Complete genome sequence data of an Antarctic bacterium Arthrobacter sp. EM1 from the freshwater lake of the King George Island.

Arthrobacter sp. EM1 is a cold-adapted bacterium isolated from the Antarctic region, which was known to exhibit mannan-degrading activity. Accordingly, this strain not only promises a cell factory for mannan-degrading enzymes, widely used in industry but also serves as a model organism to decipher its cold adaptation mechanism. Accordingly, whole genome sequencing of the EM1 strain was performed via Single Molecule Real Time sequencing under the PacBio platform, followed by genome HGAP de novo assembly and genome annotation through Rapid Annotation System Technology (RAST) server. The chromosome of this strain is 3,885,750 bp in size with a GC content of 65.8. The annotation predicted a total of 3607 protein-coding genes and 65 RNA genes, which were classified under 398 subsystems. The subsystem with the highest number of genes is carbohydrate metabolism (397 genes), which includes two genes encoding mannan-degrading enzymes (endoglucanase and α-mannosidase). This confirmed that the EM1 strain is able to produce cold-adapted mannan degrading enzymes. The complete genome sequence data have been submitted to the National Center for Biotechnology Information (NCBI) and have been deposited at GenBank (Bioproject ID Accession Number: PRJNA963062; Biosample ID Accession Number: SAMN34434776; GenBank: CP124836.1; https://www.ncbi.nlm.nih.gov/nuccore/CP124836).

Whole genome sequence data of an Antarctic bacterium, Arthrobacter sp. ES1 from the Schirmacher Oasis, East Antarctica.

Arthrobacter is a coryneform bacterium in the family of Micrococcaceae. Arthrobacter species isolated from hostile environments are capable of producing interesting bioactive compounds, some of which may be a new class of antibiotics. Here, we present the complete genome sequence of Arthrobacter sp. ES1 isolated from Schirmacher Oasis in East Antarctica. Genomic DNA sequencing was performed using the Illumina MiSeq sequencer. Arthrobacter sp. ES1 has a genome size of 3,964,927 bp and a GC content of 65.73%. The raw genome sequences have been deposited in the NCBI Sequence Read Archive database under the accession number, SRR20664316.

Functional Analysis of Conserved Hypothetical Proteins from the Antarctic Bacterium, Pedobacter cryoconitis Strain BG5 Reveals Protein Cold Adaptation and Thermal Tolerance Strategies.

Pedobacter cryoconitis BG5 is an obligate psychrophilic bacterium that was first isolated on King George Island, Antarctica. Over the last 50 years, the West Antarctic, including King George Island, has been one of the most rapidly warming places on Earth, hence making it an excellent area to measure the resilience of living species in warmed areas exposed to the constantly changing environment due to climate change. This bacterium encodes a genome of approximately 5694 protein-coding genes. However, 35% of the gene models for this species are found to be hypothetical proteins (HP). In this study, three conserved HP genes of P. cryoconitis, designated pcbg5hp1, pcbg5hp2 and pcbg5hp12, were cloned and the proteins were expressed, purified and their functions and structures were evaluated. Real-time quantitative PCR analysis revealed that these genes were expressed constitutively, suggesting a potentially important role where the expression of these genes under an almost constant demand might have some regulatory functions in thermal stress tolerance. Functional analysis showed that these proteins maintained their activities at low and moderate temperatures. Meanwhile, a low citrate synthase aggregation at 43 °C in the presence of PCBG5HP1 suggested the characteristics of chaperone activity. Furthermore, our comparative structural analysis demonstrated that the HPs exhibited cold-adapted traits, most notably increased flexibility in their 3D structures compared to their counterparts. Concurrently, the presence of a disulphide bridge and aromatic clusters was attributed to PCBG5HP1's unusual protein stability and chaperone activity. Thus, this suggested that the HPs examined in this study acquired strategies to maintain a balance between molecular stability and structural flexibility. Conclusively, this study has established the structure-function relationships of the HPs produced by P. cryoconitis and provided crucial experimental evidence indicating their importance in thermal stress response.

Sex Influences Age-Related Changes in Natural Antibodies and CD5+ B-1 Cells.

