Education in inpatient children and young people’s mental health services

<p>As a chronic disease, osteoarthritis (OA) leads to the degradation of both cartilage and subchondral bone, its development being mediated by proinflammatory cytokines like interleukin-1β. In the present study, the anti-inflammatory effect of specnuezhenide (SPN) in OA and its underlying mechanism were studied in vitro and in vivo. The results showed that SPN decreases the expression of cartilage matrix-degrading enzymes and the activation of NF-κB and wnt/β-catenin signaling, and increases chondrocyte-specific gene expression in IL-1β-induced inflammation in chondrocytes. Furthermore, SPN treatment prevents the degeneration of both cartilage and subchondral bone in a rat model of OA. To the best of our knowledge, this study is the first to report that SPN decreases interleukin-1β-induced inflammation in rat chondrocytes by inhibiting the activation of the NF-κB and wnt/β-catenin pathways, and, thus, has therapeutic potential in the treatment of OA.</p

β Cell FFA2 Deficiency Suppresses Multiple Low Dose Streptozotocin (MLDS) Induced Diabetes

Recently, the gut microbiome has been recognized as an environmental factor in the pathogenesis of type 1 diabetes (T1D). Reduced enrichment of short-chain fatty acid (SCFA)-producing pathways in the gut microbiome and SCFA levels are correlated with an increased T1D risk. We have previously shown that free fatty acid receptor 2 (FFA2), one of the main SCFA receptors, is expressed on pancreatic β cells and mediates the crosstalk between the gut microbiome and β cells in type 2 diabetes (T2D). However, the role of β cell FFA2 in T1D has not been explored. To examine this, we used our novel mouse model, a tamoxifen (TMX)-induced adult onset β cell FFA2 knockout (FFA2 βKO) and its controls (cre control and floxed control), treated with multiple low-dose streptozotocin (MLDS) to model T1D. Surprisingly, 57% of FFA2 βKO mice were protected from MLDS-induced T1D for 43 days and exhibited normal glycemic control owing to a trending higher β cell mass, whereas 100% of the control group mice developed diabetes. As transcriptome data showed marked upregulation of negative regulator of type 1 interferon (T1-IFN) signaling genes, suppressor of cytokine signaling 1/3 (Socs1/3), in FFA2 global knockout islets, we hypothesized that at the early stage of MLDS-induced T1D, when abundant IFN⍺ is presented, adulthood deletion of FFA2 in β cells attenuates T1-IFN signaling via further increase of Socs1/3, resulting protection from MLDS-induced T1D. In fact, both ex vivo and in vivo experiments showed reduced T1-IFN signaling in FFA2 βKO islets and mice. Early in the MLDS insult (7th day) FFA2 βKO mice showed higher β cell mass due to less apoptosis. Collectively, adulthood deletion of FFA2 in β cells attenuates T1-IFN signaling, leading to less apoptosis and higher β cell mass, conferring protection against MLDS-induced T1D in the early stage

AN INVESTIGATIVE ANALYSIS OF BASKETBALL INJURIES BY COLLEGE STUDENTS

ABSTRACT Introduction Basketball is a sport with strong rivalry, being, therefore, a modality with great facility to cause sports injuries along its practice. Objective Investigate the status of sports injuries in college basketball for amateur students. Methods 480 college students who were not basketball experts were selected as research volunteers. The current status of sports injuries in the 12-week optional basketball course was investigated, and the main risk factors were analyzed. Results Among amateur male college students, the average playing age of third-grade male college students is higher than that of second-grade male college students, and the weekly playing frequency is lower than that of second-grade male college students. The incidence of basketball sports injuries among male college students who do not specialize in basketball from childhood to adulthood is up to 90.7%, and 85.5% during their college years. Conclusion In basketball sports, the most vulnerable parts of amateur male college students are the wrist joint, ankle joint, knee joint, leg, head and face, shoulder, waist, and back, respectively. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.</div

Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus

<div><p>Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.</p></div

Recruitment of <i>Arabidopsis</i> Rab5 into the tombusvirus replication compartment in <i>N</i>. <i>benthamiana</i>.

