Amino Acid Sequence Homology Research Articles

Unstable Allotetraploid Tobacco Genome due to Frequent Homeologous Recombination, Segmental Deletion, and Chromosome Loss

The types of mutations and their corresponding frequencies are difficult to measure in complex genomes. In this study, a high-throughput method was developed to identify spontaneous loss-of-function alleles for the resistance gene N and the transgenic avirulence gene P50 in allotetraploid tobacco. A total of 2134 loss-of-function alleles of the N gene were identified after screening 14 million F1 hybrids. Analysis of these mutants revealed striking evolutionary patterns for genes in polyploids. Only 14 of the loss-of-function mutations were caused by spontaneous point mutations or indels, while the others were caused by homeologous recombination (with a frequency of ∼1/12 000) or chromosome loss (∼1/15 000). Loss of the chromosome with the P50 insertion occurred at a similar frequency (∼1/13 000), and the frequency of spontaneous segmental deletion in this chromosome was ∼1/16 000. Both homeologous recombination and chromosome loss considerably decreased the viability of the mutants. Our data suggest that the high mutation rate in polyploids is probably due to the occurrence of homeologous recombination and the tolerance of large mutations such as chromosome loss in polyploid genomes. Frequent mutations tend to drive polyploids to extinction unless a novel mutation helps the polyploid to effectively compete with diploids or find a new ecological niche.

STAT5b Isoform Controls IgE-mediated Mast Cell Function

Abstract Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that control survival, proliferation and many other cellular functions. This protein family consists of seven members, including the closely related STAT5A and STAT5B, which show 96% amino acid sequence homology. We previously found that Stat5B is required for IgE-mediated cytokine production by mast cells in vitro, but have not tested in vivo correlates to this finding. In the current study, we show that Stat5B deficiency (KO) reduces IgE-induced passive systemic anaphylaxis (PSA) and IL-13 secretion in vivo, without altering mast cell tissue distribution. These results were corroborated by reduced PSA in response to anti-FceRI antibody injection. However, we were surprised to find that reduced PSA was not matched by lower plasma histamine levels. Further investigation revealed that Stat5B KO mice have a >5-fold increase in basal IgE levels and are hyperresponsive to histamine-induced anaphylaxis. These data support the conclusion that Stat5B is critical for IgE-induced mast cell activation in vivo, but also indicate that it contributes to basal IgE production and suppresses vascular responses to histamine. Thus Stat5B is involved in several overlapping aspects of the systemic mast cell response that warrant further study.

Analysis of T cell antigen receptor expression by porcine natural killer T cells

Abstract Invariant natural killer T (iNKT) cells are an innate-like T cell subset expressed by some, but not all mammals. Unlike conventional T cells, iNKT cells express a semi-invariant T cell receptor (TCR) (Vα24-Jα18/Vβ11 in human and Vα14-Jα18/Vβ8.2, Vβ2, or Vβ7 chains in mice) that recognizes glycolipid antigens presented by the non-polymorphic molecule CD1d. Once activated, iNKT cells generate an extensive array of immune responses that may be useful for treating cancer, autoimmunity and infectious disease in humans. In addition, there may be potential to target iNKT cells for veterinary applications. Pigs express iNKT cells that can be detected using mouse or human CD1d-tetramer reagents loaded with the prototypic iNKT cell ligand, α-galactosylceramide (α-GalCer). We exploited this phenomenon to define the TCR repertoire of iNKT cells (CD3α+CD1d tetramer+) compared to conventional T cells (CD3α+CD1d tetramer−) sorted from the peripheral blood of commercial mixed-breed pigs. We found that iNKT cells are highly enriched for the α-chain and CDR3α loop from Vα87*-Jα60* that possess a high level of amino acid sequence homology with human Vα24 (85%) and Jα18 (86%) as well as mouse Vα14 (74%) and Jα18 (79%). Particularly striking was that the most abundant CDR3α sequence was a near perfect match for the human invariant CDR3α. Porcine iNKT cells were also biased for Vβ84* which is homologous to Vβ11 (74%) in humans, as well as Vβ8.2 (71%) and Vβ2 (80%) in mice. Our findings indicate that the porcine iNKT cell TCR is highly homologous to mouse and particularly human iNKT cell TCR, which suggests that iNKT cells among these species play a similar role in immunity and disease.

