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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. The proportion of late-onset ALS in China were low and may have distinct clinical and genetic manifestations. We aimed to investigate the natural history and remarkable psychiatric state of ALS with age at onset over 60 years in China.We collected all ALS cases from 2017 to 2020 in our center and focused on late-onset ALS patients particularly, by analyzing the clinical data, including the ALS onset and disease progression. Anxiety, depression, cognitive function, and sleep quality were assessed to reflect the psychiatric state.A total of 193 late-onset ALS patients were included in this study. The median age at onset of late-onset ALS was 65 years with the quartile from 62 to 68 years. When compared with 446 non-late-onset ALS, late-onset ALS showed distinct clinical presentation, with lower ALS Functional Rating Scale-Revised at diagnosis and faster rate of progression. Remarkably, late-onset ALS were suffering from worse psychiatric state, including serious anxiety and depression, as well as worse cognitive function with sleep quality. The abnormal psychiatric state was more pronounced in female patients of late-onset.In the current study, ALS patients with late-onset showed unique clinical features. Severe psychiatric conditions and faster progression in the early stage of the disease of late-onset ALS indicated the need for more social and psychiatric support in this population.

ObjectivesThere is significant burden on caregivers of patients with amyotrophic lateral sclerosis (ALS). However, only a few studies have focused on caregivers, and traditional research methods have obvious shortcomings in dealing with multiple influencing factors. This study was designed to explore influencing factors on caregiver burden among ALS patients and their caregivers from a new perspective.DesignCross-sectional study.SettingThe data were collected at an affiliated hospital in Guangzhou, Guangdong, China.ParticipantsFifty-seven pairs of patients with ALS and their caregivers were investigated by standardised questionnaires.Main outcome measuresThis study primarily assessed the influencing factor of caregiver burden including age, gender, education level, economic status, anxiety, depression, social support, fatigue, sleep quality and stage of disease through data mining. Statistical analysis was performed using SPSS 24.0, and least absolute shrinkage and selection operator (LASSO) regression model was established by Python 3.8.1 to minimise the effect of multicollinearity.ResultsAccording to LASSO regression model, we found 10 variables had weights. Among them, Milano-Torinos (MITOS) stage (0–1) had the highest weight (−12.235), followed by younger age group (−3.198), lower-educated group (2.136), fatigue (1.687) and social support (-0.455). Variables including sleep quality, anxiety, depression and sex (male) had moderate weights in this model. Economic status (common), economic status (better), household (city), household (village), educational level (high), sex (female), age (older) and MITOS stage (2–4) had a weight of zero.ConclusionsOur study demonstrates that the severity of ALS patients is the most influencing factor in caregiver burden. Caregivers of ALS patients may suffer less from caregiver burden when the patients are less severe, and the caregivers are younger. Low educational status could increase caregiver burden. Caregiver burden is positively correlated with the degree of fatigue and negatively correlated with social support. Hopefully, more attention should be paid to caregivers of ALS, and effective interventions can be developed to relieve this burden.

This case report is the first to describe the detection of antibodies against inositol 1,4,5-trisphosphate receptor 1 (ITPR1, I3PR) in a patient diagnosed with autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy. ITPR1 is known as one of the Purkinje cell antibodies present in autoimmune cerebellar ataxia (ACA). Here, we described the association between autoimmune GFAP astrocytopathy and autoimmune cerebellar disease (ACD).Demographic features, clinical characteristics, cerebrospinal fluid (CSF) parameters and neuroimaging findings were collected from this patient. Specifically, antibodies against GFAP and other proteins associated with neurological disorders were measured by immunofluorescence staining in both serum and CSF samples.A 52-year-old woman was diagnosed with autoimmune inflammatory meningoencephalitis. She presented with cognitive dysfunction, psychiatric/behavioral abnormalities and serious insomnia with subacute onset. Brain magnetic resonance imaging (MRI) showed bilateral hyperintensity in the semioval centers on axial images and perivascular linear enhancement oriented radially to the ventricles on sagittal images. GFAP-IgG, oligoclonal bands (OBs), N-methyl-D-aspartate receptor (NMDAR)-IgG and ITPR1-IgG co-existed in her CSF. She responded well to immunoglobulin and steroid treatments.Here, we describe the case of a patient with autoimmune GFAP astrocytopathy whose CSF was positive for ITPR1-IgG; however, she did not show typical ataxia manifestations or cerebellar lesions on her MRI scan. This suggests that ITPR1-IgG is not pathogenic, and the positivity of this antibody in CSF is probably associated with the presence of autoimmune inflammation.

Young-onset amyotrophic lateral sclerosis (ALS) refers to ALS patients with initial symptoms earlier than 45 years, representing a novel disease pattern. We aim to summarize the clinical and genetic features of 102 young-onset ALS patients in China.

