Putative Protein Research Articles

Resequencing of the TMF-1 (TATA Element Modulatory Factor) regulated protein (TRNP1) gene in domestic and wild canids

BackgroundCortical folding is related to the functional organization of the brain. The TMF-1 regulated protein (TRNP1) regulates the expansion and folding of the mammalian cerebral cortex, a process that may have been accelerated by the domestication of dogs. The objectives of this study were to sequence the TRNP1 gene in dogs and related canid species, provide evidence of its expression in dog brain and compare the genetic variation within dogs and across the Canidae. The gene was located in silico to dog chromosome 2. The sequence was experimentally confirmed by amplifying and sequencing the TRNP1 exonic and promoter regions in 72 canids (36 purebred dogs, 20 Gy wolves and wolf-dog hybrids, 10 coyotes, 5 red foxes and 1 Gy fox).ResultsA partial TRNP1 transcript was isolated from several regions in the dog brain. Thirty genetic polymorphisms were found in the Canis sp. with 17 common to both dogs and wolves, and only one unique to dogs. Seven polymorphisms were observed only in coyotes. An additional 9 variants were seen in red foxes. Dogs were the least genetically diverse. Several polymorphisms in the promoter and 3'untranslated region were predicted to alter TRNP1 function by interfering with the binding of transcriptional repressors and miRNAs expressed in neural precursors. A c.259_264 deletion variant that encodes a polyalanine expansion was polymorphic in all species studied except for dogs. A stretch of 15 nucleotides that is found in other mammalian sequences (corresponding to 5 amino acids located between Pro58 and Ala59 in the putative dog protein) was absent from the TRNP1 sequences of all 5 canid species sequenced. Both of these aforementioned coding sequence variations were predicted to affect the formation of alpha helices in the disordered region of the TRNP1 protein.ConclusionsPotentially functionally important polymorphisms in the TRNP1 gene are found within and across various Canis species as well as the red fox, and unique differences in protein structure have evolved and been conserved in the Canidae compared to all other mammalian species.

Compatible solutes determine the heat resistance of conidia

BackgroundAsexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle.ResultsIn this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance.ConclusionsHeat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

IMMU-28. TREM2 INHIBITION REPROGRAMS MYELOID CELLS TO STIMULATE ANTI-TUMOR IMMUNITY THROUGH BOTH DIRECT AND INDIRECT MECHANISMS

Abstract The prevailing view is that myeloid cells—macrophages and microglia—in the tumor microenvironment (TME) are immunosuppressive and promote glioblastoma (GBM) progression. However, myeloid cells have the functional plasticity to restrict or support tumor cell growth. TREM2 plays important roles in brain microglial function in neurodegenerative diseases, but the functional role of TREM2 in the GBM TME and whether TREM2 represents a meaningful therapeutic target remain largely unknown. To address these questions, we investigated the immune and tumor cell consequences of TREM2 inhibition by multiple orthogonal approaches—transgenic mice, antisense oligonucleotides, and blocking antibodies—in immunocompetent preclinical models (SB28, NPA C54B) and human GBM samples through bulk and single cell RNA-sequencing, flow cytometry, biochemistry, and functional assays. We found TREM2 is highly expressed in myeloid subsets, including macrophages and microglia in human and mouse GBM tumors, and that high TREM2 expression correlates with poor prognosis in GBM patients. TREM2 loss of function in human macrophages and mouse myeloid cells increased IFNγ-induced immunoactivation and proinflammatory polarization. Conditioned media from TREM2 deficient human macrophages directly killed patient-derived GBM tumor cells, and biochemical analysis identified TNF and other putative tumoricidal proteins in this conditioned media. In mouse GBM models, mice with chronic and acute Trem2 loss of function demonstrated increased tumor cell death and increased animal survival. Trem2 inhibition reprogrammed myeloid cell phenotypes and increased PD-1+CD8+ T cells in the TME. Remarkably, Trem2 deficiency enhanced the effectiveness of anti-PD-1 treatment in vivo. Trem2 inhibition suppresses tumor cell growth through direct and indirect immune cell mechanisms--by reshaping myeloid subset and T cell functions. Thus, Trem2 inhibitory strategies, in combination with immune checkpoint blockade, may represent an attractive new approach for GBM therapy.