Identification and characterization of karyotype in Passiflora hybrids using FISH and GISH

BackgroundA great interest exists in the production of hybrid plants of the genus Passiflora given the beauty and exotic features of its flowers which have ornamental value. Hybrid paternity confirmation is therefore important for assuring germplasm origin, and is typically carried out by molecular marker segregation. The aim of this study was to karyotypically characterize the chromosome heritance patterns of the progeny resultant from a cross of P. gardneri and P. gibertii using classical cytogenetics, chromosome banding, and molecular cytogenetics.ResultsAll analyzed genotypes showed the same diploid chromosome number as the genitor species: 2n = 18. Classical and CMA3 and DAPI staining allowed for chromosome counting and satellite identification (secondary constrictions). Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used to characterize subgenomes by either identifying rDNA-specific genome patterns or parental genomes, respectively.ConclusionsThe heritance of chromosomal markers presenting rDNA sites from each parent for genome identification confirmed that all obtained plants were hybrids. These results will improve breeding programs involving the species of this genus. Apart from confirming hybridization, GISH allowed the visualization of recombination between the homeologous chromosome and the introgression of sequences of interest.

In Vitro-In Vivo Extrapolation of OATP1B-Mediated Drug-Drug Interactions in Cynomolgus Monkey.

Hepatic organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are clinically relevant transporters associated with significant drug-drug interactions (DDIs) and safety concerns. Given that OATP1Bs in cynomolgus monkey share >90% degree of gene and amino acid sequence homology with human orthologs, we evaluated the in vitro-in vivo translation of OATP1B-mediated DDI risk using this preclinical model. In vitro studies using plated cynomolgus monkey hepatocytes showed active uptake Km values of 2.0 and 3.9 µM for OATP1B probe substrates, pitavastatin and rosuvastatin, respectively. Rifampicin inhibited pitavastatin and rosuvastatin active uptake in monkey hepatocytes with IC50 values of 3.0 and 0.54 µM, respectively, following preincubation with the inhibitor. Intravenous pharmacokinetics of 2H4-pitavastatin and 2H6-rosuvastatin (0.2 mg/kg) and the oral pharmacokinetics of cold probes (2 mg/kg) were studied in cynomolgus monkeys (n = 4) without or with coadministration of single oral ascending doses of rifampicin (1, 3, 10, and 30 mg/kg). A rifampicin dose-dependent reduction in i.v. clearance of statins was observed. Additionally, oral pitavastatin and rosuvastatin plasma exposure increased up to 19- and 15-fold at the highest dose of rifampicin, respectively. Use of in vitro IC50 obtained following 1 hour preincubation with rifampicin (0.54 µM) predicted correctly the change in mean i.v. clearance and oral exposure of statins as a function of mean unbound maximum plasma concentration of rifampicin. This study demonstrates quantitative translation of in vitro OATP1B IC50 to predict DDIs using cynomolgus monkey as a preclinical model and provides further confidence in application of in vitro hepatocyte data for the prediction of clinical OATP1B-mediated DDIs.

Comprehensive analysis of triacylglycerol lipases in the oleaginous diatom Fistulifera solaris JPCC DA0580 with transcriptomics under lipid degradation

Microalgal biofuels are desirable alternatives for traditional liquid fuels, but further improvements of productivity are necessary for microalgal biofuels to be economically feasible. As strategies for improving lipid productivity, repression of lipid degradation has a great potential because the lipid accumulation level is determined by a balance between lipid synthesis and degradation. However, studies of lipid degradation and its primary functioning enzyme triacylglycerol (TAG) lipases in microalgae are currently limited. In this study, we report the comprehensive analysis of TAG lipases in the oleaginous diatom Fistulifera solaris JPCC DA0580. First, we confirmed that TAGs in the lipid accumulation organelles, oil bodies, were degraded when the cells were transferred from the nutrient depleted conditions to nutrient replete condition. Further analysis revealed that, after eicosapentaenoic acid (C20:5n-3)-containing TAGs are were degraded, its molecular skeletons were likely to be recycled to produce glycolipids and phospholipids for chloroplast regeneration and cell growth, respectively. Next, we searched putative TAG lipase genes from the draft genome sequence of F.solaris, and discovered 42 candidates based on the amino acid sequence homology analysis. Subsequent transcriptome analysis revealed that 16 of the 42 lipase genes were up-regulated during lipid degradation. Among the up-regulated lipases, a number of enzymes were predicted to localize in endoplasmic reticulum which is closely associated to the lipid accumulation organelles, oil bodies. Our study provided new insights of lipid degradation in oleaginous microalgae, and putative TAG lipases which could be candidates for metabolic engineering in future study to improve microalgal lipid productivity.

