MicroRNA Lse-miR-2944c-3p regulates female reproduction in Lasioderma serricorne by targeting the heat shock protein LsHSP70-3.

Simple SummaryTudor domain-containing proteins (TDRDs) are a group of evolutionarily conserved and germline-enriched proteins, and most of them function in reproduction and the P-element-induced wimpy testis-interacting RNA (piRNA) pathway. We investigated LmTDRD5, a TDRD5 ortholog in Locusta migratoria. In males, LmTdrd5 knockdown delayed meiosis phase transition and reduced the number of elongated spermatids and sperm count. The expression levels of two haploid germ cell marker genes, LmCREM and LmACT, as well as the sperm tail marker gene LmQrich2 were downregulated. In females, LmTdrd5 knockdown decreased vitellogenin (Vg) and Vg receptor (VgR) transcript levels, thereby affecting ovarian development and oocyte maturation. Therefore, LmTdrd5 may play an important role in locust reproduction, indicative of a conserved primary function of TDRD5.Tudor family proteins exist in all eukaryotic organisms and play a role in many cellular processes by recognizing and binding to proteins with methylated arginine or lysine residues. TDRD5, a member of Tudor domain-containing proteins (TDRDs), has been implicated in the P-element-induced wimpy testis-interacting RNA (piRNA) pathway and germ cell development in some model species, but little is known about its function in other species. Therefore, we identified and characterized LmTDRD5, the TDRD5 ortholog in Locusta migratoria, a hemimetabolous pest. The LmTdrd5 gene has 19 exons that encode a protein possessing a single copy of the Tudor domain and three LOTUS domains at its N-terminus. qRT-PCR analysis revealed a high LmTdrd5 expression level in genital glands. Using RNA interference, LmTdrd5 knockdown in males led to a lag in meiosis phase transition, decreased spermatid elongation and sperm production, and downregulated the expression of the two germ cell-specific transcription factors, LmCREM and LmACT, as well as the sperm tail marker gene LmQrich2.LmTdrd5 knockdown in females reduced the expression levels of vitellogenin (Vg) and Vg receptor (VgR) and impaired ovarian development and oocyte maturation, thus decreasing the hatchability rate. These results demonstrate that LmTdrd5 is essential for germ cell development and fertility in locusts, indicating a conserved function for TDRD5.

CircSTK40 contributes to recurrent implantation failure via modulating the HSP90/AKT/FOXO1 axis

Stability evaluation of candidate reference genes for RT-qPCR normalization in Lasioderma serricorne (F.)

The p38MAPKs (mitogen-activated protein kinases) signaling contributes a pivotal role in mammalian ovarian follicular development; however, the knowledge regarding their expression in geese remains unresolved. The objective of the current study was to determine the spatio-temporal expression of heat shock protein 27 (HSP27) and mitogen- and stress-activated protein kinase 1 (MSK1) genes in the prehierarchical follicles during geese ovarian development. The prehierarchical follicles samples were harvested from 35- to 37-week-old healthy laying geese. HSP27 and MSK1 relative expression in various sized prehierachical follicles was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. Follicular wall localization of HSP27 and MSK1 was examined by using immunohistochemistry. Our results at mRNA level indicated that HSP27 was highly expressed in middle white follicles whereas MSK1 was predominantly expressed in small white follicles. The western blotting results for HSP27 and MSK1 were inconsistent with the RT-qPCR results in various stages of prehierachical follicular development but noticeably, HSP27 proteins were still expressed more in middle white follicles while MSK1 proteins were more abundant in primary follicles. At different stages of prehierarchical development, immunodetections in the granulosa and theca cells revealed that HSP27 was intensively localized in middle white follicles while strong detections of MSK1 were observed in large white follicles. These results indicate HSP27 and MSK1 might be associated to the key regulators of folliculogenesis in geese.

