Region Sequences Research Articles

Analysis of HIV transmission characteristics and intervention effects in Guangxi based on molecular networks.

This study evaluates changes in HIV transmission and the effectiveness of interventions after two rounds of the Guangxi AIDS Conquering Project (GACP) in Guangxi, China. Samples and epidemiological data from newly diagnosed people living with HIV (PLWH) between 2014-2020 were analyzed. Molecular networks were constructed using nested PCR amplification and sequencing of the pol region, and multivariable logistic regression identified factors associated with clustering and high-degree nodes. A total of 4,227 valid sequences (73.12% inclusion rate) were analyzed. Demographic changes included an increasing proportion of individuals aged ≥50 years (49.66%), with lower education (50.51%), peasants (76.82%), and heterosexual transmission (90.29%). The overall clustering rate was 86.89%, with higher clustering among individuals aged ≥50 (92.57%), those with primary school or below (89.09%), peasants (88.11%), and CRF08_BC infections (91.48%). Annual declines in cluster growth rate and clustering rates were observed, particularly among individuals aged <30, college graduates, men who have sex with men (MSM), and people who inject drugs (PWID). Key transmission hotspots were identified in Lingshan, particularly among older, less-educated individuals, and peasants. Factors associated with clustering included being male (aOR: 1.27), aged ≥50 (aOR: 3.84), and infected with CRF08_BC (aOR: 2.12). From 2017 to 2020, the risk of clustering and high-degree nodes was lower compared to 2014-2016, suggesting the effectiveness of interventions. Interventions in Guangxi effectively reduced HIV transmission among younger, high-degree populations. However, older, less-educated individuals remain at high risk, necessitating targeted strategies to address their specific needs and achieve better HIV control.

Correlation Between Cryptococcus Infection and the Nasal Mycobiota in a Population of Free-Ranging Koalas (Phascolarctos cinereus) in New South Wales, Australia

Cryptococcosis is a fungal disease in humans and animals, caused by the Cryptococcus neoformans and Cryptococcus gattii species complexes. Clinical cryptococcosis primarily manifests as upper respiratory tract disease; however, dissemination to other organs, particularly the brain, can occur. Nasal colonisation and subclinical cryptococcosis are common in koalas (Phascolarctos cinereus) due to their shared environmental niche with Cryptococcus: Eucalyptus trees. However, for reasons that remain unclear, the prevalence of clinical disease is low in koalas. Interactions between respiratory pathogens and the nasal mycobiome are thought to play a role in the development and progression of numerous respiratory diseases. As such, this study aimed to characterise the mycobiome of the nasal vestibule in koalas with and without evidence of cryptococcal colonisation and subclinical disease via the next-generation sequencing (NGS) of the ITS1 region of the fungal internal transcribed spacer (ITS) gene. Samples were collected from 47 koalas from a population of free-ranging koalas in the Liverpool Plains, NSW, Australia, with a known history of Cryptococcus exposure and nasal colonisation. Of the 47 animals tested, 6.4% were culture-positive only, 4.3% were seropositive only, and 2.1% were culture- and seropositive. C. gattii was detected in four samples via NGS. C. neoformans was not detected via NGS. There were no significant differences in the nasal mycobiomes of Cryptococcus-positive and -negative animals; thus, we could not establish a definitive association between the mycobiome and infection outcomes. We identified a number of fungal genera that were significantly more abundant in samples from Cryptococcus-positive animals, but there was no apparent relationship between these genera and the development of cryptococcosis. This study represents the first investigation of the nasal mycobiota of wild koalas. Further studies involving koalas with clinical disease are necessary to determine the role of the nasal mycobiota in the development of cryptococcosis.

