Mutations In Amino Acid Sequence Research Articles

Mutations of SARS-CoV-2 Structural Proteins in the Alpha, Beta, Gamma, and Delta Variants: Bioinformatics Analysis.

COVID-19 and Middle East Respiratory Syndrome are two pandemic respiratory diseases caused by coronavirus species. The novel disease COVID-19 caused by SARS-CoV-2 was first reported in Wuhan, Hubei Province, China, in December 2019, and became a pandemic within 2-3 months, affecting social and economic platforms worldwide. Despite the rapid development of vaccines, there have been obstacles to their distribution, including a lack of fundamental resources, poor immunization, and manual vaccine replication. Several variants of the original Wuhan strain have emerged in the last 3 years, which can pose a further challenge for control and vaccine development. The aim of this study was to comprehensively analyze mutations in SARS-CoV-2 variants of concern (VoCs) using a bioinformatics approach toward identifying novel mutations that may be helpful in developing new vaccines by targeting these sites. Reference sequences of the SARS-CoV-2 spike (YP_009724390) and nucleocapsid (YP_009724397) proteins were compared to retrieved sequences of isolates of four VoCs from 14 countries for mutational and evolutionary analyses. Multiple sequence alignment was performed and phylogenetic trees were constructed by the neighbor-joining method with 1000 bootstrap replicates using MEGA (version 6). Mutations in amino acid sequences were analyzed using the MultAlin online tool (version 5.4.1). Among the four VoCs, a total of 143 nonsynonymous mutations and 8 deletions were identified in the spike and nucleocapsid proteins. Multiple sequence alignment and amino acid substitution analysis revealed new mutations, including G72W, M2101I, L139F, 209-211 deletion, G212S, P199L, P67S, I292T, and substitutions with unknown amino acid replacement, reported in Egypt (MW533289), the United Kingdom (MT906649), and other regions. The variants B.1.1.7 (Alpha variant) and B.1.617.2 (Delta variant), characterized by higher transmissibility and lethality, harbored the amino acid substitutions D614G, R203K, and G204R with higher prevalence rates in most sequences. Phylogenetic analysis among the novel SARS-CoV-2 variant proteins and some previously reported β-coronavirus proteins indicated that either the evolutionary clade was weakly supported or not supported at all by the β-coronavirus species. This study could contribute toward gaining a better understanding of the basic nature of SARS-CoV-2 and its four major variants. The numerous novel mutations detected could also provide a better understanding of VoCs and help in identifying suitable mutations for vaccine targets. Moreover, these data offer evidence for new types of mutations in VoCs, which will provide insight into the epidemiology of SARS-CoV-2.

Relationship between the Reduced Expression of Zinc Finger Protein 668 in Bladder Cancer and Its Invasiveness.

The zinc finger protein 668 (ZNF668) gene encodes a Kruppel C2H2-type zinc-finger protein with 16 C2H2-type zinc fingers. The ZNF668 gene functions as a tumor suppressor gene in breast cancer. We histologically analyzed ZNF668 protein expression in bladder cancer and examined mutations of the ZNF668 gene in 68 cases of bladder cancer. In bladder cancer, the ZNF668 protein was expressed in the nuclei of cancer cells. In bladder cancer with submucosal and muscular infiltration, the expression of ZNF668 protein was significantly lower than that without submucosal and muscular infiltration. Eight heterozygous somatic mutations were detected in exon3 in five cases, and five of the mutations resulted in amino acid sequence mutations. Mutations resulting in amino acid sequence alterations also resulted in lower ZNF668 protein expression in bladder cancer cell nuclei, but no significant association with bladder cancer infiltration was detected. Decreased ZNF668 expression in bladder cancer was associated with submucosal and muscle invasion of cancer cells. Somatic mutations resulting in amino acid mutations in ZNF668 were found in 7.3% of the bladder cancer cases.

Drivers for mutation in amino acid sequences of two mitochondrial proteins (Cytb and COI) in Phytoseiidae mites (Acari: Mesostigmata).

