Phylogenetic inference of inter-population transmission rates for infectious diseases.

Estimating transmission rates is a challenging yet essential aspect of comprehending and controlling the spread of infectious diseases. Various methods exist for estimating transmission rates, each with distinct assumptions, data needs, and constraints. This study introduces a novel phylogenetic approach called transRate, which integrates genetic information with traditional epidemiological approaches to estimate inter-population transmission rates. The phylogenetic method is statistically consistent as the sample size (i.e. the number of pathogen genomes) approaches infinity under the multi-population susceptible-infected-recovered model. Simulation analyses indicate that transRate can accurately estimate the transmission rate with a sample size of 200 ~ 400 pathogen genomes. Using transRate, we analyzed 40,028 high-quality sequences of SARS-CoV-2 in human hosts during the early pandemic. Our analysis uncovered significant transmission between populations even before widespread travel restrictions were implemented. The development of transRate provides valuable insights for scientists and public health officials to enhance their understanding of the pandemic's progression and aiding in preparedness for future viral outbreaks. As public databases for genomic sequences continue to expand, transRate is increasingly vital for tracking and mitigating the spread of infectious diseases.

Using phylogenetics to infer HIV-1 transmission direction between known transmission pairs.

Inferring the transmission direction between linked individuals living with HIV provides unparalleled power to understand the epidemiology that determines transmission. Phylogenetic ancestral-state reconstruction approaches infer the transmission direction by identifying the individual in whom the most recent common ancestor of the virus populations originated. While these methods vary in accuracy, it is unclear why. To evaluate the performance of phylogenetic ancestral-state reconstruction to determine the transmission direction of HIV-1 infection, we inferred the transmission direction for 112 transmission pairs where transmission direction and detailed additional information were available. We then fit a statistical model to evaluate the extent to which epidemiological, sampling, genetic, and phylogenetic factors influenced the outcome of the inference. Finally, we repeated the analysis under real-life conditions with only routinely available data. We found that whether ancestral-state reconstruction correctly infers the transmission direction depends principally on the phylogeny's topology. For example, under real-life conditions, the probability of identifying the correct transmission direction increases from 32%-when a monophyletic-monophyletic or paraphyletic-polyphyletic tree topology is observed and when the tip closest to the root does not agree with the state at the root-to 93% when a paraphyletic-monophyletic topology is observed and when the tip closest to the root agrees with the root state. Our results suggest that documenting larger differences in relative intrahost diversity increases our confidence in the transmission direction inference of linked pairs for population-level studies of HIV. These findings provide a practical starting point to determine our confidence in transmission direction inference from ancestral-state reconstruction.

Population-genetic and comparative interpopulation studies of the 15 autosomal STR markers in the population living in the Northwest of Iran.

INTRODUCTION: Iran, a country in the Middle East, has several ethnic and ethno-religious groups and needs its own ethnic-specific databases for the forensic statistical parameters and allele frequency of STR markers. METHODS: We have investigated 600 unrelated Turk individuals from four northwestern provinces of Iran using the Identifiler™ system (TPOX, FGA, vWA, TH01, CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, and D21S11). Furthermore, STR allelic frequencies were compared to previously population-based data. RESULTS AND CONCLUSION: After Bonferroni correction, deviation from Hardy-Weinberg equilibrium (HWE) was observed in the FGA, TPOX, VWA, and D19S433 loci (P value < 0.05). The combined power of discrimination (CPD) and exclusion (CPE) values for all 15 STR loci were 0.9999999999999999999984 and 0.9999999, respectively. In comparison with Azerbaijani and Turkish populations, there were no significant differences on all STR markers. However, in the Chinese Han population, differences at 13 STR loci were detected. Additionally, comparisons of Fischer genetic distance indices (FST) P-values did not reveal any statistically significant difference between Northwestern Iran, Azerbaijan and Iran (Fars) populations. PCA and PCoA analyses showed that our population was grouped with different populations in different quarters, showing a positive and negative correlation, respectively. In the NJ and UPGMA phylogenetic trees, Iranian populations were grouped together. These results demonstrated that the given set of STR markers can be confidently used for all identification tests in Northwestern Iran.

Genome-wide identification and expression-pattern analysis of sulfate transporter (SULTR) gene family in cotton under multiple abiotic stresses and fiber development.

Sulfate transporter (SULTR) proteins are in charge of the transport and absorption on sulfate substances, and have been reported to play vital roles in the biological processes of plant growth and stress response. However, there were few reports of genome-wide identification and expression-pattern analysis of SULTRs in Hibiscus mutabilis. Gossypium genus is a ideal model for studying the allopolyploidy, therefore two diploid species (G. raimondii and G. arboreum) and two tetraploid species (G. hirsutum and G. barbadense) were chosen in this study to perform bioinformatic analyses, identifying 18, 18, 35, and 35 SULTR members, respectively. All the 106 cotton SULTR genes were utilized to construct the phylogenetic tree together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 8 Zea mays ones, which was divided into Group1-Group4. The clustering analyses of gene structures and 10 conserved motifs among the cotton SULTR genes showed the consistent evolutionary relationship with the phylogenetic tree, and the results of gene-duplication identification among the four representative Gossypium species indicated that genome-wide or segment duplication might make main contributions to the expansion of SULTR gene family in cotton. Having conducted the cis-regulatory element analysis in promoter region, we noticed that the existing salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) elements could have influences with expression levels of cotton SULTR genes. The expression patterns of GhSULTR genes were also investigated on the 7 different tissues or organs and the developing ovules and fibers, most of which were highly expressed in root, stem, sepal, receptacel, ovule at 10 DPA, and fiber at 20 and 25 DPA. In addition, more active regulatory were observed in GhSULTR genes responding to multiple abiotic stresses, and 12 highly expressed genes showed the similar expression patterns in the quantitative Real-time PCR experiments under cold, heat, salt, and drought treatments. These findings broaden our insight into the evolutionary relationships and expression patterns of the SULTR gene family in cotton, and provide the valuable information for further screening the vital candidate genes on trait improvement.

Evolutionary divergence of TLR9 through ancestral sequence reconstruction.

The transmembrane pattern recognition receptor, Toll-like receptor (TLR), are best known for their roles in innate immunity via recognition of pathogen and initiation of signaling response. Mammalian TLRs recognize molecular patterns associated with pathogens and initiate innate immune response. We have studied the evolutionary diversity of mammalian TLR genes for differences in immunological response. Reconstruction of ancestral sequences is a key aspect of the molecular evolution of TLR to track changes across the TLR genes. The comprehensive analysis of mammalian TLRs revealed a distinct pattern of evolution of TLR9. Various sequence-based features such as amino acid usage, hydrophobicity, GC content, and evolutionary constraints are found to influence the divergence of TLR9 from other TLRs. Ancestral sequence reconstruction analysis also revealed that the gradual evolution of TLR genes in several ancestral lineages leads to the distinct pattern of TLR9. It demonstrates evolutionary divergence with the progressive accumulation of mutations results in the distinct pattern of TLR9.

Complete plastome genomes of three medicinal heliotropiaceae species: comparative analyses and phylogenetic relationships.

BACKGROUND: Heliotropiaceae is a family of the order Boraginales and has over 450 species. The members of the family Heliotropiaceae have been widely reported to be used in traditional medicine Over time, the classification of Heliotropiaceae has remained uncertain and has moved from family to subfamily, or conversely. RESULTS: In the present study, we sequenced, analyzed, and compared the complete plastomes of Euploca strigosa, Heliotropium arbainense, and Heliotropium longiflorum with the genomes of related taxa. The lengths of the plastomes of E. strigosa, H. arbainense, and H. longiflorum were 155,174 bp, 154,709 bp, and 154,496 bp, respectively. Each plastome consisted of 114 genes: 80 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. The long repeats analysis indicated that reverse, palindromic, complement and forward repeats were all found in the three plastomes. The simple repeats analysis showed that the plastomes of E. strigosa, H. arbainense, and H. longiflorum contained 158, 165, and 151 microsatellites, respectively. The phylogenetic analysis confirmed two major clades in the Boraginales: clade I comprised Boraginaceae, while clade II included Heliotropiaceae, Ehretiaceae, Lennoaceae, and Cordiaceae. Inside the family Heliotropiaceae, E. strigosa is nested within the Heliotropium genus. CONCLUSIONS: This study expands our knowledge of the evolutionary relationships within Heliotropiaceae and offers useful genetic resources.

