DNA Sequence Of Gene Research Articles

A new genus of terraranas (Anura: Brachycephaloidea) from northern South America, with a systematic review of Tachiramantis

Since the systematics of Terrarana frogs was overhauled in 2008, five new genera have been named, including Tachiramantis from the Venezuelan Coastal Range and adjacent parts of the Cordillera Oriental of Colombia and the Sierra de Perijá along the Venezuela–Colombia border. The discovery of Tachiramantis raises questions about the relationships of several species of Pristimantis in the nearby Sierra Nevada de Santa Marta previously hypothesized to be closely related to species now referred to Tachiramantis. To test the monophyly of Tachiramantis and the relationships among its species, we generated DNA sequences for 42 individuals, and, given the variable placement of Tachiramantis in previous studies, analysed them with DNA sequences from GenBank representing 25 genera of terraranas. In total, the final matrix included DNA sequences from 414 terminals, which we analysed using tree-alignment under the parsimony optimality criterion. To identify morphological synapomorphies and diagnostic characters, we also examined cranial osteology and axial skeleton morphology. Our analyses corroborated both the placement of Tachiramantis far from Pristimantis in Craugastoridae and the monophyly of Tachiramantis. We also found that six species currently referred to Pristimantis, all endemic to the Sierra Nevada de Santa Marta, comprise the sister clade of Tachiramantis. This highly endemic clade is both well-supported by molecular data and diagnosed from Tachiramantis by seven morphological synapomorphies, leading us to recognize it as a new genus. http://zoobank.org/urn:lsid:zoobank.org:act:0036039F-F400-4CD4-A6AD-D3DD2B34BA4E

Genetic Diversity of Epichloë Endophytes Associated with Brachypodium and Calamagrostis Host Grass Genera including Two New Species.

Fungi of genus Epichloë (Ascomycota, Clavicipitaceae) are common endophytic symbionts of Poaceae, including wild and agronomically important cool-season grass species (subfam. Poöideae). Here, we examined the genetic diversity of Epichloë from three European species of Brachypodium (B. sylvaticum, B. pinnatum and B. phoenicoides) and three species of Calamagrostis (C. arundinacea, C. purpurea and C. villosa), using DNA sequences of tubB and tefA genes. In addition, microsatellite markers were obtained from a larger set of isolates from B. sylvaticum sampled across Europe. Based on phylogenetic analyses the isolates from Brachypodium hosts were placed in three different subclades within the Epichloë typhina complex (ETC) but did not strictly group according to host grass species, suggesting that the host does not always select for particular endophyte genotypes. Analysis of microsatellite markers confirmed the presence of genetically distinct lineages of Epichloë sylvatica on B. sylvaticum, which appeared to be tied to different modes of reproduction (sexual or asexual). Among isolates from Calamagrostis hosts, two subclades were detected which were placed outside ETC. These endophyte lineages are recognized as distinct species for which we propose the names E. calamagrostidis Leuchtm. & Schardl, sp. nov. and E. ftanensis Leuchtm. & A.D. Treindl, sp. nov. This study extends knowledge of the phylogeny and evolutionary diversification of Epichloë endophytes that are symbionts of wild Brachypodium and Calamagrostis host grasses.

Increased Soil Bacterial Abundance but Decreased Bacterial Diversity and Shifted Bacterial Community Composition Following Secondary Succession of Old-Field

