The SASP factor IL-6 sustains cell-autonomous senescent cells via a cGAS-STING-NFκB intracrine senescent noncanonical pathway.

Senescent cells produce a Senescence-Associated Secretory Phenotype (SASP) that involves factors with diverse and sometimes contradictory activities. One key SASP factor, interleukin-6 (IL-6), has the potential to amplify cellular senescence in the SASP-producing cells in an autocrine action, while simultaneously inducing proliferation in the neighboring cells. The underlying mechanisms for the contrasting actions remain unclear. We found that the senescence action does not involve IL-6 secretion nor the interaction with the receptor expressed in the membrane but is amplified through an intracrine mechanism. IL-6 sustains intracrine senescence interacting with the intracellular IL-6 receptor located in anterograde traffic specialized structures, with cytosolic DNA, cGAS-STING, and NFκB activation. This pathway triggered by intracellular IL-6 significantly contributes to cell-autonomous induction of senescence and impacts in tumor growth control. Inactivation of IL-6 in somatotrophic senescent cells transforms them into strongly tumorigenic in NOD/SCID mice, while re-expression of IL-6 restores senescence control of tumor growth. The intracrine senescent IL-6 pathway is further evidenced in three human cellular models of therapy-induced senescence. The compartmentalization of the intracellular signaling, in contrast to the paracrine tumorigenic action, provides a pathway for IL-6 to sustain cell-autonomous senescent cells, driving the SASP, and opens new avenues for clinical consideration to senescence-based therapies.

GDP-mannose pyrophosphorylase is an efficient target in Xanthomonas citri for citrus canker control.

Xanthomonas citri subsp. citri (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this study, we demonstrated the involvement of XanB, an enzyme with phosphomannose isomerase (PMI) and guanosine diphosphate-mannose pyrophosphorylase (GMP) activities, in Xcc pathogenicity. Additionally, we found that XanB inhibitors protect the host against Xcc infection. Besides being deficient in motility, biofilm production, and ultraviolet resistance, the xanB deletion mutant was unable to cause disease, whereas xanB complementation restored wild-type phenotypes. XanB homology modeling allowed in silico virtual screening of inhibitors from databases, three of them being suitable in terms of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, which inhibited GMP (but not PMI) activity of the Xcc recombinant XanB protein in more than 50%. Inhibitors reduced citrus canker severity up to 95%, similarly to copper-based treatment. xanB is essential for Xcc pathogenicity, and XanB inhibitors can be used for the citrus canker control. IMPORTANCE: Xcc causes citrus canker, a threat to citrus production, which has been managed with copper, being required a more sustainable alternative for the disease control. XanB was previously found on the surface of Xcc, interacting with the host and displaying PMI and GMP activities. We demonstrated by xanB deletion and complementation that GMP activity plays a critical role in Xcc pathogenicity, particularly in biofilm formation. XanB homology modeling was performed, and in silico virtual screening led to carbohydrate-derived compounds able to inhibit XanB activity and reduce disease symptoms by 95%. XanB emerges as a promising target for drug design for control of citrus canker and other economically important diseases caused by Xanthomonas sp.

STINGing Defenses: Unmasking the Mechanisms of DNA Oncovirus-Mediated Immune Escape.

DNA oncoviruses represent an intriguing subject due to their involvement in oncogenesis. These viruses have evolved mechanisms to manipulate the host immune response, facilitating their persistence and actively contributing to carcinogenic processes. This paper describes the complex interactions between DNA oncoviruses and the innate immune system, with a particular emphasis on the cGAS-STING pathway. Exploring these interactions highlights that DNA oncoviruses strategically target and subvert this pathway, exploiting its vulnerabilities for their own survival and proliferation within the host. Understanding these interactions lays the foundation for identifying potential therapeutic interventions. Herein, we sought to contribute to the ongoing efforts in advancing our understanding of the innate immune system in oncoviral pathogenesis.

