Chemolithoautotrophic diazotrophs dominate dark nitrogen fixation in mangrove sediments.

Diazotrophic microorganisms regulate marine productivity by alleviating nitrogen limitation. So far chemolithoautotrophic bacteria are widely recognized as the principal diazotrophs in oligotrophic marine and terrestrial ecosystems. However, the contribution of chemolithoautotrophs to nitrogen fixation in organic-rich habitats remains unclear. Here, we utilized metagenomic and metatranscriptomic approaches integrated with cultivation assays to investigate the diversity, distribution, and activity of diazotrophs residing in Zhangzhou mangrove sediments. Physicochemical assays show that the studied mangrove sediments are typical carbon-rich, sulfur-rich, nitrogen-limited, and low-redox marine ecosystems. These sediments host a wide phylogenetic variety of nitrogenase genes, including groups I-III and VII-VIII. Unexpectedly diverse chemolithoautotrophic taxa including Campylobacteria, Gammaproteobacteria, Zetaproteobacteria, and Thermodesulfovibrionia are the predominant and active nitrogen fixers in the 0-18 cm sediment layer. In contrast, the 18-20 cm layer is dominated by active diazotrophs from the chemolithoautotrophic taxa Desulfobacterota and Halobacteriota. Further analysis of MAGs show that the main chemolithoautotrophs can fix nitrogen by coupling the oxidation of hydrogen, reduced sulfur, and iron, with the reduction of oxygen, nitrate, and sulfur. Culture experiments further demonstrate that members of chemolithoautotrophic Campylobacteria have the nitrogen-fixing capacity driven by hydrogen and sulfur oxidation. Activity measurements confirm that the diazotrophs inhabiting mangrove sediments preferentially drain energy from diverse reduced inorganic compounds other than from organics. Overall, our results suggest that chemolithoautotrophs rather than heterotrophs are dominant nitrogen fixers in mangrove sediments. This study underscores the significance of chemolithoautotrophs in carbon-dominant ecosystems.

Single cell sequencing coupled with bioinformatics reveals PHYH as a potential biomarker in kidney ischemia reperfusion injury.

Ischemia reperfusion injury(IRI) is an important factor affecting the early function and long-term survival of transplanted kidney. Single cell RNA sequencing (scRNA-seq) is a powerful method for investigating cell-specific transcriptome changes in the kidney. This study aimed to identify the significant cell type and potential biomarkers in IRI. First, we downloaded the IRI related scRNA dataset GSE139506 from the GEO database. Then, classification of cell type was characterized and proximal tubule cell (PTC) was identified as a significant cell type. The functional enrichment analysis indicated that PTC were related to kidney function and is significant in the ferroptosis of IRI. Analyses of three-dimensional structure and iron binding substructure of protein was carried out basing on SWISS-MODEL database. Finally, we constructed the murine model with IRI and verify the higher expression of PHYH in IRI by PCR, Western blot (WB) and Immunohistochemistry (IHC) experiments. In conclusion, this study provided novel insights on the cell-type-specific expression gene biomarker in IRI pathogenesis.

Isolation of high γ-aminobutyric acid-producing lactic acid bacteria and fermentation in mulberry leaf powders.

γ-Amino butyric acid (GABA) has numerous roles in physiological processes, including neurotransmission, and induction of hypotensive, diuretic and tranquilizer effects. The present study aimed to produce GABA-enriched mulberry leaf powder by using a strain of high GABA-producing Lactobacillus pentosus SS6, which is isolated from fermented mulberry fruits. A total of 37 strains of lactic acid bacteria (LAB) were isolated from fermented mulberry fruits strains of high GABA-producing Lactobacillus pentosus were selected. The isolated LAB was analyzed using thin-layer chromatography. SS6 was used as a starter culture for the fermentation of mulberry leaf powder to produce GABA. The mulberry leaf powder was treated with 10% saccharose, 6% peptone, 1.6% K2HPO4, 1% L-sodium glutamate at 35°C for 36 h (each treatment was applied whilst the others were kept constant), in a mixture with a water content of 60%, with the respective LAB strain that was fermented by incubation at 30°C for 6 h. The results indicated that the SS6 strain produced significantly higher GABA contents in the fermentation broth compared to the other strains (P<0.05). Addition of 10% saccharose, 6% peptone, 1.6% K2HPO4 and 1% L-sodium glutamate significantly triggered the production of GABA compared with that in the groups void of those additives (P<0.05). Furthermore, the water content, treatment time, amount of LAB inoculated and the incubation temperature also significantly affected GABA production compared with untreated groups under the aforementioned conditions (P<0.05). In conclusion, 10% saccharose, 6% peptone, 1.6% K2HPO4, 1% L-sodium glutamate, and a 60% water content at 35°C significantly improved and enhanced GABA production. The present study provided a basis for the production of GABA, which may be utilized by the pharmaceutical and food industry.

