Longitudinal relationship between grip strength and cognitive function in a European population older than 50 years: A cross-lagged panel model.

OBJECTIVES: The aim of this study was to investigate the dynamic longitudinal relationship between grip strength and cognitive function. METHODS: 6175 participants aged ≥50 years were included in the study using three waves of follow-up data from the Survey of Health, Ageing, and Retirement in Europe in 2015 (T1), 2017 (T2) and 2019 (T3). Cognitive function was assessed using numeracy, verbal fluency, immediate recall, delayed recall and total. The cross-lagged panel model was used for analysis. RESULTS: There was a correlation between grip strength and cognitive function. Standardized path coefficient from numeracy T1 to grip strength T2 was 0.017 (p = 0.003), and from numeracy T2 to grip strength T3 was 0.014 (p = 0.012). Standardized path coefficient from grip strength T1 to numeracy T2 was 0.096 (p < 0.001), and from grip strength T2 to numeracy T3 was 0.113 (p < 0.001). Other indicators of cognitive function had similar relationships with grip strength. CONCLUSIONS: The study found a statistically significant longitudinal and bidirectional relationship between grip strength and cognitive function in a sample of people aged ≥50 years from several European countries.

Ultrasound-guided versus fluoroscopy-guided lumbar selective nerve root block: a retrospective comparative study.

The purpose of this study is to compare the accuracy and effectiveness of ultrasound-guided and fluoroscopy-guided lumbar selective nerve root block (SNRB), and to explore the feasibility of ultrasound-guided methods. This retrospective study included patients with lumbar radicular pain who underwent ultrasound-guided and fluoroscopy-guided selective nerve root block at Honghui Hospital Affiliated to Xi'an Jiaotong University from August 2020 to August 2022. Patients were divided into U-SNRB group and F-SNRB group according to ultrasound-guided or fluoroscopy-guided selective nerve root block. There were 43 patients in U-SNRB group and 20 patients in F-SNRB group. The pain visual analogue scale (VAS) scores, Japanese Orthopaedic Association (JOA) scores, related indexes and complications were recorded and compared between the two groups before, 30 min, 1 month and 6 months after block. To evaluate the feasibility, accuracy and effectiveness of ultrasound-guided selective nerve root block. There were no complications in the process of selective nerve root block in both groups. The operating time and the times of closing needle angle adjustment in U-SNRB group were better than those in F-SNRB group, and the difference was statistically significant (P < 0.05). The VAS score and JOA score of patients in the two groups were significantly improved 30 min after block, 1 month and 6 months after block, and the difference was statistically significant (P < 0.05). There was no significant difference between the two groups (P > 0.05). The accuracy of ultrasound-guided selective nerve root block and the degree of pain relief of patients were similar to those of fluoroscopy guidance, but the operation time and needle angle adjustment times were significantly less than that of fluoroscopy, and could effectively reduce radiation exposure. Therefore, it can be used as a better way to guide for choice.

The 3' end of the coding region of senecavirus A contains a highly conserved sequence that potentially forms a stem-loop structure required for virus rescue.

Senecavirus A (SVA) can cause a vesicular disease in swine. It is a positive-strand RNA virus belonging to the genus Senecavirus in the family Picornaviridae. Positive-strand RNA viruses possess positive-sense, single-stranded genomes whose untranslated regions (UTRs) have been reported to contain cis-acting RNA elements. In the present study, a total of 100 SVA isolates were comparatively analyzed at the genome level. A highly conserved fragment (HCF) was found to be located in the 3D sequence and to be close to the 3' UTR. The HCF was computationally predicted to form a stem-loop structure. Eight synonymous mutations can individually disrupt the formation of a single base pair within the stem region. We found that SVA itself was able to tolerate each of these mutations alone, as evidenced by the ability to rescue all eight single-site mutants from their individual cDNA clones, and all of them were genetically stable during serial passaging. However, the replication-competent SVA could not be rescued from another cDNA clone containing all eight mutations. The failure to recover SVA might be attributed to disruption of the predicted stem-loop structure, whereas introduction of a wild-type HCF into the cDNA clone with eight mutations still had no effect on virus recovery. These results suggest that the putative stem-loop structure at the 3' end of the 3D sequence is a cis-acting RNA element that is required for SVA growth.

