Shotgun metagenomic analysis of microbial communities from the Loxahatchee nature preserve in the Florida Everglades.

BackgroundCurrently, much is unknown about the taxonomic diversity and the mechanisms of methane metabolism in the Florida Everglades ecosystem. The Loxahatchee National Wildlife Refuge is a section of the Florida Everglades that is almost entirely unstudied in regard to taxonomic profiling. This short report analyzes the metagenome of soil samples from this Refuge to investigate the predominant taxa, as well as the abundance of genes involved in environmentally significant metabolic pathways related to methane production (nitrogen fixation and dissimilatory sulfite reduction).MethodsShotgun metagenomic sequencing using the Illumina platform was performed on 17 soil samples from four different sites within the Loxahatchee National Wildlife Refuge, and underwent quality control, assembly, and annotation. The soil from each sample was tested for water content and concentrations of organic carbon and nitrogen.ResultsThe three most common phyla of bacteria for every site were Actinobacteria, Acidobacteria, and Proteobacteria; however, there was variation in relative phylum composition. The most common phylum of Archaea was Euryarchaeota for all sites. Alpha and beta diversity analyses indicated significant congruity in taxonomic diversity in most samples from Sites 1, 3, and 4 and negligible congruity between Site 2 and the other sites. Shotgun metagenomic sequencing revealed the presence of biogeochemical biomarkers of particular interest (e.g., mrcA, nifH, and dsrB) within the samples. The normalized abundances of mcrA, nifH, and dsrB exhibited a positive correlation with nitrogen concentration and water content, and a negative correlation with organic carbon concentration.ConclusionThis Everglades soil metagenomic study allowed examination of wetlands biological processes and showed expected correlations between measured organic constituents and prokaryotic gene frequency. Additionally, the taxonomic profile generated gives a basis for the diversity of prokaryotic microbial life throughout the Everglades

The Value of Preoperative and Intraoperative Ultrasound in the Localization of Intrathyroidal Parathyroid Adenomas

Aim Intrathyroidal parathyroid adenoma (IPA) is rare and may easily be mistaken for thyroid nodule in ultrasonography. The aim of this study was to investigate the characteristic features of IPA and explore the value of preoperative and intraoperative ultrasound in the diagnosis and localization of IPA. Methods 13 of 216 patients who were found to have intrathyroidal parathyroid lesions underwent parathyroidectomy in our hospital because of PHPT. According to the relationship between parathyroid adenoma and thyroid gland, parathyroid adenoma was divided into extra-thyroid type or intra-thyroid type (partial or complete) and the results were compared with surgical and histopathological reports as gold standard. The sonographic features of intrathyroidal parathyroid lesions were analyzed retrospectively. Results A total of 12 intrathyroidal lesions showed profoundly hypoechoic solid nodules with well-defined border, abundant blood flow and polar feeding vessels originating from the superior or inferior thyroid artery (92.3%, 12/13). These nodules were finally confirmed as IPA (or IPAC) after surgery. Polar feeding vessel was not detected in one case of parathyroid hyperplasia confirmed by pathology (7.7%, 1/13). 12 cases were diagnosed and localized on ultrasonography before operation and 10 cases were localized on Tc-99m MIBI SPECT/CT. Conclusions The color Doppler ultrasound findings of IPA were confirmed as profoundly hypoechoic nodules with clear boundary and abundant internal blood flow. The presence of polar feeding vessels which originate from thyroid artery were identified as characteristic features of US for IPA. Preoperative and intraoperative ultrasound could be helpful in the localization and treatment of intrathyroidal parathyroid diseases

Dietary supplementation of N-carbamylglutamate promotes growth performance by modulating the homeostasis of gut microbiota in tilapia (Oreochromis niloticus)

