Differentially expressed transcripts study during pregnancy and postpartum anestrus of yak (Bos grunniens).

Background: Yak is the main livestock species in the plateau area, and its reproductive performance is low, usually two years or three years. A very few of yaks recover within a certain period of time after delivery and smoothly enter the next estrous cycle, while most of them enter the postpartum anestrus and show no estrus performance. However, the key biological factors and influencing mechanisms that cause postpartum anestrus in yaks are not clear. Objective: To study the expression of differential transcripts in ovaries of yak during pregnancy and postpartum anestrus. Methods: Each three yaks in pregnancy and anestrus under natural grazing conditions in Haiyan County, Qinghai Province were selected and slaughtered, and their ovaries were collected and sent to Biomarker Technologies. Oxford Nanopore Technologies single-molecule real-time electrical signal sequencing technology was used to perform full-length transcriptome sequencing. Astalavista software was used to identify the types of alternative splicing events in yak estrus and pregnancy, and TAPIS pipeline was used to identify alternative polyadenylation. Results: The results showed that there were 1751 differentially expressed transcripts (DETs) between pregnancy and anestrus in yak, of which 808 were upregulated and 943 were downregulated. GO analysis showed that the biological processes of DETs were mainly reproductive, reproductive and rhythmic processes. KEGG analysis showed that the DET cell junction-related adhesion junction protein (ß-catenin) and amino terminal kinase (JNK) were involved in FAs (local adhesion). Phosphatidylinositol-3-kinase (PI3K) is involved in the PI3K/AKT/mTOR signaling pathway. Circadian rhythm output cycle failure (Clock) and brain and muscle tissue aromatic hydrocarbon receptor nuclear transporter-like protein 1 (Bmal1) are involved in circadian rhythm signaling pathway. Conclusion: This study found that ß-catenin, JNK, PI3K, Clock and Bmal1 were closely related to postpartum anestrus in yak.

A comparative transcriptome and proteomics study of post-partum ovarian cycle arrest in yaks (Bos grunniens).

Post-partum ovarian cycle arrest is the main factor affecting yak reproductive efficiency. There are few reports regarding the molecular regulatory mechanism of post-partum oestrus at transcriptome and proteome levels in yaks. Our previous studies focussed on the ovaries of yaks with post-partum ovarian cycle arrest and post-partum oestrus yaks. In this study, RNA sequencing transcriptomic study was combined with quantitative proteomic analyses to identify post-partum ovarian cycle-related genes and proteins. Consequently, 1,149 genes and 24 proteins were found to be up- or downregulated during post-partum oestrus. The analysis of differentially regulated genes identified three gene or protein pairs that were synchronously upregulated and no gene or protein pairs that were synchronously downregulated, suggesting that these upregulated genes may regulate the post-partum ovarian cycle. The functional classification of these differentially expressed genes and proteins indicated their connection with the oocyte meiosis, the oestrogen signalling pathway, the progesterone-mediated oocyte maturation and the gonadotrophin-releasing hormone (GnRH) signalling pathway. In this study, a total of six genes and two proteins involved in the oocyte meiosis, the oestrogen signalling pathway, the progesterone-mediated oocyte maturation and the GnRH signalling pathway were identified. The CSNK1A1, M91_09723, M91_11326, M91_21439, M91_19073, SHC2, Atf6b, M91_03062, HSPCA and calmodulin could regulate oestrus, respectively, in the post-partum so as to control the anoestrus status.

SMART: On-Site Rapid Detection of Nucleic Acid from Plants, Animals, and Microorganisms in under 25 Minutes.

The rapid on-site nucleic acid detection method is urgently required in many fields. In this study, we report a portable and highly integrated device for DNA detection that combines ultrafast DNA adsorption and rapid DNA amplification. The device, known as silicon film mediated recombinase polymerase amplification (RPA) for nucleic acid detection (SMART), can detect target DNA in less than 25 min from plants, animals, and microbes. Utilizing SMART, transgenic maize was rapidly detected with high selectivity and sensitivity. The sensitivity threshold of the SMART for transgenic maize genomic DNA was 50 copies. The detection results of genuine samples containing plants, animals, and microbes by SMART were consistent with the conventional polymerase chain reaction (PCR) method, demonstrating the high robustness of SMART. Additionally, SMART does not require expensive equipment and is fast, affordable, and user-friendly, making it suited for the broad-scale on-site detection of nucleic acids.

Effects of melatonin on development and hormone secretion of sheep theca cells in vitro.

