Lrp13a and Lrp13b serve as vitellogenin receptors in the ovary of zebrafish†.

In oviparous animals, egg yolk is largely derived from vitellogenin, which is taken up from the maternal circulation by the growing oocytes via the vitellogenin receptor. Recently, a novel member of the lipoprotein receptor superfamily termed low-density lipoprotein receptor-related protein 13 was identified and proposed as a candidate of vitellogenin receptor in oviparous animals. However, the roles of low-density lipoprotein receptor-related protein 13 in vitellogenesis are still poorly defined. Here, we investigated the expression, vitellogenin-binding properties, and function of low-density lipoprotein receptor-related protein 13 in zebrafish. Two different lrp13 genes termed lrp13a and lrp13b were found in zebrafish. Reverse transcription polymerase chain reaction and quantitative polymerase chain reaction revealed both lrp13s to be predominantly expressed in zebrafish ovary, and in situ hybridization detected both lrp13s transcripts in the ooplasm of early stage oocytes. Two yeast hybrid studies showed that among eight vitellogenins of zebrafish, Vtg1, 2, and 3 bind to Lrp13a, while Vtg1, 2, and 5 bind to Lrp13b. We created zebrafish lrp13a and lrp13b mutant lines using CRISPR/Cas9. Knockout of lrp13a leads to a male-biased sex ratio and decreased diameter of embryo yolk, while knockout of lrp13b and double knockout of lrp13a and lrp13b leads to the delay of vitellogenesis, followed by follicular atresia. These phenotypes of mutants can be explained by the disruption of vitellogenesis in the absence of Lrp13s. Taken together, our results indicate that both Lrp13a and Lrp13b can serve as vitellogenin receptors in zebrafish among other vitellogenin receptors that are not yet described.

Evaluation of the Immunity Responses in Mice to Recombinant Bacillus subtilis Displaying Newcastle Disease Virus HN Protein Truncations.

Bacillus subtilis, a probiotic bacterium with engineering potential, is widely used for the expression of exogenous proteins. In this study, we utilized the integrative plasmid pDG364 to integrate the hemagglutinin-neuraminidase (HN) gene from Newcastle disease virus (NDV) into the genome of the B. subtilis 168 model strain. We successfully constructed a recombinant B. subtilis strain (designated B. subtilis RH) that displays a truncated HN antigen fragment on the surface of its spores and further evaluated its immunogenic effects in mice. Using ELISA, we quantified the levels of IgG in serum and secretory IgA (sIgA) in intestinal contents. The results revealed that the recombinant B. subtilis RH elicited robust specific mucosal and humoral immune responses in mice. Furthermore, B. subtilis RH demonstrated potential mucosal immune adjuvant properties by fostering the development of immune organs and augmenting the number of lymphocytes in the small intestinal villi. Additionally, the strain significantly upregulated the relative expression of inflammatory cytokines such as IL-1ß, IL-6, IL-10, TNF-α, and IFN-γ in the small intestinal mucosa. In conclusion, the B. subtilis RH strain developed in this study exhibits promising mucosal immunogenic effects. It holds potential as a candidate for an anti-NDV mucosal subunit vaccine and offers a novel preventive strategy for the poultry industry against this disease.

Co-immobilized multi-enzyme biocatalytic system on reversible and soluble carrier for saccharification of corn straw cellulose.

Herein, three enzymes (cellulase, ß-glucosidase, and pectinase) with synergistic effects were co-immobilized on the Eudragit L-100, and the recovery of co-immobilized enzymes from solid substrates were achieved through the reversible and soluble property of the carrier. The optimization of enzyme ratio overcomed the problem of inappropriate enzyme activity ratio caused by different immobilization efficiencies among enzymes during the preparation process of co-immobilized enzymes. The co-immobilized enzymes were utilized to catalytically hydrolyze cellulose from corn straw into glucose, achieving a cellulose conversion rate of 74.45% under conditions optimized for their enzymatic characteristics and hydrolytic reaction conditions. As a result of the reversibility and solubility of the carrier, the co-immobilized enzymes were recovered from the solid substrate after five cycles, retaining 54.67% of the enzyme activity. The aim of this study is to investigate the potential of co-immobilizing multiple enzymes onto the Eudragit L-100 carrier for the synergistic degradation of straw cellulose.

Gonacin: A germ cell-derived hormone with glucogenic, orexigenic, and gonadal activities.

