Correlation of distribution characteristics and dynamic changes of gut microbiota with the efficacy of immunotherapy in EGFR-mutated non-small cell lung cancer.

BACKGROUND: The effects of gut microbiota and metabolites on the responses to immune checkpoint inhibitors (ICIs) in advanced epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) have been studied. However, their effects on EGFR-mutated (EGFR +) NSCLC remain unknown. METHODS: We prospectively recorded the clinicopathological characteristics of patients with advanced EGFR + NSCLC and assessed potential associations between the use of antibiotics or probiotics and immunotherapy efficacy. Fecal samples were collected at baseline, early on-treatment, response and progression status and were subjected to metagenomic next-generation sequencing and ultra-high-performance liquid chromatography-mass spectrometry analyses to assess the effects of gut microbiota and metabolites on immunotherapy efficacy. RESULTS: The clinical data of 74 advanced EGFR + NSCLC patients were complete and 18 patients' fecal samples were dynamically collected. Patients that used antibiotics had shorter progression-free survival (PFS) (mPFS, 4.8 vs. 6.7 months; P = 0.037); probiotics had no impact on PFS. Two dynamic types of gut microbiota during immunotherapy were identified: one type showed the lowest relative abundance at the response time point, whereas the other type showed the highest abundance at the response time point. Metabolomics revealed significant differences in metabolites distribution between responders and non-responders. Deoxycholic acid, glycerol, and quinolinic acid were enriched in responders, whereas L-citrulline was enriched in non-responders. There was a significant correlation between gut microbiota and metabolites. CONCLUSIONS: The use of antibiotics weakens immunotherapy efficacy in patients with advanced EGFR + NSCLC. The distribution characteristics and dynamic changes of gut microbiota and metabolites may indicate the efficacy of immunotherapy in advanced EGFR + NSCLC.

Reproductive Ability Disparity in the Pacific Whiteleg Shrimp (Penaeus vannamei): Insights from Ovarian Cellular and Molecular Levels.

The Pacific whiteleg shrimp (Penaeus vannamei) is a highly significant species in shrimp aquaculture. In the production of shrimp larvae, noticeable variations in the reproductive capacity among female individuals have been observed. Some females experience slow gonadal development, resulting in the inability to spawn, while others undergo multiple maturations and contribute to the majority of larval supply. Despite numerous studies that have been conducted on the regulatory mechanisms of ovarian development in shrimp, the factors contributing to the differences in reproductive capacity among females remain unclear. To elucidate the underlying mechanisms, this study examined the differences in the ovarian characteristics between high and low reproductive bulks at different maturity stages, focusing on the cellular and molecular levels. Transmission electron microscopy analysis revealed that the abundance of the endoplasmic reticulum, ribosomes, mitochondria, and mitochondrial cristae in oocytes of high reproductive bulk was significantly higher than that of the low reproductive bulk in the early stages of ovarian maturation (stages I and II). As the ovaries progressed to late-stage maturation (stages III and IV), differences in the internal structures of oocytes between females with different reproductive capacities gradually diminished. Transcriptome analysis identified differentially expressed genes (DEGs) related to the mitochondria between two groups, suggesting that energy production processes might play a crucial role in the observed variations in ovary development. The expression levels of the ETS homology factor (EHF) and PRDI-BF1 and RIZ homology domain containing 9 (PRDM9), which were significantly different between the two groups, were compared using qRT-PCR in individuals at different stages of ovarian maturation. The results showed a significantly higher expression of the EHF gene in the ovaries of high reproductive bulk at the II and IV maturity stages compared to the low reproductive bulk, while almost no expression was detected in the eyestalk tissue of the high reproductive bulk. The PRDM9 gene was exclusively expressed in ovarian tissue, with significantly higher expression in the ovaries of the high reproductive bulk at the four maturity stages compared to the low reproductive bulk. Fluorescence in situ hybridization further compared the expression patterns of EHF and PRDM9 in the ovaries of individuals with different fertility levels, with both genes showing stronger positive signals in the high reproductive bulk at the four ovarian stages. These findings not only contribute to our understanding of the regulatory mechanisms involved in shrimp ovarian development, but also provide valuable insights for the cultivation of new varieties aimed at improving shrimp fecundity.