Plant secondary succession is a very effective approach for the rejuvenation of degraded ecosystems. In order to comprehend alterations and driving mechanisms of soil bacterial communities under secondary succession of old-field and reveal their subsequent impacts on the decomposition and accumulation of soil organic carbon (SOC) and nitrogen (SON), we investigated changes in soil bacterial communities following ~160 years of old-field succession on the Loess Plateau of China through analyses of quantitative polymerase chain reaction (qPCR) and Illumina MiSeq DNA sequencing of 16S rRNA genes. Our results revealed that subsequent to secondary succession of old-field, soil bacterial abundance progressively increased, while bacterial richness and diversity significantly decreased. Principal component analysis and Bray–Curtis similarity index showed that bacterial community composition gradually shifted following old-field succession. Specifically, the relative abundances of Proteobacteria, Rokubacteria, and Verrucomicrobia progressively increased, while Actinobacteria and Firmicutes slightly decreased following old-field succession. The most enriched of Proteobacteria (e.g., Rhizobiales, Xanthobacteraceae, Gammaproteobacteria, Bradyrhizobium, Rhizobiaceae, and Mesorhizobiur) were found in a climax forest, while Chloroflexi and Gemmatimonadetes had the lowest relative abundances. Further, the most enriched members of Actinobacteria, including Geodermatophilaceae, Frankiales, Blastococcus, Micrococcales, Micrococcacea, Propionibacteriales, Nocardioidaceae, Nocardioide, and Streptomycetaceae, were exhibited in the farmland stage. Our results suggested that secondary succession of old-field greatly modified soil bacterial communities via the transformation of soil nutrients levels, altering plant biomass and soil physiochemical properties. Soil bacterial community composition was transformed from oligotrophic groups to copiotrophic Proteobacteria following old-field succession, which may promote SOC and SON accumulation through increasing the utilization of labile organic carbon (C) and nitrogen (N), while decreasing decomposition of recalcitrant organic C and N from the early- to late-successional stages.

Identifying proteomic profiles as indicators of disease severity in arrhythmogenic cardiomyopathy

Abstract Introduction Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease characterized by progressive fibrofatty replacement of the myocardium and ventricular arrhythmias. Biventricular (BiV) involvement may lead to heart failure. This study aimed to investigate characteristic proteomic patterns in plasma of ACM patients, and correlated them with clinical outcome as well as physical exercise, to assess if key soluble molecules may serve as specific biomarkers for ACM, and whether mechanical stress induced by physical exercise may alter proteomic patterns in ACM patients. Methods In 38 ACM patients clinical parameters and major adverse cardiovascular events (MACE defined as presence of sustained ventricular tachycardia, ventricular fibrillation, appropriate therapy from implantable cardioverter defibrillator, sudden cardiac death, death related to end-stage heart failure or cardiac transplant) were obtained prospectively during a mean follow-up period of 36 months. All patients received genetic testing using next generation DNA sequencing. Plasma protein expression was analysed using the Proximity Extension Assay (PEA) technology, where a pair of oligonucleotide-labelled antibody probe binds to each targeted protein. In a subgroup of 11 patients blood was drawn immediately before and 3 hours after standardised bicycle exercise testing, and plasma protein expression was compared. Results 12 patients had ACM with BiV involvement, and 26 patients had isolated right ventricular (RV) involvement. During the follow-up period, 34 patients had a MACE (30% with RV and 14% with BiV). Over 360 proteins were assessed in all ACM patients and compared to 24 healthy controls. The proteomic signature of ACM patients differed significantly compared to controls, and 32 proteins were upregulated in ACM (Figure 1). The proteomic profiles of patients with RV involvement also differed from those with BiV involvement. Most importantly, after exercise, over 40 proteins were upregulated specifically in ACM patients compared to controls, including key pro-inflammatory, adipogenic molecules and also markers of cardiac fibrosis. Conclusion Our study shows that ACM patients with RV and BiV involvement have different plasma proteomic profiles compared to healthy controls. Furthermore we were able to demonstrate that, specifically in ACM patients, several pro-inflammatory pathways are upregulated after exercise compared to healthy controls, further elucidating the molecular pathways associated with arrhythmogenicity and disease progression and highlighting the key role of physical stress. Our results may enable the identification of potential future biomarkers for diagnosis and risk stratification and may pave the way for personalized patient specific treatments. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Baugarten Foundation ZurichSwiss National Foundation

The Intratumoral Bacterial Metataxonomic Signature of Hepatocellular Carcinoma.

