Integrated transcriptome and microRNA analysis reveals molecular responses to high-temperature stress in the liver of American shad (Alosa sapidissima).

BACKGROUND: Fish reproduction, development and growth are directly affected by temperature, investigating the regulatory mechanisms behind high temperature stress is helpful to construct a finer molecular network. In this study, we systematically analyzed the transcriptome and miRNA information of American shad (Alosa sapidissima) liver tissues at different cultivation temperatures of 24 â„ƒ (Low), 27 â„ƒ (Mid) and 30 â„ƒ (High) based on a high-throughput sequencing platform. RESULTS: The results showed that there were 1594 differentially expressed genes (DEGs) and 660 differentially expressed miRNAs (DEMs) in the LowLi vs. MidLi comparison group, 473 DEGs and 84 DEMs in the MidLi vs. HighLi group, 914 DEGs and 442 DEMs in the LowLi vs. HighLi group. These included some important genes and miRNAs such as calr, hsp90b1, hsp70, ssa-miR-125a-3p, ssa-miR-92b-5p, dre-miR-15a-3p and novel-m1018-5p. The DEGs were mainly enriched in the protein folding, processing and export pathways of the endoplasmic reticulum; the target genes of the DEMs were mainly enriched in the focal adhesion pathway. Furthermore, the association analysis revealed that the key genes were mainly enriched in the metabolic pathway. Interestingly, we found a significant increase in the number of genes and miRNAs involved in the regulation of heat stress during the temperature change from 24 °C to 27 °C. In addition, we examined the tissue expression characteristics of some key genes and miRNAs by qPCR, and found that calr, hsp90b1 and dre-miR-125b-2-3p were significantly highly expressed in the liver at 27 â„ƒ, while novel-m0481-5p, ssa-miR-125a-3p, ssa-miR-92b-5p, dre-miR-15a-3p and novel-m1018-5p had the highest expression in the heart at 30℃. Finally, the quantitative expression trends of 10 randomly selected DEGs and 10 DEMs were consistent with the sequencing data, indicating the reliability of the results. CONCLUSIONS: In summary, this study provides some fundamental data for subsequent in-depth research into the molecular regulatory mechanisms of A. sapidissima response to heat stress, and for the selective breeding of high temperature tolerant varieties.

Single nucleotide polymorphism SNP19140160 A > C is a potential breeding locus for fast-growth largemouth bass (Micropterus salmoides).

BACKGROUND: Largemouth bass (Micropterus salmoides) has significant economic value as a high-yielding fish species in China's freshwater aquaculture industry. Determining the major genes related to growth traits and identifying molecular markers associated with these traits serve as the foundation for breeding strategies involving gene pyramiding. In this study, we screened restriction-site associated DNA sequencing (RAD-seq) data to identify single nucleotide polymorphism (SNP) loci potentially associated with extreme growth differences between fast-growth and slow-growth groups in the F1 generation of a largemouth bass population. RESULTS: We subsequently identified associations between these loci and specific candidate genes related to four key growth traits (body weight, body length, body height, and body thickness) based on SNP genotyping. In total, 4,196,486 high-quality SNPs were distributed across 23 chromosomes. Using a population-specific genotype frequency threshold of 0.7, we identified 30 potential SNPs associated with growth traits. Among the 30 SNPs, SNP19140160, SNP9639603, SNP9639605, and SNP23355498 showed significant associations; three of them (SNP9639603, SNP9639605, and SNP23355498) were significantly associated with one trait, body length, in the F1 generation, and one (SNP19140160) was significantly linked with four traits (body weight, height, length, and thickness) in the F1 generation. The markers SNP19140160 and SNP23355498 were located near two growth candidate genes, fam174b and ppip5k1b, respectively, and these candidate genes were closely linked with growth, development, and feeding. The average body weight of the group with four dominant genotypes at these SNP loci in the F1 generation population (703.86 g) was 19.63% higher than that of the group without dominant genotypes at these loci (588.36 g). CONCLUSIONS: Thus, these four markers could be used to construct a population with dominant genotypes at loci related to fast growth. These findings demonstrate how markers can be used to identify genes related to fast growth, and will be useful for molecular marker-assisted selection in the breeding of high-quality largemouth bass.

