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Aim Tumor cell-derived microparticles (MP) can function as a targeted delivery carrier for anti-tumor drugs. Here, we aimed to generate paclitaxel-loaded microparticles (MP-PTX) from HeLa cells and examined its therapeutic potential on human cervical carcinoma. Methods MP-PTX was generated from HeLa cells by ultraviolet radiation and subsequent centrifugation. The particle size, drug loading rate, and stability of MP-PTX were examined in vitro. Flow cytometry and the MTT assay were performed to test the inhibitory effect of MP-PTX using different cell lines. Immunodeficient mice bearing HeLa cervical carcinoma were treated with 0.9% normal saline, MP, paclitaxel (PTX) (2.5 mg/kg), or MP-PTX (PTX content identical to PTX group) every day for 6 consecutive days. Tumor volume and animal survival were observed. Micro 18F-FDG PET/CT was performed to monitor the therapeutic efficacy. The proliferation activity of cells and microvessel density in tumor tissues were determined by immunohistochemical staining using Ki-67 and CD31, respectively. Results Dynamic laser scattering measurements showed that the particle size of MP-PTX was 285.58 ± 2.95 nm and the polydispersity index was 0.104 ± 0.106. And the particle size of MP-PTX was not change at 4°C for at least one week. More than 1% of PTX in the medium could be successfully encapsulated into HeLa cell-derived MP. When compared with PTX, MP-PTX treatment significantly increased apoptosis of tumor cells and reduced their proliferation. In addition, MP-PTX showed lower toxicity to normal human umbilical vein endothelial cells (HUVEC) than PTX. In vivo studies further demonstrated that MP-PTX treatment significantly inhibited the growth of cervical carcinoma, prolonged the survival of tumor-bearing mice, and reduced the toxicity of PTX. Immunohistochemical staining revealed that MP-PTX treatment led to decreased Ki-67 positive tumor cells and decreased microvessel density in tumor tissues. Conclusion Our results demonstrated that HeLa-derived MP-PTX significantly enhanced the anti-cancer effects of PTX with reduced toxicity, which may provide a novel strategy for the treatment of cervical carcinoma.

Marine hypoxia caused by nutrient enrichment in coastal waters has become a global problem for decades, especially diel-cycling hypoxia that occurs frequently in the summer season. On the contrary, sudden rainstorms, and freshwater discharge make salinity in estuarine and coastal ecosystems variable, which often occurs with hypoxia. We found mass mortality of the Hong Kong oyster Crassostrea hongkongensis in the field where hypoxia and salinity fluctuation co-occur in the summer season during the past several years. To investigate the effects of diel-cycling hypoxia and salinity changes on the hemocyte immune function of C. hongkongensis, oysters were exposed to a combined effect of two dissolved oxygen (DO) concentrations (24 h normal oxygen 6 mg/L, 12 h normal oxygen 6 mg/L, and 12 h hypoxia 2 mg/L) and three salinities (10, 25, and 35‰) for 14 days. Subsequently, all treatments were restored to constant normal oxygen (6 mg/L) and salinity under 25‰ for 3 days to study the recovery of hemocyte immune function from the combined stress. Hemocyte parameters were analyzed by flow cytometry, including hemocyte mortality (HM), total hemocyte count (THC), phagocytosis (PHA), esterase (EST) activity, reactive oxygen species (ROS), lysosomal content (LYSO), and mitochondrial number (MN). The experimental results showed that diel-cycling hypoxia and salinity changes have obvious interactive effects on various immune parameters. In detail, diel-cycling hypoxia and decreases in salinity led to increased HM, and low salinity caused heavier impacts. In addition, low salinity, and diel-cycling hypoxia also led to decreases in LYSO, EST, and THC, while the decrease of PHA only occurs in the early stage. On the contrary, ROS production increased significantly under low salinity and hypoxic conditions. After 3-day recovery, THC, PHA, EST, LYSO, and MN were basically restored to normal, while HM and ROS were still significantly affected by diel-cycling hypoxia and salinity change, indicating that the combined stress of diel-cycling hypoxia and salinity changes had latent effects on the immune function of C. hongkongensis. Our results highlight that diel-cycling hypoxia and salinity change may impair the health and survival of the Hong Kong oyster C. hongkongensis and may be the key factors for the mass mortality of this oyster in the field.

