FmHA or its successor... by FmHA or its successor agency under Public Law 103-354. (a) FmHA or its successor agency under... Governor of the State in which the real property is located. FmHA or its successor agency under Public Law...

Genetic Characterization of Circulating 2015 A(H1N1)pdm09 Influenza Viruses from Eastern India

PubMed Central

Mukherjee, Anupam; Nayak, Mukti Kant; Dutta, Shanta; Panda, Samiran; Satpathi, Biswa Ranjan; Chawla-Sarkar, Mamta

2016-01-01

In 2015, the swine derived A(H1N1)pdm09 pandemic strain outbreak became widespread throughout the different states of India. The reported cases and deaths in 2015 surpassed the previous years with more than 39000 laboratory confirmed cases and a death toll of more than 2500 people. There are relatively limited complete genetic sequences available for this virus from Asian countries. In this study, we describe the full genome analysis of influenza 2015 A(H1N1)pdm09 viruses isolated from West Bengal between January through December 2015. The phylogenetic analysis of the haemagglutinin sequence revealed clustering with globally circulating strains of genogroup 6B. This was further confirmed by the constructed concatenated tree using all eight complete gene segments of Kolkata A(H1N1)pdm09 isolates with the other strains from different timeline and lineages. A study from Massachusetts Institute of Technology (MIT) in 2015 reported novel mutations T200A and D225N in haemagglutinin gene of a 2014 Indian strain (A/India/6427/2014). However, in all the pandemic strains of 2014–2015 reported from India, so far including A(H1N1)pdm09 strains from Kolkata, D225N mutation was not observed, though the T200A mutation was found to be conserved. Neuraminidase gene of the analyzed strains did not show any oseltamivir resistant mutation H275Y suggesting continuation of Tamiflu® as drug of choice. The amino acid sequences of the all gene segments from 2015 A(H1N1)pdm09 isolates identified several new mutations compared to the 2009 A(H1N1)pdm09 strains, which may have contributed towards enhanced virulence, compared to 2009 A(H1N1)pdm09 strains. PMID:27997573

Genetic Characterization of Circulating 2015 A(H1N1)pdm09 Influenza Viruses from Eastern India.

PubMed

Mukherjee, Anupam; Nayak, Mukti Kant; Dutta, Shanta; Panda, Samiran; Satpathi, Biswa Ranjan; Chawla-Sarkar, Mamta

2016-01-01

In 2015, the swine derived A(H1N1)pdm09 pandemic strain outbreak became widespread throughout the different states of India. The reported cases and deaths in 2015 surpassed the previous years with more than 39000 laboratory confirmed cases and a death toll of more than 2500 people. There are relatively limited complete genetic sequences available for this virus from Asian countries. In this study, we describe the full genome analysis of influenza 2015 A(H1N1)pdm09 viruses isolated from West Bengal between January through December 2015. The phylogenetic analysis of the haemagglutinin sequence revealed clustering with globally circulating strains of genogroup 6B. This was further confirmed by the constructed concatenated tree using all eight complete gene segments of Kolkata A(H1N1)pdm09 isolates with the other strains from different timeline and lineages. A study from Massachusetts Institute of Technology (MIT) in 2015 reported novel mutations T200A and D225N in haemagglutinin gene of a 2014 Indian strain (A/India/6427/2014). However, in all the pandemic strains of 2014-2015 reported from India, so far including A(H1N1)pdm09 strains from Kolkata, D225N mutation was not observed, though the T200A mutation was found to be conserved. Neuraminidase gene of the analyzed strains did not show any oseltamivir resistant mutation H275Y suggesting continuation of Tamiflu® as drug of choice. The amino acid sequences of the all gene segments from 2015 A(H1N1)pdm09 isolates identified several new mutations compared to the 2009 A(H1N1)pdm09 strains, which may have contributed towards enhanced virulence, compared to 2009 A(H1N1)pdm09 strains.

Hierarchical Structure and Mechanical Improvement of an n-HA/GCO-PU Composite Scaffold for Bone Regeneration.

PubMed

Li, Limei; Zuo, Yi; Zou, Qin; Yang, Boyuan; Lin, Lili; Li, Jidong; Li, Yubao

2015-10-14

To improve the mechanical properties of bone tissue and achieve the desired bone tissue regeneration for orthopedic surgery, newly designed hydroxyapatite/polyurethane (HA/PU) porous scaffolds were developed via in situ polymerization. The results showed that the molecular modification of PU soft segments by glyceride of castor oil (GCO) can increase the scaffold compressive strength by 48% and the elastic modulus by 96%. When nano-HA (n-HA) particles were incorporated into the GCO-PU matrix, the compressive strength and elastic modulus further increased by 49 and 74%, from 2.91 to 4.34 MPa and from 95 to 165.36 MPa, respectively. The n-HA particles with fine dispersity not only improved the interface bonding with the GCO-PU matrix but also provided effective bioactivity for bonding with bone tissue. The hierarchical structure and mechanical quality of the n-HA/GCO-PU composite scaffold were determined to be appropriate for the growth of cells and the regeneration of bony tissues, demonstrating promising prospects for bone repair and regeneration.

Characterization and corrosion property of nano-rod-like HA on fluoride coating supported on Mg-Zn-Ca alloy.

PubMed

Feng, Yashan; Zhu, Shijie; Wang, Liguo; Chang, Lei; Yan, Bingbing; Song, Xiaozhe; Guan, Shaokang

2017-06-01

The poor corrosion resistance of biodegradable magnesium alloys is the dominant factor that limits their clinical application. In this study, to deal with this challenge, fluoride coating was prepared on Mg-Zn-Ca alloy as the inner coating and then hydroxyapatite (HA) coating as the outer coating was deposited on fluoride coating by pulse reverse current electrodeposition (PRC-HA/MgF 2 ). As a comparative study, the microstructure and corrosion properties of the composite coating with the outer coating fabricated by traditional constant current electrodeposition (TED-HA/MgF 2 ) were also investigated. Scanning electron microscopy (SEM) images of the coatings show that the morphology of PRC-HA/MgF 2 coating is dense and uniform, and presents nano-rod-like structure. Compared with that of TED-HA/MgF 2 , the corrosion current density of Mg alloy coated with PRC-HA/MgF 2 coatings decreases from 5.72 × 10 -5 A/cm 2 to 4.32 × 10 -7 A/cm 2 , and the corrosion resistance increases by almost two orders of magnitude. In immersion tests, samples coated with PRC-HA/MgF 2 coating always show the lowest hydrogen evolution amount, and could induce deposition of the hexagonal structure-apatite on the surface rapidly. The results show that the corrosion resistance and the bioactivity of the coatings have been improved by adopting double-pulse current mode in the process of preparing HA on fluoride coating, and the PRC-HA/MgF 2 coating is worth of further investigation.

H9N2 low pathogenic avian influenza in Pakistan (2012-2015).

PubMed

Lee, Dong-Hun; Swayne, David E; Sharma, Poonam; Rehmani, Shafqat Fatima; Wajid, Abdul; Suarez, David L; Afonso, Claudio

2016-01-01

Significant economic losses from deaths and decreased egg production have resulted from H9N2 low pathogenic avian influenza virus (LPAIV) infections in poultry across North Africa, the Middle East and Asia. The H9N2 LPAIVs have been endemic in Pakistani poultry since 1996, but no new viruses have been reported since 2010. Because novel genotypes of Pakistani H9N2 contain mammalian host-specific markers, recent surveillance is essential to better understand any continuing public health risk. Here the authors report on four new H9N2 LPAIVs, three from 2015 and one from 2012. All of the viruses tested in this study belonged to Middle East B genetic group of G1 lineage and had PAKSSR/G motif at the haemagglutinin cleavage site. The mammalian host-specific markers at position 226 in the haemagglutinin receptor-binding site and internal genes suggest that Pakistan H9N2 viruses are still potentially infectious for mammals. Continued active surveillance in poultry and mammals is needed to monitor the spread and understand the potential for zoonotic infection by these H9N2 LPAIVs.

Liver functional genomics in beef cows on grazing systems: novel genes and pathways revealed.

PubMed

Laporta, Jimena; Rosa, Guilherme J M; Naya, Hugo; Carriquiry, Mariana

2014-02-15

The adaptation of the liver to periods of negative energy balance is largely unknown in beef cattle on grazing systems. We evaluated liver transcriptome throughout gestation and early lactation of purebred and crossbred beef cows [Angus, Hereford, and their F1 crossbreeds (CR)], grazing high or low herbage allowances (HA) of native grasslands (4 and 2.5 kg dry matter/kg body wt annual mean; n = 16) using an Agilent 4 × 44k bovine array. A total of 4,661 transcripts were affected by days [272 ≥ 2.5-fold difference, false discovery rate (FDR) ≤ 0.10] and 47 pathways were altered during winter gestation (-165 to -15 days relative to calving), when cows experienced decreased body condition score, decreased insulin, and increased nonesterified fatty acid concentrations. Gluconeogenesis and fatty acid oxidation pathways were upregulated, while cell growth, DNA replication, and transcription pathways were downregulated (FDR ≤ 0.25). We observed only small changes in the liver transcriptome during early lactation (+15 to +60 days). A total of 225 genes were differentially expressed (47 ≥ 2-fold difference, FDR ≤ 0.10) between HA. The majority of those were related to glucose and pyruvate metabolism and were upregulated in high HA, reflecting their better metabolic status. Two genes were upregulated in CR cows, but 148 transcripts (74 ≥ 2-fold change difference, FDR ≤ 0.10) were affected by the HA and cow genotype interaction. The transcriptional changes observed indicated a complex and previously unrecognized, hepatic adaptive program of grazing beef cows in different nutritional environments. Novel target candidate genes, metabolic pathways, and regulatory mechanisms were reported.