Patients with combined phenotypes of Sturge–Weber syndrome and Klippel–Trenaunay syndrome have been reported, though the underlying genetic spectrum in these individuals remains to be elucidated. We reported the patient presenting with Klippel–Trenaunay and Sturge–Weber overlap syndrome in mainland China. Histopathologic study confirmed the hemangioma of vein and capillary. Co‐existence of a novel somatic KRAS c.182_183 delins TC mutation and GNAQ c.548G>A mutation was identified in the affected skin tissue rather than paired peripheral blood. The somatic mutations of GNAQ and KRAS may affect MAPK‐ERK signaling pathway, resulting in endothelial anomaly and blood vessel malformation.

Background and purpose Amyotrophic lateral sclerosis (ALS)-related genes and mutations have been increasingly discovered recently and an improved understanding of genotype-phenotype relationships may help to predict the disease course and refine genetic diagnosis. Methods We collected clinical data and blood samples from 268 patients and used next-generation sequencing to comprehensively assay genetic variations in a panel of known ALS genes from 2015 to 2019. Results Among these patients, the mean age of onset was 52.30 ± 10.42 years with a mean diagnosis delay of 15.90 ± 11.88 months. Patients with SOD1, TARDBP and FUS variants were more likely to suffer from familial ALS. Additionally, carriers of FUS variants displayed the earliest onset, followed by those with SOD1 variants. Patients with NEFH variants showed a closer link to pesticide exposure. Patients with SETX variants were prone to bulbar onset with moderate anxiety problems. No genotype-phenotype relations were found in SPG11 and ERBB4 mutants. Conclusion Our findings uncovered some genotype-phenotype relationships and may help to predict the disease course of patients with ALS in southern China.

Alcoholism is a risk factor for the development of cognitive decline and dementia. Here we demonstrated that the glymphatic function in the brain was impaired by alcohol administration. Acute moderate alcohol administration substantially retarded and reduced the entry of subarachnoid cerebrospinal fluid (CSF) via the paravascular space into the cerebral parenchyma, thus impaired CSF-interstitial fluid (ISF) exchange and parenchymal amyloid β (Aβ) peptide clearance. The elevated release of β-endorphin and reduced cerebrovascular pulsatility after acute alcohol administration may account for the impairment of the glymphatic function. Chronic moderate alcohol consumption led to pronounced activation of astrocytes and a widespread loss of perivascular AQP4 polarization in the brain, which results in an irreversible impairment of the glymphatic function. The results of the study suggest that impaired glymphatic functions and reduced parenchymal Aβ clearance found in both acute and chronic alcohol treatment may contribute to the development of cognitive decline and dementia in alcoholism.

China is stepping into the aging society. The prevalence of type 2 diabetes among elderly population is increasing in China in the last few decades. However, there is a lack of epidemiological investigations regarding to the aging-related changes of islet function in the elderly population.To investigate the islet function and glycemic conditions in the elderly population of western China.Using a complex, multistage, probability sampling design, we conducted a cross-sectional survey in a provincial representative sample of 3001 western Chinese elderly adults. A 2 h oral glucose tolerance test (OGTT) was conducted among all study participants, and islet function measurements including HOMA-β, HOMA-IR, HOMA-IS, ΔI30/ΔG30, Ip/I0 were calculated.HOMA-β, HOMA-IR and ΔI30/ΔG30 decreased gradually along with increasing age from age of 55 to age of 80 (p0.05). HOMA-IR, HOMA-β and ΔI30/ΔG30 were significantly higher while HOMA-IS lower among urban residents than rural residents or suburb residents (p0.01). FPG, 2 h plasma glucose (P2hPG) and HbA1C increased with increasing age (p0.05), while fasting insulin level (FINS) and 2 h insulin level (P2hINS) decreased with increasing age (p0.05). FPG, P2hPG and glycated hemoglobin (HbA1C) among suburb residents were lower than among urban residents and among rural residents (p0.05).Islet function decreased gradually along with increasing age with no gender difference among the elderly population in western China. Glycemic outcomes became worse with increasing age. These results indicated that strategies aimed at the prevention of diabetes in the elderly population were needed.

