Assembly of Immunogenic Protein Particles toward Advanced Synthetic Vaccines.

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.

Serum triglyceride glucose index is a valuable predictor for visceral obesity in patients with type 2 diabetes: a cross-sectional study.

BACKGROUND: Since the triglyceride glucose (TyG) index can reflect insulin resistance, it has been proven to be an efficient predictor of glycolipid-metabolism-related diseases. Therefore, this study aimed to investigate the predictive value of the TyG index for visceral obesity (VO) and body fat distribution in patients with type 2 diabetes mellitus (T2DM). METHODS: Abdominal adipose tissue characteristics in patients with T2DM, including visceral adipose area (VAA), subcutaneous adipose area (SAA), VAA-to-SAA ratio (VSR), visceral adipose density (VAD), and subcutaneous adipose density (SAD), were obtained through analyses of computed tomography images at the lumbar 2/3 level. VO was diagnosed according to the VAA (> 142 cm2 for males and > 115 cm2 for females). Logistic regression was performed to identify independent factors of VO, and receiver operating characteristic (ROC) curves were used to compare the diagnostic performance according to the area under the ROC curve (AUC). RESULTS: A total of 976 patients were included in this study. VO patients showed significantly higher TyG values than non-VO patients in males (9.74 vs. 8.88) and females (9.59 vs. 9.01). The TyG index showed significant positive correlations with VAA, SAA, and VSR and negative correlations with VAD and SAD. The TyG index was an independent factor for VO in both males (odds ratio [OR] = 2.997) and females (OR = 2.233). The TyG index ranked second to body mass index (BMI) for predicting VO in male (AUC = 0.770) and female patients (AUC = 0.720). Patients with higher BMI and TyG index values showed a significantly higher risk of VO than the other patients. TyG-BMI, the combination index of TyG and BMI, showed significantly higher predictive power than BMI for VO in male patients (AUC = 0.879 and 0.835, respectively) but showed no significance when compared with BMI in female patients (AUC = 0.865 and 0.835, respectively). CONCLUSIONS: . TyG is a comprehensive indicator of adipose volume, density, and distribution in patients with T2DM and is a valuable predictor for VO in combination with anthropometric indices, such as BMI.

[A multiplex PCR-based sensitive and specific method for detecting Y chromosome material in patients with Turner syndrome].

OBJECTIVE: To develop a multiplex PCR method for a rapid detection of Y chromosome-specific sequences in patients with Turner syndrome. METHODS: Nine genes were selected from various regions of the Y chromosome for designing the primers, which included SRY, TBL1Y, TSPY on the short arm of the Y chromosome, DDX3Y, HSFY1, RPS4Y2 and CDY1 on the long arm of Y chromosome and SHOX in the short arm and SPRY3 in the long arm of the pseudoautosomal region (PAR) of X and Y chromosomes. A multiplex PCR method for the nine genes in Y chromosome was established and optimized. The sensitivity was tested by using different amounts of genomic DNA. A total of 36 patients with Turner syndrome and a patient with male dwarfism with karyotype of 46, X, +mar were examined by the multiplex PCR method for the existence of materials from the Y chromosome. RESULTS: The optimization results of the multiplex PCR reaction system (50 µL) showed that when the final concentration of upstream and downstream of each pair of primers was 0.1 µM, the multiplex PCR reaction of the 9 pairs of primers clearly amplified the target with the expected band size, and there was no non-specific amplification. The bands were clearly visible when the amount of genomic DNA in the multiple PCR reaction system was as low as 1 ng. By using the method, we have examined the 36 patients with Turner syndrome. One patient with Turner syndrome with karyotype of 45,X[40]/47XYY[21] amplified specific seven genes on Y chromosome, 35 patients with Turner syndrome amplified only two target genes SHOX and SPRY3, but not the other seven specific genes on the Y chromosome, which was in keeping with the clinical manifestations of such patients. CONCLUSION: This study established a multiplex PCR reaction system with nine genes, which can quickly and accurately screen Y chromosome materials in patients with Turner syndrome. It has the advantages of low cost, simple operation, high specificity and rapid turn-around time, and can be used to detect Turner syndrome patients with Y chromosome material in time. The method has provided a diagnostic basis for preventive gonad resection to prevent malignant gonadal tumors.

Engineered Mycobacterium tuberculosis antigen assembly into core-shell nanobeads for diagnosis of tuberculosis.