Proteomics and metabolomics analysis of American shad (Alosa sapidissima) liver responses to heat stress.

The dramatic changes in the global climate pose a major threat to the survival of many organisms, including fish. To date, the regulatory mechanisms behind the physiological responses of fish to temperature changes have been studied, and a comprehensive analysis of the regulatory mechanisms of temperature tolerance will help to propose effective strategies for fish to cope with global warming. In this study, we investigated the expression profiles of proteins and metabolites in liver tissues of American shad (Alosa sapidissima) corresponding to different water temperatures (24 °C, 27 °C and 30 °C) at various times (1-month intervals) under natural culture conditions. Proteomic analysis showed that the expression levels of the heat shock protein family (e.g. HSPE1, HSP70, HSPA5 and HSPA.1) increase significantly with temperature and that many differentially expressed proteins were highly enriched especially in pathways related to the endoplasmic reticulum, oxidative phosphorylation and glycolysis/gluconeogenesis processes. In addition, the results of conjoint metabolomics and proteomics analysis suggested that the contents of several important amino acids and chemical compounds, including L-serine, L-isoleucine, L-cystine, choline and betaine, changed significantly under high-temperature environmental stress, affecting the metabolic levels of starch, amino acid and glucose, which is thought to represent a possible energy conservation method for A. sapidissima to cope with rapid changes in external temperature. In summary, our findings demonstrate that living under high temperatures for a long period of time leads to different physiological defense responses in A. sapidissima, which provides some new ideas for analyzing the molecular regulatory patterns of adaptation to high temperature and also provides a theoretical basis for the subsequent improvement of fish culture in response to global warming.

Molecular characterization and function of hif1a and fih1 in response to acute thermal stress in American shad (Alosa sapidissima).

In order to evaluate the function of hypoxia-inducible factor-1 alpha (hif1α) and factor inhibiting hif1α (fih1) in response to thermal stress, we first conducted a functional analysis of A. sapidissima hif1α and fih1, and determined hif1α and fih1 expressions in different tissues in response to thermal stress based on identified housekeeping genes (HKGs). The results showed that hif1α and fih1 were mainly located in the nucleus and cytoplasm. The full length cDNA sequence of hif1α and fih1 was 4073 bp and 2759 bp, respectively. The cDNA sequence of hif1α includes 15 exons encoding 750 amino acid residues, and the full length cDNA sequence of fih1 contains 9 exons encoding 354 amino acid residues. During the acute thermal stress transferring from 16 ± 0.5 °C (control) to 20 ± 0.5 °C, 25 ± 0.5 °C, and 30 ± 0.5 °C for 15 min, it was found that the expression trends of hif1α and fih1 showed an inhibitory regulation in the heart, while they consistently expressed in brain, intestine, muscle, gill, kidney and liver. In conclusion, this is the first study to identify the tissue-specific HKGs in A. sapidissima and found that ef1α and ß-actin are the most suitable HKGs. Hif1α and Fih1 are mainly the nuclear and cytoplasmic proteins, respectively, having high levels in the heart and brain. Alosa sapidissima countered a temperature increase from 16 to 25 ℃ by regulating the expressions of hif1α and fih1, but their physiological regulatory functions were unable to cope with acute thermal stress when the temperature difference was 14 ℃ (from 16 to 30 ℃).

microRNA regulation of skin pigmentation in golden-back mutant of crucian carp from a rice-fish integrated farming system.

