Long Intergenic Non-Coding RNAs in the Mammary Parenchyma and Fat Pad of Pre-Weaning Heifer Calves: Identification and Functional Analysis.

Enhanced plane of nutrition at pre-weaning stage can promote the development of mammary gland especially heifer calves. Although several genes are involved in this process, long intergenic non-coding RNAs (lincRNAs) are regarded as key regulators in the regulated network and are still largely unknown. We identified and characterized 534 putative lincRNAs based on the published RNA-seq data, including heifer calves in two groups: fed enhanced milk replacer (EH, 1.13 kg/day, including 28% crude protein, 25% fat) group and fed restricted milk replacer (R, 0.45 kg/day, including 20% crude protein, 20% fat) group. Sub-samples from the mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested from heifer calves. According to the information of these lincRNAs' quantitative trait loci (QTLs), the neighboring and co-expression genes were used to predict their function. By comparing EH vs R, 79 lincRNAs (61 upregulated, 18 downregulated) and 86 lincRNAs (54 upregulated, 32 downregulated) were differentially expressed in MFP and PAR, respectively. In MFP, some differentially expressed lincRNAs (DELs) are involved in lipid metabolism pathways, while, in PAR, among of DELs are involved in cell proliferation pathways. Taken together, this study explored the potential regulatory mechanism of lincRNAs in the mammary gland development of calves under different planes of nutrition.

Dihydropyridine Enhances the Antioxidant Capacities of Lactating Dairy Cows under Heat Stress Condition.

Heat stress (HS), a nonspecific response to environmental heat, can seriously affect dairy cow health. Feed additives may alleviate HS in dairy cows by improving rumen fermentation efficacy, stimulating feed consumption, enhancing vasodilation, and/or improving antioxidant capacity. The temperature-humidity index (THI) indicates that spring is a non-HS season, and summer is an HS season. HS results in the decrease in dairy cow antioxidant capacities. Our results indicated the decrease in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidation (T-AOC) levels and the increase in malondialdehyde (MDA) level during HS season. Meanwhile, antioxidant indexes (SOD, GSH-Px, and T-AOC) were positively correlated with milk yield (p < 0.01), whereas MDA exhibited a significant negative correlation with milk yield (p < 0.05). In addition, the effects of dihydropyridine (DHP) on antioxidant capacity and ruminal microbial communities in dairy cows under HS were investigated. During summer, dairy cows were randomly assigned into two groups under HS, including a standard diet (S-ND) group and standard diet with 3 g/day/cow DHP (S-D) group. DHP treatment significantly restored SOD and GSH-Px levels under HS. Denaturing gradient gel electrophoresis results indicated that the DHP altered ruminal bacterial community mainly composed Proteobacteria and Firmicutes in dairy cows under HS. Our results suggest that DHP can enhance the antioxidant abilities of dairy cows with favorable effects on ruminal microbial communities under HS, further alleviating HS on dairy cows.

Correction: Sodium butyrate interrupts the maturation of oocytes and enhances the development of preimplantation embryos.

[This corrects the article DOI: 10.1371/journal.pone.0220479.].

Sodium butyrate interrupts the maturation of oocytes and enhances the development of preimplantation embryos.

Histone acetylation is one of the most important posttranslational modifications that contribute to transcriptional initiation and chromatin remodeling. In the present study, we aimed to investigate the effect of sodium butyrate (NaBu), a natural histone deacetylase inhibitor (HDACi), on the maturation of oocytes, preimplantation embryonic development, and expression of important developmental genes. The results indicated that NaBu decreased the rates of GVBD and the first polar body extrusion (PBE) in vitro in a dose-dependent manner. Meanwhile, NaBu treatment led to an abnormality in the spindle apparatus in oocytes in MI. However, the ratio of phosphor-extracellular signal-regulated kinases (p-ERK)/ERK significantly decreased in oocytes treated with 2.0 mM NaBu for 8 h. Furthermore, NaBu treatment at 2.0 mM improved the quality of embryos and the mRNA expression levels of important developmental genes such as HDAC1, Sox2, and Pou5f1. These data suggest that although a high concentration NaBu will impede the meiosis of oocytes, 2.0 mM NaBu will promote the development of embryos in vitro. Further investigation is needed to clarify the direct/indirect effects of NaBu on the regulation of important developmental genes and their subsequent impacts on full-term development in mammals.

MFEprimer-3.0: quality control for PCR primers.