N-carbamylglutamate (NCG) supplementation promotes the synthesis of arginine, improves physiological parameters and modulates gut function in fish. However, its beneficial effect on the fish gut microbiota remains unclear. Thus, we investigated the beneficial effect of NCG on homeostasis of gut microbiota in tilapia. Tilapia were fed with diets supplemented with different levels of dietary NCG (Ctrl, NCGI and NCGII) for 8 weeks. The weight gain and the specific growth rate were significantly higher (p < 0.05) in NCG-supplemented groups compared to the group Ctrl. Moreover, the V3-V4 region of 16S rDNA was sequenced, and the bioinformatic analysis suggested that dietary NCG supplementation had a limited impact on the α-diversity of gut microbiota, while Non-metric multidimensional scaling analysis (NMDS) showed that dietary NCG leads to a clear separation on gut microbiota among the three groups. Furthermore, NCG supplementation reshaped the tilapia gut microbiota community, according to co-occurrence analysis and metabolic pathways prediction via PICRUSt analysis, leading to an enrichment of nutritional metabolism related functions, such as amino acid and lipid metabolisms (p < 0.05). Subsequently, the redundancy analysis (RDA) demonstrated that the structural changes of gut microbiota were determined by NCG concentration. In addition, the linear discriminant analysis effect size (LEfSe) analysis revealed 65 differentially abundant taxonomic clades were dominant in tilapia supplemented with NCG. The cross-validation and Random Forest model identified 12 key bacterial taxa in response to the NCG supplementation. Among them, four key bacterial taxa played a fundamental role in nitrogen-fixing, which may indicate that NCG increases the amino acid biosynthesis of gut microbiota and contributes to the growth promotion of tilapia

Anemoside B4 Exerts Hypoglycemic Effect by Regulating the Expression of GLUT4 in HFD/STZ Rats

Anemoside B4 (B4) is a saponin that is extracted from Pulsatilla chinensis (Bge.), and Regel exhibited anti-inflammatory, antioxidant, antiviral, and immunomodulatory activities. However, its hypoglycemic activity in diabetes mellitus has not been evaluated. Here, we explored the effect of B4 on hyperglycemia and studied its underlying mechanism of lowering blood glucose based on hyperglycemic rats in vivo and L6 skeletal muscle cells (L6) in vitro. The rats were fed a high-fat diet (HFD) for one month, combined with an intraperitoneal injection of 60 mg/kg streptozotocin (STZ) to construct the animal model, and the drug was administrated for two weeks. Blood glucose was detected and the proteins and mRNA were expressed. Our study showed that B4 significantly diminished fasting blood glucose (FBG) and improved glucose metabolism. In addition, B4 facilitated glucose utilization in L6 cells. B4 could enhance the expression of glucose transporter 4 (GLUT4) in rat skeletal muscle and L6 cells. Mechanistically, B4 elevated the inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. Furthermore, we confirmed the effect of B4 on glucose uptake involved in the enhancement of GLUT4 expression in part due to PI3K/AKT signaling by using a small molecule inhibitor assay and constructing a GLUT4 promoter plasmid. Taken together, our study found that B4 ameliorates hyperglycemia through the PI3K/AKT pathway and promotes GLUT4 initiation, showing a new perspective of B4 as a potential agent against diabetes

Association analysis of PSAP variants in Parkinson's disease patients

We read with interest the recent report by Oji and colleagues who identified three exonic mutations in the prosaposin (PSAP) gene from three families with Parkinson’s disease. In addition, a case control study involving 440 Japanese and 705 patients with sporadic Parkinson’s disease from Taiwan demonstrated that that intronic variants (rs4747203 and rs885828) located in the PSAP saposin D domain increased the risk of Parkinson’s disease (Oji et al., 2020). Since the sample size for the association study was relatively small, and independent replication is a litmus test for the reproducibility of the findings, we investigated for exome and intronic variants in a much larger sample size in our Asian population.National Medical Research Council (NMRC)National Research Foundation (NRF)Accepted versionThe authors thank the National Medical Research Council (STAR award and Parkinson's disease Large Collaborative Grant MOH-OFLCG-002) and National Research Foundation (NRF fellowship: NRF-NRFF2016-03) for their support