Abstract 18472: Plasma Proteome Changes in Heart Failure and Non-Heart Failure Patients Undergoing Cardiac Surgery

Introduction: Patients with and without heart failure display different clinical courses after cardiac surgery. Hypothesis: Unbiased analyses of the plasma proteome from extreme phenotypes in myocardial function may identify differences in candidate proteins differentially modified by cardiac surgery using cardiopulmonary bypass (CPB). Methods: We combined two prospectively-collected datasets: Left ventricular assist device (LVAD) recipients from the INSPIRE-FLO trial (N=94, NCT03081052) and coronary artery bypass graft (CABG) surgery patients with normal biventricular function (N=50). For each population, we obtained EDTA-plasma from arterial blood sampling in the operating room and on postoperative day 1. Including quality control pools, 304 total samples were digested in a 96-well format with trypsin. Peptides were analyzed by microflow liquid chromatography coupled to tandem mass spectrometry using data-independent acquisition. Proteins were identified and quantified at a 1% false discovery rate (FDR) in Spectronaut. Statistical analyses utilized a t-test with FDR correction (significance defined by log2FC>1 and q<0.05). Results: There were 943 protein groups quantified in at least one plasma sample. Patients undergoing LVAD surgery had significantly higher levels of 135 proteins including acute phase reactants (CRP, SAA1, SAA2), putative neutrophil-derived proteins (S100A8/9) and markers of renal insufficiency (B2M and REG3A) (Figure 1). Cardiac surgery using cardiopulmonary bypass increased acute phase and inflammatory markers, and cardiac proteins (ENOB, MYG and KCRM), with NGAL, TIMP1 and PAI1 among proteins with higher absolute increase after LVAD implantation versus CABG surgery ( Fig.1 ). Conclusions: Unbiased analyses of the plasma proteome identifies baseline and postoperative differences associated with extreme phenotypes of myocardial function undergoing cardiac surgery using CPB.

The YmgB-SpoT interaction triggers the stringent response in Escherichia coli

Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent from its proposed role in the two component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.

SEN1990 is a predicted winged helix-turn-helix protein involved in the pathogenicity of Salmonella enterica serovar Enteritidis and the expression of the gene oafB in the SPI-17.

Excisable genomic islands (EGIs) are horizontally acquired genetic elements that harbor an array of genes with diverse functions. ROD21 is an EGI found integrated in the chromosome of Salmonella enterica serovar Enteritidis (Salmonella ser. Enteritidis). While this island is known to be involved in the capacity of Salmonella ser. Enteritidis to cross the epithelial barrier and colonize sterile organs, the role of most ROD21 genes remains unknown, and thus, the identification of their function is fundamental to understanding the impact of this EGI on bacterium pathogenicity. Therefore, in this study, we used a bioinformatical approach to evaluate the function of ROD21-encoded genes and delve into the characterization of SEN1990, a gene encoding a putative DNA-binding protein. We characterized the predicted structure of SEN1990, finding that this protein contains a three-stranded winged helix-turn-helix (wHTH) DNA-binding domain. Additionally, we identified homologs of SEN1990 among other members of the EARL EGIs. Furthermore, we deleted SEN1990 in Salmonella ser. Enteritidis, finding no differences in the replication or maintenance of the excised ROD21, contrary to what the previous Refseq annotation of the protein suggests. High-throughput RNA sequencing was carried out to evaluate the effect of the absence of SEN1990 on the bacterium's global transcription. We found a downregulated expression of oafB, an SPI-17-encoded acetyltransferase involved in O-antigen modification, which was restored when the deletion mutant was complemented ectopically. Additionally, we found that strains lacking SEN1990 had a reduced capacity to colonize sterile organs in mice. Our findings suggest that SEN1990 encodes a wHTH domain-containing protein that modulates the transcription of oafB from the SPI-17, implying a crosstalk between these pathogenicity islands and a possible new role of ROD21 in the pathogenesis of Salmonella ser. Enteritidis.