Natural Abs are primarily produced by B-1 cells and are essential for protection against Streptococcus pneumoniae The incidence and mortality rate for pneumococcal infection increases dramatically after age 65, disproportionately affecting males in both human and murine systems. To date, there is a significant gap in our understanding of the relationship among sex, aging, natural IgM efficacy, and the natural IgM repertoire. Our investigation demonstrates that the protective capacity of serum IgM against pneumococcal infection is maintained in IgM obtained from aged female mice but absent in IgM from aged male mice. To understand this difference in protective capacity, we examined serum Ig, discovering that the protective change was not associated with shifts in levels of phosphorylcholine (PC)- or pneumococcal capsular polysaccharide serotype 3-specific IgM. Interestingly, we observed that aged females have an increase in the total number of CD5+ B-1 cells, higher serum IL-5 levels, and a larger percentage of aged female CD5+ B-1 cells that express CD86 as compared with aged males. Furthermore, single-cell IgM repertoire analysis from peritoneal PC+, splenic PC+, and bone marrow CD5+ B-1 cell subsets demonstrated greater diversity with age and a higher level of germline status in female mice than previously observed in studies of aged male mice. Aged female CD5+ B-1 cells also expressed higher levels of transcripts associated with cell activity and self-renewal, such as Nanog and Hmga2 Taken together, these data indicate that females maintain a more diverse and active CD5+ B-1 cell pool and natural IgM repertoire, which has implications for sex-related susceptibility to infection and disease.

Complete genome sequence data of tropical thermophilic bacterium Parageobacillus caldoxylosilyticus ER4B.

Parageobacillus caldoxylosilyticus, or previously identified as Geobacillus caldoxylosilyticus, is a thermophilic Gram-positive bacterium which can easily withstand growth temperatures ranging from 40 °C to 70 °C. Here, we present the first complete genome sequence of Parageobacillus caldoxylosilyticus ER4B which was isolated from an empty oil palm fruit bunch compost in Malaysia. Whole genome sequencing was performed using the PacBio RSII platform. The genome size of strain ER4B was around 3.9Mbp, with GC content of 44.31%. The genome consists of two contigs, in which the larger contig (3,909,276bp) represents the chromosome, while the smaller one (54,250bp) represents the plasmid. A total of 4,164 genes were successfully predicted, including 3,972 protein coding sequences, 26 rRNAs, 91 tRNAs, 74 miscRNA, and 1 tmRNA. The genome sequence data of strain ER4B reported here may contribute to the current molecular information of the species. It may also facilitate the discovery of molecular traits related to thermal stress, thus, expanding our understanding in the acclimation or adaptation towards extreme temperature in bacteria.

Age-related changes in antigen-specific natural antibodies are influenced by sex.

Introduction: Natural antibody (NAb) derived from CD5+ B-1 cells maintains tissue homeostasis, controls inflammation, aids in establishing long-term protective responses against pathogens, and provides immediate protection from infection. CD5+ B-1 cell NAbs recognize evolutionarily fixed epitopes, such as phosphatidylcholine (PtC), found on bacteria and senescent red blood cells. Anti-PtC antibodies are essential in protection against bacterial sepsis. CD5+ B-1 cell-derived NAbs have a unique germline-like structure that lacks N-additions, a feature critical for providing protection against infection. Previously, we demonstrated the repertoire and germline status of PtC+CD5+ B-1 cell IgM obtained from male mice changes with age depending on the anatomical location of the B-1 cells. More recently, we demonstrated serum antibody from aged female mice maintains protection against pneumococcal infection, whereas serum antibody from male mice does not provide protection. Results: Here, we show that aged female mice have significantly more splenic PtC+CD5+ B-1 cells and more PtC specific serum IgM than aged male mice. Furthermore, we find both age and biological sex related repertoire differences when comparing B cell receptor (BCR) sequencing results of PtC+CD5+ B-1 cells. While BCR germline status of PtC+CD5+ B-1 cells from aged male and female mice is similar in the peritoneal cavity, it differs significantly in the spleen, where aged females retain germline configuration and aged males do not. Nucleic acid sensing toll-like receptors are critical in the maintenance of PtC+ B-1 cells; therefore, to begin to understand the mechanism of differences observed between the male and female PtC+CD5+ B-1 cell repertoire, we analyzed levels of cell-free nucleic acids and found increases in aged females. Conclusion: Our results suggest the antigenic milieu differs between aged males and females, leading to differential selection of antigen-specific B-1 cells over time. Further elucidation of how biological sex differences influence the maintenance of B-1 cells within the aging environment will be essential to understand sex and age-related disparities in the susceptibility to bacterial infection and will aid in the development of more effective vaccination and/or therapeutic strategies specific for males and females.

Emericella nidulans (4DP5), Cladosporium herbarum (7DF12) and Bacillus subtilis improve the nutritional value of palm kernel cake (PKC) through solid-state fermentation (SSF)

Aims@#Palm kernel cake (PKC) is a high-protein, high-energy food that is widely utilized in the animal feed business. However, the high fibre and limited amino acid content of untreated PKC were the main issues for it to be used as animal feed, particularly in non-ruminants. To improve the quality of PKC, this study combined the use of solid-state fermentation (SSF) and consortia of fungi and bacteria to treat the PKC.@*Methodology and results@#Two fungi, Emericella nidulans (4DP5) and Cladosporium herbarum (7DF12) and three strains of bacteria, Bacillus subtilis, which were active mannanase producers, were used in different combinations to reduce the hemicellulose content and improve the crude protein content of PKC in a lab-scale solid-state fermentation. PKC inoculated separately with five types of mixed culture treatments were allowed to ferment. The fermentation conditions were 20% inoculum (w/v), 85-92% humidity, pH 7.0 and PKC particle size 0.8 mm. PKC treatments with two fungi, E. nidulans (4DP5) and C. herbarum (7DF12), as well as a fungus-bacterium combination, E. nidulans (4DP5) and B. subtilis, outperformed the other three treatments. The crude protein levels were increased by 3.34% and 1.86%, respectively, due to these treatments. Furthermore, the level of aflatoxins produced increased marginally but remained within the permissible limits.@*Conclusion, significance and impact of study@#The treated PKC has more sugar and crude protein and less than 20 parts per billion (ppb) of aflatoxin, making it appropriate for animal consumption. The SSF technique of combining fungi and Bacilli enhanced the nutritional and market value of PKC substantially, which can be upscaled.

Comparative Transcriptome Profiling of Kappaphycus alvarezii (Rhodophyta, Solieriaceae) in Response to Light of Different Wavelengths and Carbon Dioxide Enrichment.

Rhodophyta (red algae) comprises over 6000 species, however, there have only been a few comparative transcriptomic studies due to their under-representation in genomic databases. Kappaphycus alvarezii, a Gigartinales algae, is a valuable source of carrageenan and is extensively cultivated in many countries. The majority of seaweed farming in Southeast Asia is done in intertidal zones under varying light (i.e., spectra and irradiance) and carbon dioxide (CO2) conditions, which affects the rate of photosynthesis. This study conducted transcriptome profiling to investigate the photosynthetic mechanisms in K. alvarezii exposed to different wavelengths of light (i.e., blue, green, and red light, in comparison to white light) and CO2 availability. We analyzed the responses of photosynthetic protein complexes to light and observed that light of different wavelengths regulates a similar set of photosynthetic apparatuses. Under CO2 enrichment, genes encoding C3 and C4 enzymes were found to be actively transcribed, suggesting the likely shift in the carbon metabolism pathway or the involvement of these genes in adaptive physiological processes. This study contributes to the understanding of the regulatory mechanisms of photosynthetic carbon metabolism in red algae and has implications for the culture and commercial production of these economically valuable macroalgae.

Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia.

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients' CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.

Draft Genome Sequence of Flavobacterium sp. Strain PL002, Isolated from Antarctic Porphyra Algae.

Here, we report the draft genome sequence of Flavobacterium sp. strain PL002, isolated from Antarctic Porphyra algae. The 4,299,965-bp genome sequence is assembled into 170 contigs, has 32.92% GC content, and 3,734 predicted genes.

Effects of simulated warming on bacterial diversity and abundance in tropical soils from East Malaysia using open top chambers.

