Melatonin alleviates chronic stress-induced hippocampal microglia pyroptosis and subsequent depression-like behaviors by inhibiting Cathepsin B/NLRP3 signaling pathway in rats.

Melatonin improves chronic stress-induced hippocampal damage and depression-like behaviors, but the mechanism needs further study. This study was to explore the mechanism of melatonin inhibiting microglia pyroptosis. In virtro experiments, melatonin improved corticosterone-induced the ultrastructure and microstructure damage of HAPI cells by inhibiting pyroptosis, thereby increasing cell survival rate. Protein-protein interaction network and molecular autodocking predicted that Cathespin B might be the target of melatonin inhibition of NLRP3-mediated pyroptosis. Melatonin inhibited corticosterone-induced Cathespin B expression. Both Cathepsin B inhibitor CA-074Me and NLRP3 knockout inhibited the HAPI cells pyroptosis. Similarly, melatonin inhibited Cathepsin B agonist Pazopanib-induced activation of Cathepsin B/NLRP3 signaling pathway and HAPI cells pyroptosis. In vivo studies, melatonin inhibited chronic restraint stress (CRS)-induced activation of Cathepsin B/NLRP3 signaling pathway and alleviated hippocampal microglia pyroptosis in rats. Inhibition of microglia pyroptosis improved CRS-induced depression-like behaviors of rats. In addition, inhibition of Cathepsin B and NLRP3 alleviated hippocampal pyroptosis. Melatonin inhibited Pazopanib-induced activation of Cathepsin B/NLRP3 signaling pathway and hippocampal pyroptosis. These results demonstrated that melatonin could alleviate CRS-induced hippocampal microglia pyroptosis by inhibiting Cathepsin B/NLRP3 signaling pathway, thereby improving depression-like behaviors in rats. This study reveals the molecular mechanism of melatonin in the prevention and treatment of chronic stress-related encephalopathy.

Identification of Key Genes for Pyroptosis-Induced Salivary Gland Inflammation in Sjogren's Syndrome Based on Microarray Data and Immunohistochemistry Analysis.

Purpose: Sjogren's Syndrome (SS) is a systemic autoimmune disease primarily characterized by dysfunction of the exocrine glands. Research into the etiology and pathogenesis of salivary glands (SG) inflammation of SS is very limited. The aim of this study was to identify potential pyroptosis-related genes in SG inflammation through bioinformatics analysis and validation of the SG in SS. Methods: GSE157159 dataset and GSE159574 dataset were downloaded from Gene Expression Omnibus (GEO). Differentially Expressed Genes (DEGs) analysis was used to screen DEGs from SS and non-SS SG samples. Pyroptosis-related genes were obtained from GeneCards. After intersecting DEGs with pyroptosis-related genes, the pyroptosis-related DEGs in SS were obtained. Subsequently, ClueGO enrichment analysis, Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analysis, Protein-protein Interaction (PPI), and identification and co-expression analysis of hub genes were performed. Subsequently, we collected SG samples from 17 SS patients and 17 non-SS patients and validated the expression of two hub genes (GZMA, GBP1) and characteristic genes (GSDMD) of pyroptosis through immunohistochemistry. The accuracy of hub genes as biomarkers for predicting SS was evaluated by receiver operating characteristic (ROC) curve. Results: 834 DEGs were selected from the GSE157159 dataset, and a total of 39 pyroptosis-related DEGs were obtained. Functional analysis showed that these DEGs were significantly enriched in some inflammatory signaling pathways. Through the intersection of seven algorithms proposed by CytoHubba and validation using the GSE159574 dataset, 11 hub genes were identified, including IL18, AIM2, CCL5, CD274, GBP1, GBP5, GZMA, GZMB, TLR8, TNFS13B, and ICAM1. Finally, the results of immunohistochemistry showed that GSDMD, GZMA and GBP1 were all significantly highly expressed in SG from SS. And ROC analysis showed a high combined diagnostic value of the 3 genes (AUC=0.8858). Conclusion: Our study revealed enhanced levels of pyroptosis in the SS. GZMA and GBP1 were identified as candidate genes for pyroptosis-induced inflammation of the SG in SS, which may be used as biomarkers or potential therapeutic targets for SS.