Mutations in amino acid sequences can affect protein function. Such aspects have been poorly studied for arthropods. As recent studies have shown mutations in cytochrome b (Cytb) associated with geographic locations in several Phytoseiidae species, the present study aims at investigating (i) the mutation pattern in additional species for the Cytb fragment, (ii) the mutation pattern for another mitochondrial amino acid sequence, cytochrome c oxidase subunit 1 (COI), and (iii) factors affecting the mutations observed (taxonomy, plant support, climatic variables, wild vs. commercialised species). Mutations in amino acid sequences were assessed in seven Phytoseiidae species, with populations collected in contrasted environments. The DNA sequences were mainly obtained from published studies and some were newly obtained. Mutations were observed within and between the populations considered for both fragments, with higher mutation rates in Cytb than in COI sequences, confirming the robustness of this former fragment. Plant support and taxonomic position were not related to mutation patterns. A lower number of mutations was observed in commercialised populations than in wild ones. As preliminary tendencies, mutations in Cytb and COI sequences seem associated to temperature and moisture. Such a preliminary approach, attempting to relate mutation to functional adaptations, clearly opens new research tracks for better assessment of the drivers of mite adaptation, in a context of climate change.

Investigation on the Binding Properties of N1 Neuraminidase of H5N1 Influenza Virus in Complex with Fluorinated Sialic Acid Analog Compounds—a Study by Molecular Docking and Molecular Dynamics Simulations

Worldwide, only two types of antiviral inhibitors (M2 ion channel protein inhibitor and Neuraminidase inhibitors) are approved to treat the influenza viral infection. But the mutation of amino acid sequence in the viral membrane proteins creates the viral resistance to existing antiviral drugs or inhibitors. So the corresponding antiviral drugs have to be reformulated to match these antigenic variations. Fluorination on the carbon–based molecule significantly enriches its biological properties. Hence this study is motivated to design the fluorinated sialic acid (SIA) analog inhibitors for the neuraminidase of H5N1 influenza A virus by substituting fluorine atom at different hydroxyls (O2, O4, O7, O8, and O9) of sialic acid. 100 ns molecular dynamics simulations are carried out for each protein–ligand complex system. NAMD pair interaction energy and MM–PBSA binding free energy calculations predict two possible binding modes for N1–SIA_F2, N1–SIA_F4, and N1–SIA_F7 complexes and single binding mode for N1–SIA_F8 and N1–SIA_F9 complexes. RMSD, RMSF, and hydrogen bonding analyses are used to understand the conformational flexibility and structural stability of each complex system. It has been concluded that the fluorinated sialic acid drug candidates SIA_F2 and SIA_F7 have better inhibiting potency against the N1 neuraminidase of H5N1 influenza virus.

Effect of Genomic and Amino Acid Sequence Mutation on Virulence and Therapeutic Target of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS COV-2).

The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is one of the RNA coronaviruses which share the highest mutation rates of RNA viruses when compared with that of their hosts. The collective mutation rate of RNA viruses is up to a million times higher than their hosts and is correlated with enhanced virulence of viruses. The RNA, genomic material of SARS-CoV-2, has the capacity of showing amplified fast changes as the infection spreads. These changes were frequently observed in genes for spike glycoprotein, nucleocapsid, ORF1ab, and ORF8, together with RNA dependent RNA polymerase. In contrast, genes for envelope, membrane, ORF6, ORF7a and ORF7b showed no observable changes in terms of amino acid substitutions. Mutated SARS COV-2 at these particular sites has been associated with viral infectivity, false laboratory results and viral genome mutation and interferes with therapeutic targets. Interferences with therapeutic targets is frequently observed in genes for RdRp. Additionally, mutated viral genes for RdRp render slow fidelity of RdRp protein, resulting in a high mutation rate. Such a high mutation rate might allow new virulent forms of the virus to emerge and influence the disease profile. This review aimed to elaborate on the effect of genomic and amino acid sequence mutations on the virulence and therapeutic targets of SARS COV-2. To achieve this objective, multiple literatures have been reviewed.