Twelve newly assembled jasmine chloroplast genomes: unveiling genomic diversity, phylogenetic relationships and evolutionary patterns among Oleaceae and Jasminum species.

BACKGROUND: Jasmine (Jasminum), renowned for its ornamental value and captivating fragrance, has given rise to numerous species and accessions. However, limited knowledge exists regarding the evolutionary relationships among various Jasminum species. RESULTS: In the present study, we sequenced seven distinct Jasminum species, resulting in the assembly of twelve high-quality complete chloroplast (cp) genomes. Our findings revealed that the size of the 12 cp genomes ranged from 159 to 165 kb and encoded 134-135 genes, including 86-88 protein-coding genes, 38-40 tRNA genes, and 8 rRNA genes. J. nudiflorum exhibited a larger genome size compared to other species, mainly attributed to the elevated number of forward repeats (FRs). Despite the typically conservative nature of chloroplasts, variations in the presence or absence of accD have been observed within J. sambac. The calculation of nucleotide diversity (Pi) values for 19 cp genomes indicated that potential mutation hotspots were more likely to be located in LSC regions than in other regions, particularly in genes ycf2, rbcL, atpE, ndhK, and ndhC (Pi > 0.2). Ka/Ks values revealed strong selection pressure on the genes rps2, atpA, rpoA, rpoC1, and rpl33 when comparing J. sambac with the three most closely related species (J. auriculatum, J. multiflorum, and J. dichotomum). Additionally, SNP identification, along with the results of Structure, PCA, and phylogenetic tree analyses, divided the Jasminum cp genomes into six groups. Notably, J. polyanthum showed gene flow signals from both the G5 group (J. nudiflorum) and the G3 group (J. tortuosum and J. fluminense). Phylogenetic tree analysis reflected that most species from the same genus clustered together with robust support in Oleaceae, strongly supporting the monophyletic nature of cp genomes within the genus Jasminum. CONCLUSION: Overall, this study provides comprehensive insights into the genomic composition, variation, and phylogenetic relationships among various Jasminum species. These findings enhance our understanding of the genetic diversity and evolutionary history of Jasminum.

Comparative analysis of chloroplast genomes of Pulsatilla species reveals evolutionary and taxonomic status of newly discovered endangered species Pulsatilla saxatilis.

BACKGROUND: Pulsatilla saxatilis, a new species of the genus Pulsatilla has been discovered. The morphological information of this species has been well described, but its chloroplast genome characteristics and comparison with species of the same genus remain to be reported. RESULTS: Our results showed that the total length of chloroplast (cp.) genome of P. saxatilis is 162,659 bp, with a GC content of 37.5%. The cp. genome contains 134 genes, including 90 known protein-coding genes, 36 tRNA genes, and 8 rRNA genes. P. saxatilis demonstrated similar characteristics to other species of genus Pulsatilla. Herein, we compared cp. genomes of 10 species, including P. saxatilis, and found that the cp. genomes of the genus Pulsatilla are extremely similar, with a length of 162,322-163,851 bp. Furthermore, The SSRs of Pulsatilla ranged from 10 to 22 bp in length. Among the four structural regions of the cp. genome, most long repeats and SSRs were detected in the LSC region, followed by that in the SSC region, and least in IRA/ IRB regions. The most common types of long repeats were forward and palindromic repeats, followed by reverse repeats, and only a few complementary repeats were found in 10 cp. genomes. We also analyzed nucleotide diversity and identified ccsA_ndhD, rps16_trnK-UUU, ccsA, and rbcL, which could be used as potential molecular markers for identification of Pulsatilla species. The results of the phylogenetic tree constructed by connecting the sequences of high variation regions were consistent with those of the cp. gene phylogenetic tree, and the species more closely related to P. saxatilis was identified as the P. campanella. CONCLUSION: It was determined that the closest species to P. saxatilis is P. campanella, which is the same as the conclusion based on pollen grain characteristics, but different from the P. chinensis determined based on morphological characteristics. By revealing information on the chloroplast characteristics, development, and evolution of the cp. genome and the potential molecular markers, this study provides effective molecular data regarding the evolution, genetic diversity, and species identification of the genus Pulsatilla.