Serum Metabolomics Analysis of Skin-Involved Systemic Lupus Erythematosus: Association of Anti-SSA Antibodies with Photosensitivity.

Purpose: Systemic lupus erythematosus is a heterogeneous autoimmune disease in which skin involvement is a common manifestation. It is currently thought that the photosensitivity of SLE skin involvement is associated with anti-SSA antibodies. This study aimed to expand the current state of knowledge surrounding the molecular pathophysiology of SLE skin photosensitivity through Serum metabolomics analysis. Patients and Methods: The serum metabolites of 23 cases of skin-involved SLE (SI) group, 14 cases of no SI (NSI) group, and 30 cases of healthy controls (HC) were analyzed by using UPLC-MS/MS technology, and subgroup analysis was performed according to the expression of anti-SSA antibodies in SI. MetaboAnalyst 5.0 was used for enrichment analysis and ROC curve construction, identifying serum metabolic markers of skin-involved SLE associated with anti-SSA antibodies. Results: We identified several metabolites and metabolic pathways associated with SLE photosensitivity. Two metabolites, SM (d18:1/24:0) and gamma-CEHC can distinguish between anti-SSA antibody-positive and negative SI, with AUC of 0.829 and 0.806. These two photosensitization-related substances may be potential markers of skin involvement in SLE associated with anti-SSA antibody. Conclusion: This study provides new insights into the pathogenesis of SI patients, and provides a new molecular biological basis for the association between anti-SSA antibodies and skin photoallergic manifestations of SLE.

Genome-wide DNA methylation sequencing reveals epigenetic features and potential biomarkers of Sjögren syndrome.

AIM: Sjögren syndrome (SS) is a slowly progressive, inflammatory, autoimmune disease. The aim of this study was to construct the DNA methylation profiles of whole blood of SS patients and healthy controls (HC), and to explore the role of differentially methylated genes in the pathogenesis of the disease. METHODS: Whole-genome bisulfite sequencing was performed on three SS patients and four HC. The biological function of genes associated with differentially methylated regions (DMRs) was investigated using Gene Ontology functional analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis, using network-based key driver analysis (KDA) to find KDA genes. In clinical samples of SS patients and controls, the expression levels of KDA genes were validated by quantitative real-time polymerase chain reaction and immunohistochemical analysis. Moreover, the diagnostic value of KDA genes for SS was confirmed using receiver operating characteristic curves. RESULTS: We identified 322 DMRs, annotated as 162 associated genes. Six genes were selected via the number of networks of KDA genes. Differential expression of genes such as human leukocyte antigen (HLA) class I, ADAR, and OAS2 was observed in patients' peripheral blood mononuclear cells and the minor salivary glands, which can be used as potential diagnostic biomarkers for SS. CONCLUSION: Clinical sample validation suggested that HLA class I, ADAR, and OAS2 might play a role in the development of SS. Our study shows epigenetic regulatory mechanisms and potential disease markers associated with SS, which in turn will enable us to identify new therapeutic targets.

MS2 device: smartphone-facilitated mobile nucleic acid analysis on microfluidic device.

Mobile sensing based on the integration of microfluidic devices and smartphones, so-called MS2 technology, has enabled many applications over recent years and continues to stimulate growing interest in both research communities and industries. In particular, MS2 technology has been proven to be able to be applied to molecular diagnostic analysis and can be implemented for basic research and clinical testing. However, the currently reported MS2-based nucleic acid analysis system has limited use in practical applications, because it is not integrated with quantitative PCR, multiplex PCR, and isothermal amplification functions, and lacks temperature control, image acquisition and real-time processing units with excellent performance. To provide a more universal and powerful platform, we here developed a novel MS2 device by integrating a thermocycler, a multi fluorescence detection unit, a PCR chip, an isothermal chip, and a smartphone. The MS2 device was approximately 325 mm (L) × 200 mm (W) × 200 mm (H) in volume and only 5 kg in weight, and showed an average power consumption of about 38.4 W. The entire nucleic acid amplification and analysis could be controlled through a self-made smartphone App. The maximum heating and cooling rates were 5 °C s-1 and 4 °C s-1, respectively. The entire PCR could be completed within 65 min. The temperature uniformity was less than 0.1 °C. Besides, the temperature stability over time (30 min) was within ±0.04 °C. Four optical channels were integrated (FAM, HEX, TAMRA, and ROX) on the MS2 device. In particular, the PCR-based detection sensitivity reached 1 copy per µL, and the amplification efficiency was calculated to be 106.8%. Besides, the MS2 device also was compatible with multiplex PCR and isothermal amplification. In short, the MS2 device showed performance consistent with that of traditional commercial equipment. Thus, the MS2 device provides an easy and integrated experimental platform for molecular diagnostic-related research and potential medical diagnostic applications.