BACKGROUND: MicroRNAs (miRNAs) are endogenous small non-coding RNAs (21-25 nucleotides) that act as essential components of several biological processes. Golden-back crucian carp (GBCrC, Carassius auratus) is a naturally mutant species of carp that has two distinct body skin color types (golden and greenish-grey), making it an excellent model for research on the genetic basis of pigmentation. Here, we performed small RNA (sRNA) analysis on the two different skin colors via Illumina sequencing. RESULTS: A total of 679 known miRNAs and 254 novel miRNAs were identified, of which 32 were detected as miRNAs with significant differential expression (DEMs). 23,577 genes were projected to be the targets of 32 DEMs, primarily those involved in melanogenesis, adrenergic signaling in cardiomyocytes, MAPK signaling pathway and wnt signaling pathway by functional enrichment. Furthermore, we built an interaction module of mRNAs, proteins and miRNAs based on 10 up-regulated and 13 down-regulated miRNAs in golden skin. In addition to transcriptional destabilization and translational suppression, we discovered that miRNAs and their target genes were expressed in the same trend at both the transcriptional and translational levels. Finally, we discovered that miR-196d could be indirectly implicated in regulating melanocyte synthesis and motility in the skin by targeting to myh7 (myosin-7) gene through the luciferase reporter assay, antagomir silencing in vivo and qRT-PCR techniques. CONCLUSIONS: Our study gives a systematic examination of the miRNA profiles expressed in the skin of GBCrC, assisting in the comprehension of the intricate molecular regulation of body color polymorphism and providing insights for C. auratus breeding research.

Comparative analysis of the gut microbiota in bighead carp under different culture patterns.

AIMS: To investigate the phylogenetic composition and functional potential of bighead carp (Hypophthalmichthys nobilis) gut microbiome in two rearing patterns (bighead carp polycultured with Oreochromis niloticus in pond A and bighead carp polycultured with Cyprinus carpio in pond B, respectively), as well as the changes of plankton in the cultured water at four different time points. METHODS AND RESULTS: The intestinal contents were sequenced using Illumina HiSeq of bacterial 16S rRNA. Cyanophyta and Chlorophyta were the prevalent phytoplankton in the water, whereas Rotifers and Protozoa were the predominant zooplankton. In all, 779,563 quality-filtered sequences and 8870 amplicon sequence variants were obtained from 24 samples that numbered T1A1 to T4A3 and T1B1 to T4B3, resulting in 35 phyla, with Proteobacteria, Firmicutes, Fusobacteria and Cyanobacteria dominating. According to alpha diversity and beta diversity measurements, the bacterial communities were diverse, Chao1 richness and Pielou's evenness were significantly lower in the T2B and T4B groups. The gut bacterial communities of T1A, T1B, T2A and T2B groups differed from those of other samples, which formed distinctly clusters with principal coordinate analysis and non-metric multidimensional scaling analysis. PICRUSt2 predictive function analysis revealed that different culture patterns influenced the gut microbiota metabolic capacity. CONCLUSIONS: Intestinal bacteria belonging to the phyla Proteobacteria, Firmicutes, Cyanobacteria and Fusobacteria are better suited to inhabit in various environments and perform specific functions. Furthermore, contact with the external environment and nutrient intake also stimulate the variety of intestinal microbiotas in polycultured bighead carp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first comprehensive, high-throughput investigation of gut microbiota diversity in bighead carp during various seasons in two polycultured patterns and provide preliminary information on gut microbiome composition and changes, laying a crucial foundation for future research on fish culture patterns in various environments.

Molecular and functional analysis of the microphthalmia-associated transcription factor (mitf) gene duplicates in red tilapia.