A Multiomic, Single-Cell Measurable Residual Disease (scMRD) Assay for Phasing DNA Mutations and Surface Immunophenotypes

The small population of cancerous cells that remain following treatment, known as measurable residual disease (MRD), is the major cause of relapse in acute myeloid leukemia (AML). Usually, these refractory cells have gained additional resistance mutations or changed their surface immunophenotypes in ways that preclude detection and phasing by current gold standard flow cytometry or bulk next-generation sequencing assays. For this reason, a multiomic single-cell MRD (scMRD) assay could offer a more comprehensive indicator of relapse and the potential for faster response. Here, we present a new scMRD assay with a 0.01% limit of detection that provides single-cell clonal architecture and immunophenotyping to not only identify residual leukemia cells, but also identify putative DNA or protein targets for salvage therapy. The assay enables rare-cell detection on a standard Mission Bio Tapestri run by adding (i) an upfront bead-based protocol to enrich for blast cells, (ii) a DNA and protein panel specifically designed for AML MRD diagnosis and treatment, and (iii) a new, automated analysis pipeline to evaluate single-cell multiomics output. By utilizing Mission Bio's single-molecule DNA sensitivity for single cells, this pipeline can identify and correlate co-occurring de novo variants, thereby reducing false positive rates over bulk assays that do not correlate variants. It furthermore can create phylogenetic trees of the detected MRD cells and present their surface protein signature and arm-level copy number. In addition, the multiplexing of up to three patient samples combined in one run via germline identification further reduces per sample costs and increases throughput. To demonstrate these features on 0.01% MRD, samples were constructed by titrating diseased cells into healthy bone marrow cells before processing them with the scMRD assay. We detected 0.01% spike-in (CD34+) and 0.1% spike-in (CD117+) in 6 of 6 samples, with an average enrichment of 41x and 13x, respectively. Further testing detected CD34+ 0.1% spike-ins in 10 of 10 samples (32x average enrichment). We applied the scMRD assay to banked bone marrow aspirate samples from 3 AML patients. The scMRD assay resolved the clonal architecture identifying multiple leukemic clones with co-occurring mutations. The assay readily distinguished pre-leukemic from leukemic clones thereby increasing the specificity of MRD results. The integration of genotype and immunophenotypic further enhanced MRD detection by identifying genotype-specific protein expression patterns. By combining high sensitivity with multiomics, this assay offers a potential scalable solution for comprehensive MRD detection that guides therapeutic decision-making.

IGF‑1 inhibits palmitic acid‑induced mitochondrial apoptosis in macrophages.

Insulin growth factor‑1 (IGF‑1) is an endocrine regulator that plays an important role in normal growth and development. IGF‑1 mediated effects may result in protecting macrophages from immunometabolic response. However, it is unclear whether IGF‑1 has a protective effect on fatty acid‑induced macrophages damage. In the present study, THP‑1 cells were differentiated into macrophages and stimulated with palmitic acid (PA) in the absence or presence of IGF‑1. Macrophages apoptosis was measured by Cell Counting Kit‑8 assay, flow cytometry, Hoechst 33342 staining and western blotting. The mitochondrial damage was evaluated using JC‑1 staining and mitochondrial reactive oxygen species detection. The activation of mitophagy was assessed using immunofluorescence and western blotting. As a result, IGF‑1 significantly restored the survival rate in macrophages, while the apoptosis was inhibited through mitochondrial pathway. In addition, IGF‑1 protected the mitochondrial damage induced by PA. Furthermore, PA induced mitophagy via phosphatase and tensin homolog‑induced putative kinase protein 1/Parkin, which was reversed by IGF‑1. Taken together, the present study demonstrated the protective effect of IGF‑1 on PA‑induced mitochondrial apoptosis in macrophages, which might provide a potential therapeutic strategy for treatment of lipotoxicity.

