Utilizing transcriptomics and proteomics to unravel key genes and proteins of Oryza sativa seedlings mediated by selenium in response to cadmium stress.

BACKGROUND: Cadmium (Cd) pollution has declined crop yields and quality. Selenium (Se) is a beneficial mineral element that protects plants from oxidative damage, thereby improving crop tolerance to heavy metals. The molecular mechanism of Se-induced Cd tolerance in rice (Oryza sativa) is not yet understood. This study aimed to elucidate the beneficial mechanism of Se (1 mg/kg) in alleviating Cd toxicity in rice seedlings. RESULTS: Exogenous selenium addition significantly improved the toxic effect of cadmium stress on rice seedlings, increasing plant height and fresh weight by 20.53% and 34.48%, respectively, and increasing chlorophyll and carotenoid content by 16.68% and 15.26%, respectively. Moreover, the MDA, ·OH, and protein carbonyl levels induced by cadmium stress were reduced by 47.65%, 67.57%, and 56.43%, respectively. Cell wall metabolism, energy cycling, and enzymatic and non-enzymatic antioxidant systems in rice seedlings were significantly enhanced. Transcriptome analysis showed that the expressions of key functional genes psbQ, psbO, psaG, psaD, atpG, and PetH were significantly up-regulated under low-concentration Se treatment, which enhanced the energy metabolism process of photosystem I and photosystem II in rice seedlings. At the same time, the up-regulation of LHCA, LHCB family, and C4H1, PRX, and atp6 functional genes improved the ability of photon capture and heavy metal ion binding in plants. Combined with proteome analysis, the expression of functional proteins OsGSTF1, OsGSTU11, OsG6PDH4, OsDHAB1, CP29, and CabE was significantly up-regulated under Se, which enhanced photosynthesis and anti-oxidative stress mechanism in rice seedlings. At the same time, it regulates the plant hormone signal transduction pathway. It up-regulates the expression response process of IAA, ABA, and JAZ to activate the synergistic effect between each cell rapidly and jointly maintain the homeostasis balance. CONCLUSION: Our results revealed the regulation process of Se-mediated critical metabolic pathways, functional genes, and proteins in rice under cadmium stress. They provided insights into the expression rules and dynamic response process of the Se-mediated plant resistance mechanism. This study provided the theoretical basis and technical support for crop safety in cropland ecosystems and cadmium-contaminated areas.

Metagenomic analysis revealed N-metabolizing microbial response of Iris tectorum to Cr stress after colonization by arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) and plant growth-promoting bacteria enhance plant tolerance to abiotic stress and promote plant growth in contaminated soil. However, the interaction mechanism between rhizosphere microbial communities under chromium (Cr) stress remains unclear. This study conducted a greenhouse pot experiment and metagenomics analysis to reveal the comprehensive effects of the interaction between AMF (Rhizophagus intraradices) and nitrogen-N metabolizing plant growth promoters on the growth of Iris tectorum. The results showed that AMF significantly increased the biomass and nutrient levels of I. tectorum in contaminated soil and decreased the content of Cr in the soil. Metagenomics analysis revealed that the structure and composition of the rhizosphere microbial community involved in nitrogen metabolism changed significantly after inoculation with AMF under Cr stress. Functional genes related to soil nitrogen mineralization (gltB, gltD, gdhA, ureC, and glnA), nitrate reduction to ammonium (nirB, nrfA, and nasA), and soil nitrogen assimilation (NRT, nrtA, and nrtC) were up-regulated in the N-metabolizing microbial community. In contrast, the abundance of functional genes involved in denitrification (nirK and narI) was down-regulated. In addition, the inoculation of AMF regulates the synergies between the N-metabolic rhizosphere microbial communities and enhances the complexity and stability of the rhizosphere ecological network. This study provides a basis for improving plant tolerance to heavy metal stress by regulating the functional abundance of N-metabolizing plant growth-promoting bacteria through AMF inoculation. It helps to understand the potential mechanism of wetland plant remediation of Cr-contaminated soil.

