Ribosomal Deoxyribonucleic Acid Research Articles

Nonalcoholic Fatty Liver Disease: Changes in Gut Microbiota and Blood Lipids.

The global prevalence of nonalcoholic fatty liver disease (NAFLD) is 25%. This study aimed to explore differences in the gut microbial community and blood lipids between normal livers and those affected by NAFLD using 16S ribosomal deoxyribonucleic acid sequencing. Gut microbiome profiles of 40 NAFLD and 20 non-NAFLD controls were analyzed. Information about four blood lipids and 13 other clinical features was collected. Patients were divided into three groups by ultrasound and FibroScan, those with a normal liver, mild FL (FL1), and moderate-to-severe FL (FL2). FL1 and FL2 patients were divided into two groups, those with either hyperlipidemia or non-hyperlipidemia based on their blood lipids. Potential keystone species within the groups were identified using univariate analysis and a specificity-occupancy plot. Significant difference in biochemical parameters ion NAFLD patients and healthy individuals were identified by detrended correspondence analysis and canonical correspondence analysis. Decreased gut bacterial diversity was found in patients with NAFLD. Firmicutes/Bacteroidetes decreased as NAFLD progressed. Faecalibacterium and Ruminococcus 2 were the most representative fatty-related bacteria. Glutamate pyruvic transaminase, aspartate aminotransferase, and white blood cell count were selected as the most significant biochemical indexes. Calculation of areas under the curve identified two microbiomes combined with the three biochemical indexes that identified normal liver and FL2 very well but performed poorly in diagnosing FL1. Faecalibacterium and Ruminococcus 2, combined with glutamate pyruvic transaminase, aspartate aminotransferase, and white blood cell count distinguished NAFLD. We speculate that regulating the health of gut microbiota may release NAFLD, in addition to providing new targets for clinicians to treat NAFLD.

Alterations in nasal microbiota of patients with amyotrophic lateral sclerosis.

Links between alterations in gut microbiota composition and amyotrophic lateral sclerosis (ALS) have previously been reported. This study aimed to examine the microbiota in the nasal cavity of ALS. Sixty-six ALS patients and 40 healthy caregivers who live in close proximity with patients were enrolled. High throughput metagenomic sequencing of the 16S ribosomal deoxyribonucleic acid (rDNA) gene V3-V4 region of nasal microbiota was used to characterize the alpha and beta diversity and relative abundance of bacterial taxa, predict function, and conduct correlation analysis between specific taxa and clinical features. The nasal microbiome of ALS patients showed lower alpha diversity than that of corresponding healthy family members. Genera Gaiella , Sphingomonas , Polaribacter _1, Lachnospiraceae _NK4A136_group, Klebsiella , and Alistipes were differentially enriched in ALS patients compared to controls. Nasal microbiota composition in ALS patients significantly differed from that in healthy subjects (unweighted UniFrac P = 0.001), while Linear discriminant analysis Effect Size (LEfSe) analysis indicated that Bacteroidetes and Firmicutes dominated healthy nasal communities at the phylum level, whereas Actinobacteria was the predominant phylum and Thermoleophilia was the predominant class in ALS patients. Genus Faecalibacterium and Alistipes were positively correlated with ALS functional rating scale revised (ALSFRS-R; rs = 0.349, P = 0.020 and rs = 0.393, P = 0.008), while Prevotella -9 and Bacteroides operational taxonomic units (OTUs) were positively associated with lung function (FVC) in ALS patients ( rs = 0.304, P = 0.045, and rs = 0.300, P = 0.048, respectively). Prevotella -1 was positively correlated with white blood cell counts (WBC, rs = 0.347, P = 0.021), neutrophil percentage (Neu%, rs = 0.428, P = 0.004), and neutrophil-to-lymphocyte ratio (NLR, rs = 0.411, P = 0.006), but negatively correlated with lymphocyte percentage (Lym%, rs = -0.408, P = 0.006). In contrast, Streptococcus was negatively associated with Neu% ( rs = -0.445, P = 0.003) and NLR ( rs = -0.436, P = 0.003), while positively associated with Lym% ( rs = 0.437, P = 0.003). No significant differences in nasal microbiota richness and evenness were detected among the severe and mild ALS patients. ALS is accompanied by altered nasal microbial community composition and diversity. The findings presented here highlight the need to understand how dysbiosis of nasal microbiota may contribute to the development of ALS.