ABSTRACTMicrobiota is implicated in hepatocellular carcinoma (HCC). The spectrum of intratumoral microbiota associated with HCC progression remains elusive. Fluorescence in situ hybridization revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. Viable anaerobic or aerobic bacteria were recovered in HCC tissues by fresh tissue culture. We performed a comprehensive DNA sequencing of bacterial 16S rRNA genes in 156 samples from 28 normal liver, 64 peritumoral, and 64 HCC tissues, and the DNA sequencing yielded 4.2 million high-quality reads. Both alpha and beta diversity in peritumor and HCC microbiota were increased compared to normal controls. The most predominant phyla in HCC were Patescibacteria, Proteobacteria, Bacteroidota, Firmicutes, and Actinobacteriota. phyla of Proteobacteria, Firmicutes, and Actinobacteriota, and classes of Bacilli and Actinobacteria, were consistently enriched in peritumor and HCC tissues, while Gammaproteobacteria was especially abundant in HCC tissues compared to normal controls. Streptococcaceae and Lactococcus were the marker taxa of HCC cirrhosis. The Staphylococcus branch and Caulobacter branch were selectively enriched in HBV-negative HCCs. The abundance of Proteobacteria, Gammaproteobacteria, Firmicutes, Actinobacteriota, and Saccharimonadia were associated with the clinicopathological features of HCC patients. The inferred functions of different taxa were changed between the microbiota of normal liver and peritumor/HCC. Random forest machine learning achieved great discriminative performance in HCC prediction (area under the curve [AUC] = 1.00 in the training cohort, AUC = 0.950 for top five class signature, and AUC = 0.943 for the top 50 operational taxonomy units [OTUs] in the validation cohort). Our analysis highlights the complexity and diversity of the liver and HCC microbiota and established a specific intratumoral microbial signature for the potential prediction of HCC.IMPORTANCE Gut microbiome is an important regulator of hepatic inflammation, detoxification, and immunity, and contributes to the carcinogenesis of liver cancer. Intratumoral bacteria are supposed to be closer to the tumor cells, forming a microenvironment that may be relevant to the pathological process of hepatocellular carcinoma (HCC). However, the presence of viable intratumoral bacteria remains unclear. It is worth exploring whether the metataxonomic characteristics of intratumoral bacteria can be used as a potential marker for HCC prediction. Here, we present the first evidence of the existence of viable intratumoral bacteria in HCC using the tissue culture method. We revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. We analyzed the diversity, structure, and abundance of normal liver and HCC microbiota. We built a machine learning model for HCC prediction using intratumoral bacterial features. We show that specific taxa represent potential targets for both therapeutic and diagnostic interventions.

Difference in Intestinal Flora and Characteristics of Plasma Metabonomics in Pneumoconiosis Patients.

From the two perspectives of intestinal flora and plasma metabolomics, the mechanism of occurrence and development of pneumoconiosis was explored to provide a new target for the prevention and treatment of pneumoconiosis. In this study, 16S ribosome DNA (16SrDNA) gene sequencing technology was used to analyze the differences in intestinal flora of each research group through operational taxonomic units (OUT) analysis, cluster analysis, principal component analysis (PCA), partial least square discriminant analysis (PLS-DA), Kyoto Encyclopedia of Genes and Genomes (KEGG), and other analytical methods were used to analyze the differences in plasma metabolites between the study groups. Metabonomics analysis showed that the plasma metabolites of pneumoconiosis patients were significantly different from those of normal people. Fold change > 2; vip > 1; p < 0.05 were the screening criteria. In the positive and negative mode, we screened ten types of differential metabolites. These ten metabolites were upregulated to varying degrees in the pneumoconiosis patients. Seven metabolic pathways were obtained by analyzing the metabolic pathways of different metabolites. Among them, the aminoacyl tRNA biosynthesis pathway changed most obviously. The α diversity of two groups of intestinal flora was analyzed using the 16SrDNA technique. The results showed that there was no significant difference in ACE, Chao1, Shannon, or Simpson in the two groups (p > 0.05). Beta diversity analysis showed that there were differences in microbial communities. In pneumoconiosis patients, the abundance of Prevotellaceae increased, and the other nine species decreased. Compared to the control group, the abundance of Prevotellaceae in the intestinal flora of pneumoconiosis increased, and the abundance of the other nine species decreased. Compared to controls, ten substances in the plasma metabolites of pneumoconiosis patients were upregulated. Seven metabolic pathways were obtained by analyzing the metabolic pathways of different metabolites. Among them, the aminoacyl tRNA biosynthesis pathway changed most significantly. This provided a theoretical basis for further study on the pathogenesis, early prevention, and treatment of pneumoconiosis.

