Surfactin induces autophagy, apoptosis, and cell cycle arrest in human oral squamous cell carcinoma.

OBJECTIVES: To investigate the anticancer effects and underlying mechanisms of surfactin on human oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: The capacity of surfactin to induce apoptosis, autophagy, and cell cycle arrest of two different human OSCC cell lines was investigated by cell viability, acridine orange staining, and cell cycle regulatory protein expression, respectively. The signaling network underlying these processes were determined by the analysis of reactive oxygen species (ROS) generation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, endoplasmic reticulum (ER) stress-related protein levels, calcium release, mitogen-activated protein kinases activation, and cell cycle regulatory protein expression through corresponding reagents and experiments under various experimental conditions using specific pharmaceutical inhibitors or small interfering RNAs. RESULTS: Surfactin was able to induce apoptosis through NADPH oxidase/ROS/ER stress/calcium-downregulated extracellular signal-regulated kinases 1/2 pathway. Surfactin could also lead to autophagy that shared the common regulatory signals with apoptosis pathway until calcium node. Cell cycle arrest at G2 /M phase caused by surfactin was demonstrated through p53 and p21 accumulation combined p34cdc2 , phosphorylated p34cdc2 , and cyclin B1 inhibition, which was regulated by NADPH oxidase-derived ROS. CONCLUSION: Surfactin could induce apoptosis, autophagy, and cell cycle arrest in ROS-dependent manner, suggesting a multifaced anticancer agent for OSCC.

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK-dependent Cyclooxygenase-2.

Obesity is a world-wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro-inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS-modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase-2 (COX-2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS-stimulated gene expression of COX-2 together with the phosphorylation of p47phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase-dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS-mediated COX-2 expression and preadipocyte proliferation. Moreover, LPS-induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX-2 or PEG2 receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK-dependent COX-2 expression.

Nrf2/HO-1 partially regulates cytoprotective effects of carbon monoxide against urban particulate matter-induced inflammatory responses in oral keratinocytes.

INTRODUCTION: Exposure to airborne particulate matter (PM) increases the proportion of oral inflammatory diseases. During the formation of inflammatory conditions, the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome activation plays an important regulator. Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by heme oxygenase-1 (HO-1), has been shown to own cytoprotective effects including anti-inflammation and antioxidant. Here, we determined the novel mechanisms of carbon monoxide releasing molecule-2 (CORM-2) on PM-induced inflammatory responses in human oral keratinocytes (HOKs). METHODS: The effects of CORM-2 on the expression of various inflammatory proteins induced by PM were determined by Western blot, real-time PCR, promoter assay, and ELISA. The involvement of signaling molecules in these responses was studied by using the selective pharmacological inhibitors and siRNAs. RESULTS: We proved that PM enhanced C-reactive protein (CRP) levels, NLRP3 inflammasome and caspase-1 activation, and IL-1ß release, which were reduced by preincubation with CORM-2. Transfection with PKCα siRNA and preincubation with the ROS scavenger (N-acetyl-cysteine, NAC), an inhibitor of NADPH oxidase (diphenyleneiodonium, DPI), or the mitochondria-specific superoxide scavenger (MitoTEMPO) inhibited PM-mediated inflammatory responses. In addition, PM-regulated PKCα and NADPH oxidase activation as well as NADPH oxidase- and mitochondria-derived ROS generation were inhibited by CORM-2, but not inactivate CORM-2 (iCORM-2) pretreatment. At the end, we confirmed that CORM-2 improved PM-induced inflammatory responses via the induction of Nrf2 activation and HO-1 expression. CONCLUSION: We suggest that CORM-2 inhibits PM-induced inflammatory responses in HOKs via the inhibition of PKCα/ROS/NLRP3 inflammasome activation combined with the induction of Nrf2/HO-1 expression.

EFLA 945 restricts AIM2 inflammasome activation by preventing DNA entry for psoriasis treatment.