Burkholderia Hep_Hap autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

PubMed Central

Tiyawisutsri, Rachaneeporn; Holden, Matthew TG; Tumapa, Sarinna; Rengpipat, Sirirat; Clarke, Simon R; Foster, Simon J; Nierman, William C; Day, Nicholas PJ; Peacock, Sharon J

2007-01-01

Background The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. Results Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. Conclusion Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study. PMID:17362501

Burkholderia Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis.

PubMed

Tiyawisutsri, Rachaneeporn; Holden, Matthew T G; Tumapa, Sarinna; Rengpipat, Sirirat; Clarke, Simon R; Foster, Simon J; Nierman, William C; Day, Nicholas P J; Peacock, Sharon J

2007-03-15

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study.

The genomic evolution of H1 influenza A viruses from swine detected in the United States between 2009 and 2016.

PubMed

Gao, Shibo; Anderson, Tavis K; Walia, Rasna R; Dorman, Karin S; Janas-Martindale, Alicia; Vincent, Amy L

2017-08-01

Transmission of influenza A virus (IAV) from humans to swine occurs with relative frequency and is a critical contributor to swine IAV diversity. Subsequent to the introduction of these human seasonal lineages, there is often reassortment with endemic viruses and antigenic drift. To address whether particular genome constellations contributed to viral persistence following the introduction of the 2009 H1N1 human pandemic virus to swine in the USA, we collated and analysed 616 whole genomes of swine H1 isolates. For each gene, sequences were aligned, the best-known maximum likelihood phylogeny was inferred, and each virus was assigned a clade based upon its evolutionary history. A time-scaled Bayesian approach was implemented for the haemagglutinin (HA) gene to determine the patterns of genetic diversity over time. From these analyses, we observed an increase in genome diversity across all H1 lineages and clades, with the H1-γ and H1-δ1 genetic clades containing the greatest number of unique genome patterns. We documented 74 genome patterns from 2009 to 2016, of which 3 genome patterns were consistently detected at a significantly higher level than others across the entire time period. Eight genome patterns increased significantly, while five genome patterns were shown to decline in detection over time. Viruses with genome patterns identified as persisting in the US swine population may possess a greater capacity to infect and transmit in swine. This study highlights the emerging genetic diversity of US swine IAV from 2009 to 2016, with implications for swine and public health and vaccine control efforts.

Silicon-hydroxyapatite bioactive coatings (Si-HA) from diatomaceous earth and silica. Study of adhesion and proliferation of osteoblast-like cells.

PubMed

López-Alvarez, M; Solla, E L; González, P; Serra, J; León, B; Marques, A P; Reis, R L

2009-05-01

The aim of this study consisted on investigating the influence of silicon substituted hydroxyapatite (Si-HA) coatings over the human osteoblast-like cell line (SaOS-2) behaviour. Diatomaceous earth and silica, together with commercial hydroxyapatite were respectively the silicon and HA sources used to produce the Si-HA coatings. HA coatings with 0 wt% of silicon were used as control of the experiment. Pulsed laser deposition (PLD) was the selected technique to deposit the coatings. The Si-HA thin films were characterized by Fourier Transformed Infrared Spectroscopy (FTIR) demonstrating the efficient transfer of Si to the HA structure. The in vitro cell culture was established to assess the cell attachment, proliferation and osteoblastic activity respectively by, Scanning Electron Microscopy (SEM), DNA and alkaline phosphatase (ALP) quantification. The SEM analysis demonstrated a similar adhesion behaviour of the cells on the tested materials and the maintenance of the typical osteoblastic morphology along the time of culture. The Si-HA coatings did not evidence any type of cytotoxic behaviour when compared with HA coatings. Moreover, both the proliferation rate and osteoblastic activity results showed a slightly better performance on the Si-HA coatings from diatoms than on the Si-HA from silica.

Removal of Uranium in Drinking Water: Brimac Environmental Services, Inc. Brimac HA 216 Adsorptive Media

EPA Science Inventory

The Brimac HA 216 Adsorptive Media was tested for uranium (U) removal from a drinking water source (well water) at Grappone Toyota located in Bow, New Hampshire. The HA 216 media is a hydroxyapatite-based material. A pilot unit, consisting of a TIGG Corporation Cansorb® C-5 ste...

24 CFR 964.135 - Resident involvement in HA management operations.

Code of Federal Regulations, 2010 CFR

2010-04-01

... management operations. 964.135 Section 964.135 Housing and Urban Development Regulations Relating to Housing... Tenant Participation § 964.135 Resident involvement in HA management operations. Residents shall be... responsibility for management operations, it shall ensure strong resident participation in all issues and facets...

Combination of plasma HA and circulating M2-like monocytes may serve as a diagnostic marker for breast cancer.

PubMed

Zhang, Boke; Cao, Manlin; He, Yiqing; Liu, Yiwen; Zhang, Guoliang; Yang, Cuixia; Du, Yan; Xu, Jing; Hu, Jiajie; Gao, Feng

2017-01-01

Background: Breast cancer (BC)-derived hyaluronan (HA) can induce the formation of M2-like tumor-associated macrophages (TAMs) in tumor context. However, little is known about the correlation between circulating M2-like monocytes and plasma HA in BC patients. This study focused on evaluating the relationship between circulating M2-like monocytes and plasma HA, and further appraised the diagnostic value of them in BC. Methods: The expression of M2-like TAMs and HA was determined in pathological tissues by immunohistochemistry. Flow cytometry was used to detect the levels of circulating CD14 + CD204 + M2-like monocytes in 81 BC patients, 45 patients with breast benign diseases, and 46 healthy subjects. The levels of HA, CEA, and CA15-3 were measured in plasma samples using chemiluminescence method. Results: M2-like TAMs and HA expressions were elevated in BC tissues compared with benign tissues. In correspondence, the frequency of circulating CD14 + CD204 + M2-like monocytes and the plasma HA levels were significantly higher in patients with BC than those in control groups. Importantly, there was a positive correlation between circulating M2-like monocytes and the plasma HA (Spearman r = 0.404, p < 0.001). Area under receiver operating characteristic curve (ROC) for the combination of circulating M2-like monocytes and HA was 0.899 (95% CI: 0.853-0.946), which was higher than the panel of CEA and CA15-3. Conclusions: The frequency of circulating CD14 + CD204 + M2-like monocytes was positively correlated to plasma HA levels. The combination of circulating CD14 + CD204 + M2-like monocytes and plasma HA could provide considerable diagnostic value in BC.

Prime-boost vaccination with recombinant H5-fowlpox and Newcastle disease virus vectors affords lasting protection in SPF Muscovy ducks against highly pathogenic H5N1 influenza virus.

PubMed

Niqueux, Eric; Guionie, Olivier; Amelot, Michel; Jestin, Véronique

2013-08-28

Vaccination protocols were evaluated in one-day old Muscovy ducklings, using an experimental Newcastle disease recombinant vaccine (vNDV-H5) encoding an optimized synthetic haemagglutinin gene from a clade 2.2.1 H5N1 highly pathogenic (HP) avian influenza virus (AIV), either as a single administration or as a boost following a prime inoculation with a fowlpox vectored vaccine (vFP89) encoding a different H5 HP haemagglutinin from an Irish H5N8 strain. These vaccination schemes did not induce detectable levels of serum antibodies in HI test using a clade 2.2.1 H5N1 antigen, and only induced H5 ELISA positive response in less than 10% of vaccinated ducks. However, following challenge against a clade 2.2.1 HPAIV, both protocols afforded full clinical protection at six weeks of age, and full protection against mortality at nine weeks. Only the prime-boost vaccination (vFP89+vNDV-H5) was still fully protecting Muscovy ducks against disease and mortality at 12 weeks of age. Reduction of oropharyngeal shedding levels was also constantly observed from the onset of the follow-up at 2.5 or three days post-infection in vaccinated ducks compared to unvaccinated controls, and was significantly more important for vFP89+vNDV-H5 vaccination than for vNDV-H5 alone. Although the latter vaccine is shown immunogenic in one-day old Muscovy ducks, the present work is original in demonstrating the high efficacy of the successive administration of two different vector vaccines encoding two different H5 in inducing lasting protection (at least similar to the one induced by an inactivated reassortant vaccine, Re-5). In addition, such a prime-boost schedule allows implementation of a DIVA strategy (to differentiate vaccinated from infected ducks) contrary to Re-5, involves easy practice on the field (with injection at the hatchery and mass vaccination later on), and should avoid eventual interference with NDV maternally derived antibodies. Last, the HA insert could be updated according to

Influence of Morinda citrifolia (Noni) on Expression of DNA Repair Genes in Cervical Cancer Cells.

PubMed

Gupta, Rakesh Kumar; Bajpai, Deepti; Singh, Neeta

2015-01-01

Previous studies have suggested that Morinda citrifolia (Noni) has potential to reduce cancer risk. The purpose of this study was to investigate the effect of Noni, cisplatin, and their combination on DNA repair genes in the SiHa cervical cancer cell line. SiHa cells were cultured and treated with 10% Noni, 10 μg/dl cisplatin or their combination for 24 hours. Post culturing, the cells were pelleted, RNA extracted, and processed for investigating DNA repair genes by real time PCR. The expression of nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and base excision repair gene XRCC1 was increased 4 fold, 8.9 fold, 4 fold, and 5.5 fold, respectively, on treatment with Noni as compared to untreated controls (p<0.05). In contrast, expression was found to be decreased 22 fold, 13 fold, 16 fold, and 23 fold on treatment with cisplatin (p<0.05). However, the combination of Noni and cisplatin led to an increase of 2 fold, 1.6 fold, 3 fold, 1.2 fold, respectively (p<0.05). Noni enhanced the expression of DNA repair genes by itself and in combination with cisplatin. However, high expression of DNA repair genes at mRNA level only signifies efficient DNA transcription of the above mentioned genes; further investigations are needed to evaluate the DNA repair protein expression.