A 12-residue epitope displayed on phage T7 reacts strongly with antibodies against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a major threat to the livestock industry worldwide. Despite constant surveillance and effective vaccination, the perpetual mutations of the foot-and-mouth disease virus (FMDV) pose a huge challenge to FMD diagnosis. The immunodominant region of the FMDV VP1 protein (residues 131-170) displayed on phage T7 has been used to detect anti-FMDV in bovine sera. In the present study, the functional epitope was further delineated using amino acid sequence alignment, homology modelling and phage display. Two highly conserved regions (VP1145-152 and VP1159-170) were identified among different FMDV serotypes. The coding regions of these two epitopes were fused separately to the T7 genome and displayed on the phage particles. Interestingly, chimeric phage displaying the VP1159-170 epitope demonstrated a higher antigenicity than that displaying the VP1131-170 epitope. By contrast, phage T7 displaying the VP1145-152 epitope did not react significantly with the anti-FMDV antibodies in vaccinated bovine sera. This study has successfully identified a smaller functional epitope, VP1159-170, located at the C-terminal end of the structural VP1 protein. The phage T7 displaying this shorter epitope is a promising diagnostic reagent to detect anti-FMDV antibodies in vaccinated animals.

Expression characteristics of glutamine synthetase of wheat in Escherichia coli

Glutamine synthetase is a key enzyme in plant nitrogen assimilation. To study the structure of wheat glutamine synthetase isoenzymes, GS1, GSr, GSe, GS2 and GS2p of wheat were cloned into pET-21a, and the expression condition was optimized. Although wheat glutamine synthetase isoenzymes had 70%-80% amino acid sequence homology, the isoforms expressed with different characteristics. Induced at 30 °C, the most expression level of GSr, GSe and GS2 was after 3 h, and of GS1 was at the 7 h whereas no GS2p was expressed, and the GS isoenzymes showed different expression level, with the order of GS1 (22%)>GSr (15%)>GS2 (12%)>GSe (5%). GSe expressed as soluble protein, and GS1 expressed mainly as soluble protein whereas GSr and GS2 expressed as insoluble proteins. Induced at 30 °C for 3 h, mRNA transcript levels of GS isoforms were different, with the order of GSr (7.59)>GS2 (1.84)>GS2p (1.66)>GSe (1.46)>GS1 (1.00). The levels of mRNA transcription were not consistent with the level of the protein translation. The analysis of mRNA secondary structure showed the free energy of translation initiation region of glutamine synthetase isoforms was different, with the order of GS1 (14.4)<GSr (17.2)<GS2 (22.6) <GSe (25.4) <GS2p (31.6), the smaller freed energy, the more unstable mRNA secondary structure of translation initiation region and the higher level of protein expression. Soluble expression condition of glutamine synthetase isozymes was also different, with GS1, GSr, GSe and GS2 induced at 30 °C for 5 h, 16 °C for 15 h, 37 °C for 5 h, and 25 °C for 7 h respectively. The soluble protein showed different expression level with GS1 (20%)>GSr (13%)>GS2 (10%)>GSe (7%), and different activities with GS1>GSe>GS2, and the activity of GSr was not detected. The gene sequence of glutamine synthetase isoenzymes determines the amount, status and activity of proteins expressed in prokaryotic cells.