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a “hot-spot”-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed.Each Gc-protein with amino acid deletions within the “hot spot” of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep.In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

What a catch! Climate determinant structural differences in Atlantic Cod hemoglobin

As the climate changes, we must understand the biology of our fish stocks to better support the conservation of our global fisheries. Gadus Morhau, commonly known as Atlantic Cod, differs from other tetrameric hemoglobin species in that one of the key packing contacts, Phenylalanine 122, is replaced by Leucine, changing the interaction between the key packing contacts and thus the overall structure of the hemoglobin. Additionally, due to a hemoglobin polymorphism, Atlantic Cod has multiple subpopulations, the most common of which are the homozygous genotypes Hbl‐1 and Hbl‐2. Hbl‐1 and Hbl‐2 genotypes differ due to a mutation in amino acid sequence on the β1 chain, in the Hbl‐1 genotype, at Methionine 55, Lysine 62, and Leucine 122 (as mentioned above). However, on the Hbl‐2 genotype, the amino acids in the corresponding locations are Valine 55, Alanine 62, and Methionine 122. These mutations result in a significant divergence in structure that completely changes the optimal living conditions for the fish. tThe ideal environmental temperatures for different types of Atlantic Cod, the Hbl‐2 allele has a frequency of 99% in northern regions, whereas the Hbl‐1 allele is more common in warmer areas, suggesting that the Hbl‐2 allele helps the fish adapt to colder temperatures. Furthermore, temperature seems to impact the oxygen affinity of the two genotypes. Due to its structure, Hbl‐2 has higher oxygen bonding affinity at temperatures 10 degrees Celsius and lower, whereas the Hbl‐1 allele functions more efficiently at temperatures 14 degrees Celsius or higher. Using JMol modeling software, we compared the structures of the Hbl‐1 to Hbl‐2 Atlantic Cod hemoglobins paying close attention to the relationship between substitutional packing contacts Leucine 122 in Hbl‐1 and Methionine 122 in Hbl‐2, and the commonality of contact amino acid, Arginine B12, which is located on the α1 chain of the hemoglobin. We used JMol to analyze how amino acid substitutions in the β1 chain of the Atlantic Cod cause significant structural differences that leads to variation in optimal living conditions; we infer those adaptations may prove advantageous to their survival in their extreme environments. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine the structure‐function relationships of the Atlantic Cod hemoglobin polymorphisms. The visual model will be a valuable tool in developing our story.

Relationship between Amino Acid Sequence Mutations and Human Diseases Revealed by Piezo 1 Ion Channel Sturctural Analysis

Relationship between Amino Acid Sequence Mutations and Human Diseases Revealed by Piezo 1 Ion Channel Sturctural Analysis

Comparison of antigenic mutation during egg and cell passage cultivation of H3N2 influenza virus.

PurposeWhen influenza viruses are cultured in eggs, amino acid mutations of the hemagglutinin may occur through egg adaptation. On the other hand, when influenza viruses are cultured in animal cells, no antigenic mutation occurs unlike in eggs. Therefore, we examined whether the antigenic mutations actually occurred after passage of H3N2 (A/Texas/50/2012) virus up to 15 times in eggs and MDCK-Sky3851 cells.Materials and MethodsPrototype A/Texas/50/2012 (H3N2) influenza virus which was isolated from clinical patient, not passaged in egg, was obtained and propagated using the specific pathogen free egg and the MDCK-Sky3851 cell line up to 15 passage, and the changes in the antigen sequence of the influenza viruses were confirmed by gene sequencing and protein structure analysis.ResultsIn term of the hemagglutination titer of influenza virus, the reactivity to chicken and guinea pig red blood cell showed different results between egg propagated and cell propagated viruses. In the sequence analysis results for hemagglutinin and neuraminidase, no antigenic mutation was observed throughout all passages when cultured in MDCK-Sky3851 cells. On the other hand, mutations occurred in three amino acid sequences (H156R, G186S, S219F) in hemagglutinin up to 15 passages when cultured in eggs.ConclusionH3N2 influenza virus cultured in eggs could lead mutations in amino acid sequence of hemagglutinin, distinct from the corresponding virus cultured in cells for which no antigenic mutation was observed. These findings suggest that cell culture is a more stable and effective way of production with lower risk of antigenic mutations for the manufacture of influenza vaccines.