Analysis of Elimination Effects of Inbreeding on Genotype Frequency in Larval Stages of Chinese Shrimp.

Marine animals possess genomes of considerable complexity and heterozygosity. Their unique reproductive system, characterized by high fecundity and substantial early mortality rates, increases the risk of inbreeding, potentially leading to severe inbreeding depression during various larval developmental stages. In this study, we established a set of inbred families of Fenneropenaeus chinensis, with an inbreeding coefficient of 0.25, and investigated elimination patterns and the manifestations of inbreeding depression during major larval developmental stages. Reduced-representation genome sequencing was utilized to explore the genotype frequency characteristics across two typical elimination stages. The results revealed notable mortality in hatching and metamorphosis into mysis and post-larvae stages. Inbreeding depression was also evident during these developmental stages, with depression rates of 24.36%, 29.23%, and 45.28%. Segregation analysis of SNPs indicated an important role of gametic selection before hatching, accounting for 45.95% of deviation in the zoea stage. During the zygotic selection phase of larval development, homozygote deficiency and heterozygote excess were the main selection types. Summation of the two types explained 82.31% and 89.91% of zygotic selection in the mysis and post-larvae stage, respectively. The overall distortion ratio decreased from 22.37% to 12.86% in the late developmental stage. A total of 783 loci were identified through selective sweep analysis. We also found the types of distortion at the same locus could change after the post-larvae stage. The predominant shifts included a transition of gametic selection toward normal segregation and other forms of distortion to heterozygous excess. This may be attributed to high-intensity selection on deleterious alleles and genetic hitchhiking effects. Following larval elimination, a greater proportion of heterozygous individuals were preserved. We detected an increase in genetic diversity parameters such as expected heterozygosity, observed heterozygosity, and polymorphic information content in the post-larvae stage. These findings suggest the presence of numerous recessive deleterious alleles and their linkage and suggest a major role of the partial dominance hypothesis. The results provide valuable insights into the mechanisms of inbreeding depression in marine animals and offer guidance for formulating breeding strategies in shrimp populations.

Essential Rule Derived from Thermodynamics and Kinetics Studies of Benzopyran Compounds.

Compounds with benzopyran as the core structure play an important role in the total synthesis of antioxidants, drugs, and natural products. Herein, the thermodynamic data of benzopyran compounds and their intermediates were measured and calculated by combining thermodynamics with kinetics. The mechanism of reactions between four benzopyran compounds and organic hydride acceptors was proven to be a one-step hydride transfer. The thermodynamic properties of these compounds and their corresponding intermediates were elucidated. The rationality and accuracy of the electrochemical measurement method were proved. Furthermore, the essential rule of unique structures being present between the C-H bond and para-substituent constants on the benzene ring, as shown in previous studies, was investigated. A simultaneous correlation between thermodynamics and kinetics was found for the hydride transfer reaction, in which the reaction site is connected with the substituent through the benzene ring, a double bond, or a N atom. The likely reason for the correlation between thermodynamic and kinetic is that the benzene ring, double bond, or N atom have the role of transferring the electronic effect. This finding can be applied to the calculation of the activation energy of hydride self-exchange reactions, the prediction of kinetic isotope effects, and explorations of selective reduction processes of hydride transfer in such organic hydride compounds.

Gut microbiota signatures and fecal metabolites in postmenopausal women with osteoporosis.

