Sphingosine kinase 2 activates autophagy and protects neurons against ischemic injury through interaction with Bcl-2 via its putative BH3 domain

Our previous findings suggest that sphingosine kinase 2 (SPK2) mediates ischemic tolerance and autophagy in cerebral preconditioning. The aim of this study was to determine by which mechanism SPK2 activates autophagy in neural cells. In both primary murine cortical neurons and HT22 hippocampal neuronal cells, overexpression of SPK2 increased LC3II and enhanced the autophagy flux. SPK2 overexpression protected cortical neurons against oxygen glucose deprivation (OGD) injury, as evidenced by improvement of neuronal morphology, increased cell viability and reduced lactate dehydrogenase release. The inhibition of autophagy effectively suppressed the neuroprotective effect of SPK2. SPK2 overexpression reduced the co-immunoprecipitation of Beclin-1 and Bcl-2, while Beclin-1 knockdown inhibited SPK2-induced autophagy. Both co-immunoprecipitation and GST pull-down analysis suggest that SPK2 directly interacts with Bcl-2. SPK2 might interact to Bcl-2 in the cytoplasm. Notably, an SPK2 mutant with L219A substitution in its putative BH3 domain was not able to activate autophagy. A Tat peptide fused to an 18-amino acid peptide encompassing the native, but not the L219A mutated BH3 domain of SPK2 activated autophagy in neural cells. The Tat-SPK2 peptide also protected neurons against OGD injury through autophagy activation. These results suggest that SPK2 interacts with Bcl-2 via its BH3 domain, thereby dissociating it from Beclin-1 and activating autophagy. The observation that Tat-SPK2 peptide designed from the BH3 domain of SPK2 activates autophagy and protects neural cells against OGD injury suggest that this structure may provide the basis for a novel class of therapeutic agents against ischemic stroke

Synchronous Detection of BPV and BVDV with Duplex Taqman qPCR Method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrhea in dairy herds. BPV is a member of bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirus genus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes according to the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viral diarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the present study was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV. Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved 5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed in vitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, and then used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV. Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probe qRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were 2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplification conditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTR gene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. For clinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealing temperature was achieved in 43.2 ℃ fro duplex BPV and BPIV. Dynamic curves and standard curves were created following amplification of recombinant plasmids using the optimized duplex Taqman BPV and BVDV, with an amplification efficiency of 95.69%. Duplex Taqman qPCR could only detect DNA of BPV and cDNA of BVDV with a strong specificity. The detection limitation was as low as 2.0 × 102 copies/μL of pMD18-T-BPV plasmid and 2.0 × 101 copies/μL for pMD18-T-BVDV plasmid, respectively. Sensitivity of detection was 100-fold higher than conventional PCR. Duplex Taqman qPCR had excellent repeatability or stability with less than 1.2% of intra-assay and inter-assay. 35 and 47 positive feces samples were identified using duplex Taqman qPCR in comparison to 30 and 42 positives for universal PCR, respectively. Discussion: The bovine viral diarrhea virus (BVDV) is a key pathogenic factor in bovine diarrhea. Currently, few effective measures are available for the treatment or prevention for BVDV and BPV infections in animals. The technique was proven to be repeatable and linear over a range of at least 5 magnitudes, from 101 to 105 RNA/DNA copies, thus ensuring an accurate measurement of BPV DNA and BVDV RNA loads in clinical samples. In conclusion, a duplex Taqman qPCR was established for detecting simultaneously BPV and BVDV. Taqman qPCR method was rapid and specific assay. This assay was 100-fold sensitive than conventional PCR. It will be propitious to rapidly and differentially diagnose pathogens of viral diarrhea of dairy farms. Taqman qPCR method was rapid and specific assay and had a sensitivity of 2.0 copies/μL

Y Chromosomes of 40% Chinese Are Descendants of Three Neolithic Super-grandfathers

Demographic change of human populations is one of the central questions for delving into the past of human beings. To identify major population expansions related to male lineages, we sequenced 78 East Asian Y chromosomes at 3.9 Mbp of the non-recombining region (NRY), discovered >4,000 new SNPs, and identified many new clades. The relative divergence dates can be estimated much more precisely using molecular clock. We found that all the Paleolithic divergences were binary; however, three strong star-like Neolithic expansions at ~6 kya (thousand years ago) (assuming a constant substitution rate of 1e-9/bp/year) indicates that ~40% of modern Chinese are patrilineal descendants of only three super-grandfathers at that time. This observation suggests that the main patrilineal expansion in China occurred in the Neolithic Era and might be related to the development of agriculture.Comment: 29 pages of article text including 1 article figure, 9 pages of SI text, and 2 SI figures. 5 SI tables are in a separate ancillary fil