Quality control (QC) for lab-designed primers is crucial for the success of a polymerase chain reaction (PCR). Here, we present MFEprimer-3.0, a functional primer quality control program for checking non-specific amplicons, dimers, hairpins and other parameters. The new features of the current version include: (i) more sensitive binding site search using the updated k-mer algorithm that allows mismatches within the k-mer, except for the first base at the 3' end. The binding sites of each primer with a stable 3' end are listed in the output; (ii) new algorithms for rapidly identifying self-dimers, cross-dimers and hairpins; (iii) the command-line version, which has an added option of JSON output to enhance the versatility of MFEprimer by acting as a QC step in the 'primer design → quality control → redesign' pipeline; (iv) a function for checking whether the binding sites contain single nucleotide polymorphisms (SNPs), which will affect the consistency of binding efficiency among different samples. In summary, MFEprimer-3.0 is updated with the well-tested PCR primer QC program and it can be integrated into various PCR primer design applications as a QC module. The MFEprimer-3.0 server is freely accessible without any login requirement at: https://mfeprimer3.igenetech.com/ and https://www.mfeprimer.com/. The source code for the command-line version is available upon request.

A Genome-Wide mRNA Expression Profile in Caenorhabditis elegans under Prolonged Exposure to 1750MHz Radiofrequency Fields.

OBJECTIVE: C. elegans has been used as a biomonitor for microwave-induced stress. However, the RF (radiofrequency) fields that have been used in previous studies were weak (≤1.8W/kg), and the bio-effects on C. elegans were mostly negative or ambiguous. Therefore, this study used more intense RF fields (SAR = 3W/kg) and longer time course of exposure (60h at 25°C, L1 stage through adult stage) to investigate the biological consequences of 1750 MHz RF fields in wild-type worms. METHODS: The growth rates and lifespans of RF-exposure group and the control group were carefully recorded. RNA samples were collected at L4 (35h) and gravid adult (50h) stages for further high-throughput sequencing, focusing on differences between the RF-exposure and the sham control groups. RESULTS: The RF-exposed and sham control groups developed at almost the same rate and had similar longevity curves. In L4 stage worms, 94 up-regulated and 17 down-regulated genes were identified, while 186 up-regulated and 3 down-regulated genes were identified in adult stage worms. GO analysis showed that the differentially expressed genes at 35h were associated with growth, body morphogenesis and collagen and cuticle-based development. Genes that were linked to growth rate and reproductive development were differentially expressed at 50h. Some embryonic and larval development genes in the offspring were also differentially expressed at 50h. Ten genes were differentially expressed at both 35h and 50h, most of which were involved in both embryonic and larval developmental processes. Although prolonged RF fields did not induce significant temperature increase in RF exposure groups, the temperature inside worms during exposure was unknown. CONCLUSIONS: No harmful effects were observed in prolonged exposure to 1750 MHz RF fields at SAR of 3W/kg on development and longevity of C. elegans. Although some differentially expressed genes were found after prolonged RF exposure, these differences were ascribed to oscillating gene expression patterns in L4 and gravid adult worms. It was also difficult to rule out a weak thermal effect caused by prolonged RF exposure inside the worms.

Synthesis and analgesic effects of µ-TRTX-Hhn1b on models of inflammatory and neuropathic pain.

µ-TRTX-Hhn1b (HNTX-IV) is a 35-amino acid peptide isolated from the venom of the spider, Ornithoctonus hainana. It inhibits voltage-gated sodium channel Nav1.7, which has been considered as a therapeutic target for pain. The goal of the present study is to elucidate the analgesic effects of synthetic µ-TRTX-Hhn1b on animal models of pain. The peptide was first synthesized and then successfully refolded/oxidized. The synthetic peptide had the same inhibitory effect on human Nav1.7 current transiently expressed in HEK 293 cells as the native toxin. Furthermore, the analgesic potentials of the synthetic peptide were examined on models of inflammatory pain and neuropathic pain. µ-TRTX-Hhn1b produced an efficient reversal of acute nociceptive pain in the abdominal constriction model, and significantly reduced the pain scores over the 40-min period in the formalin model. The efficiency of µ-TRTX-Hhn1b on both models was equivalent to that of morphine. In the spinal nerve model, the reversal effect of µ-TRTX-Hhn1b on allodynia was longer and higher than mexiletine. These results demonstrated that µ-TRTX-Hhn1b efficiently alleviated acute inflammatory pain and chronic neuropathic pain in animals and provided an attractive template for further clinical analgesic drug design.

Analgesic effects of Huwentoxin-IV on animal models of inflammatory and neuropathic pain.