MetaLogo: a heterogeneity-aware sequence logo generator and aligner.

Sequence logos are used to visually display conservations and variations in short sequences. They can indicate the fixed patterns or conserved motifs in a batch of DNA or protein sequences. However, most of the popular sequence logo generators are based on the assumption that all the input sequences are from the same homologous group, which will lead to an overlook of the heterogeneity among the sequences during the sequence logo making process. Heterogeneous groups of sequences may represent clades of different evolutionary origins, or genes families with different functions. Therefore, it is essential to divide the sequences into different phylogenetic or functional groups to reveal their specific sequence motifs and conservation patterns. To solve these problems, we developed MetaLogo, which can automatically cluster the input sequences after multiple sequence alignment and phylogenetic tree construction, and then output sequence logos for multiple groups and aligned them in one figure. User-defined grouping is also supported by MetaLogo to allow users to investigate functional motifs in a more delicate and dynamic perspective. MetaLogo can highlight both the homologous and nonhomologous sites among sequences. MetaLogo can also be used to annotate the evolutionary positions and gene functions of unknown sequences, together with their local sequence characteristics. We provide users a public MetaLogo web server (http://metalogo.omicsnet.org), a standalone Python package (https://github.com/labomics/MetaLogo), and also a built-in web server available for local deployment. Using MetaLogo, users can draw informative, customized and publishable sequence logos without any programming experience to present and investigate new knowledge on specific sequence sets.

Advances, challenges and opportunities of phylogenetic and social network analysis using COVID-19 data.

Coronavirus disease 2019 (COVID-19) has attracted research interests from all fields. Phylogenetic and social network analyses based on connectivity between either COVID-19 patients or geographic regions and similarity between syndrome coronavirus 2 (SARS-CoV-2) sequences provide unique angles to answer public health and pharmaco-biological questions such as relationships between various SARS-CoV-2 mutants, the transmission pathways in a community and the effectiveness of prevention policies. This paper serves as a systematic review of current phylogenetic and social network analyses with applications in COVID-19 research. Challenges in current phylogenetic network analysis on SARS-CoV-2 such as unreliable inferences, sampling bias and batch effects are discussed as well as potential solutions. Social network analysis combined with epidemiology models helps to identify key transmission characteristics and measure the effectiveness of prevention and control strategies. Finally, future new directions of network analysis motivated by COVID-19 data are summarized.

Is the persistence of Listeria monocytogenes in food processing facilities and its resistance to pathogen intervention linked to its phylogeny?

The foodborne pathogen Listeria monocytogenes is differentiated into four distinct lineages which differ in their virulence. It remains unknown, however, whether the four lineages also differ with respect to their ability to persist in food processing facilities, their resistance to high pressure, a preservation method that is used commercially for Listeria control on ready-to-eat meats, and their ability to form biofilms. This study aimed to determine differences in the pressure resistance and biofilm formation of 59 isolates of L. monocytogenes representing lineages I and II. Furthermore, the genetic similarity of 9 isolates of L. monocytogenes that were obtained from a meat processing facility over a period of 1 year and of 20 isolates of L. monocytogenes from food processing facilities was analyzed to assess whether the ability of the lineages of L. monocytogenes to persist in these facilities differs. Analysis of 386 genomes with respect to the source of isolation revealed that genomes of lineage II are over-represented in meat isolates when compared with clinical isolates. Of the 38 strains of Lm. monocytogenes that persisted in food processing facilities (this study or published studies), 31 were assigned to lineage II. Isolates of lineage I were more resistant to treatments at 400 to 600 MPa. The thickness of biofilms did not differ between lineages. In conclusion, strains of lineage II are more likely to persist in food processing facilities while strains of lineage I are more resistant to high pressure.IMPORTANCEListeria monocytogenes substantially contributes to the mortality of foodborne disease in developed countries. The virulence of strains of four lineages of L. monocytogenes differs, indicating that risks associated with the presence of L. monocytogenes are lineage specific. Our study extends the current knowledge by documentation that the lineage-level phylogeny of L. monocytogenes plays a role in the source of isolation, in the persistence in food processing facilities, and in the resistance to pathogen intervention technologies. In short, the control of risks associated with the presence of L. monocytogenes in food is also lineage specific. Understanding the route of contamination L. monocytogenes is an important factor to consider when designing improved control measures.