Psoriasis is a chronic inflammatory skin disease that affects about 2% of the general population. Activation of the Absent in Melanoma 2 (AIM2) inflammasome is crucial for immune defense, but it can also cause inflammatory and autoimmune diseases, including psoriasis. We currently lack an AIM2 inflammasome inhibitor that could be used therapeutically. Here, we show that EFLA 945, a safe product of red grape vine leaf extracts, can restrict AIM2 inflammasome activation. Mechanistically, EFLA945 prevents DNA entry into THP-1-derived macrophages, and thereby inhibits cytoplasmic DNA-dependent apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, caspase-1 activation, and the secretion of interleukin (IL)-1ß and IL-18. The major phytochemicals of EFLA 945, resveratrol and peonidin 3-O-glucoside (P3G), appear to be the potential bioactive compounds responsible for its ability to restrict AIM2-dependent IL-1ß secretion. Importantly, in an in vivo mouse model, EFLA 945 attenuates imiquimod (IMQ)-induced psoriasis-related pro-inflammatory responses in topical psoriatic skin, including caspase-1 activation, IL-1ß maturation, and IL-17 production, and decreases the severity of psoriasis. Together, these results demonstrate that the safe natural product, EFLA 945, can restrict the AIM2 inflammasome activation through preventing DNA entry and may prove beneficial for treating psoriasis.

Haematococcus pluvialis-Derived Astaxanthin Is a Potential Neuroprotective Agent against Optic Nerve Ischemia.

Astaxanthin, a xanthophyll belonging to the family of carotenoids, is a potent antioxidant. However, much less is known about its protective effects on the oxidative stress of ischemic optic nerve. We hypothesized that astaxanthin treatment could protect retinal ganglion cells (RGCs) from death via anti-oxidative and anti-apoptotic responses. Adult male Wistar rats were fed astaxanthin (100 mg/kg/day) by daily gavage for seven consecutive days, either before or after inducing oxidative stress in the retina by photodynamic treatment. The visual function, RGC apoptosis, macrophage infiltration in the optic nerve, expression of p-Akt, p-mTOR, SGK1, pS6K, Nrf2, p62, TNFα, Il1ß in retinas were investigated. The visual function and the RGC densities were significantly higher in both pre- and post-treatment groups. The numbers of apoptotic RGCs and extrinsic macrophage infiltration in the optic nerve were significantly decreased in both astaxanthin-treated groups. Furthermore, pre- and post-treatment of astaxanthin showed a higher expression of p-Akt, p-mTOR, Nrf2 and superoxide dismutase activity, and a lower expression of cleaved caspase-3, suggesting anti-apoptotic and anti-oxidative roles. Our findings indicate that astaxanthin can preserve visual function and reduce RGC apoptosis after ischemic insults. Including astaxanthin in daily diet as a supplement may be beneficiary for ischemic optic neuropathy.

Lipopolysaccharide promoted proliferation and adipogenesis of preadipocytes through JAK/STAT and AMPK-regulated cPLA2 expression.

The proliferation and adipogenesis of preadipocytes played important roles in the development of adipose tissue and contributed much to the processes of obesity. On the other hand, lipopolysaccharide (LPS), also known as endotoxin, is a key outer membrane component of gram-negative bacteria in the gut microbiota, and has a dominant role in linking inflammation to high-fat diet-induced metabolic syndrome. Studies suggested the potential roles of LPS in hepatic steatosis and in obese mice models. However, the molecular mechanisms underlying LPS-regulated obesity remained largely unknown. Here we reported that LPS stimulated expression of cyosolic phospholipase A2 (cPLA2), one of inflammation regulators of obesity, in the preadipocytes. Pretreatment the inhibitors of JAK2, STAT3, STAT5 or AMPK significantly reduced LPS-increased mRNA and protein expression of cPLA2 together with phosphorylation of JAK2, STAT3, STAT5 and AMPK, separately. Similarly, transfection of siRNA against JAK2 or AMPK abolished expression of cPLA2 and phosphorylation of JAK2 or AMPK together with downregulated expression of JAK2 and AMPK protein. LPS enhanced activation of STAT3 and STAT5 via JAK2-dependent manner in the preadipocytes. Transfection of JAK2 or AMPK siRNA further proofed the independence of JAK2 and AMPK in LPS-treated preadipocytes. In addition, LPS-increased DNA synthesis, cell numbers and cell viability of preadipocytes were attenuated by AACOCF3, AG490, BML-275, cPLA2 siRNA, JAK2 siRNA or AMPK siRNA. Attenuation JAK2/STAT or AMPK-dependent cPLA2 expression reduced LPS-mediated adipogenesis of preadipocytes. Stimulation of arachidonic acid or AMPK activator, A-769662, increased cell numbers and cell viability and promoted differentiation of preadipocytes. Collectively, these results indicated that LPS increased preadipocytes proliferation and adipogenesis via JAK/STAT and AMPK-dependent cPLA2 expression. The mechanisms of LPS-stimulated cPLA2 expression may be a link between bacteria and obesity and provides the molecular basis for preventing metabolic syndrome or hyperplasic obesity.