[Frequency of intron 1 inversion of factor VIII gene in Chinese hemophilia A patients with case report of a female patient with heterozygous intron 1 inversion].

PubMed

Yan, Zhen-yu; Liang, Yan; Yan, Mei; Fan, Lian-kai; Xiao, Bai; Hua, Bao-lai; Liu, Jing-zhong; Zhao, Yong-qiang

2008-10-21

To investigate the frequency of intron 1 inversion (inv1) in FVIII gene in Chinese hemophilia A (HA) patients and to investigate the mechanism of pathogenesis. Peripheral blood samples were collected from 158 unrelated HA patients, aged 20 (1 - 73), including one female HA patient, aged 5, and several family members of a patient positive in inv1. One-stage method was used to assay the FVIII activity (FVIII:C). Long distance PCR and multiple PCR in duplex reactions were used to screen for the intron 22 inversion (inv22) and inv1 of the FVIII coding gene (F8). The F8 coding sequence was amplified with PCR and sequenced with an automatic sequencer. Two unrelated patients (pedigrees) were detected as inv1 positive with a positive rate of 1.26%. A rare female HA patient with inv1 was also discovered in a positive family (3 HA cases were found in this family and regarded as one case in calculating the total detection rate). The full length of FVIII was sequenced, and no other mutation was detected. There frequency of FVIII inv1 is low in Chinese HA patients compared with other populations. Female HA patients are heterozygous for FVIII inv1 and that may be resulted from nonrandom inactivation of X chromosome.

Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression

PubMed Central

Ha, Shin-Woo; Jang, Hae Lin; Nam, Ki Tae; Beck, George R.

2015-01-01

Hydroxyapatite (HA) is the primary structural component of the skeleton and dentition. Under biological conditions, HA does not occur spontaneously and therefore must be actively synthesized by mineralizing cells such as osteoblasts. The mechanism(s) by which HA is actively synthesized by cells and deposited to create a mineralized matrix are not fully understood and the consequences of mineralization on cell function are even less well understood. HA can be chemically synthesized (HAp) and is therefore currently being investigated as a promising therapeutic biomaterial for use as a functional scaffold and implant coating for skeletal repair and dental applications. Here we investigated the biological effects of nano-HAp (10×100 nm) on the lineage commitment and differentiation of bone forming osteoblasts. Exposure of early stage differentiating osteoblasts resulted in dramatic and sustained changes in gene expression, both increased and decreased, whereas later stage osteoblasts were much less responsive. Analysis of the promoter region one of the most responsive genes, alkaline phosphatase, identified the stimulation of DNA methylation following cell exposure to nano-HAp. Collectively, the results reveal the novel epigenetic regulation of cell function by nano-HAp which has significant implication on lineage determination as well as identifying a novel potential therapeutic use of nanomaterials. PMID:26141836

Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression.

PubMed

Ha, Shin-Woo; Jang, Hae Lin; Nam, Ki Tae; Beck, George R

2015-10-01

Hydroxyapatite (HA) is the primary structural component of the skeleton and dentition. Under biological conditions, HA does not occur spontaneously and therefore must be actively synthesized by mineralizing cells such as osteoblasts. The mechanism(s) by which HA is actively synthesized by cells and deposited to create a mineralized matrix are not fully understood and the consequences of mineralization on cell function are even less well understood. HA can be chemically synthesized (HAp) and is therefore currently being investigated as a promising therapeutic biomaterial for use as a functional scaffold and implant coating for skeletal repair and dental applications. Here we investigated the biological effects of nano-HAp (10 × 100 nm) on the lineage commitment and differentiation of bone forming osteoblasts. Exposure of early stage differentiating osteoblasts resulted in dramatic and sustained changes in gene expression, both increased and decreased, whereas later stage osteoblasts were much less responsive. Analysis of the promoter region one of the most responsive genes, alkaline phosphatase, identified the stimulation of DNA methylation following cell exposure to nano-HAp. Collectively, the results reveal the novel epigenetic regulation of cell function by nano-HAp which has significant implication on lineage determination as well as identifying a novel potential therapeutic use of nanomaterials. Published by Elsevier Ltd.

Performance evaluation of the Arkray Adams HA-8160 HbA1c analyser.

PubMed

Thevarajah, T Malathi; Nani, Nordin; Chew, Y Y

2008-12-01

HbA1c measurement is currently routinely used to predict long term outcome of diabetes, thus playing a fundamental role in the management of diabetes. The relationship between HbA1c value and long term diabetic complications has been established by a randomised control Diabetes Control and Complications Trial (DCCT) which used high performance liquid chromatography (HPLC) as a reference method for HbA1c assay. To ensure that HbA1c results from a variety HbA1c assay methods are similar to the DCCT values, the American Diabetes Association (ADA) recommended that all laboratories should use methods certified by the National Glycohemoglobin Standardization Programme (NGSP) with interassay coefficient variation (CV) of < 5% (ideally < 3%). The International Federation of Clinical Chemistry (IFCC) working group on HbA1c standardisation has set a CV < 2.5% as a criteria for its reference laboratories. To evaluate the performance of Arkray Adams HA-8160 HbA1c analyser which uses a cation exchange HPLC method and its correlation to HbA1c assay on Cobas Integra 800 which is an immunoturbidimetric method. For the imprecision study, patient samples and control material of two levels were analysed on HA-8160 analyser 20 times in a single run (within-run imprecision) and twice a day on five consecutive days (between-run imprecision). For the recovery study, two samples each with high and low values were selected and mixed in ratios of 1:3, 1:1 and 3:1, and were analysed by HA-8160. Sixty samples were analysed by both Cobas Integra 800 and HA-8160 for method comparison study. Ten uraemic samples and ten thalassaemic samples were assayed on Cobas Integra 800 and HA 8160 for interference study. Within-run CVs were 0.6% and 0.7% for medium and high value samples respectively, 0.6% and 0.7% for low and high level controls respectively. Between-run CVs were 0.5% and 0.4% for medium and high value samples respectively, 0.5% and 0.6% for low and high level controls respectively. The

7 CFR 1948.89 - Land condemnation by FmHA or its successor agency under Public Law 103-354.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 13 2011-01-01 2009-01-01 true Land condemnation by FmHA or its successor agency... DEVELOPMENT Section 601 Energy Impacted Area Development Assistance Program § 1948.89 Land condemnation by FmHA or its successor agency under Public Law 103-354. (a) If FmHA or its successor agency under Public...

7 CFR 1948.89 - Land condemnation by FmHA or its successor agency under Public Law 103-354.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 13 2010-01-01 2009-01-01 true Land condemnation by FmHA or its successor agency... DEVELOPMENT Section 601 Energy Impacted Area Development Assistance Program § 1948.89 Land condemnation by FmHA or its successor agency under Public Law 103-354. (a) If FmHA or its successor agency under Public...

Tradeoff between reproduction and resistance evolution to Bt-toxin in Helicoverpa armigera: regulated by vitellogenin gene expression.

PubMed

Zhang, W N; Xiao, H J; Liang, G M; Guo, Y Y; Wu, K M

2014-08-01

Evolution of resistance to insecticides usually has fitness tradeoffs associated with adaptation to the stress. The basic regulation mechanism of tradeoff between reproduction and resistance evolution to Bacillus thuringiensis (Bt) toxin in the cotton bollworm, Helicoverpa armigera (Ha), based on the vitellogenin (Vg) gene expression was analyzed here. The full-length cDNA of the Vg gene HaVg (JX504706) was cloned and identified. HaVg has 5704 base pairs (bp) with an open reading frame (ORF) of 5265 bp, which encoded 1756 amino acid protein with a predicted molecular mass of 197.28 kDa and a proposed isoelectric point of 8.74. Sequence alignment analysis indicated that the amino acid sequence of HaVg contained all of the conserved domains detected in the Vgs of the other insects and had a high similarity with the Vgs of the Lepidoptera insects, especially Noctuidae. The resistance level to Cry1Ac Bt toxin and relative HaVg mRNA expression levels among the following four groups: Cry1Ac-susceptible strain (96S), Cry1Ac-resistant strain fed on artificial diet with Bt toxin for 135 generations (BtR stands for the Cry1Ac Bt resistance), progeny of the Cry1Ac-resistant strain with a non-Bt-toxin artificial diet for 38 generations (CK1) and the direct descendants of the 135th-generation resistant larvae which were fed on an artificial diet without the Cry1Ac protein (CK2) were analyzed. Compared with the 96S strain, the resistance ratios of the BtR strain, the CK1 strain and the CK2 strain were 2917.15-, 2.15- and 2037.67-fold, respectively. The maximum relative HaVg mRNA expression levels of the BtR strain were approximately 50% less than that of the 96S strain, and the coming of maximum expression was delayed for approximately 4 days. The overall trend of the HaVg mRNA expression levels in the CK1 strain was similar to that in the 96S strain, and the overall trend of the HaVg mRNA expression levels in the CK2 strain was similar to that in the BtR strain. Our results

Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs.

PubMed

Callan, Mary Beth; Haskins, Mark E; Wang, Ping; Zhou, Shangzhen; High, Katherine A; Arruda, Valder R

2016-01-01

Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1-2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs.

Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs

PubMed Central

Callan, Mary Beth; Haskins, Mark E.; Wang, Ping; Zhou, Shangzhen; High, Katherine A.; Arruda, Valder R.

2016-01-01

Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1–2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs. PMID:27011017

Electrochemical Characteristics of Cell Cultured Ti-Nb-Zr Alloys After Nano-Crystallized Si-HA Coating.