Structural Analysis of Piezo1 Ion Channel Reveals the Relationship between Amino Acid Sequence Mutations and Human Diseases

Since first identified in 2010, Piezo proteins have been found to perform as poreforming mechanosensitive ion channels across a wide range of animals. As a Piezo ortholog primarily expressed in mammalian systems, Piezo1 has been observed to distribute mainly in nonsensory tissues, regulating osmotic homeostasis, proprioception, and light touch. With previous studies on the putative structure of Piezo1, the gating system and several mechanotransduction mechanisms have been proposed. Besides, mutations of specific amino acid sequences in Piezo1 have been linked to several human diseases such as dehydrated hereditary xerocytosis (DHS) and congenital lymphatic dysplasia (CLD). However, most of these mutations have not been well characterized. To further elucidate the relations between these mutations and diseases, UCSF Chimera is used as the tool to visualize the structural importance of each of these mutated amino acids. With the aid from UCSF Chimera, this study has recorded and interpreted clashes and contacts originated from each of the mutations. Accordingly, specific mechanisms between mutations and human diseases are proposed, which pave the way for healing.

Genome re-sequencing to identify single nucleotide polymorphism markers for muscle color traits in broiler chickens.

ObjectiveMeat quality including muscle color in chickens is an important trait and continuous selective pressures for fast growth and high yield have negatively impacted this trait. This study was conducted to investigate genetic variations responsible for regulating muscle color.MethodsWhole genome re-sequencing analysis using Illumina HiSeq paired end read method was performed with pooled DNA samples isolated from two broiler chicken lines divergently selected for muscle color (high muscle color [HMC] and low muscle color [LMC]) along with their random bred control line (RAN). Sequencing read data was aligned to the chicken reference genome sequence for Red Jungle Fowl (Galgal4) using reference based genome alignment with NGen program of the Lasergene software package. The potential causal single nucleotide polymorphisms (SNPs) showing non-synonymous changes in coding DNA sequence regions were chosen in each line. Bioinformatic analyses to interpret functions of genes retaining SNPs were performed using the ingenuity pathways analysis (IPA).ResultsMillions of SNPs were identified and totally 2,884 SNPs (1,307 for HMC and 1,577 for LMC) showing >75% SNP rates could induce non-synonymous mutations in amino acid sequences. Of those, SNPs showing over 10 read depths yielded 15 more reliable SNPs including 1 for HMC and 14 for LMC. The IPA analyses suggested that meat color in chickens appeared to be associated with chromosomal DNA stability, the functions of ubiquitylation (UBC) and quality and quantity of various subtypes of collagens.ConclusionIn this study, various potential genetic markers showing amino acid changes were identified in differential meat color lines, that can be used for further animal selection strategy.

Controlling the Biophysical Properties and Functionality of Phase-Separated Elastin-Based Droplets through Amino Acid Sequence Mutations

Liquid-liquid phase separation underlies the spontaneous formation of protein-rich droplets in a protein-poor solution phase, concomitant with a sharp increase in solution turbidity. Phase separation is typically triggered in response to a stimulus such as change in protein concentration, ionic strength, or temperature, and may be reversible or precede gelation, fibre formation, or aggregation. Phase separation is a well-known property of synthetic polymers, and is exploited for material design, for example, capsules for drug delivery and fast-setting hydrogels, but is not well understood for proteins. Some examples of phase-separating protein-based systems include lens gamma crystallin (implicated in cataract formation), ‘membraneless organelles’ (transiently formed compartments that regulate molecular interactions), and self-assembling elastomeric proteins, including some spider silks, insect resilin and vertebrate elastin, for which phase separation is on-pathway for the formation of elastic materials. Here we studied the phase separation of model polypeptides based on the protein elastin. These polypeptides contain both regions of well-defined secondary structure and intrinsic disorder. Droplet growth and biophysical properties were monitored using a variety of microscopy and optical spectroscopy techniques. We report the effect of amino acid sequence mutations on the size, stability, reversibility and interactions of phase-separated droplets, and address implications for functional tunability, including loading of small molecules.