Huwentoxin-IV (HWTX-IV), a peptide with 35 amino acid residues, was discovered in the venom of spider Ornithoctonus huwena. The peptide had an inhibitory effect on a tetrodotoxin-sensitive (TTX-S) sodium channel with highly sensitive to Nav1.7, an attractive target for pain release therapy. In this study we further demonstrated the analgesic effects of HWTX-IV using mouse and rat as an inflammatory pain model and/or a neuropathic pain models. In the both cases, the analgesic effects of the peptide were dose-dependent, and statistically significant. In the inflammatory model, 100 µg/kg of HWTX-IV produced an efficient reversal of hyperalgesia up to 63.6% after injection of formalin in rats with the efficiency equivalent to that of morphine at 50 µg/kg, and 200 µg/kg of HWTX-IV produced protective effect up to 55.6% after injection of acetic acid with the efficiency equivalent to that of morphine at 100 µg/kg. In the spinal nerve model, the peptide produced the longer and higher reversal effect on allodynia than Mexiletine. These results demonstrated that HWTX-IV released efficiently the acute inflammatory pain and chronic neuropathic pain in these animals, suggesting that HWTX-IV was a potential and efficient candidate for further clinical drug development against inflammatory and neuropathic pain.

Effect of overexpression of inhibin alpha (1-32) fragment on bovine granulosa cell proliferation, apoptosis, steroidogenesis, and development of co-cultured oocytes.

The objective was to determine the effects of an inhibin alpha (1-32) fragment gene on proliferation, apoptosis, and steroidogenesis of bovine granulosa cells (GC) isolated from medium and small follicles (diameter >4-8 and 1-4mm, respectively), and the effect of GC, previously transfected with pEGISI, on oocyte maturation and in vitro embryo development. To enhance expression of the inhibin alpha (1-32) fragment, GC were transfected with pEGISI. Transfection inhibited (P<0.05) GC proliferation (88.8+/-2.1%; mean+/-S.E.M.) compared to the control and EGFP groups (100% and 97.5+/-2.1%) from medium follicles, with no significant effect on GC from small follicles. Apoptosis was higher (P<0.01) in transfected GC than in controls. Transfection increased (P<0.05) estradiol synthesis from both medium and small follicles (0.57+/-0.13 and 0.86+/-0.13 pg/mL vs. 0.19+/-0.05 and 0.35+/-0.09 pg/mL in controls) after culturing for 48 h, with suppression (P<0.05) in transfected GC after 96 h. Transfection reduced (P<0.05) progesterone synthesis in GC from both medium and small follicles (24.5+/-3.4 and 75.4+/-4.6 ng/mL vs. 45.42+/-5.33 and 117.32+/-11.99 ng/mL in controls) after culture for 48 h, with no significant difference after 96 h. Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control (61.5+/-6.8% vs. 71.2+/-5.7%, P<0.05), with no significant effect on embryo development. In conclusion, overexpression of inhibin alpha (1-32) fragment regulated GC development; effects on subsequent oocyte maturation were both time- and stage-dependent.

Effects of iron and copper in culture medium on bovine oocyte maturation, preimplantation embryo development, and apoptosis of blastocysts in vitro.

The aim of this study was to investigate the effects of iron and copper on bovine oocyte maturation, preimplantation embryo development and apoptosis of blastocysts. The concentrations of iron in the culture media were 0 (control), 0.45, 0.81, 1.96 and 3.26 mg/l, and the concentrations of copper were 0 (control), 0.093, 0.27, 0.46 and 0.68 mg/l. The changes in the iron (1.96 mg/l) and copper concentrations (0.46 mg/l) in the culture media were measured after oocyte maturation for 22 h and after zygote culture for 48, 96, 144 and 192 h. The results showed that there were no significant differences in oocyte maturation and cleavage between media containing iron and the control, but the media containing iron had higher (P>0.05) rates of 8-cell embryos, morulae, and blastocysts than the control, and addition of 1.96 mg/l of iron increased the blastocyst rate (P>0.05). The effects of copper on oocyte maturation and cleavage were similar to iron, and addition of 0.46 and 0.68 mg/l of copper increased the rates of morulae and blastocysts (P>0.05). Addition of iron or copper significantly decreased the number of apoptotic blastomeres compared with the control (P>0.05). After oocyte maturation for 22 h and zygote culture for 48 h, the iron concentrations decreased by 3.6 and 9.2%, respectively, and the copper concentrations decreased by 6.5 and 10.9%, respectively. After zygote culture for 96, 144 and 192 h, the iron concentrations decreased by 21.4, 25.5 and 27.0%, respectively, the copper concentrations decreased by 23.9, 28.3 and 30.4%, respectively. In conclusion, iron and copper played an important role in the success of culture of 8-cell embryos, morulae, and blastocysts, and long-term lack of iron or copper increased the number of apoptotic blastomeres. Furthermore, transition of primary demand for trace amounts of iron or copper from the cytoplast to culture medium for utilization by zygotes may occur after in vitro zygote culture for 48 h.