PubMed

Jeong, Yong-Hoon; Choe, Han-Cheol

2015-01-01

The aim of this study was to investigate the electrochemical characteristics of nano crystallized Si-HA coating on Ti-Nb-Zr alloy after human osteoblast like (HOB) cell attachment. The Ti-Nb-Zr alloy was manufactured with 35 wt.% of Nb and 10 wt.% of Zr by arc melting furnace to appropriate physical properties as biomaterials. The HA and Si-substituted coatings were prepared by electron-beam physical vapor deposition method with 0.5, 0.8 and 1.2 wt.% of Si contents, and nano aging treatment was performed 500 degrees C for 1 h. The characteristics of coating surface were analyzed by field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction, respectively. To evaluate of cell attachment on cell cultured surface, the potentiodynamic test was performed on the surface using HOB cells. The results showed that the Si-HA coating surface showed higher tendency of cell attachment than that of single HA coating, corrosion resistance value was increased by dense of cell attachment.

Effects of Trichothecene Production on the Plant Defense Response and Fungal Physiology: Overexpression of the Trichoderma arundinaceum tri4 Gene in T. harzianum

PubMed Central

Cardoza, R. E.; McCormick, S. P.; Malmierca, M. G.; Olivera, E. R.; Alexander, N. J.; Monte, E.

2015-01-01

Trichothecenes are fungal sesquiterpenoid compounds, the majority of which have phytotoxic activity. They contaminate food and feed stocks, resulting in potential harm to animals and human beings. Trichoderma brevicompactum and T. arundinaceum produce trichodermin and harzianum A (HA), respectively, two trichothecenes that show different bioactive properties. Both compounds have remarkable antibiotic and cytotoxic activities, but in addition, trichodermin is highly phytotoxic, while HA lacks this activity when analyzed in vivo. Analysis of Fusarium trichothecene intermediates led to the conclusion that most of them, with the exception of the hydrocarbon precursor trichodiene (TD), have a detectable phytotoxic activity which is not directly related to the structural complexity of the intermediate. In the present work, the HA intermediate 12,13-epoxytrichothec-9-ene (EPT) was produced by expression of the T. arundinaceum tri4 gene in a transgenic T. harzianum strain that already produces TD after transformation with the T. arundinaceum tri5 gene. Purified EPT did not show antifungal or phytotoxic activity, while purified HA showed both antifungal and phytotoxic activities. However, the use of the transgenic T. harzianum tri4 strain induced a downregulation of defense-related genes in tomato plants and also downregulated plant genes involved in fungal root colonization. The production of EPT by the transgenic tri4 strain raised levels of erg1 expression and reduced squalene accumulation while not affecting levels of ergosterol. Together, these results indicate the complex interactions among trichothecene intermediates, fungal antagonists, and host plants. PMID:26150463

Low-pathogenic avian influenza virus A/turkey/Ontario/6213/1966 (H5N1) is the progenitor of highly pathogenic A/turkey/Ontario/7732/1966 (H5N9)

PubMed Central

Ping, Jihui; Selman, Mohammed; Tyler, Shaun; Forbes, Nicole; Keleta, Liya

2012-01-01

The first confirmed outbreak of highly pathogenic avian influenza (HPAI) virus infections in North America was caused by A/turkey/Ontario/7732/1966 (H5N9); however, the phylogeny of this virus is largely unknown. This study performed genomic sequence analysis of 11 avian influenza isolates from 1956 to 1979 for comparison with A/turkey/Ontario/7732/1966 (H5N9). Phylogenetic and genetic analyses included these viruses in combination with all known full-genome sequences of avian viruses isolated before 1981. It was shown that a low-pathogenic avian influenza virus, A/turkey/Ontario/6213/1966 (H5N1), that had been isolated 3 months previously, was the closest known genetic relative with six genome segments of common lineage encoding the polymerase subunits PB2, PB1 and PA, nucleoprotein (NP), haemagglutinin (HA) and non-structural (NS) proteins. The lineages of these genome segments included reassortment with other North American turkey viruses that were all rooted in North American wild waterfowl with the HA gene originating from the H5N2 serotype. The phylogenies demonstrated adaptation from North American wild birds to turkeys with the possible involvement of domestic waterfowl. The turkey isolate, A/turkey/Wisconsin/1968 (H5N9), was the second most closely related poultry isolate to A/turkey/Ontario/7732/1966 (H5N9), possessing five common lineage genome segments (PB2, PB1, PA, HA and neuraminidase). The A/turkey/Ontario/6213/1966 (H5N1) virus was more virulent than A/turkey/Wisconsin/68 (H5N9) for chicken embryos and mice, indicating a greater biological similarity to A/turkey/Ontario/7732/1966 (H5N9). Thus, A/turkey/Ontario/6213/1966 (H5N1) was identified as the closest known ancestral relative of HPAI A/turkey/Ontario/7732/1966 (H5N9), which will serve as a useful reference virus for characterizing the early genetic and biological properties associated with the emergence of pathogenic avian influenza strains. PMID:22592261

Comparative Efficacy of Hemagglutinin, Nucleoprotein, and Matrix 2 Protein Gene-Based Vaccination against H5N1 Influenza in Mouse and Ferret

PubMed Central

Rao, Srinivas S.; Kong, Wing-Pui; Wei, Chih-Jen; Van Hoeven, Neal; Gorres, J. Patrick; Nason, Martha; Andersen, Hanne; Tumpey, Terrence M.; Nabel, Gary J.

2010-01-01

Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge. PMID:20352112

An in vivo evaluation of PLLA/PLLA-gHA nano-composite for internal fixation of mandibular bone fractures.

PubMed

Peng, Weihai; Zheng, Wei; Shi, Kai; Wang, Wangshu; Shao, Ying; Zhang, Duo

2015-11-09

Internal fixation of bone fractures using biodegradable poly(L-lactic-acid) (PLLA)-based materials has attracted the attention of many researchers. In the present study, 36 male beagle dogs were randomly assigned to two groups: PLLA/PLLA-gHA (PLLA-grafted hydroxyapatite) group and PLLA group. PLLA/PLLA-gHA and PLLA plates were embedded in the muscular bags of the erector spinae and also implanted to fix mandibular bone fractures in respective groups. At 1, 2, 3, 6, 9, and 12 months postoperatively, the PLLA/PLLA-gHA and PLLA plates were evaluated by adsorption and degradation tests, and the mandibles were examined through radiographic analysis, biomechanical testing, and histological analysis. The PLLA/PLLA-gHA plates were non-transparent and showed a creamy white color, and the PLLA plates were transparent and faint yellow in color. At all time points following surgery, adsorption and degradation of the PLLA/PLLA-gHA plates were significantly less than those of the PLLA plates, and the lateral and longitudinal bending strengths of the surgically treated mandibles of the beagle dogs in the PLLA/PLLA-gHA group were significantly greater than those of the PLLA group and reached almost the value of intact mandibles at 12 months postoperatively. Additionally, relatively rapid bone healing was observed in the PLLA/PLLA-gHA group with the formation of new lamellar bone tissues at 12 months after the surgery. The PLLA/PLLA-gHA nano-composite can be employed as a biodegradable material for internal fixation of mandibular bone fractures.

Re-establishment of the IMS Hydroacoustic Station HA03, Robinson Crusoe Island, Chile

NASA Astrophysics Data System (ADS)

Haralabus, Georgios; Stanley, Jerry; Zampolli, Mario; Pautet, Lucie

2015-04-01

Water column hydrophone stations of the Comprehensive Nuclear-Test-Ban Treaty Organisation (CTBTO) International Monitoring System (IMS) comprise typically two triplets of moored hydrophones deployed on both sides of an island. Triplet distances vary approximately between 50 - 200 km from the island, with each triplet connected to the receiving shore equipment by fibre-optic submarine data cables. Once deployed, the systems relay underwater acoustic waveforms in the band 1 - 100 Hz in real time to Vienna via a shore based satellite link. The design life of hydroacoustic (HA) stations is at least 20 years, without need for any maintenance of the underwater system (UWS). The re-establishment of hydrophone station HA03 at Robinson Crusoe Island (670 km West of the Chilean mainland) is presented here. The station was destroyed in February 2010 by a Tsunami induced by an 8.8 magnitude earthquake. After a major engineering and logistical undertaking HA03 is now back in operation since April 2014. The main phases of the project are presented: (i) the installation of a shore facility for the reception of the hydrophone data from the UWS, which also relays the data back to the CTBTO International Data Center (IDC) in Vienna via a real-time satellite connection, (ii) the manufacturing and testing of the system to meet the stringent requirements of the Nuclear-Test-Ban Treaty, and (iii) the installation of the UWS with a state-of-the-art cable ship. Examples of data acquired by HA03 are also presented. These include hydroacoustic signals from the 1 April 2014 magnitude 8.2 earthquake in Northern Chile, bursting underwater bubbles from a submarine volcano near the Mariana Islands (15,000 Km away from the station), and vocalizations from the numerous marine mammals which transit in the vicinity of HA03. The use of CTBTO data for scientific purposes is possible via the virtual Data Exploitation Centre (vDEC), which is a platform that enables registered researchers to access

Nanocomposite Porous Microcarriers Based on Strontium-Substituted HA- g-Poly(γ-benzyl-l-glutamate) for Bone Tissue Engineering.

PubMed

Yan, Shifeng; Xia, Pengfei; Xu, Shenghua; Zhang, Kunxi; Li, Guifei; Cui, Lei; Yin, Jingbo

2018-05-04

Porous microcarriers have aroused increasing attention recently, which can create a protected environment for sufficient cell seeding density, facilitate oxygen and nutrient transfer, and well support the cell attachment and growth. In this study, porous microcarriers fabricated from the strontium-substituted hydroxyapatite- graft-poly(γ-benzyl-l-glutamate) (Sr10-HA- g-PBLG) hybrid nanocomposite were developed. The surface grating of PBLG, the micromorphology and element distribution, mechanical strength, in vitro degradation, and Sr 2+ ion release of the obtained Sr10-HA- g-PBLG porous microcarriers were investigated, respectively. The grafting ratio and the molecular weight of the grafted PBLG of Sr10-HA- g-PBLG could be effectively controlled by varying the initial ratio of BLG-NCA to Sr10-HA-NH 2 . The microcarriers exhibited a highly porous and interconnected microstructure with the porosity of about 90% and overall density of 1.03-1.06 g/cm 3 . Also, the degradation rate of Sr10-HA-PBLG microcarriers could be effectively controlled and long-term Sr 2+ release was obtained. The Sr10-HA-PBLG microcarriers allowed cells adhesion, infiltration, and proliferation and promoted the osteogenic differentiation of rabbit adipose-derived stem cells (ADSCs). Successful healing of femoral bone defect was proved by injection of the ADSCs-seeded Sr10-HA-PBLG microcarriers in a rabbit model.