BACKGROUND: Women suffer from various distress and disturbances after menopause, including osteoporosis, a risk factor associated with multiple diseases. Altered gut microbiota has been implicated in postmenopausal osteoporosis. In this study, to understand gut microbiota signatures and fecal metabolite changes in postmenopausal women with osteoporosis, 108 postmenopausal women were recruited for intestinal microbiota and fecal metabolite detection. Among these participants, 98 patients, who met the inclusion criteria, were divided into postmenopausal osteoporosis (PMO) and non-postmenopausal osteoporosis (non-PMO) groups based on bone mineral density (BMD). The compositions of gut bacteria and fungi were examined by 16 S rRNA gene sequencing and ITS sequencing, respectively. Meanwhile, fecal metabolites were analyzed using liquid chromatography coupled with mass spectrometry (LC-MS). RESULTS: We found that bacterial α-diversity and ß-diversity were significantly altered in PMO compared to non-PMO patients. Interestingly, fungi composition showed larger changes, and the differences in ß-diversity were more significant between PMO and non-PMO patients. Metabolomics analysis revealed that fecal metabolites, such as levulinic acid, N-Acetylneuraminic acid, and the corresponding signaling pathways were also changed significantly, especially in the alpha-Linolenic acid metabolism and selenocompound metabolism. The screened differential bacteria, fungi, and metabolites closely correlated with clinical findings between these two groups, for example, the bacterial genus, Fusobacterium, the fungal genus, Devriesia, and the metabolite, L-pipecolic acid, were significantly associated with BMD. CONCLUSIONS: Our findings indicated that there were remarkable changes in gut bacteria, fungi, and fecal metabolites in postmenopausal women, and such changes were notably correlated with patients' BMD ​​and clinical findings. These correlations provide novel insights into the mechanism of PMO development, potential early diagnostic indicators, and new therapeutic approaches to improve bone health in postmenopausal women.

Exploring the Relationship between the Gut Mucosal Virome and Colorectal Cancer: Characteristics and Correlations.

The fecal virome has been reported to be associated with CRC. However, little is known about the mucosal virome signature in CRC. This study aimed to determine the viral community within CRC tissues and their contributions to colorectal carcinogenesis. Colonic mucosal biopsies were harvested from patients with CRC (biopsies of both neoplasia and adjacent normal tissue (CRC-A)) and healthy controls (HC). The shot-gun metagenomic sequencing of virus-like particles (VLPs) was performed on the biopsies. Viral community, functional pathways, and their correlations to clinical data were analyzed. Fluorescence in situ hybridizations (FISH) for the localization of viruses in the intestine was performed, as well as quantitative PCR for the detection of Torque teno virus load in human mucosal VLP DNA. A greater number and proportion of core species were found in CRC tissues than in CRC-A and HC tissues. The diversity of the mucosal virome in CRC tissues was significantly increased compared to that in HC and CRC-A tissues. The mucosal virome signature of CRC tissues were significantly different from those of HC and CRC-A tissues at the species level. The abundances of eukaryotic viruses from the Anelloviridae family and its sub-species Torque teno virus (TTV) were significantly higher in CRC patients than in HC. Furthermore, increased levels of TTV in the intestinal lamina propria were found in the CRC group. Multiple viral functions of TTV associated with carcinogenesis were enriched in CRC tissues. We revealed for the first time that the mucosal virobiota signature of CRC is characterized by a higher diversity and more eukaryotic viruses. The enrichment of TTV species in CRC tissues suggests that they may play an oncogenic role in CRC. Targeting eukaryotic viruses in the gut may provide novel strategies for the prevention and treatment of CRC.

A Mechanism Study of Redox Reactions of the Ruthenium-oxo-polypyridyl Complex.

Over the years, RuIV(bpy)2(py)(O)2+([RuIVO]2+) has garnered considerable interest owing to its extensive use as a polypyridine mono-oxygen complex. However, as the active-site Ru=O bond changes during the oxidation process, [RuIVO]2+ can be used to simulate the reactions of various high-priced metallic oxides. In order to elucidate the hydrogen element transfer process between the Ruthenium-oxo-polypyridyl complex and organic hydride donor, the current study reports on the synthesis of [RuIVO]2+, a polypyridine mono-oxygen complex, in addition to 1H and 3H (organic hydride compounds) and 1H derivative: 2. Through 1H-NMR analysis and thermodynamics- and kinetics-based assessments, we collected data on [RuIVO]2+ and two organic hydride donors and their corresponding intermediates and established a thermodynamic platform. It was confirmed that a one-step hydride transfer reaction between [RuIVO]2+ and these organic hydride donors occurs, and here, the advantages and nature of the new mechanism approach are revealed. Accordingly, these findings can considerably contribute to the better application of the compound in theoretical research and organic synthesis.