Acute Effects of Coffee Consumption on Blood Pressure and Endothelial Function in Individuals with Hypertension on Antihypertensive Drug Treatment: A Randomized Crossover Trial.

INTRODUCTION: Coffee is a complex brew that contains several bioactive compounds and some of them can influence blood pressure (BP) and endothelial function (EF), such as caffeine and chlorogenic acids (CGAs). AIM: This study aimed to evaluate the acute effects of coffee on BP and EF in individuals with hypertension on drug treatment who were habitual coffee consumers. METHODS: This randomized crossover trial assigned 16 adults with hypertension to receive three test beverages one week apart: caffeinated coffee (CC; 135 mg caffeine, 61 mg CGAs), decaffeinated coffee (DC; 5 mg caffeine, 68 mg CGAs), and water. BP was continuously evaluated from 15 min before to 90 min after test beverages by digital photoplethysmography. Reactive hyperemia index (RHI) assessed by peripheral arterial tonometry evaluated EF before and at 90 min after test beverages. At the same time points, microvascular reactivity was assessed by laser speckle contrast imaging. Repeated-measures-ANOVA evaluated the effect of time, the effect of beverage, and the interaction between time and beverage (treatment effect). RESULTS: Although the intake of CC produced a significant increase in BP and a significant decrease in RHI, these changes were also observed after the intake of DC and were not significantly different from the modifications observed after the consumption of DC and water. Microvascular reactivity did not present significant changes after the 3 beverages. CONCLUSION: CC in comparison with DC and water neither promoted an acute increase in BP nor produced an improvement or deleterious effect on EF in individuals with hypertension on drug treatment who were coffee consumers.

Severe COVID-19 and long COVID are associated with high expression of STING, cGAS and IFN-α.

The cGAS-STING pathway appears to contribute to dysregulated inflammation during coronavirus disease 2019 (COVID-19); however, inflammatory factors related to long COVID are still being investigated. In the present study, we evaluated the association of cGAS and STING gene expression levels and plasma IFN-α, TNF-α and IL-6 levels with COVID-19 severity in acute infection and long COVID, based on analysis of blood samples from 148 individuals, 87 with acute COVID-19 and 61 in the post-COVID-19 period. Quantification of gene expression was performed by real-time PCR, and cytokine levels were quantified by ELISA and flow cytometry. In acute COVID-19, cGAS, STING, IFN-α, TNF-α, and IL-6 levels were higher in patients with severe disease than in those with nonsevere manifestations (p < 0.05). Long COVID was associated with elevated cGAS, STING and IFN-α levels (p < 0.05). Activation of the cGAS-STING pathway may contribute to an intense systemic inflammatory state in severe COVID-19 and, after infection resolution, induce an autoinflammatory disease in some tissues, resulting in long COVID.

Mitochondrial Impairment: A Link for Inflammatory Responses Activation in the Cardiorenal Syndrome Type 4.

Cardiorenal syndrome type 4 (CRS type 4) occurs when chronic kidney disease (CKD) leads to cardiovascular damage, resulting in high morbidity and mortality rates. Mitochondria, vital organelles responsible for essential cellular functions, can become dysfunctional in CKD. This dysfunction can trigger inflammatory responses in distant organs by releasing Damage-associated molecular patterns (DAMPs). These DAMPs are recognized by immune receptors within cells, including Toll-like receptors (TLR) like TLR2, TLR4, and TLR9, the nucleotide-binding domain, leucine-rich-containing family pyrin domain-containing-3 (NLRP3) inflammasome, and the cyclic guanosine monophosphate (cGMP)-adenosine monophosphate (AMP) synthase (cGAS)-stimulator of interferon genes (cGAS-STING) pathway. Activation of these immune receptors leads to the increased expression of cytokines and chemokines. Excessive chemokine stimulation results in the recruitment of inflammatory cells into tissues, causing chronic damage. Experimental studies have demonstrated that chemokines are upregulated in the heart during CKD, contributing to CRS type 4. Conversely, chemokine inhibitors have been shown to reduce chronic inflammation and prevent cardiorenal impairment. However, the molecular connection between mitochondrial DAMPs and inflammatory pathways responsible for chemokine overactivation in CRS type 4 has not been explored. In this review, we delve into mechanistic insights and discuss how various mitochondrial DAMPs released by the kidney during CKD can activate TLRs, NLRP3, and cGAS-STING immune pathways in the heart. This activation leads to the upregulation of chemokines, ultimately culminating in the establishment of CRS type 4. Furthermore, we propose using chemokine inhibitors as potential strategies for preventing CRS type 4.

