CACNA1S, 1/5), hypokalemic periodic paralysis type 2 , normokalemic periodic paralysis , and Andersen-Tawil syndrome . The basic clinical manifestations of five families were consistent with PPP, presenting with paroxysmal muscle weakness, with or without abnormal serum potassium. ATS was accompanied by ventricular arrhythmias, and skeletal and craniofacial anomalies, developing with a permanent fixed myopathy later. The electromyography showed diffuse myopathic discharge, and muscle biopsy showed tubular aggregates. Genetic testing revealed five families with PPP carried CACNA1S (R1242S), SCN4A , and KCNJ2 (R218Q) respectively. The novel heterozygous R1242S mutation in CACNA1S caused a conformational change in the protein structure, and the amino acid of this mutation site was highly conserved among different species. SCN4A mutations led to two phenotypes of HypoPP2 and NormoPP. PPPs are autosomal dominant disorders of ion channel dysfunction characterized by episodic flaccid muscle weakness secondary to abnormal sarcolemmal excitability. PPPs are caused by mutations in skeletal muscle calcium channel CaV1.1 gene (CACNA1S), sodium channel NaV1.4 gene (SCN4A), and potassium channels Kir2.1, Kir3.4 genes , including HypoPP1, HypoPP2, NormoPP, HyperPP, and ATS, which have significant clinical and genetic heterogeneity. Diagnosis is based on the characteristic clinical presentation then confirmed by genetic testing.To explore the clinical and genetic characteristics of five families with primary periodic paralysis (PPP). We reviewed clinical manifestations, laboratory results, electrocardiogram, electromyography, muscle biopsy, and genetic analysis from five families with PPP. Five families with PPP included: hypokalemic periodic paralysis type 1 (HypoPP1,  Patients usually complete recovery between the attacks, although some develop permanent fixed weakness later in life. A subset of PP patients accompany with cardiac arrhythmia or sudden cardiac death. PPs can be divided into primary periodic paralyses (PPPs) and secondary PPs by the etiology. Secondary PPs are common in hyperthyroidism, primary aldosteronism, renal tubular acidosis, and so on. Except for secondary PPs, PPPs can be suspected clinically.Periodic paralyses (PPs) are a group of skeletal muscle ion channelopathies characterized by episodic flaccid muscle weakness. The attacks may last hours to days or weeks, and often are associated with altered serum potassium (KV1.1), sodium channel (NaV1.4), and potassium channels . PPPs are classified as hyperkalemic periodic paralysis (HyperPP), normokalemic periodic paralysis (NormoPP), or hypokalemic periodic paralysis (HypoPP) based on serum K+ levels during paralytic attacks. Andersen-Tawil syndrome (ATS) is a rare PPP characterized by a triad of PP, cardiac arrhythmia, and skeletal and craniofacial anomalies, with high, low, or normal serum K+ levels.PPPs are autosomal dominant disorders caused by mutations in genes encoding the skeletal muscle calcium channel  or the skeletal muscle NaV1.4 channel gene  [V1.4 channel gene (SCN4A). ATS is caused by mutations in the inward rectifying Kir 2.1, Kir3.4 channel genes  [HypoPP is caused by mutations in the alpha subunits of either the skeletal muscle L-type CaPPPs have remarkable clinical and genetic heterogeneity. Here, we reviewed clinical manifestations, laboratory results, electrocardiogram (ECG), electromyography (EMG), muscle biopsy, and genetic analysis from five families with PPPs.+, and confirmed by genetic testing at the Third Hospital of Hebei Medical University in China between June 2016 and June 2020. All probands were examined by serum and urine K+, blood-gas analysis, aldosterone and renin-angiotensin levels on erect and supine positions, thyroid function, ultrasound analysis of the thyroid and adrenal glands to exclude secondary PPs, such as hyperthyroidism, renal tubular acidosis, primary aldosteronism and et al. Clinical, laboratory, ECG, EMG, muscle biopsy, and genetic data of these patients were collected. Written informed consent was obtained from each patient or their legal relatives before skeletal muscle biopsy and genetic testing. This study was approved by the ethics committee of the Third Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki.We retrospectively reviewed clinical and genetic features of five families with PPPs, whose main clinical manifestations were episodic flaccid weakness, with or without abnormal serum KOpen muscle biopsy was performed from the biceps brachii muscles. Biopsied skeletal muscles were snap frozen in isopentane that were cooled by liquid nitrogen and cut serially into 7 μm sections, preparing for histochemical staining and pathological analysis. Routine stains included hematoxylin and eosin (HE), modified Gomori trichrome (MGT), nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), succinic dehydrogenase (SDH), adenosine monophosphate deaminase (AMP), cytochrome c oxidase (CCO), acid phosphatase (ACID), adenosine triphospha-tase (ATPase) after incubation at pH 4.3, 4.5, and 10.1, periodic acid-Schiff (PAS), oil red O (ORO) and Sudan black B (SBB).CACNA1A, CACNA1S, CLCN1, KCNA1, KCNE3, KCNJ16, KCNJ18, KCNJ2, KCNJ5, KCNQ1, SCN4A, CHRNB1) related to ion channelopathies were sequenced by next-generation sequencing (NGS) . The probable pathogenic mutations were then predicted using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), and MutationTaster (http://mutationtaster.org/). The functional verification of novel mutations was performed by conservative analysis of amino acid and tertiary structure prediction of protein. Sanger sequencing was used to verify the variants identified by NGS and to examine the available relatives of patients. The American College of Medical Genetics and Genomics (ACMG) standards were used to evaluate the pathogenicity [Genomic DNA was extracted from peripheral blood  with a DNA Kit. All the exon regions and adjacent intron regions 50bp) of 12 genes , NormoPP (1/5), ATS (1/5).Detailed clinical, laboratory, ECG, and EMG data of the proband of five families with PPP are shown in + levels were lower at many times , 3.5–5.5 mmol/L) during the attacks. They complained of paroxysmal muscle weakness that lasted for several days, which often accompanied with muscle soreness, not muscle hypertrophy. Attacks appeared about several or dozens of times per year, which would be relieved after taking K+ supplement. Serum creatine kinase (CK) levels were elevated. Their family histories were positive  were diagnosed with HypoPP, whose serum Kpositive –c). One positive ). One prpositive ). CardiaPathological analysis of skeletal muscle biopsy was performed in four probands . The muscle pathology of patient 1 (HypoPP) showed variation of fiber diameter and scattered degenerative or necrotic muscle fibers –d). MyosCACNA1S, SCN4A, and KCNJ2, which were heterozygous missense mutations  and KCNJ2 (p.R218Q) had been reported in the literature [KCNJ2(p.R218Q) mutation was spontaneous, which was found in the proband but not his parents. A novel heterozygous R1242S mutation in CACNA1S gene was found in the proband with HypoPP and some family members. The amino acid at the R1242S mutation site was highly conserved among different species . R1242S species . The patV1.1 channel gene (CACNA1S), NaV1.4 channel gene (SCN4A), Kir 2.1 and Kir3.4 channel genes , including HypoPP1, HypoPP2, NormoPP, HyperPP, and ATS, which have remarkable clinical and genetic heterogeneity. There are five families with PPPs, whose main clinical manifestations were episodic flaccid weakness, with or without abnormal serum K+, and confirmed by genetic testing, including HypoPP1 , HypoPP2 , NormoPP  and ATS  in this study , while HypoPP2, NormoPP, and HyperPP are caused by mutations of NaV1.4 channel gene (SCN4A). ATS is caused by mutations of Kir2.1 and Kir3.4 channel genes   .V1.1 channel encoded by CACNA1S gene, caused the third positively charged arginine replaced by hydrophobic amino acid residue. Among the HypoPP1 mutations identified so far, most consist in substitutions at positively charged arginine residues in S4 segments, which could lead to the formation of a gating pore current at hyperpolarized potentials, including R528, R897, R900, R1239, and so on [V1.4 channels to cause periodic paralysis [V1.1 channel protein, considering the fact that this residue is highly conserved among different species  and mut species .V1.4 channel encoded by SCN4A gene, which causes the third positively charged arginine replaced by hydrophobic amino acid residue, leading to the formation of abnormal gating pore current. The gating pore current causes susceptibility to paradoxical depolarization during attacks. The depolarization of sarcolemma leads to inactivation of NaV1.4 channels to cause periodic paralysis [V1.4 channel, and, therefore, persistent increased sodium current and sarcolemmal depolarization, which can result in the symptom of paralysis [R675Q mutation is located at the S4 segment of domain II in the alpha subunit of the skeletal muscle voltage-gated Naaralysis ,30. It haralysis ,9 and caaralysis . T704M maralysis ,33. It haralysis .KCNJ2 encodes the Kir2.1 inward rectifier potassium channel, which is mainly expressed in skeletal muscle and cardiac myocytes. R218Q mutation is located at the carboxy-terminal  region of Kir2.1 channel protein, which reduces the amount of inward rectifier current, decelerates the action potential repolarization, and prolongs the action potential duration, thereby leading to ventricular arrhythmias associated with ATS [with ATS . Reducedwith ATS . It has with ATS ,12. Mutawith ATS . Kir2.1 with ATS .V1.1 channel gene (CACNA1S), NaV1.4 channel gene (SCN4A), Kir 2.1 and Kir3.4 channel genes . PPPs are classified as HyperPP, NormoPP, or HypoPP based on the concomitant serum K+ levels. ATS is different from the other phenotypes of PPPs, characterized by a highly variable phenotype of PP, ventricular arrhythmia, and distinctive facial and skeletal anomalies, associated with high, low, or normal serum K+ levels. Diagnosis is based on the characteristic clinical presentation then confirmed by genetic testing.PPPs are autosomal dominant disorders of ion channel dysfunction characterized by episodic flaccid weakness secondary to abnormal sarcolemmal excitability, which are caused by mutations in skeletal muscle Ca


Pyruvate kinase M2 (PKM2) is an essential regulator of the Warburg effect, but its biological function promoting immune escape of hepatocellular carcinoma (HCC) is unclear.in vitro CCK-8, colony formation, and transwell assays, and in vivo xenografts were performed to evaluate the malignancy of HCC cells. PKM2 and PD-L1 levels were examined by Western blot, qRT-PCR, and IHC. The role of PKM2 on in vivo immune response was also investigated.GEPIA web tool and immunohistochemistry (IHC) analysis were employed to evaluate the clinical relevance of PKM2 in HCC patients. Both in vitro proliferation, migration, and invasion of HCC cells, as well as in vivo tumor growth. Strikingly, PKM2 showed a strong correlation with the expression of immune inhibitory cytokines and lymphocyte infiltration in HCC. The overexpression of PKM2 sensitized HCC to immune checkpoint blockade, which enhanced IFN-γ positive CD8 T cells in HCC mice models.PKM2 was significantly upregulated in HCC and associated with a poor prognosis of HCC patients. Knockdown of PKM2 inhibited PKM2 might be a predictor and a potential therapeutic target for immune checkpoint inhibitors in HCC. As one of the most common human malignancies, hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide . Despitevia enhancing its ability to bind to the hypoxia-responsive elements (HREs)  . Accumuls (HREs) –23.Tumor cells escape from immunosurveillance in a variety of ways . PD-L1/PIn the present study, we aimed to explore the role of PKM2 in the immune escape of HCC cells, and its potential application in immune therapy of HCC.2.HEK 293T cells, human HCC cell lines  and mouse HCC cell line (Hepa1-6) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science (Shanghai China). Human HCC cell lines MHCC97-L, MHCC97-H, and HCC-LM3 were obtained from the Liver Cancer Institute at Fudan University  as previously described , 41. TheAll male mice  were purchased from Shanghai Slac Laboratory Animal Co. and maintained on standard rodent chow and water ad libitum. Prior to any surgical procedures, all mice were given intraperitoneal (i.p.) injection of Pentobarbital Sodium  and conducted in the SPF laboratory. All procedures involving animals were approved by The Animal Care and Use Committee of Fudan University .6/100 μl in 50% Matrigel and PBS per mice) were injected into the right flank of nude mice (5 mice/group) to establish the subcutaneous implantation models. The tumor growth was monitored once per week until the mice were sacrificed. Tumor volume was calculated by the formula: a × b2/2 . The tumors were collected for RNA preparation, fixed in 4% formalin, and then embedded in paraffin. Consecutive sections of tumor tissues were applied by immunohistochemistry (IHC) staining.Four human HCC cells including Sh-control MHCC97-H, Sh-PKM2 MHCC97-H, OE-vector Huh7 and OE-PKM2 Huh7 (2 × 106/100 μl in 50% Matrigel and PBS per mice) were injected into the right flank of C57BL/6 mice (5 mice/group). All the tumors were collected, and the tumor weight was measured.Four mouse HCC cells including Sh-control Hepa1-6, Sh-PKM2 Hepa1-6, OE-control Hepa1-6, and OE-PKM2 Hepa1-6 (1 × 10P = 5.91e−05)  and tumor grade (P = 0.035) rather than the other clinicopathological features including gender, age, HBV status, liver cirrhosis, AFP, ALT, tumor numbers, tumor capsule, and BCLC stage , Huh7, MHCC97-L, MHCC97-H and HCC-LM3  from TCGA data. Compared with the PKM2 low expression group, the leading enriched signatures in PKM2 high expression group included gene sets involved in cell cycle  and tumor number (P = 0.08), rather than the other clinicopathological features. Pearson product-moment correlation analysis showed a significantly positive correlation between PD-L1 and PKM2 (P = 0.0049).We further used GEPIA to analyze the correlation between PKM2 and PD-L1 expression levels, and found a close correlation between them in HCCs  at first. Either PKM2 knockdown or aPD-L1 therapy could significantly inhibit HCC is the fourth leading cause of cancer related deaths worldwide, but currently there are no effective targeted therapies for the intermediate and advanced HCC , 42, 43.via in vitro and in vivo assays. Our findings reinforce the idea that metabolic enzyme such as PKM2 is an important mediator for HCC progression. Because HCC are well known to be highly aggressive tumors, it appears that PKM2 may serve as a potential metabolic biomarker for prediction of HCC prognosis, and an effective treatment target to block HCC progression.PKM2 is an essential regulator of the Warburg effect in cancer cells which catalyzes the phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) into pyruvate and adenosine triphosphate (ATP) as fuel for proliferation and metastasis of cancer cells . Accumulvia dampened their glycolytic capacity and IFN-γ production  The studies involving human participants were reviewed and approved by Ethics Boards of Huashan Hospital of Fudan University . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Ethical Committee of Fudan University . Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Q-ZD, L-XQ, and T-EL designed the research. SW, T-EL, YiZ, and X-TS performed the research. SW, T-EL, X-TS, ZZ, MC, RW, and HW analyzed the data. T-EL and SW drafted the paper. Q-ZD, DX, and YaZ provided patient tissue samples and collected patient clinical information. Q-ZD, L-XQ, and T-EL revised the paper. All authors contributed to the article and approved the submitted version.This work was supported by the Program of Shanghai Academic Research Leader (20XD1400900), the National Key Research and Development Program of China (2017YFC1308604 and 2017YFC0908402), the National Natural Science Foundation of China , the NSFC Program of International Cooperation and Exchanges (81120108016).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to investigate the value of sirtuin 1 (SIRT1) in differentiating sepsis patients from healthy controls (HCs), and its correlation with inflammation, disease severity, as well as prognosis in sepsis patients. Serum samples were collected from 180 sepsis patients and 180 age- and gender-matched HCs. The SIRT1 level in the serum samples was detected by enzyme-linked immunoassay. The clinical data of the sepsis patients were documented, and their disease severity scores and 28-day mortality rate were assessed. SIRT1 was decreased in sepsis patients compared with HCs, and the receiver operating characteristic curve (ROC) showed that SIRT1 distinguished sepsis patients from HCs (area under the curve (AUC): 0.901; 95% confidence interval (CI): 0.868-0.934). In sepsis patients, SIRT1 negatively correlated with serum creatinine (Scr), white blood cells (WBC), C-reactive protein (CRP), acute physiology, and chronic health evaluation II (APACHE II) score, and sequential organ failure assessment (SOFA) score, while it positively correlated with albumin. No correlation of SIRT1 with primary infection site or primary organism was observed. Furthermore, SIRT1 was reduced in 28-day non-survivors compared with 28-day survivors, and subsequent ROC showed that SIRT1 predicted 28-day mortality of sepsis patients , and its prognostic value was not inferior to Scr, albumin, WBC, and CRP, but was less than SOFA score and APACHE II score. In conclusion, measurement of serum SIRT1 might assist with the optimization of disease assessment, management strategies, and survival surveillance in sepsis patients. Sepsis, the commonest cause of emergency admission to the intensive care unit (ICU) globally, develops as an overwhelming, systemic deregulated host response to a microbial infection, which leads to acute tissue damage and organ dysfunction, being associated with a high risk of mortality . It is e+ and deacetylating nearby histones (Sirtuins (SIRTs), a highly conserved family of proteins first described in yeast, are composed of seven SIRTs (SIRT1-7) that are dispersed throughout different cell compartments, and are key regulators of inflammatory stress response in immune and non-immune cells via sensing NADhistones . SIRT1 ihistones -9. As anhistones . AnotherBetween January 2017 and September 2019, 180 sepsis patients admitted to our hospital  were consecutively recruited for this study. All eligible patients were required to meet the following inclusion criteria: 1) confirmed diagnosis of sepsis in accordance with Surviving Sepsis Campaign: International Guidelines for Management of Severe Sepsis and Septic Shock, 2012 ; and 2) g for 5 min at 4°C. The serum was removed from the clot, collected in cryogenic vials, labeled accordingly, and then stored at -80°C until subsequent detection. In addition, sepsis patients' clinical data were documented, including age, gender, body mass index (BMI), smoking status, chronic comorbidities , biochemical indexes .Blood samples of sepsis patients were collected within the first 24 h of ICU admission and before antibiotic therapy, which were divided into two parts: one was used for bacteria culture and necessary biochemical tests, and the other was used for the present study. Blood samples of HCs were collected when they were undergoing the health examination. After collection, samples were allowed to clot for 30 min at room temperature followed by centrifugation at 1200 For disease severity assessment, Acute Physiology and Chronic Health Evaluation (APACHE) II score and Sequential Organ Failure Assessment (SOFA) score were assessed within 24 h after admission . The APAThe SIRT1 level in serum of sepsis patients and HCs was detected by enzyme-linked immunoassay (ELISA) using human SIRT1 ELISA Kit , according to the manufacturer's instructions. Briefly, procedures of ELISA were performed as follows: firstly, the samples were diluted with diluent in 1:1 ratio, then 50 μL of diluted samples and 50 μL of standards were added into the reaction well. Next, 50 μL biotin-labeled antibody was immediately added into the well, followed by covering with film plate, gently mixing, and incubation at 37°C for 1 h. After washing and drying with absorbent paper, 80 μL of affinity chain enzyme (HRP) was added to each well, followed by gently mixing and incubation at 37°C for 30 min. After washing and drying with absorbent paper, 50 μL of chromogenic agent was added to each well, followed by gently mixing and incubation at 37°C for 10 min in the dark. Thereafter, 50 μL termination solution was added to each well, and the absorbance value of each well was immediately measured at 450 nm wavelength using a microplate reader . Finally, the SIRT1 level was calculated according to the standard curve.All patients were managed in accordance with sepsis guidelines . Daily st-test. Correlation analysis between variables was determined by Pearson's test. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) with 95% confidence interval (CI) were used to assess the performance of variables in predicting the susceptibility to sepsis and the 28-day mortality risk. Based on the ROC analysis of SIRT1 in predicting susceptibility to sepsis, the best statistical cut-off value (maximum sum of sensitivity and specificity) was calculated. The sensitivity, specificity, false positive rate (FPR), and false negative rate (FNR) at the best cut-off point were calculated as well. Accumulating mortality was displayed using the Kaplan-Meier method, and the mortality difference between two groups was determined by the log-rank test. A P value <0.05 was considered as statistically significant.SPSS 22.0  was used for statistical analysis, and GraphPad Prism 7.01  was used for figure plotting. Data are reported as means±SD, or count (percentage). Comparison between groups was determined by the unpaired 2. Regarding complications, 25 (13.9%), 68 (37.8%), 22 (12.2%), and 39 (21.7%) sepsis patients had COPD, cardiomyopathy, chronic kidney failure, and cirrhosis, respectively. As to primary infection site, 65 (36.1%), 40 (22.2%), 33 (18.3%), 20 (11.1%), 12 (6.7%), and 10 (5.6%) sepsis patients presented with abdominal infection, respiratory infection, skin and soft tissue infection, blood stream infection, central nervous system (CNS) infection, and other infections, respectively. In addition, the mean APACHE II score was 13.8±6.3 and the mean SOFA score was 6.3±2.8. Detailed information about other clinical characteristics of sepsis patients are shown in In the total sepsis patients, the mean age was 53.7±10.9 years, and there were 108 (60.0%) males/72 (40.0%) females . The meaThe clinical characteristics were compared between septic survivors and non-survivors, which showed that non-survivors had significantly more female cases, less cases with primary infection site, more cases with anaerobes, higher Scr, WBC, CRP, APACHE II score, and SOFA score  compared with survivors, while no difference was found in age, BMI, smoking, COPD, cardiomyopathy, chronic kidney failure, cirrhosis, gram-negative bacteria, gram-positive bacteria, fungus, mycoplasmas, total negative culture, or albumin  between non-survivors and survivors .The mean level of SIRT1 was 1.947±0.983 ng/mL in HCs and 0.587±0.365 ng/mL in sepsis patients, and comparison analysis showed that SIRT1 was significantly lower in sepsis patients compared with HCs (P<0.001) . The ROCNo correlation of SIRT1 with abdominal infection, respiratory infection, skin and soft tissue infection, blood stream infection, CNS infection or other infections  was shown in sepsis patients . FurtherSIRT1 negatively correlated with Scr (r=-0.236), WBC (r=-0.202), and CRP (r=-0.405) and positively correlated with albumin (r=0.416) in sepsis patients . More im=0.006)  . The ROC=0.006) .In the present study, we found that: 1) SIRT1 was reduced in sepsis patients compared with HCs, and the ROC curve showed that SIRT1 distinguished sepsis patients from HCs; 2) SIRT1 negatively correlated with Scr, WBC, CRP, SOFA score, and APACHE II score, whereas it positively correlated with albumin in sepsis patients; and 3) SIRT1 negatively correlated with 28-day mortality, and ROC curve showed that SIRT1 predicted 28-day mortality in sepsis patients.+-dependent class III histone deacetylase, is mainly localized in the nucleus, and it modulates the immune and inflammatory responses of sepsis to control inflammatory injury in multiple organs, and ultimately protect against multiple organ dysfunction  SIRT1 probably attenuated the expression of pro-inflammatory cytokines, chemokines, and adhesion molecules in immune/endothelial cells through deacetylation and deactivation of inflammation-related factors such as NFκB p65, which inhibited inflammation in sepsis, thereby, SIRT1 was reduced in sepsis patients compared with HCs ,14; 2) SFurthermore, the present study assessed the correlation of SIRT1 with inflammation and disease severity in sepsis patients. We found that SIRT1 was associated with ameliorated inflammation and disease severity  in sepsis patients. The findings could be explained by: 1) SIRT1 probably suppressed various downstream targets , high mobility group protein box 1, and TNF-α) to exert anti-inflammatory effects and protect the body from excessive inflammation; thereby, SIRTI was associated with decreased inflammation in sepsis patients ,12,17; aConsidering our findings that SIRT1 was negatively associated with inflammation and disease severity in sepsis patients, it was speculated that SIRT1 might be related to improved prognosis as well. In the present study, we disclosed that SIRT1 was decreased in 28-day non-survivors compared with 28-day survivors, and it could predict 28-day mortality risk in sepsis patients by the ROC curve. Furthermore, Kaplan-Meier analyses exhibited that SIRT1 high was associated with lower accumulating mortality in sepsis patients. The following explanations have been proposed: 1) SIRT1 might suppress the inflammation-related signaling, attenuate oxidative stress, and repress cell apoptosis in vital organs via its downstream pathways such as NF-κB, which contributed to alleviated disease severity and lower 28-day mortality in sepsis patients ,12,; andAPACHE II and SOFA scores are commonly used prognostic scoring systems in sepsis patients, while they are complex (requiring multiple measurements) and time-consuming. Thus, it was necessary to explore novel and rapidly measurable prognostic biomarkers in sepsis patients ,19. As fThe present study was the first to evaluate the clinical value of SIRT1 for the progression and prognosis of sepsis, however, several limitations should be noted when interpreting the findings. First, the sample size was relatively small, which might impact the statistical power of the findings. Second, SIRT1 level of sepsis patients was only detected before antibiotic therapy and the variation of SIRT1 during the antibiotic therapy was not considered. Therefore, the detection of SIRT1 at different time points during the antibiotic therapy should be assessed in the future.To conclude, SIRT1 distinguished sepsis patients from HCs, and it correlated with lower disease severity and better prognosis in sepsis patients, which offers new perspectives for facilitating management strategies and survival surveillance of sepsis in clinical practice.