Thermodynamics Evaluation of Selective Hydride Reduction for α,ß-Unsaturated Carbonyl Compounds.

The selective reduction of α,ß-unsaturated carbonyl compounds is one of the core reactions and also a difficult task for organic synthesis. We have been attempting to study the thermodynamic data of these compounds to create a theoretical basis for organic synthesis and computational chemistry. By electrochemical measurement method and titration calorimetry, in acetonitrile at 298 K, the hydride affinity of two types of unsaturated bonds in α,ß-unsaturated carbonyl compounds, their single-electron reduction potential, and the single-electron reduction potential of the corresponding radical intermediate are determined. Their hydrogen atom affinity, along with the hydrogen atom affinity and proton affinity of the corresponding radical anion, is also derived separately based on thermodynamic cycles. The above data are used to establish the corresponding "Molecule ID Card" (Molecule identity card) and analyze the reduction mechanism of unsaturated carbonyl compounds. Primarily, the mixture of any carbonyl hydride ions and Ac-tempo+ will stimulate hydride transfer process and create corresponding α,ß-unsaturated carbonyl compounds and Ac-tempoH from a thermodynamic point of view.

Genomic signatures of artificial selection in fecundity of Pacific white shrimp, Penaeus vannamei.

Penaeus vannamei is the most important economic shrimp in the world. Many selective breeding programs are carried out to improve its production and performance traits. Although significant differences in the reproductive ability of female P. vannamei under artificial breeding conditions have been reported, the genome-wide adaption of the reproductive ability of domesticated female P. vannamei is less investigated. In this study, whole-genome analysis was performed along with pooled DNA sequencing on two fecundity separated bulks, high fecundity bulk (HB), and low fecundity bulk (LB). Each bulk contained 30 individuals from 3 commercial populations. A sequencing depth of >30× was achieved for each bulk, leading to the identification of 625,181 and 629,748 single nucleotide polymorphisms (SNPs) in HB and LB, respectively. Fixation index (Fst) combined with p ratio allowed for the identification of 145 selective sweep regions, with a sequence length of 14.5 Mb, accounting for 0.59% of the genome. Among the 145 selective sweep regions, a total of 64,046 SNPs were identified, and further verification was performed by genotyping 50 candidate SNPs on 60 samples from the offspring of the three populations. Furthermore, 121 genes were screened from the sweep regions. GO annotation and KEGG enrichment analyses showed that partial genes were essential for fecundity regulation. This study provides important information for in-depth investigation of genomic characteristics for long-term selective breeding on the fecundity of female P. vannamei and will also be important for genome-assisted breeding of P. vannamei in the future.

Fecal microbiota transplantation treatment of autoimmune-mediated type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is an autoimmune-mediated disease characterized by a reduced or absolute lack of insulin secretion and often associated with a range of vascular and neurological complications for which there is a lack of effective treatment other than lifestyle interventions and pharmacological treatments such as insulin injections. Studies have shown that the gut microbiota is involved in mediating the onset and development of many fecal and extrafecal diseases, including autoimmune T1DM. In recent years, many cases of gut microbiota transplantation for diseases of the bowel and beyond have been reported worldwide, and this approach has been shown to be safe and effective. Here, we conducted an experimental treatment study in two adolescent patients diagnosed with autoimmune T1DM for one year. Patients received one to three rounds of normal fecal microbiota transplants (FMT) and were followed for up to 30 weeks. Clinical outcomes were measured, including biochemical indices, medication regimen, and dosage adjustment. Fecal microbiota metagenomic sequencing after transplantation provides a reference for more reasonable and effective microbiota transplantation protocols to treat autoimmune T1DM. Our results suggest that FMT is an effective treatment for autoimmune T1DM. Clinical Trial Registration: http://www.chictr.org.cn, identifier ChiCTR2100045789.

Association of serum 25-hydroxyvitamin D (25(OH)D) levels with the gut microbiota and metabolites in postmenopausal women in China.