(1) Background: The aim of this longitudinal study was to evaluate the association between disease progression according to the Milano–Torino staging (MITOS) system and long-term survival in Chinese patients with amyotrophic lateral sclerosis (ALS). We also examined factors affecting MITOS progression. (2) Methods: Patients were enrolled and underwent follow-up at 6, 12, 18, and 24 months, and their demographic and clinical data, including the Milano–Torino stage, Amyotrophic Lateral Sclerosis Functional Rating Scale—Revised (ALSFRS-R) score and neuropsychiatric data, were evaluated. The sensitivity and specificity of predicting survival outcomes based on MITOS progression and ALSFRS-R score decline from baseline to 6 months were compared. The associations between MITOS progression from baseline to 6 months and survival outcome at 12, 18 and 24 months were examined, and factors associated with disease progression were evaluated with subgroup analyses. (3) Results: Among the 100 patients included, 74% were in stage 0 at baseline, and approximately 95% progressed to a higher stage of the MITOS system at 24 months. MITOS progression from baseline to 6 months and ALSFRS-R decline showed comparable value for predicting survival at 12, 18, and 24 months. MITOS progression from baseline to 6 months is strongly associated with death outcomes. Older age at onset and increased depression and anxiety scores may be related to disease progression. (4) Conclusions: MITOS progression during the early disease course could serve as a prognostic marker of long-term survival and may have utility in clinical trials. Age at onset and diagnosis and neuropsychiatric factors might be associated with disease progression.

Background Limb girdle muscular dystrophy type 2Y (LGMD2Y) is a rare subgroup of limb girdle muscular dystrophy featuring limb-girdle weakness, tendon contracture and cardiac involvement. It is caused by the mutation of TOR1AIP1, which encodes nuclear membrane protein LAP1 (lamina-associated polypeptide 1) and comprises heterogeneous phenotypes. The present study reported a patient with a novel homozygous TOR1AIP1 mutation that presented with selective muscle weakness, which further expanded the phenotype of LGMD2Y- and TOR1AIP1-associated nuclear envelopathies. Case presentation A 40-year-old male presented with Achilles tendon contracture and muscle weakness that bothered him from 8 years old. While the strength of his distal and proximal upper limbs was severely impaired, the function of his lower limbs was relatively spared. Muscle pathology showed dystrophic features, and electron microscopy showed ultrastructural abnormalities of disrupted muscle nuclei envelopes. Whole-exome sequencing showed a frameshift mutation in TOR1AIP1 (c.98dupC). Conclusion We reported a novel mild phenotype of LGMD2Y with relatively selective distal upper limb weakness and joint contracture and revealed the heterogeneity of LGDM2Y and the role of the LAP1 isoform by literature review.

The glymphatic system plays a central role in the clearance of extracellular wastes from the brain. Cocaine exposure can lead to pathologies that affect the entire brain, resulting in addictive disorders involving motivational and cognitive impairment. However, it remains unknown whether cocaine exposure impairs glymphatic function. In the present study, using a mouse model of noncontingent cocaine exposure, we evaluated glymphatic function including cerebrospinal fluid (CSF)-interstitial fluid (ISF) exchange and solute clearance during repeated exposures and withdrawal. We found that cocaine treatment, both during repeated exposure and withdrawal, significantly induced widespread astrogliosis and reduced cerebral blood flow (CBF), cerebrovascular pulsatility, and aquaporin-4 (AQP4) polarity. Glymphatic function was greatly impaired in mice after cocaine treatment, as evidenced by reduced CSF influx from paravascular pathways into the brain parenchyma and decreased efflux of interstitial molecules out of the parenchyma. These findings provide evidence that cocaine exposure impairs the clearance of wastes from the brain, which may contribute to the development of neurocognitive disorders in patients with drug addictions.

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4+, CD8+ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3+ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.

Long noncoding RNAs (lncRNAs) have been certified as important regulators in tumorigenesis. LncRNA GAS6-AS2 (GAS6-AS2) was a newly identified tumor-related lncRNA, and its dysregulation and oncogenic effects in melanoma and bladder cancer had been reported in previous studies. However, the expression pattern and potential function of GAS6-AS2 in osteosarcoma (OS) have not been investigated. In this study, we identified a novel OS-related lncRNA GAS6-AS2. We found that GAS6-AS2 was distinctly upregulated in both OS specimens and cell lines. Distinct up-regulation of GAS6-AS2 in OS was correlated with advanced clinical stages and shorter survivals. In addition, USF1 could directly bind to the GAS6-AS2 promoter and contribute to its overexpression. Furthermore, GAS6-AS2 knockdown caused tumor suppressive effects via reducing cellular proliferation, migration and invasion, and promoting OS cell apoptosis. Besides, GAS6-AS2 directly bound to miR-934 and downregulated its expression. Mechanistically, GAS6-AS2 positively regulated the expression of BCAT1 through sponging miR-934. Taken together, our data illustrated how GAS6-AS2 played an oncogenic role in OS and might offer a potential therapeutic target for treating OS.