In vertebrates, the microphthalmia-associated transcription factor (mitf) is at the hub of the melanin synthesis regulation network. However, little information is known about its molecular characterization, expression, location, or function in skin color differentiation and variation of red tilapia. The full-length cDNA sequences (1977 bp and 1999 bp) of mitfa and mitfb, encoding polypeptides of 491 and 514 amino acids, were effectively identified from red tilapia in this study. The Mitfa and Mitfb sequences of red tilapia clustered first with O. aureus, then with other teleost fish, according to phylogenetic analysis. Mitfa and mitfb mRNA were highly expressed in the brain, dorsal skin and eye tissues using quantitative real-time PCR. The mRNA expressions of mitfa and mitfb were the highest in the cleavage stage during the early development of red tilapia. Among three different colors of red tilapia, the expression levels of mitfa and mitfb were highest in the PB (pink with scattered black spots) dorsal skin. After overwintering, the mitfa and mitfb mRNA expressions were high in the dorsal skin of PB (color changed from pink to black). Mitfa and mitfb were mostly found in the epidermal layer of the dorsal skin, according to in situ hybridization (ISH) analysis. After injecting mitf-dsRNA duplicates along the tail vein of red tilapia, the activity of tyrosinase and the level of melanin in the dorsal skin both decreased significantly. The mRNA expressions of mitfa and its downstream genes (tyrb, tyrp1a and dct) decreased, whereas the mRNA expression of mitfb increased after mitfa-dsRNA injection. The mRNA expressions of mitfb, tyrb, tyrp1a and dct decreased, whereas the mRNA expression of mitfa increased after injecting mitfb-dsRNA. These findings suggest that mitf gene duplicates may play an important role in red tilapia skin color differentiation and variation via the melanogenesis pathway.

The stage-specific long non-coding RNAs and mRNAs identification and analysis during early development of common carp, Cyprinus carpio.

Cyprinus carpio is considered an alternative vertebrate fish model to zebrafish. However, systemic times-series research on the lncRNAs and mRNAs during early development of C. carpio has not been reported yet. This study provides the first long non-coding RNA (lncRNA)-mRNA expression profiles during six main early development stages (2 h post-fertilization hpf, 6 hpf, 12 hpf, 20 hpf, 64 hpf and 1 day post-hatching). A total of 51,979 lncRNAs were identified. We screened the top 10 abundance lncRNAs and mRNAs and stage-specific lncRNAs and mRNAs (specificity measure SPM > 0.9). We identified significant differentially expressed lncRNAs and mRNAs (|log2 (fold change)| ≥ 1 and false discovery rate FDR of <0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified numerous signaling pathways. Additionally, the lncRNA-mRNA co-regulated network analysis of two lncRNAs (lncrps25 and malat1) and two mRNAs (mitf and troponin T) were investigated. Our results provide new insight into the role of lncRNAs and mRNAs, and would advance the understanding of lncRNA-mediated mechanisms in early development of fish.

Transcriptome analysis of skin color variation during and after overwintering of Malaysian red tilapia.

The commercial value of red tilapia is hampered by variations in skin color during overwintering. In this study, three types of skin of red tilapia, including the skin remained pink color during and after overwintering (P), the skin changed from pink color to black color during overwintering and remained black color after overwintering (P-B), and the skin changed from pink color to black color during overwintering but recovered to pink color when the temperature rose after overwintering (P-B-P), were used to analyze their molecular mechanisms of color variation. The transcriptome results revealed that the P, P-B, and P-B-P libraries had 43, 42, and 43 million clean reads, respectively. The top 10 abundance mRNAs and specific mRNAs (specificity measure SPM > 0.9) were screened. After comparing intergroup gene expression levels, there were 2528, 1924, and 1939 differentially expressed genes (DEGs) between P-B-P and P-B, P-B-P and P, and P-B and P, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of color-related mRNAs showed that a number of DEGs, including tyrp1, tyr, pmel, mitf, mc1r, asip, tat, hpdb, and foxd3, might play a potential role in pigmentation. Additionally, the co-expression patterns of genes were detected within the pigment-related pathways by the PPI network from P-B vs. P group. Furthermore, DEGs from the apoptosis and autophagy pathways, such as baxα, beclin1, and atg7, might be involved in the fading of red tilapia melanocytes. The findings will aid in understanding the molecular mechanism underlying skin color variation in red tilapia during and after overwintering as well as lay a foundation for future research aimed at improving red tilapia skin color characteristics.