Theca cells (TCs) play a unique role in the structure and function of the ovary. They are not only the structural basis of the follicle but also the androgen-secreting cells in female mammals, which can affect the normal development and atresia of the follicle. The results showed that melatonin receptor (MTR) MT1 and MT2 were expressed on sheep TCs. In the present study, the effects of different concentrations of MT at 0, 10-10, 10-8, 10-6 and 10-4 M/L on sheep TCs with regards to the antioxidant levels, proliferation, apoptosis and steroid hormone secretion were investigated. The results showed that in sheep TCs, all concentrations of MT significantly decreased reactive oxygen species (ROS) concentration and BAX expression; increased Cat, Sod1, and BCL-2 expression. The proliferation viability of TCs was significantly inhibited in all groups except for 10-10 M/L MT, and the expression of cyclin D1 and CDK4 was significantly reduced. MT significantly increased StAR expression and progesterone secretion in TCs, but there was no significant effect on androgen secretion and CYP11A1, CYP17A1 and 3ß-HSD expression in all groups. MT-induced progesterone secretion was completely inhibited by Luzindole (a nonspecific MT1 and MT2 inhibitor) and partially inhibited by 4p-PDOT (specific MT2 inhibitor). MT-induced progesterone secretion can be inhibited by LY294002 (PI3K/AKT pathway inhibitor). This study indicated that MT inhibits apoptosis and proliferation of in vitro cultured sheep TCs, which has implications for slowing ovarian atresia and aging. MT activates the PI3K/Akt pathway to mediate the synthesis and secretion of progesterone by TCs. This study provides a basis for further exploration of the role of TCs on follicle development and ovarian steroid hormone secretion.

Transcriptome and proteome analysis of pregnancy and postpartum anoestrus ovaries in yak.

BACKGROUND: Domestic yaks are the most important livestock species on the Qinghai-Tibetan Plateau. Adult female yaks normally breed in the warm season (July to September) and enter anestrous in the cold season (November to April). Nevertheless, it is unclear how ovarian activity is regulated at the molecular level. OBJECTIVES: The peculiarities of yak reproduction were assessed to explore the molecular mechanism of postpartum anestrus ovaries in yaks after pregnancy and parturition. METHODS: Sixty female yaks with calves were observed under natural grazing in Haiyan County, Qinghai Province. Three yak ovaries in pregnancy and postpartum anestrus were collected. RNA sequencing and quantitative proteomics were employed to analyze the pregnancy and postpartum ovaries after hypothermia to identify the genes and proteins related to the postpartum ovarian cycle. RESULTS: The results revealed 841 differentially expressed genes during the postpartum hypoestrus cycle; 347 were up-regulated and 494 genes were down-regulated. Fifty-seven differential proteins were screened: 38 were up-regulated and 19 were down-regulated. The differential genes and proteins were related to the yak reproduction process, rhythm process, progesterone-mediated oocyte maturation, PI3K/AKT signaling pathway, and MAPK signaling pathway categories. CONCLUSIONS: Transcriptome and proteomic sequencing approaches were used to investigate postpartum anestrus and pregnancy ovaries in yaks. The results confirmed that BHLHE40, SF1IX1, FBPX1, HSPCA, LHCGR, BMP15, and ET-1R could affect postpartum hypoestrus and control the state of estrus.

Extended tacrolimus release via the combination of lipid-based solid dispersion and HPMC hydrogel matrix tablets.

The objective of this study is to evaluate the feasibility of obtaining extended release of tacrolimus by a novel combination of lipid-based solid dispersion and matrix-type extended release tablet techniques. Tacrolimus solid dispersion was prepared using glycerylbehenate (Compritol® ATO888) and Pluronic F127 as the carrier materials with hot-melt method, which was then blended with hydrogel matrix materials, such as HPMC and lactose, the powders were directly compressed into tablets. In vitro drug release tests were carried out to evaluate the performance of the solid dispersions and the tablets. The dissolution rate of tacrolimus was significantly improved by the lipid-based solid dispersion, and the incorporation of HPC into the solid dispersion obviously improved its stability after storage. Extended release tablets loaded with tacrolimus solid dispersion showed prolonged drug release patterns over 24 h, the release patterns of the tablets can be tailored by the compositions of the matrix materials, including the types and content of HPMCs. A modified processing method that directly mixed the melted solid dispersion with HPMC powders improved the uniformity of the solid dispersion inside the tablet matrix and release profile. The release data of the extended release tablet fitted well to the Korsmeyer-Peppas model with n value of 0.85, which suggested diffusion- and erosion-controlled release mechanism. The combination of lipid-based solid dispersion and HPMC hydrogel matrix may find wide applications in the extended release dosage forms of high potent, water-insoluble drugs.

Effect of the Dispersion States of Azone in Hydroalcoholic Gels on Its Transdermal Permeation Enhancement Efficacy.

The objective of this study was to investigate the effect of dispersion states of azone in gels on the transdermal permeation of levamisole hydrochloride (LH). LH hydroalcoholic gels containing azone of different dispersion states were prepared by varying the contents of azone and Tween 80, and the in vitro transdermal permeation of LH across excised rat skin was evaluated. Depending on the content of azone, mixed solvents, and solubilizer used, azone presented as dissolved molecules, solubilized in micelles, and fine or coarse emulsion droplets in gels. Dramatically increased transdermal permeation of LH within the azone contents between 0.25% and 0.75% indicated high transdermal enhancement efficiency of the molecular or micellar azone, and extra azone that existed as oil droplets did not fully exert transdermal penetration enhancement of LH. Although solubilizer (Tween 80) can greatly increase the solubility of azone, only small amount of Tween 80 (0.5%) in the gel significantly increased the steady-state flux of LH. Addition of extra amount of Tween 80 (>0.5%) reduced the amount of azone distributed in the skin, and thus decreased the transdermal drug permeation. The results partly elucidated the versatile effects of the dispersion states of azone on the transdermal permeation of hydrophilic drug from semisolid gels.