Transferrin receptor 1 ablation in satellite cells impedes skeletal muscle regeneration through activation of ferroptosis

Abstract Background Satellite cells (SCs) are critical to skeletal muscle regeneration. Inactivation of SCs is linked to skeletal muscle loss. Transferrin receptor 1 (Tfr1) is associated with muscular dysfunction as muscle‐specific deletion of Tfr1 results in growth retardation, metabolic disorder, and lethality, shedding light on the importance of Tfr1 in muscle physiology. However, its physiological function regarding skeletal muscle ageing and regeneration remains unexplored. Methods RNA sequencing is applied to skeletal muscles of different ages to identify Tfr1 associated to skeletal muscle ageing. Mice with conditional SC ablation of Tfr1 were generated. Between Tfr1SC/WT and Tfr1SC/KO (n = 6–8 mice per group), cardiotoxin was intramuscularly injected, and transverse abdominal muscle was dissected, weighted, and cryosectioned, followed by immunostaining, haematoxylin and eosin staining, and Masson staining. These phenotypical analyses were followed with functional analysis such as flow cytometry, tread mill, Prussian blue staining, and transmission electron microscopy to identify pathological pathways that contribute to regeneration defects. Results By comparing gene expression between young (2 weeks old, n = 3) and aged (80 weeks old, n = 3) mice among four types of muscles, we identified that Tfr1 expression is declined in muscles of aged mice (~80% reduction, P < 0.005), so as to its protein level in SCs of aged mice. From in vivo and ex vivo experiments, Tfr1 deletion in SCs results in an irreversible depletion of SCs (~60% reduction, P < 0.005) and cell‐autonomous defect in SC proliferation and differentiation, leading to skeletal muscle regeneration impairment, followed by labile iron accumulation, lipogenesis, and decreased Gpx4 and Nrf2 protein levels leading to reactive oxygen species scavenger defects. These abnormal phenomena including iron accumulation, activation of unsaturated fatty acid biosynthesis, and lipid peroxidation are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis further exacerbates SC proliferation and skeletal muscle regeneration. Ferrostatin‐1, a ferroptosis inhibitor, could not rescue ferroptosis. However, intramuscular administration of lentivirus‐expressing Tfr1 could partially reduce labile iron accumulation, decrease lipogenesis, and promote skeletal muscle regeneration. Most importantly, declined Tfr1 but increased Slc39a14 protein level on cellular membrane contributes to labile iron accumulation in skeletal muscle of aged rodents (~80 weeks old), leading to activation of ferroptosis in aged skeletal muscle. This is inhibited by ferrostatin‐1 to improve running time (P = 0.0257) and distance (P = 0.0248). Conclusions Satellite cell‐specific deletion of Tfr1 impairs skeletal muscle regeneration with activation of ferroptosis. This phenomenon is recapitulated in skeletal muscle of aged rodents and human sarcopenia. Our study provides mechanistic information for developing novel therapeutic strategies against muscular ageing and diseases

Trans-interaction of risk loci 6p24.1 and 10q11.21 is associated with endothelial damage in coronary artery disease