The Trachinotus ovatus (T. ovatus) reach sexual maturity as late as 4–5 years, and there are no obvious morphology differences between males and females, even at maturity, making the selection of male and female parents for selective breeding difficult. To examine the potential regulatory mechanism of sexual differentiation, we conducted a microRNAs (miRNAs) analysis on the ovaries and testes of T. ovatus. A total of 13,423,691 and 11,734,953 raw reads, representing 91,883,908 and 86,879,726 unique sequences of 18–35 nt length obtained from the ovaries and testes, respectively. After mapping to the T. ovatus transcriptome reference sequence (unpublished data), and comparing the miRNA sequences with the miRBase database, 179 known mature miRNAs and 100 novel miRNAs were identified. Expression analysis showed that, 165 miRNAs were differentially expressed between the ovary and testis. To validate the Illumina results, the expression patterns of the nine most differently expressed miRNAs (dre-miR-7a, dre-miR-7b, dre-miR-153a-3p, dre-miR-144-3p, dre-miR-301a, dre-miR-92a-3p, dre-miR-727-5p, Novel 124, and Novel 190, as well as those of five miRNAs that may relate to gonad development (dre-miR-143, dre-miR-101a, dre-miR-202-5p, dre-let-7c-5p, and dre-miR-181a-5p), were assayed with RT-qPCR. The expression trends for all the miRNAs validated with RT-qPCR were consistent with the next-generation sequencing data. The identification and characterization of the miRNAs differentially expressed between the ovary and the testis of the T. ovatus will increase our understanding of the role of miRNAs in gonad differentiation. These data also facilitate studies on miRNA regulation of teleost reproduction.

The rice flower carp (Cyprinus carpio var. Quanzhounensis) is a bony fish (superclass Osteichthyes), with very soft bones that is a significant feature unlike the other carp species. In this study, we analyzed the mRNA and microRNA (miRNA) transcriptomes in the intermuscular bones of rice flower carp and Jian carp (Cyprinus carpio var. Jian) (a typical member of common carp in China), using Illumina RNA sequencing. We identified 55,340 genes (including 47,541 known genes and 8231 predicted new genes) and 662 miRNAs (including 595 known miRNAs and 67 novel miRNAs) in the two species. By comparing the transcriptomes of the two species, we identified 1523 differentially expressed genes (DEGs) (including 576 up - and 947 downregulated DEGs) and 352 differentially expressed miRNAs (DEMs) (including 85 up- and 267 downregulated DEMs). According to the Gene Ontology (GO) annotation, 7 DEGs and 12 DEMs were found to be involved in the regulation of bone mineralization. The results of this study improve our understanding of the mRNA and miRNA profiles of carp bones, particularly those of the rice flower carp.

Intertidal and estuarine bivalves are adapted to fluctuating environmental conditions but the cellular adaptive mechanisms under combined stress scenarios are not well understood. The Hong Kong oysters Crassostrea hongkongensis experience periodic hypoxia/reoxygenation and salinity fluctuations during tidal cycles and extreme weather, which can negatively affect the respiratory organs (gills) involved in oxygen uptake and transport. We determined the effects of periodic hypoxia under different salinities on the oxidative stress response in Hong Kong oysters. Oxidative stress parameters (activities of superoxide dismutase (SOD), and catalase (CAT), tissue levels of malondialdehyde (MDA) and protein carbonyl content (PCC)) were determined in the gills of oysters exposed to diel-cycling hypoxia (hypoxia at night: 12h at 2 mg/L, reoxygenation: 12h at 6 mg/L) and normal dissolved oxygen (DO) (6 mg/L) under three salinities (10, 25, and 35‰) for 28 days. Oxygen regime in combination with salinity changes had significant interactive effects on all studied parameters except SOD. Salinity, DO and their interactions increased PCC after 14 and 28 days of exposure, and the combination of hypoxia/reoxygenation and decreased salinity showed the most severe effect. MDA content of the gills increased only after the long-term (28 days) exposure in decreased or increased salinity under normal DO treatments, showing PCC was more sensitive than MDA as biomarker of oxidative stress. Low salinity suppressed SOD activity regardless of the DO, whereas hypoxia induced SOD responses. CAT activities decreased significantly under high salinity with hypoxia/reoxygenation conditions. Our findings highlighted that periodic hypoxia/reoxygenation with salinity change induced antioxidant responses, which can impact the health of Hong Kong oyster C. hongkongensis and prolonged salinity stress may be one reason for the mortality during its aquaculture process.