Improved oral bioavailability of probucol by dry media-milling.

The polymer/probucol co-milled mixtures were prepared to improve drug dissolution rate and oral bioavailability. Probucol, a BCS II drug, was co-milled together with Copovidone (Kollidon VA64, VA64), Soluplus, or MCC using the dry media-milling process with planetary ball-milling equipment. The properties of the milled mixtures including morphology, crystal form, vitro drug dissolution and in vivo oral bioavailability in rats were evaluated. Probucol existed as an amorphous in the matrix of the co-milled mixtures containing VA64, which helped to enhance drug dissolution. The ternary mixture composed of VA64, RH40, and probucol showed increased dissolution rates in both sink and non-sink conditions. It also had a higher oral bioavailability compared to the reference formulation. Dry-media milling of binary or ternary mixtures composed of drug, polymer and surfactant possibly have wide applications to improve dissolution rate and oral bioavailability of water-insoluble drugs.

KCC2 expression changes in Diazepam-treated neonatal rats with hypoxia-ischaemia brain damage.

Hypoxia-ischaemia brain damage (HIBD) is a major type of perinatal brain injury in newborns. In this study, we investigate the short- and long-term neuroprotective effects of Diazepam on neonatal rats with HIBD and the potential mechanisms underlying its protective effects. Seven-day-old Sprague-Dawley rats were subjected to left carotid artery ligation followed by a 2-h exposure to 8% oxygen and 92% nitrogen. Diazepam was administered immediately via intraperitoneal (i.p.) injection after inducing HIBD at a dose of 10 mg kg(-1)8h(-1) for three consecutive days. Three days after HIBD, rats were decapitated, and the extent of brain injury was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Additionally, the expression of Potassium-chloride cotransporter-2 (KCC2) was analysed using real-time PCR, Western blot analysis and immunohistochemistry. Three weeks after HIBD, rats were subjected to the Morris water maze (MWM) test and the locomotor activity test to determine the long-term therapeutic effects of Diazepam. We observed that the volume of infarction in the Diazepam group was significantly less (P<0.01) compared with the HIBD group. We also observed that the learning and memory abilities of the Diazepam rats improved significantly compared with the untreated rats (P<0.05) and that the decrease in KCC2 expression was prevented (P<0.01). Early treatment with Diazepam appears to attenuate HIBD and can efficiently improve the long-term learning and memory capabilities of the animal. A potential mechanism underlying these effects may involve preventing the decrease in KCC2 expression.

Establishment and characterization of a paclitaxel­resistant human esophageal carcinoma cell line.

The aim of this study was to establish a new paclitaxel (PTX)-resistant human esophageal squamous carcinoma (ESCC) cell line and investigate its biological characteristics. The resistant cell line (EC109/Taxol) was developed in vitro by intermittent exposure of the human ESCC cell line EC109 to a high concentration of PTX with time-stepwise increment over a period of 6 months. The MTT assay was performed to test the drug resistance of EC109 and EC109/Taxol cells. The morphological features were observed using inverted microscopy and apoptosis was measured by flow cytometry (FCM) and Hoechst 33258 fluorescence staining. Cell growth curves and colony formation of EC109 and EC109/Taxol cells were compared. FCM was also used to determine the distribution of the cell cycle. The protein levels of Bcl-2, Bax, Procaspase-3 and P-gp were detected by western blotting. P-gp activity was evaluated by Rh123 accumulation and efflux assay. In vivo resistance characterization was investigated. EC109/Taxol cells were 67.2-fold resistant to PTX in comparison with EC109 cells, and also exhibited cross-resistance to 5-fluorouracil (5-FU), cisplatin (CDDP) and epirubicin (EPI). FCM and Hoechst 33258 fluorescence staining confirmed that EC109 cells treated with PTX showed significantly higher percentage of apoptotic cells compared to EC109/Taxol cells. Simultaneously, EC109/Taxol cells exhibited changes in morphology, proliferation rate, doubling time, cell cycle distribution and colony formation rate were detected as compared with EC109 cells. The resistant cell line overexpressed Bcl-2, Procaspase-3 and P-gp protein, and showed decreased Bax expression. Further, EC109/Taxol cells did not change PTX resistance in vivo. This is the first report on the establishment of an EC109/Taxol cell line with higher resistance. Bcl-2, Bax, Procaspase-3 and P-gp are involved in the resistance of cell lines to PTX, which are invaluable tools to study the resistance of anticancer drugs and to identify the methods to overcome resistance.