Genetic Diversity of Tobacco mosaic virus (TMV) isolates from tobacco growing fields of Western Anatolia, Türkiye

Tobacco mosaic virus (TMV) is an important plant virus that is common in agriculture. It is the first evidence of the existence of viruses in history. Studies on the genetic diversity of the CP gene of TMV, which plays a leading role in host interaction, are limited both in our country and around the world. Genetic diversity analyses were conducted on ten isolates of the full CP gene region of TMV obtained from the most intensive tobacco cultivation areas in our country, and compared with global isolates. TMV infection was detected in 32 out of 300 plants collected from the Aegean and Marmara regions (Çanakkale, Balıkesir, İzmir, Manisa, Uşak, Aydın and Denizli) between 2019 and 2020 using conventional molecular techniques. To genetically characterize the virus, 10 samples were selected from each region, and the complete CP gene region sequences were determined. The aligned CP gene region sequences of TMV from Türkiye and its global isolates exhibited nucleotide homology ratios ranging from 87.7% to100%, with amino acid ratios ranging from 88.7% to 100%. The Türkiye isolates displayed similarity rates of 98.5% to 100% at the nucleotide level and 98.7% and 100% at the amino acid level. In phylogenetic analysis, the 196 known isolates of TMV registered in GenBank, belong to the CP gene region, were divided into two main clades (I and II) and two subclades (Ia and Ib). Turkish isolates were clustered in the major branch with the main clade I and subclade Ia isolates. Therefore, genetic analyses were performed on the CP gene region isolates obtained from different parts of the world and a wide range of hosts, including the isolates obtained from Türkiye. The results showed high genetic stability, similar to many tobamoviruses.

Molecular and biochemical methods for species identification among the genus Cuscuta

Genus Cuscuta (Convolvulaceae) comprises of stem holoparasitic plants that are scientifically highly valuable, as they cause extensive crop damage. They exhibit high morphological similarity, which complicates their species identification. Determining the taxonomical status of obtained specimens from Bulgaria, is crucial for exploring the genetic diversity of the genus and establishing the phylogenetic relationships within the taxon. Thereby different correlations with geographic region and particular environmental conditions can be established. This study employs molecular and biochemical methods, including ITS region sequence analysis of rDNA, RAPD analysis, and HPLC/MS analysis of flavonoids, to identify Cuscuta species and conduct phylogenetic analysis. Overall, a successful identification has been made - while ITS PCR products provide more accurate species identification and phylogenic information, RAPD profiles are convenient for quick identification. Detailed investigation of the RAPD profiles is required due to differences correlating with the habitats of the species. Nevertheless, flavonoid profiles prove themselves as a valuable biochemical marker for species identification. All of the aforementioned methods are suitable and useful for species identification among the genus Cuscuta, depending on the needs and aims of the conducted analysis.

Gut bacteria Prevotellaceae related lithocholic acid metabolism promotes colonic inflammation