Cerebral microinfarcts have significant effects on the development of geriatric neurological disorders, including vascular dementia and Alzheimer's disease. However, little is known about the pathophysiological mechanisms involved in the evolution of microinfarcts and potential treatment and prevention against these microvascular ischemic lesions. In the present study, the “single cortical microinfarct model” generated via occluding a penetrating arteriole by femtosecond laser ablation and the “multiple diffuse microinfarcts model” induced by unilateral injection of cholesterol crystals through the internal carotid artery were established to investigate the pathophysiological mechanisms underlying the evolution of microinfarcts and the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on alleviating microinfarct burdens and functional deficits. The occlusion of a single penetrating arteriole led to a distinct cortical microinfarct, which manifested as neuronal loss and occupation of activated glial cells in the ischemic core. Using Fat-1 transgenic mice and fish oil supplements, we demonstrated that both endogenously-generated and exogenously-delivered ω-3 PUFAs significantly inhibited the activation of receptor-interacting serine/threonine protein kinases 1 (RIPK1) and its downstream apoptosis-associated proteins, mitigated cell apoptosis, and anatomically reduced the microinfarct size. The protective effects of ω-3 PUFAs against microinfarcts were further verified in a multiple diffuse microinfarcts model, where ω-3 PUFAs significantly attenuated cell apoptosis as revealed by TUNEL staining, alleviated the diffuse microinfarct burdens and remarkably improved the functional deficits as evidenced by reduced spontaneous anxiety, increased preference for the novel object, and improved hippocampal-based learning and short-term memory. Together, these findings demonstrate that enriched brain ω-3 PUFAs are effective for reducing microinfarct burdens and improving the function deficits, which support the clinical research and application of ω-3 PUFAs in the treatment or prophylaxis in vascular dementia., Highlights • Omega-3 PUFAs inhibit the RIPK1-mediated apoptotic pathways in cortical microinfarcts. • Omega-3 PUFAs contribute to anatomically decreasing microinfarct damage. • Omega-3 PUFAs supplements improve functional deficits in a mouse model with multiple diffuse microinfarcts. Cerebral microinfarct is defined as microscopic vascular occlusion

Summary Charcot-Marie-Tooth disease type 1A (CMT1A), one of the most frequent inherited peripheral neuropathies, is associated with PMP22 gene duplication. Previous studies of CMT1A mainly relied on rodent models, and it is not yet clear how PMP22 overexpression leads to the phenotype in patients. Here, we generated the human induced pluripotent stem cell (hiPSC) lines from two CMT1A patients as an in vitro cell model. We found that, unlike the normal control cells, CMT1A hiPSCs rarely generated Schwann cells through neural crest stem cells (NCSCs). Instead, CMT1A NCSCs produced numerous endoneurial fibroblast-like cells in the Schwann cell differentiation system, and similar results were obtained in a PMP22-overexpressing iPSC model. Therefore, despite the demyelination-remyelination and/or dysmyelination theory for CMT1A pathogenesis, developmental disabilities of Schwann cells may be considered as an underlying cause of CMT1A. Our results may have important implications for the uncovering of the underlying mechanism and the development of a promising therapeutic strategy for CMT1A neuropathy., Highlights • Modeling CMT1A disease with PMP22 duplication using hiPSC-derived NCSCs • PMP22 duplication may lead to Schwann cell developmental defect of NCSCs • PMP22-overexpressing NCSCs recapitulate the phenotype of CMT1A NCSCs, How PMP22 duplication leads to CMT1A neuropathy remains to be elucidated. In this article, Yao, Li, and colleagues show that neural crest stem cells (NCSCs) derived from CMT1A hiPSCs have developmental disabilities of Schwann cells; in contrast, CMT1A NCSCs generate numerous endoneurial fibroblast-like cells in the Schwann cell differentiation system.