The impact of zinc supplementation on assisted fertilization techniques is also described. The impact of zinc on sperm motility has been investigated in many vertebrate and invertebrate species. It has been reported that Zn2+ in human seminal plasma decreases sperm motility and that Zn2+ removal enhances motility. Reduction in the intracellular concentration of Zn2+ during epididymal transit allows the development of progressive motility and the subsequent hyper activated motility during sperm capacitation. Extracellular Zn2+ affects intracellular signaling pathways through its interaction with the Zn2+ sensing receptor (ZnR), also named GPR39. This receptor was found in the sperm tail and the acrosome, suggesting the possible involvement of Zn2+ in sperm motility and acrosomal exocytosis. Our studies showed that Zn2+ stimulates bovine sperm acrosomal exocytosis, as well as human sperm hyper-activated motility, were both mediated by GPR39. Zn2+ binds and activates GPR39, which activates the trans-membrane-adenylyl-cyclase (tmAC) to catalyze cAMP production. The NHE (Na+/H+-exchanger) is activated by cAMP, leading in increased pHi and activation of the sperm-specific Ca2+ channel CatSper, resulting in an increase in [Ca2+]i, which, together with HCO3−, activates the soluble adenylyl-cyclase (sAC). The increase in [cAMP]i activates protein kinase A (PKA), followed by activation of the Src-epidermal growth factor receptor-Pphospholipase C (Src-EGFR-PLC) cascade, resulting in inositol-triphosphate (IP3) production, which mobilizes Ca2+ from the acrosome, causing a further increase in [Ca2+]i and the development of hyper-activated motility. PKA also activates phospholipase D1 (PLD1), leading to F-actin formation during capacitation. Prior to the acrosomal exocytosis, PLC induces phosphadidylinositol-4,5-bisphosphate (PIP2) hydrolysis, leading to the release of the actin-severing protein gelsolin to the cytosol, which is activated by Ca2+, resulting in F-actin breakdown and the occurrence of acrosomal exocytosis.Several studies proposed the importance of zinc ion in male fertility. Here, we describe the properties, roles and cellular mechanisms of action of Zn It plays an essential role in the organization of DNA, RNA and proteins, as well as in the stability of cell membranes and cell division [2+ is required in diverse mechanisms, like the DNA replication, RNA transcription, differentiation, proliferation and activation of immune cells in many organs of the body [Zinc is an essential element with a wide range of biological functions. More than 200 Zn-metalloenzymes are regulated by Zndivision . We knowthe body ,4. In adthe body . Zinc iothe body .2+ in fertile men was 14.08± 2.01 and, in the infertile group, was 10.32 ± 2.98 (mg/100 mL) [2+ lack can be linked with a diminution in testis volume and testicular weight and failure of spermatogenesis [2+ is secreted mainly from the prostate [2+ to semen extenders before freezing reduces reactive oxygen species (ROS) [2+ can act as a pro-oxidant, leading to mitochondrial oxidative stress [2+ [2+ during the epididymal transit is essential for the development of progressive motility, as well as chromatin decondensation, two necessary events for successful fertilization.It has also been shown that zinc plays a significant role in the male reproductive system ,8,9,10. /100 mL) . A Zn2+ ogenesis . The preogenesis . We knowogenesis . The proogenesis . The preogenesis . Since togenesis ,19, and ogenesis . Rats trogenesis . Howeverogenesis ,22. Zincprostate . Zinc ioprostate . About 2prostate ,26, and prostate  and domeprostate ,29,30 ises (ROS) ; howevere stress ,34 and se stress ,36 are se stress . Relativress [2+ . Thus, t2+ on sperm motility, capacitation and acrosomal exocytosis, including the mechanisms of action and the impact of zinc supplementation on assisted fertilization techniques.In this review, we will focus on the effect of Zn2+ transporters are conserved in mammals, SLC39s/ZIPs and SLC30s/ZnTs, which transport Zn2+ in opposite directions through cellular and intracellular membranes . ZIP an and fertilization when mutated [spe-8gene, which encodes a protein tyrosine kinase, is involved in protein tyrosine phosphorylation [2+ may initiate the spe-8 signaling cascade, leading to sperm activation [Investigations in  mutated ,48. For rylation ,50, a knrylation . Severalrylation ,53. It htivation ,55.spe-8, regulates the release of Zn2+ from internal stores. The released Zn2+ in the cytoplasm activates zinc-regulated proteins that develop motility. Studies of zinc transporters revealed that the deletion of zipt-7.1 causes sterility [2+ transporter associated with the G-protein Gnα11, which mediates testosterone signaling in murine spermatogenic cells [2+ is transported into the nucleus [2+ acts as a second messenger that modulates sperm functions, including motility and capacitation. This suggests that intracellular Zn2+ levels should be well-controlled by zinc transporters localized at intracellular membranes and in the cell plasma membrane, which import Zn2+ from the external environment [Zipt-7.1 is a transmembrane protein localized within intracellular organelles ,57 and, terility . ZIP9 seic cells . During  nucleus , which i nucleus ,61. Thusironment .2+ in human seminal plasma decreases sperm motility [2+ removal, by binding to a protein named semenogelin, enhances motility [2+ [C. elegans, the zinc released within cells acts as a messenger in a signaling pathway to promote mobility acquisition [2+ affects sperm motility, but whether its effect is inhibitory or stimulatory seems to be species- and concentration-dependent, whereby relatively low Zn2+ concentrations stimulate motility, whereas high Zn2+ inhibits sperm motility.The impact of zinc on sperm motility has been investigated in many vertebrate and invertebrate species. It has been reported that Znmotility  and thatmotility . Howeverlity [2+ . In C. euisition . Thus, e2+ is present in sperm mitochondria and along the flagella [2+ to cysteine-sulfhydryl of ODF during spermatogenesis [2+ during epididymal transit enables sulfhydryl oxidation and stiffening of the ODF to allow the development of progressive motility [2+ (5–10 µM) stimulate hyper-activated motility in human sperm under capacitation conditions, whereas, at 30-µM Zn2+, no stimulation was observed [2+ (in the mM range) in the semen are inhibitory to sperm function, whereas, in the female reproductive tract, the concentration of Zn2+ is much lower (1.0–1.5 µM) [Furthermore, it has been shown that Znflagella ,67 and iflagella ,69. The ogenesis  protectsogenesis . Reductimotility  and the motility . Hyper-amotility . It has observed . We also–1.5 µM) . This lo2+ concentration (0.1 mM) inhibits sperm motility [2+ (0.2 mM), which compromise capacitation and hyper-activated motility, also inhibit the voltage-gated H+ channel Hv1, localized in the sperm tail and responsible for sperm cytoplasmic alkalization [2+ channel CatSper [2+ on human sperm HAM, indicating that CatSper mediates Zn2+-stimulated hyper-activated motility [2+ influx via CatSper causes membrane depolarization, leading to the activation of voltage-gated H+ channel Hv1, which is the predominant alkalization system in human sperm [v1 and preventing intracellular alkalization. The increase of intracellular pH will shift the equilibrium of the reaction: CO2+H2O-→HCO3−+H+ to the left, resulting in decreasing the concentration of bicarbonate and a reduction in sAC/PKA activities. CatSper-null sperm was produced in the mouse, in which intracellular alkalization occurs by Na+/H+ exchanger (NHE) and Hv1 does not exist. NHE-null sperm is not motile, and motility could be restored by artificial alkalization of the cytosol [2+ influx in CatSper-null sperm would not affect intracellular alkalization, allowing the activation of sAC/PKA. We suggested that Zn2+ activates the PKA-Src-EGFR cascade, which is CatSper-dependent [Motility initiation after sperm ejaculation, and the development of hyper-activated motility during sperm capacitation, are both dependent on intracellular alkalization . Excessimotility . High colization ,79 and tlization . The cyt CatSper , which i CatSper  , also named GPR39 [2+ in sperm motility and acrosomal exocytosis. Our studies showed that Zn2+ stimulates bovine sperm acrosomal exocytosis [2+ show a 40% increase in intracellular cAMP levels, which is a necessary event in the capacitation process [2+ mediates the activity of the two adenylyl-cyclase(AC) isoforms, the soluble AC (sAC), activated by bicarbonate, as well as the trans-membrane AC (tmAC), activated by GPCR, leading to an increase of intracellular cAMP  [2+. Sperm can induce Zn2+ release from the oocyte cortex [2+ inhibits sperm chemoattraction to the egg induced by oocyte-secreted progesterone in human, mouse and rabbit sperm [2+ (~0.1 mM) to bovine in vitro fertilization (IVF) medium reduces the fertilization rate [2+-dependent metalloproteases inhibit sperm passage via the cumulus oophorus in porcine IVF [It has been shown that the in vitro addition of high concentrations of Zno bovine  and humao bovine  sperm coo bovine . Zinc hao bovine ,105. It o bovine ,107; howo bovine . Thus, lo bovine ,109. The0–1.5µM)  in the f0–1.5µM)  that zone cortex ,112, leait sperm , and theion rate . Additiocine IVF .2+-dependent endopeptidase family. The isoform MMP2 is localized to the acrosomal region and tail, and MMP9 is expressed in the tail of human sperm [Matrix metalloproteinases (MMPs) are part of the Znan sperm ,117. MMPan sperm . We havean sperm .2+ (in the µM range) increase the in vitro capacitation efficiency [2+ mobilization and acrosomal exocytosis (see 2+ stimulates HAM via CatSper-dependent activation of the adenylyl-cyclase (AC)/cAMP/PKA/Src/EGFR and phospholipase C (PLC) cascade [2+ activates the EGFR during capacitation, which is mediated by the activation of tmAC, PKA and Src [2+ to capacitated bovine sperm further stimulates the EGFR and the downstream effectors PI3K, PLC and protein kinase C (PKC), leading to acrosomal exocytosis [3R) localized in the outer acrosomal membrane and in the redundant nuclear envelope (RNE) are activated by IP3 generated from the hydrolysis of phosphadidyl-inositol-4,5-bisphosphate (PIP2) by PLC, inducing the release of Ca2+ from the acrosome and RNE and promoting the development of HAM [2+-activated GPR39, leading to PLC activation, as described above.Low concentrations of Znficiency ,88 by acficiency ,121,122,osis see . We sugg cascade  . The integrity of the sperm acrosome is likely to be defective in sperm with a high rate of sAR. Accordingly, a dietary supplementation of Zn2+ to goats increases sperm acrosome integrity [2+ in preventing sAR.It is known that the occurrence of spontaneous acrosomal reaction (sAR) before the sperm reaches the proximity of the egg reduces the fertilization rate . Sperm cntegrity , supportThe effectiveness of assisted reproductive methods has been improved over the last decade. The cryopreservation of sperm under liquid nitrogen is now used in assisted reproduction centers to conserve sperm cells for extended periods of time. However, freezing and thawing processes damage the fertilizing capacity of