BackgroundThe conversion of primary bile acids to secondary bile acids by the gut microbiota has been implicated in colonic inflammation. This study investigated the role of gut microbiota related bile acid metabolism in colonic inflammation in both patients with inflammatory bowel disease (IBD) and a murine model of dextran sulfate sodium (DSS)-induced colitis.MethodsBile acids in fecal samples from patients with IBD and DSS-induced colitis mice, with and without antibiotic treatment, were analyzed using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). The composition of the microbiota in fecal samples from IBD patients and DSS-colitis mice was characterized via Illumina MiSeq sequencing of the bacterial 16S rRNA gene V3-V4 region. Metagenomic profiling further identified metabolism-related gene signatures in stool samples from DSS-colitis mice. Histological analysis, quantitative PCR (qPCR) and Western Blotting were conducted on colonic samples from DSS-induced colitis mice to assess colonic inflammation, mucosal barrier integrity, and associated signaling pathways. The multivariate analysis of bile acids was conducted using Soft Independent Modelling of Class Analogy (SIMCA, Umetrics, Sweden). The relation between the relative abundance of specific phyla/genera and bile acid concentration was assess through Spearman's correlation analyses. Finally, lithocholic acid (LCA), the key bile acid, was administered via gavage to evaluate its effect on colonic inflammation and mucosal barrier integrity.ResultsIn patients with IBD, the composition of colonic bile acids and gut microbiota was altered. Moreover, changes in the gut microbiota further modulate the composition of bile acids in the intestine. As the gut microbiota continues to shift, the bile acid profile undergoes additional alterations. The aforementioned alterations were also observed in mice with DSS-induced colitis. The study revealed a correlation between dysbiosis of the gut microbiota and modifications in the profile of colonic bile acids, notably LCA observed in both patients with IBD and mice with DSS-induced colitis. Through multivariate analysis, LCA was identified as the key bile acid that significantly affects colonic inflammation and the integrity of mucosal barrier. Subsequent experiments confirmed that LCA supplementation effectively mitigated the inhibitory effects of gut microbiota on colitis progression in mice, primarily through the activation of the sphingosine-1-phosphate receptor 2 (S1PR2)/NF-κB p65 signaling pathway. Analysis of the microbiome and metagenomic data revealed changes in the gut microbiota, notably an increased abundance of an unclassified genus within the family Prevotellaceae in DSS-induced colitis mice. Furthermore, a positive correlation was observed between the relative abundance of Prevotellaceae and bile acid biosynthesis pathways, as well as colonic LCA level.ConclusionsThese findings suggest that LCA and its positively correlated gut bacteria, Prevotellaceae, are closely associated with intestinal inflammation. Targeting colonic inflammation may involve inhibiting LCA and members of the Prevotellaceae family as potential therapeutic strategies.

Assessment of upper respiratory and gut bacterial microbiomes during COVID-19 infection in adults: potential aerodigestive transmission

SARS-CoV-2 is the viral pathogen responsible for COVID-19. Although morbidity and mortality frequently occur as a result of lung disease, the gastrointestinal (GI) tract is recognized as a primary location for SARS-CoV-2. Connections and interactions between the microbiome of the gut and respiratory system have been linked with viral infections via what has been referred to as the ‘gut-lung axis’ with potential aerodigestive communication in health and disease. This research explored the relationship between the microbiomes of the upper respiratory and GI tracts in patients with COVID-19 and examined Extraesophageal reflux (EOR), a mechanism which could contribute to dysregulated communication between the GI and respiratory tract (as identified in COVID-19). 97 patients with a laboratory diagnosis of COVID-19 infection, and 50 age-matched controls were recruited and stool, saliva and sputum were obtained from each participant. ELISA Pepsin tests and Reflux Symptom Index scores (RSI) were conducted for EOR assessment. DNA sequencing of the V4 region of the 16 S rRNA gene was performed for microbiome analysis. No differences were observed between the fecal microbiome’s alpha and Shannon diversity indices; however, a distinct microbial composition was observed in COVID-19 patients (when compared to the controls). The respiratory microbiota from individuals with COVID-19 demonstrated a statistically significant reduction in Shannon diversity and bacterial richness alongside an overall reduction in the prevalence of organisms from a typical healthy respiratory microbiome. Furthermore, the bacterial richness of the stool and sputum samples was significantly lower among COVID-19 patients admitted to ICU. A significantly higher RSI score and salivary pepsin level were detected among those with COVID-19. The data indicates that COVID-19 is associated with a dysregulation of both the gut and lung microbiome with a more marked perturbation in the lung, particularly among COVID-19 patients who had been admitted to the ICU. The presence of increased RSI scores, combined with elevated levels of Pepsin, suggests that increased micro-aspiration may occur, which is consistent with of under-recognized interactions between the GI and lung microbiomes in COVID-19 patients and requires additional study. Such studies would benefit from the insights provided by biological samples which reflect the continuum of the aerodigestive tract.

Detection of SARS-CoV-2 and a possible variant in shelter cats.