Despite recent advances in diagnosis, tuberculosis (TB) remains one of the ten leading causes of death worldwide. Here, we engineered Mycobacterium tuberculosis (Mtb) proteins (ESAT6, CFP10, and MTB7.7) to self-assemble into core-shell nanobeads for enhanced TB diagnosis. Respective purified Mtb antigen-coated polyester beads were characterized and their functionality in TB diagnosis was tested in whole blood cytokine release assays. Sensitivity and specificity were studied in 11 pulmonary TB patients (PTB) and 26 healthy individuals composed of 14 Tuberculin Skin Test negative (TSTn) and 12 TST positive (TSTp). The production of 6 cytokines was determined (IFNγ, IP10, IL2, TNFα, CCL3, and CCL11). To differentiate PTB from healthy individuals (TSTp + TSTn), the best individual cytokines were IL2 and CCL11 (>80% sensitivity and specificity) and the best combination was IP10 + IL2 (>90% sensitivity and specificity). We describe an innovative approach using full-length antigens attached to biopolyester nanobeads enabling sensitive and specific detection of human TB.

First trimester serum PAPP-A is associated with placenta accreta: a retrospective study.

PURPOSE: Our objective of this study was to investigate whether first trimester serum pregnancy-associated plasma protein-A (PAPP-A) differed amongst pregnancies with placenta previa-accreta and non-adherent placenta previa and healthy pregnancies by a retrospective cohort analysis. METHODS: A total of 177 pregnant females were included in the study, as follows: 35 cases of placenta previa-accreta, 30 cases of non-adherent placenta previa, and 112 cases of BMI and age matched, healthy pregnant controls. PAPP-A multiples of the median (MoM) were acquired from laboratory data files in 1 January 2017-30 September 2019. The probable maternal serum biochemical predictor of placenta accreta was analyzed by using multiple logistic regression analysis. RESULTS: PAPP-A MoM of placenta previa-accreta group was significantly higher than those of the non-adherent placenta previa group and control group (p = 0.009 < 0.05, p < 0.001). Serum PAPP-A was found to be significantly positively associated with placenta accreta after adjusted gestational week at time of blood sampling, BMI, age, smoking, and previous cesarean section history (OR: 3.51; 95% CI: 1.77-6.94; p = 0.0003 < 0.05). In addition, smoking (OR: 9.17; 95% CI: 1.69-49.62; p = 0.010 < 0.05) and previous cesarean section history (OR: 2.75; 95% CI: 1.23-6.17; p = 0.014 < 0.05) were also significantly associated with placenta accreta. CONCLUSION: Increased first trimester serum PAPP-A was significantly positively associated with placenta accreta, suggesting that the potential role of PAPP-A in identifying pregnancies at high risk for placenta accreta. Smoking and previous cesarean section history may be the risk factors for accreta in placenta previa patients.

Innovative antigen carrier system for the development of tuberculosis vaccines.

A major obstacle to tuberculosis (TB)-subunit-vaccine development has been the induction of inadequate levels of protective immunity due to the limited breadth of antigen in vaccine preparations. In this study, immunogenic mycobacterial fusion peptides Ag85B-TB10.4 and Ag85B-TB10.4-Rv2660c were covalently displayed on the surface of self-assembled polyester particles. This study investigated whether polyester particles displaying mycobacterial antigens could provide augmented immunogenicity (i.e., offer an innovative vaccine formulation) when compared with free soluble antigens. Herein, polyester particle-based particulate vaccines were produced in an endotoxin-free Escherichia coli strain and emulsified with the adjuvant dimethyl dioctadecyl ammonium bromide. C57BL/6 mice were used to study the immunogenicity of formulated particulate vaccines. The result of humoral immunity showed the antibodies only interacted with target antigens and not with PhaC and the background proteins of the production host. The analysis of T helper 1 cellular immunity indicated that a relatively strong production of cellular immunity biomarkers, IFN-γ and IL-17A cytokines, was induced by particulate vaccines when compared with the respective soluble controls. This study demonstrated that polyester particles have the potential to perform as a mycobacterial antigen-delivery agent to induce augmented antigen-specific immune responses in contrast to free soluble vaccines.-Chen, S., Sandford, S., Kirman, J. R., Rehm, B. H. A. Innovative antigen carrier system for the development of tuberculosis vaccines.

Brain-derived neurotrophic factor promotes proliferation and progesterone synthesis in bovine granulosa cells.