Participation of NADPH Oxidase-Related Reactive Oxygen Species in Leptin-Promoted Pulmonary Inflammation: Regulation of cPLA2α and COX-2 Expression.

Obesity is a worldwide epidemic problem and correlates to varieties of acute or chronic lung diseases such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, and pulmonary fibrosis. An increase of leptin, a kind of adipokine, in lean mice plasma has been found to impair immune responses and facilitate the infection of Klebsiella pneumoniae, resulting in increased pneumonia severity. Also, a higher leptin level is found in exhaled breath condensates of obese or asthmatic subjects, compared to healthy ones, suggesting that leptin is involved in the occurrence or exacerbation of lung injury. In previous studies, we showed that leptin stimulated cytosolic phospholipase A2-α (cPLA2α) gene expression in lung alveolar type II cells via mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB)-activated coactivator p300. Herein, we show that the in vivo application of leptin in the respiratory system upregulated the expression of inflammatory proteins cPLA2α and cyclooxygenase-2 (COX-2) together with leukocyte infiltration. Treatment with an ROS scavenger (N-acetylcysteine, NAC), an NADPH oxidase inhibitor (apocynin), or an activating protein (AP)-1 inhibitor (tanshinone IIA) attenuated leptin-mediated cPLA2α/COX-2 expression and leukocyte recruitment in the lung. Leptin increased intracellular oxidative stress in a leptin receptor (OB-R) and NADPH oxidase-dependent manner, leading to the phosphorylation of the AP-1 subunit c-Jun. In summation, leptin increased lung cPLA2α/COX-2 expression and leukocyte recruitment via the NADPH oxidase/ROS/AP-1 pathway. Understanding the inflammatory effects of leptin on the pulmonary system provides opportunities to develop strategies against lung injury related to metabolic syndrome or obesity.

Targeting ROS and cPLA2/COX2 Expressions Ameliorated Renal Damage in Obese Mice with Endotoxemia.

Obesity is associated with metabolic endotoxemia, reactive oxygen species (ROS), chronic inflammation, and obese kidney fibrosis. Although the fat-intestine-kidney axis has been documented, the pathomechanism and therapeutic targets of obese kidney fibrosis remain unelucidated. To mimic obese humans with metabolic endotoxemia, high-fat-diet-fed mice (HF group) were injected with lipopolysaccharide (LPS) to yield the obese kidney fibrosis-metabolic endotoxemia mouse model (HL group). Therapeutic effects of ROS, cytosolic phospholipases A2 (cPLA2) and cyclooxygenase-2 (COX-2) inhibitors were analyzed with a quantitative comparison of immunohistochemistry stains and morphometric approach in the tubulointerstitium of different groups. Compared with basal and HF groups, the HL group exhibited the most prominent obese kidney fibrosis, tubular epithelial lipid vacuoles, and lymphocyte infiltration in the tubulointerstitium. Furthermore, inhibitors of nonspecific ROS, cPLA2 and COX-2 ameliorated the above renal damages. Notably, the ROS-inhibitor-treated group ameliorated not only oxidative injury but also the expression of cPLA2 and COX-2, indicating that ROS functions as the upstream signaling molecule in the inflammatory cascade of obese kidney fibrosis. ROS acts as a key messenger in the signaling transduction of obese kidney fibrosis, activating downstream cPLA2 and COX-2. The given antioxidant treatment ameliorates obese kidney fibrosis resulting from a combined high-fat diet and LPS-ROS could serve as a potential therapeutic target of obese kidney fibrosis with metabolic endotoxemia.