A NEW SPECIES OF HENNEGUYA THÉLOHAN, 1892 (CNIDARIA: BIVALVULIDA: MYXOBOLIDAE) INFECTING THE SUBMUCOSA OF THE INTESTINE AND PYLORIC CECA OF RED DRUM, SCIAENOPS OCELLATUS (LINNAEUS) (PERCIFORMES: SCIAENIDAE) FROM COASTAL ALABAMA.

A new species of Henneguya Thélohan, 1892 (Bivalvulida: Myxobolidae) is described from the submucosa of the intestine and pyloric ceca of red drum (Sciaenops ocellatus [Linnaeus, 1766] [Perciformes: Sciaenidae]) from the Gulf of Mexico off Gulf Shores, Alabama. Henneguya albomaculata n. sp. differs from all congeners by the combination of myxospore dimensions, polar tubule coil count, the presence of an iodinophilic vacuole in the sporoplasm, and small-subunit ribosomal deoxyribonucleic acid (SSU rDNA) sequence. A phylogenetic analysis of the SSU rDNA recovered H. albomaculata sister to Henneguya cynosioniDykova, de Buron, Roumillat, and Fiala, 2011 in a clade composed of 11 species of Henneguya and 1 species of Myxobolus Bütschli, 1882 (Bivalvulida: Myxobolidae) that collectively infect fishes in marine or estuarine systems. Histologic sections of infected intestine and pyloric ceca revealed plasmodia of H. albomaculata n. sp. developing in the loose connective tissue of the submucosa. The new species comprises the second species of Henneguya reported from red drum.

STENOPHYSA MARMORATA (MOLLUSCA: PHYSIDADE) AS AN INTERMEDIATE HOST OF A SNAKE TREMATODE OF THE FAMILY RENIFERIDAE IN BRAZIL.

Species of the family Reniferidae are trematodes found in the oral cavity and esophagus of snakes from Nearctic and Neotropical regions. Although Renifer heterocoelium has been reported in different snake species from South America, the snails involved in its transmission remain unknown. In this study, a xiphidiocercaria emerged from the physid snail Stenophysa marmorata from Brazil and was subjected to morphological and molecular study. The general morphology, including the shape of the stylet and arrangement of penetration glands, resembles that described for reniferid trematodes from North America. Phylogenetic analysis on the basis of nuclear sequences (28S ribosomal deoxyribonucleic acid gene [1,072 base pairs {bp}] and internal transcribed spacer region [ITS, 1,036 bp]) supports identifying this larva as a member of the family Reniferidae, very possibly a species of the genus Renifer. In the 28S analysis, low molecular divergences were found to Renifer aniarum (1.4%) and Renifer kansensis (0.6%), but also concerning other 2 reniferid species, i.e., Dasymetra nicolli (1.4%) and Lechriorchis tygarti (1.0%). Regarding ITS, the divergences between this Brazilian cercaria and R. aniarum or L. tygarti were 1.9% and 8.5%, respectively. In the case of the mitochondrial marker cytochrome oxidase subunit 1 (797 bp), our Reniferidae gen. sp. differs 8.6-9.6% from Paralechriorchis syntomentera, the only reniferid with sequences available for comparison. We discuss the probable conspecificity of the larval stages here reported with R. heterocoelium, the reniferid species reported in South America.

Organically grown cannabis (Cannabis sativa L.) plants contain a diverse range of culturable epiphytic and endophytic fungi in inflorescences and stem tissues

We investigated the diversity of fungal species present on inflorescences as epiphytes and in stem tissues as endophytes in flowering plants of cannabis grown organically in British Columbia during 2019–2021. Fresh and dried inflorescence samples were obtained at various times during production while stems were obtained at harvest. Fungal species in the air were assessed by exposing Petri dishes containing potato dextrose agar + streptomycin sulfate for 1 h in the growing environment while soil samples were dilution-plated to assess soil fungal diversity. Colonies were identified from PCR-derived sequences of the ITS1-5.8S-ITS2 region of ribosomal deoxyribonucleic acid. Twenty-nine species in 26 genera were recovered from inflorescences and 17 species in 11 genera originated from stem tissues. Approximately 96% of species found on inflorescences were present in air and 45% were present in organic soil. The fungi comprised plant pathogens, saprophytes, and opportunistic human pathogens. A large proportion of the species found in air and soil in organic facilities are present on cannabis inflorescences, where they may increase total colony forming units and negatively affect product quality. Some species could contribute to allergies or secondary infections in humans. The potential benefits of endophytes within organically grown cannabis plants remain unexplored.