Genetic characteristics of measles virus strains causing two outbreaks in Guizhou province

Objective To analyze the genetic characteristics of measles virus strains causing two outbreaks in Guizhou province from November 2014 to March 2015. Methods Throat swab samples collected from measles cases in two outbreaks were inoculated into Vero/SLAM cells. Viral RNAs were extracted from positive cultures. Nucleoprotein genes were amplified by using reverse transcription-polymerase chain reaction (RT-PCR) and the PCR products were sequenced and analyzed. Results Eleven strains of wild-type measles virus were isolated from the two measles outbreaks and all of them belonged to H1a sub-genotype. Phylogenetic analysis showed that those strains were clustered into two distinct branches. Differences in nucleotide and amino acid genetic distances between the 11 strains of measles virus and the WHO reference strain of H1a sub-genotype (Chin9322) were 1.1%-1.6% and 0.7%-3.4%, respectively. Compared with the reference strain Chin9322 and Guizhou epidemic strains in recent years, six strains showed amino acid sequence mutations in 47 (G to S), 82 (S to G) and 122 (R to K) sites and two strains had a mutation in 98 (P-L) site. Conclusion H1a sub-genotype measles virus was the predominant pathogen causing two measles outbreaks in Guizhou province during 2014 to 2015. Moreover, it was also a predominant sub-genotype circulating in China and Guizhou province. Different measles virus strains of H1a sub-genotype continued to be prevalent in Guizhou province. This study provided some scientific data for the control and elimination of measles in Guizhou province. Key words: Measles virus; Outbreak; Genotype; Sequence analysis

Comparative analysis of polygalacturonase in the fruit of strawberry cultivars.

The role of polygalacturonase (PG) in the development, ripening, and softening of fruit from two strawberry cultivars with different flesh firmness and softening characteristics was compared. Changes in PG activity and gene expression during development, ripening and softening were measured. The PG genes from each cultivar were cloned and analyzed, and were classified with other PG genes using phylogenetic analysis. In Toyonoka fruit, PG activity increased gradually, reaching a peak during the pink stage, and remained at this level during post-harvest softening. Changes in PG gene expression were consistent with PG activity in these softer fruits. In the firmer Sweet Charlie fruits, PG activity was detected during the initial development stage, reaching a peak at the white stage, thereafter decreasing gradually with ripening and remaining at this lower level throughout softening. Changes in PG gene expression and PG activity were not consistent in these fruit. For both Toyonoka and Sweet Charlie PG genes (FaTPG and FaSCPG, respectively), the open reading frame was 1218 bp, encoding 405 amino acids. Five different nucleotide sites were observed between the two sequences, leading to two amino acid sequence mutations. FaTPG, FaSCPG, and PG genes from the Fragaria vesca genome were classified into three clades using phylogenetic analysis. The clade containing PG genes involved in fruit softening had functional similarity but there were no functional differences between FaTPG and FaSCPG. Differences in PG activity, gene sequence, and gene expression may have led to different roles of PG during ripening and softening in strawberries with different textures.

Study on the genotypic characteristics of Japanese encephalitis virus and serology in the healthy population of Zhejiang province, 2012-2013

To understand the genotypic characteristics and the neutralizing antibody levels of Japanese encephalitis virus (JEV) and Japanese encephalitis (JE) in both vector mosquitoes and in healthy people of Zhejiang province. Virus was isolated from mosquitos sampled from the Monitoring Stations located in Xianju county during 2012 to 2013. Phylogenetic and homological studies were carried out on the E gene. A total of 1 263 blood specimens from 642 healthy people were collected before and after the seasons of JE epidemics. JEV neutralizing antibody was detected by the micro-neutralization test. Twenty-five JEV strains were isolated from a total of 11 650 mosquitoes. The identity of nucleotide appeared as 87.8%-99.7% both from 2012 to 2013 and from 1982 to 2010 while as 87.7%-88.0% with vaccine strain SA14-14-2, in Zhejiang. The phylogeny tree of E gene indicated that the newly isolated virus belonged to genotype I but no mutation of amino acid sequence coding conformational epitope was identified in the envelop protein. Both positive rates and the geometric mean titer (GMT) of neutralizing antibody in healthy people were 31.5%-42.0% and 1 : 2.56-1 : 3.53 in Xianju county, during 2012 and 2013, respectively. Both of the two positive rates (χ(2)≤1.76, P > 0.05) and the two GMTs (u≤0.64, P > 0.5) for antibodies pre or post the epidemic season did not show significant differences. JEV isolated in Xianju during 2012 and 2013 belonged to genotype I. The positive rates of JEV neutralizing antibody from healthy people in Xianju were less than 42.0%, which showed no significant differendes pre or post JE epidemic season.