The endoplasmic reticulum (ER)-stress-induced cascade events are implicated in Parkinson’s disease (PD). The discovery of drug candidates to protect dopaminergic (DA) neurons from ER-stress-induced oxidative damage is important to resolve the pathological aspects of PD and modify its progress. In this study, we found that a recently identified unfolded protein response (UPR) modulator, azoramide, showed protective effects on patient induced pluripotent stem cells-derived midbrain DA neurons with the homozygous phospholipase A2 group 6 (PLA2G6) D331Y mutant. A series of PD-related cascade events such as ER stress, abnormal calcium homeostasis, mitochondrial dysfunction, increase of reactive oxygen species, and apoptosis were observed in PLA2G6 D331Y mutant DA neurons, whereas azoramide significantly protected PLA2G6 D331Y mutant DA neurons against these events. The beneficial effects of azoramide were abolished by treatment with a cAMP-response element binding protein (CREB) inhibitor. Our results suggest that azoramide is a potential neuroprotectant against DA neuron damage via restoring ER function and the CREB signaling.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Over 90% of cases are sporadic (sALS) and 5%-10% are familial (fALS). So far, more than 20 genes/loci have been linked to ALS. C9orf72, SOD1, TARDBP, and FUS are noted as the most common ALS genes; however, mutations of these genes explain10% of sALS cases. Recently, Rho guanine nucleotide exchange factor, encoded by ARHGEF28, has been linked to the ALS pathogenesis, possibly by binding low-molecular-weight neurofilament mRNA and affects its stability. However, a systemic screening of ARHGEF28 mutations in ALS is lacking. In this study, we sequenced the entire coding sequence of ARHGEF28 in a Chinese cohort of 399 sporadic ALS and 327 elderly controls. A total of 73 coding variants were identified, including 26 synonymous and 47 nonsynonymous. Among the nonsynonymous variants, 33 were rare (minor allele frequency [MAF]0.01), in which 18 were only identified in cases and 12 were only in controls. Three loss-of-function mutations were identified, including 2 truncations (p.Arg231Ter and p.Ser561Ter) and a frameshift deletion (p.Lys1070fs) in 2 cases and 1 control subject. The frequency of total and case-only rare variants was 7.5% (30/399) and 5.0% (20/399), respectively, in the patients. SKAT-O test suggested that the novel coding variants were marginally enriched in the cases (p = 0.049). Single-variant analysis suggested that the p.Asn1046Ser variant had a higher frequency in cases (8/399, 0.02) than in controls (1/327, 0.003) (OR: 6.67, 95% CI: 0.83-53.61; p = 0.046). By contrast, none of the low-frequency (MAF: 0.01-0.05) or common (MAF0.05) variants was associated with ALS (p0.05). Among all patients, 9 (2.3%) carried rare variants predicted to be deleterious, and the age at onset of these carriers (45.6 ± 10.9 years) was marginally younger than noncarriers (51.9 ± 10.7 years) (p = 0.11). Our results supported a possible genetic contribution of rare but not low-frequency and common coding variants to ALS. These data may have implications in the mechanisms and genetic counseling of the disease.

As40-induced vascular dysfunction has been implicated in the pathogenesis of Alzheimer׳s disease (AD). In the present study, we investigated the possible protective effects of puerarin against As40-induced vascular damage and impairment to angiogenesis in transgenic TG (fli1:EGFP) zebrafish and human endothelial cells. As40 peptides at 5 μM caused an obvious reduction of vessel branches in the subintestinal vein basket, induced NADPH oxidase-derived reactive oxygen species and impaired vascular endothelial growth factor (VEGF)-dependent angiogenesis. Pretreatment with puerarin attenuated Aβ40-induced vessel reduction and impairment to angiogenesis in a dose-dependent manner. In addition, As40 decreased VEGF-dependent phosphorylation of Akt and eNOS, whereas puerarin treatment attenuated these detrimental effects. Furthermore, the restoration of As40-induced-angiogenesis impairment by puerarin was abolished by either the PI3 kinase inhibitor LY294002 (10 μM) or eNOS inhibitor L-NAME. The present study suggests that puerarin exerts its protective action probably through reduction of NADPH oxidase-derived reactive oxygen species overproduction and activation of the PI3K/Akt/eNOS pathways.

Angiogenesis is the formation of blood vessels from pre-existing vasculature. Excessive or uncontrolled angiogenesis is a major contributor to many pathological conditions whereas inhibition of aberrant angiogenesis is beneficial to patients with pathological angiogenesis. Catunaregin is a core of novel marine compound isolated from mangrove associate. The potential anti-angiogenesis of catunaregin was investigated in human umbilical vein endothelial cells (HUVECs) and zebrafish. HUVECs were treated with different concentrations of catunaregin in the presence or absence of VEGF. The angiogenic phenotypes including cell invasion cell migration and tube formation were evaluated following catunaregin treatment in HUVECs. The possible involvement of AKT, eNOS and ERK1/2 in catunaregin-induced anti-angiogenesis was explored using Western blotting. The anti-angiogenesis of catunaregin was further tested in the zebrafish embryo neovascularization and caudal fin regeneration assays. We found that catunaregin dose-dependently inhibited angiogenesis in both HUVECs and zebrafish embryo neovascularization and zebrafish caudal fin regeneration assays. In addition, catunaregin significantly decreased the phosphorylation of Akt and eNOS, but not the phosphorylation of ERK1/2. The present work demonstrates that catunaregin exerts the anti-angiogenic activity at least in part through the regulation of the Akt and eNOS signaling pathways.

Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A isolated from mangrove fungus Xylaria sp. (no. 2508). We found that X-13 dose-dependently induced angiogenesis in zebrafish embryos and in human endothelial cells, which was accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of PI3K/Akt/eNOS by LY294002 or l-NAME suppressed X-13-induced angiogenesis. The present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.

We have previously demonstrated that dl-3n-butylphthalide (NBP) has a potential angiogenic activity. In this study, we investigated the angiogenic effect of NBP and the molecular mechanisms underlying NBP-mediated angiogenesis. Zebrafish embryos and human umbilical vein endothelial cells were treated with various doses of NBP and several signaling pathway inhibitors. NBP induced ectopic subintestinal vessel production in zebrafish embryos and induced invasion, migration, and endothelial cell tube formation of human umbilical vein endothelial cells in a dose-dependent manner. These NBP-induced angiogenic effects were partially suppressed by SU5402, a fibroblast growth factor receptor 1 inhibitor; U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor; LY294002, a phosphatidylinositol 3-kinase inhibitor; 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an Akt inhibitor; cavtratin, an endothelial nitric oxide synthase (eNOS) inhibitor and completely inhibited by a combination of U0126 and LY294002. NBP enhanced phosphorylation of ERK1/2 and fibroblast growth factor receptor 2 expression, which were inhibited by U0126. NBP increased the phosphorylation of Akt and eNOS at serine 1177, which was blocked by LY294002. NBP-stimulated nitric oxide production, which was reduced by LY294002. Our data demonstrated that (1) NBP promoted angiogenesis and (2) the angiogenic effects of NBP were mediated by the ERK1/2 and phosphatidylinositol 3-kinase/Akt-eNOS signaling pathways. Our findings suggest that NBP could be a novel agent for therapeutic angiogenesis in ischemic diseases.

The four single nucleotide polymorphisms (SNPs) rs34517613, rs3849943, rs8141797 and rs6703183, each located in a different gene, have recently been associated with risk of developing sporadic amyotrophic lateral sclerosis (SALS) in European and northern Chinese populations, but no data are yet available for other ethnic groups. Here we explored the possible association between these four SNPs and SALS in Han Chinese primarily from southern and south-western China. A total of 298 individuals with SALS from three centres in mainland China and 486 unrelated healthy controls were recruited. All subjects were successfully genotyped using the ligase detection reaction (LDR). We found no evidence that any of these SNPs are associated with risk of disease in either heterozygous or homozygous individuals (p > 0.05). Subgroup analysis based on gender showed a similar lack of association. However, subgroup analysis based on spinal- or bulbar-onset SALS revealed significant differences in the genotype distributions (p = 0.009) and minor allele frequencies of rs6703183 (p = 0.004). In conclusion, it may be premature to conclude associations between these four SNPs and SALS, which should be examined in additional ethnic groups.

High levels of cell surface glucose regulated protein 78 (sGRP78) have been implicated in cancer growth, survival, metastasis, and chemotherapy resistance. However, the underlying mechanism remains largely unknown. Here we report that the level of sGRP78 expression in human breast tumors gradually increases during cancer progression. Overexpression of GRP78 significantly enhanced its membrane distribution in human MCF-7 breast cancer cells, but had no effect on endoplasmic reticulum (ER) stress. High levels of sGRP78 facilitated cell proliferation and migration, as well as suppressed cell apoptosis. Neutralization of sGRP78 by a specific antibody against GRP78 alleviated sGRP78-induced cell growth and migration. Importantly, high phosphorylation levels of the signal transducer and activator of transcription 3 (STAT3) were found in human breast tumors that express sGRP78 and MCF-7 cells infected with adenovirus encoding human GRP78. Pretreatment with a GRP78 antibody suppressed STAT3 phosphorylation. Furthermore, genetic and pharmacological inhibition of STAT3 reversed the impacts of GRP78 on cell proliferation, apoptosis, and migration. These findings indicate that STAT3 mediates sGRP78-promoted breast cancer cell growth and migration.

Background and purpose: The H63D polymorphism in the hemochromatosis (HFE) gene has been reported as a risk factor for amyotrophic lateral sclerosis (ALS) in Europe and America, but no data have been reported for Asia. Here, we investigated the possible association between H63D and sporadic ALS (sALS) in a Chinese Han population. Methods: A total of 195 individuals with sALS from three centers in China and 405 unrelated healthy controls were recruited. All subjects were genotyped by restriction fragment length polymorphism (RFLP) analysis. Results: Sporadic ALS was significantly related to the H63D polymorphism in heterozygous carriers (odds ratio 3.10, 95%CI: 1.49–6.47, P = 0.002). Conclusions: The HFE H63D polymorphism may contribute to the development of sALS in Chinese.