Identification of potentially zoonotic parasites in captive orangutans and semi-captive mandrills: Phylogeny and morphological comparison.

Cysts and trophozoites of vestibuliferid ciliates and larvae of Strongyloides were found in fecal samples from captive orangutans Pongo pygmaeus and P. abelii from Czech and Slovak zoological gardens. As comparative material, ciliates from semi-captive mandrills Mandrillus sphinx from Gabon were included in the study. Phylogenetic analysis of the detected vestibuliferid ciliates using ITS1-5.8s-rRNA-ITS2 and partial 18S ribosomal deoxyribonucleic acid(rDNA) revealed that the ciliates from orangutans are conspecific with Balantioides coli lineage A, while the ciliates from mandrills clustered with Buxtonella-like ciliates from other primates. Morphological examination of the cysts and trophozoites using light microscopy did not reveal differences robust enough to identify the genera of the ciliates. Phylogenetic analysis of detected L1 larvae of Strongyloides using partial cox1 revealed Strongyloides stercoralis clustering within the cox1 lineage A infecting dogs, humans, and other primates. The sequences of 18S rDNA support these results. As both B. coli and S. stercoralis are zoonotic parasites and the conditions in captive and semi-captive settings may facilitate transmission to humans, prophylactic measures should reflect the findings.

MORPHOLOGIC OBSERVATION AND FIRST MOLECULAR CHARACTERIZATION OF ENCYCLOMETRA JAPONICA (DIGENEA: ENCYCLOMETRIDAE) IN THE DIGESTIVE TRACT OF WILD TIGER KEELBACKS (RHABDOPHIS TIGRINUS) IN JAPAN.

Encyclometra japonica Yoshida and Ozaki, 1929 is a fluke that parasitizes the digestive tract of snakes. When the species was first reported, it was originally characterized on the basis of morphologic features, namely, the characteristics of the 2 testes located obliquely in the anterior-posterior direction, and the position of the ventral sucker one-fourth to one-third from the anterior extremity. Thereafter, more specimens with other morphologic variations were reported, and a new morphologic feature for species discrimination was proposed. However, the proposal is uncertain, and the criteria for morphologic discrimination between E. japonica and other Encyclometra species remain debatable. Thus, morphologically discriminable characteristics on the basis of a sufficient number of specimens are required, and molecular methods for species identification that allow for a more objective assessment need to be established. Therefore, in the present study, nucleotide sequences of 18S, 28S ribosomal deoxyribonucleic acid and cytochrome c oxidase subunit I (COI) of E. japonica were determined along with detailed morphologic observations for the first time. Key characteristics were observed in all specimens, whereas some morphologic variations were observed in the symmetry of the cecum. The COI sequences, which are known to be variable genetic regions, were identical among all specimens; therefore, all of them were considered the same species. This result clarifies the high morphologic variation in E. japonica. The 18S and 28S sequences were 99.78% and 99.11-99.19% similar to those of Encylometra colubrimurorum. The high degree of homology between these genes indicates that E. japonica and E. colubrimurorum are closely related but independent species. The present data will be used for the identification of E. japonica and to evaluate the relationships within the genus Encyclometra.