SARS-CoV-2 is the cause of mild to severe acute respiratory disease that led to significant loss of human lives worldwide between 2019 and 2022. The virus has been detected in various animals including cats and dogs making it a major public health concern and a One Health issue. In this study, conjunctival and pharyngeal swabs (n = 350) and serum samples (n = 350) were collected between July and December 2020 from cats that were housed in an animal shelter and tested for the infection of SARS-CoV-2 using real time reverse-transcription polymerase chain reaction (rRT-PCR) that targeted the N1 and N2 genes, and a SARS-CoV-2 surrogate virus neutralization Test (sVNT), respectively. 203 (58%) swab samples were negative (N1 and N2 not detected), 2 (0.6%) were positive (N1 and N2 detected) and 145 (41%) were inconclusive (only N1 detected). Analysis of the N2 region and multiple sequence alignment revealed base-pair deletions and substitutions in the N2 probe binding region of the feline samples RNA extracts in comparison with the positive control and human SARS-CoV-2 sequences in the GenBank database. Substituting the N2 probe with a probe derived from the cat sample amplicon sequences, 123 of 127 (96.9%) of the N2 negative samples returned positive. All but one of the 350 serum samples were negative for SARS-CoV-2 antibody. These observations indicated that although detection of SARS-CoV-2 infection was low in the samples tested, pet cats can harbor the virus and serve as potential source for virus spread that may lead to human infections. Additionally, cats may harbor a yet-to-be described virus that is somewhat related to SARS-CoV-2.

Molecular Characterization, Oxidative Stress-Mediated Genotoxicity, and Hemato-Biochemical Changes in Domestic Water Buffaloes Naturally Infected with Trypanosoma evansi Under Field Conditions

(1) Background: Surra is a debilitating disease of wild and domestic animals caused by Trypanosoma evansi (T. evansi), resulting in significant mortality and production losses in the affected animals. This study is the first to assess the genetic relationships of T. evansi in naturally affected buffaloes from Multan district, Pakistan, using ITS-1 primers and evaluating the effects of parasitemia and oxidative stress on DNA damage and hematobiochemical changes in infected buffaloes. (2) Methods: Blood samples were collected from 167 buffaloes using a multi-stage cluster sampling strategy, and trypomastigote identification was performed through microscopy and PCR targeting RoTat 1.2 and ITS-1 primers. Molecular characterization involved ITS-1 via neighbor-joining analysis. The impact of parasitemia loads was correlated with oxidative stress markers, genotoxicity, and hematobiochemical parameters using Pearson correlation and multivariable regression models. (3) Results: Field-stained thin blood film microscopy and molecular identification revealed 8.98% and 10.18% infection rates, respectively. Phylogenetic analysis based on ITS-1 region sequences of the identified isolates showed close genetic associations with Indian isolates. The mean trypomastigote count observed in the infected buffaloes was 5.15 × 106 (±5.3 × 102)/µL of blood. The parasitemia loads were significantly correlated with the alterations in oxidative stress markers, DNA damage, and changes in hematobiochemical parameters. Infected animals exhibited significant (p &lt; 0.05) alterations in oxidative stress biomarkers, including catalase, nitric oxide, and malondialdehyde concentrations. Noteworthily, a comet assay revealed a significantly (p &lt; 0.0001) higher mean genetic damage index in the infected buffaloes (0.7 ± 0.04) compared with the healthy ones (0.196 ± 0.004). Alongside significant (p &lt; 0.05) reductions in red cell indices, a marked elevation in leukocyte counts and serum hepatic enzyme levels was recorded in the affected buffaloes. (4) Conclusion: T. evansi isolates of buffaloes from Multan, Pakistan, have genetic similarities to Indian isolates. This study also revealed that higher parasitemia loads induce genotoxicity in the infected animals through oxidative stress and cause hematobiochemical alterations under natural field conditions.