AHEAD. Advate in HaEmophilia A outcome Database.

PubMed

Oldenburg, J; Kurnik, K; Huth-Kühne, A; Zimmermann, R; Abraham, I; Klamroth, R

2010-11-01

The clinical picture of haemophilia A patients is often characterised by recurrent bleedings, in particular joint bleeds. Thus far, long-term data on the outcome of haemophilia A patients are scarce as regards the development of target joints, joint replacement, lost days from school or work due to bleedings, and the quality of life, as most previous studies were limited to the aspects of safety and efficacy. The Baxter-initiated AHEAD (Advate in HaEmophilia A outcome Database) study is a multi-centre, prospective, non-interventional observational study of haemophilia A patients. All patients with a residual FVIII activity of £5% who are being treated with ADVATE are eligible. There are no limitations in terms of patient age or treatment regimen. AHEAD is scientifically supported by a renowned interdisciplinary steering board and is intended to yield data on 500 patients in up to 30 haemophilia centres, collected during a period of four years. The large patient population has been chosen in order to ensure a valid database. The objective of the study is to record haemophilia-related arthropathies, which will be defined based on imaging techniques (e. g. MRI, X-ray, ultrasound) and the judgment of the attending physician. In addition, extensive data will be collected on joint replacement surgeries, pseudotumour development, bleeding-related pain, quality of life (age-related questionnaires: Haem-A-QoL, Haemo-QoL, SF10, SF12v2), risk factors (diabetes mellitus, arterial hypertension, nicotine abuse), blood group, gene mutation, physical activity, and on the efficacy and safety of Advate. The patient data will be entered into an electronic CRF system at the centres. Plausibility checks during data entry, regular monitoring visits, and the option of auditing all serve to ensure a high data quality for AHEAD. The first patient was enrolled in the study in early June 2010; recruitment is planned to continue until the end of 2011. The Ethics Committee of the University

Simultaneous detection of eight avian influenza A virus subtypes by multiplex reverse transcription-PCR using a GeXP analyser.

PubMed

Li, Meng; Xie, Zhixun; Xie, Zhiqin; Liu, Jiabo; Xie, Liji; Deng, Xianwen; Luo, Sisi; Fan, Qing; Huang, Li; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Wang, Sheng

2018-04-18

Recent studies have demonstrated that at least eight subtypes of avian influenza virus (AIV) can infect humans, including H1, H2, H3, H5, H6, H7, H9 and H10. A GeXP analyser-based multiplex reverse transcription (RT)-PCR (GeXP-multiplex RT-PCR) assay was developed in our recent studies to simultaneously detect these eight AIV subtypes using the haemagglutinin (HA) gene. The assay consists of chimeric primer-based PCR amplification with fluorescent labelling and capillary electrophoresis separation. RNA was extracted from chick embryo allantoic fluid or liquid cultures of viral isolates. In addition, RNA synthesised via in vitro transcription was used to determine the specificity and sensitivity of the assay. After selecting the primer pairs, their concentrations and GeXP-multiplex RT-PCR conditions were optimised. The established GeXP-multiplex RT-PCR assay can detect as few as 100 copies of premixed RNA templates. In the present study, 120 clinical specimens collected from domestic poultry at live bird markets and from wild birds were used to evaluate the performance of the assay. The GeXP-multiplex RT-PCR assay specificity was the same as that of conventional RT-PCR. Thus, the GeXP-multiplex RT-PCR assay is a rapid and relatively high-throughput method for detecting and identifying eight AIV subtypes that may infect humans.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

A Genome-wide Association Study Identifies Risk Alleles in Plasminogen and P4HA2 Associated with Giant Cell Arteritis.

PubMed

Carmona, F David; Vaglio, Augusto; Mackie, Sarah L; Hernández-Rodríguez, José; Monach, Paul A; Castañeda, Santos; Solans, Roser; Morado, Inmaculada C; Narváez, Javier; Ramentol-Sintas, Marc; Pease, Colin T; Dasgupta, Bhaskar; Watts, Richard; Khalidi, Nader; Langford, Carol A; Ytterberg, Steven; Boiardi, Luigi; Beretta, Lorenzo; Govoni, Marcello; Emmi, Giacomo; Bonatti, Francesco; Cimmino, Marco A; Witte, Torsten; Neumann, Thomas; Holle, Julia; Schönau, Verena; Sailler, Laurent; Papo, Thomas; Haroche, Julien; Mahr, Alfred; Mouthon, Luc; Molberg, Øyvind; Diamantopoulos, Andreas P; Voskuyl, Alexandre; Brouwer, Elisabeth; Daikeler, Thomas; Berger, Christoph T; Molloy, Eamonn S; O'Neill, Lorraine; Blockmans, Daniel; Lie, Benedicte A; Mclaren, Paul; Vyse, Timothy J; Wijmenga, Cisca; Allanore, Yannick; Koeleman, Bobby P C; Barrett, Jennifer H; Cid, María C; Salvarani, Carlo; Merkel, Peter A; Morgan, Ann W; González-Gay, Miguel A; Martín, Javier

2017-01-05

Giant cell arteritis (GCA) is the most common form of vasculitis in individuals older than 50 years in Western countries. To shed light onto the genetic background influencing susceptibility for GCA, we performed a genome-wide association screening in a well-powered study cohort. After imputation, 1,844,133 genetic variants were analyzed in 2,134 case subjects and 9,125 unaffected individuals from ten independent populations of European ancestry. Our data confirmed HLA class II as the strongest associated region (independent signals: rs9268905, p = 1.94 × 10 -54 , per-allele OR = 1.79; and rs9275592, p = 1.14 × 10 -40 , OR = 2.08). Additionally, PLG and P4HA2 were identified as GCA risk genes at the genome-wide level of significance (rs4252134, p = 1.23 × 10 -10 , OR = 1.28; and rs128738, p = 4.60 × 10 -9 , OR = 1.32, respectively). Interestingly, we observed that the association peaks overlapped with different regulatory elements related to cell types and tissues involved in the pathophysiology of GCA. PLG and P4HA2 are involved in vascular remodelling and angiogenesis, suggesting a high relevance of these processes for the pathogenic mechanisms underlying this type of vasculitis. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Construction of surface HA/TiO2 coating on porous titanium scaffolds and its preliminary biological evaluation.

PubMed

Chen, Hongjie; Wang, Chunli; Yang, Xiao; Xiao, Zhanwen; Zhu, Xiangdong; Zhang, Kai; Fan, Yujiang; Zhang, Xingdong

2017-01-01

A simple approach to fabricating hydroxyxapatite/titanium dioxide (HA/TiO 2 ) coating on porous titanium (Ti) scaffolds was developed in the present study. Surface TiO 2 layer was firstly formed on porous Ti scaffolds with multi-scale pores by acid-alkali (AA) treatment. The outer HA layer was then formed on the TiO 2 layer by subsequent pulse electrochemical deposition (ED) technique. All the three main process parameters, i.e. deposition times, current density and mass transfer mode affected the properties of the HA coating notably. Under the conditions of 90 deposition cycles, -10mA/cm 2 of pulse current density and stirring, a thin layer of homogeneous and nanorod-like HA sediments was formed on the substrate surface of porous Ti scaffolds. The results of protein adsorption and cellular experiments showed that compared to the single TiO 2 surface, the HA/TiO 2 surface allowed more adsorption of serum proteins and further enhanced the alkaline phosphatase (ALP) activity of MC3T3-E1 osteoblasts. Copyright © 2016 Elsevier B.V. All rights reserved.

RGO/AuNR/HA-5FU nanocomposite with multi-stage release behavior and efficient antitumor activity for synergistic therapy.

PubMed

Yang, Ying; Wang, Yunlong; Zhu, Manzhou; Chen, Yan; Xiao, Yazhong; Shen, Yuhua; Xie, Anjian

2017-05-02

A reduced graphene oxide (RGO)/gold nanorod (AuNR)/hydroxyapatite (HA) nanocomposite was designed and successfully synthesized for the first time. An anticancer drug, 5-fluorouracil (5FU), was chosen as a model drug to be loaded in RGO/AuNR/HA. The fabricated RGO/AuNR/HA-5FU showed robust, selective targeting and penetrating efficiency against HeLa cells due to the good compatibility and nontoxicity of HA, and showed excellent synergetic antitumor effects through combined chemotherapy (CT) by 5FU and photothermal therapy (PTT) by both RGO and AuNRs under near-infrared (NIR) laser irradiation. More importantly, this synergistic dual therapy based on RGO/AuNR/HA can also minimize side effects in normal cells and exhibits greater antitumor activity because of a multi-stage drug release ability triggered by the pH sensitivity of HA in the first stage and the combined photothermal conversion capabilities of RGO and AuNRs by means of the NIR laser irradiation in the second stage. This study suggests that the novel RGO/AuNR/HA multi-stage drug delivery system may represent a promising potential application of multifunctional composite materials in the biomedical field.

Relocation of a rust resistance gene R 2 and its marker-assisted gene pyramiding in confection sunflower (Helianthus annuus L.).