AdipoR-increased intracellular ROS promotes cPLA2 and COX-2 expressions via activation of PKC and p300 in adiponectin-stimulated human alveolar type II cells.

Adiponectin, an adipokine, accumulated in lung system via T-cadherin after allergens/ozone challenge. However, the roles of adiponectin on lung pathologies were controversial. Here we reported that adiponectin stimulated expression of inflammatory proteins, cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of reactive oxygen species (ROS) in human alveolar type II A549 cells. AdipoR1/2 involved in adiponectin-activated NADPH oxidase and mitochondria, which further promoted intracellular ROS accumulation. Protein kinase C (PKC) may involve an adiponectin-activated NADPH oxidase. Similarly, p300 phosphorylation and histone H4 acetylation occurred in adiponectin-challenged A549 cells. Moreover, adiponectin-upregulated cPLA2 and COX-2 expression was significantly abrogated by ROS scavenger (N-acetylcysteine) or the inhibitors of NADPH oxidase (apocynin), mitochondrial complex I (rotenone), PKC (Ro31-8220, Gö-6976, and rottlerin), and p300 (garcinol). Briefly, we reported that adiponectin stimulated cPLA2 and COX-2 expression via AdipoR1/2-dependent activation of PKC/NADPH oxidase/mitochondria resulting in ROS accumulation, p300 phosphorylation, and histone H4 acetylation. These results suggested that adiponectin promoted lung inflammation, resulting in exacerbation of pulmonary diseases via upregulating cPLA2 and COX-2 expression together with intracellular ROS production. Understanding the adiponectin signaling pathways on regulating cPLA2 and COX-2 may help develop therapeutic strategies on pulmonary diseases.

Optimizing a Male Reproductive Aging Mouse Model by D-Galactose Injection.

The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8-10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging.

Endothelin-1 induces VCAM-1 expression-mediated inflammation via receptor tyrosine kinases and Elk/p300 in human tracheal smooth muscle cells.

The elevated level of endothelin-1 (ET-1) has been detected in the bronchoalveolar lavage of patients with severe asthma, acute lung injury, acute respiratory distress syndrome, and sepsis. ET-1 may affect vessel tone together with lung physiology and pathology. Vascular cell adhesion molecule-1 (VCAM-1) is one kind of adhesion molecules participating in the process of polymorphonuclear leukocyte transmigration and regulating the occurrence and amplification of tissue inflammation. However, the molecular mechanisms underlying ET-1-mediated expression of VCAM-1 on human tracheal smooth muscle cells (HTSMCs) were largely unknown. Here we reported that ET-1 stimulated expression of VCAM-1 gene on HTSMCs, which was blocked by pretreatment with the inhibitors of ET receptors, Src, matrix metalloproteinases (MMPs), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), phosphatidylinositol 3-kinase (PI3K), AKT, MEK1/2, and p300, suggesting the participation of these signaling components in ET-1-regulated HTSMC responses. Furthermore, transfection with small-interfering RNA (siRNA) of Src, AKT, p42 mitogen-activated protein kinase (MAPK), or p300 downregulated the respective proteins and significantly attenuated ET-1-induced VCAM-1 expression. ET-1 also stimulated phosphorylation of Src, EGFR, PDGFR, AKT, p42/p44 MAPK, and Elk-1 and acetylation of histone H4 on HTSMCs. Immunoprecipitation assay showed the association between Elk-1 and p300 in the nucleus. Adhesion assay revealed that the adhesion of THP-1 to HTSMCs challenged with ET-1 was increased, which was attenuated by the inhibitors of ET receptors, Src, MMPs, EGFR, PDGFR, PI3K, AKT, p42/p44 MAPK, and p300. Taken together, these data suggested that ET-1 promotes occurrence and amplification of pathology-related airway inflammation via enhancing VCAM-1 expression in an ET receptor/Src/MMP/EGFR, PDGFR/PI3K/AKT/p42/p44 MAPK/Elk-1/p300 pathway in HTSMCs.

Leptin Promotes cPLA2 Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade.