Cells at early and late stages of infection with Senecavirus A: Comparative analysis of N6-methyladenosine modification on mRNAs.

Infection with Senecavirus A (SVA) causes differential phenotypes in cells. In this study, cells were inoculated with SVA for culture. At 12 and 72 h post infection, cells were independently harvested for high-throughput RNA sequencing, and further methylated RNA immunoprecipitation sequencing. The resultant data were comprehensively analyzed for mapping N6-methyladenosine (m6A)-modified profiles of SVA-infected cells. More importantly, m6A-modified regions were identified in the SVA genome. A dataset of m6A-modified mRNAs was generated for screening out differentially m6A-modified mRNAs, further subjected to a series of in-depth analyses. This study not only showed statistical differentiation of m6A-modified sites between two SVA-infected groups, but also demonstrated that SVA genome, as a positive-sense, single-stranded mRNA, itself could be modified through the m6A pattern. Out of the six samples of SVA mRNAs, only three were identified to be m6A-modified, implying that the epigenetic effect might not be a crucial driving force for SVA evolution.

Structural and nonstructural proteins of Senecavirus A: Recent research advances, and lessons learned from those of other picornaviruses.

Senecavirus A (SVA) is an emerging virus, causing vesicular disease in swine. SVA is a single-stranded, positive-sense RNA virus, which is the only member of the genus Senecavirus in the family Picornaviridae. SVA genome encodes 12 proteins: L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C and 3D. The VP1 to VP4 are structural proteins, and the others are nonstructural proteins. The replication of SVA in host cells is a complex process coordinated by an elaborate interplay between the structural and nonstructural proteins. Structural proteins are primarily involved in the invasion and assembly of virions. Nonstructural proteins modulate viral RNA translation and replication, and also take part in antagonizing the antiviral host response and in disrupting some cellular processes to allow virus replication. Here, we systematically reviewed the molecular functions of SVA structural and nonstructural proteins by reference to literatures of SVA itself and other picornaviruses.

Identification of cis-acting replication element in VP2-encoding region of Senecavirus A genome.

Picornavirus possesses one positive-sense, single-stranded RNA genome, in which a cis-acting replication element (cre) is located. The cre is a stem-loop structure that harbors a conserved AAACA motif within its loop region. This motif functions as a template for adding two U residues to the viral VPg, therefore generating a VPg-pUpU that is required for viral RNA synthesis. Senecavirus A (SVA) is an emerging picornavirus. Its cre has not been identified as yet. In the present study, one putative cre containing a typical AAACA motif was computationally predicted to exist within the VP2-encoding sequence of SVA. To test the role of this putative cre, 22 SVA cDNA clones with different point mutations in their cre-formed sequences were constructed in an attempt to rescue replication-competent SVAs. A total of 11 viruses were rescued from their individual cDNA clones, implying that some mutated cres exerted lethal impacts on SVA replication. To eliminate these impacts, an intact cre was artificially inserted into those SVA cDNA clones without ability of recovering virus. The artificial cre was proven to be able of compensating for some, but not all, defects caused by mutated cres, leading to successful recovery of SVAs. These results indicated that the putative cre of SVA was functionally similar to those of other picornaviruses, perhaps involved in the uridylylation of VPg.

Translation of Senecavirus A polyprotein is initiated from the IRES-proximal initiation codon.

To clarify whether Senecavirus A (SVA) has the potential of alternative translation, an extra G residue was inserted into an SVA cDNA clone, resultantly generating an "AUGAUG" motif. The second AUG is the authentic SVA initiation codon, whereas the first AUG is a putative one. Subsequently, eighteen nucleotides were inserted one by one between AUG and AUG for reconstructing cDNA clones. The test of virus recovery showed that three replication-competent SVAs, whose AUG/AUG-flanked sequences were not multiples of three nucleotides, were successfully rescued from their individual cDNA clones. The wild-type SVA possesses a UUUUU motif within the polyprotein-encoding region. Sanger sequencing showed that these three replication-competent SVAs harbored one or two extra U residues in the UUUUU motif, implying that polyprotein translation was initiated from the putative AUG, and the authentic AUG would be inactivated. This is probably attributed to the lack of ribosome scanning along an SVA genome.