Identification of miR-4644 as a suitable endogenous normalizer for circulating miRNA quantification in hepatocellular carcinoma.

Background: Circulating microRNAs (miRNAs) have proved to be promising biomarkers for early diagnosis and therapeutic monitoring in cancers. Particularly for hepatocellular carcinoma (HCC), detection of circulating miRNA biomarkers as a new diagnostic approach has been written into the latest Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China (2019 edition). However, no general consensus on an ideal endogenous normalizer for circulating miRNAs quantification has been reached, so it will affect the accuracy of quantitative results. In this study, we aim to identify a stable endogenous normalizer for analyzing circulating miRNAs. Methods: Candidate miRNAs were selected by screening dataset GSE104310, as well as data statistics and analysis. Five commonly reference genes were chosen for further comparison and verification. Then, the expression levels of these genes in serum were analyzed by quantitative reverse transcription PCR (RT-qPCR) among four groups, including patients diagnosed with HCC, chronic hepatitis B (CHB), liver cirrhosis, and healthy subjects. Furthermore, the stability of target genes was evaluated using geNorm, NormFinder, comparative ΔCq programs, and validated by database. We also explored the availability of the miRNA combination, and compared the performance difference between combination and individuals, as well as the selectivity of miRNA references in the combinations. Results: 11 candidate miRNAs were obtained, and miR-4644 stood out among these miRNAs, and proved to be much more stable than other endogenous miRNAs. Further study showed that miR-4644 exhibited higher stability and expression abundance than other commonly miRNA reference controls. Finally, we discovered the combination of miR-4644 and miR-16 revealed high performance in stability when compared to miRNA individuals. Furthermore, the combination consisted of references with closer nature could give rise to amplification effects in stability. Conclusions: Our findings demonstrated that miR-4644 is an ideal endogenous normalizer for circulating microRNA quantification in hepatocellular carcinoma. Besides, combining miR-4644 with miR-16 into a whole as a reference control would greatly improve the accuracy of quantification.

A Novel Microfluidic Device Integrated with Chitosan-Modified Capillaries for Rapid ZIKV Detection.

The outbreak of Zika virus (ZIKV) has posed a great challenge to public health in recent years. To address the urgent need of ZIKV RNA assays, we integrate the microfluidic chip embedded with chitosan-modified silicon dioxide capillaries, smartphone-based detection unit to be a C3-system for the rapid extraction and detection of ZIKV RNA. The C3-system is characterized by: (1) four chitosan-modified silicon dioxide capillaries integrated in the microfluidic chip for target ZIKV RNA enrichment and "in situ PCR" (polymerase chain reaction) amplification; (2) smartphone-based point of care (POC) device consisting of a pneumatic subsystem for controlling the nucleic acid extraction processes in the microfluidic chip, a heating subsystem for sample lysis and PCR amplification, and an optical subsystem for signal acquisition. The entire detection processes including sample lysis, ZIKV RNA enrichment, and reverse-transcription polymerase chain reaction (RT-PCR) is achieved in the microfluidic chip. Moreover, PCR buffers can be directly loaded into the chitosan-modified silicon dioxide capillaries for "in situ PCR", in which the captured ZIKV RNA is directly used for downstream PCR without any loss. ZIKV RNA extracted by the C3-system can be successfully recovered at very low concentrations of 50 transducing units (TU)/mL from crude human saliva. This means that our method of detecting viremia in patients infected with ZIKV is reliable.

The Functional Impact of Alternative Splicing on the Survival Prognosis of Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is a type of breast cancer (BC) showing a high recurrence ratio and a low survival probability, which requires novel actionable molecular targets. The involvement of alternative splicing (AS) in TNBC promoted us to study the potential roles of AS events in the survival prognosis of TNBC patients. METHODS: A total of 150 TNBC patients from The Cancer Genome Atlas (TCGA) were involved in this work. To study the effects of AS in the recurrence-free survival (RFS) prognosis of TNBC, we performed the analyses as follows. First, univariate Cox regression model was applied to identify RFS-related AS events. Their host genes were analyzed by Metascape to discover the potential functions and involved pathways. Next, least absolute shrinkage and selection operator (LASSO) method was used to select the most informative RFS-related AS events to constitute an AS risk factor for RFS prognosis, which was evaluated by Kaplan-Meier (KM) and receiver operating characteristic (ROC) curves in all the data and also in different clinical subgroups. Furthermore, we analyzed the relationships between splicing factors (SFs) and these RFS-related AS events to seek the possibility that SFs regulated AS events to influence RFS. Then, we evaluated the potential of these RFS-related AS events in the overall survival (OS) prognosis from all the above aspects. RESULTS: We identified a total of 546 RFS-related AS events, which were enriched in some splicing and TNBC-associated pathways. Among them, seven RFS-related events were integrated into a risk factor, exhibiting satisfactory RFS prognosis alone and even better performance when combined with clinical tumor-node-metastasis stages. Furthermore, the correlation analysis between SFs and the seven AS events revealed the hypotheses that SRPK3 might upregulate PCYT2_44231_AA to have an effect on RFS prognosis and that three other SFs may work together to downregulate FLAD1_7874_RI to influence RFS prognosis. In addition, the seven RFS-related AS events were validated to be promising in the OS prognosis of TNBC as well. CONCLUSION: The abnormal AS events regulated by SFs may act as a kind of biomarker for the survival prognosis of TNBC.

A Lab-on-a-Chip Device Integrated DNA Extraction and Solid Phase PCR Array for the Genotyping of High-Risk HPV in Clinical Samples.

Point-of-care (POC) molecular diagnostics play a crucial role in the prevention and treatment of infectious diseases. It is necessary to develop portable, easy-to-use, inexpensive and rapid molecular diagnostic tools. In this study, we proposed a lab-on-a-chip device that integrated DNA extraction, solid-phase PCR and genotyping detection. The ingenious design of the pneumatic microvalves enabled the fluid mixing and reagent storage to be organically combined, significantly reducing the size of the chip. The solid oligonucleotide array incorporated into the chip allowed the spatial separation of the primers and minimized undesirable interactions in multiplex amplification. As a proof-of-concept for POC molecular diagnostics on the device, five genotypes of high-risk human papillomavirus (HPV) (HPV16/HPV18/HPV31/HPV33/HPV58) were examined. Positive quality control samples and HPV patient cervical swab specimens were analyzed on the integrated microdevice. The platform was capable of detection approximately 50 copies of HPV virus per reaction during a single step, including DNA extraction, solid-phase PCR and genotype detection, in 1 h from samples being added to the chip. This simple and inexpensive microdevice provided great utility for the screening and monitoring of HPV genotypes. The sample-to-result platform will pave the way for wider application of POC molecular testing in the fields of clinical diagnostics, food safety, and environmental monitoring.

iTRAQ proteomic analysis of the hippocampus in a rat model of nicotine-induced conditioned place preference.

Repeated exposures to nicotine are known to result in persistent changes in proteins expression in addiction-related brain regions, such as the striatum, nucleus accumbens and prefrontal cortex, but the changes induced in the protein content of the hippocampus remain poorly studied. This study established a rat model of nicotine-induced conditioned place preference (CPP), and screened for proteins that were differentially expressed in the hippocampus of these rats using isobaric tags for relative and absolute quantitation labeling (iTRAQ) coupled with 2D-LC MS/MS. The nicotine-induced CPP was established by subcutaneously injecting rats with 0.2 mg/kg nicotine. Relative to the control (saline) group, the nicotine group showed 0.67- and 1.5-fold changes in 117 and 10 hippocampal proteins, respectively. These differentially expressed proteins are mainly involved in calcium-mediated signaling, neurotransmitter transport, GABAergic synapse function, long-term synaptic potentiation and nervous system development. Furthermore, RT-PCR was used to confirmed the results of the proteomic analysis. Our findings identify several proteins and cellular signaling pathways potentially involved in the molecular mechanisms in the hippocampus that underlie nicotine addiction. These results provide insights into the mechanisms of nicotine treatment in hippocampus.