H3K9me3 Levels Affect the Proliferation of Bovine Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) possess the characteristics of self-renewal and differentiation, as well as the ability to generate functional sperm. Their unique stemness has broad applications in male infertility treatment and species preservation. In rodents, research on SSCs has been widely reported, but progress is slow in large livestock such as cattle and pigs due to long growth cycles, difficult proliferation in vitro, and significant species differences. Previously, we showed that histone 3 (H3) lysine 9 (K9) trimethylation (H3K9me3) is associated with the proliferation of bovine SSCs. Here, we isolated and purified SSCs from calf testicular tissues and investigated the impact of different H3K9me3 levels on the in vitro proliferation of bovine SSCs. The enriched SSCs eventually formed classical stem cell clones in vitro in our feeder-free culture system. These clones expressed glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1, specific marker for SSCs), NANOG (pluripotency protein), C-KIT (germ cell marker), and strong alkaline phosphatase (AKP) positivity. qRT-PCR analysis further showed that these clones expressed the pluripotency genes NANOG and SOX2, and the SSC-specific marker gene GFRα1. To investigate the dynamic relationship between H3K9me3 levels and SSC proliferation, H3K9me3 levels in bovine SSCs were first downregulated using the methyltransferase inhibitor, chaetocin, or transfection with the siRNA of H3K9 methyltransferase suppressor of variegation 3-9 homologue 1 (SUV39H1). The EDU (5-Ethynyl-2'-deoxyuridine) assay revealed that SSC proliferation was inhibited. Conversely, when H3K9me3 levels in bovine SSCs were upregulated by transfecting lysine demethylase 4D (KDM4D) siRNA, the EDU assay showed a promotion of cell proliferation. In summary, this study established a feeder-free culture system to obtain bovine SSCs and explored its effects on the proliferation of bovine SSCs by regulating H3K9me3 levels, laying the foundation for elucidating the regulatory mechanism underlying histone methylation modification in the proliferation of bovine SSCs.

Bovine Pluripotent Stem Cells: Current Status and Prospects.

Pluripotent stem cells (PSCs) can differentiate into three germ layers and diverse autologous cell lines. Since cattle are the most commonly used large domesticated animals, an important food source, and bioreactors, great efforts have been made to establish bovine PSCs (bPSCs). bPSCs have great potential in bovine breeding and reproduction, modeling in vitro differentiation, imitating cancer development, and modeling diseases. Currently, bPSCs mainly include bovine embryonic stem cells (bESCs), bovine induced pluripotent stem cells (biPSCs), and bovine expanded potential stem cells (bEPSCs). Establishing stable bPSCs in vitro is a critical scientific challenge, and researchers have made numerous efforts to this end. In this review, the category of PSC pluripotency; the establishment of bESCs, biPSCs, and bEPSCs and its challenges; and the application outlook of bPSCs are discussed, aiming to provide references for future research.

Effect of Serotonin (5-Hydroxytryptamine) on Follicular Development in Porcine.

5-Hydroxytryptamine (5-HT) is an inhibitory neurotransmitter widely distributed in mammalian tissues, exerting its effects through binding to various receptors. It plays a crucial role in the proliferation of granulosa cells (GCs) and the development of follicles in female animals, however, its effect on porcine follicle development is not clear. The aim of this study is to investigate the expression of 5-HT and its receptors in various parts of the pig ovary, as well as the effect of 5-HT on porcine follicular development by using ELISA, quantitative real-time PCR (qPCR) and EdU assays. Firstly, we examined the levels of 5-HT and its receptors in porcine ovaries, follicles, and GCs. The findings revealed that the expression of different 5-HT receptors varied among follicles of different sizes. To investigate the relationship between 5-HT and its receptors, we exposed the GCs to 5-HT and found a decrease in 5-HT receptor expression compared to the control group. Subsequently, the treatment of GCs with 0.5 µM, 5 µM, and 50 µM 5-HT showed an increase in the expression of cell cycle-related genes, and EdU results indicated cell proliferation after the 0.5 µM 5-HT treatment. Additionally, the expression of genes involved in E2 synthesis was examined after the treatment of granulosa cells with 0.5 µM 5-HT. The results showed that CYP19A1 and HSP17ß1 expression was decreased. These results suggest that 5-HT might affect the development of porcine follicle by promoting the proliferation of GCs and inhibiting the synthesis of estrogen. This provides a new finding for exploring the effect of 5-HT on follicular development, and lays a foundation for further research on the mechanism of 5-HT in follicles.