Multiple founding paternal lineages inferred from the newly-developed 639-plex Y-SNP panel suggested the complex admixture and migration history of Chinese people.

BACKGROUND: Non-recombining regions of the Y-chromosome recorded the evolutionary traces of male human populations and are inherited haplotype-dependently and male-specifically. Recent whole Y-chromosome sequencing studies have identified previously unrecognized population divergence, expansion and admixture processes, which promotes a better understanding and application of the observed patterns of Y-chromosome genetic diversity. RESULTS: Here, we developed one highest-resolution Y-chromosome single nucleotide polymorphism (Y-SNP) panel targeted for uniparental genealogy reconstruction and paternal biogeographical ancestry inference, which included 639 phylogenetically informative SNPs. We genotyped these loci in 1033 Chinese male individuals from 33 ethnolinguistically diverse populations and identified 256 terminal Y-chromosomal lineages with frequency ranging from 0.0010 (singleton) to 0.0687. We identified six dominant common founding lineages associated with different ethnolinguistic backgrounds, which included O2a2b1a1a1a1a1a1a1-M6539, O2a1b1a1a1a1a1a1-F17, O2a2b1a1a1a1a1b1a1b-MF15397, O2a2b2a1b1-A16609, O1b1a1a1a1b2a1a1-F2517, and O2a2b1a1a1a1a1a1-F155. The AMOVA and nucleotide diversity estimates revealed considerable differences and high genetic diversity among ethnolinguistically different populations. We constructed one representative phylogenetic tree among 33 studied populations based on the haplogroup frequency spectrum and sequence variations. Clustering patterns in principal component analysis and multidimensional scaling results showed a genetic differentiation between Tai-Kadai-speaking Li, Mongolic-speaking Mongolian, and other Sinitic-speaking Han Chinese populations. Phylogenetic topology inferred from the BEAST and Network relationships reconstructed from the popART further showed the founding lineages from culturally/linguistically diverse populations, such as C2a/C2b was dominant in Mongolian people and O1a/O1b was dominant in island Li people. We also identified many lineages shared by more than two ethnolinguistically different populations with a high proportion, suggesting their extensive admixture and migration history. CONCLUSIONS: Our findings indicated that our developed high-resolution Y-SNP panel included major dominant Y-lineages of Chinese populations from different ethnic groups and geographical regions, which can be used as the primary and powerful tool for forensic practice. We should emphasize the necessity and importance of whole sequencing of more ethnolinguistically different populations, which can help identify more unrecognized population-specific variations for the promotion of Y-chromosome-based forensic applications.

Lysobacter stagni sp. nov. and Limnohabitans lacus sp. nov., isolated from a pond.