Hyperplasia or hypertrophy of adipose tissues plays a crucial role in obesity, which is accompanied by the release of leptin. Recently, obesity was determined to be associated with various pulmonary diseases including asthma, acute lung injury, and chronic obstructive pulmonary disease. However, how obesity contributes to pulmonary diseases and whether leptin directly regulates lung inflammation remains unclear. We used cell and animal models to study the mechanisms of leptin mediation of pulmonary inflammation. We found that leptin activated de novo synthesis of cytosolic phospholipase A2-α (cPLA2-α) in vitro in the lung alveolar type II cells, A549, and in vivo in ICR mice. Upregulated cPLA2-α protein was attenuated by pretreatment with an OB-R blocking antibody, U0126, SB202190, SP600125, Bay11-7086, garcinol, and p300 siRNA, suggesting roles of p42/p44 MAPK, p38 MAPK, JNK1/2, NF-κB, and p300 in leptin effects. Leptin enhanced the activities of p42/p44 MAPK, p38 MAPK, JNK1/2, and p65 NF-κB in a time-dependent manner. Additional studies have suggested the participation of OB-R, p42/p44 MAPK, and JNK1/2 in leptin-increased p65 phosphorylation. Furthermore, p300 phosphorylation and histone H4 acetylation were reduced by blockage of OB-R, p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB in leptin-stimulated cells. Similarly, blockage of the MAPKs/NF-κB/p300 cascade significantly inhibited leptin-mediated cPLA2-α mRNA expression. Our data as a whole showed that leptin contributed to lung cPLA2-α expression through OB-R-dependent activation of the MAPKs/NF-κB/p300 cascade.

Explainable machine learning model for identifying key gut microbes and metabolites biomarkers associated with myasthenia gravis.

Diagnostic markers for myasthenia gravis (MG) are limited; thus, innovative approaches are required for supportive diagnosis and personalized care. Gut microbes are associated with MG pathogenesis; however, few studies have adopted machine learning (ML) to identify the associations among MG, gut microbiota, and metabolites. In this study, we developed an explainable ML model to predict biomarkers for MG diagnosis. We enrolled 19 MG patients and 10 non-MG individuals. Stool samples were collected and microbiome assessment was performed using 16S rRNA sequencing. Untargeted metabolic profiling was conducted to identify fecal amplicon significant variants (ASVs) and metabolites. We developed an explainable ML model in which the top ASVs and metabolites are combined to identify the best predictive performance. This model uses the SHapley Additive exPlanations method to generate both global and personalized explanations. Fecal microbe-metabolite composition differed significantly between groups. The key bacterial families were Lachnospiraceae and Ruminococcaceae, and the top three features were Lachnospiraceae, inosine, and methylhistidine. An ML model trained with the top 1 % ASVs and top 15 % metabolites combined outperformed all other models. Personalized explanations revealed different patterns of microbe-metabolite contributions in patients with MG. The integration of the microbiota-metabolite features and the development of an explainable ML framework can accurately identify MG and provide personalized explanations, revealing the associations between gut microbiota, metabolites, and MG. An online calculator employing this algorithm was developed that provides a streamlined interface for MG diagnosis screening and conducting personalized evaluations.

Impact of prior bariatric surgery on outcomes of hospitalized patients with systemic lupus erythematosus: a propensity score-matched analysis of the U.S. nationwide inpatient sample.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease that primarily affects young women, has many different manifestations, and the disease severity can wax and wane. OBJECTIVES: This study aims to determine the association between prior bariatric surgery and SLE outcomes. SETTING: A population-based, retrospective study using data from the U.S. Nationwide Inpatient Sample (NIS) database between 2005 and 2018. METHODS: Data of hospitalized patients with SLE and morbid obesity were extracted. Exclusion criteria were age <18 years and missing information on mortality. Patients were separated into 2 groups: with or without prior bariatric surgery. One: 4 propensity score matching (PSM) were performed to balance the characteristics between the groups. Associations between prior bariatric surgery and inpatient outcomes were determined by logistic regressions. RESULTS: A total of 38,286 hospitalized patients with SLE and morbid obesity were identified. After exclusions and PSM, there remained 9050 subjects in the study sample (with prior bariatric surgery: 1810; without prior bariatric surgery: 7240), representing 44,952 U.S. inpatients. After adjusting for confounders in multivariable analysis, prior bariatric surgery was significantly associated with lower odds for prolonged length of stay (LOS), unfavorable discharge, and SLE-related complications. Specifically, prior bariatric surgery was significantly associated with lower risks of acute myocardial infarction (AMI)/stroke (aOR = .44, 95% CI: .30-.65), venous thromboembolism (VTE) (aOR = .74, 95% CI: .57-.96), pneumonia (aOR = .60, 95% CI: .47-.76), sepsis (aOR = .62, 95% CI: .51-.75), acute and chronic glomerulonephritis (aOR = .53, 95% CI: .38-.76), acute kidney injury (AKI) (aOR = .64, 95% CI: .52-.77), and anemia (aOR = .77, 95% CI: .68-.87). CONCLUSIONS: Prior bariatric surgery is associated with favorable in-hospital outcomes in hospitalized patients with SLE and decreased likelihood for several SLE-related complications.