BACKGROUND: Vitamin D insufficiency or deficiency is associated with an altered microbiota in older men. However, the relationship between the gut microbiota and 25-hydroxyvitamin D (25(OH)D) levels remains unknown in postmenopausal women. In this study, fecal microbiota profiles for 88 postmenopausal women in the high 25(OH)D (HVD) group (n = 44) and the low 25(OH)D (LVD) group (n = 44) were determined. An integrated 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach was applied to explore the association of serum 25(OH)D levels with the gut microbiota and fecal metabolic phenotype. Adjustments were made using several statistical models for potential confounding variables identified from the literature. RESULTS: The results demonstrated that the community diversity estimated by the Observe, Chao1 and ACE indexes was significantly lower in the LVD group than in the HVD group. Additionally, two kinds of characteristic differences in the microflora were analyzed in the HVD group, and ten kinds of characteristic differences in the microflora were analyzed in the LVD group. We observed that some bacteria belonging to the genera Bifidobacterium, Bacillus, F0332 and Gemella, were enriched in the LVD group, as were other genera, including Lachnoclostridium, UC5_1_2E3, Ruminococcus_gnavus_group and un_f_Lachnospiraceae. Christensenellaceae, Eggerthellaceae and Cloacibacillus were enriched in the HVD group. The L-pyroglutamic acid, inosine, and L-homocysteic acid levels were higher in the HVD group and were negatively correlated with the 1,2-benzenedicarboxylic acid and cholic acid metabolic levels. CONCLUSIONS: These observations provide a better understanding of the relationships between serum 25(OH)D levels and the fecal microbiota and metabolites in postmenopausal women.

Fecal microbiota transplantation reverses insulin resistance in type 2 diabetes: A randomized, controlled, prospective study.

Objectives: Recent studies have shown that fecal microbiota transplantation (FMT) improved the metabolic profiles of patients with type 2 diabetes mellitus (T2DM), yet the effectiveness in reversing insulin resistance and increasing metformin sensitivity in T2DM patients have not been reported. In this study, we evaluated the improvements of T2DM patients and their gut microbiota by FMT alone and FMT plus metformin. Methods: A total of 31 patients with newly diagnosed T2DM were randomized to intervention by metformin, FMT, or FMT plus metformin in the study. Patients were followed up at baseline and week 4 after treatment. Blood and stool samples were collected and subject to analyze clinical parameters and microbial communities by metagenomic sequencing, respectively. Results: FMT alone and FMT plus metformin significantly improved the clinical indicators HOMA-IR and BMI in T2DM, besides fasting blood glucose, postprandial blood glucose, and hemoglobin A1c that were also controlled by metformin. Donor microbiota effectively colonized in T2DM with slightly higher colonization ration in FMT than FMT plus metformin within 4 weeks, resulting in increased microbial diversity and community changes from baseline after treatment. A total of 227 species and 441 species were significantly alerted after FMT and FMT plus metformin, respectively. FMT were significantly associated with the clinical parameters. Among them, Chlorobium phaeovibrioides, Bifidibacterium adolescentis and Synechococcus sp.WH8103 were potential due to their significantly negative correlations with HOMA-IR. Conclusions: FMT with or without metformin significantly improve insulin resistance and body mass index and gut microbial communities of T2DM patients by colonization of donor-derived microbiota.

Identification of two genes potentially related to myogenesis and muscle growth in Fenneropenaeus chinensis: Activin receptor II and Follistatin-like protein.