Divergent impacts on the gut microbiome and host metabolism induced by traditional Chinese Medicine with Cold or Hot properties in mice

BackgroundTraditional Chinese Medicine (TCM) has been practiced and developed in China over thousands of years under the guidance of a series of complicated traditional theories. Herbs within TCM usually are classified according to their different properties ranging from cold, cool, warm to hot, which are simplified as Cold and Hot properties. TCM with either Cold or Hot properties are used in various formulae designed for the purpose of restoring the balance of patients. Emerging evidence has highlighted that an altered gut microbiota or host metabolism are critically involved in affecting the healing properties of TCM. However, at present the exact influences and crosstalk on the gut microbiota and host metabolism remain poorly understood.MethodsIn the present study, the divergent impacts of six TCMs with either Cold or Hot properties on gut microbiome and host metabolism during short- or long-term intervention in mice were investigated. Six typical TCMs with Hot or Cold properties including Cinnamomi Cortex (rougui, RG), Zingiberis Rhizoma (ganjiang, GJ), Aconiti Lateralis Radix Praeparata (fuzi, FZ), Rhei Radix et Rhizoma (dahuang, DH), Scutellariae Radix (huangqin, HQ), and Copitdis Rhizoma (huanglian, HL) were selected and orally administered to male C57BL/6J mice for a short- or a long-term (7 or 35 days). At the end of experiments, serum and cecal contents were collected for metabolomic and gut microbiome analyses using gas chromatography–tandem mass spectrometry (GC–MS) or 16S ribosomal deoxyribonucleic acid (16S rDNA) sequencing.ResultsThe results revealed that the gut microbiome underwent divergent changes both in its composition and functions after short-term intervention with TCM possessing either Cold or Hot properties. Interestingly, the number of changed genus and bacteria pathways was reduced in Hot_LT, but was increased in Cold_LT, especially in the HL group. Increased α diversity and a reduced F/B ratio revealed the changes in Hot_ST, but a reduced Shannon index and increased altered bacteria function was evident in Cold_LT. The serum metabolic profile showed that the influence of TCM on host metabolism was gradually reduced over time. Glycolipid metabolism related pathways were specifically regulated by Hot_ST, but also surprisingly by Cold_LT. Reduced lactic acid in Cold_ST, increased tryptophan concentrations and decreased proline and threonine concentrations in Cold_LT perhaps highlighting the difference between the two natures influence on serum metabolism. These metabolites were closely correlated with altered gut microbiota shown by further correlation analyses.ConclusionThe results indicated that TCM properties could be, at least partially characterized by an alteration in the gut microbiota and metabolic profile, implying that the divergent responses of gut microbiome and host metabolism are involved in different responses to TCM.Graphic

Aspergillus violaceofuscus alleviates cadmium and chromium stress in Okra through biochemical modulation.

Endophytic fungi from the Chilli were used to help okra plants exposed to cadmium (Cd) or chromium (Cr) stress. Initially, the strain Ch06 produced higher amounts of indole acetic acid (IAA) (230.5 μg/mL), sugar (130.7 μg/mL), proteins (128.2 μg/mL), phenolics (525.6 μg/mL) and flavonoids (98.4 μg/mL) in Czapek broth supplemented with Cd or Cr. The production of IAA and other metabolites in such a higher concentration suggested that Ch06 might improve plant growth under heavy metal stress. For this reason, an experiment was designed, in which biomass of Ch06 (at 2g/100g of sand) were applied to the okra plants exposed to Cd or Cr stress (at 100 or 500 μg/g). The results exhibited that Ch06 improved the total chlorophyll (36.4±0.2 SPAD), shoot length (22.6±0.2 cm), root length (9.1±0.6 cm), fresh weight (5±0.6 g), dry weight (1.25±0.01 g), sugars (151.6 μg/g), proteins (114.8 μg/g), proline (6.7 μg/g), flavonoids (37.9 μg/g), phenolics (70.7 μg/g), IAA (106.7 μg/g), catalase (0.75 enzyme units/g tissue) and ascorbic acid oxidaze (2.2 enzyme units/g tissue) of the associated okra plants. Similar observations have been recorded in Ch06 associated okra plants under Cd and Cr stress. Also, Ch06 association reduced translocation of Cd (35% and 45%) and Cr (47% and 53%) to the upper parts of the okra plants and thus reduced their toxicity. The internal transcribed spacer (ITS) region amplification of 18S rDNA (ribosomal deoxyribo nucleic acid) exhibited that the potent strain Ch06 was Aspergillus violaceofuscus. The results implied that A. violaceofuscus has the ability to promote host species growth exposed to Cd and Cr. Moreover, it helped the host plants to recover in Cd and Cr polluted soils, hence can be used as biofertilizer.