the sperm due to osmotic effects and oxidative stress. Under cryopreservation, sperm is deprived of the seminal plasma, which contains antioxidants and vitamins that act against free radicals and, thereby, protect the sperm cells ,130.Cryopreservation affects sperm cells in many ways: by diminishing the fertilization capacity, reducing motility, altering morphology (such as coiled tails), reducing viability , damaginThe addition of antioxidants, such as zinc, in the freezing medium to improve the fertility capacity and preserve the sperm from oxidative damage is becoming increasingly widespread ,137. StuRecent studies demonstrated the beneficial effects of zinc addition to human ejaculate before cryopreservation on sperm viability and motility after thawing ,139. TheZinc oxide nanoparticles (ZnONPs), initially developed for drug delivery in cancer research , were apZinc-deficient nutrition causes a low quality of sperm and male infertility , and the2+ concentration in the seminal plasma is needed for normal sperm function and fertilization; however, highly the toxic content of Zn2+ may have a negative effect on sperm quality. Although many studies prove the association between seminal plasma Zn2+ concentration and sperm physiology, it certainly cannot be said that seminal Zn2+ deficiency causes infertility. The addition of zinc in the micromolar range to a sperm medium in vitro can contribute to the amelioration of sperm motility and capacitation to fertilize the oocyte. Zinc supplementation should be considered as a good player for the improvement of in vitro-assisted reproduction procedures, although Zn2+ dietary supplementation has not already been proved to ameliorate pregnancy rate in humans. Altogether, the currently available data revealed the importance of zinc ions for male fertility, which could be important to improve human reproductive health and the reproductive efficiency in agriculturally important livestock species. Further studies in fertile and infertile men are required to prove this claim, and the determination of the seminal Zn2+ level in these men will help to solve this question.An adequate Zn


Transient receptor potential (TRP) or transient receptor potential channels are a highly diverse family of mostly non-selective cation channels. In the mammalian genome, 28 members can be identified, most of them being expressed predominantly in the plasma membrane with the exception of the mucolipins or TRPMLs which are expressed in the endo-lysosomal system. In mammalian organisms, TRPMLs have been associated with a number of critical endo-lysosomal functions such as autophagy, endo-lysosomal fusion/fission and trafficking, lysosomal exocytosis, pH regulation, or lysosomal motility and positioning. The related non-selective two-pore cation channels (TPCs), likewise expressed in endosomes and lysosomes, have also been found to be associated with endo-lysosomal trafficking, autophagy, pH regulation, or lysosomal exocytosis, raising the question why these two channel families have evolved independently. We followed TRP/TRPML channels and TPCs through evolution and describe here in which species TRP/TRPMLs and/or TPCs are found, which functions they have in different species, and how this compares to the functions of mammalian orthologs. The earliest evidence for life on Earth dates back some 3.5 billion years . First, Drosophila melanogaster [Capitella teleta), indicates that TRP channels have been somewhat plastic during metazoan evolution [The founding member of the TRP family was initially discovered as a receptor-operated sensory cation channel in a blind strain of nogaster ,7,8, whinogaster ,10. Beyonogaster . The exivolution .A common feature of all TRPs is their homotetrameric assembly of six-transmembrane subunits or domains (S1–S6). Unlike other described ion channel families, activation and regulatory mechanisms of TRP channels cannot be reliably predicted based on their subfamily memberships. Often TRPs are activated by multiple stimuli, suggesting that the physiologically relevant stimulus for any given TRP will be governed by the specific cellular context ,16,17,18Accidental discoveries are often the most significant ones. Such a fortunate accident occurred in 1955, when Christian de Duve discovered the lysosome (Greek: “digestive body”) while attempting to localize the enzyme glucose-6-phosphatase . Traditi−, K+, Na+, and Ca2+ ions are embedded within lysosomal membranes. Activation of, e.g., the TRPMLs facilitates lysosomal cation efflux, decreasing the lysosomal membrane potential and facilitating v-ATPase proton pumping. Resultantly, the lysosomal resting membrane potential lies between −40 and −20 mV [Regulated ion fluxes across the lysosomal membrane are a crucial driving force for a number of processes, providing the ionic environment necessary for nutrient homeostasis, osmotic adaptation, or lysosomal trafficking and fusion . Severald −20 mV ,24,25. Td −20 mV .2+ into the cytosol from the endo-lysosomal lumen [2, a major constituent of endo-lysosomal membranes, and by the luminal pH/proton concentration which differs between various endosomal organelles and lysosomes [The endo-lysosomal channels have recently gained increasing attention as drug targets implicated in various pathologies ,32,33,34al lumen . All thrysosomes ,32,39. Tysosomes ,41. Geneysosomes . On the ysosomes ,44,45,46ysosomes .MCOLN1) is highly conserved in the animal kingdom. Furthermore, many species have more than one TRPML gene. All known genomes of tetrapods have three copies of TRPML channels, with the exception of the domesticated pig (Sus scrofa) where Mcoln3 was found to be a severely truncated pseudogene, lacking five of the six transmembrane domains and the pore-coding region [MCOLN1 gene is localized on chromosome 19, distinct from MCOLN2 and MCOLN3, which are closely located on chromosome 1 [MCOLN2 gene is located downstream in the same orientation as MCOLN3, in the genomes of most jawed vertebrates (Gnathostomes), but not pigs, Xenopus tropicalis, and zebrafish [The number of TRPML channels expressed varies between different species. Thus, studying their evolution may help to better understand their physiological roles and functions. The TRPML1 gene  contain one Mcoln gene ortholog each  genome was sequenced [Mcoln homolog  , where a total of five Mcoln homologs can be found: mcoln2 and two mcoln1- and mcoln3-like genes each  results in neurodegeneration and motor defects, resulting from impaired clearance of apoptotic cells from the fly brain. Mirroring MLIV, 1trpml flies exhibited impaired autophagy marked by accumulating macromolecules [1trpml pupae exhibit impaired amphisome/lysosomal fusion and impaired mTORC1 activity, attributable to decreased fly viability [1trpml and MLIV is exciting, it should be noted that primary mouse Mcoln1−/− neurons isolated do not exhibit abnormal mTORC1 activity, underscoring potential differences between species or neuronal subpopulations [The functional overlap between elopment . Similarolecules . It was iability . The deciability . Excitiniability . Althougulations . It woulIxodes scapularis and Tetranychus urticae express two and three TRPML channels, respectively, implying potential functional divergence [Caenorhabditis elegans Mcoln-homolog is termed cup-5 and appears ubiquitously expressed in lysosomes. The null mutation of cup-5 causes maternal effect lethality and lamellar structures similar to those observed in human MLIV cells. Transfection of cup-5 mutant nematodes with human TRPML1 partially rescues their decreased viability, albeit not as well as overexpression of cup-5 itself [Lottia gigantea and Capitella teleta) appear to be the first species to possess all six metazoan TRP channel subfamilies [Amphimedon queenslandica), Placozoa (Trichoplax adhaerens), and Cnidaria (Nematostella vectensis), contain only a few cell types, but encode at least one Mcoln gene, suggesting the Mcoln family already started growing around 600 million years ago [Insects in most cases express a single TRPML channel; however, vergence . The Cae5 itself . Lophotrfamilies . Primitiears ago ,68,69,70Monosiga brevicollis and Salpingoeca rosetta), free-living unicellular and colonial flagellate eukaryotes considered the closest extant relatives of animals, express five TRP subfamilies, among them TRPML [Capsaspora owczarzaki, demonstrating that many metazoan-type TRP channels emerged already in the unicellular common ancestors of Metazoa. In 2012, a study showed that the apusozoan protist Thecamonas trahens expresses TRPP and TRPV channels, suggesting these might represent some of the most ancient metazoan-type TRP channels [Dictyostelium discoideum located in the evolutionary tree close to Thecamonas trahens has both of them. Of note, Dictyostelium TRPML is, in contrast to mammalian TRPMLs, required for lysosomal Ca2+ uptake [Leishmania  and Trypanosoma and the alveolate protist Paramecium tetraurelia, which evolved around 700 million years ago. In another unicellular eukaryotic protist Lingulodinium polyedra TRPMs, TPPMLs and TRPPs have been identified [Two choanoflagellates P2, resulting in the release of ions and solutes from the lumen of the vacuole. However, there is no proof for direct activation of Trpy1p via PIP2 [2+, which triggers its activation and Ca2+ efflux in response to oxidative stress [Lysosomes and vacuoles share many similarities in molecular composition and function, e.g., degradation of cellular substances, ion accumulation and storage, and residency of luminal hydrolases, membrane transporters, the vacuolar proton pump and ion channels ,75,76. Frganisms ,55. Trpyrganisms . The welIP2 ,77. Trpye stress .Ulva compressa) and brown (Ectocarpus siliculosus) algae, application of mammalian TRP channel agonists and antagonists leads to a positive and negative regulation of cellular Ca2+ uptake, respectively [Chlamydomonas reinhardtii, commonly found in soil and fresh water, has a fine-tuned navigation system based on Ca2+ conductance [Chlamydomonas reinhardtii [Thecamonas trahens [Saccharomyces cerevisiae Trpy1p , and pharmacological block by BCTC or gadolinium [TRPs are found in mammals, worms, insects, and yeast, but appear mysteriously absent from land plants . In greeectively ,81, suggectively . Furtherductance ,84. In 2nhardtii  and multnhardtii . Arias-Dnhardtii . Interes trahens . The TRP trahens . CrTRPP2e Trpy1p . Two indesponses ,87. TRP dolinium ,86,87,88Arabidopsis thaliana) and marine plants  [In contrast to TRPML channels, TPCs (two-pore channels), the other group of non-selective endo-lysosomal cation channels in mammalian genomes, are widely found in terrestrial  . All pla nitens) ,90,91. T+ and Na+ [2+ and Ca2+ [TPC1 activity was first shown by Hedrich and Neher in barley mesophyll vacuoles . Upon ac and Na+ . Plant T and Na+ , 14-3-3  and Na+ , kinases and Na+ ,97, pH [ and Na+ ,98, redo and Na+ , and Mg2and Ca2+ ,100. In and Ca2+ ,102) andand Ca2+  have beetpc1-2 mutant compared to WT plants [2+ signals in the root were attenuated in tpc1-2 mutants, but increased in TPC1 overexpression lines [2+ signals, generated upon wounding, were gone upon loss of TPC1 function. This observation pointed to a role