Machine learning strategy for identifying altered gut microbiomes for diagnostic screening in myasthenia gravis.

Myasthenia gravis (MG) is a neuromuscular junction disease with a complex pathophysiology and clinical variation for which no clear biomarker has been discovered. We hypothesized that because changes in gut microbiome composition often occur in autoimmune diseases, the gut microbiome structures of patients with MG would differ from those without, and supervised machine learning (ML) analysis strategy could be trained using data from gut microbiota for diagnostic screening of MG. Genomic DNA from the stool samples of MG and those without were collected and established a sequencing library by constructing amplicon sequence variants (ASVs) and completing taxonomic classification of each representative DNA sequence. Four ML methods, namely least absolute shrinkage and selection operator, extreme gradient boosting (XGBoost), random forest, and classification and regression trees with nested leave-one-out cross-validation were trained using ASV taxon-based data and full ASV-based data to identify key ASVs in each data set. The results revealed XGBoost to have the best predicted performance. Overlapping key features extracted when XGBoost was trained using the full ASV-based and ASV taxon-based data were identified, and 31 high-importance ASVs (HIASVs) were obtained, assigned importance scores, and ranked. The most significant difference observed was in the abundance of bacteria in the Lachnospiraceae and Ruminococcaceae families. The 31 HIASVs were used to train the XGBoost algorithm to differentiate individuals with and without MG. The model had high diagnostic classification power and could accurately predict and identify patients with MG. In addition, the abundance of Lachnospiraceae was associated with limb weakness severity. In this study, we discovered that the composition of gut microbiomes differed between MG and non-MG subjects. In addition, the proposed XGBoost model trained using 31 HIASVs had the most favorable performance with respect to analyzing gut microbiomes. These HIASVs selected by the ML model may serve as biomarkers for clinical use and mechanistic study in the future. Our proposed ML model can identify several taxonomic markers and effectively discriminate patients with MG from those without with a high accuracy, the ML strategy can be applied as a benchmark to conduct noninvasive screening of MG.

Novel five nucleotide deletion in dysferlin leads to autosomal recessive limb-girdle muscular dystrophy.

Muscular dystrophy (MD) is a genetic disorder that causes progressive muscle weakness and degeneration. Limb-girdle muscular dystrophy (LGMD) is a type of MD that mainly causes muscle atrophy within the shoulder and pelvic girdles. LGMD is classified into autosomal dominant (LGMD-D) and autosomal recessive (LGMD-R) inheritance patterns. Mutations in the Dysferlin gene (DYSF) are common causes of LGMD-R. However, genetic screening of DYSF mutations is rare in Taiwan. Herein, we identified a novel c.2867_2871del ACCAG deletion and a previously reported c.937+1G>A mutation in DYSF from a Taiwanese family with LGMD. The primary symptoms of both siblings were difficulty climbing stairs, walking on the toes, and gradually worsening weakness in the proximal muscles and increased creatine kinase level. Through pedigree analysis and sequencing, two siblings from this family were found to have compound heterozygous DYSF mutations (c. 937+1G>A and c. 2867_2871del ACCAG) within the separated alleles. These mutations induced early stop codons; if translated, truncated DYSF proteins will be expressed. Or, the mRNA products of these two mutations will merit the nonsense-mediated decay, might result in no dysferlin protein expressed. To our knowledge, this is the first report of a novel c.2867_2871del ACCAG deletion in DYSF. Further research is required to examine the effects of the novel DYSF mutation in Taiwanese patients with LGMD.