Comprehensive physiology and proteomics analysis revealed the resistance mechanism of rice (Oryza sativa L) to cadmium stress.

Cadmium contamination can lead to a decrease in crop yield and quality. However, Cd-tolerant rice can improve rice resistance genes, improve crop tolerance to heavy metals, and protect plants from oxidative damage. In this study, Japonica rice: Chunyou 987 and Indica rice: Chuanzhong you 3607 were used to reveal the molecular response mechanism of Cd-tolerant rice under cadmium concentration of 3 mg/kg through comparative experiments combined with physiology and proteomics. The results showed that compared with indica rice, japonica rice showed more robust resistance to Cd stress and effectively retained many Cd ions in roots. Moreover, it enhanced its enzymatic and non-enzymatic anti-oxidative stress mechanism, which increased the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) by 47.37%, 21.75%, and 55.42%, respectively. The contents of non-enzymatic antioxidant substances ascorbic acid (AsA), glutathione (GSH), cysteine (Cys), proline (PRO), anthocyanins (OPC), and flavonoids were increased by 25.32%, 42.67%, 21.43%, 50.81%, 33.23%, and 72.16%, respectively. Through proteomics analysis, it was found that in response to the damage caused by cadmium stress, Japonica rice makes Photosynthesis functional proteins (psbO and PetH), Photosynthesis antenna proteins (LHCA and ASCAB9), Carbon fixation functional proteins (PEPC and OsAld), Porphyrin metabolism functional proteins (OsRCCR1 and SE5), Glyoxylate and dicarboxylate The expression of metabolism functional proteins (CATC and GLO4.) and Glutathione metabolism functional proteins (APX8 and OsGSTU13) were significantly up-regulated, which stimulated the antioxidant stress mechanism and photosynthetic system, and constructed a robust energy supply system to ensure the normal metabolic activities of life. Strengthening the mechanisms of plant homeostasis. In summary, this study revealed the molecular mechanism of tolerance to Cd stress in japonica rice, and the results of this study will provide a possible way to improve Cd-resistant rice seedlings.

In vitro and in silico analyses reveal the toxicity of metolachlor to grass carp hepatocytes and the antagonism of melatonin.

Due to the widespread use of metolachlor (MET), the accumulation of MET and its metabolites in the environment has brought serious health problems to aquatic organisms. At present, the toxicity of MET on the physiological metabolism of aquatic animals mainly focused on the role of enzymes. There is still a lack of research on the molecular mechanisms of MET hepatotoxicity, especially on antagonizing MET toxicity. Therefore, this study focuses on grass carp hepatocytes (L8824 cells) closely related to toxin accumulation. By establishing a MET exposed L8824 cells model, it is determined that MET exposure induces pyrolytic inflammation of L8824 cells. Subsequent mechanistic studies found that MET exposure induces pyroptosis in L8824 cells through mitochondrial dysfunction, and siCaspase-1 inhibits the MET induced ROS production, suggesting a regulation of ROS-NLRP3- Caspase-1 pyroptotic inflammation cycling center in MET induced injury to L8824 cells. Molecular docking revealed a strong binding energy between melatonin (MT) and Caspase-1. Finally, a model of L8824 cells with MT intervention in MET exposure was established. MT can antagonize the pyroptosis induced by MET exposure in L8824 cells by targeting Caspase-1, thereby restoring mitochondrial function and inhibiting the ROS-pyroptosis cycle. This study discovered targets and mechanisms of MT regulating pyroptosis in MET exposed-L8824 cells, and the results are helpful to provide new targets for the design of MET antidotes.

Molecular Regulation of Thermogenic Mechanisms in Beige Adipocytes.