CD8+ T cells with characteristic T cell receptor beta motif are detected in blood and expanded in synovial fluid of ankylosing spondylitis patients

The risk of AS is associated with genomic variants related to antigen presentation and specific cytokine signalling pathways, suggesting the involvement of cellular immunity in disease initiation/progression. The aim of the present study was to explore the repertoire of TCR sequences in healthy donors and AS patients to uncover AS-linked TCR variants. Using quantitative molecular-barcoded 5'-RACE, we performed deep TCR β repertoire profiling of peripheral blood (PB) and SF samples for 25 AS patients and 108 healthy donors. AS-linked TCR variants were identified using a new computational approach that relies on a probabilistic model of the VDJ rearrangement process. Using the donor-agnostic probabilistic model, we reveal a TCR β motif characteristic for PB of AS patients, represented by eight highly homologous amino acid sequence variants. Some of these variants were previously reported in SF and PB of patients with ReA and in PB of AS patients. We demonstrate that identified AS-linked clones have a CD8+ phenotype, present at relatively low frequencies in PB, and are significantly enriched in matched SF samples of AS patients. Our results suggest the involvement of a particular antigen-specific subset of CD8+ T cells in AS pathogenesis, confirming and expanding earlier findings. The high similarity of the clonotypes with the ones found in ReA implies common mechanisms for the initiation of the diseases.

Comparative approach sex cromosomes in Belostoma species by genomic in situ hibridization (GISH) regarding to the evolutionary process

All Belostoma species studied possess holokinetic chromosomes and an inverse relationship between chromosome number and chromosome size because autosomal and/or sex chromosome fusions and sex chromosome fragmentations have seemingly played the most important role in their karyotype evolution. The modal diploid chromosome number 2n=26+X1X2Y/X1X1X2X2 (male/female) observed in ten species and reduced chromosome numbers (16, 14, and 8) with a simple sex chromosome system XY/XX (male/female) detected in other six species could be indicating two paths of karyotype evolution. In species with a multiple sex chromosome system (X1X2Y), the NOR is located on an autosomal bivalent, whereas in species with a simple sex chromosome system (XY) it is placed on both sex chromosomes. Genomic in situ hybridization (GISH) assay could be a useful tool to compare the genome of Belostoma species, which have different karyotype numbers and sex chromosome systems, providing information on the genome organization and distinctive location of DNA fractions on sex chromosomes. We examined the composition of sex chromosomes from B. elegans (2n=26+X1X2Y/X1X1X2X2), B. micantulum (2n=14+XY/XX), and B. oxyurum (2n=6+XY/XX) by means GISH due to they can be considered as model species to ascertain the possible trends of karyotype evolution. We hybridized labelled male genomic probe of each species to each corresponding male chromosome preparations with an excess of unlabelled female genomic DNA of the same species. Belostoma micantulum and B. oxyurum presented larger Y chromosomes than B. elegans, and the genomic probes displayed strong binding in 40-60% of the total length of Ys. However, the male probe of B. elegans highlighted to its whole Y chromosome. Based on these results and our previous data, we may conclude that the Y sex chromosome constitution in B. micantulum and B. oxyurum was originated by fusion between the ancestral NOR-autosomal bivalent and the original sex chromosomes. The advantage of this approach allowed us to study chromosome rearrangements as well as to identify homeologous chromosomes of related species. Therefore, application of molecular cytogenetic techniques constitutes for elucidating chromosome evolution in this group.

Molecularly Imprinted Nanocavities Capable of Ligand-Binding Domain and Size/Shape Recognition for Selective Discrimination of Vascular Endothelial Growth Factor Isoforms.

Vascular endothelial growth factor 165 (VEGF165) is known to be predominantly expressed in the first stage of vascularization; therefore, the detection of VEGF165 is important in the stage diagnosis of cancers. Molecularly imprinted nanocavities, capable of the selective discrimination of VEGF165 from other VEGF isoforms, were prepared by surface-initiated atom transfer radical polymerization. VEGF165 was immobilized on a gold-coated glass substrate by anchored heparin moieties, where the immobilized heparin was able to capture VEGF165 by binding with the heparin-binding domain (HBD) on VEGF165. Molecular imprinting was conducted on the immobilized VEGF165 by using methacrylic acid (MAA) as a functional monomer to interact with basic amino acids outside of the HBD of VEGF165 by electrostatic interaction. After the removal of VEGF165 from the obtained polymer thin layer (ca. 7 nm), VEGF165-imprinted nanocavities remained, in which the heparin moiety and MAA residues were located in suitable positions for VEGF165 recognition. The molecularly imprinted polymer (MIP) thin layer showed a binding affinity for VEGF165 (dissociation constant: 3.4 nM) that was ten times higher than that of the substrate before polymerization (heparin-immobilized substrate). A much lower binding affinity for VEGF121, which contains no heparin-binding domain, was observed. Moreover, the MIP thin layer distinguished VEGF165 from VEGF189, which possesses a larger molecular size than VEGF165, an amino acid sequence homology of 87%, and contains HBDs, whereas the heparin-immobilized substrate showed almost no selectivity. These results suggested that the heparin moiety within the nanocavity provided HBD selectivity and the polymer matrix composed of the molecularly imprinted nanocavity provided size/shape selectivity, which resulted in the highly selective discrimination of VEGF isoforms.

Comparative Oligo-FISH Mapping: An Efficient and Powerful Methodology To Reveal Karyotypic and Chromosomal Evolution.

Developing the karyotype of a eukaryotic species relies on identification of individual chromosomes, which has been a major challenge for most nonmodel plant and animal species. We developed a novel chromosome identification system by selecting and labeling oligonucleotides (oligos) located in specific regions on every chromosome. We selected a set of 54,672 oligos (45 nt) based on single copy DNA sequences in the potato genome. These oligos generated 26 distinct FISH signals that can be used as a "bar code" or "banding pattern" to uniquely label each of the 12 chromosomes from both diploid and polyploid (4× and 6×) potato species. Remarkably, the same bar code can be used to identify the 12 homeologous chromosomes among distantly related Solanum species, including tomato and eggplant. Accurate karyotypes based on individually identified chromosomes were established in six Solanum species that have diverged for >15 MY. These six species have maintained a similar karyotype; however, modifications to the FISH signal bar code led to the discovery of two reciprocal chromosomal translocations in Solanum etuberosum and S. caripense We also validated these translocations by oligo-based chromosome painting. We demonstrate that the oligo-based FISH techniques are powerful new tools for chromosome identification and karyotyping research, especially for nonmodel plant species.

Hybrids of Passiflora: P. gardneri versus P. gibertii, confirmation of paternity, morphological and cytogenetic characterization

Two new varieties of interspecific hybrids of Passiflora have been developed from the cross between P. gardneri versus P. gibertii, both registered under the Passiflora Society International. Twelve putative hybrids were analyzed. Hybridization was confirmed using RAPD and SSR markers. Primer UBC11 (5′-CCGGCCTTAC-3′) generated informative bands. Primer SSR Pe75 has amplified species-specific fragments and a heterozygote status was observed with two parent bands 300 and 350 bp. The molecular markers generated have been analyzed for the presence or absence of specific informative bands. Based on the morphological characterization, we have identified two hybrid varieties: P. ‘Gabriela’ and P. ‘Bella’. P. ‘Gabriela’ produced flowers in bluish tones, bluish petals on the adaxial and abaxial faces, light blue sepals on the adaxial and light green on the abaxial faces, corona with the base of filaments in intense lilac color and white apex. P. ‘Bella’ produced flowers in lilac tones, intense lilac petals on the adaxial and abaxial faces, dark lilac sepals with whitish edges on the adaxial and light green on the abaxial faces, corona with the base of filaments in intense lilac color and white apex. The cytogenetic analysis verified that the hybrids have the same chromosomal number as the parents (2n = 18); the formation of bivalents between the homeologous chromosomes (n = 9) was observad, leading to regular meiosis, which allows the sexual reproduction and use of these hybrids in breeding programs.

