Impact of high-altitude acclimatization and de-acclimatization on the intestinal microbiota of rats in a natural high-altitude environment.

Introduction: Intestinal microorganisms play an important role in the health of both humans and animals, with their composition being influenced by changes in the host's environment. Methods: We evaluated the longitudinal changes in the fecal microbial community of rats at different altitudes across various time points. Rats were airlifted to high altitude (3,650 m) and acclimatized for 42 days (HAC), before being by airlifted back to low altitude (500 m) and de-acclimatized for 28 days (HADA); meanwhile, the control group included rats living at low altitude (500 m; LA). We investigated changes in the gut microbiota at 12 time points during high-altitude acclimatization and de-acclimatization, employing 16S rRNA gene sequencing technology alongside physiological indices, such as weight and daily autonomous activity time. Results: A significant increase in the Chao1 index was observed on day 14 in the HAC and HADA groups compared to that in the LA group, indicating clear differences in species richness. Moreover, the principal coordinate analysis revealed that the bacterial community structures of HAC and HADA differed from those in LA. Long-term high-altitude acclimatization and de- acclimatization resulted in the reduced abundance of the probiotic Lactobacillus. Altitude and age significantly influenced intestinal microbiota composition, with changes in ambient oxygen content and atmospheric partial pressure being considered key causal factors of altitude-dependent alterations in microbiota composition. High-altitude may be linked to an increase in anaerobic bacterial abundance and a decrease in non-anaerobic bacterial abundance. Discussion: In this study, the hypobaric hypoxic conditions at high-altitude increased the abundance of anaerobes, while reducing the abundance of probiotics; these changes in bacterial community structure may, ultimately, affect host health. Overall, gaining a comprehensive understanding of the intestinal microbiota alterations during high-altitude acclimatization and de-acclimatization is essential for the development of effective prevention and treatment strategies to better protect the health of individuals traveling between high- and low-altitude areas.

Transcriptome analysis reveals molecular pathways in the iron-overloaded Tibetan population.

Iron overload can lead to chronic complications, serious organ dysfunction or death in the body. Under hypoxic conditions, the body needs more iron to produce red blood cells to adapt to the hypoxic environment. The prevalence of iron overload in the Tibetan population is higher than that in the Han population. To explore the molecular mechanism of iron-overload in the Tibetan population, this study investigated the transcriptome of the Tibetan iron overload population to obtain differentially expressed genes (DEGs) between the iron-overloaded population and the normal iron population. Functional enrichment analysis identified key related pathways, gene modules and coexpression networks under iron-overload conditions, and the 4 genes screened out have the potential to become target genes for studying the development of iron overload. A total of 28 pathways were screened to be closely related to the occurrence and development of iron overload, showing that iron overload is extremely related to erythrocyte homeostasis, cell cycle, oxidative phosphorylation, immunity, and transcriptional repression.

Salidroside Affects Gut Microbiota Structure in db/db Mice by Affecting Insulin, Blood Glucose and Body Weight.

Purpose: The purpose of this study was to investigate the regulatory effect of salidroside on the intestinal flora of mice with type 2 diabetes (T2DM) and its protective effect in the body. Patients and Methods: We acclimated 8-week-old mice for 7 days, divided them into 4 groups, and continued dosing for 8 weeks. We recorded weekly blood glucose levels and body weight for each mouse. After the completion of the feeding cycle, the 16S rRNA of the intestinal flora in the mice was sequenced, and the insulin and C-peptide levels in each group of mice were measured. Four samples were taken from each group for liver and kidney section staining. Results: Our results showed that gut microbiota diversity and function were significantly different between the diabetic mice and healthy mice and that insulin levels, body weight, and blood glucose levels could significantly influence gut microbiota changes at the genus level. The gut microbiota diversity and function of db/db mice were also altered after salidroside administration. Salidroside could attenuate inflammatory damage, lipid accumulation and inflammatory changes in the diabetic liver, as well as diabetic kidney damage. Candidatus arthromitus and Odoribacter are important species of the microbiota during diabetes and may serve as potential therapeutic targets. Conclusion: Our investigation of the associated pathological conditions and fecal microbiota in db/db mice provides new insights into the pathogenesis of T2DM and provides implications for the diagnosis and treatment of T2DM.

