Hydrogen sulfide (H2S) acts as an energy source, a toxin, and a gasotransmitter across diverse biological contexts. We use the robust locomotory responses of Caenorhabditis elegans to high levels of H2S to elucidate the molecular mechanisms underlying its acute and adaptive responses. We find that the H2S-evoked behavioral response is shaped by multiple environmental factors including oxygen (O2) levels and nutritional state and is modulated by various pathways such as insulin, TGF-β, and HIF-1 signaling, as well as by input from O2-sensing neurons. Prolonged exposure to H2S activates HIF-1 signaling, leading to the upregulation of stress-responsive genes, including those involved in H2S detoxification. This promotes an adaptive state in which locomotory speed is reduced in H2S, while responsiveness to other stimuli is preserved. In mutants deficient in HIF-1 signaling, iron storage, and detoxification mechanisms, animals display a robust initial response but rapidly enter a sleep-like behavior characterized by reduced mobility and diminished responsiveness to subsequent sensory stimuli. Furthermore, while acute production of mitochondria-derived reactive O2 species (ROS) appears to initiate the avoidance response to H2S, persistently high ROS promotes an adaptive state, likely by activating various stress-response pathways, without substantially compromising cellular H2S detoxification capacity. Taken together, our study provides comprehensive molecular insights into the mechanisms through which C. elegans modulates and adapts its response to H2S exposure.

The Shaker/Kv1 subfamily of voltage-gated potassium (K+) channels is essential for modulating membrane excitability. Their loss results in prolonged depolarization and excessive calcium influx. These channels have also been implicated in a variety of other cellular processes, but the underlying mechanisms remain poorly understood. Through comprehensive screening of K+ channel mutants in C. elegans, we discovered that shk-1 mutants are highly susceptible to bacterial pathogen infection and oxidative stress. This vulnerability is associated with reduced glycogen levels and substantial mitochondrial dysfunction, including decreased ATP production and dysregulated mitochondrial membrane potential under stress conditions. SHK-1 is predominantly expressed and functions in body wall muscle to maintain glycogen storage and mitochondrial homeostasis. RNA-sequencing data reveal that shk-1 mutants have decreased expression of a set of cation-transporting ATPases (CATP), which are crucial for maintaining electrochemical gradients. Intriguingly, overexpressing catp-3, but not other catp genes, restores the depolarization of mitochondrial membrane potential under stress and enhances stress tolerance in shk-1 mutants. This finding suggests that increased catp-3 levels may help restore electrochemical gradients disrupted by shk-1 deficiency, thereby rescuing the phenotypes observed in shk-1 mutants. Overall, our findings highlight a critical role for SHK-1 in maintaining stress tolerance by regulating glycogen storage, mitochondrial homeostasis, and gene expression. They also provide insights into how Shaker/Kv1 channels participate in a broad range of cellular processes.

Urochordate Ciona spp. are ideal marine model organisms for studying embryogenesis and developmental and evolutionary biology. However, the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging. This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp. The destination vector sets contained two Gateway cassettes flanked by Minos sites, allowing the N- or C-terminal tagging of a protein of interest with various fluorescent markers. In addition, we optimized the CRISPR/Cas9 and CRISPR/dCas9 systems by incorporating P2A-mCherry, a fluorescent indicator for Cas9 expression at cellular resolution. We demonstrated the effective destruction or inhibition of target genes when CRISPR constructs were introduced into fertilized eggs. Furthermore, we engineered a dual fluorescence sensor system that helps visualize successful gene knockouts at the cellular level in specific tissues. The genetic tools developed in this study offer a robust method for gene expression, cell tracking, and subcellular protein localization while also facilitating tissue-specific functional analysis in Ciona embryos and other model systems.