Mutations in the TARDBP gene, which encodes the Tar DNA binding protein, have been shown to causes of both familial amyotrophic lateral sclerosis (FALS) and sporadic ALS (SALS). Recently, several novel TARDBP exon 6 mutants have been reported in patients with ALS in Europe and America but not in Asia. To further examine the spectrum and frequency of TARDBP exon 6 mutations, we investigated their frequency in ethnic Chinese patients with sporadic ALS. TARDBP exon 6 was screened by direct sequencing in 207 non-SOD1 SALS patients and 230 unrelated healthy controls but no mutations were identified. Our data indicate that exon 6 mutations in TARDBP are not a common cause of SALS in Han Chinese population from Southern Mainland China.

This study was undertaken to observe the effects and possible mechanism of CC chemokine ligand 5 (CCL5) on invasion, proliferation and percentage of CD44+/CD24- subpopulation of human breast cancer line MCF-7 and to investigate the correlation of expression levels of CCL5 and its receptors with the progression of breast cancer. We used real-time RT-PCR to detect the expression levels of CCL5 and its receptors CCR5, CCR1 and CCR3 in 36 breast cancer specimens of different TNM stage and their corresponding normal breast tissue. CCL5 expression and invasive ability of four human breast cancer cell lines MCF-7, SK-BR-3, T-47D and MDA-MB-231 were analyzed by real-time RT-PCR and cell invasion assay, respectively. Effects of recombinant human CCL5 (rhCCL5) on cell proliferation and percentage of the CD44+/CD24- subpopulation in MCF-7 cells were analyzed respectively by MTT assay and flow cytometry. We also used cell invasion assay to detect the invasive ability of both CD44+/CD24- and CD44+/CD24+ subpopulations of MCF-7 cells treated with rhCCL5 and/or CCR5 monoclonal antibody. Our results revealed that CCL5 and CCR5 expression were higher in breast cancer tissue than those in their corresponding normal tissue and breast cancer tissue with higher TNM stage contained more CCL5 mRNA. In addition, CCR5 expression and invasive ability of CD44+/CD24- subpopulation were higher than those of CD44+/CD24+ subpopulation of MCF-7 cells. Moreover, treatment of rhCCL5 increased the proportion of CD44+/CD24- cells and the proliferation of MCF-7 cells. Induction of rhCCL5 increased the cell invasive ability of both CD44+/CD24- and CD44+/CD24+ cells, which could be partially antagonized by CCR5 monoclonal antibody. Collectively, our data show that CCL5 increased the proportion of CD44+/CD24- subpopulation and induced invasion and proliferation of MCF-7 cells, and expression of CCL5 and CCR5 in breast cancer tissue was positively correlated with breast cancer progression.

To study the genes change of hMSC before and after differentiation into neuron-like cells.hMSC were separated from marrow, cultured and expanded in culture medium. After hMSC being induced to differentiate with Shenqiye, Neuron-specific enolase (MSE), neurofilament (NF), and glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry respectively. Using semi-quantitiative RT-PCR to detect ten genes change in hMSC before and after differentiation. Two genes were used to detect the gene sequence.When treated with the inducer--Shenqiye, hMSC exhibited neuronal phenotype. The expressions of NSE and NF in the neuron-like cells were positive, but the glial astrocyte marker GFAP was not found. hMSC expressed germline, ectodermal, endodermal and mesodermal genes prior to neurogenasis. After differentiation, germline genes did not express, while the expressions of endodermal and mesodermal genes were weakened. Ectodermal and neuronal genes expressed stronger.hMSC express multipotentiality, and the neural differentiation comprises quantitative modulation of gene expression rather than simple on-off switching of neural specific genes.

Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) share several clinical and neuropathologic features, and studies suggest that several gene mutations and polymorphisms are involved in both conditions. Matrix metalloproteinase-9 (MMP-9) is implicated in the pathogenesis of PD and ALS, and the C(−1562)T polymorphism in the MMP-9 gene leads to higher promoter activity. We therefore investigated whether this polymorphism predisposes to both PD and sporadic ALS (sALS). Samples from 351 subjects with PD and 351 healthy controls from two major cities in China were compared, while samples from 226 subjects with sALS were compared to the same number of controls from three centers in China. A possible association between the C(−1562)T polymorphism in the MMP-9 gene and PD or sALS was assessed by restriction fragment length polymorphism (RFLP) analysis. Our results show a significant association between the C(−1562)T polymorphism in the MMP-9 gene and risk of PD (odds ratio = 2.268, 95% CI 1.506–3.416, p