Differentially expressed microRNAs in diapausing versus HCl-treated Bombyx embryos.

Differentially expressed microRNAs were detected to explore the molecular mechanisms of diapause termination. The total small RNA of diapause-destined silkworm eggs and HCl-treated eggs was extracted and then sequenced using HiSeq high-throughput method. 44 novel miRNAs were discovered. Compared to those in the diapause-destined eggs, 61 miRNAs showed significant changes in the acid-treated eggs, with 23 being up-regulated and 38 being down-regulated. The potential target genes of differentially expressed miRNAs were predicted by miRanda. Gene Ontology and KEGG pathway enrichment analysis of these potential target genes revealed that they were mainly located within cells and organelles, involved in cellular and metabolic processes, and participated in protein production, processing and transportation. Two differentially expressed genes, Bombyx mori SDH and Bmo-miR-2761-3p, were further analyzed with qRT-PCR. BmSDH was significantly up-regulated in the HCl-treated eggs, while Bmo-miR-2761-3p was down-regulated. These results suggested that these two genes were well coordinated in silkworm eggs. Dual luciferase reporter assay demonstrated that Bmo-miR-2761-3p inhibited the expression of BmSDH.

Shotgun proteomic analysis on the diapause and non-diapause eggs of domesticated silkworm Bombyx mori.

To clarify the molecular mechanisms of silkworm diapause, it is necessary to investigate the molecular basis at protein level. Here, the spectra of peptides digested from silkworm diapause and non-diapause eggs were obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) and were analyzed by bioinformatics methods. A total of 501 and 562 proteins were identified from the diapause and non-diapause eggs respectively, of which 309 proteins were shared commonly. Among these common-expressed proteins, three main storage proteins (vitellogenin precursor, egg-specific protein and low molecular lipoprotein 30 K precursor), nine heat shock proteins (HSP19.9, 20.1, 20.4, 20.8, 21.4, 23.7, 70, 90-kDa heat shock protein and heat shock cognate protein), 37 metabolic enzymes, 22 ribosomal proteins were identified. There were 192 and 253 unique proteins identified in the diapause and non-diapause eggs respectively, of which 24 and 48 had functional annotations, these unique proteins indicated that the metabolism, translation of the mRNA and synthesis of proteins were potentially more highly represented in the non-dipause eggs than that in the diapause eggs. The relative mRNA levels of four identified proteins in the two kinds of eggs were also compared using quantitative reverse transcription PCR (qRT-PCR) and showed some inconsistencies with protein expression. GO signatures of 486 out of the 502 and 545 out of the 562 proteins identified in the diapause and non-diapause eggs respectively were available. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed the Metabolism, Translation and Transcription pathway were potentially more active in the non-dipause eggs at this stage.

Inactivation and mechanisms of chlorine dioxide on Nosema bombycis.

Biological tests demonstrated that the inactivation of Nosema bombycis (N. bombycis) spores by chlorine dioxide (ClO(2)) occurs very fast and is highly sensitive. The lowest effective inactivation dosage and time was 15mg/mL for 30min. The inactivation of spores was additionally verified by using double color fluorescence stain and spore germination testing. A series of biological changes, including a large number of substrates that were leaked out from the spores included proteins, DNA, polysaccharide, K(+), and Ca(2+), occurred a short time after N. bombycis spores were treated with ClO(2). In addition, the lipid of spores was disrupted and ATPase activity was inhibited, which resulted in the destruction of the inner structure of the spores.