One important goal of fish genetic breeding is to identify valuable loci and genes that can facilitate growth and thereby productivity. Few such loci or genes have been identified in golden pompano (Trachinotus ovatus), a species of significant economic value. In this study, we produced a high-quality chromosome-level genome assembly of the golden pompano by de novo sequencing and assemblies for 2 parents and 200 F1 offspring by genome re-sequencing. We exploited these assemblies to identify loci and genes by QTL mapping, Kompetitive Allele Specific PCR (KASP) genotyping, and haplotype-based regional association analysis based on growth records of a 64 biparental and 147 individuals from a naturally occurring population. At a locus 291kb from BSNP21031, we identified a somatostatin receptor type 1-like (designated as gpsstr1) gene in which the BSNP1369 of the promoter region was highly associated with growth. Loss of sstr1a, the homolog of gpsstr1 in zebrafish, caused growth retardation. Sstr1a mediated growth via sstr2 and Wnt-gsk-3β signaling pathways. Our findings provide new insights into the underlying mechanisms controlling growth. Our strategy can serve as an effective way to uncover novel genomic information and facilitate improvement of fish growth.

It is difficult to distinguish the sexes of Trachinotus ovatus based on appearance, and little data about sex-determining genes are available for this species. Here, we generated 200 F2 individuals using the parents R404 and R403. DNA samples were collected from 50 individuals of each sex and aggregated into sex-specific DNA pools. Specific-locus amplified fragment sequencing was integrated with bulked segregant analysis to detect candidate sex-associated genes. Approximately 3,153,153 high-quality single-nucleotide polymorphism (SNP) markers and 135,363 high-quality insertion-deletion (Indel) markers were generated. Six candidate regions within chromosome 14, encompassing 132 candidate genes, were identified as closely related to sex. Based on annotations, six genes (EVM0019817, EVM0004192, EVM0001445, EVM0005260, EVM0014734, and EVM0009626) were predicted to be closely associated with sex. These results present an efficient genetic mapping approach that lays a foundation for molecular sex discrimination in T. ovatus.

Aim Tumor cell-derived microparticles (MP) can function as a targeted delivery carrier for anti-tumor drugs. Here, we aimed to generate paclitaxel-loaded microparticles (MP-PTX) from HeLa cells and examined its therapeutic potential on human cervical carcinoma. Methods MP-PTX was generated from HeLa cells by ultraviolet radiation and subsequent centrifugation. The particle size, drug loading rate, and stability of MP-PTX were examined in vitro. Flow cytometry and the MTT assay were performed to test the inhibitory effect of MP-PTX using different cell lines. Immunodeficient mice bearing HeLa cervical carcinoma were treated with 0.9% normal saline, MP, paclitaxel (PTX) (2.5 mg/kg), or MP-PTX (PTX content identical to PTX group) every day for 6 consecutive days. Tumor volume and animal survival were observed. Micro 18F-FDG PET/CT was performed to monitor the therapeutic efficacy. The proliferation activity of cells and microvessel density in tumor tissues were determined by immunohistochemical staining using Ki-67 and CD31, respectively. Results Dynamic laser scattering measurements showed that the particle size of MP-PTX was 285.58 ± 2.95 nm and the polydispersity index was 0.104 ± 0.106. And the particle size of MP-PTX was not change at 4°C for at least one week. More than 1% of PTX in the medium could be successfully encapsulated into HeLa cell-derived MP. When compared with PTX, MP-PTX treatment significantly increased apoptosis of tumor cells and reduced their proliferation. In addition, MP-PTX showed lower toxicity to normal human umbilical vein endothelial cells (HUVEC) than PTX. In vivo studies further demonstrated that MP-PTX treatment significantly inhibited the growth of cervical carcinoma, prolonged the survival of tumor-bearing mice, and reduced the toxicity of PTX. Immunohistochemical staining revealed that MP-PTX treatment led to decreased Ki-67 positive tumor cells and decreased microvessel density in tumor tissues. Conclusion Our results demonstrated that HeLa-derived MP-PTX significantly enhanced the anti-cancer effects of PTX with reduced toxicity, which may provide a novel strategy for the treatment of cervical carcinoma.