Two Gram-negative bacteria, designated as strains LF1T and HM2-2T, were isolated from an artificial pond in a honey farm at Hoengseong-gun, Gangwon-do, Republic of Korea. The 16S rRNA sequence analysis results revealed that strain LF1T belonged to the genus Lysobacter and had the highest sequence similarity to Lysobacter niastensis GH41-7T (99.0 %), Lysobacter panacisoli CJ29T (98.9 %), and Lysobacter prati SYSU H10001T (98.2 %). Its growth occurred at 20-37 °C, at pH 5.0-12.0, and in the presence of 0-2% NaCl. The major fatty acids were iso-C15 : 0, iso-C16 : 0, and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C content was 67.5 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain LF1T and species of the genus Lysobacter were 79.1-84.4% and 22.0-27.5 %, respectively. The 16S rRNA sequence analysis results revealed that strain HM2-2T belonged to the genus Limnohabitans and was most closely related to Limnohabitans planktonicus II-D5T (98.9 %), Limnohabitans radicicola JUR4T (98.4%), and Limnohabitans parvus II-B4T (98.4 %). Its growth occurred at 10-35 °C, at pH 5.0-11.0, and in the presence of 0-2% NaCl. The major fatty acids were C16 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). The major polar lipid was phosphatidylethanolamine. The DNA G+C content was 59.9 mol%. The ANI and dDDH values between strain HM2-2T and its closely related strains were 75.1-83.0% and 20.4-26.4 %, respectively. Phenotypic, genomic, and phylogenetic data revealed that strains LF1T and HM2-2T represent novel species in the genera Lysobacter and Limnohabitans, for which the names Lysobacter stagni sp. nov. and Limnohabitans lacus sp. nov. are proposed, respectively. The type strain of Lys. stagni is LF1T (=KACC 23251T=TBRC 17648T), and that of Lim. lacus is HM2-2T (=KACC 23250T=TBRC 17649T).

Roseibium algae sp. nov., isolated from a marine alga (Grateloupia sp.).

A novel Gram-stain-negative, rod-shaped, non-spore-forming, aerobic, motile bacterium with a single polar or subpolar flagellum, designated strain H3510T, was isolated from marine alga collected on sea shore of Yantai, PR China. The organism grew optimally at 28 °C and pH 7.0 and in presence of 3.0 % (w/v) NaCl. The strain exhibited positive catalase activity but negative oxidase and nitrate reduction activities. The predominant cellular fatty acids were C18 : 1 ω7c and/or C18 : 1 ω6c, 11-methyl C18 : 1 ω7c, and C16 : 0. Additionally, the major polar lipids were phosphatidylglycerol, phosphatidylmonomethylethanolamine, diphosphatidylglycerol, and phosphatidylethanolamine; the respiratory quinone was ubiquinone 10 (Q-10). The genomic DNA G+C content of strain H3510T was 54.2%. The novel strain showed the closest relationship with Roseibium polysiphoniae KMM 9699T with 98.2 % 16S rRNA gene sequence similarity. The calculated values for average nucleotide identity and DNA-DNA hybridization between strain H3510T and the phylogenetically related Roseibium species were in the range of 71.3-74.9 % and 13.7-19.9 %, respectively. Based on polyphasic analyses, strain H3510T was identified as representing a novel species of the genus Roseibium, for which the name Roseibium algae sp. nov. is proposed. The type strain is H3510T (=KCTC 8206T=MCCC 1K04325T). The heterologously expressed inositol 2-dehydrogenase gene from strain H3510T displayed high oxidation activity on myo-inositol and showed potential in the production of rare stereoisomers of inositol, such as scyllo-inositol.

Methylobacterium amylolyticum sp. nov. and Methylobacterium ligniniphilum sp. nov., isolated from soil.