Heat-Resistant, Robust, and Hydrophilic Separators Based on Regenerated Cellulose for Advanced Supercapacitors.

Separators in supercapacitors (SCs) typically suffer from defects of low mechanical property, limited ion transport, and electrolyte wettability, and poor thermal stability, impeding the development of SCs. Herein, high-performance regenerated cellulose (RC) based separators are designed that are fabricated by effective hydrolytic etching of inorganic CaCO3 nanoparticles from a filled RC membrane. The as-prepared RC separator displays excellent comprehensive performances such as higher tensile strength (75.83 MPa) and thermal stability (200 °C), which is superior to commercial polypropylene-based separator (Celgard 2500) and sufficient to maintain their structural integrity even at temperatures in excess of 200 °C. Benefiting from its hydrophilicity, high porosity, and outstanding electrolyte uptake rate (208.5%), the RC separator exhibits rapid transport and permeability of ions, which is 2.5× higher than that of the commercial nonwoven polypropylene separator (NKK -MPF30AC-100) validated by electrochemical tests in the 1.0 m Na2 SO4 electrolyte. Results show that porous RC separator with unique advantages of superior electrolyte wettability, mechanical robustness, and high thermal stability, is a promising separator for SCs with high-performance and safety.

Tumor cell vaccine combined with Newcastle disease virus promote immunotherapy of lung cancer.

Lung cancer is a fatal disease with the highest worldwide morbidity and mortality rates. Despite recent advances in targeted therapy and immune checkpoint inhibitors for cancer, their efficacy remained limited. Therefore, we designed a Newcastle disease virus (NDV)-modified tumor whole-cell vaccine as a therapeutic vaccine and identified its antigen presentation level to develop effective immunotherapy. Then, we calculated the therapeutic and immune-stimulating effects of NDV-modified lung cancer cell vaccine and intratumoral NDV injection combination on tumor-bearing mice. The results showed that the immunogenic cell death (ICD) expression in NDV-modified lung cancer cell vaccine stimulates dendritic cell maturation and T cell activation in vivo and in vitro. Moreover, NDV-modified lung cancer cell vaccine combined with intratumoral NDV injection could significantly inhibit tumor growth and enhance the differentiation of Th1 cells and Inflammatory cell infiltration in vivo, leading to an excellent immunotherapeutic effect. Therefore, our results revealed that NDV-modified lung cancer cell vaccine combined with intratumoral NDV injection could promote antigen presentation and induce a strong antitumor immune response, which provided a promising combined therapy strategy for tumor immunotherapy.

Method for Diagnosing Bearing Faults in Electromechanical Equipment Based on Improved Prototypical Networks.

Due to the complexity of electromechanical equipment and the difficulties in obtaining large-scale health monitoring datasets, as well as the long-tailed distribution of data, existing methods ignore certain characteristics of health monitoring data. In order to solve these problems, this paper proposes a method for the fault diagnosis of rolling bearings in electromechanical equipment based on an improved prototypical network-the weight prototypical networks (WPorNet). The main contributions of this paper are as follows: (1) the prototypical networks, which perform well on small-sample classification tasks, were improved by calculating the different levels of influence of support sample distributions in order to achieve the prototypical calculation. The change in sample influence was calculated using the Kullback-Leibler divergence of the sample distribution. The influence change in a specific sample can be measured by assessing how much the distribution changes in the absence of that sample; and (2) The Gramian Angular Field (GAF) algorithm was used to transform one-dimensional time series into two-dimensional vibration images, which greatly improved the application effect of the 2D convolutional neural network (CNN). Through experiments on MAFAULDA and CWRU bearing datasets, it was shown that this network effectively solves the shortcomings of a small number of valid samples and a long-tail distribution in health monitoring data, it enhances the dependency between the samples and the global data, it improves the model's feature extraction ability, and it enhances the accuracy of model classification. Compared with the prototypical network, the improved network model increased the performance of the 2-way 10-shot, 2-way 20-shot, and 2-way 50-shot classification tasks by 5.23%, 5.74%, and 4.37%, respectively, and it increased the performance of the 4-way 10-shot, 4-way 20-shot, and 4-way 50-shot classification tasks by 12.02%, 10.47%, and 4.66%, respectively. Experimental results show that the improved prototypical network model has higher sample classification accuracy and stronger anti-interference ability compared with traditional small-sample classification models.