Brain-derived neurotrophic factor (BDNF) is involved in regulating the growth of ovarian follicles, maturation of the oocyte, and development of the early embryo through its receptor, tyrosine kinase receptor B (TrkB). However, it is still unclear as to how BDNF influences proliferation and steroidogenesis of bovine granulosa cells (GCs). In this paper, we confirmed that BDNF and TrkB were expressed in bovine GCs, and that proliferation and steroidogenesis by bovine GCs were reduced by knockdown of BDNF or inhibition of TrkB. With respect to GC proliferation, BDNF enhanced cellular viability and the percentage of cells in the S phase. BDNF also activated both protein kinase B (PKB, also known as AKT) and the extracellular signal-regulated protein kinase 1/2 (ERK1/2)-signaling pathway. Through the AKT-signaling pathway, BDNF increased the expression of proliferation-related genes, including cyclin A1 (CCNA1), cyclin E2 (CCNE2), cyclin D1 (CCND1), and cyclin-dependent kinase 1 (CDK1). However, through the ERK1/2 signaling pathway, BDNF only increased the expression of CCNA1 and CCNE2. Regarding steroidogenesis by bovine GCs, BDNF promoted progesterone (P 4 ) synthesis, but had no effect on estradiol; it also activated the AKT-signaling pathway and increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase, 3ß- and steroid δ-isomerase 1 (HSD3B1). In summary, our data are the first to show that BDNF promotes the proliferation of bovine GCs through TrkB-AKT and ERK1/2 signaling pathways and increases P4 synthesis by bovine GCs through the TrkB-AKT signaling pathway.

Brain-derived neurotrophic factor: A steroidogenic regulator of Leydig cells.

The brain-derived neurotrophic factor (BDNF) was first recognized for its roles in the peripheral and central nervous systems, and its complex functions on mammalian organs have been extended constantly. However, to date, little is known about its effects on the male reproductive system, including the steroidogenesis of mammals. The purpose of this study was to elucidate the effects of BDNF on testosterone generation of Leydig cells and the underlying mechanisms. We found that BDNF-induced proliferation of TM3 Leydig cells via upregulation of proliferating cell nuclear antigen ( Pcna) and promoted testosterone generation as a result of upregulation of steroidogenic acute regulatory protein ( Star), 3b-hydroxysteroid dehydrogenase ( Hsd3b1), and cytochrome P450 side-chain cleavage enzyme ( Cyp11a1) both in primary Leydig cells and TM3 Leydig cells, which were all attenuated in Bdnf knockdown TM3 Leydig cells. Furthermore, the possible mechanism of testosterone synthesis was explored in TM3 Leydig cells. The results showed that BDNF enhanced extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation, and the effect was disrupted by Bdnf deletion. Moreover, PD98059, a potent selective inhibitor of ERK1/2 activation, compromised BDNF-induced testosterone generation and upregulation of Star, Hsd3b1, and Cyp11a1. The Bdnf knockdown assay, on the other hand, indicated the autocrine effect of BDNF on steroidogenesis in TM3 Leydig cells. On the basis of these results, we concluded that BDNF, acting as an autocrine factor, induced testosterone generation as a result of the upregulation of Star, Hsd3b1, and Cyp11a1 via stimulation of the ERK1/2 pathway.

Polyester as Antigen Carrier toward Particulate Vaccines.

Spherical polyhydroxyalkanoate (PHA) inclusions are naturally self-assembled inside bacteria. These PHA beads are shell-core structures composed of a hydrophobic PHA core surrounded by proteins, such as PHA synthase (PhaC). PhaC is covalently attached and serves as an anchor protein for foreign protein vaccine candidate antigens. PHA beads displaying single and multiple antigens showed enhanced immunological properties when compared to respective soluble vaccines. This review highlights the unique design space of the PHA bead-based vaccines toward the development of safe and synthetic particulate vaccines. The PHA bead technology will be compared with chemically synthesized polyesters, such as polylactic acids, formulated to deliver vaccine antigens. The performance of PHA bead vaccine candidates to induce specific immune responses and protective immunity against bacterial and viral pathogens in animal trials will be summarized. We propose that the PHA bead technology offers a versatile vaccine platform for design and cost-effective manufacture of synthetic multivalent vaccines.

Effective and economical column-based method for RNA isolation from animal cells.