A New Genus of Diplostomids (Digenea: Diplostomoidea) from the Nile Crocodile in South Africa with a Key to Diplostomid Genera

The Diplostomidae Poirier, 1886 is a large family of digeneans within the superfamily Diplostomoidea Poirier, 1886. Members of the family are distributed worldwide and parasitize a diversity of tetrapod definitive hosts. Notably, only 2 mature diplostomids are known from crocodilians and both are suggested to be accidental infections. In this study, we use morphological and molecular data to describe Neofibricola n. gen. from a Nile crocodile Crocodylus niloticus collected in South Africa. We provide a description of adults and metacercariae of the type species, Neofibricola smiti n. sp., and metacercariae of a likely congeneric species. We generated partial 28S and internal transcribed spacer region ribosomal deoxyribonucleic acid (DNA) and cytochrome c oxidase 1 subunit mitochondrial DNA for both species and utilized the newly generated 28S sequences to examine phylogenetic affinities of these new taxa. In addition, we provide a new key to diplostomid genera, considering the substantial systematic changes and newly erected genera since the previously published key to diplostomid genera.

Characteristics of gut microbiota of term small gestational age infants within 1 week and their relationship with neurodevelopment at 6 months

IntroductionSmall for gestational age (SGA) infants are at a higher risk of neurodevelopmental delay than infants appropriate for gestational age (AGA). Previous studies have confirmed that gut microbiota in early life influences subsequent neurodevelopment. However, few studies have reported corresponding data in SGA populations.ObjectiveWe aimed to evaluate the characteristics of the gut microbiota of term SGA infants and the associations between the gut microbiota in SGA infants and neurodevelopmental outcomes at 6 months of age.MethodsFecal samples were collected on days 1, 3, 5, and 7 from term SGA and AGA infants born between June 2020 and June 2021 at the Peking University First Hospital. 16S ribosomal deoxyribonucleic acid amplicon sequencing was used to analyze the fecal microbiota. We followed up for 6 months and used the Ages and Stages Questionnaires-3 (ASQ-3) to evaluate the neurodevelopmental outcomes among SGA infants.ResultsA total of 162 neonates were enrolled, with 41 SGA infants (25.3%) in the study group and 121 AGA infants (74.7%) in the control group. The gut microbial diversity in the SGA group was lower than that in the AGA group on days 1, 3, 5, and 7. Non-metric multidimensional scaling and analysis of similarities showed significant differences between the two groups. The SGA group had increased relative abundances of Ralstonia (3, 5, and 7 days) and Clostridium (3 and 7 days). The dominant microorganisms of the SGA group were Ralstonia on day 1, Escherichia_Shigella on days 3 and 7, and Clostridia on day 5. We found that the gut microbial diversity of SGA infants with poor communication scores was higher than that of SGA infants with good communication scores on day 3. Fine motor scores were negatively correlated with the relative abundance of Bacteroides_fragilis on day 1. A negative correlation was observed between gross motor scores and relative abundance of Clostridium_saccharobutylicum on day 7. Bacteroidota, Bacteroidia, Bacteroides, and Bacteroides_fragilis were the dominant microorganisms in the good communication score group on day 7. Communication scores were positively correlated with the relative abundance of Bacteroidota, Bacteroides, and Bacteroides_fragilis on day 7.ConclusionThe gut microbial diversity of term SGA infants was significantly lower in the first week of life than that of term AGA infants. Certain pathogenic and conditional pathogenic bacteria, such as Escherichia_Shigella, Ralstonia and Clostridium increased or formed the dominant microbiota in SGA infants. Alpha diversity, Bacteroidota, Bacteroides, Bacteroides_fragilis, and Clostridium_saccharobutylicum found in SGA infants may be associated with neurodevelopmental outcomes at 6 months of age, indicating possible therapeutic targets for clinical intervention.

Replacement of Trypsin by Proteases for Medical Applications.