DNA methylation plays an important role in iron-overloaded Tibetans.

The prevalence of iron overload in Tibetans in Tibet is higher than that in Han. DNA methylation (DNAm) is closely related to iron metabolism and iron level. Nevertheless, the epigenetic status of Tibetans with iron overload is unknown, and we therefore aimed to explore whether the phenomenon observed in the Tibetan population is regulated by epigenetics. The results showed that 2.26% of cytosine was methylated in the whole genome, and that the rate of CG cytosine methylation was higher in individuals in the iron overload (TH) group than in those in the iron normal (TL) group. We analyzed differentially methylated genes (DMGs) in whole-genome bisulfite sequencing data from the TH and TL groups of high-altitude Tibetans. Protein-protein interaction and pathway analyses of candidate DMGs related to iron uptake and transport showed that epigenetic changes in 10 candidate genes (ACO1, CYBRD1, FLVCR1, HFE, HMOX2, IREB2, NEDD8, SLC11A2, SLC40A1 and TFRC) are likely to relate to iron overload. This work reveals, for the first time, changes of DNAm in Tibetan people with iron overload, which suggest that DNAm is a mechanism underlying differences in iron content between individuals in the high-altitude Tibetan population. Our findings should contribute to the study of iron metabolism and the overall health status of Tibetans.

Key regulatory genes and signaling pathways involved in islet culture: a bioinformatic analysis.

Type 1 diabetes (T1D) is characterized by non-ideal mass and low survival rate of islets. Therefore, it is necessary to find intrinsic factors that prolong the survival of islets. This study aimed to track out hub genes and pathways in the process of islet culture by bioinformatic analysis. We downloaded the gene expression microarray of GSE42591 from the Gene Expression Omnibus (GEO). Aberrant Differentially methylated genes (DMGs) were obtained using the GEO2R tool. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses were performed on selected genes by using the Database for Annotation Visualization and Integrated Discovery (DAVID). A protein-protein interaction (PPI) network was constructed with the Retrieval of Interacting Genes (STRING) and visualized in Cytoscape 3.7.2. A total of 434 genes were overexpressed and 114 genes underexpressed in fresh to cultured 4 h tissue. KEGG pathway enrichment analyses revealed the TGF-beta signaling pathway, MAPK signaling pathway, or VEGF signaling pathway. The genes FN1, MKI67, IGF1, MAPK14, COL1A1 might be involved in islet culture. In general, this work scrutinized islet culture-relevant knowledge and provided insight into the regulation and mediation of islet survival.

De novo transcriptome analysis of Tibetan medicinal plant Dysphania schraderiana.

Dysphania schraderiana is widely distributed in Lhasa (Tibet, China) and used as a traditional medicine. However, the lack of genetic information hinders the understanding of its physiological processes, such as the biosynthesis of secondary metabolites. Herein, we used Illumina Hiseq4000 platform to sequence the transcriptome of flower and leaf tissues from D. schraderiana for the first time. Totally, 40,142 unigenes were assembled from approximately 5.2 million clean reads. All unigenes underwent gene prediction and were subsequently annotated in a NR (NCBI non-redundant protein) database, COG (Clusters of Orthologous Groups of proteins) database, and KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Among the 40,142 unigenes, 2,579 genes were identified as differentially expressed between flowers and leaves, and used in further enrichment analysis. Also, 2,156 unigenes were annotated as transcription factors. Furthermore, our transcriptome analysis resulted in the identification of candidate unigenes annotated to enzymes involved in terpenoid biosynthesis. Taken together, this work has laid the foundation for the investigation of secondary metabolite biosynthesis and other physiological processes of D. schraderiana.

Identification of gene-phenotype connectivity associated with flavanone naringenin by functional network analysis.