Combination of oncolytic adenovirus and luteolin exerts synergistic antitumor effects in colorectal cancer cells and a mouse model

In recent years, oncolytic viruses have attracted increasing interest due to their potent antitumor effects. Luteolin, a natural product, has additionally been observed to exhibit various pharmacological antitumor activities. Previously, a novel dual-targeting oncolytic adenovirus, complement decay-accelerating factor (CD55)-tumor necrosis factor ligand superfamily member 10 (TRAIL), was constructed, which exhibited significant growth inhibitory effects in various types of tumor cell. The present study investigated whether the combination of luteolin and CD55-TRAIL was able to exert a synergistic antitumor effect in colorectal carcinoma (CRC) cells. The cytotoxicity and tumor cell apoptosis mediated by combination treatment in CRC cells were detected via an MTT assay, Hoechst staining and western blotting, respectively. Tumor growth in vivo was examined in a CRC mouse xenograft model following various treatments. The results demonstrated that the addition of luteolin enhanced oncolytic adenovirus-mediated enhanced green fluorescent protein, early region 1A and TRAIL expression. The combination of CD55-TRAIL with luteolin synergistically inhibited tumor growth and promoted CRC cellular apoptosis in vitro and in vivo. Additionally, the combination of CD55-TRAIL with luteoli n significa ntly decrea sed cy totoxicit y in lung/bronchial normal epithelial cells, compared with single treatment

Effects of vitamin C on inhalation anesthetic isoflurane-induced developmental, neuronal apoptosis in neonatal rats

Developmental abnormalities, neuronal apoptosis and associated cognitive impairment following isoflurane exposure in neonatal rodents have been reported. The study was undertaken to investigate the effect of vitamin C supplementation against isoflurane-induced neurotoxicity. Seven day old rats were exposed to 1.1% isoflurane, or air for 6 hours. Treatment groups were administered with vitamin C (30 mg/kg, orally) from postnatal day 1 (P1) to P10 and were exposed to isoflurane on P7. Isoflurane exposure induced apoptosis was determined by Fluoro-Jade C and terminal deoxynucleotidyl-transferase-mediated 2-deoxyuridine 5-triphosphate nick-end labeling assay. Vitamin C considerably improved memory and learning impairments, modulated neuroapoptosis and improved expressions of brain-derived neurotrophic factor, nerve growth factor, Bcl-xL and decreased activated caspase-3 expressions. Thus, vitamin C effectively offered protection against isoflurane-induced neuronal apoptosis, learning and memory disturbances

Clinical study of Medpor Titan implantation for orbital blowout fracture with nasal endoscopy

AIM: To investigate the clinical effect of reconstruction of orbital blowout fracture by conjunctival incision combined with Medpor Titan implantation under nasal endoscopy. METHODS: Sixteen patients(16 eyes)diagnosed with orbital blowout fracture, include medial fractures, floor fractures and extended fractures, were performed reconstruction by transconjunctival approach with implant material- Medpor Titan under nasal endoscopic-assisted; observation of postoperative visual acuity, eyeball protrusion, extraocular movement limitations, diplopia, orbital CT, occurrence of implanted material rejection was taken. RESULTS: A 3-month follow-up was performed and the therapeutic efficacies of anatomic and functional recovery were evaluated. No further vision loss or infection occurred postoperatively. In the 16 patients, the average postoperative observation was 3mo, enophthalmos were fully corrected, diplopia disappeared. Orbital(CT)did not reveal implant displacement and rejection postoperatively. CONCLUSION: Endoscopic-assisted reconstruction of orbital blowout fracture by conjunctival incision combined with Medpor Titan implantation is a safe and effective surgical method with the characteristics of with direct operation, clear range of fracture exposure and safe and reliable operation, not only restores the patient's visual function and appearance, but also reduces the incidence of complications, avoiding postoperative facial scar