PubMed

Qi, L L; Ma, G J; Long, Y M; Hulke, B S; Gong, L; Markell, S G

2015-03-01

The rust resistance gene R 2 was reassigned to linkage group 14 of the sunflower genome. DNA markers linked to R 2 were identified and used for marker-assisted gene pyramiding in a confection type genetic background. Due to the frequent evolution of new pathogen races, sunflower rust is a recurring threat to sunflower production worldwide. The inbred line Morden Cross 29 (MC29) carries the rust resistance gene, R 2 , conferring resistance to numerous races of rust fungus in the US, Canada, and Australia, and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments and SSR marker analyses on the 117 F2 individuals derived from a cross of HA 89 with MC29 (USDA), R 2 was mapped to linkage group (LG) 14 of the sunflower, and not to the previously reported location on LG9. The closest SSR marker HT567 was located at 4.3 cM distal to R 2 . Furthermore, 36 selected SNP markers from LG14 were used to saturate the R 2 region. Two SNP markers, NSA_002316 and SFW01272, flanked R 2 at a genetic distance of 2.8 and 1.8 cM, respectively. Of the three closely linked markers, SFW00211 amplified an allele specific for the presence of R 2 in a marker validation set of 46 breeding lines, and SFW01272 was also shown to be diagnostic for R 2 . These newly developed markers, together with the previously identified markers linked to the gene R 13a , were used to screen 524 F2 individuals from a cross of a confection R 2 line and HA-R6 carrying R 13a . Eleven homozygous double-resistant F2 plants with the gene combination of R 2 and R 13a were obtained. This double-resistant line will be extremely useful in confection sunflower, where few rust R genes are available, risking evolution of new virulence phenotypes and further disease epidemics.

Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes.

PubMed

Bastida, Jose Maria; González-Porras, Jose Ramon; Jiménez, Cristina; Benito, Rocio; Ordoñez, Gonzalo R; Álvarez-Román, Maria Teresa; Fontecha, M Elena; Janusz, Kamila; Castillo, David; Fisac, Rosa María; García-Frade, Luis Javier; Aguilar, Carlos; Martínez, María Paz; Bermejo, Nuria; Herrero, Sonia; Balanzategui, Ana; Martin-Antorán, Jose Manuel; Ramos, Rafael; Cebeiro, Maria Jose; Pardal, Emilia; Aguilera, Carmen; Pérez-Gutierrez, Belen; Prieto, Manuel; Riesco, Susana; Mendoza, Maria Carmen; Benito, Ana; Hortal Benito-Sendin, Ana; Jiménez-Yuste, Víctor; Hernández-Rivas, Jesus Maria; García-Sanz, Ramon; González-Díaz, Marcos; Sarasquete, Maria Eugenia

2017-01-05

Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.

H1N1 viral proteome peptide microarray predicts individuals at risk for H1N1 infection and segregates infection versus Pandemrix® vaccination

PubMed Central

Ambati, Aditya; Valentini, Davide; Montomoli, Emanuele; Lapini, Guilia; Biuso, Fabrizio; Wenschuh, Holger; Magalhaes, Isabelle; Maeurer, Markus

2015-01-01

A high content peptide microarray containing the entire influenza A virus [A/California/08/2009(H1N1)] proteome and haemagglutinin proteins from 12 other influenza A subtypes, including the haemagglutinin from the [A/South Carolina/1/1918(H1N1)] strain, was used to gauge serum IgG epitope signatures before and after Pandemrix® vaccination or H1N1 infection in a Swedish cohort during the pandemic influenza season 2009. A very narrow pattern of pandemic flu-specific IgG epitope recognition was observed in the serum from individuals who later contracted H1N1 infection. Moreover, the pandemic influenza infection generated IgG reactivity to two adjacent epitopes of the neuraminidase protein. The differential serum IgG recognition was focused on haemagglutinin 1 (H1) and restricted to classical antigenic sites (Cb) in both the vaccinated controls and individuals with flu infections. We further identified a novel epitope VEPGDKITFEATGNL on the Ca antigenic site (251–265) of the pandemic flu haemagglutinin, which was exclusively recognized in serum from individuals with previous vaccinations and never in serum from individuals with H1N1 infection (confirmed by RNA PCR analysis from nasal swabs). This epitope was mapped to the receptor-binding domain of the influenza haemagglutinin and could serve as a correlate of immune protection in the context of pandemic flu. The study shows that unbiased epitope mapping using peptide microarray technology leads to the identification of biologically and clinically relevant target structures. Most significantly an H1N1 infection induced a different footprint of IgG epitope recognition patterns compared with the pandemic H1N1 vaccine. PMID:25639813

Inactivation of ceramide synthase 2 catalytic activity in mice affects transcription of genes involved in lipid metabolism and cell division.

PubMed

Bickert, Andreas; Kern, Paul; van Uelft, Martina; Herresthal, Stefanie; Ulas, Thomas; Gutbrod, Katharina; Breiden, Bernadette; Degen, Joachim; Sandhoff, Konrad; Schultze, Joachim L; Dörmann, Peter; Hartmann, Dieter; Bauer, Reinhard; Willecke, Klaus

2018-07-01

The replacement of two consecutive histidine residues by alanine residues in the catalytic center of ceramide synthase 2 in a new transgenic mouse mutant (CerS2 H/A) leads to inactivation of catalytic activity and reduces protein level to 60% of the WT level. We show here by qRT-PCR and transcriptome analyses that several transcripts of genes involved in lipid metabolism and cell division are differentially regulated in livers of CerS2 H/A mice. Thus, very long chain ceramides produced by CerS2 are required for transcriptional regulation of target genes. The hepatocellular carcinomata previously described in old CerS2 KO mice were already present in 8-week-old CerS2 H/A animals and thus are caused by the loss of CerS2 catalytic activity already during early life. Copyright © 2018 Elsevier B.V. All rights reserved.

The use of sodium hyaluronate-carboxymethylcellulose (HA-CMC) barrier in gynecologic malignancies: a retrospective review of outcomes.

PubMed

Tan, Annie; Argenta, Peter; Ramirez, Rose; Bliss, Robin; Geller, Melissa

2009-02-01

Concerns exist regarding the safety of sodium hyaluronate-carboxymethylcellulose (HA-CMC, Seprafilm) adhesion barrier in regard to cancer survival as a result of in vitro data demonstrating that hyaluronan, a component of HA-CMC, may promote tumor growth. We sought to determine whether use of HA-CMC is associated with duration of disease-free or overall survival and rates of immediate complication in patients with gynecologic malignancies. We identified 202 consecutive patients with epithelial ovarian, fallopian tube, and primary peritoneal cancer who underwent initial surgical staging or interval debulking at the University of Minnesota between January 2001 and December 2004. Information on patients' demographics, medical history, surgical procedures, postoperative complications, disease stage, histology, adjuvant therapy, and disease-free and overall survival was collected from medical records. Survival curves were compared between patients receiving or not receiving HA-CMC by stratified Cox regression models, log rank, and Wilcoxon tests. The level of significance was set to alpha = .05 for each test. Eighty patients received intraoperative placement of HA-CMC and 122 did not. Immediate postoperative complication rates were equivalent in both groups. Median follow-up was 2.1 years. There was no difference in disease-free survival (5-year estimate 23.6% vs. 33.3%, P = .80) or overall survival (5-year estimate 29.7% vs. 40.3%, P = .75) between those who received HA-CMC and those who did not. Our retrospective analysis suggests that HA-CMC adhesion barrier does not affect disease-free survival or overall survival; nor does it increase the immediate postoperative complication rates in patients undergoing abdominal surgery for ovarian, fallopian tube, and primary peritoneal carcinomas.

In Vivo Bio-distribution and Efficient Tumor Targeting of Gelatin/Silica Nanoparticles for Gene Delivery

NASA Astrophysics Data System (ADS)

Zhao, Xueqin; Wang, Jun; Tao, SiJie; Ye, Ting; Kong, Xiangdong; Ren, Lei

2016-04-01

The non-viral gene delivery system is an attractive alternative to cancer therapy. The clinical success of non-viral gene delivery is hampered by transfection efficiency and tumor targeting, which can be individually overcome by addition of functional modules such as cell penetration or targeting. Here, we first engineered the multifunctional gelatin/silica (GS) nanovectors with separately controllable modules, including tumor-targeting aptamer AGRO100, membrane-destabilizing peptide HA2, and polyethylene glycol (PEG), and then studied their bio-distribution and in vivo transfection efficiencies by contrast resonance imaging (CRI). The results suggest that the sizes and zeta potentials of multifunctional gelatin/silica nanovectors were 203-217 nm and 2-8 mV, respectively. Functional GS-PEG nanoparticles mainly accumulated in the liver and tumor, with the lowest uptake by the heart and brain. Moreover, the synergistic effects of tumor-targeting aptamer AGRO100 and fusogenic peptide HA2 promoted the efficient cellular internalization in the tumor site. More importantly, the combined use of AGRO100 and PEG enhanced tumor gene expression specificity and effectively reduced toxicity in reticuloendothelial system (RES) organs after intravenous injection. Additionally, low accumulation of GS-PEG was observed in the heart tissues with high gene expression levels, which could provide opportunities for non-invasive gene therapy.

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1).

PubMed

Yang, Shi-gui; Wo, Jian-er; Li, Min-wei; Mi, Fen-fang; Yu, Cheng-bo; Lv, Guo-liang; Cao, Hong-Cui; Lu, Hai-feng; Wang, Bao-hong; Zhu, Hanping; Li, Lan-Juan

2009-12-09

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4(+) T cells and the IFN-gamma expression of CD8(+) T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-gamma expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.

Self-assembling HA/PEI/dsRNA-p21 ternary complexes for CD44 mediated small active RNA delivery to colorectal cancer.