Follow-up of intraocular retinoblastoma through the quantitative analysis of conserved nuclear DNA sequences in aqueous humor from patients.

Fundoscopy is the standard method for diagnosis and follow-up of intraocular retinoblastoma, but it is sometimes insufficient to discern whether tumors are inactivated following treatments. In this work, we hypothesized that the amount of conserved nuclear DNA sequences in the cell-free DNA (cfDNA) fraction of the aqueous humor (AH) might complement fundoscopy for retinoblastoma follow-up. To address our hypothesis, we developed highly sensitive droplet digital polymerase chain reaction (ddPCR) methods to quantify highly conserved DNA sequences of nucleus-encoded genes (GAPDH and B4GALNT1) and of a mitochondrial gene, MT-ATP6. We obtained AH samples during intravitreal treatments. We analyzed 42 AH samples from 25 patients with intraocular retinoblastoma and 11 AH from controls (non-cancer patients). According to clinical criteria, we grouped patients as having progression-free or progressive retinoblastoma. cfDNA concentration in the AH was similar in both retinoblastoma groups. Copy counts for nucleus-derived sequences of GAPDH and B4GALNT1 were significantly higher in the AH from patients with progressive disease, compared to the AH from progression-free patients and control non-cancer patients. The presence of mitochondrial DNA in the AH explained that both retinoblastoma groups had similar cfDNA concentration in AH. The optimal cut-off point for discriminating between progressive and progression-free retinoblastomas was 108 GAPDH copies per reaction. Among patients having serial AH samples analyzed during their intravitreal chemotherapy, GAPDH copies were high and decreased below the cut-off point in those patients responding to chemotherapy. In contrast, one non-responder patient remained with values above the cut-off during follow-up, until enucleation. We conclude that the measurement of conserved nuclear gene sequences in AH allows follow-up of intraocular retinoblastoma during intravitreal treatment. The method is applicable to all patients and could be relevant for those in which fundoscopy evaluation is inconclusive.

P487 Basidiobolus omanensis, an emerging pathogen to watch out for?

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Introduction Basidiobolus species can be found in amphibian, reptile, lizard, insectivorous bat, and soil, as well as decaying vegetables, and fruits. Basidiobolus species infect both adults and children, with the majority of cases reported from the Middle East, including Oman. Basidiobolomycosis is a chronic subcutaneous infection of the trunk and limbs that typically manifests as subcutaneous or gastrointestinal lesions with only a few cases of systemic involvement. Basidiobolus haptosporus, B. heterosporus, B. magnus, B. meristosporus, B. microsporus, B. omanensis and B. ranarum are the seven species in the genus Basidiobolus. Four of these species have been linked to gastrointestinal infections in humans. Basidiobolus ranarum is the most commonly reported species from humans, followed by B. omanensis. We recently described B. omanensis as a novel species from a patient in Oman. It was isolated from a boy with type 1 diabetes who died as a result of basidiobolmycosis complications. Four more fatal human cases have been documented since then (unpublished data), but little is known about its role as a pathogen in humans.ObjectiveThe goal of this paper is to present four cases of basidiobolomycosis caused by B. omanensis in young children and adults, each with unique diagnostic and treatment challenges.MethodsWe collected four cases of basidiobolomycosis caused by B. omanensis from various tertiary hospitals in Oman. All identifications were based on ribosomal DNA gene sequencing of the internal transcribed spacers (ITS) and partial large subunit (LSU). The CLSI method was used for testing the antifungal drug susceptibility in vitro.ResultsWe present four new cases of Basidiobolus infection in humans (two from children and two from adults). The underlying condition, site of infection, clinical presentation, treatment, and outcomes are all summarized in Table 1. Basidiobolus omanensis strains isolated from these cases were molecularly identified using (ITS and LSU) gene sequencing. The minimum inhibitory concentration (MIC) for amphotericin B (AMB) is 1 µg/ml, isavuconazole (ISC) is 4 µg/ml, itraconazole (ITC), posaconazole (PSC), and voriconazole (VRC) is > 16 µg/ml respectively. These were the first cases of the fungus B. omanensis ever diagnosed globally at various hospitals in Oman. All of the cases presented diagnostic and treatment challenges. Despite the atypical nature of the disease, clinical response was obtained following surgical excision and antifungal treatment, and the mortality rate among the four cases was 50%.ConclusionThis new species may cause problems with identification and antifungal susceptibility, which is why clinicians should be aware of infections with uncommon mould species and consider DNA sequencing for confirmation. Any fungus can cause infection in an immunocompromised host and should never be dismissed as a contaminant out of hand.