of TPC1 in systemic Ca2+ signaling [Arabidopsis thaliana TPC1 fou2 variant (fatty acid oxygenation upregulated 2) point mutation D454N leads to an increased production of the stress hormone jasmonate, even under non-stressed conditions. The fou2 plants exhibit a strong growth retardation phenotype [+ release due to TPC1 hyperactivity [2+ wave, it seems to be reasonable to suggest a supreme trigger, regulating Ca2+ and K+ fluxes [Since loss of TPC1 function does not drastically impair plant growth , its phyT plants . In contT plants . Intereson lines . Furtherignaling . The Arahenotype ,108, proactivity . Since T+ fluxes . Vacuola+ fluxes , thus tr2+-induced Ca2+ release , initially proposed by Fabiato et al. (1985) in the animal field, was adapted by Ward and Schroeder (1994) to plant research [2+ signals generated by TPC1 in Vicia faba guard cell vacuoles. However, the ionic condition used in this study was far away from the physiological concentration for Ca2+ and K+. By applying non-physiological ionic conditions, TPC1 channel-mediated Ca2+ currents were also recorded in other species, but only in the opposite direction, from cytosol to vacuole [2+ was postulated [2+-binding motifs of TPC1 [Arabidopsis thaliana TPC1 channel variant (fou2) shows increased vacuolar Ca2+ and slightly lower resting cytosolic Ca2+ levels compared to WT, which would be difficult to reconcile with TPC1 releasing Ca2+ under physiological conditions [2+ signals remains debated and needs to be further evaluated. A similar complex debate exists in the field of mammalian TPC research, where the role of TPCs in endo-lysosomal Ca2+ release remains controversially discussed.The concept of Caresearch ,112,113. vacuole ,115,116.stulated , likely  of TPC1 ,118. To  et al. 15 in the nditions . In sum,Monosiga brevicollis [Capsaspora owczarzaki and Thecamonas trahens, respectively [Drosophila melanogaster and Caenorhabditis elegans, other Protostomes such as Lophotrochozoans (Lottia gigantea and Capitella teleta) encode three orthologs of human TPCs. Deuterostomes such as sea urchin (Strongylocentrotus purpuratus) also possess three TPC isoforms  and nicotinic acid adenine dinucleotide phosphate (NAADP) in animals [2+ release from intracellular acidic stores in animals [2+ release from acidic stores can induce downstream Ca2+ release from the ER through Ca2+ induced Ca2+ release (CICR) [2+ release channels activated by NAADP, but rather Na+ release channels activated by PIP2 [2+. Furthermore, agonists were found to differentially affect the Ca2+/Na+ permeability ratio, suggesting that the cation permeability of the channel can be modulated in a ligand-dependent manner, in turn resulting in distinct effects on key lysosomal functions such as lysosomal pH and exocytosis [The general structure of metazoan TPCs is very similar to their green orthologs. The unique TPC1 gene structure (2 × 6 TMDs) contrasts the 1 × 6 TMD channels  and the 4 × 6 TMD voltage-gated calcium or sodium channels ,120. Thivicollis ) and eveectively . The oldmembrane ,123. TPC animals ,124,125.e (CICR) . It has IP2 . The disIP2 ,128 partocytosis .Loss of TPCs seems to not affect viability, as double TPC1/2 KO mice age normally . There a2+ signaling is also found in other species. Kelu et al. (2018) demonstrated that in zebrafish Ca2+ release via TPC2 from acidic stores/endo-lysosomes is required for the establishment of synchronized activity in primary motor neurons (PMNs), and postulated a role of NAADP/TPC/Ca2+ signaling in skeletal muscle differentiation [2+ signaling during zebrafish myogenesis. Knock down or pharmacological inhibition of ARC1-like leads to an attenuation of Ca2+ signaling and disruption of slow muscle cell development [Dictyostelium discoideum TPC2 disruption leads to delayed development and prolonged growth in culture, delaying expression of early developmental genes. Extracellular cAMP-induced Ca2+ signals are delayed in tpc2-disrupted cells, and sensitivity to weak bases is increased, consistent with an increase in vesicular pH [Evidence for a role of TPCs in Cantiation . Furtherelopment . In singcular pH .2+ signaling, several questions remain surrounding TPC function. It remains to be further elucidated how NAADP activates TPCs, directly or indirectly, and what the physiological consequences of ligand-dependent cation permeability changes in mammalian TPCs are. Furthermore, the physiological role of Na+ release from mammalian TPCs remains unclear, and the up- and downstream signaling pathways of TPCs need to be further elucidated.Despite extensive support for the physiological relevance of TPCs in endo-lysosomal CaCaenorhabditis elegans and Drosophila melanogaster, while they are in contrast to TRPMLs present in most plants. Nevertheless, coexistence is highly conserved throughout the Metazoa kingdom and beyond. Unlike mammalian TPCs, plant TPC1 is a Ca2+-regulated, nonselective cation channel, insensitive to PIP2 and NAADP [Thecamonas trahens and six in Amphimedon queenslandica to zero in insects and only two in humans, the number of Mcoln genes seems to increase, reaching three and even five isoforms late in evolution , suggestive of a possible increasing physiological relevance of TRPML channels during evolution.Both TRPMLs and TPCs are comparatively old proteins. They coexist in many species with some remarkable exceptions, e.g., TPCs are missing from nd NAADP . The rea


Arabidopsis thaliana and several other plant species including maize, rice, and Populus.Nitrogen (N) is an essential nutrient for plant growth and development; therefore, N deficiency is a major limiting factor in crop production. Plants have evolved mechanisms to cope with N deficiency, and the role of protein-coding genes in these mechanisms has been well studied. In the last decades, regulatory non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and long ncRNAs (lncRNAs), have emerged as important regulators of gene expression in diverse biological processes. Recent advances in technologies for transcriptome analysis have enabled identification of N-responsive ncRNAs on a genome-wide scale. Characterization of these ncRNAs is expected to improve our understanding of the gene regulatory mechanisms of N response. In this review, we highlight recent progress in identification and characterization of N-responsive ncRNAs in  Plants require 17 essential elements  to complete their life cycle . AvailabArabidopsis thaliana, expression of genes encoding high-affinity NRTs  is induced in roots under N deficiency [AtNRT2 genes are down-regulated, and nitrate absorption is switched to low-affinity mode via the AtNRT1 family [A. thaliana, several transcription factors are responsible for transcriptional regulation of these genes [NR and NiR expression in response to nitrate supply [AtNRT2 and suppress high-affinity nitrate transport under N sufficiency [NIGT1s is negatively autoregulated and is also regulated by NLPs. Integration of the NLP–NIGT1 cascade and NIGT1 autoregulation seems to orchestrate the nitrate response [Plants optimize the activities of N absorption and assimilation in response to N availability in soils by regulating expression of genes involved in these processes. In 1 family . Genes e1 family ,8 and fase genes . NIN-like supply ,11,12. Nficiency ,14. Exprresponse .A. thaliana. Lateral root (LR) growth is stimulated under moderate N deficiency (275 or 550 µM in media [TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (TAR2), an auxin biosynthetic gene involved in development of LR primordia [CLE genes  are induced in response to N deficiency in root cells, and accumulated CLE peptides interact with CLV1, resulting in repression of genes involved in LR development [Modification of root architecture is also important for plant adaptation to changing N availability ,16,17,18in media ), enablirimordia . By contrimordia ,21,22, wrimordia . CLE genelopment . RepressA. thaliana, Populus (Populus tomentosa), rice (Oryza sativa), and maize (Zea mays) [Despite these advances in research on plant response to N availability, we are still far from understanding the molecular mechanisms underlying complex spatiotemporal regulation of hundreds of genes in response to N availability. Over the last decade, a large number of non-coding RNAs (ncRNAs) have been discovered that are distinct from well-known housekeeping RNAs such as ribosomal RNA, transfer RNA, small nuclear RNA, and small nucleolar RNA . The proea mays) ,41,42,43ncRNAs can be either housekeeping or regulatory. The former group has a housekeeping role in transcription and translation, whereas the latter regulates genes . RegulatMIR genes), predominantly by RNA polymerase II, as precursors called primary miRNAs (pri-miRNAs). Mammalian miRNA host genes often encode multiple miRNAs and are frequently nested within protein-coding genes, whereas their plant counterparts are independent transcription units with a single miRNA [The major difference between miRNAs and siRNAs is their biogenesis: miRNAs are processed from transcripts that can form local RNA hairpin precursor structures, whereas siRNAs are processed from long bimolecular RNA duplexes or extended hairpins . Initialle miRNA . Pri-miRle miRNA ,47,48,49le miRNA ,51,52.trans-acting siRNAs (tasiRNAs) are best studied and, unlike many other siRNAs, act in trans to direct post-transcriptional repression of their mRNA targets [cis-antisense gene pairs, which are transcribed from opposite DNA strands of the same genomic loci (cis-NATs) [Depending on the organism and the cellular context, siRNA precursors, double-stranded RNAs (dsRNAs), may arise from bidirectional transcription (sense and antisense transcripts) of a chromosomal locus or from unidirectional transcription followed by RNA-dependent RNA polymerase activity . Endogen targets . Precursis-NATs) . Similaris-NATs) ,65,66,67In addition to their role in transcriptional silencing, intercellular mobility of small RNAs is another important aspect to be considered. Small RNAs can move either locally (cell-to-cell) or systemically (organ-to-organ) and play a role as mobile silencing signals. Several nutrient-responsive miRNAs are known to be accumulated in the phloem during starvation, indicating that those miRNAs are systemically translocated in the plant to amplify silencing signals (reviewed in ). Such aIPS1 [ASCO [PIF3 and represses its expression to regulate photomorphogenesis [COLDAIR, COLDWRAP, and COOLAIR are transcribed from FLOWERING LOCUS C (FLC) and interact with Polycomb proteins, thereby controlling epigenetic silencing of FLC mediated by histone modifications [LncRNAs are transcribed from intergenic, intronic, and coding regions, and regulate gene expression ,69. SomeIPS1  and ASCOS1 [ASCO . LncRNAsogenesis . The welications ,74,75.A. thaliana small RNA project (ASRP) database contains 218,585 unique small RNA sequences [Although mammalian ncRNAs remain the best studied, with the help of recent advances in sequencing technologies, identification of plant ncRNAs has almost caught up with the mammalian field. Several databases for plant ncRNAs have been established ; the A. equences , and GREequences . Many plequences ,28,30,78equences .A. thaliana, miRNAs have