Adipose tissue is conventionally recognized as a metabolic organ responsible for storing energy. However, a proportion of adipose tissue also functions as a thermogenic organ, contributing to the inhibition of weight gain and prevention of metabolic diseases. In recent years, there has been significant progress in the study of thermogenic fats, particularly brown adipose tissue (BAT). Despite this progress, the mechanism underlying thermogenesis in beige adipose tissue remains highly controversial. It is widely acknowledged that beige adipose tissue has three additional thermogenic mechanisms in addition to the conventional UCP1-dependent thermogenesis: Ca2+ cycling thermogenesis, creatine substrate cycling thermogenesis, and triacylglycerol/fatty acid cycling thermogenesis. This paper delves into these three mechanisms and reviews the latest advancements in the molecular regulation of thermogenesis from the molecular genetic perspective. The objective of this review is to provide readers with a foundation of knowledge regarding the beige fats and a foundation for future research into the mechanisms of this process, which may lead to the development of new strategies for maintaining human health.

Efficacy of vitamin and antioxidant supplements for treatment of diabetic peripheral neuropathy: systematic review and meta-analysis of randomized controlled trials.

ABSTRACTThe results of treatment effect of vitamin or antioxidant intake on diabetic peripheral neuropathy (DPN) was inconsistent. Therefore, we performed a meta-analysis of randomized controlled trials (RCTs) to examine whether these supplements are effective in DPN treatment. We searched seven databases from inception to October 2021. All RCTs of DPN treatments with vitamin and antioxidant supplements were included. We performed sensitivity and subgroup analysis, and also tested for publication bias by the funnel plot and Egger's test. A total of 14 studies with 1384 patients were included in this systematic review. Three high-quality trials showed that vitamin and antioxidant supplements significantly increased sensory nerve conduction velocity (SNCV) of the sural nerve (MD = 2.66, 95%CI (0.60, 4.72), P < 0.05, I2 = 0%). Seven studies (758 participants) suggested that these supplements might have improvement on motor nerve conduction velocity (MNCV) of the peroneal nerve in DPN patients with the random-effect model (MD = 0.60, 95%CI (0.28, 0.92), P < 0.05, I2 = 65%). In four studies, these supplements could have improved on MNCV of the median nerve with the fixed-effect model (MD = 4.22, 95%CI (2.86, 5.57), P < 0.05, I2 = 0%). However, ten studies (841 participants) have suggested that vitamin and antioxidant supplements have not decreased glycosylated haemoglobin (HbA1c). Vitamin and antioxidant supplements may improve the conduction velocity of nerves, including median, sural and peroneal nerves of patients with DPN. But these supplements have not decreased HbA1c in DPN patients. Several trials with a large sample size are needed to provide evidence support for clinical practice in the future.

Response characteristics of rhizosphere microbial community and metabolites of Iris tectorum to Cr stress.

Chromium (Cr) is a toxic heavy element that interferes with plant metabolite biosynthesis and modifies the plant rhizosphere microenvironment, affecting plant growth. However, the interactions and response mechanisms between plants and rhizosphere bacteria under Cr stress still need to be fully understood. In this study, we used Iris tectorum as a research target and combined physiology, metabolomics, and microbiology to reveal the stress response mechanism of I. tectorum under heavy metal chromium stress. The results showed that Cr stress-induced oxidative stress inhibited plant growth and development and increased malondialdehyde and oxygen free radicals content. Also, it increased ascorbate peroxidase, peroxidase activity, and superoxide dismutase activity, as well as glutathione and soluble sugar content. Microbiome analysis showed that Cr stress changed the rhizosphere bacterial community diversity index by 33.56%. Proteobacteria, Actinobacteriota, and Chloroflexi together accounting for 71.21% of the total sequences. Meanwhile, the abundance of rhizosphere dominant and plant-promoting bacteria increased significantly with increasing time of Cr stress. The improvement of the soil microenvironment and the recruitment of bacteria by I. tectorum root secretions were significantly enhanced. By metabolomic analysis, five vital metabolic pathways were identified, involving 89 differentially expressed metabolites, divided into 15 major categories. In summary, a multi-omics approach was used in this study to reveal the interaction and stress response mechanisms between I. tectorum and rhizosphere bacterial communities under Cr stress, which provided theoretical basis for plant-microbial bioremediation of Cr-contaminated soils in constructed wetlands. This may provide more valuable information for wetland remediation of heavy metal pollution.

