Dirofilaria immitis and Onchocercidae spp. in wild felids from Brazil.

Among the species described within the Onchocercidae family, Dirofilaria immitis is regarded as the most common worldwide, causing severe and often fatal conditions in dogs, cats, and occasionally humans. Dirofilaria spp. are vectored by mosquitoes, simulids, and culicoids, with their epidemiology dependent on the geographical distribution of competent vectors. Eight species of Dirofilaria have been reported so far in Brazil, of which six parasitize non-human primates, deer, procyonids, and marsupials. Here, we investigated the occurrence of Onchocercidae in wild felids (i.e., Panthera onca, Puma concolor, Herpailurus yagouaroundi, Leopardus geoffroyi, Leopardus guttulus, Leopardus pardalis, Leopardus wiedii, Leopardus munoai) from different locations in Brazil. Overall, 82 samples (n = 63 blood; n = 19 tissues) were molecularly screened for cytochrome c oxidase subunit-1 (cox1) gene. Four (i.e., 4.8%) wild felid samples were positive, and at BLAST analysis, the obtained sequences showed varying percentage of nucleotide identity with the genera Brugia (i.e., 87-88%), Setaria (i.e., 89%), and D. immitis (i.e., 94.4%). Phylogenetic analyses clustered sequences obtained into three distinct clades, one with D. immitis and the remaining two with other Onchocercidae spp. Data herein obtained highlight the need for a more comprehensive understanding of the diversity and biology of Onchocercidae in South America in order to assess the potential impact that these species may have for domestic and wild animals, as well as humans.

[Cloning and functional analysis of AcFMO from onion during alliine biosynthesis].

Flavin-containing monooxygenase (FMO) is the key enzyme in the biosynthesis pathway of CSOs with sulfur oxidation. In order to explore the molecular regulatory mechanism of FMO in the synthesis of onion CSOs, based on transcriptome database and phylogenetic analysis, one AcFMO gene that may be involved in alliin synthesis was obtained, the AcFMO had a cDNA of 1 374 bp and encoded 457 amino acids, which was evolutionarily closest to the AsFMO of garlic. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) indicated that AcFMO was the highest in the flowers and the lowest in the leaf sheaths. The results of subcellular localization showed that the AcFMO gene product was widely distributed throughout the cell A yeast expression vector was constructed, and the AcFMO gene was ecotopically overexpressed in yeast to further study the enzyme function in vitro and could catalyze the synthesis of alliin by S-allyl-l-cysteine. In summary, the cloning and functional identification of AcFMO have important reference value for understanding the biosynthesis of CSOs in onions.

Morphological and molecular characterization of Stomachicola muraenesocis Yamaguti, 1934 (Digenea: Hemiuridae) from the daggertooth pike conger Muraenesox cinereus (Forsskål).

Hemiurid digeneans conspecific with Stomachicola muraenesocis Yamaguti, 1934 (the type species of the genus Stomachicola Yamaguti, 1934) were collected from the stomach of the daggertooth pike conger Muraenesox cinereus (Forsskål) off the Persian Gulf of Iran. This study aimed to provide a detailed characterization of Stom. muraenesocis, including measurements, illustrations and scanning electron microscopy (s.e.m.) representations. Comparisons with the original and previous descriptions revealed morphological and metrical variations in several features (i.e. body size and shape, arrangement of reproductive organs, soma to ecsoma length ratio, position of genital opening, number of vitelline tubules and extension of uterine coils) between Stom. muraenesocis from different hosts and localities. This study presents the first molecular sequence data associated with the small (18S) and large (28S) subunit nuclear ribosomal RNA genes (rDNA) for Stom. muraenesocis. Phylogenetic analyses of the 18S dataset placed Stom. muraenesocis as sister lineage to a clade formed of a group of species of Lecithaster Lühe, 1901 (Lecithasteridae Odhner, 1905). In contrast, phylogenetic analyses based on the 28S consistently recovered a sister relationship between Stom. muraenesocis and representatives of the Hemiuridae Looss, 1899. Further comprehensive phylogenetically based classification in light of morphology and taxonomic history of the Hemiuridae and Lecithasteridae is required to infer phylogenetic affinities and historical biogeography of Stomachicola. A comprehensive list of previously reported species of Stomachicola together with their associated hosts, localities and morphometric data is provided.