Cell culture has a crucial role in many applications in biotechnology. The production of vaccines, recombinant proteins, tissue engineering, and stem cell therapy all need cell culture. Most of these activities needed adherent cells to move, which should be trypsinized several times until received on a large scale. Although trypsin is manufactured from the bovine or porcine pancreas, the problem of contamination by unwanted animal proteins, unwanted immune reactions, or contamination to pathogen reagents is the main problem. This study investigated microbial proteases as a safe alternative for trypsin replacement in cell culture experiments for the detachment of adherent cells. The bacteria were isolated from the leather industry effluent based on their protease enzymes. After sequencing their 16S ribosomal deoxyribonucleic acid, their protease enzymes were purified, and their enzyme activities were assayed. The alteration of enzymatic activities using different substrates and the effect of substrate concentrations on enzyme activities were determined. The purified proteases were evaluated for cell detachment in the L929 fibroblast cells compared to trypsin. The separated cells were cultured again, and cell proliferation was determined by the MTT assay. The results showed that the isolated bacteria were Bacillus pumilus, Stenotrophomonas sp., Klebsiella aerogenes, Stenotrophomonas maltophilia, and Bacillus licheniformis. Among the isolated bacteria, the highest and the lowest protease activity belonged to Stenotrophomonas sp. and K. aerogenes, with 60.34 and 11.09 U/mL protease activity, respectively. All the isolated microbial proteases successfully affected L929 fibroblast cells' surface proteins and detached the cells. A significant induction in cell proliferation was observed in the cells treated with K. aerogenes protease and B. pumilus protease, respectively (P < 0.05). The obtained results suggested that microbial proteases can be used as safe and efficient alternatives to trypsin in cell culture in biopharmaceutical applications.

Evaluating methylation of human ribosomal DNA at each CpG site reveals its utility for cancer detection using cell-free DNA.

Ribosomal deoxyribonucleic acid (DNA) (rDNA) repeats are tandemly located on five acrocentric chromosomes with up to hundreds of copies in the human genome. DNA methylation, the most well-studied epigenetic mechanism, has been characterized for most genomic regions across various biological contexts. However, rDNA methylation patterns remain largely unexplored due to the repetitive structure. In this study, we designed a specific mapping strategy to investigate rDNA methylation patterns at each CpG site across various physiological and pathological processes. We found that CpG sites on rDNA could be categorized into two types. One is within or adjacent to transcribed regions; the other is distal to transcribed regions. The former shows highly variable methylation levels across samples, while the latter shows stable high methylation levels in normal tissues but severe hypomethylation in tumors. We further showed that rDNA methylation profiles in plasma cell-free DNA could be used as a biomarker for cancer detection. It shows good performances on public datasets, including colorectal cancer [area under the curve (AUC) = 0.85], lung cancer (AUC = 0.84), hepatocellular carcinoma (AUC = 0.91) and in-house generated hepatocellular carcinoma dataset (AUC = 0.96) even at low genome coverage (<1×). Taken together, these findings broaden our understanding of rDNA regulation and suggest the potential utility of rDNA methylation features as disease biomarkers.

Analysis of microflora in dentinal tubule by denaturing gradient gel electrophoresis

This study was performed to apply denaturing gradient gel electrophoresis to microbiological examination of endodontic infections. The method was used to assess the bacterial communities in dentinal tubules. Samples were collected using #15, #35, and #60 K-type files from five infected root canals during treatment. Deoxyribonucleic acid(DNA) was extracted from the samples, and 16S ribosomal DNA was amplified by PCR using universal primers. The polymerase chain reaction(PCR) products were separated in the denaturing gel and band patterns were compared between the deep (#60 file) and superficial layers (#15 file) of the dentin. The major bands were then excised and DNA fragments in the gel were cloned and sequenced. The sequence data were subjected to BLAST search in the GenBank database for determination of bacterial species or closest relatives. In three root canals, similar band patterns were observed in both superficial and deep layers of the dentin, while several specific bands remained in the deep layer in two roots. The bacteria isolated from the deep layers were Porphyromonas gingivalis, Olsenella profuse, Atopobium rimae, and Prevotella, Flexistipes, Firmicutes, Peptostreptococcus, Dialister, and Eubacterium spp. Unlike previous studies, gram-negative anaerobic rods were isolated from the deep layers. Clone library analysis was simultaneously performed and similar results were obtained. The method utilized here will be useful for microbiological examination of endodontic infections. In addition, although it is still unknown whether they were viable, this study demonstrated the presence of gram-negative rods in dentinal tubules.