High-throughput sequencing revealed low-efficacy genome editing using Cas9 RNPs electroporation and single-celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis.

High-throughput sequencing revealed low-efficacy genome editing using Cas9 RNPs electroporation and single-celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis.

Molecular biology analysis of ABO blood group variants caused by natural chimaerism.

The chimaerism phenomenon constitutes a significant mechanism underlying ABO phenotype discrepancies; however, its detection has technical challenges. In the current study, we explored different techniques to establish the chimaeric status of ABO blood types. Fifteen individuals with possible chimaeric ABO blood type, as suggested by standard tube or column agglutination method and RBC adsorption-elution test, were enrolled in the study. The red blood cells from 11 investigated subjects showed mix-field agglutination with anti-A or anti-B in blood typing; weak A or B antigens on the other four individuals' RBCs were detected by adsorption-elution tests. The genetic study was conducted with PCR-SSP genotype, DNA sequencing of the ABO gene, STR analysis and ddPCR. The genetic chimaeric status was confirmed in four (27%) individuals by SSP test alone. The ABO gene sequencing identified an additional ABO allele and enabled chimaerism detection in 10 (67%) subjects. The STR analyses established the chimaerism status in 13 (87%) individuals. In the two cases where neither of the tests mentioned above had positive findings, the ddPCR was adopted, and microchimaerism, with an extremely low degree of chimaerism (0.77% and 0.12%), was revealed. The ddPCR revealed the unequal haplotypes (29.5% B vs. 70.5% O) in one subject and distinguished this B/O-O/O chimaera from certain B subgroups (B/O genotype without any mutation) like B3 . The ABO blood type chimaerism can be genetically established by comprehensive molecular methods, including PCR-SSP/DNA sequencing, STR and ddPCR, which is particularly sensitive for the detection of microchimaerism.

Association of HLA-DRB1*15:02:01, DQB1*05:01:24 and DPB1*13:01:01 in Thai patients with systemic sclerosis.

HLA studies in patients with systemic sclerosis (SSc) have shown variable results. This study aimed to examine the association of HLA class I and II risk alleles in Thai SSc patients, and clarify the contribution of risk HLA alleles to the pathogenesis and clinical manifestations. Blood samples from 92 SSc patients and 135 healthy controls (HCs) were collected. Eleven loci of the HLA class I (HLA-A, B, and C) and class II (HLA-DR, DP, and DQ) genes were determined by a 3-field (6-digit) analysis using the Next Generation DNA Sequencing (NGS) method. Anti-topoisomerase-I antibodies (ATA) and anti-centromere antibodies (ACA) were identified by ELISA methods. Allele frequencies (AFs) of HLA-DRB1*15:02:01, DRB5*01:02:01, DQB1*05:01:24, DPB1*13:01:01, and DQA1*01:01:01 were increased significantly in the whole SSc and SSc patients with positive ATA, but with negative ACA (SSc/ATA+/ACA-). Of these, DPB1*13:01:01 was the most susceptible allele. The DRB1*15:02:01, DQB1:05:01:24, and DPB1*13:01:01 alleles were estimated to locate on the unique haplotype, and haplotype frequency was estimated to be significantly higher than those in the HCs (p=0.002). The linkage analysis of DRB1*15/16 revealed that most of the DRB1*15:02:01 alleles were linked to DRB5*01:02:01 or DRB5*01:08:01N. The linkage of DRB1*16:02:01 to DRB5*01:01:01 was observed frequently. The associations of risk alleles with several SSc clinical features were observed. HLA-DRB1*15:02:01, DRB5*01:02:01, DQB1*05:01:24, and DPB1*13:01:01 on the unique haplotype were associated with the pathogenesis and clinical features of SSc in Thai patients. The linkage of DRB1*15:02:01 to DRB5*01:08:01N was observed commonly in northern Thai patients.