Expression of Phospholipase D Family Member 6 in Bovine Testes and Its Molecular Characteristics.

Spermatogonial stem cells (SSCs) are the only primitive spermatogonial cells in males that can naturally transmit genetic information to their offspring and replicate throughout their lives. Phospholipase D family member 6 (PLD6) has recently been found to be a surface marker for SSCs in mice and boars; however, it has not been validated in cattle. The results of reversed transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) found that the relative expression of the PLD6 gene in the testicular tissues of two-year-old Simmental calves was significantly higher than that of six-month-old calves. Immunofluorescent staining further verified the expression of PLD6 protein in bovine spermatogenic cells like germ cell marker DEAD box helicase 4 (DDX4, also known as VASA). Based on multiple bioinformatic databases, PLD6 is a conservative protein which has high homology with mouse Q5SWZ9 protein. It is closely involved in the normal functioning of the reproductive system. Molecular dynamics simulation analyzed the binding of PLD6 as a phospholipase to cardiolipin (CL), and the PLD6-CL complex showed high stability. The protein interaction network analysis showed that there is a significant relationship between PLD6 and piwi-interacting RNA (piRNA) binding protein. PLD6 acts as an endonuclease and participates in piRNA production. In addition, PLD6 in bovine and mouse testes has a similar expression pattern with the spermatogonium-related genes VASA and piwi like RNA-mediated gene silencing 2 (PIWIL2). In conclusion, these analyses imply that PLD6 has a relatively high expression in bovine testes and could be used as a biomarker for spermatogenic cells including SSCs.

Effects of dimethyl sulfoxide (DMSO) on DNA methylation and histone modification in parthenogenetically activated porcine embryos.

Epigenetic mechanisms play an important role in oogenesis and early embryo development in mammals. Dimethyl sulfoxide (DMSO) is frequently used as a solvent in biological studies and as a vehicle for drug therapy. Recent studies suggest that DMSO detrimentally affects porcine embryonic development, yet the mechanism of the process in parthenogenetically activated porcine embryos has not been reported. In this study, we found that treatment of embryos with 1.5% DMSO significantly decreased the cleavage and blastocyst rates, total cell number of blastocysts and the anti-apoptotic gene BCL-2 transcription level; however, the percentage of apoptotic cells and the expression levels of the pro-apoptotic gene BAX were not changed. Treatment with DMSO significantly decreased the expression levels of DNMT1 , DNMT3a , DNMT3b , TET1 , TET2 , TET3 , KMT2C , MLL2 and SETD3 in most of the stages of embryonic development and increased 5-mC signals, while the staining intensity for 5-hmC had no change in porcine preimplantation embryos from 2-cell to the blastocyst stages. Meanwhile, DMSO decreased the level of H3K4me3 during the development of parthenogenetically activated porcine embryos. After treatment with DMSO, expression levels of the pluripotency-related genes POU5F1 and NANOG decreased significantly (P <0.01), whereas the imprinted gene H19 did not change (P >0.05). In conclusion, these results suggest that DMSO can affect genome-wide DNA methylation and histone modification by regulating the expression of epigenetic modification enzymes, and DMSO also influences the expression level of pluripotent genes. These dysregulations lead to defects in embryonic development.

Surface-Plasmon-Enhanced Transmetalation between Copper and Palladium Nanoparticle Catalyst.

Surface-plasmon-mediated phenylacetylide intermediate transfer from the Cu to the Pd surface affords a novel mechanism for transmetalation, enabling wavelength-tunable cross-coupling and homo-coupling reaction pathway control. C-C bond forming Sonogashira coupling and Glaser coupling reactions in O2 atmosphere are efficiently driven by visible light over heterogeneous Cu and Pd nanoparticles as a mixed catalyst without base or other additives. The reaction pathway can be controlled by switching the excitation wavelength. Shorter wavelengths (400-500 nm) give the Glaser homo-coupling diyne, whereas longer wavelength irradiation (500-940 nm) significantly increases the degree of cross-coupling Sonogashira coupling products. The ratio of the activated intermediates of alkyne to the iodobenzene is wavelength dependent and this regulates transmetalation. This wavelength-tunable reaction pathway is a novel way to optimize the product selectivity in important organic syntheses.

The A30P α-synuclein mutation decreases subventricular zone proliferation.