The 5'-end motif of Senecavirus A cDNA clone is genetically modified in 36 different ways for uncovering profiles of virus recovery.

Senecavirus A (SVA) is an emerging picornavirus. Its genome is one positive-sense, single-stranded RNA. The viral protein (VPg) is covalently linked to the extreme 5' end of the SVA genome. A complex hairpin-pseudoknot-hairpin (HPH) RNA structure was computationally predicted to form at the 5' end of the SVA genome. A total of three extra "U" residues (UUU) served as a linker between the HPH structure and the VPg, causing putative UUU-HPH formation at the extreme 5' end of the SVA genome. It is unclear how the UUU-HPH structure functions. One SVA cDNA clone (N0) was constructed previously in our laboratory. Here, the N0 was genetically tailored for reconstructing a set of 36 modified cDNA clones (N1 to N36) in an attempt to rescue replication-competent SVAs using reverse genetics. The results showed that a total of nine viruses were successfully recovered. Out of them, five were independently rescued from the N1 to N5, reconstructed by deleting the first five nucleotides (TTTGA) one by one from the extreme 5' end of N0. Interestingly, these five viral progenies reverted to the wild-type or/and wild-type-like genotype, suggesting that SVA with an ability to repair nucleotide defects in its extreme 5' end. The other four were independently rescued from the N26 to N29, containing different loop-modifying motifs in the first hairpin of the HPH structure. These four loop-modifying motifs were genetically stable after serial passages, implying the wild-type loop motif was not a high-fidelity element in the first hairpin during SVA replication. The other genetically modified sequences were demonstrated to be lethal elements in the HPH structure for SVA recovery, suggesting that the putative HPH formation was a crucial cis-acting replication element for SVA propagation.

Tolerance of Senecavirus A to Mutations in Its Kissing-Loop or Pseudoknot Structure Computationally Predicted in 3' Untranslated Region.

Senecavirus A (SVA) is an emerging virus that belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense and single-stranded RNA, containing two untranslated regions (UTRs). The 68-nt-long 3' UTR is computationally predicted to possess two higher-order RNA structures: a kissing-loop interaction and an H-type-like pseudoknot, both of which, however, cannot coexist in the 3' UTR. In this study, we constructed 17 full-length SVA cDNA clones (cD-1 to -17): the cD-1 to -7 contained different point mutations in a kissing-loop-forming motif (KLFM); the cD-8 to -17 harbored one single or multiple point mutations in a pseudoknot-forming motif (PFM). These 17 mutated cDNA clones were independently transfected into BSR-T7/5 cells for rescuing recombinant SVAs (rSVAs), named rSVA-1 to -17, corresponding to cD-1 to -17. The results showed that the rSVA-1, -2, -3, -4, -5, -6, -7, -9, -13, and -15 were successfully rescued from their individual cDNA clones. Moreover, all mutated motifs were genetically stable during 10 viral passages in vitro. This study unveiled viral abilities of tolerating mutations in the computationally predicted KLFM or PFMs. It can be concluded that the putative kissing-loop structure, even if present in the 3' UTR, is unnecessary for SVA replication. Alternatively, if the pseudoknot formation potentially occurs in the 3' UTR, its deformation would have a lethal effect on SVA propagation.

Experimental evidence for occurrence of putative copy-choice recombination between two Senecavirus A genomes.