Fish require abundant nutrients to generate a large number of eggs for spawning. Based on the evolutionary conservation of human FBN2 and its C-terminal placensin-like sequences in fish, we identified a peptide hormone gonacin (GONAdal Cell placensIN) and found its high expression in early-stage germ cells in the ovary and testis of zebrafish. We demonstrated that gonacin is essential for food intake, glucose release, and ovarian development in zebrafish. Similar expression patterns and functions of gonacin were also demonstrated in rainbow trout. Gonacin represents the first hormone secreted by germ cells with endocrine functions in vertebrates, bridging the energy homeostasis and reproduction.

Pacsin 2-dependent N-cadherin internalization regulates the migration behaviour of malignant cancer cells.

Collective cell migration is the coordinated movement of multiple cells connected by cadherin-based adherens junctions and is essential for physiological and pathological processes. Cadherins undergo dynamic intracellular trafficking, and their surface level is determined by a balance between endocytosis, recycling and degradation. However, the regulatory mechanism of cadherin turnover in collective cell migration remains elusive. In this study, we show that the Bin/amphiphysin/Rvs (BAR) domain protein pacsin 2 (protein kinase C and casein kinase substrate in neurons protein 2) plays an essential role in collective cell migration by regulating N-cadherin (also known as CDH2) endocytosis in human cancer cells. Pacsin 2-depleted cells formed cell-cell contacts enriched with N-cadherin and migrated in a directed manner. Furthermore, pacsin 2-depleted cells showed attenuated internalization of N-cadherin from the cell surface. Interestingly, GST pull-down assays demonstrated that the pacsin 2 SH3 domain binds to the cytoplasmic region of N-cadherin, and expression of an N-cadherin mutant defective in binding to pacsin 2 phenocopied pacsin 2 RNAi cells both in cell contact formation and N-cadherin endocytosis. These data support new insights into a novel endocytic route of N-cadherin in collective cell migration, highlighting pacsin 2 as a possible therapeutic target for cancer metastasis.

Effects of Antimicrobial Peptides Gal-13 on the Growth Performance, Intestinal Microbiota, Digestive Enzyme Activities, Intestinal Morphology, Antioxidative Activities, and Immunity of Broilers.

To evaluate the application effect of antimicrobial peptides Gal-13 (AMP Gal-13) instead of antibiotic feed additives, 90 7-day-old Ross 308 broilers were randomly divided into 3 groups. Group A was fed a basic diet as the control, and Groups B and C were supplemented with AMP Gal-13 (100 mg/kg and 200 mg/kg, respectively). After a 35-day feeding experiment, the weight and average daily gain (ADG) of the broilers in Group B were significantly higher than those of the broilers in Group A. The Enterococcus sp. and Escherichia coli counts in the ileum and cecum in Group A were significantly higher than those in Groups B and C, while the Lactic acid bacteria (LAB) and Bifidobacterium sp. counts were significantly lower. The amylase activity of the jejunum in Group B was significantly higher than that in Group A. The villus length (VL): crypt depth (CD) ratios of the jejunum and ileum in Group B were significantly higher than those in Group A. The glutathione peroxidase (GSH-Px) activities in the liver and serum in Groups B and C were significantly higher than those in Group A, while the malondialdehyde (MDA) activity was significantly lower. The titers of Newcastle disease virus (NDV)-specific antibodies were elevated significantly in Group B at the age of 42 days. Additionally, the weights of the spleen and thymus were significantly increased. The expression levels of Il-2, Il-6, Tgf-ß4, Tnf-α, and Mif in the spleen in Groups B and C were significantly downregulated to different degrees; Il-4 expression in Group B was significantly upregulated, while Ifn-γ expression in Group C was significantly upregulated. The results suggested that adding AMP Gal-13 to the diet could improve intestinal digestion, the antioxidant capacity, and immune function, ultimately promoting the growth of broilers.

Surface Display of porcine circovirus type 2 antigen protein cap on the spores of bacillus subtilis 168: An effective mucosal vaccine candidate.

The oral mucosal vaccine has great potential in preventing a series of diseases caused by porcine circovirus type 2 (PCV2) infection. This study constructed a recombinant Bacillus subtilis RB with PCV2 Capsid protein (Cap) on its spore surface and cotB as a fusion partner. The immune properties of the recombinant strain were evaluated in a mouse model. IgA in intestinal contents and IgG in serum were detected by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that recombinant spores could activate strong specific mucosal and humoral immune responses. In addition, spores showed good mucosal immune adjuvant function, promoting the proliferation of CD3+, CD4+ and CD8+ T cells and other immune cells. We also found that the relative expression of inflammatory cytokines such as IL-1ß, IL-6, IL-10, TNF-α and IFN in the small intestinal mucosa was significantly up-regulated under the stimulation of recombinant bacteriophage. These effects are important for the balance of Th1/Th2-like responses. In summary, our results suggest that recombinant B. subtilis RB as a feed additive provides a new strategy for the development of novel and safe PCV2 mucosal subunit vaccines.