The Litopenaeus vannamei (L. vannamei) is one of the most widely cultured shrimp species in the world, with low temperature being one of the most serious threats to its growth and survival. To examine the potential regulatory mechanism of cold adaptation, we conducted a microRNAs (miRNAs) analysis on the hepatopancreas of L. vannamei under normal temperature 28 °C (M28), cold acclimation 16 °C for 6 days (M16), and recovered under normal temperature (MR). In total 14,754,823, 14,945,246 and 15,880,093 raw reads representing 10,690,259, 8,587,144, and 11,512,941 unique sequences of 18–32 nt length were obtained from the M28, M16 and MR libraries, respectively. After comparing the miRNA sequences with the miRBase database, 68 known mature miRNAs and 47 novel miRNAs were identified. Expression analysis showed that 34 miRNAs were significantly differential expressed in response to cold adaptation. Compared to the M28 library, 21 miRNAs were upregulated and 13 miRNAs were downregulated significantly in the M16 library. After recovery to normal temperature, there are 16 miRNAs upregulated and 15 miRNAs downregulated significantly compared to M28 library. Then, five significantly differential expressed miRNAs under cold acclimation including three known miRNAs (mja-miR-6491, mja-miR-6494, and Bta-miR-2478) and two newly-identified miRNAs (novel_68 and novel_5) were selected for validation by RT-qPCR in the hepatopancreas and muscle tissues of cold treated shrimps. The expression trend of most the miRNAs from RT-qPCR were consistent with the next-generation sequencing data. Further, the Gene Ontology (GO) annotation showed that the metabolic process GO term was significantly enriched with target genes of the differentially expressed miRNAs. Additionally, KEGG pathway analysis suggested that the fatty acid degradation and glycerolipid metabolism pathways etc. are significantly enriched with the target genes. These findings may contribute to a better understanding of the molecular mechanisms governing the responses to low temperature in L. vannamei.

Although shrimp are of great economic importance, few full-length shrimp transcriptomes are available. Here, we used Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology to generate transcripts from the Pacific white shrimp (Litopenaeus vannamei). We obtained 322,600 full-length non-chimeric reads, from which we generated 51,367 high-quality unique full-length transcripts. We corrected errors in the SMRT sequences by comparison with Illumina-produced short reads. We successfully annotated 81.72% of all unique SMRT transcripts against the NCBI non-redundant database, 58.63% against Swiss-Prot, 45.38% against Gene Ontology, 32.57% against Clusters of Orthologous Groups of proteins (COG), and 47.83% against Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Across all transcripts, we identified 3,958 long non-coding RNAs (lncRNAs) and 80,650 simple sequence repeats (SSRs). Our study provides a rich set of full-length cDNA sequences for L. vannamei, which will greatly facilitate shrimp transcriptome research.

The present study was performed to determine the molecular mechanism of calcium gluconate (CG) in alleviating the toxic effect of hydrofluoric (HF) acid on human dermal fibroblasts (HDFs). HDF morphology was observed by optical microscopy and the vimentin immunofluorescence assay. Cell viability and apoptosis were evaluated by the Cell Counting Kit-8 and Annexin V/propidium iodide assays, respectively. The levels of apoptosis-associated factors, as well as Wnt2, Wnt3a and β-catenin were detected by reverse transcription-quantitative polymerase chain reaction and western blotting. Levels of matrix metalloproteinase (MMP)-1 and basic fibroblast growth factor (bFGF) were detected by ELISA and western blotting. Carboxyterminal propeptide of type I collagen (CICP) was detected by ELISA, while L-Hydroxyproline (L-HYP) was detected by colorimetry. First, the morphology of normal HDFs was observed. Cell viability was inhibited and apoptosis was increased in a dose- and time-dependent manner following treatment with HF acid [0, 2, 4, 6, 8, 10 and 20% (v/v)] for 0, 2, 4, 6, 8, 10 and 20 min. The effects were blocked by CG at different doses (50, 100 and 200 µmol/l) and time points (6, 12 and 24 h), following treatment with 8% (v/v) HF acid for 6 min. The levels of Caspase-3, B-cell lymphoma (Bcl)-2 associated X protein, Wnt2, Wnt3a and β-catenin were decreased, whereas Bcl-2 was increased by CG treatment dose-dependently, when compared with HF control. CG promoted the expression of MMP-1, bFGF and L-HYP, and inhibited CICP, when compared with HF control. Based on the present results, CG alleviated the toxic effect of HF acid on HDFs by regulating the Wnt/β-catenin signaling pathway.