Senecavirus A (SVA), formerly known as Seneca Valley virus, belongs to the genus Senecavirus in the family Picornaviridae. SVA has a single-stranded, positive-sense RNA genome, which is actually an mRNA that initiates translation via its own internal ribosome entry site (IRES). The SVA IRES has been demonstrated to be the hepatitis C virus (HCV)-like IRES, containing eight stem-loop domains: domain (D)II, DIIIa, DIIIb, DIIIc, DIIId1, DIIId2, DIIIe and DIIIf. In this study, stem-forming motifs (SFMs) in the eight domains were independently subjected to site-directed mutagenesis (SDM) to construct eight SVA minigenomes for dual-luciferase reporter assay. The result suggested that except the DII, the other seven domains were closely evolved in the IRES activity. Subsequently, a full-length SVA cDNA clone tagged with a reporter gene was genetically modified to construct eight SFM-mutated ones, separately transfected into BSR-T7/5 cells in an attempt to rescue replication-competent SVAs. Nevertheless, no virus was successfully rescued from its own cDNA clone, implying each of the putative domains necessary in SVA IRES for viral replication. Further, we attempted to rescue replication-competent SVA via pairwise transfection of cDNA clones. Out of 28 combinations of co-transfection, four were demonstrated to be able to rescue replication-competent SVAs. Sanger sequencing showed that all four viruses had the wild-type IRES genotype, suggesting the occurrence of putative copy-choice recombination between two IRES-modifying genomes.

Fraxetin alleviates microglia-mediated neuroinflammation after ischemic stroke.

Background: Neuroinflammation, which is mainly mediated by excessive microglia activation, plays a major role in ischemic stroke. Overactivated microglia secrete numerous inflammatory cytokines, causing excessive inflammatory responses and ultimately exacerbating ischemic brain injury. Hence, compounds that attenuate neuroinflammation could become promising drug candidates for ischemic stroke. Fraxetin has an anti-inflammatory effect in many inflammatory diseases. However, whether it possesses an anti-inflammatory capacity in microglia-mediated neuroinflammation after ischemic brain injury is unknown. Our study aimed to investigate the suppression effect of fraxetin on neuroinflammation in lipopolysaccharide (LPS)-activated microglia and establish whether fraxetin could alleviate ischemic brain injury in a rodent model of ischemic stroke. Methods: For the in vitro experiment, primary microglia were obtained from 1-day-old C57/BL6J mice. The cells were activated with LPS and treated with fraxetin at a non-cytotoxic concentration. Real-time PCR, enzyme-linked immunosorbent assays, and immunofluorescence staining were used to evaluate the anti-inflammatory effects of fraxetin. The potential molecular mechanisms were explored and verified through RNA-sequencing analysis, western blotting and real-time PCR. For the in vivo experiment, focal ischemia was induced by middle cerebral artery occlusion (MCAO) in 8-week-old male C57/BL6J mice. Fraxetin (5 mg/kg) or an equal volume of saline was injected into mice intraperitoneally after MCAO, and 2% 2,3,5-triphenyltetrazolium chloride staining was applied to measure infarct volume. Behavioral tests were conducted to measure neurological deficits in the mice. Real-time PCR, western blotting, and immunofluorescence staining were used to examine the expression of inflammatory cytokines and microglia activation in the ischemic penumbra. Results: Fraxetin effectively inhibited the expression of proinflammatory cytokines including inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-1 beta, and interleukin-6 in LPS-activated microglia. Fraxetin also suppressed the PI3K/Akt/NF-κB signaling pathway in activated microglia, which contributed to its anti-inflammatory effects. Furthermore, the administration of fraxetin attenuated ischemic brain injury and behavioral deficits after stroke. Finally, fraxetin was found to attenuate the activation of microglia both in vitro and in vivo. Conclusions: Our results suggest that fraxetin has a suppression effect on microglia-mediated neuroinflammation, and this effect is associated with the PI3K/Akt/NF-κB signaling pathway. Fraxetin may therefore have potential neuroprotective properties for ischemic stroke.

Synthetic VSMCs induce BBB disruption mediated by MYPT1 in ischemic stroke.

Vascular smooth muscle cells (VSMCs) have been widely recognized as key players in regulating blood-brain barrier (BBB) function, and their roles are unclear in ischemic stroke. Myosin phosphatase target subunit 1 (MYPT1) is essential for VSMC contraction and maintaining healthy vasculature. We generated VSMC-specific MYPT1 knockout (MYPT1SMKO) mice and cultured VSMCs infected with Lv-shMYPT1 to explore phenotypic switching of VSMCs and the accompanied impacts on BBB integrity. We found that MYPT1 deficiency induced phenotypic switching of synthetic VSMCs, which aggravated BBB disruption. Proteomic analysis identified evolutionarily conserved signaling intermediates in Toll pathways (ECSIT) as a downstream molecule that promotes activation of synthetic VSMCs and contributed to IL-6 expression. Knocking down ECSIT rescued phenotypic switching of VSMCs and BBB disruption. Additionally, inhibition of IL-6 decreased BBB permeability. These findings reveal that MYPT1 deficiency activated phenotypic switching of synthetic VSMCs and induced BBB disruption through ECSIT-IL-6 signaling after ischemic stroke.