Regulation of microRNAs (miRNAs) on various biological processes has been a surprising and exciting field. Identification of miRNAs is the first step to comprehensively understand their functions. However, attempts on global identification and functional verification of miRNAs are very limited in penaeid shrimp Marsupenaeus japonicus , an economically important aquatic species. By performing an integrated analysis of transcriptomic profile from gastrula embryos of M. japonicus , 21 conserved miRNAs in M. japonicas (mja-miRNAs), belonging to 19 miRNA families, were identified and characterized. Of the 21 mja-miRNAs, 15 miRNAs were successfully verified to be predominantly expressed in gastrula stage, where they displayed dynamic expression patterns compared with those in naupliuin stage. Based on perfect or near-perfect match to target region, 120 target genes were predicted at transcriptome-wide level. Noteworthy, gene ontology (GO) classification and metabolic pathway annotation revealed eight targets that were actively involved in developmental processes. Of the predicted miRNA-mRNA pairs, six targets were then randomly selected and experimentally validated by dual luciferase reporter assay, where three pairs were proved with potential targeting activity. Overall, to search for conserved miRNAs potentially involved in early development of M. japonicus , we combined in silico and experimental methods, which can be applied in other organisms as well. Our data implied important roles of miRNAs in the early embryonic development and also suggested the presence of complex miRNA-mRNA functional networks in M. japonicus.

Hydrogen sulfide (H 2 S) can act as an energy source, a poison and a gasotransmitter in organisms. We used the robust locomotory responses to H 2 S in Caenorhabditis elegans to delineate the molecular mechanisms governing sensory and adaptive responses to H 2 S exposure. We found that C. elegans exhibited transiently increased locomotory activity and turning behavior as a strategy to escape the noxious H 2 S stimulation. The behavioral responses to H 2 S were modulated by a complex network of signaling pathways, including cyclic GMP signaling in ciliated sensory neurons, calcineurin, nuclear hormone receptors, to the major starvation regulators such as insulin and TGF-β signaling. The response to H 2 S was substantially affected by the ambient O 2 levels and their prior experience in low O 2 environments, suggesting an intricate interplay between O 2 and H 2 S sensing mechanisms. Prolonged exposure to H 2 S robustly evoked H 2 S detoxification coupled with reduced locomotory response to the subsequent H 2 S challenges. Intriguingly, the expression of genes involved in iron homeostasis, including ftn-1 and smf-3 , was substantially modified in exposure to H 2 S, implying that labile iron levels are affected by H 2 S. In support of this, iron supplement significantly bolstered the behavioral response to H 2 S. In addition, mitochondria, one of the central hubs for H 2 S metabolism, played a crucial role in adaptive responses to H 2 S. In summary, our study provides molecular insights into the mechanisms through which C. elegans detects, modulates, and adapts its response to H 2 S.

Hypoxia alters eating behavior in different animals. In C. elegans , hypoxia induces a strong food leaving response. We found that this behavior was independent of the known O 2 response mechanisms including acute O 2 sensation and HIF-1 signaling of chronic hypoxia response. Mutating egl-3 and egl-21 , encoding the neuropeptide pro-protein convertase and carboxypeptidase, led to defects in hypoxia induced food leaving, suggesting that neuropeptidergic signaling was required for this response. However, we failed to identify any neuropeptide mutants that were severely defective in hypoxia induced food leaving, suggesting that multiple neuropeptides act redundantly to modulate this behavior.

G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C. elegans . These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts.

Certain sets of genes are derived from gene duplication and share substantial sequence similarity in

In human beings, there is a ∼16,569 bp circular mitochondrial DNA (mtDNA) encoding 22 tRNAs, 12S and 16S rRNAs, 13 polypeptides that constitute the central core of ETC/OxPhos complexes, and some non-coding RNAs. Recently, mtDNA has been shown to have some covalent modifications such as methylation or hydroxylmethylation, which play pivotal epigenetic roles in mtDNA replication and transcription. Post-translational modifications of proteins in mitochondrial nucleoids such as mitochondrial transcription factor A (TFAM) also emerge as essential epigenetic modulations in mtDNA replication and transcription. Post-transcriptional modifications of mitochondrial RNAs (mtRNAs) including mt-rRNAs, mt-tRNAs and mt-mRNAs are important epigenetic modulations. Besides, mtDNA or nuclear DNA (n-DNA)-derived non-coding RNAs also play important roles in the regulation of translation and function of mitochondrial genes. These evidences introduce a novel concept of mitoepigenetics that refers to the study of modulations in the mitochondria that alter heritable phenotype in mitochondria itself without changing the mtDNA sequence. Since mitochondrial dysfunction contributes to carcinogenesis and tumor development, mitoepigenetics is also essential for cancer. Understanding the mode of actions of mitoepigenetics in cancers may shade light on the clinical diagnosis and prevention of these diseases. In this review, we summarize the present study about modifications in mtDNA, mtRNA and nucleoids and modulations of mtDNA/nDNA-derived non-coding RNAs that affect mtDNA translation/function, and overview recent studies of mitoepigenetic alterations in cancer.