BackgroundInsulin/insulin-like growth peptide signaling (IIS) down-regulates hemolymph sugar level and facilitates larval growth in the soybean pod borer, Maruca vitrata. The objective of this study is to determine whether IIS of M. vitrata can mediate ovarian development of adult females.ResultsA pair of ovaries consists of 8 ovarioles, each of which is separated into distal germarium and proximal vitellarium in M. vitrata. In the germarium, oocyte development occurred with active mitotic activity which was visible by incorporating bromodeoxyribose uridine. Previtellogenic development and subsequent vitellogenesis began soon after adult emergence. They continued with increase of female age. Oocyte development was facilitated by up-regulation of vitellogenin (Vg) and Vg receptor (VgR) gene expression. Larval diets significantly influenced on ovarian development of M. vitrata because oocyte development varied with pupal size derived from larvae treated with different nutritional diets. Its ovarian development was dependent on endocrine signal(s) from the head because decapitation soon after adult emergence prevented oogenesis and subsequent vitellogenesis along with marked reduction of Vg and VgR expression. Topical application of juvenile hormone (JH) significantly recovered its ovarian development whereas farnesoic acid (a precursor of JH biosynthesis) or 20-hydroxyecdysone treatment did not. JH stimulated vitellogenesis and choriogenesis, but not previtellogenic development. In contrast, insulin injection to decapitated females stimulated oocyte differentiation and vitellogenesis along with increase of Vg and VgR expression. To further analyze the effect of insulin on ovarian development, expression of four IIS components (InR, FOXO, Akt, and TOR) genes was manipulated by RNA interference. Hemocoelic injection of gene-specific double stranded RNAs significantly reduced their target gene mRNA levels and interfered with ovarian development. An addition of insulin to JH treatment against decapitated females enhanced the gonadotropic effect of JH by stimulating oogenesis.ConclusionsIIS plays crucial role in mediating previtellogenic development of M. vitrata in response to nutrient signal. It also enhances the gonadotropic effect of JH II on vitellogenesis.

Although both the heat shock protein 70 (HSP70) and the activating transcription factor 5 (ATF5) have been shown to promote cell survival of transformed cells but not survival of non-transformed cells, the relationship of the two molecules is unknown. Here we show that HSP70 and ATF5 are concomitantly up-regulated upon transient but down-regulated over prolonged cellular stress and apoptotic stimulation in the rat C6 glioma and human U87 glioma cells. HSP70 interacts strongly with the N-terminal activation domain of ATF5, which is expected to be rigid and uniquely structured under physiological conditions because of extraordinary high concentration (over 25%) of proline residues. Binding of HSP70 to ATF5 is an ATP-driven process and requires functional ATPase on the nucleotide binding domain of the HSP70 molecule. Overexpression of HSP70 dramatically stabilizes the ATF5 protein, which is otherwise subject to rapid degradation, facilitated by both proteasome-dependent and caspase-dependent processes, whereas HSP70 depletion leads to acceleration of ATF5 degradation and transcription repression of Bcl-2 and Egr-1, which are downstream targets of ATF5 in C6 and U87 glioma cells. Our data reveal an essential role for HSP70 in maintaining high levels of ATF5 expression in glioma cells and support the conclusion that ATF5 is an important substrate protein of HSP70 that mediates HSP70-promoted cell survival in glioma cells.

Heat shock proteins (HSPs), a family of conserved proteins that are produced by cells in response to stresses, are known as molecular chaperones with a range of housekeeping and cellular protective functions. The 40 kD heat shock protein (HSP40) is a co-chaperone for HSP70 in the regulation of ATP hydrolysis. Unlike its well-documented cofactor HSP70, little is currently known regarding the biological functions of HSP40 in crustacean species such as penaeid shrimp. In the present study, the cDNA encoding HSP40 (Lv-HSP40) was identified from the Pacific white shrimp Litopenaeus vannamei, a highly significant commercial culture species. The structural organization indicates that Lv-HSP40 belongs to the type-I HSP40s. The muscle, gill, and hepatopancreas are the main sites of Lv-HSP40 transcript expression. Within these tissues, Lv-HSP40 mRNA were predominantly exhibited in the myocytes, epithelial cells and hepatopancreatic cells, respectively. Under acute thermal stress in the culture environment, Lv-HSP40 transcript levels are significantly induced in these three tissues, while low pH stress only upregulates Lv-HSP40 mRNA in the hepatopancreas and gill. During ontogenesis, Lv-HSP40 transcript levels are high at early embryonic stages and drop sharply at late embryonic and early larval stages. The ovary is another major organ of Lv-HSP40 mRNA expression in female shrimp, and Lv-HSP40 transcripts were mainly presented in the follicle cells but only weekly detected in the oocytes. Ovarian Lv-HSP40 mRNA levels increase continuously during gonadal development. Silencing of the Lv-HSP40 gene by RNA interference may effectively delay ovarian maturation after unilateral eyestalk ablation. The roles of Lv-HSP40 in ovarian development are speculated to be independent of its cofactor HSP70, and the vitellogenesis factor vitellogenin (Vg) and vitellogenin receptor (VgR). Our study, as a whole, provides new insights into the roles of HSP40 in multiple physiological processes in L. vannamei: (1) HSP40 is a responding factor during stressful conditions; and (2) HSP40 participates in embryonic and ovarian development.