been relatively well studied. Several miRNAs have been characterized as associated with N stresses [AUXIN RESPONSE FACTOR 8 (ARF8); nitrate influx suppresses expression of miR167, allowing ARF8 to accumulate in the root pericycle cells and to initiate LR outgrowth [ARF10/16/17 and SCARECROW-LIKE PROTEIN 6 (SCL6), respectively [AUXIN-SIGNALING F-BOX PROTEIN 3 (AFB3) also control the responses of root system architecture to both external and internal N [AFB3 is induced by nitrate and promotes LR formation, whereas miR393 is induced by N metabolites, thus allowing plants to respond appropriately to N conditions [NUCLEAR FACTOR Y SUBUNIT A5 (NFYA5), a positive regulator of nitrate transporter genes AtNRT2.1 and AtNRT1.1 [ALKENYL HYDROXALKYL PRODUCING 2 (AOP2), which encodes 2-oxoglutarate-dependent dioxygenase involved in synthesis of N-containing metabolites called glucosinolates [AOP2 is post-transcriptionally repressed by miR826 and miR5090, decreasing glucosinolate synthesis and thus enabling plants to distribute N to metabolites required for survival [AOP2, implying coevolution of miR826, miR5090, and AOP2 in adaptation to N deprivation [Among N-responsive ncRNAs in stresses ,81,82,83utgrowth . N-starvectively ,86. miR3ternal N ,88. Exprnditions . miR169 AtNRT1.1 . miR826 inolates . Under Nsurvival . Interesrivation ,92.N-responsive miRNAs have been identified by isolation of small RNAs from total RNA by size fractionation followed by sequencing. Initially, a genome-wide search of nitrate-induced miRNAs using 454-sequencing technology identified miR393  reportedT5120 and TAS3 have been reported to be involved in N response [T5120 was significantly induced by 2h treatment of A. thaliana roots with nitrate [NRT2.1, NIR, NRT2.2, and NIA, and orchestrates early response to nitrate [A. thaliana, overexpression of T5120 significantly increases nitrate reductase activity, amino acid content, seedling biomass, and expression of nitrate-assimilatory genes including NIA1, NIA2, NIR, and GLN1.1, whereas nitrate uptake is not affected [T5120 gene is positioned adjacent to NIA1 on the chromosome and might regulate its expression by affecting chromatin structure or modification [APOLO, and COOLAIR [T5120-mediated control of N assimilation is required for T5120 engineering to develop crops with improved N use efficiency.Recently, response ,40. Expr nitrate . In tran COOLAIR ,96. FurtTAS3 is a precursor of tasiRNAs called tasiARFs, which target ARF2, ARF3, and ARF4 [TAS3 transcripts are bound and cleaved by miR390, an evolutionarily conserved miRNA, which triggers tasiARF production [TAS3/ARF pathway is part of the auxin-mediated molecular network that orchestrates LR formation in A. thaliana [TAS3 is significantly reduced under severe N deficiency [TAS3 positively regulates LR growth [TAS3 down-regulation in response to N deficiency suppresses LR outgrowth by increasing expression of ARFs. Consistent with this hypothesis, expression of ARF2–4, especially ARF4, is up-regulated by N deficiency, whereas TAS3 overexpression down-regulates ARF4 and significantly increases LR length, even under severe N deficiency [TAS3 decreases not only expression of ARFs but also that of NRT2.4 [TAS3-derived small RNA and the NRT2.4 transcript [TAS3-dependent cleavage of the NRT2.4 transcript by rapid amplification of cDNA ends [TAS3 as a source of multiple tasiRNAs that regulate multiple signaling pathways depending on N availability. Inhibition of LR growth in response to severe N deficiency has been explained by NRT1.1-mediated auxin transport [TAS3-derived tasiARFs  [A. thaliana roots exposed to 12 different nutrient starvation conditions  for 4 days and identified 60 lncRNAs differentially expressed (|log2 fold change| ≥ 1) under at least one nutrient deficiency in comparison with nutrient-sufficient controls [AT1G11185, AT1G67105, and TAS3 have been also reported to be N responsive by Liu et al. [AT1G67105 was predicted to bind the mRNA of an ammonium transporter gene AMT1;2 [AT1G67105 is down-regulated, whereas AMT1;2 is up-regulated, indicating the possible role of AT1G67105 in regulation of ammonium uptake by controlling expression of AMT1;2 [RNA-Seq data have provided a catalog of N-responsive lncRNAs. Liu et al.  analyzedontrols) . We havecontrols ,101. Amou et al. . For fure AMT1;2 ,102. Undf AMT1;2 . Our stuARF8 [COX [AGO1 [Genome-wide microarray-based analysis identified 14 differentially expressed miRNAs in maize  under chronic (15 days) or transient (2 h) N deficiency . They weARF8 ), (ii) gRF8 [COX ), and . UsOX [AGO1 ,109 charOX [AGO1 . Deep seOX [AGO1 ,111. AnoOX [AGO1 .Of 7245 lncRNAs identified by RNA-Seq analysis in maize leaves under N sufficiency or N deficiency, 637 were responsive to low N . Coexprecis-NAT pairs, AMT1.1–cis-NATAMT1.1 and AMT1.2–cis-NATAMT1.2. In general, mRNA–cis-NAT pairs whose partial or entire sequences are complementary to other transcripts form dsRNAs that can be cleaved by a Dicer endonuclease to produce small RNAs [AMT1 mRNA itself rather than from dsRNAs, indicating a different mechanism of small RNA biogenesis [cis-NATs are transcribed from the AMT gene loci of other species such as A. thaliana, barley (Hordeum vulgare), and maize, cis-NATAMT might have an evolutionally conserved role in N response.Expression of miRNAs in rice roots exposed to different N sources and concentrations has been comprehensively analyzed by small RNA-Seq . In N-stall RNAs ,115. Howogenesis . As cis-Populus is a fast-growing tree of high economic and environmental value, especially in timber production and forestry. Expression profiling of ncRNAs in P. tomentosa plantlets under N deficiency has recently been performed [Populus and will help to engineer woody plants with improved N-use efficiency. There is no report available on genome-wide identification of N-responsive ncRNAs in other tree species at this time.erformed ,116,117.erformed  identifiGlycine max) [Kufri Jyoti) [Camellia sinensis) [Hordeum vulgare) [Triticum aestivum and Triticum turgidum) [Further to the plant species described above, N-responsive miRNAs have also been identified from dicot crop species including soybean (ine max) , potato i Jyoti) , tea plainensis) , and frovulgare)  and wheaurgidum) ,124,125,MIR genes have originated mainly by duplication of pre-existing MIR genes or protein-coding genes [MIR genes in a lineage-specific manner, which may have allowed organisms to develop more complex gene regulatory networks to adapt to various stress conditions. From a practical perspective, ncRNAs are attractive targets of genome engineering to enhance the stress tolerance of crops without disrupting any essential genes.It is now obvious that the plant genome produces tens of thousands of lncRNAs . The seqng genes . In adding genes , suggestN response in plants has been extensively studied from multiple perspectives such as morphology, development, fertility, metabolites, hormonal signaling, and gene expression. However, how the expression of N-responsive genes is differentially and appropriately regulated depending on N conditions remains to be further investigated. ncRNAs are important regulators of gene expression, and molecular and functional characterization of N-responsive ncRNAs will extend our understanding of gene regulatory mechanisms in N response. Although a large number of N-responsive ncRNAs have been identified in several different plant species over the last decades, dissecting their role and regulation remains a major challenge. Further studies on ncRNAs that provide experimental validation of their biological function are of great importance in the coming decades. Moreover, in silico approaches to predict ncRNA functions using multiple sources of different high-throughput data are necessary to accelerate ncRNA characterization . Enginee


In this review article, we found the most common acupoints for the relief of nervous system disorders including GV20, GV14, ST36, ST37, and LI4. Acupuncture exhibits dual regulatory functions of activating or inhibiting different p38 MAPK pathways, contributing to an overall improvement of clinical symptoms and function in several nervous system diseases.Acupuncture is clinically used to treat various diseases and exerts positive local and systemic effects in several nervous system diseases. Advanced molecular and clinical studies have continually attempted to decipher the mechanisms underlying these effects of acupuncture. While a growing understanding of the pathophysiology underlying several nervous system diseases shows it to be related to inflammation and impair cell regeneration after ischemic events, the relationship between the therapeutic mechanism of acupuncture and the p38 MAPK signal pathway has yet to be elucidated. This review discusses the latest advancements in the identification of the effect of acupuncture on the p38 signaling pathway in several nervous system diseases. We electronically searched databases including PubMed, Embase, and the Cochrane Library from their inception to April 2020, using the following keywords alone or in various combinations: “acupuncture”, “p38 MAPK pathway”, “signaling”, “stress response”, “inflammation”, “immune”, “pain”, “analgesic”, “cerebral ischemic injury”, “epilepsy”, “Alzheimer’s disease”, “Parkinson’s disease”, “dementia”, “degenerative”, and “homeostasis”. Manual acupuncture and electroacupuncture confer positive therapeutic effects by regulating proinflammatory cytokines, ion channels, scaffold proteins, and transcription factors including TRPV1/4, Na Acupuncture is a form of therapy that has been practiced for more than 3000 years in Asia ,2. MedicAcupuncture is widely used to treat various diseases and exerts positive effects, including analgesia, at both local and systemic levels ,10,11,12MAPKs are a large group of evolutionarily conserved proteins in the plant and animal kingdoms. MAPKs have been implicated in diverse cellular processes including cell survival, proliferation, differentiation, and migration. dP-consensus) designates a MAPK protein to its cognate subfamily, and the p38 MAPK subfamily bears the Thr-Gly-Tyr (TGY) dual-phosphorylation domain [Three major subfamilies of MAPK proteins have been defined: ERK, JNK, and the p38 MAPKs . The midn domain ,78.The most extensively studied MAPK pathways are activated by dual-specificity serine-threonine/tyrosine kinases (STK) termed MAPK kinases , which are activated by MAPKK kinases (MAPKKKs or MAP3Ks). For the p38 MAPK pathway, MKK3 and MKK6 serve as MKKs, and the identified MAPKKKs include MAPKKK2, MAPKKK3, ASK-1, tumor progression locus-2 (Tpl2), and transforming growth factor-β-activated kinase 1 (TAK-1) of the non-canonical transforming growth factor-β (TGFβ) pathway. In general, ERK proteins are primarily activated by growth factors; JNKs are activated by stress-, differentiation-, and growth-related factors; and p38 is activated by stress-related factors ,82,83,84The p38 MAPK family comprises p38α, p38β, p38γ, and p38δ , and p38δ is expressed in endocrine glands ,114,115.Nine members of the dual-specificity phosphatases specific for MAPKs, termed MKPs, have been reported. Each member has specific substrates, tissue distribution, and