Molecular mechanism of enhancing the immune effect of the Newcastle disease virus vaccine in broilers fed with Bacillus cereus PAS38.

The aim of this study was to explore the molecular mechanism of enhancing the immune effect of the Newcastle disease virus (NDV) vaccine in broilers fed with Bacillus cereus PAS38. The results showed that the NDV antibody titer of broilers in the treatment group supplemented with B. cereus PAS38 was higher than that of the control group, and the difference was significant at 28 days of age (P < 0.05). The spleen, thymus and bursa of fabricius of 42-day-old broilers were quickly collected to construct a differentially expressed gene library of suppression subtractive hybridization (SSH). A total of 31 immune-related differentially expressed genes were screened from three immune organs, of which 15 were up-regulated and 16 were down-regulated. After silencing the up-regulated genes MIF, CD74, DOCK2 and KLHL6, the expression levels of cytokines (Akirin2, NF-κB, IL-2, IL-4, IL-6, IFN-γ and TNF-α) in lymphocytes were reduced to varying degrees. B. cereus PAS38 might be involved in the proliferation, differentiation, activation, migration of B lymphocytes and vaccine antigen presentation by up-regulating the expression of MIF, CD74, DOCK2, KLHL6 and other genes. Moreover, it also stimulated plasma cells to produce immunoglobulins and specific antibodies, thereby improving the humoral immune function of broilers and enhancing the immune effect of the NDV vaccine.

Dynamin 2 and BAR domain protein pacsin 2 cooperatively regulate formation and maturation of podosomes.

Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells.

Relationship Between Proactive Personality and Job Performance of Chinese Nurses: The Mediating Role of Competency and Work Engagement.

Background: As one of the main participants in health care, nurses are esteemed an important driving force for the vigorous health care development. Studies report that nurses' proactive personality has positive effects on their job performance; however, this relationship acquires further understanding. Objective: A cross-sectional study was performed to explore the relationship between nurses' proactive personality and job performance; the mediating role of nurses' competency and work engagement in this relationship was also evaluated. Methods: The study was performed in a large third-degree general hospital in October 2019, Xi'an, PR, China. A sample of 246 nurses participated in this cross-sectional study. Proactive personality was assessed with the Proactive Personality Questionnaire (PPS), job performance was assessed by Heilman three-item measurements, nurse competence was estimated with Nurse Competency Scale (NCS), and work engagement was assessed with the Utrecht Work Engagement Scale (UWES). The structural equation model was used to test the main hypotheses. Results: Structural equation model analysis revealed that work engagement partially mediated the association between proactive personality and job performance. The serial two-mediator model which was used to explore the association between proactive personality and job performance through competency and work engagement, in sequence, was demonstrated. Conclusion: This study demonstrates that work engagement partially mediated the association between nurses' proactive personality and their job performance. The serial two-mediator model demonstrated that proactive personality was associated with job performance via competency and work engagement. This study also revealed the critical role of nursing managers in understanding the nurses' proactive personality, which would facilitate them to enhance the latter's competency and promote their work engagement. All these will in turn constantly improve the overall quality of nursing and advance professional development of nursing and benefits for patients.

Glucose metabolism is required for oocyte maturation of zebrafish.

Glucose is an essential source of energy production for animal cells. The importance of glucose metabolism in oocyte maturation has been studied extensively in mammals. However, such roles in non-mammalian species are still largely unknown. Here, we used zebrafish as a model, which is phylogenetically distant from mammals, and analyzed the role of glucose metabolism in oocyte maturation. Major glucose transporters (GLUT/Slc2A) were analyzed in zebrafish, two Slc2a1 (Slc2a1a and Slc2a1b), one Slc2a2, and two Slc2a3 (Slc2a3a and Slc2a3b) were identified. Among these five Slc2a genes, slc2a1b exhibited the highest expression level in fully grown follicles. The expression of slc2a1b gradually increased during folliculogenesis, and also significantly increases during the oocyte maturation process. Consistently, the glucose concentration increases during natural oocyte maturation. By using a fluorescent glucose derivative (6-NBDG) to trace glucose transport, the uptake of glucose by ovarian follicles in a time-dependent manner could be observed. Intriguingly, by treatment of glucose in vitro, oocyte maturation could be induced in a time-, dose- and stage-dependent manner. Glucose can be metabolized by glycolysis, the pentose phosphate pathway (PPP), the hexosamine biosynthesis pathway (HBP), and the polyol pathway. Using the inhibitors for these pathways, we found only PPP but not glycolysis, HBP or polyol pathway is essential for oocyte maturation. All these results clearly demonstrate for the first time that the glucose metabolism is required for oocyte maturation of zebrafish, suggesting the highly conserved role of glucose metabolism in control of oocyte maturation between fish and mammals.