Activin receptor (ActR) and follistatin-like (FSTL) genes, which are involved in the Myostatin (Mstn) related TGF-ß/Smad signaling pathway, play important roles in regulating the muscle generation, development and growth of muscle in vertebrate. Our previous studies have confirmed that Mstn negatively regulates muscle development and growth in Fenneropenaeus chinensis as that in vertebrate. However, the roles of ActR and FSTL in muscle development and growth in invertebrate remains unclear. In the present study, type II ActR(FcActRII) and FSTL (FcFSTL) genes from F. chinensis were cloned and characterized, and their functions on muscle development and growth were investigated. The full-length cDNAs of FcActRII and FcFSTL were 2366 bp that encoded 572 amino acids and 2474 bp that encoded 717 amino acids, respectively. Sequence analysis revealed that the overall protein sequences of the two genes shared 97% and 96% identities with Penaeus vannamei and 50%-59% and 35%-36% identities with vertebrates, respectively. In the early development stages, muscles firstly appeared in nauplius stage and developed gradually until post larval, and the mRNA expressions of FcActRII increased from gastrula to zoea stage and then decreased from zoea stage to post larval stage while that of FcFSTL was lowest in gastrula stage and increased rapidly in nauplius stage and then expressed stably from nauplius stage to post-larval stage. In the adult shrimp, the two genes were widely distributed in the examined tissues. The FcActRII expression in muscle of L group was significantly lower than that of S group, but the FcFSTL expression showed an opposite result. After down-regulating the expression of FcMstn by RNAi, FcActRII expression was significantly down-regulated while that of FcFSTL was up-regulated. The present study suggested that FcActRII and FcFSTL, regulated by FcMstn, might be involved in myogenesis and muscle growth.

Segregation distortion: high genetic load suggested by a Chinese shrimp family under high-intensity selection.

Segregation distortion is a common phenomenon found in most genetic mapping studies and is an important resource to dissect the mechanism of action in gene loci that cause deviation. Marine animals possess high genetic diversity and genomic heterozygosity, they therefore are ideal model organisms to study segregation distortion induced by selection. In the present study, we constructed a full-sib family of Fenneropenaeus chinensis and exerted high-intensity selection on 10,000 incipient progenies. 2b-RAD method was employed in remaining 273 individuals to develop genome-wide SNPs for segregating analysis and 41,612 SNPs were developed. 50.77% of 32,229 high-quality representative markers deviated from the expected Mendelian ratio. Results showed that most of these distorted markers (91.57%) were influenced at zygotic level. Heterozygote excess (53.07%) and homozygous deletions (41.96%) may both play an important role, sum of which explained 95.03% of distortion after fertilization. However, further results identified highly probable linkage among deleterious alleles, which may account for a considerable portion of heterozygote excess rather than single locus with heterozygote advantage. Results of this study support a major role of deleterious alleles in genetic load, thus in favor of partial dominance hypothesis. It would also offer necessary recommendations for the formulation of breeding strategy in shrimps.

Feed competition reduces heritable variation for body weight in Litopenaeus vannamei.

BACKGROUND: Competition is a common social interaction among shrimp and depending on its intensity, it can affect heritable variation and response to selection. Little is known about the variance of indirect genetic effects (IGE) under competitive and non-competitive conditions in shrimp. In this study, we used extended mixed linear models to estimate genetic parameters for the direct genetic effect (DGE) and IGE on body weight in Litopenaeus vannamei raised under ad libitum (AF, non-competitive environment) and restricted (RF, competitive environment) feeding regimes. RESULTS: Estimates of heritabilities for body weight obtained with a traditional animal model (i.e. without accounting for IGE) were 0.11 ± 0.09 under AF and 0.25 ± 0.11 under RF. With extended animal models that accounted for IGE, the corresponding estimates for body weight were 0.07 ± 0.08 and 0.34 ± 0.11. Thus, heritabilities were higher under the RF regime than under the AF regime, regardless of whether IGE was accounted for or not. The log-likelihood ratio test revealed significant IGE under the RF regime. Although estimates of indirect genetic variance were low (0.0023 ± 0.0013 for AF and 0.0028 ± 0.0012 for RF), they contributed substantially to the total heritable variance: 66.8% for AF and 692.2% for RF. The total heritable variance was smaller under the RF regime (0.7 ± 1.3) than under the AF regime (5.8 ± 2.6) because of the high contribution of the negative covariance between DGE and IGE (- 7.03). Estimates of the correlation between DGE and IGE were 0.32 ± 0.47 under AF and - 0.93 ± 0.15 under RF, those of DGE and IGE for body weight between both regimes were 0.94 ± 0.07 and 0.67 ± 0.20, respectively, and those of IGE for body weight with DGE for survival were - 0.12 ± 0.22 under AF and - 0.58 ± 0.20 under RF. CONCLUSIONS: These results indicate that strong competitive interactions occurred under the RF regime in L. vannamei. Significant reranking and variation in IGE of individuals were observed between the two feeding regimes. Strong competitive interactions reduced the total heritable variation for body weight when food was restricted. These results indicate that the extent of competition among L. vannamei depends on the feeding regime applied and that this competition affects the genetic basis of body weight.