Antimicrobial peptides (AMPs) are the most important players in the innate immune system, providing a principal first-line of defense against the invading pathogens. Crustin, a type of whey acidic protein (WAP) domain-containing and cationic cysteine-rich AMP, can function in a protease inhibition or an effector molecule manner. In the present study, a new Crustin was cloned and identified from Pacific white shrimp Litopenaeus vannamei and designated as LvCrustinA. The full-length cDNA of LvCrustinA was 687 bp, with a 519 bp open reading frame (ORF) that encoded a peptide of 172 amino acids. Domain analysis indicated that LvCrustinA contained a Glycine-rich region in the N-terminal and a single WAP domain within eight cysteines in the C-terminal. The 5' upstream regulatory sequence of 1249 bp (promoter) was obtained using a genome walking method, and it contained several conserved transcription factors binding motifs including NF-κB, AP-1 and STAT (Signal transducers and activators of transcription). Dual-reporter assay showed that NF-κB transcription factors LvDorsal and LvRelish, and AP-1 transcription factor Lvc-Jun could up-regulate the promoter activity of LvCrustinA, suggesting that NF-κB and JNK-c-Jun pathways could be involved in regulating the expression of LvCrustinA. Moreover, LvCrustinA was abundantly expressed in immune related tissues such as gill, hemocyte and epithelium, and its expression was up-regulated in response to Vibrio parahaemolyticus and White spot syndrome virus (WSSV) challenges in gill tissue, suggesting that LvCrustinA could be involved in the host defense against bacterial and viral infection. Additionally, RNAi mediated knockdown of LvCrustinA resulted in shrimps with the higher cumulative mortality during V. parahaemolyticus and WSSV infection. Taken together, these results provided some insight into the expression and transcriptional regulatory role of LvCrustinA, and its defensive role against pathogenic infection.

Red claw crayfish (Cherax quadricarinatus) has recently attracted widespread attention as an emerging candidate for sustainable aquaculture production in Australia and abroad. Although they are of great economic importance, few full-length transcriptomes are available. Here, transcripts of C. quadricarinatus were generated by using Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology. With SMRT, 289,232 full-length nonchimeric reads were identified, resulting in 14,089 unigene, with an N50 length of 4363 bp and a mean read length of 3913 bp. The error rate of SMRT sequences by comparison with Illumina-produced short reads was only 0.011%. A total of 13,449 transcripts (95.4%) were annotated against the protein database. Across all transcripts, 4952 long noncoding RNAs (lncRNAs), 1090 putative TF members and 21,108 simple sequence repeats (SSRs) were identified. After Vibrio alginolyticus infection, a total of 252 DEGs were screened out after a comparative analysis between samples. Of the 252 DEGs, Hsps, IAPs and Gpx were upregulated during V. alginolyticus infection, many genes were associated with V. cholerae infection, the NF-kappa B signalling pathway, apoptosis-multiple species, the TNF signalling pathway and antigen processing and presentation pathways. Our study provides a rich set of full-length cDNA sequences for C. quadricarinatus, which will greatly facilitate crayfish transcriptome research.

Ocean acidification and hypoxia have become increasingly severe in coastal areas, and their co-occurrence poses emerging threats to coastal ecosystems. Here, we investigated the combined effects of ocean acidification and hypoxia on the reproductive capacity of the thick-shelled mussel Mytilus coruscus. Our results demonstrated low pH but not low oxygen induced decreased gonadosomatic index (GSI) in mussels. Male mussels had a lower level of sex steroids (estradiol, testosterone, and progesterone) when kept at low pH. Expression of genes related to reproduction were also impacted by low pH with a downregulation of genes involved in gonad development in males (β-catenin and Wnt-7b involved in males) and an upregulation of testosterone synthesis inhibition-related gene (Wnt-4) in females. Overall, our results suggest that ocean acidification has an impact on the gonadal development through an alternation of gene expression and level of steroids while hypoxia had no significant effect.