Molecular phylogenetics of sessile Dolium sedentarium, a petalomonad euglenid.

The euglenids are a species-rich group of flagellates with varying modes of nutrition that can be found in diverse habitats. Phagotrophic members of this group gave rise to phototrophs and hold the key to understanding the evolution of euglenids as a whole, including the evolution of complex morphological characters like the euglenid pellicle. Yet to understand the evolution of these characters, a comprehensive sampling of molecular data is needed to correlate morphological and molecular data, and to estimate a basic phylogenetic backbone of the group. While the availability of SSU rDNA and, more recently, multigene data from phagotrophic euglenids has improved, several "orphan" taxa remain without any molecular data whatsoever. Dolium sedentarium is one such taxon: It is a rarely-observed phagotrophic euglenid that inhabits tropical benthic environments and is one of few known sessile euglenids. Based on morphological characters, it has been thought of as part of the earliest branch of euglenids, the Petalomonadida. We report the first molecular sequencing data for Dolium using single-cell transcriptomics, adding another small piece in the puzzle of euglenid evolution. Both SSU rDNA and multigene phylogenies confirm it as a solitary branch within Petalomonadida.

Birth-and-death evolution of ribonuclease 9 genes in Cetartiodactyla.

RNase9 plays a reproductive function and has been recognized as an important member of the ribonuclease (RNase) A superfamily, a gene family that is widely used as a model for molecular evolutionary studies. Here, we identified 178 RNase9 genes from 95 Cetartiodactyla species that represent all four lineages and 21 families of this clade. Unexpectedly, RNase9 experienced an evolutionary scenario of "birth and death" in Ruminantia, and expression analyses showed that duplicated RNase9A and RNase9B genes are expressed in reproductive tissues (epididymis, vas deferens or prostate). This expression pattern combined with the estimate that these genes duplicated during the middle Eocene, a time when Ruminantia become a successful lineage, suggests that the RNase9 gene duplication might have been advantageous for promoting sperm motility and male fertility as an adaptation to climate seasonality changes of this period. In contrast, all RNase9 genes were lost in the Cetacean lineage, which might be associated with their high levels of prostatic lesions and lower reproductive rates as adaptations to a fully aquatic environment and a balance to the demands of ocean resources. This study reveals a complex and intriguing evolutionary history and functional divergence for RNase9 in Cetartiodactyla, providing new insights into the evolution of the RNaseA superfamily and molecular mechanisms for organismal adaptations to the environment.

Impact of molecular sequence data completeness on HIV cluster detection and a network science approach to enhance detection.

Detection of viral transmission clusters using molecular epidemiology is critical to the response pillar of the Ending the HIV Epidemic initiative. Here, we studied whether inference with an incomplete dataset would influence the accuracy of the reconstructed molecular transmission network. We analyzed viral sequence data available from ~ 13,000 individuals with diagnosed HIV (2012-2019) from Houston Health Department surveillance data with 53% completeness (n = 6852 individuals with sequences). We extracted random subsamples and compared the resulting reconstructed networks versus the full-size network. Increasing simulated completeness was associated with an increase in the number of detected clusters. We also subsampled based on the network node influence in the transmission of the virus where we measured Expected Force (ExF) for each node in the network. We simulated the removal of nodes with the highest and then lowest ExF from the full dataset and discovered that 4.7% and 60% of priority clusters were detected respectively. These results highlight the non-uniform impact of capturing high influence nodes in identifying transmission clusters. Although increasing sequence reporting completeness is the way to fully detect HIV transmission patterns, reaching high completeness has remained challenging in the real world. Hence, we suggest taking a network science approach to enhance performance of molecular cluster detection, augmented by node influence information.