Genome resequencing and bioinformatic analysis of SNP containing candidate genes in the autoimmune vitiligo Smyth line chicken model.

BackgroundThe Smyth line (SL) chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. To understand the genetic components underlying the susceptibility to develop SL vitiligo (SLV), whole genome resequencing analysis was performed in SLV chickens compared with non-vitiliginous parental Brown line (BL) chickens, which maintain a very low incidence rate of vitiligo.ResultsIllumina sequencing technology and reference based assembly on Red Jungle Fowl genome sequences were used. Results of genome resequencing of pooled DNA of each 10 BL and SL chickens reached 5.1x and 7.0x coverage, respectively. The total number of SNPs was 4.8 and 5.5 million in BL and SL genome, respectively. Through a series of filtering processes, a total of ~1 million unique SNPs were found in the SL alone. Eventually of the 156 reliable marker SNPs, which can induce non-synonymous-, frameshift-, nonsense-, and no-start mutations in amino acid sequences in proteins, 139 genes were chosen for further analysis. Of these, 14 randomly chosen SNPs were examined for SNP verification by PCR and Sanger sequencing to detect SNP positions in 20 BL and 70 SL chickens. The results of the analysis of the 14 SNPs clearly showed differential frequencies of nucleotide bases in the SNP positions between BL and SL chickens. Bioinformatic analysis showed that the 156 most reliable marker SNPs included genes involved in dermatological diseases/conditions such as ADAMTS13, ASPM, ATP6V0A2, BRCA2, COL12A1, GRM5, LRP2, OBSCN, PLAU, RNF168, STAB2, and XIRP1. Intermolecular gene network analysis revealed that candidate genes identified in SLV play a role in networks centered on protein kinases (MAPK, ERK1/2, PKC, PRKDC), phosphatase (PPP1CA), ubiquitinylation (UBC) and amyloid production (APP).ConclusionsVarious potential genetic markers showing amino acid changes and potential roles in vitiligo development were identified in the SLV chicken through genome resequencing. The genetic markers and bioinformatic interpretations of amino acid mutations found in SLV chickens may provide insight into the genetic component responsible for the onset and the progression of autoimmune vitiligo and serve as valuable markers to develop diagnostic tools to detect vitiligo susceptibility.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-707) contains supplementary material, which is available to authorized users.

Understanding the Relationship Between Biotherapeutic Protein Stability and Solid–Liquid Interfacial Shear in Constant Region Mutants of IgG1 and IgG4

Relative stability of therapeutic antibody candidates is currently evaluated primarily through their response to thermal degradation, yet this technique is not always predictive of stability in manufacture, shipping, and storage. A rotating disk shear device is proposed that produces defined shear conditions at a known solid–liquid interface to measure stability in this environment. Five variants of IgG1 and IgG4 antibodies were created using combinations of two discrete triple amino acid sequence mutations denoted TM and YTE. Antibodies were ranked for stability based on shear device output (protein decay coefficient, PDC), and compared with accelerated thermal stability data and the melting temperature of the CH2 domain (Tm1) from differential scanning calorimetry to investigate technique complimentarity. Results suggest that the techniques are orthogonal, with thermal methods based on intramolecular interaction and shear device stability based on localized unfolding revealing less stable regions that drive aggregation. Molecular modeling shows the modifications’ effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability. The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability.