Evolution of Intestinal Microbiota of Asphyxiated Neonates Within 1 Week and Its Relationship With Neural Development at 6 Months.

Introduction: Asphyxia is an emergent condition in neonates that may influence the function of the nervous system. Research has shown that intestinal microbiota is very important for neurodevelopment. Studies regarding the association between gut microbiota and neurodevelopment outcome in asphyxiated newborns remain scarce.Objective: To study the microbial characteristics of asphyxiated neonates within 1 week of life and to investigate their relationship with neural development at 6 months.Methods: The feces produced on days 1, 3, and 5, and the clinical data of full-term neonates with asphyxia and without asphyxia, delivered from March 2019 to October 2020 at Peking University First Hospital, were collected. We used 16S ribosomal deoxyribonucleic acid amplicon sequencing to detect the intestinal microbiota of asphyxiated neonates and neonates in the control group. We followed up asphyxiated neonates for 6 months and used the Ages and Stages Questionnaires-3 (ASQ-3) to evaluate their development.Results: A total of 45 neonates were enrolled in the study group and 32 were enrolled in the control group. On day 1, the diversity and richness of the microflora of the study group were more than those of the control group. Non-metric multidimensional scaling analysis showed significant differences in the microbiota of the two groups on days 1, 3, and 5. At the phylum level, the main microflora of the two groups were not different. At the genus level, the study group had increased relative abundance of Clostridium_sensu_stricto_1, Lachnoclostridium, Fusicatenibacter, etc. on day 1. On day 3, the relative abundance of Clostridium_sensu_stricto_1, Fusicatenibacter, etc. was still greater than that of the control group, and the relative abundance of Staphylococcus was less than that of the control group. On day 5, the relative abundance of Clostridium_sensu_stricto_1 and Lachnoclostridium was still higher than that of the control group, and the relative abundance of Dubosiella in the study group was significantly increased. At the species level, on day 3, the relative abundance of Staphylococcus caprae in the study group was less than that in the control group. Linear discriminant analysis effect size showed that the microbiota of the study group mainly consisted of Lachnospiraceae and Clostridia on day 1 and Clostridia on day 3. In the control group, Staphylococcus was the dominant bacterium on day 3. Neonates in the study group were followed up for 6 months, and the communication score of ASQ-3 was negatively correlated with the relative abundance of Lachnospiraceae and Clostridia on day 1.Conclusion: The diversity and richness of the microbiota of asphyxiated neonates on the first day of life were significantly increased and mainly consisted of pathogenic flora. Lachnospiraceae and Clostridia found in neonates with asphyxia on day 1 of life may be related to neural development at 6 months.

DESCRIPTION AND PHYLOGENETIC AFFINITIES OF A NEW SPECIES OF NEOPSILOTREMA (DIGENEA: PSILOSTOMIDAE) FROM LESSER SCAUP, AYTHYA AFFINIS (ANSERIFORMES: ANATIDAE).

Neopsilotrema is a small genus of psilostomid digeneans parasitic in the intestine of birds in the Palearctic and Nearctic. At present, the genus includes 4 species: Neopsilotrema lisitsynae from the Palearctic and Neopsilotrema affine, Neopsilotrema lakotae, and Neopsilotrema marilae from the Nearctic. Herein, we describe a new species, Neopsilotrema itascae n. sp., from lesser scaup Aythya affinis collected in Minnesota. The species can be distinguished from congeners on the basis of the ventral sucker:oral sucker width ratio, body width:length ratio, and cirrus sac size, along with other characters. We generated new 28S ribosomal deoxyribonucleic acid (DNA) and NADH dehydrogenase (ND1) mitochondrial DNA sequence data of a variety of psilostomids from the Palearctic and Nearctic along with sequences of the ribosomal internal transcribed spacer (ITS) region (ITS1 + 5.8S + ITS2) from 3 Neopsilotrema species. The molecular phylogenetic affinities of a variety of psilostomid taxa were studied using 28S sequence data. The 28S sequences of psilostomids demonstrated 1-7.9% intergeneric divergence, whereas the sequences of ND1 had 17.7-34.1% intergeneric divergence. The interspecific divergence among members of Neopsilotrema was somewhat lower (0.2-0.5% in 28S; 0.3-0.4% in ITS; 12-15.7% in ND1). Our comparison of DNA sequences along with morphologic study suggests Holarctic distribution of N. lisitsynae.