Active promoters are urgently needed for shrimp cells which are hard to be immortalized. Translationally controlled tumor protein (TCTP) is a widely and abundantly expressed, growth-related protein. In this study we successfully isolated the promoter (P tctp ) and ORF (= open reading frame) of the TCTP gene from Litopenaeus vannamei and analysed the promoter activity of P tctp and the growth-promoting effects of over-expressed TCTP protein in the primary Oka organ (lymphoid tissue) cells. It was found, that cytomegavirus (CMV) promoter, highly active in mammalian and fish cells, had a driving activity too low to be detected in shrimp cells. However, shrimp P tctp had a higher driving activity than P cmv in the shrimp cells and an obvious fluorescent signal was observed in the shrimp cells transfected by a P cmv -P tctp -driven plasmid. However, no obvious growth-promoting effects were observed in the eGFP/TCTP [eGFP = enhanced green fluorescent protein] or TCTP-transfected shrimp cells possibly due to the relatively low expression efficiency.

Study on shrimp miRNAs was limited and just 7 mature miRNA sequences of Marsupenaeus japonicus are deposited in mirBase database. In this study, miRNAs and their target gene candidates were computationally identified from shrimp Penaeu s monodon and then experimentally validated. Using 39 908 expressed sequence tags (ESTs) and 21 124 genome survey sequences (GSSs) of P. monodon (pmo) as reference dataset, a comprehensive approach based on inter-species homolog search was employed to investigate the candidate miRNAs (i.e. pmo-miRNA). A total of eight miRNAs belonging to 7 families were computationally identified and five out of them were subsequently validated by PCR and sequencing. Of these, pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-3819 were first identified from shrimps. Both the mature pmo-miRNAs and the corresponding precursors were conserved among different species. Based on perfect or near-perfect match to the target region, the target gene candidates of pmomiRNAs were predicted from 10 331 mRNA sequences of P. monodon. A total of 20 genes were predicted as the targets of pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-6492. Experimental validation by dual luciferase reporter assay confirmed the targeting between 3 pmo-miRNAs and one or two of their target genes, especially the pmo-miR-4979 which could significantly down-regulate the expression of target gene (JR226772). This study updates the miRNAs and their targets in P. monodon and lays a solid foundation for future RNAi study.