Intestinal microbiota analysis of different Blastocystis subtypes and Blastocystis-negative individuals in Taiwan.

BACKGROUND: Intestinal parasitic infections are the most common infectious diseases among Southeast Asian migrant workers in Taiwan, especially for infections with Blastocystis hominis. However, little is known about the impact of Blastocystis subtypes (STs) on the gut microbiota. MATERIAL AND METHODS: We retrospectively evaluated the prevalence of intestinal parasites in a teaching hospital in Northern Taiwan in the period of 2015 to 2019. Blastocystis-positive stool specimens were collected for ST analysis by polymerase chain reaction in 2020. Intestinal microbiota analyses of different Blastocystis STs and Blastocystis-free individuals were conducted by 16S rRNA sequencing. RESULTS: A total of 13,859 subjects were analyzed, of which 1,802 cases (13%) were diagnosed with intestinal parasitic infections. B. hominis infections were the most prevalent (n = 1546, 85.7%). ST analysis of Blastocystis-positive samples (n=150) indicated that ST1 was the most common type, followed by ST3, ST4, ST2, ST7, and ST5. Different Blastocystis STs (ST1, ST3, and ST4) were associated with distinct richness and diversity of the microbiota. Taxonomic profiles revealed that Akkermansia muciniphila was significantly enriched for all analyzed Blastocystis STs, whereas Holdemanella biformis was more abundant in the Blastocystis-free group. Additionally, Succinivibrio dextrinosolvens and Coprococcus eutactus were specifically more abundant in ST3 carriers than in non-infected individuals. CONCLUSIONS: This study demonstrates that A. muciniphila is positively associated with all Blastocystis STs, while H. biformis was negatively associated with them. Several bacteria were enriched in specific STs, highlighting the need for further microbiota analysis at the ST level to elucidate the pathogenicity of Blastocystis.

Transactivation of EGFR/PI3K/Akt involved in ATP-induced inflammatory protein expression and cell motility.

Phenotype transition of vascular smooth muscle cells (VSMCs) is important in vascular diseases, such as atherosclerosis and restenosis. Once released, ATP may promote activation of VSMCs by stimulating cyclooxygenase-2 (COX-2), cytosolic phospholipase A(2) (cPLA(2)) expression and prostaglandin (PG)E(2) synthesis via activation of MAPKs and NF-κB. However, whether alternative signaling pathways participated in regulating COX-2 and cPLA(2) expression associated with cell migration were investigated in rat VSMCs. Western blot analysis, RT-PCR, promoter assay and PGE(2) ELISA were used to determine expression of COX-2, cPLA(2) and PGE(2). Specific inhibitors and siRNAs against various protein kinases or transcription factors were used to investigate the related signaling components in inflammatory protein induction by ATPγS. We found that ATPγS-induced COX-2 and cPLA(2) expression and PGE(2) release was attenuated by the pharmacological inhibitors or transfection with siRNA against PKCδ, c-Src, EGFR, PI3-K, Akt, p44/p42 MAPK or Elk-1. Moreover, ATPγS-stimulated phosphorylation of PKCδ, c-Src, EGFR, Akt, p42/p44 MAPK and Elk-1, suggesting the participation of PKCδ/c-Src/EGFR/PI3-K/Akt/p42/p44 MAPK cascade in mediating Elk-1 activities in VSMCs. In addition, migration assay revealed that ATPγS promoted cell mobility through up-regulation of COX-2 and cPLA(2) expression and PGE(2) release, which was attenuated by pretreatment with PGE(2) receptor antagonists. Taken together, these data showed that ATPγS up-regulated the expression of COX-2 and cPLA(2) through transactivation of PKCδ/c-Src/EGFR/PI3K/Akt/Elk-1 pathway. Newly synthesized PGE(2) acted on its receptors to promote cell motility of ATPγS-stimulated VSMCs.

Adenosine triphosphate regulates NADPH oxidase activity leading to hydrogen peroxide production and COX-2/PGE2 expression in A549 cells.