One Porphyrin Per Chain Self-Assembled Helical Ion-Exchange Channels for Ultrahigh Osmotic Energy Conversion.

Ion-exchange membranes (IEMs) convert osmotic energy into electricity when embedded in a reverse electrodialysis cell. IEMs with both high permselectivity and ionic conductivity are highly needed to increase the energy conversion efficiency. The ionic conductivity can be improved by increasing the content of immobile charge carriers, but it is always accompanied by undesirable permselectivity decrease due to excess swelling. Until now, breaking the permselectivity-conductivity tradeoff still has remained a challenge. Here, we demonstrate a membrane with the least ion-exchange capacity (∼10-2 mequiv g-1), generating an ultrahigh power density of 19.3 W m-2 at a 50-fold concentration ratio. The membrane is made of a porphyrin-core four-star block copolymer (p-BCP), forming the high-density helical porphyrin channels (∼1011 cm-2) under the synergistic effect of BCP self-assembly and porphyrin π-π stacking. The porphyrin channel shows high Cl- selectivity and high conductivity, benefiting high-performance osmotic energy conversion. This economic and facile membrane design strategy provides a promising approach to developing a new generation of IEMs.

Transcriptional regulation of the porcine miR-17-92 cluster.

The miR-17-92 cluster has been involved in the cell cycle, apoptosis, and signaling. However, its transcriptional regulation has not been fully characterized. To elucidate the transcriptional regulation, the promoter of miR-17-92 was analyzed in detail in pig here. We found that, as an intronic miRNA, porcine miR-17-92 cluster was regulated by two independent promoters, an A/T-rich region directly upstream of the miR-17-92 coding sequence, and a G/C-rich region corresponding to the host gene promoter of the human miR-17-92 cluster. Several cis-regulatory elements were identified including sites for c-Myc, NFY, E2F3, and SP1, among which NFY and c-Myc sites were present in both A/T- and G/C-rich regions, while E2F3 and SP1 sites only existed in G/C-rich region. Sites for c-Myc, E2F3, and SP1 were positive for regulating transcription. NFY sites played bipartite roles, functioning as a repressor for the A/T-rich region, and as an activator for the G/C-rich region. Additionally, we found that levels of individual miRNAs in the cluster were not promoted completely in parallel with each other or with pri-miR-17-92 by the A/T-rich region, through using a self-made vector by modifying pGL3-basic in which firefly luciferase gene was replaced with an miR-17-92 cluster and a direct upstream A/T-rich region. The expression regulation of miR-17-92 is complicated and the results will contribute to further revealing the regulatory mechanisms under the expression of the miR-17-92 cluster.

The Nrf2 pathway is required for intracellular replication of Toxoplasma gondii in activated macrophages.

Reactive oxygen species (ROS) produced by oxidases and nonenzymatic sources are important for host defence against intracellular pathogens. In this study, we knocked out the Nrf2 gene in RAW264.7 cells using the CRISPR/Cas9 system and investigated the antioxidant effects of the Nrf2 pathway in the cells stimulated by IFN-γ and TNF-α. The results indicated that the Nrf2 signalling pathway is necessary for maintaining redox homeostasis in activated RAW264.7 cells. Inactivation of Nrf2 impaired parasite growth. We also found that p62 contributes to Nrf2-mediated pathways involved in T gondii infection. These findings suggest that the Nrf2/Keap1 pathway may be targeted to prevent and treat toxoplasmosis.

Identification of porcine CTLA4 gene polymorphism and their association with piglet diarrhea and performance traits.