Massive Sequencing of V3-V4 Hypervariable Region in Pyogenic Liver Abscesses Reveals the Presence of Unusual Bacteria Not Detected by Classical Culture Methods

Massive Sequencing of V3-V4 Hypervariable Region in Pyogenic Liver Abscesses Reveals the Presence of Unusual Bacteria Not Detected by Classical Culture Methods

Genetic Diversity and Population Structure of Macrobrachium nipponense Populations in the Saline–Alkaline Regions of China

The oriental river prawn Macrobrachium nipponense is of great economic importance in China. However, its culture in the saline–alkaline regions is limited. This study used D-loop region sequences to analyze the genetic diversity and population structure across the saline–alkaline regions of China. A total of 264 individuals were successfully sequenced from nine sites, including Daqing in Heilongjiang Province (HLJ), Songyuan in Jilin Province (JL), Ulanhot in the east of inner Mongolia (NMG), Jingtai in Gansu Province (GS), Dongying in Shandong Province (SD), Dongtai in Jiangsu Province (JS), Nanchang in Jiangxi province (JX), Tianjin in mainland China (TJ), and Yinchuan in Ningxia Province (NX). In addition, 89 haplotypes were defined. The haplotype diversity (h) and nucleotide diversity π showed remarkable genetic diversity in the JS, NX, JL, and TJ sites. It was found that the genetic variation within sites was relatively greater. The genetic distance (D) and the pairwise genetic differentiation index (FST) revealed that SD and GS are closely related. The FST values among the nine sites were significant except for the one between SD and HLJ (p-value &gt; 0.05). The cluster analyses and the phylogenetic tree identified two main groups. There is an association among sites by geographic location, and the JS site shows higher diversity. The results of this study provide basic information for the protection and development of M. nipponense resources in the saline–alkaline regions of China.

Characterization of an Invisible HIV-1 Circulating Recombinant Form (CRF149_01B) in China.

In this study, by analyzing the available near full-length genome (NFLG) sequences of CRF55_01B, it was found that two of the NFLG sequences could not be clustered with other NFLG sequences. Recombination analysis and phylogenetic analysis suggested that these two NFLG sequences arose by recombination with subtype B based on CRF55_01B, rather than by recombination directly derived from CRF01_AE and subtype B. In addition, two other HIV-1 partial gene fragments found in the database shared the same characteristics as these two NFLG sequences in the key recombination region. These sequences may therefore represent a previously unrecognized circulating recombinant form (CRF), which has been named CRF149_01B. Evolutionary analyses suggested that CRF149_01B emerged between approximately 2005 and 2007. The discovery of CRF149_01B highlights the complexity of HIV recombinant evolution and advances the refinement of the HIV genotyping system. A deeper understanding of HIV-1 genetics will facilitate molecular tracing and provide a basis for studying the biological properties of HIV.

Persistence of Ancestral KhoeSan Mitochondrial Patterns in Contemporary South African Populations.

Southern Africa has been inhabited by hunter-gatherers for at least 20,000 years and has received diverse immigration flows in the last 2000 years. The original inhabitants have interacted with the pastoralist migrants from Eastern Africa (∼2000 ybp), followed by the southern Bantu migration arriving some 1000 ybp, and more recently with the European and Asian settlers after the 17th century. Many of the original Khoekhoe and San inhabitants have either become extinct or have disappeared through admixture in South Africa (SA), in a sex-biased manner involving KhoeSan women. In this study, we generated mitochondrial DNA (mtDNA) control region (CR) sequences for 247 South African individuals. The sampling effort was concentrated in regions and populations with historical links to the KhoeSan population groups: admixed (Coloured, Griqua), Nama (Khoekhoe) and Bantu in three provinces. Here we evaluate the composition and extent of connectivity between population groups and regions, and to assess the distribution of haplotypes for the practical application of mtDNA CR data in forensic identifications. The analysis of the newly generated sequences revealed 142 distinct haplotypes, of which 122 were unique. Haplogroup L0 was predominant (overall 71.7%). A high-frequency L0d2a haplotype dominated the pool of the admixed groups with 10%-12.5% incidence overall or per region. Comparative analysis with 545 extant mtDNA CR sequences from South African KhoeSan and admixed descendants revealed extensive population structure and high within-group haplotype sharing. The observed population and regional variations, combined with the prevalence of high-frequency haplotypes, align with patterns of matrilocality. These findings highlight the limitations of using mtDNA control region analysis for forensic applications in South Africa.