Naringenin, extracted from grapefruits and citrus fruits, is a bioactive flavonoid with antioxidative, anti-inflammatory, antifibrogenic, and anticancer properties. In the past two decades, the growth of publications of naringenin in PubMed suggests that naringenin is quickly gaining interest. However, systematically regarding its biological functions connected to its direct and indirect target proteins remains difficult but necessary. Herein, we employed a set of bioinformatic platforms to integrate and dissect available published data of naringenin. Analysis based on DrugBank and the Search Tool for the Retrieval of Interacting Genes/Proteins revealed seven direct protein targets and 102 indirect protein targets. The protein-protein interaction (PPI) network of total 109 naringenin-mediated proteins was next visualized using Cytoscape. What's more, all naringenin-mediated proteins were subject to Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis by the Database for Annotation, Visualization and Integrated Discovery, which resulted in three ESR1-related signaling pathways and prostate cancer pathway. Refined analysis of PPI network and KEGG pathway identified four genes (ESR1, PIK3CA, AKT1, and MAPK1). Further genomic analysis of four genes using cBioPortal indicated that naringenin might exert biological effects via ESR1 signaling axis. In general, this work scrutinized naringenin-relevant knowledge and provided an insight into the regulation and mediation of naringenin on prostate cancer.

Venomics of the spider Ornithoctonus huwena based on transcriptomic versus proteomic analysis.

The spider Ornithoctonus huwena is a venomous spider found in southern China. Its venom is a complex mixture of numerous biologically active components. In this study, 41 novel unique transcripts encoding cellular proteins or other possible venom components were generated from the previously constructed cDNA library. These proteins were also annotated by KOG (eukaryotic orthologous group) and GO (gene ontology) terms. A novel cellular transcript contig encoding an EF-hand protein (named HWEFHP1) was found, which might be involved in the secretion of toxins in the venom glands. In order to have an overview of the molecular diversity of the O. huwena venom, the datasets of all the transcripts, peptides and proteins known so far were analyzed. A comparison of the data obtained through a proteomic versus a transcriptomic approach, revealed that only 15 putative cystine knot toxins (CKTs) were identified by both approaches, 29 transcripts coding for CKTs were found in the transcriptome but not as translated peptides in the venom proteome. However, no cellular protein with identical molecular weight was identified by both approaches. Our data may contribute to a deeper understanding of the biology and ecology of O. huwena and the relationship between structure and function of individual toxins.

Molecular diversification of peptide toxins from the tarantula Haplopelma hainanum (Ornithoctonus hainana) venom based on transcriptomic, peptidomic, and genomic analyses.

The tarantula Haplopelma hainanum (Ornithoctonus hainana) is a very venomous spider found widely in the hilly areas of Hainan province in southern China. Its venom contains a variety of toxic components with different pharmacological properties. In the present study, we used a venomic strategy for high-throughput identification of tarantula-venom peptides from H. hainanum. This strategy includes three different approaches: (i) transcriptomics, that is, EST-based cloning and PCR-based cloning plus DNA sequencing; (ii) peptidomics, that is, off-line multiple dimensional liquid chromatography coupled with mass spectrometry (MDLC-MS) plus peptide sequencing (direct Edman sequencing and bottom-up mass spectrometric sequencing); (iii) genomics, that is, genomic DNA cloning plus DNA sequencing. About 420 peptide toxins were detected by mass spectrometry, and 272 peptide precursors were deduced from cDNA and genomic DNA sequences. After redundancy removal, 192 mature sequences were identified by three approaches. This is the largest number of peptide toxin sequences identified from a spider species so far. On the basis of precursor sequence identity, peptide toxins from the tarantula H. hainanum venom can be classified into 11 superfamilies (and related families). Our results revealed that gene duplication and focal hypermutation may be responsible for the enormous molecular diversity in spider peptide toxins. The current work is an initial overview for the study of tarantula-venom peptides in parallel transcriptomic, peptidomic, and genomic analyses. It is hoped that this work will also provide an effective guide for high-throughput identification of peptide toxins from other spider species, especially tarantula species.

Transcriptome analysis of the venom glands of the Chinese wolf spider Lycosa singoriensis.