The objective of this study was to evaluate the association between the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) gene and piglet diarrhea. In this study, the mRNA expression of the CTLA4 gene increased significantly in IPEC-J2 cells after Escherichia coli K88 infection. Single nucleotide polymorphisms (SNPs) located in the 5' flanking region (SNPs g.107281989C>T) and 3'-untranslated region (3'-UTR; SNPs g.107288753C>A) were identified, and they were in linkage disequilibrium in both Min pigs and the Landrace population. Association analysis showed that Landrace piglets with a TT or AA genotype had a lower diarrhea index, and AA animals had higher average daily gain when compared to CC pigs, respectively (p < 0.05). However, the relationship between SNPs and diarrhea and performance traits in the Min population was not significant. Haplotype analysis indicated that the TC haplotype had the lowest diarrhea index. The 5' flanking deletion assay suggested that SNP g.107281989C>T was a molecular marker instead of the functional marker. This research demonstrated that genetic variances in the CTLA4 gene had significant effects on Landrace piglet diarrhea resistance.

Molecular identification and transcriptional regulation of porcine IFIT2 gene.

IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) plays important roles in host defense against viral infection as revealed by studies in humans and mice. However, little is known on porcine IFIT2 (pIFIT2). Here, we performed molecular cloning, expression profile, and transcriptional regulation analysis of pIFIT2. pIFIT2 gene, located on chromosome 14, is composed of two exons and have a complete coding sequence of 1407 bp. The encoded polypeptide, 468 aa in length, has three tetratricopeptide repeat motifs. pIFIT2 gene was unevenly distributed in all eleven tissues studied with the most abundance in spleen. Poly(I:C) treatment notably strongly upregulated the mRNA level and promoter activity of pIFIT2 gene. Upstream sequence of 1759 bp from the start codon which was assigned +1 here has promoter activity, and deltaEF1 acts as transcription repressor through binding to sequences at position - 1774 to - 1764. Minimal promoter region exists within nucleotide position - 162 and - 126. Two adjacent interferon-stimulated response elements (ISREs) and two nuclear factor (NF)-κB binding sites were identified within position - 310 and - 126. The ISRE elements act alone and in synergy with the one closer to start codon having more strength, so do the NF-κB binding sites. Synergistic effect was also found between the ISRE and NF-κB binding sites. Additionally, a third ISRE element was identified within position - 1661 to - 1579. These findings will contribute to clarifying the antiviral effect and underlying mechanisms of pIFIT2.

A haplotype variant of porcine IFIT2 increases poly(I:C)-induced activation of NF-κB and ISRE-binding factors.

Interferon-induced protein with tetratricopeptide repeats (IFIT) 2 is associated with various viral infections and pathogenesis in humans and mice. However, there are few reports on IFIT2 in pigs and the polymorphic information remains unclear. Here, by using a direct PCR sequencing method, we identified four single nucleotide polymorphisms (SNPs), c.259G>A (p.Gly87Ser), c.520T>G (p.Phe174Val), c.571C>T (p.Pro191Ser), and c.879A>G (p.Glu293Glu), for the first time in the coding sequence of the porcine (p) IFIT2 gene from a Chinese local breed (Hebao pig), Western commercial pig breeds (Yorkshire and Landrace), and a Chinese developed breed (Beijing Black pig). SNP c.520T>G (p.Phe174Val) leads to the addition of a tetratricopeptide repeat motif, characteristic structure of the IFIT family. SNPs c.259G>A and c.520T>G are medium polymorphic loci (0.25 < polymorphic information content < 0.5) and distributed differently in Western pig breeds and the Chinese local pig, Hebao, which is well known for its strong resistance to disease. Additionally, they are completely linked. The four SNPs constituted five haplotypes with GTCA and AGCA as dominant. The haplotype variant AGCA, which is mainly present in Hebao pigs, significantly synergized the poly(I:C)-induced activation of transcription factors, including NF-κB and IFN-stimulated response element (ISRE)-binding factors, and the expression of interferon ß, indicating that the variant contributes to the induction or magnitude of the immune response upon viral infection. The data showed that variant AGCA might be useful in improving the resistance of pigs to viruses through marker-assisted selection.