Three novel homozygous ITGB2 mutations among two patients with leukocyte adhesion defect type-1: Two case reports

BACKGROUNDA leukocyte adhesion defect (LAD) is a rare primary immunodeficiency disorder. LAD type 1 (LAD-1) is the most common, which is caused by ITGB2 mutation resulting in dysfunction of β2 integrin, which impairs leukocyte adherence to the endothelium.CASE SUMMARYThe first two cases of LAD-1 in Thailand presented with recurrent omphalitis, soft tissue infection, marked leukocytosis, and neutrophilia. One patient experienced delayed umbilical cord separation. Mutation analysis was performed by direct DNA sequencing of the ITGB2 gene. The results revealed two novel homozygous missense mutations, c.920C>T (p.Leu307Pro) in exon 8 and c.758G>A (p.Arg253His) in exon 7, and one novel homozygous nonsense mutation, c.262C>T (p.Gln88Ter) in exon 4, in the genomic DNA of the first and second patients, respectively. Heterozygous mutations were identified in the parents of both patients, suggesting a carrier status. The patients were administered intravenous antibiotics for infections with good clinical responses. Hematopoietic stem cell transplantation could not be performed due to the unavailability of matched donors. However, a significant decline in infections was observed after antibiotic prophylaxis. Several follow-up visits were conducted for both patients. They are currently 6 years old.CONCLUSIONMolecular analysis is essential for definitive diagnosis, early treatment implementation, and prevention of LAD-1 in future pregnancy.

Study of Biodeterioration Potential of Microorganisms Isolated in the Paintings Storeroom of Mouze Makhsus Museum, Golestan Palace, Tehran

ABSTRACT Paintings are essential components of cultural heritage all over the world. They consist of organic molecules such as oils, waxes, gums, sugars, polysaccharides, and proteins that can support the growth of various microorganisms. In this study, in order to identify the microbial community possibly involved in deterioration of paintings in the storeroom of Mouze Makhsus, culturable bacteria and fungi colonizing the paintings were isolated. The identification of isolated bacteria and fungi was performed through morphological analyses and either PCR amplification and DNA sequencing of 16srRNA gene, for bacteria, and ITS genomic region, for fungi. Based on the obtained 16S rRNA gene sequence of bacteria and ITS region sequence of fungi, a phylogenetic tree was constructed using the Neighbor-Joining method. The study also evaluated, in vitro, the cellulolytic activity of the bacterial and fungal isolates. The results highlighted the diverse array of microbial groups colonizing the sampled paintings. Culture-based analyses revealed 27 isolates of bacteria and fungi. Detected fungi fell in seven genera including Aspergillus, Penicillium, Cladosporium, Alternaria, Curvularia, Chaetomium, and Trametes. The majority of isolated bacteria belong to Bacillus genus, whereas to a lesser extent Staphylococcus, Micrococcus, Paenibacillus, Arthrobacter, Heyndrickxia, Priestia, and Rathayibacter were isolated. Qualitative and quantitative assessment of cellulolytic enzyme production showed that the fungal isolates present important cellulolytic activity, and they are potentially dangerous to the paintings.

Livoneca redmanii Leach, 1818 (Cymothoidae) a parasitic isopod infesting the gills of the European seabass, Dicentrarchus labrax (Linnaeus, 1758): morphological and molecular characterization study

BackgroundPrevalence, morphology, and molecular characteristics of isopodiosis in the European seabass, Dicentrarchus labrax, in Egypt were assessed using light and electron microscopy and polymerase chain reaction targeting the mitochondrial COI (cytochrome oxidase c) gene.ResultsAdult parasites were found mainly in the branchial cavity between gill arches and to a lesser extent in the buccal cavity. They were morphologically identified as the Cymothoidae Livoneca redmanii Leach, 1818 (Crustacea: Isopoda). Obviously, a 23% prevalence rate of isopods was reported in D. labrax from Egyptian Mediterranean waters. Destructive and degenerative necrotic alterations with complete sloughing of gill lamellae of the infested fish were observed. DNA sequencing of the mitochondrial COI gene confirmed the identification of the parasite which was deposited in the GenBank under accession numbers MW600099, MZ208984, and MZ208985. Furthermore, the phylogenetic analysis demonstrated that parasites emerged from a monophyletic clade closely affiliated with L. redmanii and were clearly distinguished from other isopod genospecies.ConclusionThe present investigation addresses L. redmanii infestation in D. labrax in Egypt and affirmed morphological properties via the scanning electron microscopy (SEM) and molecular characteristics of this isopod species. The drastic effects of this parasite on the infected fish were proven both clinically and histopathologically.