subcellular localization. For example, MAPK phosphatase 7 (MKP-7) binds to and inactivates p38α and p38β through dephosphorylation; however, it does not interact with p38γ and p38δ; MKP-5 and CL100/MKP-1 also bind to p38α and p38β, but not to p38γ or p38δ  Figure .Several studies have revealed a direct interaction of the N-terminal region of the MAPKK with a docking groove present on the surface of the MAPK distant from the catalytic active site. The second determinant of MAPKK specificity is the structure of the MAPK activation loop harboring the Thr-Xxx-Tyr dual-phosphorylation motif. The specificity of these interactions partly mediates the potential of an individual MAPKK to selectively activate a particular MAPK. A recent study emphasized that dynamic changes are necessary for enzyme activity . For insMkk3, Mkk4, and Mkk6 genes on the p38 MAPK signaling pathway [In yeast, only a single MAPKK appears to activate each MAPK, whereas mammalian MAPK signaling modules include more than one MAPKK. The MAPKKs responsible for activating the p38 MAPK pathways appear to be specific to cell type and stimulus ,114,116. pathway ; they fo pathway ,122,123.Several MAPKKKs have been implicated in the regulation of p38 MAPK signaling, including the mixed-lineage kinases (MLKs), apoptosis signal-regulating kinase 1 (ASK-1), TAK-1, and some members of the MAPK/ERK kinase kinase (MEKK) family. Low-molecular-weight guanosine-5′-triphosphate (GTP)-binding proteins of the Rho subfamily, including Ras-related C3 botulinum toxin substrate 1 (Rac1), the erythrocyte membrane glycoprotein Cd242, the nonlipid modified Ras-related protein (Rit), the transcription termination factor Rho, and heterotrimeric G-protein-coupled receptors (GPCRs) contribute to p38 activation upstream of MAPKKKs ,134,135.MKK-independent activation is achieved through autophosphorylation and activation of p38α after interaction with TGFβ-activated kinase 1 (TAB1), which appears to activate p38α via the 5’ AMP-activated protein kinase (AMPK) in ischemic heart tissue . TAB1 seAnother MKK-independent mechanism underlying p38α activation has been observed in T-cell stimulation, wherein p38α is activated by T-cell antigen receptor (TCR)-mediated stimulation through p38α phosphorylation on a noncanonical activating residue, Tyr323. This activated p38α alters its structural conformation, phosphorylating third-party substrates and its TGY motif .Transient receptor potential vanilloid receptors (TRPVs) are mechanosensitive channels highly associated with nervous system functions including pain, memory, and mechanical sensations. Furthermore, capsaicin receptor TRPV1 is a key regulator of pain and inflammation and is upregulated in microglia in the brain, especially in the anterior cingulate cortex ,139,140.Furthermore, several studies have examined biomarkers of nerve damage, including astrocytic marker glial fibrillary acidic protein (GFAP), microglial marker ionized calcium-binding adapter molecule 1 (Iba-1), S100 calcium-binding protein B (S100B), and the receptor for advanced glycation end-products (RAGE), revealing marked upregulation of these molecules in the dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) of Complete Freund’s adjuvant (CFA)-treated mice. This inflammatory effect was reversed through electroacupuncture, which achieved an equivalent result to that of TRV1 gene deletion .Microglia cells are resident macrophages in the central nervous system (CNS) with a small soma with thin and branched processes. In the case of neural injury, their processes rapidly migrate toward the site of injury. In peripheral nerve injury, spinal cord microglia are activated. Spinal P2X4 receptors (P2X4Rs), phosphorylated p38 MAPK (p-p38-MAPK), and brain-derived neurotrophic factor (BDNF) are upregulated in spared nerve injury (SNI) rats . BDNF siThese results suggest that increasing the expression of BDNF synthesis, the downregulation of P2X4Rs, and the inhibition of p38 phosphorylation might account for the therapeutic effects induced by acupuncture in nerves injury. Previous in vitro and a few in vivo studies have reported positive nerve growth-promoting effects of electric fields where the cathode was placed toward the distal end of the injured nerve stumps ,153,154.Together with several previous clinical studies ,157,158,A). Administering EA at 2 Hz at acupoint ST36-37 reportedly relieved neuropathic pain through long-term depression (LTD) of the C-fiber [B) and the serotonergic inhibitory systems through modulation of μ-opioid and 5-hydroxytryptamine 1 (5-HT1) receptors [Furthermore, the analgesic effect of low-frequency (2 Hz) EA is exerted on the noradrenergic descending pathway, involving the modulation of spinal GABAergic nerves  is rapidly activated by heat or bradykinin and translocates, assisted by scaffolding proteins, from intracellular compartments to the plasma membrane. In rat DRG, the TRPV1 signaling pathways involve the phosphatidylinositol 3-kinases (PI3K), PKC, and calmodulin-dependent protein kinase II (CaMKII) ,163. TheIn contrast to physiological pain, pathological pain does not depend on the presence of tissue-damaging stimuli. Neuropathic pain can be agonizing, potentially persisting over long periods, and it is often resistant to known painkillers. Increasing evidence indicates that spinal microglia react and undergo a series of changes that directly influence the establishment of painful peripheral neuropathy ,169,170.Since MAPKs significantly contribute to the development of hyperalgesia, the inhibition of any of the three pathways, namely the ERK, p38, and JNK pathways, can rescue inflammatory or neuropathic pain. Several studies have reported that 2- and 15-Hz EA can downregulate cerebral TRPV4 expression and attenuate chronic constriction injury (CCI)-induced neuropathic pain in an animal model ,178,179.Presurgical sham-, low-, and high-frequency EA treatments were reported to significantly reduce the postoperative patient-controlled analgesia morphine requirement; moreover, both low- and high-frequency EA decreased opioid-related side effects including nausea and dizziness throughout the first postoperative day ,177. Amov), TRPV1, and acid-sensing ion channel 3 (ASIC3) in DRG neurons in response to proinflammatory cytokines is a key component of inflammatory pain [Furthermore, overexpression of the voltage-gated sodium channel . The origin of recurrent headache accompanied by visual or sensory symptoms is speculated to involve the hypothalamus, brain stem, and cortex. Current theories suggest that migraine is a neurovascular disorder involving cortical spreading depression, neurogenic inflammation, and vasodilation ,195. OwiA randomized controlled trial investigated the efficacy and tolerability of acupuncture in comparison with topiramate treatment in chronic migraine prophylaxis. The effectiveness of acupuncture was similar to or greater than that of prophylactic pharmacotherapy, with fewer side effects in migraine . The selThe difference between transcutaneous electrical nerve stimulation (TENS) and EA is that TENS involves the application of the cathode at the affected area rather than at acupoints. Furthermore, TENS is used to reduce mechanical hyperalgesia in knee joint inflammatory pain ,197, altAlthough proinflammatory cytokines are regulated at different levels, the role of the p38α MAPK pathway in posttranscriptional regulation has received increasing attention. Numerous p38 MAPK–regulated mRNAs often contain an adenylate-uridylate-rich element (termed AU-rich element or ARE) in the 3′ untranslated region (3′UTR) ,201. TheThese results are consistent with those of several previous animal and clinical studies reporting that EA improves analgesia during the acute phase posttraumatic healing and pain after major surgery ,177. NumDuring a cerebral ischemia (CI) event, hypoxia is induced in the brain, followed by the upregulation of various cytokines and potentially neurotoxic molecules. Nitric oxide reportedly influences cerebral oxygen vasoreactivity during severe hypoxia . Both maHeat shock proteins (Hsp) significantly contribute to cellular regenerative processes in injured tissues because Hsp is upregulated during stem cell differentiation; however, the depletion or inhibition of Hsp70/Hsc70 impairs this process . Hsp27 aAnother study administered 2 Hz EA the GV20 and ST36 acupoints in rat models of cerebral I/R injury and observed lowered peak levels of adrenocorticotrophic hormone and HSP70, suggesting that EA may inhibit excessive stress, reduce inflammation, and promote neural repair, thus facilitating healing in ischemic stroke . The mecUncaria rhynchophylla is a herb used in traditional Chinese medicine for epilepsy management; its therapeutic effect was reportedly associated with attenuated mossy fiber sprouting, astrocyte proliferation, S100B protein overexpression, and increased hippocampal neuronal survival [Epilepsy is characterized by an abnormal electric discharge of the brain, especially in the hippocampus. Epilepsy is usually associated with neuronal loss, astrocyte proliferation, mossy fiber sprouting, and synaptic reorganization in the hippocampus ,218,219.Acupuncture has been applied for clinically managing epilepsy. Previous studies have reported that auricular acupuncture positively influences drug-resistant epilepsy patients, potentially through vagus nerve stimulation, attenuating the hyperactive hippocampus, regulating inflammatory cytokine pathways, protecting the hippocampus from apoptosis, and ameliorating the sprouting of mossy fibers in the hippocampus ,220,221.Acupuncture exerts antiepileptic effects by inducing anatomical changes and changes in neurotransmitter, inflammatory cytokine, and transcription factor levels. Regarding studies to date, the related mechanisms primarily involve the suppression of TRPA1/pERK and TLR4/ERK pathways and activation of the PI3K/Akt pathway. Although some studies have reported the inactivation of the TLR4 pathway, accompanied by a reduction in pCaMKIIα, pERK, pp38, pJNK, and pNFκB levels , the rolExternal noxious stimulation alters intracellular signal transduction pathways in primary afferents, potentially contributing to pain hypersensitivity. This pathway proceeds via TRPVs, especially TRPV1 and TRPV4, leading to the rapid phosphorylation of p38 MAPK in the afferent neurons and the induction of hyperalgesia. Recent studies have revealed that thermal stimulation may not be the only trigger for TRPV activation ,223. KaoMicroglia, the endogenous macrophages of the CNS, monitor their territory through a constant movement of their elaborated thin processes and responses to local stressors and immune disruptions. Clinical studies and preclinical animal models have implicated the dysregulation and overproduction of proinflammatory cytokines from activated microglia in the CNS as contributors to the pathophysiology of chronic neurodegenerative disorders including Alzheimer’s disease , ParkinsFew studies have focused on the effects of androgens on neuroinflammation. Yang et al. investigated the neuroprotective role of androgens  in lipopolysaccharide (LPS)-induced neuroinflammation, neuronal damage, and behavioral dysfunction . DHT