Molecular characterization of myostatin and its inhibitory function on myogenesis and muscle growth in Chinese Shrimp, Fenneropenaeus chinensis.

Myostatin (Mstn) inhibits muscle growth in vertebrates with endoskeleton, but it is still inconclusive that Mstn is a positive or negative regulator in crustacean with exoskeleton, and little information was available for its function on myogenesis. In this study, we identified and characterized the Mstn from Fenneropenaeus chinensis (FcMstn), and investigated its function on myogenesis and muscle growth. Two different cDNA sequences (2628 bp and 2604 bp) encoding for slightly different sizes of proteins were obtained for FcMstn, containing 86 bp of 5' untranslated regions (UTR) and 1258 bp of 3' UTR. The open reading frame of the long sequence and the short sequence contain 1284 bp and 1260 bp cDNA, encoding 427 and 419 amino acid sequence, respectively. Sequence analysis revealed that the overall protein sequence and specific functional sites of FcMstn were highly conserved with those in other crustacean species. In the early development stage, the muscle firstly appeared in nauplius stage and developed gradually until post larval, but the expression of FcMstn at mRNA and protein levels decreased from nauplius stage to post larval stage, indicating that Mstn involved in myogenesis as a negative regulator in shrimp. In the adult shrimp, the expression of FcMstn at mRNA and protein levels in muscle were significantly lower in the larger group than in the smaller group, and the diameter and number of muscle fiber of the muscle were significantly different between the two groups. Moreover, the shrimp with reduced level of FcMstn by RNAi displayed a dramatic faster growth rate compared with the control group. The present study demonstrates that FcMstn involved in myogenesis and muscle growth probably also as a negative regulator in shrimp like in vertebrates.

Integrative phenotypic and gene expression data identify myostatin as a muscle growth inhibitor in Chinese shrimp Fenneropenaeus chinensis.

Growth traits, largely determined by muscle growth, are the most critical economic traits in shrimp breeding. Myostatin (Mstn) is a conserved inhibitor of muscle growth in vertebrates, but until now solid evidence supporting a similar function of Mstn in invertebrates has been lacking. In the present study, we examined the Mstn expression along with growth trait data in a Fenneropenaeus chinensis population, to establish a potential correlation between Mstn and growth. The heritabilities of FcMstn expression, body weight at 190 days of culture, body weight and length at 230 days of culture, and average daily gain were estimated using 773 individuals and a thirteen-generation pedigree. The results showed FcMstn expression was negatively correlated with the growth traits, and the mean FcMstn expression in females was significantly lower than that of males, indicating Mstn negatively regulates muscle growth in shrimp, and its lower expression may underscore the faster growth of females. Low heritabilities were detected for FcMstn expression, suggesting that the expression of Mstn might be heritable in shrimp. These results provide strong support for a growth inhibitory function of Mstn in F. chinensis, and suggest a potential method for selective breeding of this species without substantial experimental resources and labor force.

Screening of the candidate genes related to low-temperature tolerance of Fenneropenaeus chinensis based on high-throughput transcriptome sequencing.

In order to screen the candidate genes of Fenneropenaeus chinensis related to low-temperature tolerance, this research takes juvenile prawns of F. chinensis (P40) in low temperature stress group (4°C) and normal temperature group (18°C) as experimental materials. The results showed that a total of 127,939 Unigenes with average length of 1,190 bp were obtained by assembly, of which 46% were annotated in the Nr database. A total of 1,698 differentially expressed genes were screened by differential gene expression analysis, of which 920 genes showed up-regulated expression and 778 genes showed down-regulated expression. Both GO and KEGG enrichment analysis revealed that differentially expressed genes were enriched in spliceosomes, ribosomes, bile secretion, ABC transport pathways, and cellular nitrogen compound synthesis. A further in-depth analysis obtained 8 genes that may be associated with low-temperature traits of F. chinensis. Five of them displayed up-regulated expression, including ATP-binding cassette protein C, acid ceramidase, glutathione transferase, C-type lectin and heat shock protein HSP70. The remaining three genes, γ-butyl betaine hydroxylase, ß-hexosaminidase A and long chain fatty acid-CoA ligase displayed down-regulated expression. Eight differentially expressed genes were randomly selected and the real time RT-PCR verification showed that their expression levels were consistent with the sequencing results, demonstrating the accuracy of the sequencing results. The results of this study provide basic data for revealing the molecular mechanisms of F. chinensis in response to low temperature stress and the molecular assisted breeding of F. chinensis in low temperature.