Application of phosphoric acid plus hydrogen peroxide pretreatment and Aspergillus species enzymatic decomposition on rice straw for bioethanol production

Cellulose is one of the most important of natural renewable sources. Cellulase enzymes convert it into oligosaccharides and glucose. These enzymes have several applications in industries and widely used in second generation of biofuel production process. The aim of this study was to use rice straw as a carbon source of Saccharomyces cerevisiae cells to produce bioethanol. Phosphoric acid plus hydrogen peroxide chemical pretreatment was performed to obtain a cellulose-rich source of rice straw. Mesophilic filamentous fungal isolates collected from cattle manure samples, the best isolates were selected based on cellulase activities at optimal reaction conditions. These isolates were identified by DNA sequencing of ITS region. Aspergillus fumigatus and A. niger showed the highest activities of endoglucanase, exoglucanase and total cellulase enzymes at temperature 35 °C, pH 5.5, and temperature 30 °C, pH 4.5, respectively. Pretreated rice straw was hydrolyzed by enzymatic solution of two fungal isolates. The obtained sugars (8.654 g/L for A. fumigatus and 7.412 g/L for A. niger hydrolysates) were fermented by Saccharomyces cerevisiae yeast cells and converted to the bioethanol. The highest amounts of ethanol were obtained from A. fumigatus and A. niger lysates after 72 h of fermentation as 11.2 g/L and 8.29 g/L, respectively.

Vishniacozyma siamensis sp. nov., a new anamorphic tremellomycetous yeast species isolated from a mangrove forest in Thailand.

Six strains (DMKU-SG26, DMKU-SG42, DMKU-SYM22, DMKU-RG41, DMKU-RX317 and DMKU-RGM25) representing a novel basidiomycetous yeast species were isolated from leaf surfaces of mangrove plants collected in Thailand. Pairwise sequence analysis indicated that the six strains either had identical nucleotide substitution in the D1/D2 domains of the large subunit (LSU) rRNA gene sequences or differed by one to three nucleotide(s). They also had identical or differed by one to five nucleotide substitution(s) in the internal transcribed spacer (ITS) regions. blastn searches of the GenBank database revealed that the six strains were closely related to the holotype of type strains of Vishniacozyma peneaus, V. terrae, V. phoenicis, V. taiwanica and V. europaea, but with 6-15 (1.14-2.48%) and 16-26 (5.4-8.8%) nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and the ITS regions, respectively. Phylogenetic analysis based on the concatenated sequences of the ITS regions and D1/D2 domains of the LSU rRNA gene showed that these strains are placed in the Vishniacozyma clade but were at a distinctly different position from the other recognized species of the genus. Based on the phylogenetic analysis and phenotypic characteristics, these six strains are a novel species of the genus Vishniacozyma, for which the name Vishniacozyma siamensis sp. nov. is proposed to accommodate them. The holotype is TBRC 18499T and the ex-type culture is PYCC 10042 (=DMKU-SG26). The MycoBank number of the novel species is MB 855838.

Protein CREATE enables closed-loop design of de novo synthetic protein binders.