Streptomyces qinzhouensis sp. nov., a mangrove soil actinobacterium.

A novel Streptomyces strain (SSL-25T) was isolated from mangrove soil sampled at QinzhouBay, PR China. The isolate was observed to be Gram-stain-positive and to form greyish-white aerial mycelia that differentiated into straight spore chains with smooth-surfaced spores on International Streptomyces Project 2 medium. The cell-wall peptidoglycan was determined to contain ll-diaminopimelicacid. The cell-wall sugars were glucose and mannose. The predominant menaquinones were MK-9 (H6), MK-9 (H8) and MK-9 (H4). The major polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and several unidentified phospholipids. The predominant cellular fatty acids were C16:0, iso-C16:0 and summed feature 3 (C16:1ω7c/C16:1ω6c). The genome size of strain SSL-25T was 8.1 Mbp with a G+C content of 71.5 mol%. Phylogenetic analysis indicated that strain SSL-25T is closely related to Streptomyces tsukubensis NRRL 18488T (99.4 % sequence similarity). However, the digital DNA-DNA hybridization (39.8 %) and average nucleotide identity (91.3 %) values between them showed that it represents a distinct species. Furthermore, the results of morphological, physiological and biochemical tests allowed further phenotypic differentiation of strain SSL-25T from S. tsukubensis NRRL 18488T. Therefore, based on these results, it is concluded that strain SSL-25T represents a novel Streptomyces species, for which the name Streptomyces qinzhouensis sp. nov. is proposed. The type strain is SSL-25T (=CICC 11054T=JCM33585T).

Dynamic transcriptome sequencing and analysis during early development in the bighead carp (Hypophthalmichthys nobilis).

BACKGROUND: Early development is a key process of the life history of fish. However, the relationship between the transcriptome and the dynamic regulation of early development is still uncharacterized in the bighead carp (Hypophthalmichthys nobilis). In the present study, we performed transcriptome analysis of six development stages in H. nobilis, aiming to understand the dynamic molecular regulation of early development in this fish. RESULTS: A total of 76,573 unigenes were assembled from clean sequence reads, with an average length of 1768 base. Among which, 41,742 (54.54%) unigenes were annotated to public protein databases, and an additional 59,014 simple sequence repeat (SSR) loci were identified among the unigenes. Furthermore, 30,199 differentially expressed transcripts (DETs) (fold change > 4 or < 0.25, and the false discovery rate FDR < 0.01) were observed in comparisons between the adjacent developmental stages, and nine expression patterns (profiles) were simulated using series-cluster analysis across six developmental stages. The unigenes expression level markedly increased after the DS1 stage (early blastula), and the numbers of DETs gradually decreased during subsequent development. The largest transcriptomic change (up- or down-regulated) was detected during the period from DS1 to DS2 (6-somite stage), which was enriched for many biological processes and metabolic pathways related to maternal to zygotic transition (MZT). Distinctly protein-protein interaction (PPI) networks were plotted for DETs during the period from DS1 to DS2. The genes (or proteins) from the same pathways were integrated together, and showed with obvious co-regulation patterns. In the series-cluster analysis, a remarkable profile of gene expression (profile_48) was identified that is probably related to the hatching during H. nobilis development, and the strict co-expression of a hatching enzyme gene (hce1) with 33 other annotated genes was identified from this profile. CONCLUSIONS: The results indicated that strict dynamic regulation occurs during the early development in H. nobilis, especially in embryogenesis before hatching. This study provides valuable new information and transcriptomic resources related to H. nobilis early development, and for certain events such as MZT and hatching.

Integrated analysis of long non-coding RNA and mRNA expression in different colored skin of koi carp.