Cloning, expression, purification and structure analysis of protein tyrosine phosphatase of Bombyx mori

Protein tyrosine phosphatase (PTP, EC 3.1.3.48) specifically catalyzes the removal of phosphate groups from phosphorylated tyrosine residues, resulting in protein dephosphorylation, thus regulates life activities such as cell growth, proliferation, differentiation and immunization. Protein tyrosine phosphatase h of Bombyx mori (BmPTP-h) is involved in the replication of nucleopolyhedrovirus (NPV) in Bombyx mori, but the structure and properties of BmPTP-h are little known so far. In this study, the coding sequence of BmPTP-h gene was cloned from the midgut of Bombyx mori, and its genomic structure, mRNA structure, sequence signature, secondary structure and the state in solution were analyzed. Homologous amino acid sequences alignment analysis indicated that BmPTP-h had a high similarity to PTP sequences of numbers of insect NPVs, implying that they may have a common ancestor and similar function. We constructed a prokaryotic expression vector, expressed and obtained the soluble recombinant BmPTP-h in Escherichia coli at 25 ℃, and purified BmPTP-h using Ni-NTA affinity chromatography. Gel filtration analysis showed that BmPTP-h was able to form aggregate and monomer in solution. Circular dichroism spectroscopy analysis showed that the recombinant BmPTP-h contained α-helix structure. Increasing temperature resulted in the unfolding of the α-helix structure of BmPTP-h and the decrease of the α-helix structure content of BmPTP-h. These studies provide a basis to better study the structure and regulation mechanism of BmPTP-h.

An ultrasensitive electrochemical immunosensor for Cry1Ab based on phage displayed peptides

The rapid and widespread adoption of Bacillus thuringiensis (Bt) proteins in genetically modified (GM) crops has raised concerns about the impact of GM crops on environment and food safety. A sensitive and specific method for detecting Bt proteins in GM crops is of great significance for environment and food risk assessment. In this study, using Cry1Ab as a model Bt protein, an ultrasensitive electrochemical immunosensor for Cry1Ab protein has been developed based on phage displayed peptide. Phage displayed peptides against Cry1Ab protein were obtained from a phage displayed peptide library without animal immunization process through biopanning-elution strategy. After modification of electrode with gold nanoparticles, selected phage displayed peptide was applied to electrochemical immunosensor for Cry1Ab. Under the optimized conditions, the peptide-based immunosensor showed a dynamic range of 0.01–100ng/mL and a limit detection of 7pg/mL. Specific measurement of this established method was conducted by testing cross-reactivity of Cry1Ac (88% amino acid sequence homology to Cry1Ab protein), and the result showed that peptide-based immunosensor has negligible cross-reactivity with analogue. In addition, the accuracy and reproducibility of this established immunosensor was evaluated by testing the recovery of spiked samples and assay coefficients of variation, respectively. The results showed that the average recovery of corn and wheat sample was 90–120% and 86.7–120%, respectively; the intra-assay coefficient of variation was 7.4% (n = 6), and the inter-assay coefficient of variation was 6.9% (n = 6) at 1ng/mL Cry1Ab solution. Furthermore, the novel concept of peptide-based immunosensor may provide a potential application in general method for the ultrasensitive detection of various Bt proteins.

Sub genome anchored physical frameworks of the allotetraploid Upland cotton (Gossypium hirsutum L.) genome, and an approach toward reference-grade assemblies of polyploids

Like those of many agricultural crops, the cultivated cotton is an allotetraploid and has a large genome (~2.5 gigabase pairs). The two sub genomes, A and D, are highly similar but unequally sized and repeat-rich, which pose significant challenges for accurate genome reconstruction using standard approaches. Here we report the development of BAC libraries, sub genome specific physical maps, and a new-generation sequencing approach that will lead to a reference-grade genome assembly for Upland cotton. Three BAC libraries were constructed, fingerprinted, and integrated with BAC-end sequences (BES) to produce a de novo whole-genome physical map. The BAC map was partitioned by sub genomes through alignment to the diploid progenitor D-genome reference sequence with densely spaced BES anchor points and computational filtering. The physical maps were validated with FISH and genetic mapping of SNP markers derived from BES. Two pairs of homeologous chromosomes, A11/D11 and A12/D12, were used to assess multiplex sequencing approaches for completeness and scalability. The results represent the first sub genome anchored physical maps of Upland cotton, and a new-generation approach to the whole-genome sequencing, which will lead to the reference-grade assembly of allopolyploid cotton and serve as a general strategy for sequencing other polyploid species.

A pipeline for proteome-scale identification and studies on hormone sensitive lipases in Mycobacterium tuberculosis

Hormone sensitive lipases (HSLs) play an important role in the survival of M. tuberculosis during dormancy. They help in the utilization of fatty acids from stored lipids. The objective of the current study was to identify all HSLs from the proteome of M. tuberculosis H37Rv. We have developed a novel HSL identification pipeline, based on amino acid sequence homology, presence of conserved motifs and other sequence features deciphered from known HSL dataset. Through this pipeline, we identified 10 proteins as putative HSLs in M. tuberculosis. We have annotated a lipase LipT, as putative p-nitrobenzyl esterase and also identified a new motif “PGG” which is a possible characteristic motif of a subfamily of HSLs.

Modular endolysin of Burkholderia AP3 phage has the largest lysozyme-like catalytic subunit discovered to date and no catalytic aspartate residue

Endolysins are peptidoglycan-degrading enzymes utilized by bacteriophages to release the progeny from bacterial cells. The lytic properties of phage endolysins make them potential antibacterial agents for medical and industrial applications. Here, we present a comprehensive characterization of phage AP3 modular endolysin (AP3gp15) containing cell wall binding domain and an enzymatic domain (DUF3380 by BLASTP), both widespread and conservative. Our structural analysis demonstrates the low similarity of an enzymatic domain to known lysozymes and an unusual catalytic centre characterized by only a single glutamic acid residue and no aspartic acid. Thus, our findings suggest distinguishing a novel class of muralytic enzymes having the activity and catalytic centre organization of DUF3380. The lack of amino acid sequence homology between AP3gp15 and other known muralytic enzymes may reflect the evolutionary convergence of analogous glycosidases. Moreover, the broad antibacterial spectrum, lack of cytotoxic effect on human cells and the stability characteristics of AP3 endolysin advocate for its future application development.

Design, synthesis, biological evaluation and molecular modeling of novel 2-amino-4-(1-phenylethoxy) pyridine derivatives as potential ROS1 inhibitors

With the aim of discovering potential and selective inhibitors targeting ROS1 kinase, we rationally designed, synthesized and evaluated two series of novel 2-amino-pyridine derivatives with 1-phenylethoxy at C-3 and C-4 position. The enzymic assays results indicated that six of the new compounds 13b-13d and 14a-14c showed remarkably higher inhibitory activities against ROS1 kinase. The most promising compounds, 13d and 14c displayed the most desired ROS1 inhibitory activity with IC50 values of 440 nM and 370 nM respectively. Furthermore, 13d and 14c displayed ROS1 inhibitory selectivity of about 7-fold and 12-fold, relative to that of ALK sharing about 49% amino acid sequence homology in the kinase domains. They also showed good anti-proliferative effects against ROS1-addicted HCC78 cell lines with the IC50 values of 8.1 μM and 65.3 μM, respectively. Moreover, molecular docking and molecular dynamics simulation studies disclosed that compound 14c and 13d shared similar binding poses with Crizotinib except the selective binding site of ROS1. It also gave a probable molecular explanation for their activity and selectivity, which the methoxyl group in benzene ring was the crucial to the selectivity to ROS1 versus ALK.

T Cell Immunity to Zika and Dengue Viral Infections.

The 2015-2016 Zika virus (ZIKV) outbreak in the Americas has brought this relatively unknown flavivirus into the spotlight as the etiologic agent of congenital Zika syndrome in newborns and, potentially, of Guillain-Barré syndrome in adults. ZIKV shares a high degree of amino acid sequence homology with the closely related flavivirus Dengue virus (DENV). Due to the paucity of research investigating T cell-mediated immune responses to ZIKV, the precise roles of T cells in protection against and pathogenesis of ZIKV infection are unclear. Nevertheless, knowledge gained from studies of T cell responses to other flaviviruses, such as the four DENV serotypes, provides a framework for investigating and understanding anti-ZIKV T cell responses. In this review, we summarize our current understanding of how T cells respond to primary and secondary infections with DENV and ZIKV. A thorough understanding of the role of ZIKV-specific and DENV cross-reactive T cells in controlling infection is essential for developing ZIKV- and DENV-specific vaccines with maximal safety and efficacy.