BACKGROUNDAngiogenesis is closely associated with angiogenesis-dependent diseases including cancers and ocular diseases. Anti-angiogenic therapeutics have been focusing on the (VEGF)/VEGFR signaling axis. However, the clinical resistance, high cost and frequent administration of anti-VEGF drugs make it urgent to discover novel angiogenic pathways.VE-PTP (ptprb) is a novel target with great anti-angiogenic potential. However, it is unclear whether upstream signaling pathways targeting VE-PTP exist in angiogenesis.METHODSWhole genome and embryo transcriptome sequencing were applied to discover the new genenxhl. Transgenic zebrafish model, morpholino knockdown and small interfering RNA were used to explore the role ofnxhlin angiogenesis bothin vitroandin vivo. RNA pulldown, RIP and ChIRP-MS were used to identify interactions between RNA and protein.RESULTSWe discovered a novel zebrafish genenxhlwhich is a homologue of the conserved genenxhthat co-expressed with some key genes essential for embryo development in vertebrate.Nxhldeletion causes angiogenesis defects in embryo. Moreover,nxhlis essential to mediate effects of angiogenesisin vivoandin vitro, andptprbdepletion duplicates the phenotypes ofnxhldeficiency. Importantly,nxhlacts upstream ofptprband regulates many extreme importantptprb-linked angiogenic genes by targeting VE-PTP (ptprb) through interactions with NCL. Notably,nxhldeletion decreases the phosphorylation of NCL T76 and increases the acetylation of NCL K88, suggestingnxhlmay regulate downstream VE-PTP signaling pathways by mediation of NCL posttranslational modification. This is the first description of the interaction betweennxhland NCL, NCL and VE-PTP (ptprb), uncovering a novelnxhl-NCL-VE-PTP signaling pathway on angiogenesis regulation.CONCLUSIONSOur study identifiesnxhlcontrolling angiogenesis by targeting VE-PTP through interactions with NCL, uncovering novel upstream controllers of VE-PTP. Thisnxhl-NCL-VE-PTP pathway may be a therapeutic target in the treatment of angiogenesis-dependent diseases.Clinical PerspectiveWhat Is New?We report a novelnxhl-NCL-VE-PTP signaling pathway that controls angiogenesis.We for the first time demonstrate thatnxhlinteracts with NCL which simultaneously binds to VE-PTP that plays key roles on EC adherens junction, integrity and vascular homeostasis.Nxhlalso controls some other crucial VE-PTP-linked downstream angiogenic genes (such as Tie2, VEGFaa, VEGFR2, Erbb2, S1pr1 and Hey2) which explain the phenotypes induced by thenxhldeficiency.Our study indicates the key role ofnxhlon controlling angiogenesis as an upstream regulator of VE-PTP.What Are the Clinical Implications?There are several ongoing researches investigating the utility of VE-PTP or NCL inhibitors on treatment of angiogenesis-dependent diseases including a range of cancers and nonneoplastic diseases, such as AMD, DME, RA and atherosclerosis.Targeting thenxhl-NCL-VE-PTP pathway may facilitate therapeutic angiogenesis in patients with cancers or ocular diseases such as DME.Our study highlights the great potential ofnxhlon anti-angiogenic therapeutics by targeting VE-PTP.

Crassostrea hongkongensis is a popular and important native oyster species that is cultured mainly along the coast of the South China Sea. However, the absence of a reference genome has restricted genetic studies and the development of molecular breeding schemes for this species. Here, we combined PacBio and 10 × Genomics technologies to create a C. hongkongensis genome assembly, which has a size of 610 Mb, and is close to that estimated by flow cytometry (~650 Mb). Contig and scaffold N50 are 2.57 and 4.99 Mb, respectively, and BUSCO analysis indicates that 95.8% of metazoan conserved genes are completely represented. Using a high-density linkage map of its closest related species, C. gigas, a total of 521 Mb (85.4%) was anchored to 10 haploid chromosomes. Comparative genomic analyses with other molluscs reveal that several immune- or stress response-related genes extensively expanded in bivalves by tandem duplication, including C1q, Toll-like receptors and Hsp70, which are associated with their adaptation to filter-feeding and sessile lifestyles in shallow sea and/or deep-sea ecosystems. Through transcriptome sequencing, potential genes and pathways related to sex determination and gonad development were identified. The genome and transcriptome of C. hongkongensis provide valuable resources for future molecular studies, genetic improvement and genome-assisted breeding of oysters.