Analysis of ecdysone-pulse responsive region of BMWCP2 in wing disc of Bombyx mori.

The present study was undertaken to clarify the regulation of cuticle-protein gene expression. Bombyx BAC library was screened to obtain the sequences of regulatory regions of cuticle protein genes isolated from wing discs of Bombyx mori. Two kb upstream of BMWCP2 was cloned into a reporter plasmid, and a reporter assay was operated. Plasmids were introduced into wing discs and wing tissues using a gene gun. DNA introduction into wing discs was confirmed with plasmid pA3GFP. The upstream region of BMWCP2 showed stage-specific activity: strongest at P0. EMSA analysis indicated the binding of BmbetaFTZ-F1. Ecdysone pulse-responsive sequences were examined in vitro. A luciferase assay was performed using reporter plasmids that contained different length upstream-regions of BMWCP2. With this method, we identified the ecdysone-responsive region. With deletion of the BMWCP2 upstream region, mutagenesis of the BmbetaFTZ-F1 binding site and EMSA analysis, it was confirmed that the BMWCP2 expression was regulated by BmbetaFTZ-F1 through the ecdysone pulse. This is first to apply the introduction of reporter plasmids into small organs to examine the developmental and hormonal regulation of the cuticle protein gene expression. We demonstrated that the binding of BmbetaFTZ-F1 facilitated the promoter activity of the BMWCP2 cuticle protein gene in vitro.

Genome-wide identification of cuticular protein genes in the silkworm, Bombyx mori.

Many kinds of cuticular proteins are found in a single insect species and their numbers and features are diversified among insects. Because there are so many cuticular proteins and so much sequence variation among them, an overview of cuticular protein gene is needed. Recently, a complete silkworm genome sequence was obtained through the integration of data from two whole genome sequence projects performed independently in 2004. To identify cuticular protein genes in the silkworm Bombyx mori exhaustively, we searched both the Bombyx whole genome sequence as well as various EST libraries, and found 220 putative cuticular protein genes. We also revised the annotation of the gene model, and named each identified cuticular protein based on its motif. The phylogenetic tree of cuticular protein genes among B. mori, Drosophila melanogaster, and Apis mellifera revealed that duplicate cuticular protein clusters have evolved independently among insects. Comparison of EST libraries and northern blot analyses showed that the tissue- and stage-specific expression of each gene was intricately regulated, even between adjacent genes in the same gene cluster. This study reveals many novel cuticular protein genes as well as insights into cuticular protein gene regulation.

Glycine-rich protein genes, which encode a major component of the cuticle, have different developmental profiles from other cuticle protein genes in Bombyx mori.

Three types of GRP (glycine-rich proteins) cDNAs were identified in the EST database of Bombyx mori. These came from 21 ESTs in the W3-stage wing disc EST library. We named them BmGRP1, BmGRP2 and BmGRP3. BmGRP1 and BmGRP2 had 57% identity in deduced amino acid sequences. Expression of all BmGRPs was observed in the epidermis at the fourth molting stage, and in the wing at pupation and mid-pupal stage. It is suggested that BmGRPs contribute to larval, pupal and adult cuticles together with other cuticle proteins. Transcripts of BmGRP2 increased after 7 days of pupal stage. BmGRP2 is suggested to construct adult trachea in the wing. Hormonal response of BmGRPs was compared with that of another group of cuticular protein genes, BMWCPs. BmGRPs were induced by a pulse of 20E. Induction of BmGRP3 was observed in W1 wing discs in the presence of JHA which was added with 20E, whereas that of BMWCP2 was inhibited in the presence of JHA. Induction of BmGRPs was observed in the wing discs of V3 and W1 stages, while that of BMWCP2 was not observed in the V3 wing discs. These differences between BMWCPs and BmGRPs in response to hormones at different developmental stages are discussed.