<p>(A) Confocal laser microscopy shows partial co-localization of TBSV RFP-tagged p33 replication protein or CIRV RFP-tagged p36 with the GFP-AtRab5B protein in <i>N</i>. <i>benthamiana</i> cells. Expression of the above proteins from the 35S promoter was achieved after agro-infiltration into <i>N</i>. <i>benthamiana</i> leaves. Scale bars represent 20 μm. (B) Partial re-localization of RFP-AtRab5B protein to the peroxisomes (marked by GFP-SKL) in <i>N</i>. <i>benthamiana</i> cells infected with either TBSV or CNV. The bottom image shows the absence of re-localization of RFP-AtRab5B protein to the peroxisome in the mock-infected plant leaves. Scale bars represent 20 μm. (C) Partial re-localization of RFP-AtRab5B protein to the mitochondria (marked by mito-EGFP) in <i>N</i>. <i>benthamiana</i> cells infected with CIRV. The bottom image shows the absence of re-localization of RFP-AtRab5B protein to the mitochondria in the mock-infected plant leaves. Scale bars represent 20 μm. (D) TBSV infection induces membrane proliferation, which is occasionally visualized as aggregated circle-like structures. These membranous structures are enriched in PE in plant cells. The confocal laser microscopy image shows the enrichment of PE and its co-localization with the TBSV p33/p92 replication proteins, which were detected with p33-specific primary antibody and secondary antibody conjugated with Alexa Fluor488. Localization of PE is detected by using biotinylated duramycin peptide and streptavidin conjugated with Alexa Fluor 405. DIC images are shown on the right. Scale bars represent 20 μm. (E) Top image: <i>In planta</i> interaction between TBSV p33-cYFP replication protein and the nYFP-AtRab5B protein. Expression of the above proteins from the 35S promoter was done after agro-infiltration into <i>N</i>. <i>benthamiana</i> leaves. Note that p33-cYFP and the nYFP-AtRab5B protein were detected by bimolecular fluorescence complementation (BiFC). Control BiFC experiments included nYFP-MBP protein. Bottom images: The interaction between p33 replication protein and AtRab5B occurs in the replication compartment decorated by RFP-p33. As expected, the enlarged replication compartment (highlighted via RFP-SKL) also contained the viral dsRNA replication intermediate only in TBSV-infected cells (second panel form the bottom) but not in the mock-inoculated cells (bottom panel). Scale bars represent 20 μm. (F) The corresponding experiments with the CIRV p36 protein and AtRab5B (see panel E for details). Scale bars represent 20 μm.</p

Rab5 is partly co-localized with PE-enriched tombusvirus replication compartment in plant cells.

<p>(A) Confocal laser microscopy images show the co-localization of GFP-AtRab5B with the TBSV p33-RFP replication protein in subcellular areas enriched with PE in <i>N</i>. <i>benthamiana</i> protoplasts. Scale bars represent 20 μm in each panel. (B) Confocal laser microscopy images confirm that these subcellular areas are derived from aggregated peroxisomes based on co-localization with either Pex13-GFP peroxisomal membrane marker protein or GFP-SKL peroxisomal luminal marker protein. (C) Control images show the lack of PE enrichment in peroxisomes in the absence of viral components. Note the absence of aggregated peroxisomes in these cells. (D) Confocal laser microscopy images show the co-localization of GFP-AtRab5B with the CIRV p36-RFP replication protein in subcellular areas enriched with PE. (E) Confocal laser microscopy images confirm that these subcellular areas are derived from aggregated mitochondria based on co-localization with Tim21-GFP marker protein. (F) Control images show the lack of PE enrichment in mitochondria in the absence of viral components. Note the absence of aggregated mitochondria in these cells.</p

A model on the roles of p33-mediated recruitment of Rab5-positive endosomes in the formation of large tombusviral replication compartments.

<p>At the early stage of tombusvirus replication, TBSV-induced spherule formation may take place in the existing peroxisomal membranes. At the peak time of replication, occurring at a late stage at which point the viral components are much more abundant due to ongoing translation of viral RNAs, however, a portion of p33 molecules co-opt the PE-rich Rab5-positive endosomes via p33–GTP-Rab5 interaction using the actin cables. These processes lead to the formation of large replication compartments containing aggregated peroxisomes fused with PE-rich Rab5-positive endosomes, providing the suitable microenvironment for building numerous spherules harboring the active tombusvirus VRCs. We envision similar early and peak/late stages with CIRV, except the involvement of mitochondria in building the viral replication compartment.</p

Interaction between p33 replication protein and yeast Rab5 ortholog Vps21p.