Relative performance of Bayesian morphological clock and parsimony methods for phylogenetic reconstructions: Insights from the case of Myomiminae and Dryomyinae glirid rodents.

Extinct organisms provide crucial information about the origin and time of origination of extant groups. The importance of morphological phylogenetics for rigorously dating the tree of life is now widely recognized and has been revitalized by methodological developments such as the application of tip-dating Bayesian approaches. Traditionally, molecular clocks have been node calibrated. However, node calibrations are often unsatisfactory because they do not allow the fossil age to inform about phylogenetic hypothesis. The introduction of tip calibrations allow fossil species to be included alongside their living relatives, and the absence of molecular sequence data for these taxa to be remedied by supplementing the sequence alignments for living taxa with phenotype character matrices for both living and fossil taxa. Therefore, only phylogenetic analyses that take into account morphological characters can incorporate both fossil and extant species. Herein we present an unprecedented morphological dataset for a vast group of glirid rodents, to which different phylogenetic methodologies have been applied. We have compared the tree topologies resulting from traditional parsimony and Bayesian phylogenetic approaches and calculate stratigraphic congruence indices for each. Bayesian tip-dated clock methods seem to outperform parsimony with our dataset. The strict consensus tree recovered by tip dating invalidates the classic classification and allows dates to be proposed for the divergence and origin of the different clades.

Proposal for the Sungminiaceae fam. nov. (Ralfsiales, Phaeophyceae) for Sungminia gen. nov. with three new species based on molecular and morphological analyses.

Based on morphological and molecular analyses, five families have been recognized within the crustose brown algal order Ralfsiales. Our morphological and molecular sequence data were used to assess the establishment and phylogenetic relationship of Sungminia gen. nov. Phylogenies based on rbcL and concatenated rbcL and COI-5P genes support the recognition of Sungminia composed of three distinct lineages, Sungminia gladiata sp. nov., S. pyriformis sp. nov., and S. asiatica sp. nov. We consider that the Sungminia group is clearly distinct at the family level and propose to place Sungminia in a new family, the Sungminiaceae fam. Nov. Our phylogenetic analyses show that the Sungminiaceae forms a strongly supported monophyletic clade with probable sister relationship to the Mesosporaceae. The Sungminiaceae is characterized by perithallial erect filaments moderately adhered, the rod-shaped perithallial erect filaments, plurangia terminated with single sterile cell, and unangia terminally inserted on 1-2 celled stalk that is lateral-basal or sessile to a paraphysis.

Advancing genomic epidemiology by addressing the bioinformatics bottleneck: Challenges, design principles, and a Swiss example.

The SARS-CoV-2 pandemic led to a huge increase in global pathogen genome sequencing efforts, and the resulting data are becoming increasingly important to detect variants of concern, monitor outbreaks, and quantify transmission dynamics. However, this rapid up-scaling in data generation brought with it many IT infrastructure challenges. In this paper, we report about developing an improved system for genomic epidemiology. We (i) highlight key challenges that were exacerbated by the pandemic situation, (ii) provide data infrastructure design principles to address them, and (iii) give an implementation example developed by the Swiss SARS-CoV-2 Sequencing Consortium (S3C) in response to the COVID-19 pandemic. Finally, we discuss remaining challenges to data infrastructure for genomic epidemiology. Improving these infrastructures will help better detect, monitor, and respond to future public health threats.

Computational characterization and analysis of molecular sequence data of Elizabethkingia meningoseptica.

OBJECTIVE: Elizabethkingia meningoseptica is a multidrug resistance strain which primarily causes meningitis in neonates and immunocompromised patients. Being a nosocomial infection causing agent, less information is available in literature, specifically, about its genomic makeup and associated features. An attempt is made to study them through bioinformatics tools with respect to compositions, embedded periodicities, open reading frames, origin of replication, phylogeny, orthologous gene clusters analysis and pathways. RESULTS: Complete DNA and protein sequence pertaining to E. meningoseptica were thoroughly analyzed as part of the study. E. meningoseptica G4076 genome showed 7593 ORFs it is GC rich. Fourier based analysis showed the presence of typical three base periodicity at the genome level. Putative origin of replication has been identified. Phylogenetically, E. meningoseptica is relatively closer to E. anophelis compared to other Elizabethkingia species. A total of 2606 COGs were shared by all five Elizabethkingia species. Out of 3391 annotated proteins, we could identify 18 unique ones involved in metabolic pathway of E. meningoseptica and this can be an initiation point for drug designing and development. Our study is novel in the aspect in characterizing and analyzing the whole genome data of E. meningoseptica.