IL-37 Represses the Autoimmunity in Myasthenia Gravis via Directly Targeting Follicular Th and B Cells.

IL-37 is a newly identified immune-suppressive factor; however, the function, cellular sources, and mechanism of IL-37 in humoral immunity and Myasthenia gravis (MG) are still unclear. In this study, we found IL-37 were substantially downregulated in the serum and PBMCs of MG patients compared with healthy controls. The lower IL-37 was associated with severer disease (quantitative MG score) and higher follicular Th (Tfh)/Tfh17 and B cell numbers. Flow cytometry analysis revealed that IL-37 was mainly produced by CD4+ T cells without overlapping with Th1, Th17, and Tfh subsets in MG patients. Regulatory IL-37+ T cell rarely expressed Foxp3 and CD25 but produced numerous IL-4. Tfh and B cell expressed high levels of SIGIRR, the receptor of IL-37, in MG patients. Mechanically, IL-37 directly bond to SIGIRR, repressed the proliferation, cytokine production of Tfh and B cells, and the secretion of autoantibody via inhibition of STAT3 signaling in Tfh and B cells.

FasL-PDPK1 Pathway Promotes the Cytotoxicity of CD8+ T Cells During Ischemic Stroke.

CD8+ T cells are recognized as key players in exacerbation of ischemic stroke; however, the underlying mechanism in modulating the function of CD8+ T cells has not been completely elucidated. Here, we uncovered that FasL enhanced the cytotoxicity of CD8+ T cells to neurons after ischemic stroke. Inactivation of FasL specific on CD8+ T cells protected against brain damage and neuron loss. Proteomic analysis identified that PDPK1 functioned downstream of FasL signaling and inhibition of PDPK1 effectively reduced cytotoxicity of CD8+ T cells and improved ischemic neurological deficits. Taken together, these results highlight an intrinsic FasL-PDPK1 pathway regulating the cytotoxicity of CD8+ T cells in ischemic stroke.

Double-negative T cells remarkably promote neuroinflammation after ischemic stroke.

CD3+CD4-CD8- T cells (double-negative T cells; DNTs) have diverse functions in peripheral immune-related diseases by regulating immunological and inflammatory homeostasis. However, the functions of DNTs in the central nervous system remain unknown. Here, we found that the levels of DNTs were dramatically increased in both the brain and peripheral blood of stroke patients and in a mouse model in a time-dependent manner. The infiltrating DNTs enhanced cerebral immune and inflammatory responses and exacerbated ischemic brain injury by modulating the FasL/PTPN2/TNF-α signaling pathway. Blockade of this pathway limited DNT-mediated neuroinflammation and improved the outcomes of stroke. Our results identified a critical function of DNTs in the ischemic brain, suggesting that this unique population serves as an attractive target for the treatment of ischemic stroke.

Mitochondrial dysfunction and cerebral metabolic abnormalities in patients with mitochondrial encephalomyopathy subtypes: Evidence from proton MR spectroscopy and muscle biopsy.