potLigusticum chuanxiong and Angelica sinensis) and is speculated to help treat cardiovascular and cerebrovascular diseases by preventing thrombosis. Furthermore, sodium ferulate prevents Aβ-induced MKK3/MKK6-p38-Hsp27 signaling and reduces apoptosis in the rat hippocampus [Sodium ferulate is used in traditional Chinese medicine  are the accumulation of extracellular plaques and intracellular neurofibrillary tangles that are composed of filaments of β-amyloid polymers and the neuronal microtubule-associated protein Tau, respectively. Elevated β-amyloid levels in an AD brain are speculated to induce microglial activation and the consequent release of proinflammatory cytokines induced by the p38 MAPK pathway, potentially contributing to AD pathogenesis together with other disorders including neuronal injury, trauma, ischemia, and the accumulation of oxidants associated with brain aging ,18,224.Since more than half of the phosphorylation sites in paired helical filament–Tau are serine and threonine residues followed by proline, members of the MAPK family, especially p38α, potentially play an important role in phosphorylating Tau . ConsideOther studies have focused on the receptor for advanced glycation end products (RAGE), a multiligand member of the immunoglobulin superfamily of cell surface molecules, which serves as a receptor for Aβ on neurons, microglia, astrocytes, and endothelial cells of blood vessels. RAGE is upregulated in brain regions affected by AD. A murine transgenic model revealed that RAGE is a potential therapeutic target for AD .Several studies have reported that EA and manual acupuncture have a therapeutic effect on AD. For example, EA at acupoint GV20 suppresses Aβ generation . StimulaParkinson’s disease is a neurodegenerative disorder that causes severe motor impairment owing to loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Several studies have focused on PD pathophysiology and reported potential therapeutic targets for PD.One of the direct neuroprotective effects is exerted by the protein deglycase DJ-1, which inhibits ASK1 through the nuclear sequestration of death domain–associated protein (Daxx) ,231,232.A recent study reported that EA at acupoint KI3 reduced the excitotoxicity of dopaminergic neurons by regulating the NMDA receptor function and thus may potentially serve as a novel therapeutic approach for PD . The levFibromyalgia is a common chronic pain syndrome characterized by chronic widespread mechanical pain. TRPV1 and TRPV4 are believed to play a crucial role in the pathophysiology of fibromyalgia. EA at 2 Hz at bilateral ST36 acupoints was reported to reduce long-lasting mechanical hyperalgesia by downregulating TRPV4, p-p38, p-JNK, and pCREB in the peripheral nervous system and CNS ,239.Taken together, acupuncture exerts anti-inflammation, anti-apoptosis, and neuroprotection effects via modulating proinflammatory cytokines, increasing the levels of neurotrophic factors and modulating signaling pathways, such as p38 MAPKs, Raf/MAPK/ERK1/2, TLR4/ERK, PI3K/AKT, AC/cAMP/PKA, ASK1–JNK/p38, and downstream CREB, JNK, m-TOR, NF-κB, and Bcl-2/Bax balance. EA relieves acute pain through inhibiting the TRPV1 signaling and PI3K/PKC/CaMKII pathway. EA also promote the release of opiates to activate μ-, δ-, and κ-opioid receptors, therefore producing immediate and persistent analgesic effects. Furthermore, EA can downregulate TRPV1-mediated inflammation and the release of proinflammatory cytokines, GFAP, Iba-1, and S100B by inhibiting the phosphorylation of p38 MAPK, therefore inhibiting microglial activation that triggers nerve damage and pain. Combining acupuncture to the treatment of a cerebral ischemic/hemorrhagic event can have a positive effect; an upregulation of BDNF synthesis, downregulation of P2X4Rs, and inhibition of p38 phosphorylation leads to the activation of the Raf-1/MEK1/2/ERK1/2/p90RSK/Bad signaling pathway, which contributes to the downregulation of caspase-3-dependent neuronal apoptosis. Acupuncture also inhibits the TNF-α/TRADD/FADD/cleaved caspase-8/cleaved caspase-3 apoptotic pathway in the ischemic cortical penumbra after reperfusion. In the case of treating epilepsy and seizure, several antiepileptic mechanisms have been proposed, including the downregulation of JNK or ERK1/2 and proinflammatory factors  in the cerebral cortex and hippocampus. Auricular EA can reduce hippocampal hyperactivity through TRPA1 and PKCε/PKCα/pERK1/2 signaling pathways. Furthermore, acupuncture can regulate p38γ and p38δ and reduce Tau hyperphosphorylation in neurodegenerative diseases. Acupuncture also exhibits a therapeutic effect that was reported to involve the regulation of DJ-1, ASK1, Daxx, phosphorylated PKA, PKC, α-Ca2+/CaMKII, pERK, and CREB. Acupuncture exerts curative effects in migraine through local analgesia, anti-inflammation, neuropeptide regulation, cytoskeleton remodeling, cell repair, and improvement in overall homeostasis. Furthermore, the downregulation of TRPV4, p-p38, p-JNK, and pCREB in the peripheral nervous system and CNS may account for the therapeutic effect of acupuncture in fibromyalgia.This literature review based on the results of previous studies where we electronically searched databases including PubMed, Embase, and the Cochrane Library from their inception to April 2020, using the following keywords alone or in various combinations: “acupuncture”, “p38 MAPK pathway”, “signaling”, “stress response”, “inflammation”, “immune”, “pain”, “analgesic”, “cerebral ischemic injury”, “epilepsy”, “Alzheimer’s disease”, “Parkinson’s disease”, “dementia”, “degenerative”, and “homeostasis”. The schematic explanation of the searching process is presented as Among the aforementioned cell pathways, the p38 MAPK signaling pathway plays a role in the therapeutic effect of acupuncture in several nervous system diseases. EA inhibits the ascending pain pathway and intracellular p38-mediated inflammatory pathway by stimulating peripheral opioid receptors. Furthermore, EA promotes endogenic analgesic mechanisms, thus exerting immediate local analgesic effects and rescuing CNS-induced chronic pain. Acupuncture also counteracts p38 MAPK signal transduction, reduces Aβ deposits in the brain, and improves learning and memory in AD patients.Acupuncture exhibits a dual regulatory function through the activation or inhibition of different p38 MAPK pathways and contributes to an overall improvement of clinical symptoms and physiological functions in several nervous system diseases. The p38 MAPK pathway potentially induces both protective and inhibitory effects in highly similar systems. Further studies are required to identify and characterize the various substrates of these kinases involved in cell differentiation/cell repair, neurotoxicity, and neurodegeneration, thus potentially furthering the current understanding of the mechanism underlying acupuncture in treating these diseases.


Diffuse large B cell lymphoma (DLBCL) is a heterogenous disease that has been distinguished into at least two major molecular entities, the germinal center-like B cell (GCB) DLBCL and activated-like B cell (ABC) DLBCL, based on transcriptome expression profiling. A recurrent ch11q24.3 gain is observed in roughly a fourth of DLBCL cases resulting in the overexpression of two ETS transcription factor family members, ETS1 and FLI1. Here, we knocked down ETS1 expression by siRNA and analyzed expression changes integrating them with ChIP-seq data to identify genes directly regulated by ETS1. ETS1 silencing affected expression of genes involved in B cell signaling activation, B cell differentiation, cell cycle, and immune processes. Integration of RNA-Seq (RNA sequencing) data and ChIP-Seq (chromatin immunoprecipitation sequencing) identified 97 genes as bona fide, positively regulated direct targets of ETS1 in ABC-DLBCL. Among these was the Fc receptor for IgM, FCMR , which showed higher expression in ABC- than GCB-DLBCL clinical specimens. These findings show that ETS1 is contributing to the lymphomagenesis in a subset of DLBCL and identifies FCMR as a novel target of ETS1, predominantly expressed in ABC-DLBCL. Diffuse large B cell lymphoma (DLBCL) constitutes the most common type of lymphoma, accounting for 30–40% of new cases each year . It is aETS1 and FLI1, two transcription factors belonging to the ETS family of proteins  and AffGSE10846  and GSE2GSE10846 ) were usTo identify genes and pathways regulated by ETS1 in ABC-DLBCL, we performed microarray-based gene expression profiling in three ABC-DLBCL cell lines  after silencing of the transcription factor using siRNA . Functiop value < 0.05) , this time performing RNA-Seq after ETS1 silencing by siRNA . Functio < 0.05) : 174 gened genes . The CD5 biology ,34,35, Cnockdown . In addin , as well for other genes  that had been identified only in the RNA-Seq analysis  as examples.To identify direct targets of ETS1, we took advantage of the ChIP-Seq data available at Cistrome database . A list d a GSEA B. FigureExploiting three additional ChiP-Seq datasets  while no expression was seen in GCB-DLBCL (n = 7) cell lines  across different series of DLBCL clinical specimens  . HoweveSince FCMR can have a modulatory effect on BCR signaling pathways ,44,45, tWe presented a full investigation of networks regulated by the ETS1 transcription factor in ABC-DLBCL, performing transcriptome profiling after gene silencing, followed by functional annotation and integration with Chip-Seq data.bona fide ETS1 direct targets. ETS1 appeared to regulate important biologic pathways: BCR activation, B cell differentiation, proliferation, and antiapoptotic pathways. These features can all be related to the main phenotype of ABC-DLBCL , indicating that ETS1 contributes to the molecular pathogenesis of this subset. The effect was largely due to a direct regulation of genes involved in these pathways, as shown by their identification as true CD79A, ARHGAP9, GPSM3, PTPN7, SASH3, HCST, RGS1, CD52, and FCMR, which are part of signaling pathways known to promote B cell proliferation and survival. Together with PAX5, ETS1 interacts with the mouse mb-1 promoter coding for CD79 [Among the ETS1 positively regulated targets there were for CD79 . Accordifor CD79 ,5,6. Ourfor CD79 . ARHGAP9 codes for a Rho GTPase, playing a role in adhesion processes of hematopoietic cells to extracellular matrix, including an inhibitory activity on MAPK signaling . Due to FCMR gene codes for an immunoglobulin receptor that is highly selective for IgM and can contribute to B cell activation [As already mentioned, other transcription factors important for normal and neoplastic B cells are BCL6, BLIMP1, and PAX5. The integration of ETS1 data with publicly available ChiP-Seq datasets ,39,40 intivation ,33. Howetivation . Mouse mtivation ,57. Althtivation , in the tivation . We obsetivation . Howevertivation . Our study showed that ETS1 regulates pathways that are fundamental for ABC-DLBCL, indicating that the gene contributes to the pathogenesis of this lymphoma subtype. Among ETS1 direct targets, we identified FCMR, which was significantly more expressed in ABC-DLBCL than GCB-DLBCL and contributes to an increased cell growth with an upregulation of pETS1 and pAKT.