OBJECTIVES: To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. RESULTS: Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120 ng/5 × 106 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. CONCLUSIONS: The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20 min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.

Association of RBP-4 gene polymorphisms with follicular cysts in large white sows.

Follicular cysts, which is a common infertility disease, can cause financial losses in pig breeding programmes. The pathogenesis and mechanisms of the formation of follicular cysts are not understood clearly. In our previous study, the concentration of retinol-binding protein 4 (RBP-4) in the follicular fluid (FF) of the ovary with follicular cysts was found to be significantly higher than that of normal ovary, thereby suggesting that RBP-4 may be a candidate biomarker for porcine follicular cysts. To study the association of RBP-4 and follicular cysts further, we detected the polymorphisms of the RBP-4 gene and the presence of follicular cysts by PCR-Restriction fragment length polymorphism (RFLP) assay. In this study, we screened the mutations of RBP-4 gene in 79 sows with follicular cysts and 100 normal sows without cysts. Results showed that +249-63G>C polymorphisms were significantly associated with follicular cysts, and sows with CC genotype in RBP-4 gene had a high risk of developing follicular cysts. Hence, our findings further proved that RBP-4 may be a novel biomarker for follicular cysts, which may be valuable for the diagnosis of follicular cysts and molecular breeding of pigs.

Astragaloside IV Alleviates Ammonia-Induced Apoptosis and Oxidative Stress in Bovine Mammary Epithelial Cells.

Ammonia is one of the major toxic components of metabolites in blood and tissues of high-producing dairy cows and could affect the health of bovine mammary glands. Bovine mammary epithelial cells are sensitive to oxidative stress induced by intensive cell metabolism. In our previous study, we found that ammonia could induce oxidative stress, apoptosis and inflammatory responses in bovine mammary epithelial cells. In the present study, the cytoprotective effects of astragaloside IV against ammonia in vitro were explored. The results demonstrated that pretreatment of MAC-T cells with astragaloside IV could potently suppress the increase in the level of intracellular reactive oxygen species (ROS) and the rate of cell apoptosis, inhibit the ammonia-induced inflammatory responses, and rescue the decrease of cell viability. Astragaloside IV prevented ammonia-induced endoplasmic reticulum stress. Astragaloside IV also significantly suppressed the levels of BAX, caspase 3 and p53 phosphorylation in ammonia-induced MAC-T cells. Nuclear factor erythroid 2-related factor 2(Nrf2) was essential for cytoprotective effects of astragaloside IV in MAC-T cells, as knockdown of Nrf2 dramatically abolished the prosurvival effects of astragaloside IV on treated cells. Furthermore, the PI3K/AKT and ERK/MAPK pathways were responsible for the induction of Nrf2 by astragaloside IV. In conclusion, astragaloside IV played a beneficial role against ammonia-induced damage of MAC-T cells. This provides a cue for future study to use astragaloside IV as a protective and curative agent against ammonia exposure of mammary glands in dairy cows.

Protective Effects of Astragaloside IV against LPS-Induced Endometritis in Mice through Inhibiting Activation of the NF-κB, p38 and JNK Signaling Pathways.

Endometritis, inflammation of the endometrium, is a common reproductive obstacle disease that can lead to infertility in female animals. Astragaloside IV (AS IV), one of the major and active components of the Astragalus membranaceus (Fisch.) Bunge, is known for its anti-inflammatory effects. In the present study, the effects and mechanisms of AS IV on lipopolysaccharide (LPS)-induced endometritis were investigated using a mouse model. Female mice were prepared with AS IV (0.01 mg/g) by gavage for six days before being stimulated with LPS. The results showed that the histopathological changes, levels of inflammatory cytokines (IL-1ß and TNF-α), concentration of NO, and myeloperoxidase (MPO) activity in LPS-induced uteri were attenuated significantly by pretreatment with AS IV. Furthermore, LPS-induced activations of NF-κB, p38, and JNK signal pathways were suppressed by pretreatment with AS IV. In conclusion, the data provided new evidence that AS IV effectively attenuates LPS-induced endometritis through inhibition of TLR4-mediated NF-κB, p38, and JNK signaling pathways, implying that AS IV might become a promising potential anti-inflammatory agent for endometritis and other inflammatory diseases.

Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary.