The wolf spider Lycosa singoriensis is a hunting spider with a widespread distribution in northwest China. The venom gland of spiders, which is a very specialized secretory tissue, can secrete abundant and complex toxin components. To extensively examine the transcripts expressed in the venom glands of L. singoriensis, we generated 833 expressed sequence tags (ESTs) from a directional cDNA library. Toxin-like sequences account for 69.1% of these ESTs, 17.3% are similar to cellular transcripts and 13.6% have no significant similarity to any known sequences. Here, we identified 223 novel toxin-like sequences, which can be classified into six different superfamilies; that means a novel potential source of ligands for varied ion channels was discovered. With the aid of Gene Ontology terms and homology to eukaryotic orthologous groups, the annotation of cellular transcripts revealed some cellular processes important for the toxin secretion of venom glands including protein synthesis, protein folding, tuned post-translational processing and trafficking, etc.

Expression and characterization of jingzhaotoxin-34, a novel neurotoxin from the venom of the tarantula Chilobrachys jingzhao.

Jingzhaotoxin-34 (JZTX-34) is a 35-residue polypeptide from the venom of Chinese tarantula Chilobrachys jingzhao. Our previous work reported its full-length cDNA sequence encoding a precursor with 87 residues. In this study we report the protein expression and biological function characterization. The toxin was efficiently expressed by the secretary pathway in yeast. Under whole-cell patch-clamp mode, the expressed JZTX-34 was able to inhibit tetrodotoxin-sensitive (TTX-S) sodium currents (IC(50) approximately 85 nM) while having no significant effects on tetrodotoxin-resistant (TTX-R) sodium currents on rat dorsal root ganglion neurons. The inhibition of TTX-S sodium channels was completely reversed by strong depolarization (+120 mV). Toxin treatment altered neither channel activation and inactivation kinetics nor recovery rate from inactivation. However, it is interesting to note that in contrast to huwentoxin-IV, a recently identified receptor site-4 toxin from Ornithoctonus huwena venom, 100 nM JZTX-34 caused a negative shift of steady-state inactivation curve of TTX-S sodium channels by approximately 10 mV. The results indicated that JZTX-34 might inhibit mammalian sensory neuronal sodium channels through a mechanism similar to HWTX-IV by trapping the IIS4 voltage sensor in the resting conformation, but their binding sites should not overlay completely.

Expression, purification and characterization of a group of lectin-like peptides from the spider Ornithoctonus huwena.

By sequencing random clones from the venom gland cDNA library of the spider Ornithoctonus huwena, a transcript, named SHL-Ib1, encoding a lectin-like peptide was cloned. The amino acid sequence of the putative mature peptide of SHL-Ib1 is identical, except for seven different residues, with that of SHL-I, a lectin found in the venom of O. huwena. The mature peptides of SHL-Ib1b and SHL-Ib1c are the mutants of SHL-Ib1 with two or three amino acid residues truncated at the C-terminal. The recombinant SHL-Ib1b and SHL-Ib1c were expressed successfully by the yeast expression system and purified by using a combination of ion-exchange and reverse phase high performance liquid chromatography (HPLC). The molecular masses of the two expressed peptides were identified by mass spectrometry, indicating that the C-terminals of the two peptides were not amidated. The two peptides can agglutinate human erythrocytes at minimal concentrations of 0.75 and 1.475mg/ml, respectively. Structure modeling of SHL-Ib1 has given a clue to the low agglutination bioactivities of these recombinant toxins. These lectin-like peptides, due to the small molecular sizes, may have the advantage to investigate the binding mechanism of the lectin and have the potential to be the carrier for drug delivery.

JZTX-IV, a unique acidic sodium channel toxin isolated from the spider Chilobrachys jingzhao.