Molecular characteristics of acute gastroenteritis outbreaks caused by norovirus, in Jiangsu province

To study both the epidemiologic and molecular characteristics of outbreaks caused by norovirus (NoV) with its variants, in Jiangsu. 67 specimens from seven gastroenteritis outbreaks were collected from October 2012 to March 2013 in Jiangsu. NoV gene group was detected by Real-Time RT-PCR. NoV portions of RdRp gene and VP1 gene were amplified under RT-PCR. Seven gastroenteritis outbreaks were caused by NoV. Among all the fecal specimens,45 (67.2%) showed positive to NoV G II. Study on the genotype was conducted through analyzing the nucleotide sequence of RdRp gene. Based on the RdRp region, 7 strains appeared to be G II, with 3 and 38 strains belonged to G II.4--Sydney variants. Results from phylogenetic analysis confirmed that 38 variants shared 99% identity with G II.4--Sydney. We also amplified the VP1 genes from 6 variants and comparing with 9 epidemic strains on the sequence amino acid sequence. All the strains showed mutation in amino acid sequence at some key sites which were closely related to the forming of neutralizing epitopes. The short interval periods between all 7 NoV outbreaks with identical viral strain indicated the emergence of a new NoV variant in Jiangsu province,that had caused a number of epidemics abroad. Results from our study suggested that the development of monitoring programs on this novel G II.4--Sydney variant should be a part of the NoV surveillance in Jiangsu province or even in the country.

PHYLOGENETIC ANALYSIS OF HUMAN INFLUENZA VIRUSES А(H3N2), ISOLATED IN UKRAINE DURING THE 2011–2012 EPIDEMIC SEASON

Aim. To perform the phylogenetic analysis of segments, encodes hemagglutinin (HA) and neuraminidase (NA) of A (H3N2) influenza viruses, isolated in Ukraine during the 2011–2012 epidemic season. Methods. In this study there were used the real-time polymerase chain reaction (realtime RT-PCR), sequencing and phylogenetic analysis methods. Results. The genetic segments HA and NA of Ukrainian influenza isolates of A (H3N2) influenza were analyzed. Appropriate phylogenetic trees were constructed by Neighbor-Joining method using the Kimura 2-parameter model. The statistical evaluation of phylogenetic analysis has been performed by uping the bootstrap test (1000 replicates). The genetic similarity of ukrainian isolates, vaccine strain and viruses all over the world were analyzed. All genetic groups of Victoria/208 cluster included our isolates were described. The key mutations in amino acid sequences in HA and NA proteins were identified. Conclusions. The multiple ways of entering the epidemic strains of influenza A (H3N2) viruses 2011–2012 season in Ukraine were identified as well. The mutations in hemagglutinin of Ukrainian isolates were located in highly variable regions and weren’t associated with significant changes in viruses. No selection of resistant mutations (E119V, E119V + I222V, R292K and N294S) to oseltamivir and zanamivir in NA proteins was observed.

N-Glycan Moieties in Neonatal Fc Receptor Determine Steady-state Membrane Distribution and Directional Transport of IgG

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule known to protect IgG and albumin from catabolism and transport IgG across polarized epithelial cells in a bidirectional manner. Previous studies have shown species-specific differences in ligand binding, IgG transport direction, and steady-state membrane distribution when expressed in polarized epithelial cells. We hypothesized that these differences may be due to the additional N-glycans expressed on the rat FcRn, because N-glycans have been proposed to function as apical targeting signals, and that two of the N-glycan moieties have been shown to contribute to the IgG binding of rat FcRn. A panel of mutant human FcRn variants was generated to resemble the N-glycan expression of rat FcRn in various combinations and subsequently transfected into Madin-Darby canine kidney II cells together with human beta2-microglobulin. Mutant human FcRn clones that contained additional N-glycan side-chain modifications, including that which was fully rodentized, still exhibited specificity for human IgG and failed to bind to mouse IgG. At steady state, the mutant human FcRn with additional N-glycans redistributed to the apical cell surface similar to that of rat FcRn. Furthermore, the rodentized human FcRn exhibited a reversal of IgG transport with predominant transcytosis from an apical-to-basolateral direction, which resembled that of the rat FcRn isoform. These studies show that the N-glycans in FcRn contribute significantly to the steady-state membrane distribution and direction of IgG transport in polarized epithelia.