Detecting Selection in the HIV-1 Genome during Sexual Transmission Events.

Little is known about whether and how variation in the HIV-1 genome affects its transmissibility. Assessing which genomic features of HIV-1 are under positive or negative selection during transmission is challenging, because very few virus particles are typically transmitted, and random genetic drift can dilute genetic signals in the recipient virus population. We analyzed 30 transmitter-recipient pairs from the Zurich Primary HIV Infection Study and the Swiss HIV Cohort Study using near full-length HIV-1 genomes. We developed a new statistical test to detect selection during transmission, called Selection Test in Transmission (SeTesT), based on comparing the transmitter and recipient virus population and accounting for the transmission bottleneck. We performed extensive simulations and found that sensitivity of detecting selection during transmission is limited by the strong population bottleneck of few transmitted virions. When pooling individual test results across patients, we found two candidate HIV-1 genomic features for affecting transmission, namely amino acid positions 3 and 18 of Vpu, which were significant before but not after correction for multiple testing. In summary, SeTesT provides a general framework for detecting selection based on genomic sequencing data of transmitted viruses. Our study shows that a higher number of transmitter-recipient pairs is required to improve sensitivity of detecting selection.

Drug resistance pattern among ART-naive clients attending an HIV testing and counseling center in Dhaka, Bangladesh.

In Bangladesh, antiretroviral therapy (ART) is provided without screening drug resistance-associated mutations (DRM) among people living with HIV, while DRM might emerge and transmit to the newly infected individual. The present study was aimed to identify DRM among ART-naive clients from an HIV testing and counseling (HTC) center in the initial stages of ART programs. Randomly selected (n = 64) archived plasma samples were used for the pol gene amplification and sequencing by sanger technology. Recovered sequences (n = 10) were genotyped using HIV genotyping tools of NCBI and analyzed using the Stanford University HIV drug resistance database (hivdb.stanford.edu). Various genotypes with a number of DRM were identified in HTC clients, who belonged to different risk groups based on behavioral data. The drug resistance algorithm showed that all samples were fully resistant to tipranavir/ritonavir drugs except for one intermediate resistance. Despite the small sample size, our understanding from this study warrants an ART policy with a DRM monitoring system for the country.

SSBP1-Disease Update: Expanding the Genetic and Clinical Spectrum, Reporting Variable Penetrance and Confirming Recessive Inheritance.

Purpose: To report novel genotypes and expand the phenotype spectrum of SSBP1-disease and explore potential disease mechanism. Methods: Five families with previously unsolved optic atrophy and retinal dystrophy underwent whole genome sequencing as part of the National Institute for Health Research BioResource Rare-Diseases and the UK's 100,000 Genomes Project. In silico analysis and protein modelling was performed on the identified variants. Deep phenotyping including retinal imaging and International Society for Clinical Electrophysiology of Vision standard visual electrophysiology was performed. Results: Seven individuals from five unrelated families with bilateral optic atrophy and/or retinal dystrophy with extraocular signs and symptoms in some are described. In total, 6 SSBP1 variants were identified including the previously unreported variants: c.151A>G, p.(Lys51Glu), c.335G>A p.(Gly112Glu), and c.380G>A, p.(Arg127Gln). One individual was found to carry biallelic variants (c.380G>A p.(Arg127Gln); c.394A>G p.(Ile132Val)) associated with likely autosomal recessive SSBP1-disease. In silico analysis predicted all variants to be pathogenic and Three-dimensional protein modelling suggested possible disease mechanisms via decreased single-stranded DNA binding affinity or impaired higher structure formation. Conclusions: SSBP1 is essential for mitochondrial DNA replication and maintenance, with defects leading to a spectrum of disease that includes optic atrophy and/or retinal dystrophy, occurring with or without extraocular features. This study provides evidence of intrafamilial variability and confirms the existence of an autosomal recessive inheritance in SSBP1-disease consequent upon a previously unreported genotype.