Neurotoxins are important tools to explore the structure and function relationship of different ion channels. From the venom of Chinese spider Chilobrachys jingzhao, a novel toxin, Jingzhaotoxin-IV (JZTX-IV), is isolated and characterized. It consists of 34 amino acid residues including six acidic residues clustered with negative charge (pI=4.29). The full-length cDNA of JZTX-IV encodes an 86-amino acid precursor containing a signal peptide of 21 residues, a mature peptide of 34 residues and an intervening sequence of 29 residues with terminal Lys-Gly as the signal of amidation. Under whole-cell patch clamp conditions, JZTX-IV inhibits current and slows the inactivation of sodium channels by shifting the voltage dependence of activation to more depolarized potentials on DRG neurons, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. In addition, JZTX-IV shows a slowing inactivation of sodium channel with a hyperpolarizing shift of the steady-state inactivation on acutely isolated rat cardiac cell and DRG neurons, differs from the classic site 3 toxins that do not affect the steady-state of inactivation. At high concentration, JZTX-IV has no significant effect on tetrodotoxin-resistant (TTX-R) sodium channels on rat DRG neurons and tetrodotoxin-sensitive (TTX-S) sodium channels on hippocampal neurons. Our data establish that, contrary to known toxins, JZTX-IV neither binds to the previously characterized classic site 4, nor site 3 by modifying channel gating, thus making it a novel probe of channel gating in sodium channels with potential to shed new light on this process.

Transcriptome analysis revealed novel possible venom components and cellular processes of the tarantula Chilobrachys jingzhao venom gland.

The tarantula Chilobrachys jingzhao is one of the most venomous spiders with a specialized organ, venom gland, which synthesizes and secretes the complex and abundant toxin proteins. The components of the venom have been extensively studied. As far as the molecular mechanism of toxin secretion and metabolism is concerned, we still knew a little. To obtain a comprehensive view of function of its venom gland we constructed a non-normalized cDNA library of the venom gland and generated 788 expressed sequence tags (ESTs). All ESTs were assembled into 356 non-redundancy sequences including 85 clusters and 271 singlets, of which 31.4% of total unique sequences belong to secretion protein coding sequences including cystine knot toxins (29.1%) and other secretion proteins (2.3%); 54.0% are similar to common cellular transcripts; and 14.6% have no significant similarity to any known sequences. Annotation of these ESTs revealed some novel possible venom components and cellular processes important for venom gland functions, including protein posttranslation processing, cell motility, protein synthesis, energy supply, etc. This study contains a transcriptome analysis of spider venom gland focusing on its cellular structural and functional aspects, and confirms the very specialized nature of the spider venom gland as toxin producer.

Genomic organization and cloning of novel genes encoding toxin-like peptides of three superfamilies from the spider Orinithoctonus huwena.

The bird spider Ornithoctonus huwena is one of the most venomous spiders in China. Its venom is a mixture of various compounds with diverse bioactivities. Ninety proteins and 47 peptides have been identified, and 67 cDNA sequences encoding different toxin precursors have been cloned. However, the genomic DNA of them is seldom reported. To characterize the genomic DNA structure of huwentoxins, the genomic DNA encoding toxins of three superfamilies were cloned by using sequence specific or partially degenerate primers based on their cDNA sequences. An unexpected finding was that the intron was lacking in the genomic sequences of three superfamilies. The genomic DNA information has predictive value for better understanding the relationship of spider toxin evolution. In addition, we have cloned and analyzed 19 novel genes encoding toxin-like precursors by using the genomic DNA of the spider O. huwena.

Molecular diversification based on analysis of expressed sequence tags from the venom glands of the Chinese bird spider Ornithoctonus huwena.

The bird spider Ornithoctonus huwena is one of the most venomous spiders in China. Its venom has been investigated but usually only the most abundant components have been analyzed. To characterize the primary structure of O. huwena toxins, a list of transcripts within the venom gland were made using the expressed sequence tag (EST) strategy. We generated 468 ESTs from a directional cDNA library of O. huwena venom glands. All ESTs were grouped into 24 clusters and 65 singletons, of which 68.00% of total ESTs belong to toxin-like sequences, 13.00% are similar to body peptide transcripts and 19.00% have no significant similarity to any known sequences. Precursors of all toxin-like sequences can be classified into eight different superfamilies (HWTX-I superfamily, HWTX-II superfamily, HWTX-X superfamily, HWTX-XIV superfamily, HWTX-XV superfamily, HWTX-XVI superfamily, HWTX-XVII superfamily, HWTX-XVIII superfamily) except HWTX-XI and HWTX-XIII, according to the identity of their precursor sequences. The results have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms, and for understanding the relationship of spider toxin evolution based on the diversification of cDNA sequences, primary structure of precursor peptides, three-dimensional structure motifs and biological functions.