Serum microRNAs are non-invasive biomarkers for the presence and progression of subarachnoid haemorrhage

Correspondence: miRNAs are important regulators of translation and have been associated with the pathogenesis of a number of cardiovascular diseases including stroke and may be possible prognostic biomarkers. The purpose of the present study was to determine the expression levels of miRNAs in the sera of subarachnoid haemorrhage (SAH) patients and to evaluate their relationships with the severity and clinical outcome of SAH. Serum samples on day 3 after the onset of SAH were subjected to microarray analysis with Exqion miRCURY TM LNA array and quantitative PCR analysis. Serum samples from SAH patients (n=60) and healthy controls (n=10) were subjected to quantitative PCR analysis. The severities and clinical outcomes of the SAH patients were evaluated with the WFNS grade and the Modified Rankin Scale (mRS). Three miRNAs, miR-502-5p, miR-1297 and miR-4320 were significantly up-regulated in the sera of SAH patients when compared with the healthy controls. The serum miR-502-5p and miR-1297 levels were significantly higher in the patients with severe SAH and a poor outcome than in those with mild SAH and a good outcome (P<0.05). The areas under the receiver operating characteristic (ROC) curves (AUCs) of miR-502-5p, miR-1297 and miR-4320 to distinguish the SAH patients from the healthy controls were 0.958 (P<0.001), 0.950 (P<0.001) and 0.843 (P<0.001) respectively. Taken together, these results indicate that miR-502-5p and miR-1297 are potentially valuable indicators of the diagnosis, severity and prognosis of SAH, and miR-4320 was a potentially valuable indicator of the diagnosis of SAH

Effects of climate change on the potential distribution of the threatened relict Dipentodon sinicus of subtropical forests in East Asia: Recommendations for management and conservation

Dipentodon sinicus Dunn. (Dipentodonaceae) is a rare and threatened relict plant species usually found co-dominating with other relict plants in subtropical forest patches in highly fragmented habitats of southwestern China, northern Vietnam and northeastern Myanmar of East Asia. To date, its management and conservation strategies in the light of climate change have not been explored. We evaluated effects of climate change on the distribution of climatically suitable areas of D. sinicus as found prevailing during the last glacial maximum (LGM), the mid-Holocene and the present time, and predicted the distribution of climatically suitable habitats in 2070 throughout East Asia. The results as derived from ecological niche modeling (ENM) show the current distribution to be limited to the prehistoric (the mid-Holocene and LGM) refugia, and to indicate decreasing probability of presence and a reducing range of distribution for 2070. In addition, the suitable areas predicted with high probability (0.5–1) only account for on average 9.8% of the total area of potential habitats (threshold‒1) among the models for the year 2070, thereby indicating that D. sinicus is highly vulnerable. Under all the future scenarios for the year 2070, 69–74.2% of potential habitats in China would be outside protected areas. We assess and propose priorities for protected areas, and provide suggestions for conservation management strategies.This study received financial support from Science and Technology Department of Yunnan University, China (2019YNU002), the Ministry of Science and Technology of China (2015FY210200-15), Ajuts a Grups de Recerca Consolidats” (grants nos. 2014-SGR514-GREB and 2017-SGR1116) from the Generalitat de Catalunya (Spain), Applied Basic Research Foundation of Yunnan Province, China (Grant No. 2019FB058), the Environment Research and Technology Development Fund (JPMEERF15S11407) of the Environmental Restoration and Conservation Agency of Japan, and the Kakenhi Grant Number 15H02833.Highlights Abstract Keywords 1. Introduction 2. Material and methods 2.1. Species 2.2. Occurrence data and ecological niche modeling 3. Results 3.1. Model performance and present potential distribution 3.2. Projected distribution during the mid-Holocene (ca. 6000 yr BP) and LGM (ca. 21,000 yr BP) 3.3. Projected distribution under future climate (2070) 4. Discussion 4.1. Effects of climate change on spatial distribution patterns of D. sinicus 5. Recommendations for future conservation efforts and management Declaration of competing interest Acknowledgements Appendix A. Supplementary data Reference

Sensory Neuron-Specific GPCR Mrgprs Are Itch Receptors Mediating Chloroquine-Induced Pruritus

The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side effect of this widely used antimalarial drug. Here, we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and coexpress gastrin-releasing peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with the MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics