Exploring the conformational dynamics and thermodynamics of EGFR S768I and G719X + S768I mutations in non-small cell lung cancer: An in silico approaches.

Non-small cell lung cancer (NSCLC) is often driven by mutations in the epidermal growth factor receptor (EGFR) gene. However, rare mutations such as G719X and S768I lack standard anti-EGFR targeted therapies. Understanding the structural differences between wild-type EGFR and these rare mutants is crucial for developing EGFR-targeted drugs. We performed a systematic analysis using molecular dynamics simulations, essential dynamics (ED), molecular mechanics Poisson-Boltzmann surface area, and free energy calculation methods to compare the kinetic properties, molecular motion, and free energy distribution between wild-type EGFR and the rare mutants' structures G719X-EGFR, S768I-EGFR, and G719X + S768I-EGFR. Our results showed that S768I-EGFR and G719X + S768I-EGFR have higher global and local conformational flexibility and lower thermal and global structural stability than WT-EGFR. ED analysis revealed different molecular motion patterns between S768I-EGFR, G719X + S768I-EGFR, and WT-EGFR. The A-loop and αC-helix, crucial structural elements related to the active state, showed a tendency toward active state development, providing a molecular mechanism explanation for NSCLC caused by EGFR S768I and EGFR G719C + S768I mutations. The present study may be helpful in the development of new EGFR-targeted drugs based on the structure of rare mutations. Our findings may aid in developing new targeted treatments for patients with EGFR S768I and EGFR G719X + S768I mutations.

Genetic and Molecular Evidence of a Tetrapolar Mating System in the Edible Mushroom Grifola frondosa.

Grifola frondosa is a valuable edible fungus with high nutritional and medicinal values. The mating systems of fungi not only offer practical strategies for breeding, but also have far-reaching effects on genetic variability. Grifola frondosa has been considered as a sexual species with a tetrapolar mating system based on little experimental data. In the present study, one group of test crosses and six groups of three-round mating experiments from two parental strains were conducted to determine the mating system in G. frondosa. A chi-squared test of the results of the test-cross mating experiments indicated that they satisfied Mendelian segregation, while a series of three-round mating experiments showed that Mendelian segregation was not satisfied, implying a segregation distortion phenomenon in G. frondosa. A genomic map of the G. frondosa strain, y59, grown from an LMCZ basidiospore, with 40.54 Mb and 12 chromosomes, was generated using genome, transcriptome and Hi-C sequencing technology. Based on the genomic annotation of G. frondosa, the mating-type loci A and B were located on chromosomes 1 and 11, respectively. The mating-type locus A coded for the ß-fg protein, HD1, HD2 and MIP, in that order. The mating-type locus B consisted of six pheromone receptors (PRs) and five pheromone precursors (PPs) in a crossed order. Moreover, both HD and PR loci may have only one sublocus that determines the mating type in G. frondosa. The nonsynonymous SNP and indel mutations between the A1B1 and A2B2 mating-type strains and the reference genome of y59 only occurred on genes HD2 and PR1/2, preliminarily confirming that the mating type of the y59 strain was A1B2 and not A1B1. Based on the genetic evidence and the more reliable molecular evidence, the results reveal that the mating system of G. frondosa is tetrapolar. This study has important implications for the genetics and hybrid breeding of G. frondosa.

Heat-induced antigen retrieval utilizing modified Tris-EDTA buffer for reprobing of Western blots on nitrocellulose paper.

The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10-25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.

Expression of expanded GGC repeats within NOTCH2NLC causes behavioral deficits and neurodegeneration in a mouse model of neuronal intranuclear inclusion disease.

GGC repeat expansions within NOTCH2NLC have been identified as the genetic cause of neuronal intranuclear inclusion disease (NIID). To understand the molecular pathogenesis of NIID, here, we established both a transgenic mouse model and a human neural progenitor cells (hNPCs) model. Expression of the NOTCH2NLC with expanded GGC repeats produced widespread intranuclear and perinuclear polyglycine (polyG), polyalanine (polyA), and polyarginine (polyR) inclusions, leading to behavioral deficits and severe neurodegeneration, which faithfully mimicked the clinical and pathological features associated with NIID. Furthermore, conserved alternative splicing events were identified between the NIID mouse and hNPC models, among which was the enrichment of the binding motifs of hnRNPM, an RNA binding protein known as alternative splicing regulator. Expanded NOTCH2NLC-polyG and NOTCH2NLC-polyA could interact with and sequester hnRNPM, while overexpression of hnRNPM could ameliorate the cellular toxicity. These results together suggested that dysfunction of hnRNPM could play an important role in the molecular pathogenesis of NIID.

Sensitivity of Sniffer Dogs for a Diagnosis of Parkinson's Disease: A Diagnostic Accuracy Study.

BACKGROUND: The diagnostic criteria for Parkinson's disease (PD) remain complex, which is especially problematic for nonmovement disorder experts. A test is required to establish a diagnosis of PD with improved accuracy and reproducibility. OBJECTIVE: The study aimed to investigate the sensitivity and specificity of tests using sniffer dogs to diagnose PD. METHODS: A prospective, diagnostic case-control study was conducted in four tertiary medical centers in China to evaluate the accuracy of sniffer dogs to distinguish between 109 clinically established medicated patients with PD, 654 subjects without PD, 37 drug-naïve patients with PD, and 185 non-PD controls. The primary outcomes were sensitivity and specificity of sniffer dog's identification. RESULTS: In the study with patients who were medicated, when two or all three sniffer dogs yielded positive detection results in a sample tested, the index test sensitivity, specificity, and positive and negative likelihood ratios were 91% (95% CI: 84%-96%), 95% (95% CI: 93%-97%), and 19.16 (95% CI: 13.52-27.16) and 0.10 (95% CI: 0.05-0.17), respectively. The corresponding sensitivity, specificity, and positive and negative likelihood ratios in patients who were drug-naïve were 89% (95% CI: 75%-96%), 86% (95% CI: 81%-91%), and 6.6 (95% CI: 4.51-9.66) and 0.13 (95% CI: 0.05-0.32), respectively. CONCLUSIONS: Tests using sniffer dogs may be a useful, noninvasive, fast, and cost-effective method to identify patients with PD in community screening and health prevention checkups as well as in neurological practice. © 2022 International Parkinson and Movement Disorder Society.

Associations of multiple visual rating scales based on structural magnetic resonance imaging with disease severity and cerebrospinal fluid biomarkers in patients with Alzheimer's disease.

The relationships between multiple visual rating scales based on structural magnetic resonance imaging (sMRI) with disease severity and cerebrospinal fluid (CSF) biomarkers in patients with Alzheimer's disease (AD) were ambiguous. In this study, a total of 438 patients with clinically diagnosed AD were recruited. All participants underwent brain sMRI scan, and medial temporal lobe atrophy (MTA), posterior atrophy (PA), global cerebral atrophy-frontal sub-scale (GCA-F), and Fazekas rating scores were visually evaluated. Meanwhile, disease severity was assessed by neuropsychological tests such as the Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and Clinical Dementia Rating (CDR). Among them, 95 patients were tested for CSF core biomarkers, including Aß1-42, Aß1-40, Aß1-42/Aß1-40, p-tau, and t-tau. As a result, the GCA-F and Fazekas scales showed positively significant correlations with onset age (r = 0.181, p < 0.001; r = 0.411, p < 0.001, respectively). Patients with late-onset AD (LOAD) showed higher GCA-F and Fazekas scores (p < 0.001, p < 0.001). With regard to the disease duration, the MTA and GCA-F were positively correlated (r = 0.137, p < 0.05; r = 0.106, p < 0.05, respectively). In terms of disease severity, a positively significant association emerged between disease severity and the MTA, PA GCA-F, and Fazekas scores (p < 0.001, p < 0.001, p < 0.001, p < 0.05, respectively). Moreover, after adjusting for age, gender, and APOE alleles, the MTA scale contributed to moderate to severe AD in statistical significance independently by multivariate logistic regression analysis (p < 0.05). The model combining visual rating scales, age, gender, and APOE alleles showed the best performance for the prediction of moderate to severe AD significantly (AUC = 0.712, sensitivity = 51.5%, specificity = 84.6%). In addition, we observed that the MTA and Fazekas scores were associated with a lower concentration of Aß1-42 (p < 0.031, p < 0.022, respectively). In summary, we systematically analyzed the benefits of multiple visual rating scales in predicting the clinical status of AD. The visual rating scales combined with age, gender, and APOE alleles showed best performance in predicting the severity of AD. MRI biomarkers in combination with CSF biomarkers can be used in clinical practice.

Unique characteristics of G719X and S768I compound double mutations of epidermal growth factor receptor (EGFR) gene in lung cancer of coal-producing areas of East Yunnan in Southwestern China.

BACKGROUND: The principal objective of this project was to investigate the Epidermal Growth Factor Receptor (EGFR) gene mutation characteristics of lung cancer patients, which can provide a molecular basis for explaining the clinicopathological features, epidemiology and use of targeted therapy in lung cancer patients in the coal-producing areas of East Yunnan. METHODOLOGY: We collected 864 pathologically confirmed lung cancer patients' specimens in First People's Hospital of Qujing City of Yunnan Province from September 2016 to September 2021. We thereafter employed Next Generation Sequencing (NGS) technology to detect all exons present in the EGFR gene. RESULTS: The overall mutation frequency of the EGFR gene was 47.22%. The frequency of EGFR gene mutations in the tissue, plasma, and cytology samples were found to be 53.40%, 23.33%, and 62.50%, respectively. Univariate analysis indicated that the coal-producing areas and Fuyuan county origin were significantly associated with relatively low EGFR gene mutation frequency. Female, non-smoking history, adenocarcinoma, non-brain metastasis, and tissue specimens were found to be related to high EGFR gene mutation frequency. Multivariate logistic regression analysis suggested the lung cancer patients in the central area of Qujing City, stage Ia, non-coal-producing areas, non-Fuyuan origin, and non-Xuanwei origin were more likely to develop EGFR gene mutations. The most common mutations were L858R point mutation (33.09%) and exon 19 deletion (19-del) (21.32%). Interestingly, the mutation frequency of G719X (p = 0.001) and G719X + S768I (p = 0.000) in the coal-producing areas were noted to be more significant than those in non-coal-producing regions. CONCLUSION: This findings of this study might be important in establishing the correlation between routine using NGS for EGFR gene mutation diagnosis and clinical practice in the lung cancer patients.

Genetic Polymorphisms of Pneumocystis jirovecii in HIV-Positive and HIV-Negative Patients in Northern China.

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and ß-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China.

Mutations in GBA, SNCA, and VPS35 are not associated with Alzheimer's disease in a Chinese population: a case-control study.

SNCA, GBA, and VPS35 are three common genes associated with Parkinson's disease. Previous studies have shown that these three genes may be associated with Alzheimer's disease (AD). However, it is unclear whether these genes increase the risk of AD in Chinese populations. In this study, we used a targeted gene sequencing panel to screen all the exon regions and the nearby sequences of GBA, SNCA, and VPS35 in a cohort including 721 AD patients and 365 healthy controls from China. The results revealed that neither common variants nor rare variants of these three genes were associated with AD in a Chinese population. These findings suggest that the mutations in GBA, SNCA, and VPS35 are not likely to play an important role in the genetic susceptibility to AD in Chinese populations. The study was approved by the Ethics Committee of Xiangya Hospital, Central South University, China on March 9, 2016 (approval No. 201603198).

Total protein staining with Congo red as an alternative loading control for western blot analysis.

For western blot analysis, a housekeeping protein, such as ß-actin or glyceraldehyde-3-phosphate dehydrogenase, is used as loading control with the assumption that these proteins are stable. In practice, these internal loading control proteins vary with different cell states and tissue types. These internal standards are not appropriate for use with serum, extracellular secretion, cerebrospinal fluid analysis or for protein purification. We investigated total protein measurement using Congo red staining and found it to be a superior alternative to routine loading controls. Advantages include lower cost, technical simplicity and improved linear regression. We propose using Congo red staining for total protein immunoblotting to evaluate protein loading in western blots.

Association Between Serum Vitamins and the Risk of Alzheimer's Disease in Chinese Population.

BACKGROUND: Alzheimer's disease (AD) is a chronic and fatal neurodegenerative disease; accumulating evidence suggests that vitamin deficiency is associated with the risk of AD. However, studies attempting to elucidate the relationship between vitamins and AD varied widely. OBJECTIVE: This study aimed to investigate the relationship between serum vitamin levels and AD in a cohort of the Chinese population. METHODS: A total of 368 AD patients and 574 healthy controls were recruited in this study; serum vitamin A, B1, B6, B9, B12, C, D, and E were measured in all participants. RESULTS: Compared with the controls, vitamin B2, B9, B12, D, and E were significantly reduced in AD patients. Lower levels of vitamin B2, B9, B12, D, and E were associated with the risk of AD. After adjusting for age and gender, low levels of vitamin B2, B9, and B12 were still related to the risk of AD. In addition, a negative correlation was determined between vitamin E concentration and Activity of Daily Living Scale score while no significant association was found between serum vitamins and age at onset, disease duration, Mini-Mental State Examination, and Neuropsychiatric Inventory Questionnaire score. CONCLUSION: We conclude that lower vitamin B2, B9, B12, D, and E might be associated with the risk of AD, especially vitamin B2, B9, and B12. And lower vitamin E might be related to severe ability impairment of daily activities.

Short-term high-fat diet favors the appearances of apoptosis and gliosis by activation of ERK1/2/p38MAPK pathways in brain.

High-fat diet (HFD) has been associated with neuroinflammation and apoptosis in distinct brain regions. To explore the effect of short-term (7, 14 and 21 days) high-fat overfeeding on apoptosis, inflammatory signaling proteins, APP changes and glial cell activities in cerebral cortex and cerebellum. Mice were fed with HFD for different lengths (up to 21 days) and after each time body weights of mice was tested, then the apoptotic proteins, IL-1ß, APP, BACE1and MAPKs, Akt and NF-κB signaling activity were evaluated by western blots. Results demonstrate that short period of high-fat overnutrition significantly promotes apoptosis, APP expression at day 21 of cerebral cortex and at day 7 of cerebellum compared to chow diet. In addition, increased GFAP+astrocytes, Iba-1+microglia and IL-1ß 30 were observed in cerebral cortex after 21 days HFD, but no changes for 7 days overfeeding of cerebellum. Serendipitously, ERK1/2 pathway was activated both in cerebral cortex and cerebellum for different time course of HFD. Furthermore, increased phospho-p38 MAPK level was observed in cerebellum only. In consistent with in vivo results, SH-SY5Y cells treatment with cholesterol (50 µM, 100 µM) for 48 h culture in vitro demonstrated that pro-apoptotic proteins were enhanced as well. In brief, short-term HFD consumption increases sensitivity to apoptosis, APP and IL-1ß production as well as gliosis in cerebral cortex and cerebellum, which may be related to enhancement of ERK1/2 and p38 MAPK activation.

Viral loads, lymphocyte subsets and cytokines in asymptomatic, mildly and critical symptomatic patients with SARS-CoV-2 infection: a retrospective study.

BACKGROUND: Tens of million cases of coronavirus disease-2019 (COVID-19) have occurred globally. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attacks the respiratory system, causing pneumonia and lymphopenia in infected individuals. The aim of the present study is to investigate the laboratory characteristics of the viral load, lymphocyte subset and cytokines in asymptomatic individuals with SARS-CoV-2 infection in comparison with those in symptomatic patients with COVID-19. METHODS: From January 24, 2020, to April 11, 2020, 48 consecutive subjects were enrolled in this study. Viral loads were detected by RT-PCR from throat-swab, sputum and feces samples. Lymphocyte subset levels of CD3 + , CD4 + , and CD8 + T lymphocytes, B cells and NK cells were determined with biological microscope and flow cytometric analysis. Plasma cytokines (IL2, IL4, IL5, IL6, IL8, IL10, TNF-α, IFN-α and IFN-γ) were detected using flow cytometer. Analysis of variance (ANOVA), Chi-square or Fisher's exact test and Pearson's Correlation assay was used for all data. RESULTS: Asymptomatic (AS), mild symptoms (MS) and severe or critical cases (SCS) with COVID-19 were 11 (11/48, 22.9%), 26 (54.2%, 26/48) and 11 cases (11/48, 22.9%), respectively. The mean age of AS group (47.3 years) was lower than SCS group (63.5 years) (P < 0.05). Diabetes mellitus in AS, MS and SCS patients with COVID-19 were 0, 6 and 5 cases, respectively, and there was a significant difference between AS and SCS (P < 0.05). No statistical differences were found in the viral loads of SARS-CoV-2 between AS, MS and SCS groups on admission to hospital and during hospitalization. The concentration of CD 3 + T cells (P < 0.05), CD3 + CD4 + T cells (P < 0.05), CD3 + CD8 + T cells (P < 0.01), and B cells (P < 0.05) in SCS patients was lower than in AS and MS patients, while the level of IL-5 (P < 0.05), IL-6 (P < 0.05), IL-8 (P < 0.01) and IL-10 (P < 0.01), and TNF-α (P < 0.05) was higher. The age was negatively correlated with CD3 + T cells (P < 0.05), CD3 + CD4 + T cells (P < 0.05), and positively correlated with IL-2 (P < 0.001), IL-5 (P < 0.05), IL-6 (P < 0.05) IL-8 (P < 0.05), and IL-10 (P < 0.05). The viral loads were positively correlated with IL-2 (P < 0.001), IL-5 (P < 0.05), IL-6 (P < 0.05) IL-8 (P < 0.05) and IL-10 (P < 0.05), while negatively correlated with CD 3 + T cells (P < 0.05) and CD3 + CD4 + T cells (P < 0.05). CONCLUSIONS: The viral loads are similar between asymptomatic, mild and severe or critical patients with COVID-19. The severity of COVID-19 may be related to underlying diseases such as diabetes mellitus. Lymphocyte subset and plasma cytokine levels may be as the markers to distinguish severely degrees of disease, and asymptomatic patients may be as an important source of infection for the COVID-19.

Nogo-A-Δ20/EphA4 interaction antagonizes apoptosis of neural stem cells by integrating p38 and JNK MAPK signaling.

Nogo-A protein consists of two main extracellular domains: Nogo-66 (rat amino acid [aa] 1019-1083) and Nogo-A-Δ20 (extracellular, active 180 amino acid Nogo-A region), which serve as strong inhibitors of axon regeneration in the adult CNS (Central Nervous System). Although receptors S1PR2 and HSPGs have been identified as Nogo-A-Δ20 binding proteins, it remains at present elusive whether other receptors directly interacting with Nogo-A-Δ20 exist, and decrease cell death. On the other hand, the key roles of EphA4 in the regulation of glioblastoma, axon regeneration and NSCs (Neural Stem Cells) proliferation or differentiation are well understood, but little is known the relationship between EphA4 and Nogo-A-Δ20 in NSCs apoptosis. Thus, we aim to determine whether Nogo-A-Δ20 can bind to EphA4 and affect survival of NSCs. Here, we discover that EphA4, belonging to a member of erythropoietin-producing hepatocellular (Eph) receptors family, could be acting as a high affinity ligand for Nogo-A-Δ20. Trans-membrane protein of EphA4 is needed for Nogo-A-Δ20-triggered inhibition of NSCs apoptosis, which are mediated by balancing p38 inactivation and JNK MAPK pathway activation. Finally, we predict at the atomic level that essential residues Lys-205, Ile-190, Pro-194 in Nogo-A-Δ20 and EphA4 residues Gln-390, Asn-425, Pro-426 might play critical roles in Nogo-A-Δ20/EphA4 binding via molecular docking.

Mutation analysis of CAPN1 in Chinese populations with spastic paraplegia and related neurodegenerative diseases.

BACKGROUND: Mutations in CAPN1 have recently been reported to cause the spastic paraplegia 76 (SPG76) subtype of hereditary spastic paraplegia (HSP). To investigate the role of CAPN1 in spastic paraplegia and other neurodegenerative diseases, including spinocerebellar ataxia (SCA), early-onset Parkinson's disease (EOPD), and amyotrophic lateral sclerosis (ALS) we conducted a mutation analysis of CAPN1 in a cohort of Chinese patients with SPG, SCA, EOPD, and ALS. METHODS: Variants of CAPN1 were detected in the three cohorts by Sanger or whole-exome sequencing, and all exons and exon-intron boundaries of CAPN1 were analysed. RESULTS: A novel CAPN1 splicing variant (NM_001198868: c.338-1G > A) identified in a familial SPG/SCA showed a complex phenotype, including spastic paraplegia, ataxia, and extensor plantar response. This mutation was confirmed by Sanger sequencing and completely co-segregated with the phenotypes. Sequencing of the cDNA from the three affected patients detected a guanine deletion (c.340_340delG) that was predicted to result in an early stop codon after 61 amino acids (p. D114Tfs*62). No CAPN1 pathogenic mutation was found in the EOPD or ALS groups. CONCLUSION: Our data reveal a novel CAPN1 mutation found in patients with SPG/SCA and emphasize the spastic and ataxic phenotypes of SPG76, but CAPN1 may not play a major role in EOPD and ALS.

A sensitive and reversible staining of proteins on blot membranes.

A modified, sensitive and reversible method for protein staining on nitrocellulose (NC) and polyvinylidine fluoride (PVDF) membranes was developed in Western blotting. The method employed Congo red staining to visualize proteins on different blot membranes. Staining of proteins with Congo red dye is more faster procedures. According to the experimental results, approximate 20 ng proteins could be detected in 3 min in room temperature. The staining on the proteins is easily reversible with Congo red destaining solution for NC and PVDF membranes, so that the blot membranes can be reused for Western blotting. In addition, we confirmed that the staining method is fully compatible with Western blot detection. NC and PVDF membranes treatment with Congo red staining does not interfere with conventional chemiluminescent substrates of peroxidase. As compared to MemCode reversible protein stain kits from Pirece Biotechnology, the staining technique is more sensitive, lower of cost, convenient and not adversely affecting subsequent Western blotting results. On the other hand, the stain is more sensitive than the Ponceau S staining. Therefore, Congo red staining is a promising and ideal alternative for current protein stain. Besides, the binding modes of Congo red or Ponceau S stain were investigated using various 2D and 3D molecular docking and demonstrated potential molecular basis for sensitivity of Congo red staining are higher than Ponceau S.

Astrocytes autophagy in aging and neurodegenerative disorders.

Astrocytes can serve multiple functions in maintaining cellular homeostasis of the central nervous system (CNS), and normal functions for autophagy in astrocytes is considered to have very vital roles in the pathogenesis of aging and neurodegenerative diseases. Autophagy is a major intracellular lysosomal (or its yeast analog, vacuolar) clearance pathways involved in the degradation and recycling of long-lived proteins, oxidatively damaged proteins and dysfunctional organelles by lysosomes. Current evidence has shown that autophagy might influence inflammation, oxidative stress, aging and function of astrocytes. Although the interrelation between autophagy and inflammation, oxidative stress, aging or neurological disorders have been addressed in detail, the influence of astrocytes mediated-autophagy in aging and neurodegenerative disorders has yet to be fully reviewed. In this review, we will summarize the most up-to-date findings and highlight the role of autophagy in astrocytes and link autophagy of astrocytes to aging and neurodegenerative diseases. Due to the prominent roles of astrocytic autophagy in age-related neurodegenerative diseases, we believe that we can provide new suggestions for the treatment of these disorders.

Integrated analysis highlights multiple long non­coding RNAs and their potential roles in the progression of human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a prevalent aggressive malignant tumor with poor prognosis. Investigations into the molecular changes that occur as a result of the disease, as well as identification of novel biomarkers for its diagnosis and prognosis, are urgently required. Long non­coding RNAs (lncRNAs) have been reported to play a critical role in tumor progression. The present study performed data mining analyses for ESCC via an integrated study of accumulated datasets and identification of the differentially expressed lncRNAs from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. The identified intersection of differentially expressed genes (lncRNAs, miRNAs and mRNAs) in ESCC tissues between the GEO and TCGA datasets was investigated. Based on these intersected lncRNAs, the present study constructed a competitive endogenous RNA (ceRNA) network of lncRNAs in ESCC. A total of 81 intersection lncRNAs were identified; 67 of these were included in the ceRNA network. Functional analyses revealed that these 67 key lncRNAs primarily dominated cellular biological processes. The present study then analyzed the associations between the expression levels of these 67 key lncRNAs and the clinicopathological characteristics of the ESCC patients, as well as their survival time using TCGA. The results revealed that 31 of these lncRNAs were associated with tumor grade, tumor­node­metastasis (TNM) stage and lymphatic metastasis status (P<0.05). In addition, 15 key lncRNAs were demonstrated to be associated with survival time (P<0.05). Finally, 5 key lncRNAs were selected for validation of their expression levels in 30 patients newly diagnosed with ESCC via reverse transcription­quantitative PCR (RT­qPCR). The results suggested that the fold changes in the trends of up­ and downregulation between GEO, TCGA and RT­qPCR were consistent. In addition, it was also demonstrated that a select few of these 5 key lncRNAs were significantly associated with TNM stage and lymph node metastasis (P<0.05). The results of the clinically relevant analysis and the aforementioned bioinformatics were similar, hence proving that the bioinformatics analysis used in the present study is credible. Overall, the results from the present study may provide further insight into the functional characteristics of lncRNAs in ESCC through bioinformatics integrative analysis of the GEO and TCGA datasets, and reveal potential diagnostic and prognostic biomarkers for ESCC.