AIMS: Accumulated evidence indicates that cerebral metabolic features, evaluated by proton magnetic resonance spectroscopy (1 H-MRS), are sensitive to early mitochondrion dysfunction associated with mitochondrial encephalomyopathy (ME). The metabolite ratios of lactate (lac)/Cr, N-acetyl aspartate (NAA)/creatine (Cr), total choline (tCho)/Cr, and myoinositol (mI)/Cr are measured in the infarct-like lesions by 1 H-MRS and may reveal metabolic changes associated with ME. However, the application of this molecular imaging technique in the investigation of the pathology of ME subtypes is unknown. METHODS: In this study, cerebral metabolic features of pathologically diagnosed ME cases, that is, 19 mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); nine chronic progressive external ophthalmoplegia (CPEO); and 23 healthy controls, were investigated using 1 H-MRS. Receiver operating characteristics (ROC) analysis was used to evaluate the diagnostic power of the cerebral metabolites. Histochemical evaluation was carried out on muscle tissues derived from biopsy to assess the abnormal mitochondrial proliferation. The association between cerebral metabolic and mitochondrial cytopathy was examined by correlation analysis. RESULTS: Patients with MELAS or CPEO exhibited a significantly higher Lac/Cr ratio and a lower NAA/Cr ratio compared with controls. The ROC curve of Lac/Cr ratio indicated prominent discrimination between MELAS or CPEO and healthy control subjects, whereas the NAA/Cr ratio may present diagnostic power in the distinction of MELAS from CPEO. Lower NAA/Cr ratio was associated with higher Lac/Cr in MELAS, but not in CPEO. Furthermore, higher ragged-red fibers (RRFs) percentages were associated with elevated Lac/Cr and reduced NAA/Cr ratios, notably in MELAS. This association was not noted in the case of mI/Cr ratio. CONCLUSIONS: Mitochondrial cytopathy (lactic acidosis and RRFs on muscle biopsy) was associated with neuronal viability but not glial proliferation, notably in MELAS. Mitochondrial neuronopathy and neuronal vulnerability are considered significant causes in the pathogenesis of MELAS, particularly with regard to stroke-like episodes.

Proteomic analysis of the effects of Nur77 on lipopolysaccharide-induced microglial activation.

Microglia are critical components of the immune response in the central nervous system. Our study aims to explore potential role of Nur77 in lipopolysaccharide (LPS)-induced microglial activation. Primary wild-type and Nur77-/- microglia were stimulated with LPS and protein extracts were detected via mass spectrometry. Q-PCR and western blotting were performed to validate candidate proteins. A total of 2004 proteins were identified, with 749 and 677 significantly differentially expressed proteins in wild-type and Nur77-/- microglia in resting and activated states, respectively. Signaling pathway analysis showed that significantly differentially expressed proteins in LPS-treated Nur77-/- microglia were present in important signaling pathways of microglial activation, including the Toll-like receptor signaling pathway, the MAPK signaling pathway, FcγR-mediated phagocytosis, and chemokine signaling pathways. Furthermore, we found that Nur77 could be the upstream protein of the vav1 and ERK1/2 signaling pathway. This study provided new insights into the understanding the mechanisms of the effects of Nur77 on LPS-activated microglia.

Effect of histone modifications on hMLH1 alternative splicing in gastric cancer.

hMLH1 is one of the mismatch genes closely related to the occurrence of gastric cancer. Epigenetic regulation may play more important roles than gene mutations in DNA damage repair genes to drive carcinogenesis. In this article, we discuss the role of epigenetic changes, especially histone modifications in the regulation of hMLH1 alternative splicing. Our results showed that hMLH1 delEx10, delEx11, delEx10-11, delEx16 and delEx17 transcripts were ubiquitous in sporadic Chinese gastric cancer patients and gastric cancer cell lines. Lower level of H4K16ac and H3ac was detected in hMLH1 exon 10-11 region in gastric cancer cell lines when compared with human gastric mucosal epithelial cell line GES-1. A significant decrease of hMLH1 delEx11 and delEx10-11 was observed in gastric cancer cell lines after trichostatin A treatment. H3K36me3 and H3K4me2 levels were lower in hMLH1 exon 10-11 and exon 16-17 regions in gastric cancer lines when compared with GES-1. Aberrant transcripts such as hMLH1 delEx11 and delEx10-11 were significantly higher in gastric cancer cell lines after small interfering RNA-mediated knockdown of SETD2 (the specific methyltransferase of H3K36). The hMLH1 delEx10 and delEx10-11 transcripts were increased after interference of SRSF2. Taken together, our study demonstrates that lower level of histone acetylation and specific histone methylation such as H3K36me3 correlate with aberrant transcripts in hMLH1 exon 10-11 region. SRSF2 may be involved in these specific exons skipping as well.