The effects of global warming are increasingly evident, where global surface temperatures and atmospheric concentration of carbon dioxide have increased in past decades. Given the role of terrestrial bacteria in various ecological functions, it is important to understand how terrestrial bacteria would respond towards higher environmental temperatures. This study aims to determine soil bacterial diversity in the tropics and their response towards in situ warming using an open-top chamber (OTC). OTCs were set up in areas exposed to sunlight throughout the year in the tropical region in Malaysia. Soil samples were collected every 3 months to monitor changes in bacterial diversity using V3-V4 16S rDNA amplicon sequencing inside the OTCs (treatment plots) and outside the OTCs (control plots). After 12 months of simulated warming, an average increase of 0.81 to 1.15 °C was recorded in treatment plots. Significant changes in the relative abundance of bacterial phyla such as Bacteroidetes and Chloroflexi were reported. Increases in the relative abundance of Actinobacteria were also observed in treatment plots after 12 months. Substantial changes were observed at the genus level, where most bacterial genera decreased in relative abundance after 12 months. This study demonstrated that warming can alter soil bacteria in tropical soils from Kota Kinabalu.

Subclonal STAT3 mutations solidify clonal dominance.

T large granular lymphocyte leukemia (T-LGLL) is a clonal lymphoproliferative disorder that can arise in the context of pathologic or physiologic cytotoxic T-cell (CTL) responses. STAT3 mutations are often absent in typical T-LGLL, suggesting that in a significant fraction of patients, antigen-driven expansion alone can maintain LGL clone persistence. We set out to determine the relationship between activating STAT3 hits and CTL clonal selection at presentation and in response to therapy. Thus, a group of patients with T-LGLL were serially subjected to deep next-generation sequencing (NGS) of the T-cell receptor (TCR) Vß complementarity-determining region 3 (CDR3) and STAT3 to recapitulate clonal hierarchy and dynamics. The results of this complex analysis demonstrate that STAT3 mutations produce either a sweeping or linear subclone within a monoclonal CTL population either early or during the course of disease. Therapy can extinguish a LGL clone, silence it, or adapt mechanisms to escape elimination. LGL clones can persist on elimination of STAT3 subclones, and alternate STAT3-negative CTL clones can replace therapy-sensitive CTL clones. LGL clones can evolve and are fueled by a nonextinguished antigenic drive. STAT3 mutations can accelerate this process or render CTL clones semiautonomous and not reliant on physiologic stimulation.

Fanconi Anemia germline variants as susceptibility factors in aplastic anemia, MDS and AML.

Using next generation sequencing we have systematically analyzed a large cohort of 489 patients with bone marrow failure (BMF), including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), aplastic anemia (AA), and related conditions for the presence of germline (GL) alterations in Fanconi Anemia (FA) and telomerase genes. We have detected an increased frequency of heterozygous FA gene mutations in MDS and to lesser degree in AML suggesting that the presence of one normal allele may not be completely protective and indeed heterozygous FA lesions may have a long latency period before hematologic manifestation. In contrast, GL telomerase gene mutations were not associated with increased disease risk. When compared to large control cohorts, we have not detected an increased frequency of damaging variants among telomerase complex genes, including those previously believed to be involved in the pathogenesis of AA. Our results may suggest that while low penetrance and delayed disease onset can confound identification of genetic predisposition factors, GL FA alterations can be also associated with MDS.

Clinical features and treatment outcomes in large granular lymphocytic leukemia (LGLL).

Large granular lymphocytic leukemia (LGLL) represents a clonal/oligoclonal lymphoproliferation of cytotoxic T and natural killer cells often associated with STAT3 mutations. When symptomatic, due to mostly anemia and neutropenia, therapy choices are often empirically-based, because only few clinical trials and systematic studies have been performed. Incorporating new molecular and flow cytometry parameters, we identified 204 patients fulfilling uniform criteria for LGLL diagnoses and analyzed clinical course with median follow-up of 36 months, including responses to treatments. While selection of initial treatment was dictated by clinical features, the initial responses, as well as overall responses to methotrexate (MTX), cyclosporine (CsA), and cyclophosphamide (CTX), were similar at 40-50% across drugs. Sequential use of these drugs resulted in responses in most cases: only 10-20% required salvage therapies such as ATG, Campath, tofacitinib, splenectomy or abatacept. MTX yielded the most durable responses. STAT3-mutated patients required therapy more frequently and had better overall survival.

Public health campaigns and their effect on stroke knowledge in a high-risk urban population: A five-year study.

Background The level of knowledge of stroke risk factors and stroke symptoms within a population may determine their ability to recognize and ultimately react to a stroke. Independent agencies have addressed this through extensive awareness campaigns. The aim of this study was to determine the change in baseline knowledge of stroke risk factors, symptoms, and source of stroke knowledge in a high-risk Toronto population between 2010 and 2015. Methods Questionnaires were distributed to adults presenting to cardiovascular clinics at the University of Toronto in Toronto, Canada. In 2010 and 2015, a total of 207 and 818 individuals, respectively, participated in the study. Participants were identified as stroke literate if they identified (1) at least one stroke risk factor and (2) at least one stroke symptom. Results A total of 198 (95.6%) and 791 (96.7%) participants, respectively, completed the questionnaire in 2010 and 2015. The most frequently identified risk factors for stroke in 2010 and 2015 were, respectively, smoking (58.1%) and hypertension (49.0%). The most common stroke symptom identified was trouble speaking (56.6%) in 2010 and weakness, numbness or paralysis (67.1%) in 2015. Approximately equal percentages of respondents were able to identify ≥1 risk factor (80.3% vs. 83.1%, p = 0.34) and ≥1 symptom (90.9% vs. 88.7%, p = 0.38). Overall, the proportion of respondents who were able to correctly list ≥1 stroke risk factors and stroke symptoms was similar in both groups.(76.8% vs. 75.5%, p = 0.70). The most commonly reported stroke information resource was television (61.1% vs. 67.6%, p = 0.09). Conclusion Stroke literacy has remained stable in this selected high-risk population despite large investments in public campaigns over recent years. However, the baseline remains high over the study period. Evaluation of previous campaigns and development of targeted advertisements using more commonly used media sources offer opportunities to enhance education.

Molecular features of early onset adult myelodysplastic syndrome.

Myelodysplastic syndromes are typically diseases of older adults. Patients in whom the onset is early may have distinct molecular and clinical features or reflect a demographic continuum. The identification of differences between "early onset" patients and those diagnosed at a traditional age has the potential to advance understanding of the pathogenesis of myelodysplasia and may lead to formation of distinct morphological subcategories. We studied a cohort of 634 patients with various subcategories of myelodysplastic syndrome and secondary acute myeloid leukemia, stratifying them based on age at presentation and clinical parameters. We then characterized molecular abnormalities detected by next-generation deep sequencing of 60 genes that are commonly mutated in myeloid malignancies. The number of mutations increased linearly with age and on average, patients >50 years of age had more mutations. TET2, SRSF2, and DNMT3A were more commonly mutated in patients >50 years old compared to patients ≤50 years old. In general, patients >50 years of age also had more mutations in spliceosomal, epigenetic modifier, and RAS gene families. Although there are age-related differences in molecular features among patients with myelodysplasia, most notably in the incidence of SRSF2 mutations, our results suggest that patients ≤50 years old belong to a disease continuum with a distinct pattern of early onset ancestral events.

Recurrent genetic defects on chromosome 5q in myeloid neoplasms.

BACKGROUND: Deletion of chromosome 5q (del(5q)) is the most common karyotypic abnormality in myeloid neoplasms. MATERIALS AND METHODS: To define the pathogenic molecular features associated with del(5q), next-generation sequencing was applied to 133 patients with myeloid neoplasms (MDS; N = 69, MDS/MPN; N = 5, sAML; N = 29, pAML; N = 30) with del(5q) as a sole abnormally or a part of complex karyotype and results were compared to molecular features of patients diploid for chr5. FINDINGS: A number of 5q genes with haploinsufficient expression and/or recurrent somatic mutations were identified; for these genes, CSNK1A1 and G3BP1 within the commonly deleted 5q region and DDX41 within a commonly retained region were most commonly affected by somatic mutations. These genes showed consistent haploinsufficiency in deleted cases; low expression/mutations of G3BP1 or DDX41 were associated with poor survival, likely due to decreased cellular function. The most common mutations on other chromosomes in patients with del(5q) included TP53, and mutations of FLT3 (ITD or TKD), NPM1 or TET2 and were mutually exclusive. Serial sequencing allowed for definition of clonal architecture and dynamics, in patients with exome sequencing allelic imbalance for informative SNPs facilitated simultaneous approximation of clonal size of del(5q) and clonal burden for somatic mutations. INTERPRETATION: Our results illuminate the spectrum of molecular defects characteristic of del(5q), their clinical impact and succession of stepwise evolution.