Parkinson's disease (PD) is associated with olfactory defects in addition to dopaminergic degeneration. Dopaminergic signalling is necessary for subventricular zone (SVZ) proliferation and olfactory bulb (OB) neurogenesis. Alpha-synuclein (α-syn or Snca) modulates dopaminergic neurotransmission, and SNCA mutations cause familial PD, but how α-syn and its mutations affect adult neurogenesis is unclear. To address this, we studied a bacterial artificial chromosome transgenic mouse expressing the A30P SNCA familial PD point mutation on an Snca-/- background. We confirmed that the SNCA-A30P transgene recapitulates endogenous α-syn expression patterns and levels by immunohistochemical detection of endogenous α-syn in a wild-type mouse and transgenic SNCA-A30P α-syn protein in the forebrain. The number of SVZ stem cells (BrdU+GFAP+) was decreased in SNCA-A30P mice, whereas proliferating (phospho-histone 3+) cells were decreased in Snca-/- and even more so in SNCA-A30P mice. Similarly, SNCA-A30P mice had fewer Mash1+ transit-amplifying SVZ progenitor cells but Snca-/- mice did not. These data suggest the A30P mutation aggravates the effect of Snca loss in the SVZ. Interestingly, calbindin+ and calretinin (CalR)+ periglomerular neurons were decreased in both Snca-/-, and SNCA-A30P mice but tyrosine hydroxylase+ periglomerular OB neurons were only decreased in Snca-/- mice. Cell death decreased in the OB granule layer of Snca-/- and SNCA-A30P mice. In the same region, CalR+ numbers increased in Snca-/- and SNCA-A30P mice. Thus, α-syn loss and human A30P SNCA decrease SVZ proliferation, cell death in the OB and differentially alter interneuron numbers. Similar disruptions in human neurogenesis may contribute to the olfactory deficits, which are observed in PD.

Dopamine Imaging in Living Cells and Retina by Surface-Enhanced Raman Scattering Based on Functionalized Gold Nanoparticles.

Retinal dopamine is believed to be involved in the development of myopia, which is projected to affect almost half of the world population's visual health by 2050. Direct visualization of dopamine in the retina with high spatial precision is essential for understanding the biochemical mechanism during the development of myopia. However, there are very few approaches for the direct detection of dopamine in the visual system, particularly in the retina. Here, we report surface-enhanced Raman scattering (SERS)-based dopamine imaging in cells and retinal tissues with high spatial precision. The surface of gold nanoparticles is modified with N-butylboronic acid-2-mercaptoethylamine and 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester), which shows excellent specific reaction with dopamine. The existence of dopamine triggers the aggregation of gold nanoparticles that subsequently form plasmonic hot spots to dramatically increase the Raman signal of dopamine. The as-synthesized SERS nanoprobes have been evaluated and applied for dopamine imaging in living cells and retinal tissues in form-deprivation (FD) myopia guinea pigs, followed by further investigation on localized dopamine levels in the FD-treated mice. The results suggest a declined dopamine level in mice retina after 2-week FD treatment, which is associated with the development of myopia. Our approach will greatly contribute to better understanding the localized dopamine level associated with myopia and its possible treatments. Furthermore, the imaging platform can be utilized to sensing other important small molecules within the biological samples.

Survivable potential of germ cells after trehalose cryopreservation of bovine testicular tissues.

Germplasm preservation of livestock or endangered animals and expansion of germline stem cells are important. The purpose of this study is to investigate whether supplementation of trehalose to the freezing medium (FM) reduces tissular damage and improves the quality of testicular cells in the cryopreserved bovine testicular tissues. We herein established an optimized protocol for the cryopreservation of bovine testicular tissues, and the isolation as well as culture of bovine germ cells containing spermatogonial stem cells (SSCs) from these tissues. The results showed that FM containing 10% dimethyl sulfoxide (Me2SO/DMSO), 10% knockout serum replacement (KSR) and 20% trehalose (FM5) combined with the uncontrolled slow freezing (USF) procedures has the optimized cryoprotective effect on bovine testicular tissues. The FM5 + USF protocol reduced the cell apoptosis, maintained high cell viability, supported the structural integrity and seminiferous epithelial cohesion similar to that in the fresh tissues. Viable germ cells containing SSCs were effectively isolated from these tissues and they maintained germline marker expressions in the co-testicular cells and co-mouse embryonic fibroblasts (MEF) feeder culture systems respectively, during the short-term culture. Additionally, upregulated transcriptions of spermatogenic differentiation marker C-KIT and meiotic marker SYCP3 were detected in these cells after retinoic acid-induced differentiation. Together, FM5 + USF is suitable for the cryopreservation of bovine testicular tissues, with benefits of reducing the apoptosis, maintaining the cell viability, supporting the testicular structure integrity, and sustaining the survival and differentiation potential of bovine germ cells containing SSCs.