Crassostrea hongkongensis is one of the three most-commonly cultivated oyster species in China. Although microRNAs (miRNAs) expression in the gonads have been widely investigated, few studies of miRNAs in mollusk gonads are available, particularly in oyster. In the present study, we analyzed the miRNAs expressed in the ovaries and testes of C. hongkongensis. We obtained 14,166,409 and 15,133,900 raw reads from the ovaries and testes, respectively, yielding 13,634,997 (ovarian) and 14,494,149 (testicular) 18-35-nt sequences. We mapped these sequences to the C. hongkongensis genome reference sequence, and identified 8,771,717 (ovarian) and 9,926,014 (testicular) sequences corresponding to miRNAs in the Rfam database. After blasting the miRNA sequences against the miRBase database, we identified 50 known mature miRNAs and 53 novel miRNAs. Of these, 27 miRNAs were significantly upregulated in ovaries as compared to the testes, and 43 miRNAs were significantly upregulated in the testes as compared to the ovaries. To validate the differential expression results generated by Illumina sequencing, we used RT-real-time quantitative PCR (RT-qPCR) to characterize the expression patterns of the six most differently expressed miRNAs (lgi-miR-1990, lgi-miR-1986, lgi-miR-263b, lgi-miR-279, lgi-miR-1992, and novel_98) as well as two miRNAs associated with gonad development (lgi-miR-29 and lgi-miR-8). Most of the RT-qPCR miRNA expression patterns were similar to those recovered by high-throughput sequencing with the exceptions of novel_98 and lgi-miR-1992. Gene Ontology (GO) annotations indicated that the multi-organism cellular process GO category was enriched with the target genes of the differentially expressed miRNAs. Additionally, the target genes were enriched in several KEGG pathways, including the ECM-receptor interaction, galactose metabolism, phagosome, and notch signaling pathway. These pathways are involved in gonadal differentiation and the maintenance of gonad function. This identification and characterization of the miRNAs differentially expressed between the ovaries and testes of C. hongkongensis will increase our understanding of the role of miRNAs in gonad differentiation in the oyster.

Min Wei,* Jinxia Peng,* Peng Wu, Ping Chen, Hongru Yang, Yongxia Cui, Linglin Yang Department of Oncology, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, People’s Republic of China *These authors contributed equally to this work Purpose: Autophagy, the process responsible for degrading cytoplasmic organelles to sustain cellular metabolism, has been associated with cancer initiation and progression. As TP53-induced glycolysis and apoptosis regulator (TIGAR) is among the important genes that can regulate autophagy, we aimed to investigate the correlation between the expression levels of TIGAR and the autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3B), as well as their association with clinical outcomes, in nasopharyngeal carcinoma (NPC) patients.Methods: We detected the expressions of TIGAR and LC3B in 182 NPC tissue samples via immunohistochemical staining.Results: A significant correlation between TIGAR and LC3B expressions was identified (P=0.045). Moreover, survival analysis showed that TIGAR– or LC3B+ expression was associated with improved overall survival, local regional failure-free survival, distant failure-free survival, and failure-free survival rates, compared with TIGAR+ or LC3B– expression, respectively. Meanwhile, when combining TIGAR with LC3B expression in terms of prognostic value, patients with TIGAR+/LC3B– expression were significantly disadvantaged with regard to overall survival, local regional failure-free survival , distant failure-free survival, and failure-free survival compared with other groups based on the log-rank test and Cox regression analyses (all P

The Pacific white shrimp (Litopenaeus vannamei), one of the most widely cultured shrimp species in the world, often suffers from cold stress. To understand the molecular mechanism of cold tolerance in Pacific white shrimp, we conducted a proteomic analysis on two contrasting shrimp cultivars, namely, cold-tolerant Guihai2 (GH2) and cold-sensitive Guihai1 (GH1), under normal temperature (28°C), under cold stress (16°C), and during recovery to 28°C. In total, 3,349 proteins were identified, among which 2,736 proteins were quantified. Based on gene ontology annotations, differentially expressed proteins largely belonged to biological processes, cellular components, and molecular functions. KEGG pathway annotations indicated that the main changes were observed in the lysosome, ribosomes, and oxidative phosphorylation. Subcellular localization analysis showed a significant increase in proteins present in cytosol, extracellular regions, and mitochondria. Combining enrichment-based clustering analysis and qRT-PCR analysis, we found that glutathione S-transferase, zinc proteinase, m7GpppX diphosphatase, AP2 transcription complex, and zinc-finger transcription factors played a major role in the cold stress response in Pacific white shrimp. Moreover, structure proteins, including different types of lectin and DAPPUDRAFT, were indispensable for cold stress tolerance of the Pacific white shrimp. Results indicate the molecular mechanisms of the Pacific white shrimp in response to cold stress and provide new insight into breeding new cultivars with increased cold tolerance.