Background and aims: Single nucleotide polymorphism rs6903956 has been identified as one of the genetic risk factors for coronary artery disease (CAD). However, rs6903956 lies in a non-coding locus on chromosome 6p24.1. We aim to interrogate the molecular basis of 6p24.1 containing rs6903956 risk alleles in endothelial disease biology. Methods and Results: We generated induced pluripotent stem cells (iPSCs) from CAD patients (AA risk genotype at rs6903956) and non-CAD subjects (GG non-risk genotype at rs6903956). CRISPR-Cas9-based deletions (Δ63- 89bp) on 6p24.1, including both rs6903956 and a short tandem repeat variant rs140361069 in linkage disequilibrium, were performed to generate isogenic iPSC-derived endothelial cells. Edited CAD endothelial cells, with removal of ‘A’ risk alleles, exhibited a global transcriptional downregulation of pathways relating to abnormal vascular physiology and activated endothelial processes. A CXC chemokine ligand on chromosome 10q11.21, CXCL12, was uncovered as a potential effector gene in CAD endothelial cells. Underlying this effect was the preferential inter-chromosomal interaction of 6p24.1 risk locus to a weak promoter of CXCL12, confirmed by chromatin conformation capture assays on our iPSC-derived endothelial cells. Functionally, risk genotypes AA/AG at rs6903956 were associated significantly with elevated levels of circulating damaged endothelial cells in CAD patients. Circulating endothelial cells isolated from patients with risk genotypes AA/AG were also found to have 10 folds higher CXCL12 transcript copies/cell than those with non-risk genotype GG. Conclusions: Our study reveals the trans-acting impact of 6p24.1 with another CAD locus on 10q11.21 and is associated with intensified endothelial injury.Ministry of Education (MOE)Nanyang Technological UniversityNational Research Foundation (NRF)Published versionThe National Research Foundation, Singapore (Project Number 370062002) funded the Singapore Coronary Artery Disease Genetics Study (SCADGENS) and genotyping of the participants. The team from Nanyang Technological University Singapore was funded by an Academic Research Fund Tier 1 grant (2018-T1-001-030) from the Ministry of Education, Singapore, Human Frontier Science Program Research Grant (RGY0069/2019), and the Nanyang Assistant Professorship. K.Y. T. is supported by NTU Research Scholarship. H.H.L. is supported by the Institute of Molecular and Cell Biology (IMCB) Scientific Staff Development Award (SSDA) for her part-time Ph.D. A.K.K.T. is supported by IMCB, A*STAR, Precision Medicine and Personalised Therapeutics Joint Research Grant 2019, the 2nd A*STAR-AMED Joint Grant Call 192B9002 and NUHSRO/2021/035/NUSMed/04/NUS-IMCB Joint Lab/LOA

Shotgun metagenomic analysis of microbial communities from the Loxahatchee nature preserve in the Florida Everglades.

BackgroundCurrently, much is unknown about the taxonomic diversity and the mechanisms of methane metabolism in the Florida Everglades ecosystem. The Loxahatchee National Wildlife Refuge is a section of the Florida Everglades that is almost entirely unstudied in regard to taxonomic profiling. This short report analyzes the metagenome of soil samples from this Refuge to investigate the predominant taxa, as well as the abundance of genes involved in environmentally significant metabolic pathways related to methane production (nitrogen fixation and dissimilatory sulfite reduction).MethodsShotgun metagenomic sequencing using the Illumina platform was performed on 17 soil samples from four different sites within the Loxahatchee National Wildlife Refuge, and underwent quality control, assembly, and annotation. The soil from each sample was tested for water content and concentrations of organic carbon and nitrogen.ResultsThe three most common phyla of bacteria for every site were Actinobacteria, Acidobacteria, and Proteobacteria; however, there was variation in relative phylum composition. The most common phylum of Archaea was Euryarchaeota for all sites. Alpha and beta diversity analyses indicated significant congruity in taxonomic diversity in most samples from Sites 1, 3, and 4 and negligible congruity between Site 2 and the other sites. Shotgun metagenomic sequencing revealed the presence of biogeochemical biomarkers of particular interest (e.g., mrcA, nifH, and dsrB) within the samples. The normalized abundances of mcrA, nifH, and dsrB exhibited a positive correlation with nitrogen concentration and water content, and a negative correlation with organic carbon concentration.ConclusionThis Everglades soil metagenomic study allowed examination of wetlands biological processes and showed expected correlations between measured organic constituents and prokaryotic gene frequency. Additionally, the taxonomic profile generated gives a basis for the diversity of prokaryotic microbial life throughout the Everglades