Culture bovine prospermatogonia with 2i medium.

Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3ß (2i) can enhance pluripotency maintenance, effects of 2i-based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα-1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF+ /GFRα-1+ prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll-based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα-1, PLZF, anti-apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c-kit and pro-apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H2 O2 -induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2-mediated H3K9me3 through Mek1/2 and Gsk3ß signalling, evidencing successful cryopreservation and expansion of bovine germplasm.

[Research progress on chemical compositions and pharmacological action of Drynariae Rhizoma].

Drynariae Rhizoma is warm in nature and bitter in taste, mainly acting on liver and kidney systems. It is a common Chinese herbal medicine for the treatment of fracture and bone injury. The chemical compositions of Drynariae Rhizoma mainly include flavonoids, triterpenoids, phenylpropanoids and lignans. At present, modern pharmacological and clinical studies have shown that Drynariae Rhizoma has the effects of anti osteoporosis, promoting fracture healing, kidney protection, anti-inflammatory, promoting tooth growth, preventing and treating aminoglycoside ototoxicity and lowering blood lipid. In addition, the toxicity evaluation experiment of Drynariae Rhizoma has also shown that it has no obvious toxic and side effects. Naringin is a kind of dihydroflavone in Drynariae Rhizoma. Many studies have shown that naringin and other total flavonoids play an important role in anti-osteoporosis, promoting fracture healing, anti-inflammation, promoting tooth growth and lowering blood lipid. In this study, the research progresses on chemical consti-tuents and pharmacological activities of Drynariae Rhizoma in recent years were reviewed, and some mechanisms of action were summarized, to provide references for the further research and development of Drynariae Rhizoma.

Mild Acetylation and Solubilization of Ground Whole Plant Cell Walls in EmimAc: A Method for Solution-State NMR in DMSO-d6.

Lignocellulosic biomass is mainly composed of polysaccharides and lignin. The complexity and diversity of the plant cell wall polymers makes it difficult to isolate the components in pure form for characterization. Many current approaches to analyzing the structure of lignocellulose, which involve sequential extraction and characterization of the resulting fractions, are time-consuming and labor-intensive. The present study describes a new and facile system for rationally derivatizing and dissolving coarsely ground plant cell wall materials. Using ionic liquids (EmimAc) and dichloroacetyl chloride as a solvent/reagent produced mildly acetylated whole cell walls without significant degradation. The acetylated products were soluble in DMSO-d6 from which they can be characterized by solution-state two-dimensional nuclear magnetic resonance (2D NMR) spectrometry. A distinct advantage of the procedure is that it realizes the dissolution of whole lignocellulosic materials without requiring harsh ball milling, thereby allowing the acquisition of high-resolution 2D NMR spectra to revealing structural details of the main components (lignin and polysaccharides). The method is therefore beneficial to understanding the composition and structure of biomass aimed at its improved utilization.

TET3 overexpression facilitates DNA reprogramming and early development of bovine SCNT embryos.

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

Cryopreservation of calf testicular tissues with knockout serum replacement.

To enrich bovine gonocytes from cryopreserved testicular tissues, the cryoprotection effects of the freezing media containing knockout serum replacement (KSR) were examined. Using Minimum essential medium (MEM) + 10% dimethyl sulfoxide (Me2SO) as the basic medium, calf testicular tissues were cryopreserved in media containing 0, 5, 10, 20, 40, 90% KSR and 5% fetal bovine serum (FBS) respectively. Morphologically, the seminiferous cords and interstitium were well preserved in all groups. The gonocytes were all glial cell line-derived neurotrophic factor (GDNF) family receptor α-1 (GFRα-1) positive. The recovery rates in all KSR groups were higher than that of the 10% Me2SO group, while comparable to the 5% FBS group. The enriched gonocytes expressed gonocyte marker GFRα-1 typically. Collectively, supplementation of 5-10% KSR can achieve comparable cryoprotective effects with using 5% FBS, which is useful in future study due to its defined formulation that is more consistent in quality and stable in supply.

MiR-155 aggravates impaired autophagy of pancreatic acinar cells through targeting Rictor.