Generation of Chimeric RNAs by cis-splicing of adjacent genes (cis-SAGe) in mammals.

Chimeric RNA molecules, possessing exons from two or more independent genes, are traditionally believed to be produced by chromosome rearrangement. However, recent studies revealed that cis-splicing of adjacent genes (cis- SAGe) is one of the major mechanisms underlying the formation of chimeric RNAs. cis-SAGe refers to intergenic splicing of directly adjacent genes with the same transcriptional orientation, resulting in read-through transcripts, termed chimeric RNAs, which contain sequences from two or more parental genes. cis-SAGe was first identified in tumor cells, since then its potential in carcinogenesis has attracted extensive attention. More and more scientists are focusing on it. With the development of research, cis-SAGe was found to be ubiquitous in various normal tissues, and might make a crucial contribution to the formation of novel genes in the evolution of genomes. In this review, we summarize the splicing pattern, expression characteristics, possible mechanisms, and significance of cis-SAGe in mammals. This review will be helpful for general understanding of the current status and development tendency of cis-SAGe.

Angular non-critical phase-matching second-harmonic-generation characteristics of RECOB (RE = Tm, Y, Gd, Sm, Nd and La) crystals.

For the first time, the angular non-critical phase-matching (A-NCPM) second-harmonic-generation (SHG) characteristics of a family of monoclinic oxoborate crystals, RECa4O(BO3)3 (RECOB, RE = Tm, Y, Gd, Sm, Nd and La), were comprehensively investigated. For all of the realizable A-NCPM SHG styles, the feature parameters including PM wavelength, angular, wavelength and temperature acceptance bandwidths, have been derived from the theory and verified by the experiments. We discovered that the closer the ion radius between RE3+ and Ca2+, the smaller the birefringence, and the better the A-NCPM SHG properties. As a result, for the Type-I SHG on Y-axis which has the largest effective nonlinear optical coefficient (deff) among the three realizable A-NCPM styles, NdCOB crystal presents the longest PM wavelength (927 nm), the largest angular acceptance bandwidth (Δθ⋅l1/2 = 84.3 mrad·cm1/2, Δϕ⋅l1/2 = 58.8 mrad·cm1/2), and the broadest wavelength acceptance bandwidth (8.7 nm). This discovery will contribute to the design of new NCPM materials, at the same time the parameter formula will be helpful for the theoretical prediction of NCPM performance.

Transcriptomics Analysis on Excellent Meat Quality Traits of Skeletal Muscles of the Chinese Indigenous Min Pig Compared with the Large White Breed.

The Min pig (Sus scrofa) is a well-known indigenous breed in China. One of its main advantages over European breeds is its high meat quality. Additionally, different cuts of pig also show some different traits of meat quality. To explore the underlying mechanism responsible for the differences of meat quality between different breeds or cuts, the longissimus dorsi muscle (LM) and the biceps femoris muscle (BF) from Min and Large White pigs were investigated using transcriptome analysis. The gene expression profiling identified 1371 differentially expressed genes (DEGs) between LM muscles from Min and Large White pigs, and 114 DEGs between LM and BF muscles from the same Min pigs. Gene Ontology (GO) enrichment of biological functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the gene products were mainly involved in the IRS1/Akt/FoxO1 signaling pathway, adenosine 5'-monophosphate-activated protein kinase (AMPK) cascade effects, lipid metabolism and amino acid metabolism pathway. Such pathways contributed to fatty acid metabolism, intramuscular fat deposition, and skeletal muscle growth in Min pig. These results give an insight into the mechanisms underlying the formation of skeletal muscle and provide candidate genes for improving meat quality. It will contribute to improving meat quality of pigs through molecular breeding.

Enhanced inflammatory damage by microRNA-136 targeting Klotho expression in HK-2 cells by modulating JAK/STAT pathway.