The pantropic retroviral expression system has been widely used to stably introduce foreign genes into the genomeof dividing cells fromnon-mammalian systems. However, we found that the current commercial retrovirus expression systemdid notwork in inactively dividing primary shrimp cells. To overcomethis, we successfully developed a triple-pseudotyped retroviral expression system that can be used for effective gene transfer and expression of reporter genes in both mammalian and penaeid shrimp cells by improving the commercial pantropic retrovirus system (VPK-305, Cell Biolabs) in two ways. First, the promoter regions of translationally controlled tumor protein (TCTP) genes were cloned from the shrimps Metapenaeus ensis and Marsupenaeus japonicus, named Pme-tctp and Pmj-tctp, respectively. We inserted them into the retroviral reporter vectors of pMCs-P-LTR-GFP or eGFP, immediately after the retroviral promoter P-LTR (5' LTR, long terminal repeat sequence). We then analyzed their expression efficiencies in mammalian and shrimp cells by the lipofection method. It was found that P-LTR was a strong promoter in mammalian cells, but failed to effectively drive the reporter genes to express in the primary lymphoid cells (LCOO) derived from Oka organs of shrimp (M. ensis). However, inclusion of the shrimp promoters (Pme-tctp and Pmj-tctp) significantly improved the expression efficiency of the retroviral vectors in shrimp cells. Next, the revised retroviral vectors of pMCs-P-LTR-Pme-tctp (or Pmj-tctp)-GFP or eGFP were pseudotyped with envelope protein VSV-G alone, or triplywith VSV-G, VP19 and VP28 (two envelope proteins of shrimp white spot syndrome virus). And we then examined the corresponding infection and expression efficiencies in shrimp primary LCOO cells and embryo cells. We found that only the triple-pseudotyped retrovirus containing the shrimp-derived promoter could successfully mediate the delivery and fluorescent expression of GFP and eGFP genes, but VSV-G-pseudotyped retrovirus had poor tropism to shrimp LCOO cells. Confocal microscopic observation further confirmed the hypothesis that the improved tropism of the triplepseudotyped retrovirus to shrimp cells might be attributed to the inclusion of VP19 and VP28 into the envelope of the packaged retrovirus. In spite of this, the expression efficiency and infection efficiency (20%-30%) of reporter genes in shrimp cells was still lower than those in mammalian cells. In infected shrimp primary embryo cells, strong green fluorescent signal was detected only in neuron-like cells, not in fibroblast-like cells. This triplepseudotyped retroviral expression system ultimately may prove useful for immortalization of cultured shrimp cells and production of transgenic shrimps.Statement of relevance:We developed a novel triple-pseudotyped retroviral expression systemthat can be used in effective gene transfer and expression of foreign genes in shrimp cells.This system may prove useful for the immortalization of cultured shrimp cells which are needed in the study and control of shrimp viral diseases.This system may prove useful for the production of transgenic shrimps with new traits. (C) 2016 Elsevier B. V. All rights reserved.

MicroRNAs(miRNAs) are short(20-23-nucleotide), non-coding and single-stranded RNA molecules which regulate target gene expression at the post-transcriptional level to affect organisms' growth, development and cancer occurence. The maturity of miRNAs need a series of collaborative process mediated by large protein complexes and researches of miRNAs biosynthesis and activity controlare still in initial stage at present. This review mainly discusses the influencing factors of miRNAs biosynthesis and activity and relevant regulation mechanism. Cancer occurrence is usually accompanied by abnormal miRNAs expression profile, and further study for regulation mechanism of miRNAs biosynthesis will surely provide important theoretical basis and guidance for gene therapy of cancer and development of new technology.

Single factor test of concentration of dNTP,Mg2+,Taq DNA polymerase,primer and DNA template was used to optimize the ISSR-PCR amplification system which has been established initially.The results showed that suitable ISSR-PCR reaction system for Odontotermes formosanus was 20 μl reaction system containing 2.5 U Taq DNA polymerase,2.0 mmol·L-1 Mg2+,0.1 mmol·L-1 dNTP,1.0 μmol·L-1 primer and 20 ng DNA template.

Objective To obtain strains with cellulose degradation ability from the digestive tract of Reticulitermes chinensis Snyder and optimize the culture conditions for bacteria enzyme production.Methods Screening medium was used for isolation of endoglucanase-producing bacteria and the result of strain identification was based on microscopic observation,culture characteristics and phylogenetic analysis of 16S rDNA PCR.The optimal medium for endoglucanase production was optimized by orthogonal test and optimal conditions were optimized by single factor test.Results Strain B03 with activity of endoglucanase was identified as Citrobacter.The optimal carbon source and nitrogen source for enzyme production were CMC-Na and peptone.The optimal medium for enzyme production was composed of CMC-Na 5.0 g/L,peptone 4.0 g/L,NH4Cl 0.6 g/L,KH2PO4 0.5 g/L and MgSO4 0.9 g/L.The optimal enzyme-producing conditions were initial pH 5.0,35 °C and liquid volume 20-30 mL per 150 mL shake flask.Conclusion After optimization,the enzyme activity can reach up to 0.311 U/mL from 0.184 U/mL.The result of this study has certain guiding significance to industrialization of cellulose.