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Blocking estrogen-induced AMH expression is crucial for normal follicle formation.

In mammals, primordial follicles assembled in fetuses or during infancy constitute the oocyte resources for life. Exposure to 17beta-estradiol and phytogenic or endocrine-disrupting chemicals during pregnancy and/or the perinatal period leads to the failure of normal follicle formation. However, the mechanisms underlying estrogen-mediated abnormal follicle formation and physiological follicle formation in the presence of endogenous natural estrogen are not well understood. Here, we reveal that estrogen receptor 1, activated by estrogen, binds to the 5' region of the anti-Mullerian hormone (Amh) gene and upregulates its transcription before follicle formation in cultured mouse fetal ovaries. Ectopic expression of AMH protein was observed in pregranulosa cells of these explants. Furthermore, the addition of AMH to the culture medium inhibited normal follicle formation. Conversely, alpha-fetoprotein (AFP) produced in the fetal liver reportedly blocks estrogen action, although its role in follicle formation is unclear. We further demonstrated that the addition of AFP to the medium inhibited ectopic AMH expression via estrogen, leading to successful follicle formation in vitro Collectively, our in vitro experiments suggest that upon estrogen exposure, the integrity of follicle assembly in vivo is ensured by AFP.

Igf3: a novel player in fish reproduction†.

As in other vertebrates, fish reproduction is tightly controlled by gonadotropin signaling. One of the most perplexing aspects of gonadotropin action on germ cell biology is the restricted expression of gonadotropin receptors in somatic cells of the gonads. Therefore, the identification of factors conveying the action of gonadotropins on germ cells is particularly important for understanding the mechanism of reproduction. Insulin-like growth factors (Igfs) are recognized as key factors in regulating reproduction by triggering a series of physiological processes in vertebrates. Recently, a novel member of Igfs called Igf3 has been identified in teleost. Different from the conventional Igf1 and Igf2 that are ubiquitously expressed in a majority of tissues, Igf3 is solely or highly expressed in the fish gonads. The role of Igf3 in mediating the action of gonadotropin through Igf type 1 receptor on several aspects of oogenesis and spermatogenesis have been demonstrated in several fish species. In this review, we will summarize existing data on Igf3. This new information obtained from Igf3 provides insight into elucidating the molecular mechanism of fish reproduction, and also highlights the importance of Igf system in mediating the action of gonadotropin signaling on animal reproduction.

Igf3 is essential for ovary differentiation in zebrafish†.

Zebrafish gonadal sexual differentiation is an important but poorly understood subject. Previously, we have identified a novel insulin-like growth factor (Igf) named insulin-like growth factor 3 (Igf3) in teleosts. The importance of Igf3 in oocyte maturation and ovulation has been recently demonstrated by us in zebrafish. In this study, we have further found the essential role of Igf3 in gonadal sexual differentiation of zebrafish. A differential expression pattern of igf3 between ovary and testis during sex differentiation (higher level in ovary than in testis) was found in zebrafish. An igf3 knockout zebrafish line was established using TALENs-mediated gene knockout technique. Intriguingly, all igf3 homozygous mutants were males due to the female-to-male sex reversal occurred during sex differentiation. Further analysis showed that Igf3 did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells. Oocyte development was arrested at primary growth stage, and the ovary was gradually sex-reversed to testis before 60 day post fertilization (dpf). Such sex reversal was likely due to decreased germ cell proliferation by suppressing PI3K/Akt pathway in early ovaries of igf3 mutants. Estrogen is considered as a master regulator in fish sex differentiation. Here, we found that igf3 expression could be upregulated by estrogen in early stages of ovarian follicles as evidenced in in vitro treatment assays and cyp19a1a mutant zebrafish, and E2 failed to rescue the defects of igf3 mutants in ovarian development, suggesting that Igf3 may serve as a downstream factor of estrogen signaling in sex differentiation. Taken together, we demonstrated that Igf3 is essential for ovary differentiation in zebrafish.

Co-immobilization of multi-enzyme on reversibly soluble polymers in cascade catalysis for the one-pot conversion of gluconic acid from corn straw.