Molecular study of Nucleophosmin 1(NPM1) gene in acute myeloid leukemia in Kurdish population.

BACKGROUND: In patients with Acute Myeloid Leukemia (AML) the most frequent acquired molecular abnormalities and important prognostic indicators is nucleophosmin-1 (NPM1) mutations. Our study aims was molecular study of Nucleophosmin -1 gene in Acute Myeloid Leukemia in Kurdish population. PATIENTS &METHODS: A total of 50 patients with AML, (36) of them attended Nanakaly Hospital and (14) attended Hiwa Hospital and 30 healthy subjects as control were selected randomly, all were matched of age and gender. Polymerase chain reaction (PCR) was used for detection of NPM1 gene mutation. Three samples of PCR product for NPM1 gene mutations were sequenced, and mutations were determined by comparison with the normal NPM1 sequence NCBI (GenBank accession number NM_002520). RESULTS: Out of 50 patients with AML, 5 (10%) of them were NPM1 gene mutation positive, and 45 (90%) were negative. The mutation were a base substitution (C to A), (G to C), (G to T), transversion mutation in addition of frame shift mutation and all mutated cases were heterozygous and retained a wild type allele. CONCLUSION: Identification of NPM1 mutations in AML are important for prognostication, treatment decision and optimization of patient care.

Identification and biochemical characterization of an ATP-dependent dihydroxyacetone kinase from Trypanosoma cruzi.

Dihydroxyacetone (DHA) can be used as an energy source by many cell types; however, it is toxic at high concentrations. The enzyme dihydroxyacetone kinase (DAK) has shown to be involved in DHA detoxification and osmoregulation. Among protozoa of the genus Trypanosoma, T. brucei, which causes sleeping sickness, is highly sensitive to DHA and does not have orthologous genes to DAK. Conversely, T. cruzi, the etiological agent of Chagas Disease, has two putative ATP-dependent DAK (TcDAKs) sequences in its genome. Here we show that T. cruzi epimastigote lysates present a DAK specific activity of 27.1 nmol/min/mg of protein and that this form of the parasite is able to grow in the presence of 2 mM DHA. TcDAK gene was cloned and the recombinant enzyme (recTcDAK) was expressed in Escherichia coli. An anti-recTcDAK serum reacted with a protein of the expected molecular mass of 61 kDa in epimastigotes. recTcDAK presented maximal activity using Mg+2, showing a Km of 6.5 µM for DHA and a K0.5 of 124.7 µM for ATP. As it was reported for other DAKs, recTcDAK activity was inhibited by FAD with an IC50 value of 0.33 mM. In conclusion, TcDAK is the first DAK described in trypanosomatids confirming another divergent metabolism between T. brucei and T. cruzi.

Complex History of Aerobic Respiration and Phototrophy in the Chloroflexota Class Anaerolineae Revealed by High-Quality Draft Genome of Ca. Roseilinea mizusawaensis AA3_104.

Roseilinea is a novel lineage of Chloroflexota known only from incomplete metagenome-assembled genomes (MAGs) and preliminary enrichments. Roseilinea is notable for appearing capable of anoxygenic photoheterotrophy despite being only distantly related to well-known phototrophs in the Chloroflexia class such as Chloroflexus and Roseiflexus. Here, we present a high-quality MAG of a member of Roseilinea, improving our understanding of the metabolic capacity and phylogeny of this genus, and resolving the multiple instances of horizontal gene transfer that have led to its metabolic potential. These data allow us to propose a candidate family for these organisms, Roseilineaceae, within the Anaerolineae class.