Molecular epidemiology characteristics and detecting transmission of carbapenemase-producing enterobacterales in southwestern China

BackgroundTo investigate the genotype and clinical characteristics of carbapenem-resistant Enterobacteriaceae (CRE) strains in southwest China and provide information on the treatment stopping the spread of the infection. MethodsThe clinical information of CRE isolates was collected from 19 hospitals in 12 cities across Sichuan Province, China, between June 2018 and April 2019. The isolates were detected by DNA sequencing of genes encoding carbapenem enzymes and multilocus sequence types (MLSTs). ResultsA total of 166 nonrepetitive CRE isolates were isolated during the study period from sputum, blood, urine, and other samples. Klebsiella pneumoniae carbapenemase (KPC) was dominant in Klebsiella pneumoniae (53.9%), followed by New Delhi metallo-β-lactamase (NDM) (42.1%). A total of 43 STs were detected. The most common ST of K. pneumoniae was ST11, and that of Escherichia coli was ST410. Pairwise single nucleotide polymorphism (SNP) distances and the likelihood of local transmission by epidemiology were plotted for each species. About 65% of these pairs had ≤ 20 pairwise SNPs. ConclusionA large number of CRE strains carried carbapenemase. Although NDM-ST12 K. pneumoniae should not be disregarded, KPC-ST11is the predominant strain. Thus, the possibility of transmission between E. coli and K. pneumoniae could not be ignored.

Discovery of Aedes (Ochlerotatus) pionips Dyar, 1919 (Diptera, Culicidae) in Germany

A group of black-legged mosquito species within the subgenus Ochlerotatus Lynch Arribalzaga, 1891 of the genus Aedes Meigen, 1818 is difficult to identify based on their morphology. This group includes Aedes pionips Dyar, 1919, whose first record in the Erzgebirge (Germany) is reported in this study. The objective was to compile and add species specific characters for the morphological discrimination of Ae. pionips from similar mosquitoes in order to facilitate species identification. Generated cytochrome c oxidase subunit I gene DNA sequences of Ae. pionips from Germany were compared to sequences from related species, using a phylogenetic tree.

The value of metagenomic next-generation sequencing in bronchoalveolar lavage fluid samples of anti-MDA5-positive dermatomyositis patients.

Dear Editor, Anti-melanoma differentiation-associated gene 5 antibody-positive DM (anti-MDA5-positive DM) is a special subtype of myositis often combined with rapidly progressive interstitial lung disease, which has received increasing attention due to its poor prognosis and high mortality. A recent study reported a close relationship between anti-MDA5-positive DM and infection [1]. However, real-world clinical information is limited in those patients. Metagenomic next-generation sequencing (mNGS) is a sensitive detection technology that combines high-throughput sequencing with bioinformatic analysis for investigating an entire pathogen genome through RNA or DNA gene sequencing in a given sample. We retrospectively collected the data on 22 anti-MDA5-positive DM patients who completed mNGS testing with bronchoalveolar lavage fluid (BALF). Demographic data, including sex, age, detected pathogens, CD4 T cell counts and treatments, are shown in Table 1. Twenty-two patients were divided into two groups. In group 1, 10 patients underwent BALF mNGS testing prior to any treatment. Of these, four patients had bacterial infection, six had Pneumocystis jirovecii infection, four had viral infection and one had parasitic infection. In group 2, 12 patients showed aggravated dyspnoea symptoms after received glucocorticoids and immunosuppressants, underwent chest CT examination, and showed newly appearing pulmonary infiltrates compared with baseline. The BALF mNGS testing demonstrated six patients with bacterial infection. Of the seven patients with fungal infection, five were infected with P. jirovecii (71.4%). Eight patients had viral infection, with human betaherpesvirus 5 (cytomegalovirus, CMV) and human gammaherpesvirus 4 (Epstein–Barr virus; EBV) showing the highest detection rate (CMV: 87.5% and EBV: 50%). Finally, 11 patients had P. jirovecii infection, and for them the level of CD4 T cells was 283.1 (50.1) cells/µl, which was significantly lower than the level (452.5 (140.6) cells/µl) in patients without the P. jirovecii infection (P < 0.01, independent-sample t-test).