BACKGROUND: Long non-coding RNAs (lncRNAs) perform crucial roles in biological process involving complex mechanisms. However, information regarding their abundance, characteristics and potential functions linked to fish skin color is limited. Herein, Illumina sequencing and bioinformatics were conducted on black, white, and red skin of Koi carp (Cyprinus carpio L.). RESULTS: A total of 590,415,050 clean reads, 446,614 putative transcripts, 4252 known and 72,907 novel lncRNAs were simultaneously obtained, including 92 significant differentially expressed lncRNAs and 722 mRNAs. Ccr_lnc5622441 and Ccr_lnc765201 were up-regulated in black and red skin, Ccr_lnc14074601 and Ccr_lnc2382951 were up-regulated in white skin, and premelanosome protein a (Pmela), Pmelb and tyrosinase (Tyr) were up-regulated in black skin. The expression patterns of 18 randomly selected differentially expressed genes were validated using the quantitative real-time PCR method. Moreover, 70 lncRNAs acting on 107 target mRNAs in cis and 79 lncRNAs acting on 41,625 target mRNAs in trans were investigated. The resulting co-expression networks revealed that a single lncRNA can connect with numerous mRNAs, and vice versa. To further reveal their potential functions, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed, and membrane, pigment cell development, cAMP signaling, melanogenesis and tyrosine metabolism appear to affect skin pigmentation. Additionally, three lncRNAs (Ccr_lnc142711, Ccr_lnc17214525 and Ccr_lnc14830101) and three mRNAs (Asip, Mitf and Tyr) involved in the melanogenesis pathway were investigated in terms of potential functions in embryogenesis and different tissues. CONCLUSIONS: The findings broaden our understanding of lncRNAs and skin color genetics, and provide new insight into the mechanisms underlying lncRNA-mediated pigmentation and differentiation in Koi carp.

Population structure, demographic history and local adaptation of the grass carp.

BACKGROUND: Genetic diversity within a species reflects population evolution, ecology, and ability to adapt. Genome-wide population surveys of both natural and introduced populations provide insights into genetic diversity, the evolutionary processes and the genetic basis underlying local adaptation. Grass carp is the most important freshwater foodfish species for food and water weed control. However, there is as yet no overall picture on genetic variations and population structure of this species, which is important for its aquaculture. RESULTS: We used 43,310 SNPs to infer the population structure, evidence of local adaptation and sources of introduction. The overall genetic differentiation of this species was low. The native populations were differentiated into three genetic clusters, corresponding to the Yangtze, Pearl and Heilongjiang River Systems, respectively. The populations in Malaysia, India and Nepal were introduced from both the Yangtze and Pearl River Systems. Loci and genes involved in putative local selection for native locations were identified. Evidence of both positive and balancing selection was found in the introduced locations. Genes associated with loci under putative selection were involved in many biological functions. Outlier loci were grouped into clusters as genomic islands within some specific genomic regions, which likely agrees with the divergence hitchhiking scenario of divergence-with-gene-flow. CONCLUSIONS: This study, for the first time, sheds novel insights on the population differentiation of the grass carp, genetics of its strong ability in adaption to diverse environments and sources of some introduced grass carp populations. Our data also suggests that the natural populations of the grass carp have been affected by the aquaculture besides neutral and adaptive forces.

Characterization and functional analysis of slc7a11 gene, involved in skin color differentiation in the red tilapia.