Acartia arbruta (previously A. tonsa) in British Columbia: a bioindicator of climate-driven ecosystem variability in the northeast Pacific Ocean

Abstract Climate change is driving biogeographic shifts in marine zooplankton. In the northeast (NE) Pacific, the distribution of the estuarine copepod, Acartia tonsa ( Dana, 1849), is generally limited to California except during marine warming events where it is found as far north as British Columbia (BC). Documentation of such events relies on accurate species identification. Phylogenetic reconstruction of the genus Acartia using 768 mitochondrial cytochrome oxidase subunit I Deoxyribonucleic Acid (DNA) sequences collected worldwide demonstrates that NE Pacific A. tonsa is distinct from all conspecifics and congeners. Males of NE Pacific A. tonsa are larger with slight differences in the fifth pair of legs as compared to geographically isolated conspecifics. Accordingly, we propose NE Pacific A. tonsa be reclassified to Acartia arbruta. Analysis of mitochondrial 16S ribosomal RNA DNA sequences from 154 specimens of A. arbruta collected from California, Oregon and BC suggests that A. arbruta detected in BC is a mixture of southern populations. Southern populations are likely driven north during “warm” phases of the oceanographic processes indexed by the Pacific Decadal Oscillation, which is positively correlated with A. arbruta biomass anomalies on the continental shelf of Vancouver Island, BC. The presence of A. arbruta in BC waters is a useful bioindicator of marine warming events.

Molecular identification and subtyping of Blastocystis sp. in hospital patients in Central China

Blastocystis is a common enteric protist that inhabits the gastrointestinal tract of approximately 1 billion people worldwide. In this study, a total of 1,070 patients from two hospitals in Zhengzhou, Central China were enrolled to know molecular characteristics of Blastocystis sp. The microorganism was identified and subtyped with a PCR amplification and sequencing of the small subunit ribosomal deoxyribonucleic acid (SSU-rDNA). The overall minimum prevalence of Blastocystis sp. in participants was 3.1% (33/1070). Although there were no significant differences on Blastocystis sp. infections among study sites, age groups, and gender, the higher infection was observed in the patients with gastrointestinal diseases (8.8%, 15/170). Sequence analysis of the 33 isolates revealed three known subtypes, such as ST1 (n = 7), ST3 (n = 23), and ST7 (n = 3). Among them, ST3 was the dominant subtype being detected in 23 isolates (69.7%), followed by ST1 (21.2%, 7/33) and ST7 (9.1%, 3/33). The phylogenetic analysis demonstrated that three subtypes (ST1, ST3 and ST7) were clustered with their reference sequences with good bootstrap support. The subtype determination of Blastocystis sp. isolates by the phylogenetic analysis was well supported by online platform. The present study provides the first molecular report of Blastocystis sp. infections in hospital patients in Central China.

Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.

There and Back Again from Monotypy: A New Species of the Casque-Headed Corythomantis Boulenger 1896 (Anura, Hylidae) from the Espinhaço Mountain Range, Brazil

The genus Corythomantis was monotypic for over 100 yr, encompassing only the species C. greeningi. In 2012, a second species, C. galeata, was described, but this species was recently reassigned to Nyctimantis, rendering Corythomantis once again monotypic. The geographic distribution of C. greeningi covers the Caatinga and Cerrado biomes from northeast Brazil, with a western limit in Tocantins state and a southern limit in Minas Gerais state. Here we demonstrate the existence of a second species of Corythomantis through molecular, acoustic, and morphological data. The new species differs in morphology from C. greeningi in head shape and tibia coloration. The new species has an advertisement call with shorter duration, higher pulse rate, and different acoustic structure. Mitochondrial deoxyribonucleic acid (DNA) data show a genetic divergence from C. greeningi of 2.88% in the 16S ribosomal DNA gene and 14.06% in the cytochrome oxidase I gene. The geographic distribution of the new species is restricted to the Espinhaco Mountain Range at elevations from 315 to 930 m above sea level.