Evaluation of MALDI Biotyper Mycobacteria Library for Identification of Nontuberculous Mycobacteria.

The Bruker Biotyper matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) platform was assessed on its ability to accurately identify 314 nontuberculous mycobacteria (NTM) representing 73 species. All NTM isolates, representing 183 rapidly growing and 131 slowly growing organisms, were previously identified by Sanger DNA sequencing of the full-length 16S rRNA gene, and region V of the rpoB gene. An optimized version of the Bruker bead-beating procedure for protein extraction of NTM isolates was used to ensure high quality spectra for all NTM isolates, including less frequently encountered species. NTM spectra were analyzed using Bruker's research use only, Mycobacteria Library v6.0, supplemented by the MicrobeNet database. Identification of NTM by MALDI-TOF had an accuracy of 94% (296/314). The identification accuracy for rapidly growing mycobacteria was higher at 99% (182/183) than it was for slowly growing mycobacteria at 87% (114/131). While MALDI-TOF performed well against Sanger sequencing of the 16S rRNA gene alone, there were 11 species that required additional sequencing of rpoB. Most discrepancies between MALDI-TOF and sequencing results are likely due to underrepresentation of some species in the libraries used. Overall, the results of this study support Bruker's MALDI-TOF platform as an accurate and reliable method for the identification of NTM.

Morphological and molecular characterization of Lernanthropus kroyeri, a copepod infesting the gills of European seabass Dicentrarchus labrax

The European seabass Dicentrarchus labrax, Europe's first commercially farmed non-salmonid fish and the most farmed Mediterranean fish, with Egypt as a major producer, is susceptible to several diseases, one of which being copepod infestation. Lernanthropus kroyeri (Beneden 1851) is a parasitic copepod that can be found across the Mediterranean Sea. Little research on L. kroyeri has been done along the Mediterranean Sea off the Egyptian coasts. In the current investigation, gill filaments, particularly those of the second-gill arch, were found to be infested with parasites with a prevalence rate of 23%. They were morphologically identical and confirmed as members of the family Lernanthropidae, having the characteristic features of L. kroyeri. Scanning electron microscopy (SEM) was used to examine morphological traits. Clinical and histological evidence of the parasite's catastrophic impact on infected fish has been described. DNA sequencing of the parasite's mitochondrial COI gene proved its identity, and it was the first of L. kroyeri to be added to the GenBank database, with the accession number MZ726776. The phylogenetic analysis revealed that the parasites arose from a monophyletic group that is distinct from other Lernanthropidae family members.

An insertion mutation in the &lt;i&gt;Apoe&lt;/i&gt; gene associated with spontaneous hyperlipidemia in mice

IntroductionSpontaneously hyperlipidemic (SHL) mice, a mouse strain derived from an inbred strain of Japanese wild (original)-type mice (KOR; Mus musculus molossinus), show high plasma cholesterol concentrations with disruption of the apolipoprotein E (Apoe) gene. However, the details of the Apoe gene of SHL mice have yet to be described.Material and methodsThe DNA sequence of the Apoe gene of SHL mice was compared to that of control KOR mice in genomic DNA and cDNA analyses.ResultsIn the DNA analysis, a 4700-bp fragment was found to be inserted into exon 4 of the Apoe gene of SHL mice. The insertion contained two 365-bp repeats at each terminal and was flanked by a 6-bp target duplication at each side. The inserted fragment produced a frameshift of an early stop codon, resulting in a protein product that consisted of 87 amino acids in SHL mice compared to 311 amino acids in control KOR mice.ConclusionsThese findings provide useful information about the molecular basis of SHL mice and related lipid disorders.