Red tilapia has become more popular for aquaculture production in China in recent years. However, the pigmentation differentiation that has resulted from the process of genetic breeding and skin color variation during the overwintering period are the main problems limiting the development of commercial culture. The genetic basis of skin color differentiation is still not understood. Solute carrier family 7 member 11 (slc7a11) has been identified to be a critical genetic regulator of pheomelanin synthesis in the skin of mammals. However, little information is available about its molecular characteristics, expression, location and function in skin color differentiation of fish. In this study, three complete cDNA sequences (2159 bp, 2190 bp and 2249 bp) of slc7a11 were successfully isolated from Malaysian red tilapia, encoding polypeptides of 492, 525 and 492 amino acids respectively. Quantitative real-time PCR demonstrated that slc7a11 mRNA expression is high in the ventral skin of PR (pink with scattered red spots) fish. Immunofluorescence analysis revealed that xCT (the protein encoded by slc7a11) was concentrated mainly in the cytoplasm and nucleus of both the dorsal and ventral skin cells of fish. After RNA interference of slc7a11, slc7a11 and cbs mRNA expressions decreased, but the tyr mRNA expression increased in the skin of fish. Results suggest that slc7a11 plays an important role in skin color formation and differentiation of red tilapia through the melanogenesis pathway.

Identification and characterization of skin color microRNAs in Koi carp (Cyprinus carpio L.) by Illumina sequencing.

BACKGROUND: MicroRNAs (miRNAs) are endogenous, small (21-25 nucleotide), non-coding RNAs that play important roles in numerous biological processes. Koi carp exhibit diverse color patterns, making it an ideal subject for studying the genetics of pigmentation. However, the influence of miRNAs on skin color regulation and variation in Koi carp is poorly understood. RESULTS: Herein, we performed small RNA (sRNA) analysis of the three main skin colors in Koi carp by Illumina sequencing. The results revealed 330, 397, and 335 conserved miRNAs (belonging to 81 families) and 340, 353, and 351 candidate miRNAs in black, red, and white libraries, respectively. A total of 164 differentially expressed miRNAs (DEMs) and 14 overlapping DEMs were identified, including miR-196a, miR-125b, miR-202, miR-205-5p, miR-200b, and etc. Target prediction and functional analysis of color-related miRNAs such as miR-200b, miR-206, and miR-196a highlighted putative target genes, including Mitf, Mc1r, Foxd3, and Sox10 that are potentially related to pigmentation. Determination of reference miRNAs for relative quantification showed that let-7a was the most abundant single reference gene, and let-7a and miR-26b was the most abundant combination. CONCLUSIONS: The findings provide novel insight into the molecular mechanisms determining skin color differentiation in Koi carp, and serve as a valuable reference for future studies on tissue-specific miRNA abundance in Koi carp.

ZDHHC7-mediated S-palmitoylation of Scribble regulates cell polarity.

Scribble (SCRIB) is a tumor-suppressor protein, playing critical roles in establishing and maintaining epithelial cell polarity. SCRIB is frequently amplified in human cancers but does not localize properly to cell-cell junctions, suggesting that mislocalization of SCRIB disrupts its tumor-suppressive activities. Using chemical reporters, here we showed that SCRIB localization was regulated by S-palmitoylation at conserved cysteine residues. Palmitoylation-deficient mutants of SCRIB were mislocalized, leading to disruption of cell polarity and loss of their tumor-suppressive activities to oncogenic YAP, MAPK and PI3K/AKT pathways. We further found that ZDHHC7 was the major palmitoyl acyltransferase regulating SCRIB. Knockout of ZDHHC7 led to SCRIB mislocalization and YAP activation, and disruption of SCRIB's suppressive activities in HRas(V12)-induced cell invasion. In summary, we demonstrated that ZDHHC7-mediated SCRIB palmitoylation is critical for SCRIB membrane targeting, cell polarity and tumor suppression, providing new mechanistic insights of how dynamic protein palmitoylation regulates cell polarity and tumorigenesis.

Baseline Hepatitis B Virus Titer Predicts Initial Postpartum Hepatic Flare: A Multicenter Prospective Study.