Biological and therapeutic implications of RKIP in Gastrointestinal Stromal Tumor (GIST): an integrated transcriptomic and proteomic analysis

BackgroundGastrointestinal stromal tumors (GIST) represent a significant clinical challenge due to their metastatic potential and limited treatment options. Raf kinase inhibitor protein (RKIP), a suppressor of the MAPK signaling pathway, is downregulated in various cancers and acts as a metastasis suppressor. Our previous studies demonstrated low RKIP expression in GIST and its association with poor outcomes. This study aimed to expand on the previous findings and investigate the biological and therapeutic implications of RKIP loss on GIST.MethodsTo validate the RKIP prognostic significance, its expression was evaluated by immunohistochemistry in 142 bona fide GIST cases. The functional role of RKIP was evaluated in vitro, using the GIST-T1 cell line, which was knocked out for RKIP. The biological and therapeutic implications of RKIP were evaluated by invasion, migration, apoptosis, and 2D / 3D viability assays. Additionally, the transcriptome and proteome of RKIP knockout cells were determined by NanoString and mass spectrometry, respectively.ResultsImmunohistochemical analysis revealed the absence of RKIP in 25.3% of GIST cases, correlating with a tendency toward poor prognosis. Functional assays demonstrated that RKIP knockout increased GIST cells’ invasion and migration potential by nearly 60%. Moreover, we found that RKIP knockout cells exhibited reduced responsiveness to Imatinib treatment and higher cellular viability in 2D and 3D in vitro models, as assessed by apoptosis-related protein expression. Through comprehensive genetic and proteomic profiling of RKIP knockout cells, we identified several putative RKIP-regulated proteins in GIST, such as COL3A1.ConclusionsUsing a multidimensional integrative analysis, we identified, for the first time in GIST, molecules and pathways modulated by RKIP that may potentially drive metastasis and, consequently, poor prognosis in this disease.

Bioinformatics analysis and prokaryotic expression of a cystatin analogue from Spirometra erinaceieuropaei

Cystatin plays a crucial role in immune evasion by parasites. It regulates diverse immune response processes, such as antigen presentation, cytokine and NO production, and phagocytosis. In recent years, an increasing number of parasite cystatins have been identified and studied for the treatment of inflammatory diseases. In contrast, cystatin from Spirometra erinaceieuropaei has received limited research attention. The objective of this study was to utilize bioinformatics tools and molecular biology techniques to predict the biological properties and obtain a recombinant cystatin analogue from Spirometra erinaceieuropaei (SeCystatin). The SeCystatin gene consists of 417 bp and encodes a putative 100-amino acid protein. The predicted molecular weight and isoelectric point of SeCystatin were 10.89 kDa and 6.82, respectively; SeCystatin possesses 12 phosphorylation sites and five post-translational modification sites but has no signal peptide and transmembrane region. The secondary structure of SeCystatin consists of one α-helix, four β-folds and six coils. It also possesses a cystatin-specific conserved domain, QxVxG, which is positioned at the boundary between the first and second β-folds, thereby demonstrating the characteristic features of type I cystatin. In the context of molecular evolution, SeCystatin demonstrates the highest level of evolutionary similarity to Schistocephalus solidus. The SeCystatin gene was fully synthesized, and the recombinant plasmid pET-30a (+)-SeCystatin was constructed to express the target protein in Escherichia coli BL 21. Recombinant SeCystatin was successfully induced for expression by IPTG and subsequently purified using affinity chromatography (Ni-IDA). Our study provides a solid foundation for further studies on the biological functions of SeCystatin.