PubMed

Feng, Chen-Lin; Han, Yan-Xing; Guo, Hui-Hui; Ma, Xiao-Lei; Wang, Zhi-Qiang; Wang, Lu-Lu; Zheng, Wen-Sheng; Jiang, Jian-Dong

2017-11-01

Our previous work proved that sequence specific double strand RNA (dsRNA-p21) effectively activated p21 gene expression of colorectal cancer (CRC) cells and consequently suppressed CRC growth. However, efficient delivery system is a significant challenge to achieve sufficient therapy. In this study, a self-assembled HA/PEI/dsRNA-p21 ternary complex (TC-dsRNA-p21) was developed for the tumor-target delivery of dsRNA-p21 into CRC cells. Hyaluronic acid (HA) was introduced to shield the PEI/dsRNA-p21 binary complexes (BC-dsRNA-p21) for reducing the cytotoxicity of PEI and for increasing the tumor-targeted intracellular uptake by cancer cells through HA-CD44 mediated endocytosis. Comparing to the BC-dsRNA-p21, the TC-dsRNA-p21 showed increase in size, decrease in zeta potential, low cytotoxicity as well as high stability in physiological conditions due to the anionic shielding. Confocal microscopy analysis and flow cytometry confirmed that TC-dsRNA-p21 had high transfection efficiency in the CD44-abundant Lovo cells, as compared with binary complex. In vitro physiological experiment showed that, comparing to the control group, the TC-dsRNA-p21 effectively activated the expression of p21 mRNA and P21 protein, causing blockage of cell cycle at G 0 /G 1 phase and suppression of cancer cell proliferation as well as colony formation. Furthermore, in vivo distribution experiment demonstrated that the TC-dsRNA-p21 could effectively accumulate at rectal wall for up to 10 h, following in situ application. These findings indicated that TC-dsRNA-p21 might hold great potential for delivering dsRNA-p21 to treat CRC.

An interpenetrating HA/G/CS biomimic hydrogel via Diels-Alder click chemistry for cartilage tissue engineering.

PubMed

Yu, Feng; Cao, Xiaodong; Zeng, Lei; Zhang, Qing; Chen, Xiaofeng

2013-08-14

In order to mimic the natural cartilage extracellular matrix, a novel biological degradable interpenetrating network hydrogel was synthesized from the gelatin (G), hyaluronic acid (HA) and chondroitin sulfate (CS) by Diels-Alder "click" chemistry. HA was modified with furylamine and G was modified with furancarboxylic acid respectively. (1)H NMR spectra and elemental analysis showed that the substitution degrees of HA-furan and G-furan were 71.5% and 44.5%. Then the hydrogels were finally synthesized by cross-linking furan-modified HA and G derivatives with dimaleimide poly(ethylene glycol) (MAL-PEG-MAL). The mechanical and degradation properties of the hydrogels could be tuned simply through varying the molar ratio between furan and maleimide. Rheological, mechanical and degradation studies demonstrated that the Diels-Alder "click" chemistry is an efficient method for preparing high performance biological interpenetrating hydrogels. This biomimic hydrogel with improved mechanical properties could have great potential applications in cartilage tissue engineering. Copyright © 2013 Elsevier Ltd. All rights reserved.

Employing the cyclophosphate to accelerate the degradation of nano-hydroxyapatite/poly(amino acid) (n-HA/PAA) composite materials.

PubMed

Jing, Linjing; Chen, Li; Peng, Haitao; Ji, Mizhi; Xiong, Yi; Lv, Guoyu

2017-12-01

Owing to the good degradability and biocompatibility of polyphosphoesters (PPEs), the aim of the current study was to investigate a novel degradable composite of nano-hydroxyapatite/poly(amino acid) (n-HA/PAA) with cyclophosphate (CPE) via in situ melting polymerization to improve the degradation of n-HA/PAA. The structure of each composite was characterized via Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The degradation properties were studied in terms of the weight loss and pH in a phosphate-buffered saline (PBS) solution, while the surface morphology was examined using a scanning electron microscope-energy dispersive spectrometer (SEM-EDS) after soaking the surface in simulated body fluid (SBF). The cell proliferation, cell adhesion, and alkaline phosphatase (ALP) activity were used for the analysis of cytocompatibility. The weight loss results showed that the n-HA/PAA composite was 9.98 wt%, weighed after soaking in the PBS solution for 12 weeks, whereas the nano-hydroxyapatite/polyphosphoester-amino acid (n-HA/PPE-AA) composite was 46.94 wt%. The pH of the composites was in a suitable range between 6.64 to 7.06 and finally stabilized at 7.39. The SEM and EDS results revealed the formation of an apatite-like layer on the surface of the n-HA/PPE-AA composites after soaking in SBF for one week. The cell counting Kit 8 (CCK-8) assay of the cell culture in the leaching liquid of the n-HA/PPE-AA composites exhibited non-cytotoxicity and high-proliferation, and the cell adhesion showed the well spreading and normal phenotype extension of the cells on the n-HA/PPE-AA composites surface. Concurrently, the co-culture results of the composites and cells confirmed that the n-HA/PPE-AA composites exhibited a higher ALP activity. In summary, the results demonstrated that the n-HA/PPE-AA composites had a controllable degradation property, good bioactivity, and cytocompatibility.

Laminated electrospun nHA/PHB-composite scaffolds mimicking bone extracellular matrix for bone tissue engineering.

PubMed

Chen, Zhuoyue; Song, Yue; Zhang, Jing; Liu, Wei; Cui, Jihong; Li, Hongmin; Chen, Fulin

2017-03-01

Electrospinning is an effective means to generate nano- to micro-scale polymer fibers resembling native extracellular matrix for tissue engineering. However, a major problem of electrospun materials is that limited pore size and porosity may prevent adequate cellular infiltration and tissue ingrowth. In this study, we first prepared thin layers of hydroxyapatite nanoparticle (nHA)/poly-hydroxybutyrate (PHB) via electrospinning. We then laminated the nHA/PHB thin layers to obtain a scaffold for cell seeding and bone tissue engineering. The results demonstrated that the laminated scaffold possessed optimized cell-loading capacity. Bone marrow mesenchymal stem cells (MSCs) exhibited better adherence, proliferation and osteogenic phenotypes on nHA/PHB scaffolds than on PHB scaffolds. Thereafter, we seeded MSCs onto nHA/PHB scaffolds to fabricate bone grafts. Histological observation showed osteoid tissue formation throughout the scaffold, with most of the scaffold absorbed in the specimens 2months after implantation, and blood vessels ingrowth into the graft could be observed in the graft. We concluded that electrospun and laminated nanoscaled biocomposite scaffolds hold great therapeutic potential for bone regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Biopolymers for Medical Applications: Polyglycerol Sebacate (PGS) doped Hydroxyapatite (HA)

NASA Astrophysics Data System (ADS)

Teruel, Maria; Kuthirummal, Narayanan; Levi, Nicole; Wake College Team

2011-04-01

In the investigation to engineer the ideal scaffolding device for cleft palate repair, polyglycerol sebacate (PGS) doped with hydroxyapatite (HA) were chosen for their elastomeric and biodegradable properties, as well as their cost-effective synthesis. Hydroxyapatite was integrated into the PGS to form a composite with high porosity and improved mechanical properties yielding a good substrate for cell attachment during the repair process. FT-IR scans were performed to characterize the composite polymer. Differential Scanning Calorimetry (DSC) was utilized to identify an acceptable glass transition temperature (Tg), between -18 and - 21°C. At this Tg, it was determined that the material was sufficiently polymerized to a point where it was durable yet pliable enough to use for cleft palate devices. In the synthesis of PGS 3% and 5% HA, a Tg of - 20.10°C and - 21.72°C, respectively, was achieved and further analytical tests were then performed on the polymers. Methods of analysis included X-Ray Diffraction and Tensile Strength Testing. Acknowledgements to the Research Department of Plastic and Reconstructive Surgery, Wake Forest University and College of Charleston.

In Silico Prediction and Experimental Confirmation of HA Residues Conferring Enhanced Human Receptor Specificity of H5N1 Influenza A Viruses.

PubMed

Schmier, Sonja; Mostafa, Ahmed; Haarmann, Thomas; Bannert, Norbert; Ziebuhr, John; Veljkovic, Veljko; Dietrich, Ursula; Pleschka, Stephan

2015-06-19

Newly emerging influenza A viruses (IAV) pose a major threat to human health by causing seasonal epidemics and/or pandemics, the latter often facilitated by the lack of pre-existing immunity in the general population. Early recognition of candidate pandemic influenza viruses (CPIV) is of crucial importance for restricting virus transmission and developing appropriate therapeutic and prophylactic strategies including effective vaccines. Often, the pandemic potential of newly emerging IAV is only fully recognized once the virus starts to spread efficiently causing serious disease in humans. Here, we used a novel phylogenetic algorithm based on the informational spectrum method (ISM) to identify potential CPIV by predicting mutations in the viral hemagglutinin (HA) gene that are likely to (differentially) affect critical interactions between the HA protein and target cells from bird and human origin, respectively. Predictions were subsequently validated by generating pseudotyped retrovirus particles and genetically engineered IAV containing these mutations and characterizing potential effects on virus entry and replication in cells expressing human and avian IAV receptors, respectively. Our data suggest that the ISM-based algorithm is suitable to identify CPIV among IAV strains that are circulating in animal hosts and thus may be a new tool for assessing pandemic risks associated with specific strains.