Non-small cell lung carcinoma (NSCLC) accounts for most of all lung cancers, which is the leading cause of mortality in human beings. High level of cyclooxygenase-2 (COX-2) is one of the features of NSCLC and related to the low survival rate of NSCLC. However, whether extracellular nucleotides releasing from stressed resident tissues contributes to the expression of COX-2 remains unclear. Here, we showed that stimulation of A549 cells by adenosine 5'-O-(3-thiotriphosphate) (ATPγS) led to an increase in COX-2 gene expression and prostaglandin E(2) (PGE(2)) synthesis, revealed by Western blotting, RT-PCR, promoter assay, and enzyme-linked immunosorbent assay. In addition, ATPγS induced intracellular reactive oxygen species (ROS) generation through the activation of NADPH oxidase. The increase of ROS level resulted in activation of the c-Src/epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor (NF)-κB cascade. We also found that activated Akt was translocated into the nucleus and recruited with NF-κB and p300 to form a complex. Thus, activation of p300 modulated the acetylation of histone H4 via the NADPH oxidase/c-Src/EGFR/PI3K/Akt/NF-κB cascade stimulated by ATPγS. Our results are the first to show a novel role of NADPH oxidase-dependent Akt/p65/p300 complex formation that plays a key role in regulating COX-2/PGE(2) expression in ATPγS-treated A549 cells. Taken together, we demonstrated that ATPγS stimulated activation of NADPH oxidase, resulting in generation of ROS, which then activated the downstream c-Src/EGFR/PI3K/Akt/NF-κB/p300 cascade to regulate the expression of COX-2 and synthesis of PGE(2) in A549 cells. Understanding the regulation of COX-2 expression and PGE(2) release by ATPγS on A549 cells may provide potential therapeutic targets of NSCLC.

Translational Medicine in Uremic Vascular Calcification: Scavenging ROS Attenuates p-Cresyl Sulfate-Activated Caspase-1, NLRP3 Inflammasome and Eicosanoid Inflammation in Human Arterial Smooth Muscle Cells.

We formerly proved that uremic vascular calcification (UVC) correlates tightly with oxidative elastic lamina (EL) injury and two cell fates (apoptosis and osteocytic conversion) in smooth muscle cells (SMC) of chronic kidney disease (CKD) patients and eliminating p-cresyl sulfate (PCS)-activated intracellular ROS ameliorates the MAPK signaling pathway in a human arterial SMC (HASMC) model. Nonetheless, whether ROS scavenger attenuates PCS-triggered inflammasome activation and eicosanoid inflammation in the UVC process remains unknown. Patients with lower extremity amputation were categorized into CKD and normal control group according to renal function. We used immunohistochemistry stain to analyze UVC in arterial specimens, including oxidative injury (8-hydroxy-2'-deoxyguanosine (8-OHdG) and internal EL disruption), cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX2), interleukin-1 beta (IL-1ß), caspase-1 and NLRP3. To simulate the patho-mechanism of human UVC, the therapeutic effects of ROS scavenger on PCS-triggered inflammatory pathways was explored in a HASMC model. We found CKD patients had higher circulating levels of PCS and an increase in medial arterial calcification than the control group. In CKD arteries, the severity of UVC corresponded with expressions of oxidative EL disruption and 8-OHdG. Furthermore, coupling expressions of cPLA2 and COX2 were accentuated in CKD arteries, indicative of eicosanoid inflammation. Notably, tissue expressions of IL-1ß, caspase-1 and NLRP3 were enhanced in parallel with UVC severity, indicative of inflammasome activation. From bedside to bench, ROS scavenger attenuates PCS-activated expressions of cPLA2/COX2, pro-caspase-1 and NLRP3 in the HASMC model. UVC as an inevitable outcome is predictive of death in CKD patients. Nonetheless, UVC remain pharmacoresistant despite the evolution of treatment for mineral-parathyroid hormone-vitamin D axis. Beyond the mineral dysregulation, the stimulation of pro-oxidant PCS alone results in eicosanoid inflammation and inflammasome activation. Concerning the key role of Caspase-1 in pyroptosis, cell fates of HASMC in uremic milieu are not limited to apoptosis and osteogenesis. In view of this, reducing ROS and PCS may act as a therapeutic strategy for UVC-related cardiovascular events in CKD patients.