BACKGROUND: Ovarian retinoid homeostasis plays an important role in the physiological function of the ovary. Retinol-binding protein 4 (RBP4) acts as the mediator for the systemic and intercellular transport of retinol and is heavily involved in cellular retinol influx, efflux, and exchange. However, the expression patterns and regulatory mechanisms of Rbp4 in the ovary remain unclear. METHODS: The expression pattern of ovarian Rbp4 was examined in immature mice during different developmental stages and in adult mice during different stages of the estrous cycle. The potential regulation and mechanisms of ovarian Rbp4 expression by estrogen and related gonadotropins in mouse ovaries were also investigated. RESULTS: The present study demonstrated that the ovarian expression of Rbp4 remained constant before puberty and increased significantly in the peripubertal period. In adult female mice, the expression of Rbp4 increased at proestrus and peaked at estrus at both the mRNA and protein levels. The protein distribution of RBP4 was mainly localized in the granulosa cell and theca cell layer in follicles. In addition, the expression of Rbp4 was significantly induced by follicle-stimulating hormone (FSH) or FSH + luteinizing hormone (LH) in combination in immature mouse (3 weeks old) ovaries in vivo and in granulosa cells cultured in vitro, both at the mRNA and protein levels. In contrast, treatment with LH or 17ß-estradiol did not exhibit any observable effects on ovarian Rbp4 expression. Transcription factors high-mobility group AT-hook 1 (HMGA1), steroidogenic factor 1 (SF-1), and liver receptor homolog 1 (LRH-1) (which have been previously shown to be involved in activation of Rbp4 transcription), also responded to FSH stimulation. In addition, H-89, an inhibitor of protein kinase A (PKA), and the depletion of HMGA1, SF-1, and LRH-1 by small interfering RNAs (siRNAs), resulted in a dramatic loss of the induction of Rbp4 expression by FSH at both the mRNA and protein levels. CONCLUSIONS: These data indicate that the dynamic expression of Rbp4 is mainly regulated by FSH through the cAMP-PKA pathway, involving transcriptional factors HMGA1, SF-1, and LRH-1, in the mouse ovary during different stages of development and the estrous cycle.

Follicle-stimulating hormone (FSH) promotes retinol uptake and metabolism in the mouse ovary.

BACKGROUND: Retinoids (retinol and its derivatives) are required for the development and maintenance of normal physiological functions of the ovary. However, the mechanisms underlying the regulation of ovarian retinoid homeostasis during follicular development remain unclear. METHODS: The present study determined retinoid levels and the expression levels of genes involved in the retinol uptake and its metabolic pathway in the ovaries of follicle-stimulating hormone (FSH)-treated mice and in granulosa cells treated with FSH using ultra performance liquid chromatography (UPLC) combined with quadrupole time-of-flight high-sensitivity mass spectrometry (Q-TOF/HSMS) and real-time PCR analysis. RESULTS: The levels of total retinoids and retinoic acid (RA) and expressions of retinol-oxidizing enzyme genes alcohol dehydrogenase 1 (Adh1) and aldehyde dehydrogenase (Aldh1a1) are increased in the ovaries of mice treated with FSH; in contrast, the retinyl ester levels and retinol-esterifying enzyme gene lecithin: retinol acyltransferase (Lrat) expression are diminished. In FSH-treated granulosa cells, the levels of retinyl esters, retinaldehyde, and total retinoids are augmented; and this is coupled with an increase in the expressions of stimulated by retinoic acid 6 (Stra6) and cellular retinol-binding protein 1 (Crbp1), genes in the retinol uptake pathway, and Adh1, Adh7, and Aldh1a1 as well as a diminution in Lrat expression. CONCLUSIONS: These data suggest that FSH promotes retinol uptake and its conversion to RA through modulating the pathways of retinol uptake and metabolism in the mouse ovary. The present study provides a possible mechanism for the regulation of endogenous RA signaling in the developing follicles.

Effects of ammonia on apoptosis and oxidative stress in bovine mammary epithelial cells.

Ammonia, produced mainly from the deamination of amino acids and glutamine, is one of the major toxic components in blood and tissues that may affect bovine health. However, the physiological and pathological roles of ammonia in the mammary glands are not understood clearly. In the present study, the bovine mammary epithelial cell line (MAC-T) was utilised as an in vitro model to determine the effects of ammonia on bovine mammary gland. We demonstrated that ammonia stimulated the production of intracellular reactive oxygen species, decreased mitochondrial membrane potential, interrupted intracellular calcium ion (Ca2+) homeostasis and induced cell apoptosis. Ammonia also significantly reduced cell viability and increased the proportion of apoptotic cells through enhancing the level of p53 phosphorylation and increasing the expressions of BAX, caspase 8, caspase 9, caspase 3. Interestingly, bumetanide, a specific Na+ K+ 2Cl--cotransporter inhibitor, dramatically abolished the damaging effects of ammonia on the cells. These data suggest that ammonia exposure induces apoptosis in bovine mammary epithelial cells via activation of the p53 pathway and the mitochondrial apoptotic pathway, and that these effects involved the Na+ K+ 2Cl--cotransporter.