Insights into the molecular basis of immunosuppression and increasing pathogen infection severity of ammonia toxicity by transcriptome analysis in pacific white shrimp Litopenaeus vannamei.

The high concentration of ammonia resulting from intensive culture system and environmental pollution could cause disease occurrence in shrimp, but little information is available on its molecular mechanisms. In this study, we performed comparative transcriptome analysis among WSSV-infected shrimp under ammonia stress (LAV), WSSV-infected shrimp under normal water (LV), and normal shrimp under ammonia stress (LA) groups to identify the key genes and pathways involved in immunosuppression and increasing pathogen infection severity caused by ammonia toxicity in Litopenaeus vannamei. Totally, 526 significantly differential expressed genes (DEGs) were identified in LAV group compared to LV and LA groups, among which 270 genes were lost expressed and 67 genes uniquely expressed in the LAV group. According to the public functional reports for the annotated DEGs, they potentially involved in the following functions: (1) accelerating pathogen adhesion, invasion and multiplication; (2) reducing the ability for pathogen defense and immune response; (3) inhibiting positive regulation of apoptotic and antioxidant defense for host homeostasis; (4) inhibiting transcription and protein transport; (5) and increasing protein methylation and ubiquitination, etc. A total of 13 pathways were obtained mainly involving in this process, which mainly led to the following changes: (1) increasing the immunosuppression, anemia, endocrine dysfunction, neurotoxic effect and neuroinvasion, atherosclerosis and thrombogenesis, blood-brain barrier penetration, thyroid disorder, necrosis, inflammation, and circadian disturbance; (2) reducing the ability of vascular remodeling, angiogenesis, cell survival, migration, apoptosis, and lymph transferred to blood stream; (3) leading to cell hypertrophy, cellular shape changes, and mesangial matrix expansion. The present results firstly supplied molecular mechanisms for the ammonia toxicity inhibiting the immune system and increasing pathogen infection severity in shrimp, which is a prerequisite for better understanding the pathogenesis caused by ammonia toxicity.

Isolation and characterization of a Raf gene from Chinese shrimp Fenneropenaeus chinensis in response to white spot syndrome virus infection.

Raf is a member in the Ras/Raf/MAPKK/MAPK signaling transduction pathway. To obtain a better understanding of Raf in the interaction between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), the sequence of cDNA of Raf from F. chinensis (FcRaf) was obtained. The FcRaf gene contained a 2421 bp open reading frame (ORF). The FcRaf shared most characteristic of Raf protein, such as the Raf-like Ras-binding domain (RBD), phorbol esters/diacylglycerol binding domain (C1 domain), and catalytic domain of the serine/threonine kinases, Raf (STKc_Raf). The sequence of functional domains of Raf protein was relatively conserved. The FcRaf mRNA was detected in the tissues of gill, muscle, and hepatopancreas from normal F. chinensis. The mRNA abundance level of FcRaf in the gill was the highest, which was 2.7-fold the level in the hepatopancreas. The expression level of FcRaf was significantly (P < 0.05) up-regulated in the tissues of gill, muscle, and hepatopancreas post WSSV-infection, which suggested that FcRaf might be involved in the interaction between F. chinensis and WSSV. Two SNP loci were identified in the ORF, one of which was a C-T mis-sense mutation, where an Ala was replaced by a Val, and induced the predicted protein secondary structure change. Considering the relatively low MAF (0.07), whether this mis-sense mutation was a detrimental mutation needs further investigation.