The aim of this study was to investigate the role and mechanism of miR-155 in regulating autophagy in a caerulein-induced acute pancreatitis (AP) cellular model. GFP-LC3 immunofluorescence assay was performed to detect autophagy vesicle formation in pancreatic acinar cell line AR42J. AR42J cells were transfected with miR-155 mimic, inhibitor, and corresponding controls to explore the effect of miR-155 on autophagy. The protein levels of LC3-I, LC3-II, Beclin-1, and p62 were analyzed by western blot analysis. Dual-luciferase reporter assay was performed to verify the interaction between miR-155 and Rictor (RPTOR independent companion of MTOR complex 2). The results showed that caerulein treatment induced impaired autophagy as evidenced by an increase in the accumulation of p62 together with LC3-II in AR42J cells, accompanied by miR-155 upregulation. Furthermore, miR-155 overexpression aggravated, whereas miR-155 silencing reduced the caerulein-induced impairment of autophagy. Mechanistically, Rictor was confirmed to be a direct target of miR-155, which could rescue the miR-155 overexpression-mediated aggravation of impaired autophagy. Collectively, these findings indicate that miR-155 aggravates impaired autophagy in caerulein-treated pancreatic acinar cells by targeting Rictor.

Parasympathectomy increases resting secretion of the submandibular gland in minipigs in the long term.

Parasympathectomy leads to retrogressive alteration and dysfunction of the submandibular gland (SMG) within 1 month, but its long-term effect is unclear. Excessive secretion is observed in half of the patients 4-6 months after SMG transplantation, which completely denervates the gland. Here, we investigated the long-term effect of parasympathectomy on the secretion of SMGs in minipigs. The results showed that the resting salivary secretion of SMGs decreased by 82.9% of that in control at 2 months after denervation, but increased by 156% at 6 months. Although experiencing an atrophic period, the denervated glands regained their normal morphology by 6 months. The expression of the function-related proteins, including muscarinic acetylcholine receptor (mAChR) 3, aquaporin 5 (AQP5), tight junction protein claudin-3, and claudin-4 was decreased at 2 months after denervation. Meanwhile, the protein expression of stem cell markers, including sex-determining region Y-box 2 and octamer-binding transcription factor 4, and the number of Ki67+ cells were significantly increased. However, at 6 months after denervation, the expression of mAChR3, AQP5, claudin-1, claudin-3, and claudin-4 was significantly raised, and the membrane distribution of these proteins was increased accordingly. The autonomic axonal area of the glands was reduced at 2 months after denervation but returned to the control level at 6 months, suggesting that reinnervation took place in the long term. In summary, parasympathectomy increases resting secretion of the SMGs in the long term with a possible mechanism involving improved transepithelial fluid transport. This finding may provide a new strategy for xerostomia treatment.

Tet3 is required for normal in vitro fertilization preimplantation embryos development of bovine.

DNA methylation is a central epigenetic event that regulates cellular differentiation, reprogramming, and pathogenesis. DNA demethylation occurs in preimplantation embryos and primordial germ cells. Recent studies suggest that TET3-mediated oxidation of 5-methylcytosine (5-mC) contributes to genome-wide loss of DNA methylation, yet the mechanism of this process in bovine preimplanted embryos has remained unknown. In this study, we analyzed the expression of Tet gene family at different stages of embryo development. The results revealed that Tet3 was highly expressed in bovine oocytes and in vitro fertilization preimplantation embryos. Knockdown of Tet3 by injection of siRNA in germinal vesicle oocytes was used to assess its role in epigenetic remodeling and embryo development. The results showed that knockdown of Tet3 significantly inhibited oocyte development, maturation, fertilization, and decreased subsequently cleavage and blastocyst rates. Tet3 knockdown significantly increased 5-mC levels, whereas the 5-hmC levels slightly declined. The quantitative polymerase chain reaction data showed that expression levels of the pluripotency genes (POU5F1 and NANOG) were significantly decreased, but the imprinted gene H19 did not change in the Tet3 knockdown group. In addition, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) promoter regions showed hypermethylation in the Tet3 knockdown group, except the imprinted gene H19. Furthermore, the percentage of apoptotic cells and the expression levels of the proapoptotic gene BAX were significantly increased, whereas the antiapoptotic gene BCL-2 messenger RNA levels were decreased in the Tet3 knockdown group. Our results indicated that Tet3 could influence the expression level of the pluripotency genes through regulating the methylation status of the promoter region, thus affect embryonic development.