Investigating the physiological role of S199A and S199D mutants of PHF6 protein in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a form of leukemia characterized by the proliferation of immature T lymphocytes. NOTCH1 is one of the most frequently mutated genes in T-ALL. NOTCH1 expression in T-cell development depends on plant homeodomain finger protein 6 (PHF6), which plays a tumor suppressor role in T-ALL. Several studies have shown that PHF6 expression is essential for NOTCH1 expression. Therefore, whether posttranslational modification of PHF6 plays a role in the regulation of NOTCH1 expression and T-ALL cell line proliferation was investigated herein. The amino acid sequence of PHF6 was analyzed and it was found that a putative protein kinase A (PKA) phosphorylation motif RDRS199 was conserved in several vertebrate species and the S199 site was expected to be phosphorylated according to the PhosphoSite database. Therefore, an eukaryotic expression vector of human PHF6 was constructed, and the codon 199 was changed to the codon encoding the nonphosphorylatable alanine and the phosphorylation-mimicking aspartic acid via site-directed mutagenesis. After confirming the ectopic expressions of the PHF6 vectors by western blot analysis, the effects of these proteins were identified on the NOTCH1 expression using western blot analysis, leukemic cell proliferation using MTT assay, and expressions of the cell surface markers of T-cells using flow cytometry. The ectopic expression of wild-type PHF6 stimulated the formation of CD4 + T-cells. While the expression of the wild-type PHF6 suppressed the growth of the leukemic cell line, this effect was diminished in both the alanine and aspartic acid mutants of PHF6. In addition, both mutants also seemed to negatively affect the NOTCH1 expression, although the effect of the alanine mutant was more severe. Taken together, the different biological activities exerted by the conserved S199 phosphorylation-site mutants shown in this study implicate that signaling pathway(s) leading to differential phosphorylation of this residue may have a substantial effect on the activity of PHF6, and thus may constitute a potential therapeutic target in T-ALL.

Screening Plasma Proteins for the Putative Drug Targets for Carpal Tunnel Syndrome.

Carpal tunnel syndrome (CTS) is one of the most common work-related musculoskeletal disorders. The present study sought to identify putative causal proteins for CTS. We conducted a two-sample Mendelian randomization (MR) analysis to evaluate the causal association between 2859 plasma proteins (N = 35,559) and CTS (N = 1,239,680) based on the published GWAS summary statistics. Then we replicated the significant associations using an independent plasma proteome GWAS (N = 10,708). Sensitivity analyses were conducted to validate the robustness of MR results. Multivariate MR and mediation analyses were conducted to evaluate the mediation effects of body mass index (BMI), type 2 diabetes (T2D), and arm tissue composition on the association between putative causal proteins and CTS. Colocalization analysis was used to examine whether the identified proteins and CTS shared causal variant(s). Finally, we evaluated druggability of the identified proteins. Ten plasma proteins were identified as putative causal markers for CTS, including sCD14, PVR, LTOR3, CTSS, SIGIRR, IFNL3, ASPN, TM11D, ASIP, and ITIH1. Sensitivity analyses and reverse MR analysis validated the robustness of their causal effects. Arm tissue composition, BMI, and T2D may play a fully/partial mediating role in the causal relationships of ASIP, TM11D, IFNL3, PVR, and LTOR3 with CTS. The association of ASPN and sCD14with CTS were supported by colocalization analysis. Druggability assessment demonstrated that sCD14, CTSS, TM11D, and IFNL3 were potential drug therapeutic targets. The present study identified several potential plasma proteins that were causally associated with CTS risk, providing new insights into the pathogenesis of protein-mediated CTS and offering potential targets for new therapies.

Isolation and genome sequence annotation of KillerTomato, a newly discovered cluster EE bacteriophage infecting Microbacterium.

Discovered in Pocatello, Idaho, soil near a tomato garden, siphovirus KillerTomato infects Microbacterium foliorum NRRL B-24224. KillerTomato is a lytic cluster EE phage with a 17,442-bp genome and 68.6% GC content. Of 25 genes, 20 were assigned putative functions, including a putative tail assembly chaperone protein with a programmed frameshift and an endolysin.

An Orphan VrgG Auxiliary Module Related to the Type VI Secretion Systems from Pseudomonas ogarae F113 Mediates Bacterial Killing.