Proteins have proven to be useful agents in a variety of fields, from serving as potent therapeutics to enabling complex catalysis for chemical manufacture. However, they remain difficult to design and are instead typically selected for using extensive screens or directed evolution. Recent developments in protein large language models have enabled fast generation of diverse protein sequences in unexplored regions of protein space predicted to fold into varied structures, bind relevant targets, and catalyze novel reactions. Nevertheless, we lack methods to characterize these proteins experimentally at scale and update generative models based on those results. We describe Protein CREATE (Computational Redesign via an Experiment-Augmented Training Engine), an integrated computational and experimental pipeline that incorporates an experimental workflow leveraging next generation sequencing and phage display with single-molecule readouts to collect vast amounts of quantitative binding data for updating protein large language models. We use Protein CREATE to generate and assay thousands of designed binders to IL-7 receptor α and insulin receptor with parallel positive and negative selections to identify on-target binders. We discover not only individual novel binders but also features of ligand-receptor binding, including preservation of the IL7Rα - ligand hydrophobic interface specifically and existence of multiple approaches to contact the insulin receptor. We also demonstrate the importance of structural features, such as the lack of unpaired cysteine residues, toward design fidelity and find computational pre-screening metrics, such as interchain predicted TM scoring (iPTM), while useful, are imperfect predictors as they neither guarantee experimental binding nor rule it out. We use the data collected from Protein CREATE to score designs from the initial generative models. Globally, Protein CREATE will power future closed-loop design-build-test cycles to enable fine-grained design of protein binders.

Modifications to rhesus macaque TCR constant regions improve TCR cell surface expression.

T cell immunotherapy success is dependent on effective levels of antigen receptor expressed at the surface of engineered cells. Efforts to optimize surface expression in T cell receptor (TCR)-based therapeutic approaches include optimization of cellular engineering methods and coding sequences, and reducing the likelihood of exogenous TCR α and β chains mispairing with the endogenous TCR chains. Approaches to promote correct human TCR chain pairing include constant region mutations to create an additional disulfide bond between the two chains, full murinization of the constant region of the TCR α and β sequences, and a minimal set of murine mutations to the TCR α and β constant regions. Preclinical animal models are valuable tools to optimize engineering designs and methods, and to evaluate the potential for off-target tissue injury. To further develop rhesus macaque models for TCR based cellular immunotherapy, we tested methods for improving cell surface expression of rhesus macaque TCR in rhesus macaque primary cells by generating five alternative TCRαβ constant region constructs in the context of a SIV Gag-specific TCR: 1. human codon optimized rhesus macaque (RH); 2. RH TCR with an additional disulfide linkage; 3. rhesus macaque constant sequences with minimal murine amino acid substitutions; 4. murinized constant sequences; and 5. murinized constant sequences with a portion of the exposed FG loop in the β constant sequence replaced with rhesus macaque sequence to reduce potential immunogencity. Murinization or mutation of a minimal set of amino acids to the corresponding murine sequence of the constant region resulted in the greatest increase in rhesus macaque TCR surface expression relative to wild type. All novel TCR constructs retained the ability to induce production of cytokines in response to cognate peptide antigen specific stimulation. This work can inform the design of TCRs selected for use in rhesus macaque models of TCR-based cellular immunotherapy.

Identification of Starmerella aleppica f.a., sp. nov. and large indels in the rRNA cistron that split the Starmerella genus.

Yeast strains representing a novel asexual ascomycetous species were isolated from seven Malva sylvestris flowers. Sequencing of the chromosomal regions coding for the D1/D2 domains of the large subunit ribosomal RNA, the ITS1-5.8S-ITS2 segments and parts of the gene coding for the small subunit ribosomal RNA showed that the isolates were conspecific. Comparative analysis of these sequences and the corresponding sequences of the type strains of ascomycetous yeasts revealed that the strains represent a hitherto undescribed species belonging to the sensu stricto subclade of the genus Starmerella. The new species is osmotolerant and can develop invasive pseudohyphae, but does not form spores. For the new species, the name Starmerella aleppica f.a. (forma asexualis) is proposed. The holotype, preserved in a metabolically inactive state, is CBS 12960T (extype cultures: 2-1361 and CCY 90-2-1, NCAIM Y.02123). The GenBank accession numbers of barcode sequences are JX515983 (D1/D2 domain), JX515985 (ITS1-5.8S-ITS2 and partial 18S rRNA gene), PQ613837 (TEF1 partial sequence) and PQ613838 (RPB2 partial sequence). MycoBank: MB855459. The analysis of the D1/D2 and internal transcribed spacer (ITS) sequences of the type strains of species of the genus identified multiple multinucleotide indels that can be used as taxonomic markers (InDel markers). The indel patterns of the subclades are very different and homogeneous within the subclades. This result reinforces the idea raised, but also refuted, in previous studies that the Starmerella subclades may represent different genera.