Role of lncRNAs as Novel Biomarkers and Therapeutic Targets in Ovarian Cancer.

Ovarian cancer (OC) is the leading cause of death among all gynecological malignancies in the world and its underlying mechanism is still unclear. Compared with research on microRNAs, research on long non-coding RNAs (lncRNAs) is still in its infancy. Studies in recent years have demonstrated that lncRNAs exhibit multiple biological functions in various stages of OC development. In this review, we conclude that lncRNAs are closely involved in the pathogenesis of OC. The expression of lncRNAs indicates the early diagnosis, prognosis, and response to chemotherapy of OC. An attractive approach to treatment of OC is lncRNA small interfering RNA or acting as a plasmid targeting the expression of toxic genes, which is a novel step toward a major breakthrough in the treatment of human OC. E2-regulated lncRNA and its polymorphism, methylation, are also involved in OC. Further research efforts are needed before fully identifying, characterizing, and elucidating the actual functions of lncRNAs in OC at the molecular level and putting them into clinical practice.

Differential expression of microRNAs in TM3 Leydig cells of mice treated with brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that can promote the development and proliferation of neurons. BDNF has been found to be involved in male reproduction. Leydig cells in testicular interstitial tissues can secrete testosterone in a luteinizing hormone-dependent manner. We showed that BDNF and its receptor TrkB were expressed in mice TM3 Leydig cells in the present study. Furthermore, BDNF can promote proliferation of mouse TM3 Leydig cells in vitro. Results of microRNA (miRNA) deep sequencing showed that BDNF can alter the expression profile of miRNAs in TM3 Leydig cells. Eighty-three miRNAs were significantly different in the BDNF-treated and control groups (fold change of >2.0 or <0.5, P < 0.05) wherein 40 were upregulated and 43 were downregulated. The expression levels of miR-125a-5p, miR-22-5p, miR-342-59, miR-451a, miR-148a-5p, miR-29b-3p, miR-199b-5p, and miR-145a-5p were further confirmed by quantitative real-time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs participate in the pathways involved in signal transduction, cancer, metabolism, endocrine system, immune system, and nerve system. This study indicated that miRNAs might be involved in the BDNF-regulated cellular functions of Leydig cells.

MicroRNA-16 Modulates Melatonin-Induced Cell Growth in the Mouse-Derived Spermatogonia Cell Line GC-1 spg Cells by Targeting Ccnd1.

Melatonin exerts a range of physiological effects. However, the functional significance of melatonin in spermatogenesis and the underlying mechanisms remain unclear. MicroRNAs (miRNAs) are essential in the initiation and progression of testicular development, including spermatogenesis. Thus far, limited information is known about the role of miRNAs in melatonin-mediated spermatogenesis. In this study, the expression levels of testicular miRNA machinery genes, namely, Dgcr8 and Xpo5, were significantly increased by melatonin. The miRNA expression profile was identified in the testes of melatonin-treated mice. Ten miRNAs were significantly up-regulated, and 15 miRNAs were down-regulated. Melatonin (25 µM) enhanced cell growth and reduced apoptosis in GC-1 spg cells. Among the down-regulated miRNAs, miR-16 expression was influenced by melatonin in GC-1 spg cells. The miR-16 mimics in GC-1 spg cells significantly suppressed cell growth and promoted cell apoptosis. Conversely, transfection of the miR-16 inhibitor significantly increased cell growth and decreased cell apoptosis. The protein expression level of CCND1 (Cyclin D1) in GC-1 spg cells was decreased by the miR-16 mimics and increased by knockdown of miR-16. Moreover, bioinformatics and reporter activity analyses showed that Ccnd1 was a potential target of miR-16. These results suggested that miR-16 may function as a novel regulator of testicular functions during melatonin stimulation by targeting Ccnd1.

Enzyme Engineering for In Situ Immobilization.

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.