BACKGROUND AND GOALS: A series of changes in the immune system occur during pregnancy and puerperium. Currently, we aim to characterize both the natural changes in liver inflammation and its association with hepatitis B viremia during this special period. PATIENTS AND METHODS: Chronic hepatitis B (CHB) gravidas were recruited and followed up to 52 weeks postpartum. Virological and biochemical parameters were assessed throughout the period. RESULTS: A total of 1097 CHB mothers had finished the entire follow-up including 451 accepting telbivudine, 178 accepting tenofovir, and 468 without antiviral therapy. Among the mothers, 11.94% went through hepatic flare in the first trimester and the rate decreased to 2.1% at the time of delivery. Nevertheless, a much higher frequency (19.78%) was observed in the early postpartum. Interestingly, alanine aminotransferase level decreased along with the development of pregnancy and then suddenly increased in the first month of puerperium. In addition, a downward trend was observed on the titer of HBsAg and HBeAg after delivery. Of note, an obvious higher frequency of alanine aminotransferase flare was revealed in mothers with high viremia (>6 log10 IU/mL). With multivariate analysis, only hepatitis B virus titer at baseline was strongly associated with hepatic flare during early postpartum (95% confidence interval, 1.012-3.049, P=0.045). The predictive rates of hepatic flare at baseline viral load of 6, 7, and 8 log10 IU/mL were 16.67%, 28.30%, and 30.60%, respectively. CONCLUSIONS: CHB gravidas with high viremia should be monitored closely during entire pregnancy, and extended antiviral therapy is recommend to those mothers with baseline viremia >7 log10 IU/mL.

Comparative microRNA-seq Analysis Depicts Candidate miRNAs Involved in Skin Color Differentiation in Red Tilapia.

Differentiation and variation in body color has been a growing limitation to the commercial value of red tilapia. Limited microRNA (miRNA) information is available on skin color differentiation and variation in fish so far. In this study, a high-throughput Illumina sequencing of sRNAs was conducted on three color varieties of red tilapia and 81,394,491 raw reads were generated. A total of 158 differentially expressed miRNAs (|log2(fold change)| ≥ 1 and q-value ≤ 0.001) were identified. Target prediction and functional analysis of color-related miRNAs showed that a variety of putative target genes—including slc7a11, mc1r and asip—played potential roles in pigmentation. Moreover; the miRNA-mRNA regulatory network was illustrated to elucidate the pigmentation differentiation, in which miR-138-5p and miR-722 were predicted to play important roles in regulating the pigmentation process. These results advance our understanding of the molecular mechanisms of skin pigmentation differentiation in red tilapia.

Total Synthesis and Stereochemical Assignment of Delavatine A: Rh-Catalyzed Asymmetric Hydrogenation of Indene-Type Tetrasubstituted Olefins and Kinetic Resolution through Pd-Catalyzed Triflamide-Directed C-H Olefination.

Delavatine A (1) is a structurally unusual isoquinoline alkaloid isolated from Incarvillea delavayi. The first and gram-scale total synthesis of 1 was accomplished in 13 steps (the longest linear sequence) from commercially available starting materials. We exploited an isoquinoline construction strategy and developed two reactions, namely Rh-catalyzed asymmetric hydrogenation of indene-type tetrasubstituted olefins and kinetic resolution of ß-alkyl phenylethylamine derivatives through Pd-catalyzed triflamide-directed C-H olefination. The substrate scope of the first reaction covered unfunctionalized olefins and those containing polar functionalities such as sulfonamides. The kinetic resolution provided a collection of enantioenriched indane- and tetralin-based triflamides, including those bearing quaternary chiral centers. The selectivity factor (s) exceeded 100 for a number of substrates. These reactions enabled two different yet related approaches to a key intermediate 28 in excellent enantiopurity. In the synthesis, the triflamide served as not only an effective directing group for C-H bond activation but also a versatile functional group for further elaborations. The relative and absolute configurations of delavatine A were unambiguously assigned by the syntheses of the natural product and its three stereoisomers. Their cytotoxicity against a series of cancer cell lines was evaluated.