Comparative Analysis of Protist Communities in Oilsands Tailings Using Amplicon Sequencing and Metagenomics.

The Canadian province of Alberta contains substantial oilsands reservoirs, consisting of bitumen, clay and sand. Extracting oil involves separating bitumen from inorganic particles using hot water and chemical diluents, resulting in liquid tailings waste with ecotoxicologically significant compounds. Ongoing efforts aim to reclaim tailings-affected areas, with protist colonisation serving as one assessment method of reclamation progress. Oilsands-associated protist communities have mainly been evaluated using amplicon sequencing of the 18S rRNA V4 region; however, this barcode may overlook important protist groups. This study examined how community assessment methods between the V4 and V9 regions differ in representing protist diversity across four oilsands-associated environments. The V9 barcode identified more operational taxonomical units (OTUs) for Discoba, Metamonada and Amoebozoa compared with the V4. A comparative shotgun metagenomics approach revealed few eukaryotic contigs but did recover a complete Paramicrosporidia mitochondrial genome, only the second publicly available from microsporidians. Both V4 and V9 markers were informative for assessing community diversity in oilsands-associated environments and are most effective when combined for a comprehensive taxonomic estimate, particularly in anoxic environments.

Airway total bacterial density, microbiota community composition and relationship with clinical parameters in bronchiectasis

Background and ObjectiveThis study explored the relationship between total bacterial density, airway microbiota composition and clinical parameters in bronchiectasis. We determined changes with time during clinical stability and following antibiotic treatment of a pulmonary exacerbation. MethodsWe conducted a multicentre longitudinal cohort study of UK participants with CT confirmed bronchiectasis. Sputum samples and clinical parameters [FEV1% predicted, lung clearance index, C-reactive protein, white cell count and Quality of Life] were collected when participants were clinically stable and pre/post-antibiotic treatment of an exacerbation. Total bacterial density and microbiota community composition was measured by quantitative polymerase chain reaction and sequencing of the V4 region of bacterial 16S rRNA, respectively. ResultsAmong 105 participants at baseline, 65 (62%) were female with a mean age of 65 years and FEV1 at 69% predicted. In participants who remained clinically stable (n=15), no significant changes were observed in bacterial density, microbiota diversity, richness, evenness, and dominance (p=0.30, 0.45, 0.54, 0.23 and 0.43; respectively) across four time points over a 1-year period. Similarly, for participants with paired pre/post-antibiotic treatment samples (n=19), no significant changes were observed (p=0.30, 0.46, 0.44, 0.71 and 0.58; respectively). However, considerable fluctuation in community composition between samples was apparent for most patients. Total bacterial density and microbiota composition did not correlate with clinical parameters at baseline (n=75). ConclusionsStability in bacterial density and microbiota diversity, richness, evenness and dominance was observed over time at a population level but considerable fluctuation was apparent in samples from individual patients.

Collection and Processing of Samples for Next-Generation Sequencing to Study the Gut Microbiome.

16S rRNA gene sequencing is commonly used for identification and quantitation of microorganisms in complex biological mixtures, such as the human gut microbiome. The 16S rRNA gene is an excellent target gene for sequencing DNA in a heterogenous sample, as it is a highly conserved part of the transcriptional machinery. Universal PCR primers are used to amplify the conserved regions of 16S. Thus, it is possible to amplify the gene in a wide range of different microorganisms from a single sample. As the 16S rRNA gene consists of both conserved and variable regions, sequencing of the variable regions can be used to differentiate between different bacterial species.