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder mainly affecting motor neurons. Mutations in superoxide dismutase-1 (SOD-1) account for about 20% of familial ALS patients. A robust supply of motoneurons carrying the mutated gene would help understand the causes of motoneuron death and develop new therapeutics for the disease. Here, we established induced pluripotent stem (iPS) cell lines from SOD1G93A mice and compared their potency in motoneuron generation with normal iPS cells and mouse embryonic stem cells (E14). Our results showed that iPS cells derived from SOD1G93A mice possessed the similar potency in neuronal differentiation to normal iPS cells and E14 cells and can be efficiently driven to motoneuron-like phenotype. These cells exhibited typical neuronal morphology, expressed key motoneuron markers, including ChAT and HB9, and generated repetitive trains of action potentials. Furthermore, these neurons highly expressed human SOD-1 and exhibited shorter neurites compared to controls. The present study provides evidence that ALS-iPS cells can be used as disease models in high-throughput screening and mechanistic studies due to their ability to efficiently differentiate into specific neuronal subtypes.

Induced pluripotent stem cells (iPSCs) hold promise for the treatment of motoneuron diseases because of their distinct features including pluripotency, self-derivation and potential ability to differentiate into motoneurons. However, it is still unknown whether human iPSC-derived motoneurons can functionally innervate target muscles in vivo, which is the definitive sign of successful cell therapy for motoneuron diseases. In the present study, we demonstrated that human iPSCs derived from mesenchymal cells of the umbilical cord possessed a high yield in neural differentiation. Using a chemically-defined in vitro system, human iPSCs efficiently differentiated into motoneurons which displayed typical morphology, expressed specific molecules, and generated repetitive trains of action potentials. When transplanted into the injured musculocutaneous nerve of rats, they survived robustly, extended axons along the nerve, and formed functional connections with the target muscle (biceps brachii), thereby protecting the muscle from atrophy. Our study provides evidence for the first time that human iPSC-derived motoneurons are truly functional not only in vitro but also in vivo, and they have potential for stem cell-based therapies for motoneuron diseases.

Background Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting 2% of the population aged over 65 years old. Mitochondrial defects and oxidative stress actively participate in degeneration of dopaminergic (DA) neurons in PD. Paeonolum, a main component isolated from Moutan cortex, has potent antioxidant ability. Here, we have examined the effects of paeonolum against MPP+-induced neurotoxicity in zebrafish and PC12 cells. Methods The overall viability and neurodegeneration of DA neurons was assessed in ETvmat2:green fluorescent protein (GFP) transgenic zebrafish, in which most monoaminergic neurons are labeled by GFP. Damage to PC12 cells was measured using a cell viability assay and assessment of nuclear morphology. Intracellular reactive oxygen species (ROS) and the level of total GSH were assessed. The mitochondrial cell death pathway including mitochondrial membrane potential, cytochrome C release and caspase-3 activity were also examined in PC12 cells. Results Paeonolum protected against MPP+-induced DA neurodegeneration and locomotor dysfunction in zebrafish in a concentration-dependent manner. Similar neuroprotection was replicated in the PC12 cellular model of MPP+ toxicity. Paeonolum attenuated MPP+-induced intracellular ROS accumulation and restored the level of total GSH in PC12 cells. Furthermore, paeonolum significantly inhibited the mitochondrial cell death pathway induced by MPP+. Conclusions Collectively, the present study demonstrates that paeonolum protects zebrafish and PC12 cells against MPP+-induced neurotoxicity.

Purpose High glucose level in diabetic patients may lead to advanced glycation end product (AGE) modified proteins. This study investigated AGE modified proteins in tears and compared their levels in diabetic patients (DM) with non-diabetic controls (CTL). Methods Basal tears were collected from DM with (DR) or without (DNR) retinopathy and CTL. Total AGE modified proteins were detected quantitatively by a dot immunobinding assay. The AGE modified proteins were separated in 1D- and 2D-SDS gels and detected by western-blotting. The individual AGE modified proteins were also compared between groups using densitometry. Results Compared with the CTL group, tear concentrations of AGE modified proteins were significantly elevated in DR and DNR groups. The concentration of AGE modified proteins in diabetic tears were positively correlated with AGE modified hemoglobin (HbA1c) and postprandial blood glucose level (PBG). Western blotting of AGE modified proteins from 1D-SDS gels showed several bands, the major one at around 60 kDa. The intensities of AGE modified protein bands were higher in DM tears than in CTL tears. Western blotting from 2D-SDS gels showed a strongly stained horizontal strip, which corresponded to the major band in 1D-SDS gels. Most of the other AGE modified protein species were within molecular weight of 30–60 kDa, PI 5.2–7.0. Densitometry analysis demonstrated several AGE modified proteins were elevated in DR or DNR tears. Conclusions Total and some individual AGE modified proteins were elevated in DM tears. AGE modified proteins in tears may be used as biomarkers to diagnose diabetes and/or diabetic retinopathy.