Two pink-pigmented bacteria, designated strains NEAU-140T and NEAU-KT, were isolated from field soil collected from Linyi, Shandong Province, PR China. Both isolates were aerobic, Gram-stain-negative, rod-shaped, and facultatively methylotrophic. 16S rRNA gene sequences analysis showed that these two strains belong to the genus Methylobacterium. Strain NEAU-140T exhibited high 16S rRNA gene sequence similarities to Methylobacterium radiotolerans NBRC 15690T (97.43 %) and Methylobacterium phyllostachyos NBRC 105206T (97.36 %). Strain NEAU-KT exhibited high 16S rRNA gene sequence similarities to M. phyllostachyos NBRC 105206T (99.00 %) and Methylobacterium longum DSM 23933T (98.72 %). A phylogenetic tree based on 16S rRNA gene sequences showed that strain NEAU-140T formed a clade with Methylobacterium aerolatum (95.94 %), Methylobacterium persicinum (95.66 %) and Methylobacterium komagatae (96.87 %), and strain NEAU-KT formed a cluster with M. phyllostachyos and M. longum. The predominant fatty acid in both strains was C18 : 1 ω7c. Both strains contained ubiquinone Q-10 as the only respiratory quinone. The polar lipid profiles of both strains contained diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. Whole-genome phylogeny showed that strains NEAU-140T and NEAU-KT formed a phyletic line with M. aerolatum, M. persicinum, Methylobacterium radiotolerans, Methylobacterium fujisawaense, Methylobacterium oryzae, Methylobacterium tardum, M. longum and M. phyllostachyos. The orthologous average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain NEAU-140T and its closely related strains were lower than 82.62 and 25.90  %, respectively. The ANI and dDDH values between strain NEAU-KT and its closely related strains were lower than 86.29 and 31.7 %, respectively. The genomic DNA G+C contents were 71.63 mol% for strain NEAU-140T and 69.08 mol% for strain NEAU-KT. On the basis of their phenotypic and phylogenetic distinctiveness and the results of dDDH and ANI hybridization, these two isolates represent two novel species within the genus Methylobacterium, for which the names Methylobacterium amylolyticum sp. nov. (type strain NEAU-140T=MCCC 1K08801T=DSM 110568T) and Methylobacterium ligniniphilum sp. nov. (type strain NEAU-KT=MCCC 1K08800T=DSM 110567T) are proposed.

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches.

BACKGROUND: Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. RESULTS: In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. CONCLUSIONS: Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

Hepatitis C virus subtype diversity and transmission clusters characteristics among drug users in Zhuhai, South China.

BACKGROUND: Hepatitis C virus (HCV) infection poses a major public health challenge globally, especially among injecting drug users. China has the world's largest burden of HCV infections. However, little is known about the characteristics of transmission networks among drug user populations. This study aims to investigate the molecular epidemiology and transmission characteristics of HCV infections among drug users in Zhuhai, a bustling port city connecting Mainland China and its Special Administrative Regions. METHODS: Participants enrolled in this study were drug users incarcerated at Zhuhai's drug rehabilitation center in 2015. Their sociodemographic and behavioral information, including gender, promiscuity, drug use method, and so forth, was collected using a standardized questionnaire. Plasmas separated from venous blood were analyzed for HCV infection through ELISA and RT-PCR methods to detect anti-HCV antibodies and HCV RNA. The 5'UTR fragment of the HCV genome was amplified and further sequenced for subtype identifications and phylogenetic analysis. The phylogenetic tree was inferred using the Maximum Likelihood method based on the Tamura-Nei model, and the transmission cluster network was constructed using Cytoscape3.8.0 software with a threshold of 0.015. Binary logistic regression models were employed to assess the factors associated with HCV infection. RESULTS: The overall prevalence of HCV infection among drug users was 44.37%, with approximately 19.69% appearing to clear the HCV virus successfully. Binary logistic regression analysis revealed that those aged over 40, engaging in injecting drug use, and being native residents were at heightened risk for HCV infection among drug user cohorts. The predominant HCV subtypes circulating among those drug users were 6a (60.26%), followed by 3b (16.7%), 3a (12.8%), 1b (6.41%) and 1a (3.85%), respectively. Molecular transmission network analysis unveiled the presence of six transmission clusters, with the largest propagation cluster consisting of 41 individuals infected with HCV subtype 6a. Furthermore, distinct transmission clusters involved eight individuals infected with subtype 3b and seven with subtype 3a were also observed. CONCLUSION: The genetic transmission networks revealed a complex transmission pattern among drug users in Zhuhai, emphasizing the imperative for a targeted and effective intervention strategy to mitigate HCV dissemination. These insights are pivotal for shaping future national policies on HCV screening, treatment, and prevention in port cities.

Molecular based identification and phylogenetic relationship of the leech Hirudinaria manillensis from India by using mitochondrial cytochrome c oxidase subunit I gene.