Identification of microRNAs as novel biomarkers for esophageal squamous cell carcinoma: a study based on The Cancer Genome Atlas (TCGA) and bioinformatics.

BACKGROUND: MicroRNAs (miRNAs) have played important roles in the regulation of gene expression in many cancers, but their roles in esophageal squamous cell carcinoma (ESCC) are still unclear. The aim of this study was to determine the potential ESCC-specific key miRNAs from a large sample dataset in The Cancer Genome Atlas (TCGA). METHODS: Integrative bioinformatics analysis was used to identify key ESCC-specific miRNAs related to the ESCC patients' tumor histological grade and lymphatic metastasis from TCGA. Next, these key miRNA potential gene regulatory functions and relationships with ESCC patients' clinical characteristics and overall survival were analyzed. Finally, three key miRNAs were selected randomly and quantificational real-time polymerase chain reaction (qRT-PCR) was used to validate in 51 newly diagnosed ESCC patients' tissues samples (collected from Nov. 2017 to Feb. 2019, in Wuwei, China) whether the bioinformatics analyses results were reliable and valid. Two-tailed Student's t test, Pearson Chi-squared test and Kaplan-Meier survival analysis were used in this study. RESULTS: Thirty-five ESCC-specific miRNAs from TCGA database were investigated (fold-change > 2.0, P < 0.05), and 28 participated in the miRNAs-mRNAs co-expression network construction, while 17 were related with ESCC patients' tumor histological grade, TNM stage, and lymphatic metastasis (P < 0.05). Meanwhile, six miRNAs (including miR-200b-3p, miR-31-5p, miR-15b-5p, miR-141-3p, miR-135b-5p, and miR-195-5p) were correlated with overall survival of ESCC patients (log-rank, P < 0.05). MiR-135b-5p, miR-15b-5p, and miR-195-5p were selected for verification of the expression levels in 51 ESCC patients' tissue samples by using qRT-PCR. We found that the fold-changes between qRT-PCR and TCGA were completely consistent. The results also suggested that miR-135b-5p, miR-15b-5p, and miR-195-5p were significantly correlated with tumor differentiation degrees (P < 0.05), miR-195-5p was significantly correlated with tumor TNM stage (P < 0.05), and miR-135b-5p was significantly correlated with lymph-node metastasis (P < 0.05). MiR-135b-5p, miR-15b-5p, and miR-195-5p expression levels, ESCC patient clinical features association analysis results and the aforementioned TCGA bioinformatics analyses were similar. CONCLUSION: This study identified key ESCC-related miRNAs. The key miRNAs are worthy of further investigation as potential novel biomarkers for diagnosis, classification, and prognosis of ESCC.

Expansion of Human-Specific GGC Repeat in Neuronal Intranuclear Inclusion Disease-Related Disorders.

Neuronal intranuclear inclusion disease (NIID) is a slowly progressing neurodegenerative disease characterized by eosinophilic intranuclear inclusions in the nervous system and multiple visceral organs. The clinical manifestation of NIID varies widely, and both familial and sporadic cases have been reported. Here we have performed genetic linkage analysis and mapped the disease locus to 1p13.3-q23.1; however, whole-exome sequencing revealed no potential disease-causing mutations. We then performed long-read genome sequencing and identified a large GGC repeat expansion within human-specific NOTCH2NLC. Expanded GGC repeats as the cause of NIID was further confirmed in an additional three NIID-affected families as well as five sporadic NIID-affected case subjects. Moreover, given the clinical heterogeneity of NIID, we examined the size of the GGC repeat among 456 families with a variety of neurological conditions with the known pathogenic genes excluded. Surprisingly, GGC repeat expansion was observed in two Alzheimer disease (AD)-affected families and three parkinsonism-affected families, implicating that the GGC repeat expansions in NOTCH2NLC could also contribute to the pathogenesis of both AD and PD. Therefore, we suggest defining a term NIID-related disorders (NIIDRD), which will include NIID and other related neurodegenerative diseases caused by the expanded GGC repeat within human-specific NOTCH2NLC.