Heat shock protein 90 (HSP90) is a highly conserved protein and plays an important role in maintaining the structure of protein, participating in the immunity and regulating the cell cycle. Using the rapid amplification of cDNA ends (RACE) techniques, the cDNA sequence of HSP90 gene (designated Sp- HSP90) was cloned and characterized from the mud crab Scylla paramamosain . The full-length cDNA of Sp-HSP90 is 2677 bp with a complete open reading frame (ORF) of 2166 bp, which encodes a polypeptide of 721 amino acids. Five conserved blocks defining HSP90 protein family were found in the deduced amino acid sequence of Sp-HSP90. It contains an adenosine-5’-triphosphatase (ATPase) domain in the N-terminal and a conserved signature sequence MEEVD in the C-terminal. Quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) analyses revealed the distribution of Sp-HSP90 mRNA in different tissues and its temporal expression in haemocytes of the crabs challenged with Vibrio parahaemolyticus. Different levels of Sp-HSP90 mRNA were detected in heart, hepatopancreas, muscle, haemocytes, testis and ovary except eyestalk. The expression level of Sp-Hsp90 mRNA in hemocytes was found to be obviously up-regulated after live and heat-killed bacterial challenge and significantly higher in live bacteria group than that in heat-killed bacteria group. These results suggest that Sp-HSP90 gene might act on immunity and resistance to infection in S. paramamosain. Key words: Heat shock protein 90, quantitative real-time polymerase chain reaction (PCR), Scylla paramamosain, Vibrio parahaemolyticus .

We report the first cloning and characterization of cDNA encoding a putative vitellogenin (Vg) receptor (VgR) from the shrimp, Penaeus monodon. The shrimp VgR cDNA is 6.8 kb; the deduced protein has 1943 amino acids with a molecular weight of 211 kDa. VgR is ovary specific and consists of conserved cysteine-rich domains, epidermal growth factor-like domains, and YWTD motifs similar to the low-density lipoprotein, very low-density lipoprotein, and VgR of insects and vertebrates. VgR expression level in the ovary is low during early vitellogenesis and increases to maximum levels in females with a gonadosomatic index of 3-4, presumably when needed for receptor-mediated endocytosis during the rapid phase of extraovarian Vg production by the hepatopancreas. A peptide from the C-terminal end of VgR was synthesized for antibody production. Anti-VgR antibody recognized an ovarian membrane protein, and the level of this protein was high when extraovarian production of Vg reached peak levels. By immunohistochemical analysis, VgR was detected strongly in the membranes of larger oocytes. VgR expression was knocked down after the shrimp were injected with VgR double-stranded RNA, leading to a decrease in VgR protein content in the ovary, but an increase in the hemolymph level of Vg. This study represents the first report of the functional analysis of a putative VgR in a crustacean.

Tu1928 Tissue Expression of Heat Shock Protein 27, 70 and 90 Might Be Utilized As a Diagnostic and Prognostic Biomarker in Patients With Gastric Adenocarcinoma

Tu1932 Endoscopic Features of Food Protein-Induced Proctocolitis in Neonates and Infants

Tu1927 Heat Shock Protein 27 Expression Is Inversely Correlated With Intraepithelial Neoplasia and Positively Correlated With Poor Differentiation of Gastric Cancer

To establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte). Based on the structural differences between HBVcccDNA and HBV rcDNA, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. According to the standard preparation, cccDNA levels of specimen were calculated. We have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative. The nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.

Objective To investigate the expression of heat shock protein 60 (HSP60) and apoptosis related protein caspase 3 in testis of rat varicocele (VC) , and the role of HSP60 in apoptosis of testicular germ cells in rats. Methods Thirty-two SD rats were randomly divided into the control group (n= 8) , sham operation group (n=8) and VC model group (n=16). VC rat models were prepared by ligation of the left renal vein. Real-time quantitative PCR and Western blot assay were used to determine the mRNA and protein expression levels of HSP60 in testicular tissues of VC rats; immunoblot assay was used to determine the expression of caspase 3 (activated type) in VC rat testis. We analyzed the relationship between HSP60 and caspase 3 expressions in VC rat testis. Results Compared with the control group and the sham operation group, VC rats had increased testis mRNA and protein levels of HSP60 and caspase 3 (HSP60 mRNA: 2.67±0.42 vs 1.18±0.30, 0.98±0.16, HSP60 protein: 12.02±1.78 vs 2.31±0.34, 3.15±0.60; caspase 3 mRNA: 4.28±0.47 vs 1.45±0.17, 1.38±0.24, caspase 3 protein: 5.44±2.06 vs 1.32±0.84, 1.16±0.76, all P 0.05). In the VC model group, HSP60 and cleaved caspase 3 expression in rat testis tissue were positively correlated (R=0.95, P<0.05). Conclusion HSP60 expression is increased in testis of VC rats, and may be involved in testicular germ cell apoptosis, which in turn essentially contributes to male infertility in VC. Key words: Varicocele; Heat shock protein 60; Testis; Apoptosis

HBO suppressed nitric oxide and apoptosis in articular cartilage defect via up-regulation of hsp 70 expression —In vitro and in vivo study