Mitochondria have been described as 'the powerhouse of the cell' as the organelle generates the majority of adenosine triphosphate (ATP) in cells to support life. Mitochondria can be damaged due to stress, for example by reactive oxygen species (ROS). TP53-induced glycolysis and apoptosis regulator (TIGAR) serves a role in suppressing ROS damage and may protect mitochondria integrity. In the present study, the localization of TIGAR on mitochondria in 5-8F cells was demonstrated. Furthermore, it was indicated that the knockdown of TIGAR using lentivirus-short hairpin RNA induces the loss of mitochondrial membrane potential and cytochrome c leakage. However, these damaged mitochondria were not degraded in cells, but exhibited an abnormal appearance as indicated by mitochondrial swelling, crista collapse and vacuolization, with physiological dysfunction marked by reduced ATP production. Therefore, TIGAR maybe an indispensable protein for mitochondrial protection and degradation following cellular damage.

The Pacific white shrimp (Litopenaeus vannamei) is one of the most widely cultured shrimp species in the world. Despite L. vannamei having tropical origins, it is being reared subtropically, with low temperature stress being one of the most severe threats to its growth, survival and distribution. To unravel the molecular basis of cold tolerance in L. vannamei, the suppression subtractive hybridization (SSH) platform was employed to identify cold responsive genes in the hepatopancreas of L. vannamei. Both forward and reverse cDNA libraries were constructed, followed by dot blot hybridization, cloning, sequence analysis and quantitative real-time PCR. These approaches identified 92 cold induced and 48 cold inhibited ESTs to give a total of 37 cold induced and 17 cold inhibited contigs. Some of the identified genes related to stress response or cell defense, such as tetraspanins (TSPANs), DEAD-box helicase, heat shock proteins (HSPs) and metallothionein (MT), which were more abundant in the forward SSH library than in the reverse SSH library. The most abundant Est was a tetraspanin-8 (TSPAN8) homolog dubbed LvTSPAN8. A multiple sequence alignment and transmembrane domain prediction was also performed for LvTSPAN8. LvTSPAN8 expression was also examined in the gills, muscle, heart and hepatopancreas following cold exposure and showed the highest expression levels in the hepatopancreas. Overall, this study was able to identify several known genes and novel genes via SSH that appear to be associated with cold stress and will help to provide further insights into the molecular mechanisms regulating cold tolerance in L. vannamei.

Background The Pacific white shrimp (Litopenaeus vannamei) is the world’s most prevalent cultured crustacean species. However, the supply of high-quality broodstocks is limited and baseline information related to its reproductive activity and molecular issues related to gonad development are scarce. In this study, we performed transcriptome sequencing on the gonads of adult male and female L. vannamei to identify sex-related genes. Results A total of 25.16 gigabases (Gb) of sequences were generated from four L. vannamei gonadal tissue libraries. After quality control, 24.11 Gb of clean reads were selected from the gonadal libraries. De-novo assembly of all the clean reads generated a total of 65,218 unigenes with a mean size of 1021 bp and a N50 of 2000 bp. A search of all-unigene against Nr, SwissProt, KEGG, COG and NT databases resulted in 26,482, 23,062, 20,659, 11,935 and 14,626 annotations, respectively, providing a total of 30,304 annotated unigenes. Among annotated unigenes, 12,320 unigenes were assigned to gene ontology categories and 20,659 unigenes were mapped to 258 KEGG pathways. By comparing the ovary and testis libraries, 19,279 testicular up-regulated and 3,529 ovarian up-regulated unigenes were identified. Enrichment analysis of differentially expressed unigenes resulted in 1060 significantly enriched GO terms and 34 significantly enriched KEGG pathways. Nine ovary-specific, 6 testis-specific, 45 testicular up-regulated and 39 ovarian up-regulated unigenes were then confirmed by semi-quantitative PCR and quantitative real-time PCR. In addition, using all-unigenes as a reference, a total of 13,233 simple sequence repeats (SSRs) were identified in 10,411 unigene sequences. Conclusions The present study depicts the first large-scale RNA sequencing of shrimp gonads. We have identified many important sex-related functional genes, GO terms and pathways, all of which will facilitate future research into the reproductive biology of shrimp. We expect that the SSRs detected in this study can then be used as genetic markers for germplasm evaluation of breeding and imported populations. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2219-4) contains supplementary material, which is available to authorized users.