BACKGROUND: A molecular approach for the identification of unknown species by the using mitochondrial cox1 gene is an effective and reliable as compared with morphological-based identification. Hirudinaria manillensis referred to as Asian Buffalo Leech, is found in South Asia and traditionally used as medicine owing to its medicinal properties. METHODS AND RESULTS: The study aimed to isolate and identify the leech species using cox1 gene sequencing and their phylogenetic relationships. The nucleotide sequences of cytochrome c oxidase subunit I (cox1) mitochondrial genes were analyzed for species identification and the phylogenetic relationship of crucial therapeutic leech Hirudinaria manillensis. The isolated DNA from the leech sample was amplified with cox1 gene-specific primers. BLAST results with the H. manillensis sequence showed 89.24% homology with H. manillensis and phylogenetic tree analysis revealed the genetic relationship with other GenBank submitted sequences. CONCLUSION: The present study concluded that the cox1 gene could be an effective way to identify the leech H. manillensis and provided sufficient phylogenetic information to distinguish H. manillensis indicating a significant mtDNA-based approach to species identification.

Exploring DNA barcode for accurate identification of threatened Aconitum L. species from Western Himalaya.

BACKGROUND: Aconitum species, belonging to Ranunculaceae, have high medicinal importance but due to their overexploitation come under IUCN (International Union for Conservation of Nature) red list. The precise identification of the Aconitum species is equally important because they are used in herbal formulations. The present study aimed to develop an efficient DNA barcode system for the authentic identification of Aconitum species. METHODS AND RESULTS: A set of 92 barcode gene sequences (including 12 developed during the present study and 80 retrieved from NCBI) of 5 Aconitum species (A. heterophyllum, A. vialoceum, A. japonicum, A. napellus, and A. stapfianum) were analyzed using three methods (tree-based, distance-based, and similarity-based) for species discrimination. The PWG-distance method was found most effective for species discrimination. The discrimination rate of PWG- distance ranged from 33.3% (rbcL + trnH-psbA) to 100% (ITS, rbcL + ITS, ITS + trnH-psbA and rbcL + ITS + trnH-psbA). Among DNA barcodes and their combinations, the ITS marker had the highest degree of species discrimination (NJ-40%, PWG-100% and BLAST-40%), followed by trnH-psbA (NJ-20%, PWG-60% and BLAST-20%). ITS also had higher barcoding gap as compared to other individual barcodes and their combinations. Further, we also analyzed six Aconitum species (A. balfourii, A. ferox, A. heterophyllum, A. rotundifolium, A. soongaricum and A. violaceum) existing in Western Himalaya. These species were distinguished clearly through tree-based method using the ITS barcode gene with 100% species resolution. CONCLUSION: ITS showed the best species discrimination power and was used to develop species-specific barcodes for Aconitum species. DNA barcodes developed during the present study can be used to identify Aconitum species.

Evaluating the effects of virulence genotype, swarming motility, and multi-locus sequence types of Escherichia coli on layer chicken embryos.

AIMS: To determine the effects of swarming motility (SM) and multi-locus sequence types (MLST) on the main effect of virulence genotype of Escherichia coli through an embryos lethality assay between the 12th and 18th days of incubation. METHODS AND RESULTS: We collected 58 E. coli isolates from asymptomatic commercial hens (n = 42) and lesions of colibacillosis cases (n = 16), then classified their virulence genotype as avirulent, moderately virulent, virulent-healthy, and virulent-colibacillosis categories by the presence of five virulence-associated genes (iroN, ompT, hlyF, iutA, and iss). These isolates were further classified as non-motile, motile, or hyper-motile by SM assay. From the 58 isolates, we selected 29 for ELA and determined their MLST. Each isolate was inoculated into 15 embryonated eggs through the allantoic cavity. We found the avirulent isolates reduced the relative embryo weight compared to virulent-colibacillosis and moderately virulent isolates (37.49 vs. 41.51 and 40.34%, P = 0.03). Among the moderately virulent and virulent-colibacillosis categories, embryo lethality was lower when isolates were non-motile. Yolk retention was unaffected by virulence categories, motility, or MLST. CONCLUSION: Interaction between virulence genotype and SM substantially influenced the embryo lethality assay of E. coli isolates.