<p>(A) Confocal laser microscopy images show the partial co-localization of TBSV BFP-tagged p33 or the CIRV BFP-tagged p36 replication proteins with the RFP-tagged Vps21p protein in wt yeast cells. Differential interference contrast (DIC) images are shown on the right. Scale bars represent 2 μm. (B) The split ubiquitin assay was used to test binding between TBSV p33 or CIRV p36 and Vps21p in wt (NMY51) yeast. The bait p33/p36 was co-expressed with the shown prey proteins. The mutant Vps21Q₆₆L is locked into the active GTP-bound stage, whereas the mutant Vps21S₂₁L is locked into the inactive GDP-bound form. <i>SSA1</i> (HSP70 chaperone) and the empty prey vector (NubG) were used as positive and negative controls, respectively. The left panel shows p33:Vps21p interactions; the right panel demonstrates that comparable amounts of yeasts were used for these experiments. (C) Pull-down assay including the 10xHis-tagged yeast Vps21 and the <i>Arabidopsis</i> Rab5B proteins with the C-terminal (soluble) portion of the TBSV p33 (T33C) and CIRV p36 (C36C) replication proteins. Top panel: western blot analysis of the captured cellular proteins with the maltose-binding protein (MBP)-affinity purified p33C/p36C was performed with anti-His antibody. The negative control was MBP. Lane 7 shows molecular weight markers. Bottom panel: comassie blue-staining of protein gel with the purified MBP-p33C (the C-terminal soluble portion of TBSV p33), MBP-p36C (the C-terminal soluble portion), and MBP. (D) Decreased TBSV repRNA accumulation in <i>vps21Δypt52Δypt53Δ</i> yeast. To launch TBSV repRNA replication, we expressed His₆-p33 and His₆-p92 from the galactose-inducible <i>GAL1</i> promoter, and DI-72(+) repRNA from the galactose-inducible <i>GAL10</i> promoter in the parental (BY4741) and in <i>vps21Δypt52Δypt53Δ</i> yeast strains. FLAG-tagged Vps21 or its mutants were expressed from the copper-inducible <i>CUP1</i> promoter. The yeast cells were pre-cultured for 12 h at 29°C in 2% glucose SC minimal media, and then for 22 h at 23°C in 2% galactose SC minimal media supplemented with 50 μM CuSO₄. Northern blot analysis was used to detect DI-72(+) repRNA accumulation. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels (second panel from top). Bottom panels: western blot analysis of the accumulation level of His₆-tagged p33, His₆-p92, and FLAG-Vps21 proteins using anti-His and anti-FLAG antibodies, respectively. Note that FLAG-Vps21 forms a double band due to prenylation (a lipidation type of posttranslational modification) that is required for binding to the endosomal membrane. The faster migrating band represents the prenylated form of Vps21 (depicted by an arrow), whereas the unmodified form is depicted by an open arrowhead. The total protein samples were stained with coomassie blue. Each experiment was performed three times. (E) Decreased CIRV repRNA accumulation in <i>vps21Δypt52Δypt53Δ</i> yeast. See further details in panel D. (F) Decreased TBSV repRNA accumulation in <i>vps9Δmuk1Δ</i> yeast. See further details in panel D. (G) Decreased CIRV repRNA accumulation in <i>vps9Δmuk1Δ</i> yeast. See further details in panel D. (H) Comparable activities of the tombusvirus replicases assembled in cell-free extracts (CFEs) prepared from either wt or from <i>vps21Δypt52Δypt53Δ</i> yeast. Denaturing PAGE analysis of in vitro tombusvirus replicase activity in the CFEs. Note that this image shows the repRNAs made by a full cycle of replicase activity, producing both (-) and (+)-strands in vitro. The CFEs contained the same amount of total yeast proteins. Each experiment was performed three times. (I) Comparable activities of the tombusvirus replicases assembled in CFE membrane fractions prepared from either wt or from <i>vps21Δypt52Δypt53Δ</i> yeast. Note that the supernatant fraction was obtained from wt yeast CFE in each sample. See further details in panel H.</p

The early endosomal membranes are enriched with PE in yeast and plant cells.

<p>(A) Confocal laser microscopy images show the enrichment of PE and its co-localization with the early endosomal RFP-tagged Vps21p expressed from <i>TEF1</i> promoter in the absence of tombusviral components in wt yeast cells (top two images). DIC images are shown on the right. Localization of PE is detected by using biotinylated duramycin peptide and streptavidin conjugated with Alexa Fluor 405. The bottom image shows the lack of PE enrichment in trans-Golgi network marked by RFP-Tlg1. Scale bars represent 2 μm. (B) Confocal laser microscopy images show the enrichment of PE with the endosomal GFP-tagged AtRab5B expressed from 35S promoter in the absence of tombusviral components in <i>N</i>. <i>benthamiana</i> cells. Scale bars represent 20 μm. (C) Enrichment of exogenous PE in early endosomes labeled with RFP-Vps21 protein in wt yeast cells. Yeast cells were cultured (initial 0.3 OD₆₀₀) with 80 μM NBD-PE for 12–14 h. Scale bars represent 2 μm. (D) The control panel shows minimal level of NBD-PE enrichment in the trans-Golgi network labeled with RFP-Tlg1 in wt yeast cells. Scale bars represent 2 μm.</p

The role of actin filaments in recruitment of Rab5-positive endosomes to the large tombusviral replication compartments in plant cells.

<p>(A–B) Confocal laser microscopy images of CIRV-infected <i>N</i>. <i>benthamiana</i> cells expressing AtTim21-GFP mitochondrial marker and the RFP-tagged active GTP-locked AtRab5B mutant. Note the large aggregated mitochondria-containing area (marked by a white arrowhead) and the actin-like filamentous structure (pointed at by a white arrow). Scale bars represent 20 μm. (C) An enlarged subcellular area showing the aggregated mitochondria and the RFP-AtRab5 mutant. Scale bar represents 5 μm. (D–G) Still images from a movie taken from plant cells co-expressing RFP-AtRab5B with TBSV p33-BFP (D–E) or CIRV p36-BFP (F–G) in transgenic plants expressing GFP-mTalin (an actin filament marker). Scale bars represent 20 μm. (D and F) All three channels from 0s are shown. White arrow depicts the direction of Rab5-positive endosomes (red) moving towards the replication compartment (blue) via actin filaments (green). (E and G) Merged images of RFP-AtRab5B and p33-BFP/p36-BFP. White arrow shows the movement of Rab5-positive endosomes. Scale bars represent 20 μm. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000128#pbio.2000128.s014" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000128#pbio.2000128.s015" target="_blank">S3</a> Videos.</p