The model rhizobacterium Pseudomonas ogarae F113, a relevant plant growth-promoting bacterium, encodes three different Type VI secretion systems (T6SS) in its genome. In silico analysis of its genome revealed the presence of a genetic auxiliary module containing a gene encoding an orphan VgrG protein (VgrG5a) that is not genetically linked to any T6SS structural cluster, but is associated with genes encoding putative T6SS-related proteins: a possible adaptor Tap protein, followed by a putative effector, Tfe8, and its putative cognate immunity protein, Tfi8. The bioinformatic analysis of the VgrG5a auxiliary module has revealed that this cluster is only present in several subgroups of the P. fluorescens complex of species. An analysis of the mutants affecting the vgrG5a and tfe8 genes has shown that the module is involved in bacterial killing. To test whether Tfe8/Tfi8 constitute an effector-immunity pair, the genes encoding Tfe8 and Tfi8 were cloned and expressed in E. coli, showing that the ectopic expression of tfe8 affected growth. The growth defect was suppressed by tfi8 ectopic expression. These results indicate that Tfe8 is a bacterial killing effector, while Tfi8 is its cognate immunity protein. The Tfe8 protein sequence presents homology to the proteins of the MATE family involved in drug extrusion. The Tfe8 effector is a membrane protein with 10 to 12 transmembrane domains that could destabilize the membranes of target cells by the formation of pores, revealing the importance of these effectors for bacterial interaction. Tfe8 represents a novel type of a T6SS effector present in pseudomonads.

AI-assisted structural consensus-proteome prediction of human monkeypox viruses isolated within a year after the 2022 multi-country outbreak.

The monkeypox virus (MPX) belongs to the Orthopoxvirus genus of the Poxviridae family, is endemic in parts of Africa and causes a disease in humans similar to smallpox. The most recent outbreak of MPX is already affecting 110 countries, with 86,956 confirmed cases since May 2022 and has consequently become a focus of interest. In particular, a molecular understanding of the virus is essential to study infection processes and pathogen-host interactions, predict tropism changes, or guide drug development and drug discovery as well as vaccine development or vaccine adaptation at a very early stage. Herein, we present a study of the structural proteome of the currently circulating MPX: Our consensus analysis of 3,713 genome sequences sampled within a year after the outbreak revealed 10,580 characteristic candidate open reading frames (ORFs). A search in the non-redundant protein database reduced the number of suspected ORFs to 1,079, of which 210 are representative proteins in typical MPX reference genomes. This should serve as a collection of putative proteins within the currently spreading MPX, a compound of information that could support timely drug discovery, mutational analyses, and vaccine development. We, herein, present the so far most comprehensive structural proteome by providing atomistic 3D models of 210 proteins, generated with three state-of-the-art structure prediction methods, including a mutational analysis of the proteome, with a particular focus on the drug-binding sites of tecovirimat and brincidofovir. IMPORTANCE The 2022 outbreak of the monkeypox virus already involves, by April 2023, 110 countries with 86,956 confirmed cases and 119 deaths. Understanding an emerging disease on a molecular level is essential to study infection processes and eventually guide drug discovery at an early stage. To support this, we provide the so far most comprehensive structural proteome of the monkeypox virus, which includes 210 structural models, each computed with three state-of-the-art structure prediction methods. Instead of building on a single-genome sequence, we generated our models from a consensus of 3,713 high-quality genome sequences sampled from patients within 1 year of the outbreak. Therefore, we present an average structural proteome of the currently isolated viruses, including mutational analyses with a special focus on drug-binding sites. Continuing dynamic mutation monitoring within the structural proteome presented here is essential to timely predict possible physiological changes in the evolving virus.

Non-clinical evaluation of local and systemic immunity induced by different vaccination strategies of the candidate tuberculosis vaccine M72/AS01

A new efficacious tuberculosis vaccine targeting adolescents/adults represents an urgent medical need. The M72/AS01E vaccine candidate protected half of the latently-infected adults against progression to pulmonary tuberculosis in a Phase IIb trial (NCT01755598). We report that three immunizations of mice, two weeks apart, with AS01-adjuvanted M72 induced polyfunctional, Th1-cytokine-expressing M72-specific CD4+/CD8+ T cells in blood and lungs, with the highest frequencies in lungs. Antigen-dose reductions across the three vaccinations skewed pulmonary CD4+ T-cell profiles towards IL-17 expression. In blood, reducing antigen and adjuvant doses of only the third injection (to 1/5th or 1/25th of those of the first injections) did not significantly alter CD4+ T-cell/antibody responses; applying a 10-week delay for the fractional third dose enhanced antibody titers. Delaying a full-dose booster enhanced systemic CD4+ T-cell and antibody responses. Cross-reactivity with PPE and non-PPE proteins was assessed, as Mycobacterium tuberculosis (Mtb) virulence factors and evasion mechanisms are often associated with PE/PPE proteins, to which Mtb39a (contained in M72) belongs. In silico/in vivo analyses revealed that M72/AS01 induced cross-reactive systemic CD4+ T-cell responses to epitopes in a non-vaccine antigen (putative latency-associated Mtb protein PPE24/Rv1753c). These preclinical data describing novel mechanisms of M72/AS01-induced immunity could guide future clinical development of the vaccine.

The ELOVL6 homolog in Penaeus vannamei plays a dual role in fatty acid metabolism and immune response

In mammals, elongation of very long chain fatty acid protein 6 (ELOVL6), a key enzyme in long chain fatty acids elongation, has been reported to regulate other metabolism processes and immunity, including inflammation in vertebrates. However, little is currently known about the ELOVL6 homolog in invertebrates, especially its role in immune response. In this study, the ELOVL6 ortholog in Penaeus vannamei (designated PvELOVL6) was cloned and found to have an open reading frame (ORF) of 435 bp and encode a putative protein of 144 amino acids. Transcripts of PvELOVL6 are constitutively expressed in all shrimp tissues tested and induced in the hepatopancreas and hemocytes by Vibrio parahaemolyticus and Streptococcus iniae. Besides, PvELOVL6 knockdown followed by Vibrio parahaemolyticus challenge revealed that PvELOVL6 regulates the expression of several genes involved in fatty acid metabolism and immunity, including PvLGBP, PvLectin, PvMnSOD, PvProPO, PvFABP, PvLipase, PvCOX and PvGPDH. Moreover, transcript levels of PvELOVL6, fatty acids metabolism-related genes (i.e., PvGPDH, PvFABP, PvPERO and PvSPLA2), and immune-related genes (i.e., PvProPO, PvLectin, PvLGBP, PvLysozyme and PvCatalase) increased after silencing of the sterol regulatory element binding protein (PvSREBP). Thus, PvELOVL6 is involved in immune response and regulated by PvSREBP through an unknown mechanism in penaeid shrimp.

Characterization of the Metallothionein Gene Family in Avena sativa L. and the Gene Expression during Seed Germination and Heavy Metal Stress.

Metallothioneins (MTs) are a family of small proteins rich in cysteine residues. The sulfhydryl group of metallothioneins can bind to metal ions, maintaining metal homeostasis and protecting the cells from damage caused by toxic heavy metals. Moreover, MTs can function as reactive oxygen species scavengers since cysteine thiols undergo reversible and irreversible oxidation. Here, we identified 21 metallothionein genes (AsMTs) in the oat (Avena sativa L.) genome, which were divided into four types depending on the amino acid sequences of putative proteins encoded by identified genes. Analysis of promoter sequences showed that MTs might respond to a variety of stimuli, including biotic and abiotic stresses and phytohormones. The results of qRT-PCR showed that all four types of AsMTs are differentially expressed during the first 48 hours of seed germination. Moreover, stress induced by the application of zinc, cadmium, and a mixture of zinc and cadmium affects the expression of oat MTs variously depending on the MT type, indicating that AsMT1-4 fulfil different roles in plant cells.

Unravelling the nsSNP mediated mechanism of antibiotic resistance in Salmonellaenterica serovar Typhi and identification of potential therapeutic alternatives: A genomics and structural bioinformatics study

Problem consideredTyphoid fever caused by Salmonella enterica serovar Typhi (Salmonella Typhi) strains with multi-drug resistance (MDR) has become a significant global threat, as existing therapeutic options are limited. To combat this issue, efficient approaches are required to understand the resistance mechanism and identify potential therapeutic alternatives. MethodsIn this study, we employed concerted genomics and structural investigation to analyze single nucleotide polymorphisms (SNPs) in putative drug targets from whole genomes of Salmonella Typhi. The impact of these SNPs on the stability of encoded proteins and their effect on functional domains were analyzed to gain better insights into emerging MDR patterns. ResultsFrom the analysis of 360 putative drug target proteins in 60 whole genomes, we identified both prevalent and new mutations. Despite a relatively high conservation (>98 % identity) of protein targets and their functional domains in the MDR variants, INDEL mutations were found to induce local changes. The mutations were profiled as stabilizing or destabilizing based on their structural impact on the respective proteins. Interestingly, the MDR strains showed a preference for stabilizing mutations that altered local flexibility, thus compromising drug-susceptibility. ConclusionTo mitigate the risks associated with antibiotic stress-induced stabilizing mutations in drug targets, we performed virtual screening to identify over 500 potential phytocompounds. Through the screening process, Nimbolide emerged as a promising alternative anti-Typhoid candidate for future experimental validations due to its favourable pharmacokinetic properties, high target affinity (despite mutations), and greater interactive dynamics stability compared to conventional antibiotics.

Phenotypic and Genomic Characterization of Streptomyces pakalii sp. nov., a Novel Species with Anti-Biofilm and Anti-Quorum Sensing Activity in ESKAPE Bacteria.

The increasing number of infections caused by antimicrobial multi-resistant microorganisms has led to the search for new microorganisms capable of producing novel antibiotics. This work proposes Streptomyces pakalii sp. nov. as a new member of the Streptomycetaceae family. The strain ENCB-J15 was isolated from the jungle soil in Palenque National Park, Chiapas, Mexico. The strain formed pale brown, dry, tough, and buried colonies in the agar with no diffusible pigment in GAE (glucose-asparagine-yeast extract) medium. Scanning electron micrographs showed typical mycelium with long chains of smooth and oval-shaped spores (3-10 m). The strain grew in all of the International Streptomyces Project (ISP)'s media at 28-37 °C with a pH of 6-9 and 0-10% NaCl. S. pakalii ENCB-J15 assimilated diverse carbon as well as organic and inorganic nitrogen sources. The strain also exhibited significant inhibitory activity against the prodigiosin synthesis of Serratia marcescens and the inhibition of the formation and destruction of biofilms of ESKAPE strains of Acinetobacter baumannii and Klebsiella pneumoniae. The draft genome sequencing of ENCB-J15 revealed a 7.6 Mb genome with a high G + C content (71.6%), 6833 total genes, and 6746 genes encoding putative proteins. A total of 26 accessory clusters of proteins associated with carbon sources and amino acid catabolism, DNA modification, and the antibiotic biosynthetic process were annotated. The 16S rRNA gene phylogeny, core-proteome phylogenomic tree, and virtual genome fingerprints support that S. pakalii ENCB-J15 is a new species related to Streptomyces badius and Streptomyces globisporus. Similarly, its average nucleotide identity (ANI) (96.4%), average amino acid identity (AAI) (96.06%), and virtual DNA-DNA hybridization (67.3%) provide evidence to recognize it as a new species. Comparative genomics revealed that S. pakalli and its closest related species maintain a well-conserved genomic synteny. This work proposes Streptomyces pakalii sp. nov. as a novel species that expresses anti-biofilm and anti-quorum sensing activities.