In Silico Prediction and Experimental Confirmation of HA Residues Conferring Enhanced Human Receptor Specificity of H5N1 Influenza A Viruses

NASA Astrophysics Data System (ADS)

Schmier, Sonja; Mostafa, Ahmed; Haarmann, Thomas; Bannert, Norbert; Ziebuhr, John; Veljkovic, Veljko; Dietrich, Ursula; Pleschka, Stephan

2015-06-01

Newly emerging influenza A viruses (IAV) pose a major threat to human health by causing seasonal epidemics and/or pandemics, the latter often facilitated by the lack of pre-existing immunity in the general population. Early recognition of candidate pandemic influenza viruses (CPIV) is of crucial importance for restricting virus transmission and developing appropriate therapeutic and prophylactic strategies including effective vaccines. Often, the pandemic potential of newly emerging IAV is only fully recognized once the virus starts to spread efficiently causing serious disease in humans. Here, we used a novel phylogenetic algorithm based on the informational spectrum method (ISM) to identify potential CPIV by predicting mutations in the viral hemagglutinin (HA) gene that are likely to (differentially) affect critical interactions between the HA protein and target cells from bird and human origin, respectively. Predictions were subsequently validated by generating pseudotyped retrovirus particles and genetically engineered IAV containing these mutations and characterizing potential effects on virus entry and replication in cells expressing human and avian IAV receptors, respectively. Our data suggest that the ISM-based algorithm is suitable to identify CPIV among IAV strains that are circulating in animal hosts and thus may be a new tool for assessing pandemic risks associated with specific strains.

Genetic diversities of MT-ND1 and MT-ND2 genes are associated with high-altitude adaptation in yak.

PubMed

Shi, Yu; Hu, Yongsong; Wang, Jie; Elzo, Mauricio A; Yang, Xue; Lai, Songjia

2018-04-01

Tibetan yak (Bos grunniens) inhabiting the Qinghai-Tibet Plateau (QTP) where the average altitude is 4000 m, is specially adapted to live at these altitudes. Conversely, cattle (B. taurus) has been found to suffer from high-altitude hypertension or heart failure when exposed to these high altitudes. Two mitochondrial genes, MT-ND1 and MT-ND2, encode two subunits of NADH dehydrogenase play an essential role in the electron transport chain of oxidative phosphorylation (OXPHOS). We sequenced these two mitochondrial genes in two bovine groups (70 Tibetan yaks and 70 Xuanhan cattle) and downloaded 300 sequences of B. taurus (cattle), 93 sequences of B. grunniens (domestic yak), and 2 sequences of B. mutus (wild yak) from NCBI to increase our understanding of the mechanisms of adaptability to hypoxia at high altitudes in yaks compared to cattle. MT-ND1 SNP m.3907 C > T, present in all Tibetan yaks, was positively associated with high-altitude adaptation (p < .0006). Specially, mutation m.3638 A > G present in all cattle, resulting in the termination of transcription, was negatively associated with high-altitude adaptation (p < .0006). Additionally, MT-ND2 SNPs m.4351 G > A and m.5218 C > T also showed positive associations with high-altitude adaptation (p < .0004). MT-ND1 haplotypes H2, H3, H4, H6, and H7 showed positive associations but haplotype H20 had a negative association with high-altitude adaptation (p < .0008). Similarly, MT-ND2 haplotypes Ha1 Ha8, Ha10, and Ha11 were positively associated whereas haplotype Ha2 was negatively associated with adaptability to high-altitudes (p < .0008). Thus, MT-ND1 and MT-ND2 can be considered as candidate genes associated with adaptation to high-altitude environments.

TRB3 is elevated in psoriasis vulgaris lesions and mediates HaCaT cells proliferation in vitro.

PubMed

Yu, Xiao-Jing; Song, Tie-Jun; Zhang, Lu-Wei; Su, Ying; Wang, Ke-Yu; Sun, Qing

2017-10-01

Psoriasis is a chronic skin disease characterized by abnormal keratinocyte proliferation and differentiation, inflammation, and angiogenesis. Overexpression of tribbles homolog3 (TRB3), which belongs to the tribbles family of pseudokinases, has been found in several human tumors and metabolic diseases, but its role in psoriasis has not been fully clarified. The aim of this study is to investigate the expression of TRB3 in psoriasis and explore its roles in the proliferation of keratinocytes. Twenty-four patients with psoriasis vulgaris were recruited for the study. Diagnosis of psoriasis was based on clinical and histologic examinations. Immunohistochemistry and real-time reverse transcription PCR (RT-PCR) were performed to determine protein and messenger RNA (mRNA) expression of TRB3 in psoriasis lesions. 5-Bromo-2-deoxyUridine (BrdU) incorporation assay were performed for cell proliferation. Cell cycle distribution was assessed by flow cytometry analysis. The levels of TRB3 is elevated in psoriatic lesions compared with psoriatic non-lesions. The HaCat cells expressed the TRB3 gene. We found TRB3 silencing to significantly inhibit HaCat cell proliferation. Furthermore, the specific knockdown of TRB3 slowed down the cell cycle at the gap 0/first gap phase. In conclusion, our data suggest that TRB3 is overexpressed in lesions of patients with psoriasis and may be involved in the abnormal proliferation of keratinocytes. Therefore, TRB3 may be a potential therapeutic target for psoriasis. © American Federation for Medical Research (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Hanford Facility Dangerous Waste Closure Plan - Plutonium Finishing Plant Treatment Unit Glovebox HA-20MB

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

PRIGNANO, A.L.

2003-06-25

This closure plan describes the planned activities and performance standards for closing the Plutonium Finishing Plant (PFP) glovebox HA-20MB that housed an interim status ''Resource Conservation and Recovery Act'' (RCRA) of 1976 treatment unit. This closure plan is certified and submitted to Ecology for incorporation into the Hanford Facility RCRA Permit (HF RCRA Permit) in accordance with Hanford Federal Facility Agreement and Consent Order (Tri-Party Agreement; TPA) Milestone M-83-30 requiring submittal of a certified closure plan for ''glovebox HA-20MB'' by July 31, 2003. Glovebox HA-20MB is located within the 231-5Z Building in the 200 West Area of the Hanford Facility.more » Currently glovebox HA-20MB is being used for non-RCRA analytical purposes. The schedule of closure activities under this plan supports completion of TPA Milestone M-83-44 to deactivate and prepare for dismantlement the above grade portions of the 234-5Z and ZA, 243-Z, and 291-Z and 291-Z-1 stack buildings by September 30, 2015. Under this closure plan, glovebox HA-20MB will undergo clean closure to the performance standards of Washington Administrative Code (WAC) 173-303-610 with respect to all dangerous waste contamination from glovebox HA-20MB RCRA operations. Because the intention is to clean close the PFP treatment unit, postclosure activities are not applicable to this closure plan. To clean close the unit, it will be demonstrated that dangerous waste has not been left at levels above the closure performance standard for removal and decontamination. If it is determined that clean closure is not possible or is environmentally impractical, the closure plan will be modified to address required postclosure activities. Because dangerous waste does not include source, special nuclear, and by-product material components of mixed waste, radionuclides are not within the scope of this documentation. Any information on radionuclides is provided only for general knowledge. Clearance form only

Resveratrol protects HaCaT cells from ultraviolet B-induced photoaging via upregulation of HSP27 and modulation of mitochondrial caspase-dependent apoptotic pathway.

PubMed

Zhou, Fen; Huang, Xin; Pan, Yun; Cao, Di; Liu, Chuan; Liu, Yiyi; Chen, Aijun

2018-05-15

The skin is the outermost protective barrier between the internal and external environment in humans. Chronic exposure to ultraviolet (UV) radiation is a major cause of photoaging. Evidence suggests that resveratrol suppresses UVB-induced photoaging. In this study, we aimed to investigate the protective effects of resveratrol against UVB-induced photoaging in HaCaT cells and to determine the underlying mechanisms. Apoptosis of normal or HSP27-overexpressing HaCaT cells in the presence of UVB was analyzed by flow cytometry. The mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Resveratrol inhibited UVB-induced apoptosis by upregulating the expression of HSP27, reducing the production of proapoptotic proteins such as p65, Bax, and cleaved caspase-3, and promoting the expression of anti-apoptotic protein Bcl-2. However, UVB irradiation on HaCaT cells pretreated with resveratrol led to the upregulation of Bax, downregulation of Bcl-2, and promotion of p65 and caspase-3 activation after silencing of HSP27 gene. These findings suggest that the inhibition of HSP27 expression can partially reverse the anti-apoptotic effect of resveratrol and confirm that resveratrol can regulate HSP27 and thus control p65 and caspase-3 activation. In summary, resveratrol plays a role in photoprotection by upregulating HSP27 expression, increasing Bcl-2/Bax ratio, and inhibiting caspase-3 activity and p65 expression. Copyright © 2018 Elsevier Inc. All rights reserved.

Structure, mechanical property and corrosion behaviors of (HA+β-TCP)/Mg-5Sn composite with interpenetrating networks.

PubMed

Wang, X; Li, J T; Xie, M Y; Qu, L J; Zhang, P; Li, X L

2015-11-01

In this paper, a novel (Hydroxyapatite+β-tricalcium phosphate)/Mg-5Sn ((HA+β-TCP)/Mg-5Sn) composite with interpenetrating networks was fabricated by infiltrating Mg-5Sn alloy into porous HA+β-TCP using suction casting technique. The structure, mechanical property and corrosion behaviors of the composite have been evaluated by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical testing, electrochemical and immersion test. It is shown that the molten Mg-5Sn alloy has infiltrated not only into the pores but also into the struts of the HA+β-TCP scaffold to forming a compact composite. The microstructure observation also shows that the Mg alloy contacts to the HA+β-TCP closely, and no reaction layer can be found between Mg-5Sn alloy and scaffold. The ultimate compressive strength of the composite is as high as 176MPa, which is about four fifths of the strength of the Mg-5Sn bulk alloy. The electrochemical and immersion tests indicate that the corrosion resistance of the composite is better than that of the Mg-5Sn bulk alloy. The corrosion products on the composite surface are mainly Mg(OH)2, Ca3(PO4)2 and HA. Appropriate mechanical and corrosion properties of the (HA+β-TCP)/Mg-5Sn composite indicate its possibility for new bone tissue implant materials. Copyright © 2015 Elsevier B.V. All rights reserved.

Bioactive and biocompatible pieces of HA/sol-gel glass mixtures obtained by the gel-casting method.