Sporadic isolation of Tetratrichomonas species from the cattle urogenital tract.

Tritrichomonas foetus is a venereal trichomonad parasite which causes reproductive issues in cattle. No other trichomonads are known to be urogenital pathogens in cattle, but there are several reports of Tetratrichomonas and Pentatrichomonas isolates of unclear origin from the cattle urogenital tract (UGT) in the Americas. This study reports the first case of a non-T. foetus cattle urogenital trichomonad isolate in Europe. Molecular analysis of the internal transcribed spacer (ITS) 1-5.8S ribosomal RNA-ITS 2 and 18S ribosomal RNA loci suggest that the isolate is a Tetratrichomonas species from a lineage containing other previously described bull preputial isolates. We identified close sequence similarity between published urogenital and gastrointestinal Tetratrichomonas spp., and this is reviewed alongside further evidence regarding the gastrointestinal origin of non-T. foetus isolates. Routine screening for T. foetus is based on culture and identification by microscopy, and so considering other trichomonad parasites of the bovine UGT is important to avoid misdiagnosis.

Changes in the Gut Microbiota Following Bariatric Surgery Are Associated with Increased Alcohol Intake in a Female Rat Model.

AIMS: We aimed to investigate if differences in gut microbiota diversity and composition are associated with post-operative alcohol intake following bariatric surgery in a rat model. METHODS: Twenty-four female rats were randomized to three treatment groups: sham surgery, vertical sleeve gastrectomy (VSG) or Roux-en-Y gastric bypass (RYGB). Stool was collected pre- and post-operatively and 16S rRNA gene amplification and sequencing was performed. Analysis focused on correlating microbial diversity, type of surgery and alcohol (EtOH) intake. RESULTS: Pre-operative stools samples on regular diet showed similar taxonomic composition and Shannon diversity among the three treatment groups. There was a significant decrease in Shannon diversity and a change in taxonomic composition of the gut microbiota after rats was fed high fat diet. Post-operatively, the RYGB group showed significantly lower taxonomic diversity than the VSG and sham groups, while the VSG and sham groups diversity were not significantly different. Taxonomic composition and function prediction based on PICRUSt analysis showed the RYGB group to be distinct from the VSG and sham groups. Shannon diversity was found to be negatively associated with EtOH intake. CONCLUSIONS: Changes in the taxonomic profile of the gut microbiota following bariatric surgery, particularly RYGB, are associated with increased EtOH intake and may contribute to increased alcohol use disorder risk through the gut-brain-microbiome axis.

Extraction, purification, physicochemical properties and antioxidant activity of a new polysaccharide from Ocimum album L. seed.

In this study, a novel polysaccharide fraction from Ocimum album seed was extracted and then purified by Cellulose DEAE-52 and Sephadex G-200 anion exchange chromatography. The structural, physicochemical and antioxidant properties of the main polysaccharide fraction (OAP-1A) were evaluated. The purified polysaccharide contained 94.3% carbohydrate, 3.56% moisture and 2.14% ash and result of gel permeation chromatography (GPC) showed average molecular weight of 593 kDa. The results of high-performance liquid chromatography (HPLC) showed that OAP-1A was a neutral hetero-polysaccharide composed of mannose (35.7%), glucose (33.32%), galactose (19.6%) and rhamnose (11.38%). In addition, GC-MS data, nuclear magnetic resonance (NMR) spectrum and Fourier transform infrared (FT-IR) analysis revealed that the backbone of OAP-1A consists of →3)-ß-D-Manp-(1→, →3,4)-ß-D-Manp-(1→, →3,6)-ß-D-Manp-(1→, →3)-α-D-Glcp-(1→, →6)-ß-D-Galp-(1→, →4)-α-L-Rhap-(1→ and α-D-Glcp-(1→. X-ray diffraction (XRD) analysis showed semi-crystalline structure in OAP-1A. Differential scanning colorimeter (DSC) and thermo-gravimetry analysis (TGA) indicated that OAP-1A had relatively high thermal stability. Moreover, OAP-1A showed strong scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.