MiR-136 acts as a tumor suppressor by promoting cell apoptosis and downregulating Bcl-2 in glioma cells. Hence, an attempt has been made to evaluate the role of miR-136 in regulation of inflammatory damage in HK-2 cells. HK-2 cells were cultured and assessed for viability. The cells were then transfected with miR-136 mimic, si- miR-136, si-Klotho, and NC. Dual luciferase test was performed to confirm the target of miR-136 which was assumed to be Klotho. Cell viability, apoptosis, expressions of inflammatory cytokines like TNF-α, IL-1ß, IL-6 and IL-8 were assessed in HK-2 cells with overexpressing miR-136 or with knocked down miR-136 activities, following exposure to LPS. LPS induced inflammatory damage decreased cell viability, induced cell apoptosis, and increased the expression of different inflammatory cytokines. It was found that LPS decreased the expression of miR-136. Over-expression of miR-136 inhibited cell viability, enhanced apoptosis, and increased expression of inflammatory cytokines while knockdown of miR-136 showed opposite results with p-values < 0.05. MiR-136 negatively regulated the expression of Klotho with p-value < 0.05. Over-expression of miR-136 inhibited the expression of Klotho and activated JAK/STAT and mTOR signaling pathways and vice versa. Hence, it can be concluded that miR-136 enhances inflammatory damage probably by targeting klotho as has been observed in luciferase assay by inactivation of JAK/STAT and mTOR signaling pathways.

Molecular characterization of Sp110 gene in pigs.

Speckled 110 kDa (Sp110) plays an important role in infectious diseases, as revealed by studies in humans. However, little is known regarding porcine Sp110. To elucidate its potential role in porcine resistance to viral diseases, here, the complete coding sequence of porcine Sp110 gene and its 26 alternatively spliced isoforms were isolated using reverse transcription (RT)-polymerase chain reaction (PCR), and another seven splicing patterns were obtained using a minigene construct. Subcellular distribution of 11 representative isoforms was characterized in PK-15 cells transiently transfected with their respective GFP fusion constructs, and only isoforms (R and V) bearing all functional domains were localized in nucleus, indicating all the other isoforms lose normal functions of Sp110 owing to alternative splicing. Real-time quantitative PCR and competitive RT-PCR showed that both isoforms R and V had similar tissue expression profile, half-life and response to poly(I:C), a synthetic analog of viral double-stranded RNA, while the longer one (isoform R) was transcribed at a higher level. The results indicated that porcine Sp110 has a role in viral infection and that isoform R is the dominant active form. Overall the data provide potential resource for molecular breeding of pig resistant to diseases and contributes to breeding pigs resistant to viral infection.

High glucose induces rat mesangial cells proliferation and MCP-1 expression via ROS-mediated activation of NF-κB pathway, which is inhibited by eleutheroside E.

Glomerular hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy (DN). High glucose-induced oxidative stress is implicated in the etiology of DN. This study aims to investigate the effect of eleutheroside E (EE) on high glucose mediated rat mesangial cells (MCs) proliferation and monocyte chemoattractant protein-1 (MCP-1) expression and the underlying mechanism. MCs proliferation was assessed by MTT assay. Reactive oxygen species (ROS) level and MCP-1 expression were evaluated by ELISA kit. The protein expression of p47, NF-κB p65, p-NF-κB p65, IκBα, p-IκBα, IKKß and p-IKKß were determined by Western blot. The results showed that treatment with EE markedly attenuated high glucose induced MCs proliferation and in a dose-dependent manner. Intervention with EE also significantly blocked high glucose induced intracellular ROS production by decreasing NADPH oxidase activity. Meanwhile, EE administration could effectively alleviate the high glucose-stimulated activation of NF-κB, the degradation of IκBα and the expression of MCP-1. These results demonstrate that high glucose enhances MCs proliferation and MCP-1 expression by activating the ROS/NF-κB pathway and can be inhibited by EE. Our findings provide a new perspective for the clinical treatment of DN.

Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.

Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.