The difficulties in the process of cellulose cascade conversion based on immobilization technology lies in the recycling enzymes from rich solid-containing straw hydrolysate and the incompatibility of conventional immobilization with this process. In this study, three types of enzyme (cellulase, glucose oxidase and catalase) were successfully immobilized on a reversible soluble Eudragit L-100. Through the determination of the preparation conditions, enzymatic properties and catalytic conditions, the co-immobilized enzyme was applied to the catalytic reaction of one-pot conversion of corn straw to gluconic acid. The yield of gluconic acid achieved 0.28 mg/mg, conversion rate of cellulose in corn straw to gluconic acid reached 61.41%. The recovery of co-immobilized enzyme from solid substrate was achieved by using reversible and soluble characteristics of the carrier. After 6 times of recycling, the activity of co-immobilized enzyme was maintained at 52.38%, confirming the feasibility of multi-enzyme immobilization strategy using reversible soluble carrier in cascade reactions.

Genetic Deletion of miR-430 Disrupts Maternal-Zygotic Transition and Embryonic Body Plan.

MiR-430 is considered an important regulator during embryonic development, but genetic loss-of-function study is still lacking. Here we demonstrated that genetic deletion of the miR-430 cluster resulted in developmental defects in cell movement, germ layer specification, axis patterning and organ progenitor formation in zebrafish. Transcriptome analysis indicated that the maternally provided transcripts were not properly degraded whereas the zygotic genome expressed genes were not fully activated in the miR-430 mutants. We further found that a reciprocal regulatory loop exists between miR-430 and maternally provided transcripts: the maternally provided transcripts (Nanog, Dicer1, Dgcr8, and AGOs) are required for miR-430 biogenesis and function, whereas miR-430 is required for the clearance of these maternally provided transcripts. These data provide the first genetic evidence that miR-430 is required for maternal-zygotic transition and subsequent establishment of embryonic body plan.

Dual roles of PDE9a in meiotic maturation of zebrafish oocytes.

The essential role of cyclic guanosine monophosphate (cGMP) signaling in regulating the oocyte meiotic cell cycle has been established. However, control of the level of cGMP in ovarian follicles is unclear. The cGMP-hydrolyzing phosphodiesterases (PDEs) are important in regulating the cellular cGMP level. We used zebrafish as a model to study the role of a cGMP-hydrolyzing phosphodiesterase-9a (PDE9a) in meiotic maturation of oocytes. Three PDE9a coding genes (PDE9aa, PDE9ab, and PDE9ac) were identified in zebrafish. Both pde9aa and pde9ac are expressed in most adult tissues including the ovary, but pde9ab is only expressed in the ovary, kidney, pituitary, and brain. All three pde9as mRNA exhibited different expression profiles during folliculogenesis. All of them are highly expressed in the oocyte but not in the follicular cell. The expression of both pde9aa and pde9ab, but not pde9ac, in ovarian follicles increases during oocyte maturation either in natural ovulatory cycle or induced by administration of hCG in vivo. We overexpressed pde9aa by injection of capped pde9aa mRNA into the oocytes. The cGMP level was decreased, and oocyte maturation was stimulated. When the activity of PDE9a was blocked by a specific inhibitor, Bay736691, the oocyte maturation was also stimulated. The stimulatory effect could be blocked by a gap junction blocker. However, the spontaneous oocyte maturation of denuded oocytes was not largely affected after treatment with Bay736691. All of the mature oocytes obtained by either treatment of Bay736691 or injection of pde9aa mRNA, could be fertilized in vitro. These results demonstrate the dual roles of PDE9a in oocyte maturation. The basal level of PDE9a is responsible for maintaining the meiotic arrest, and the increased level of PDE9a induced by LH signaling is helpful for stimulating meiotic maturation by hydrolyzing cGMP in oocytes.

Zebrafish as a model for studying ovarian development: Recent advances from targeted gene knockout studies.

Ovarian development is a complex process controlled by precise coordination of multiple factors. The targeted gene knockout technique is a powerful tool to study the functions of these factors. The successful application of this technique in mice in the past three decades has significantly enhanced our understanding on the molecular mechanism of ovarian development. Recently, with the advent of genome editing techniques, targeted gene knockout research can be carried out in many species. Zebrafish has emerged as an excellent model system to study the control of ovarian development. Dozens of genes related to ovarian development have been knocked out in zebrafish in recent years. Much new information and perspectives on the molecular mechanism of ovarian development have been obtained from these mutant zebrafish. Some findings have challenged conventional views. Several genes have been identified for the first time in vertebrates to control ovarian development. Focusing on ovarian development, the purpose of this review is to briefly summarize recent findings using these gene knockout zebrafish models, and compare these findings with mammalian models. These established mutants and rapid development of gene knockout techniques have prompted zebrafish as an ideal animal model for studying ovarian development.