Role of the cGAS/STING pathway in the control of Brucella abortus infection acquired through the respiratory route.

Despite the importance of the respiratory route for Brucella transmission, the lung immune response to this pathogen is scarcely characterized. We investigated the role of the cGAS/STING pathway of microbial DNA recognition in the control of respiratory Brucella infection. After in vitro B. abortus infection, CFU numbers were significantly higher in alveolar macrophages (AM) and lung explants from STING KO mice than in samples from wild type (WT) mice, but no difference was observed for cGAS KO samples. CFU were also increased in WT AM and lung epithelial cells preincubated with the STING inhibitor H151. Several proinflammatory cytokines (TNF-α, IL-1ß, IL-6, IP-10/CXCL10) were diminished in Brucella-infected lung explants and/or AM from STING KO mice and cGAS KO mice. These cytokines were also reduced in infected AM and lung epithelial cells pretreated with H151. After intratracheal infection with B. abortus, STING KO mice exhibited increased CFU in lungs, spleen and liver, a reduced expression of IFN-ß mRNA in lungs and spleen, and reduced levels of proinflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF) and lung homogenates. Increased lung CFU and reduced BALF cytokines were also observed in cGAS KO mice. In summary, the cGAS/STING pathway induces the production of proinflammatory cytokines after respiratory Brucella infection, which may contribute to the STING-dependent control of airborne brucellosis.

The ubiquitin E3 ligase TRIM10 promotes STING aggregation and activation in the Golgi apparatus.

STING is an endoplasmic reticulum-resident protein regulating innate immunity. After binding with cyclic guanosine monophosphate-AMP (cGAMP), STING translocates from the endoplasmic reticulum (ER) to the Golgi apparatus to stimulate TBK1 and IRF3 activation, leading to expression of type I interferon. However, the exact mechanism concerning STING activation remains largely enigmatic. Here, we identify tripartite motif 10 (TRIM10) as a positive regulator of STING signaling. TRIM10-deficient macrophages exhibit reduced type I interferon production upon double-stranded DNA (dsDNA) or cGAMP stimulation and decreased resistance to herpes simplex virus 1 (HSV-1) infection. Additionally, TRIM10-deficient mice are more susceptible to HSV-1 infection and exhibit faster melanoma growth. Mechanistically, TRIM10 associates with STING and catalyzes K27- and K29-linked polyubiquitination of STING at K289 and K370, which promotes STING trafficking from the ER to the Golgi apparatus, formation of STING aggregates, and recruitment of TBK1 to STING, ultimately enhancing the STING-dependent type I interferon response. Our study defines TRIM10 as a critical activator in cGAS-STING-mediated antiviral and antitumor immunity.

cGAS-STING pathway as a potential trigger of immunosenescence and inflammaging.

Aging is associated with an increased incidence of autoimmune diseases, despite the progressive decline of immune responses (immunosenescence). This apparent paradox can be explained by the age-related chronic low-grade systemic inflammation (inflammaging) and progressive dysregulation of innate signaling. During cellular aging, there is an accumulation of damaged DNA in the cell's cytoplasm, which serves as ubiquitous danger-associated molecule, promptly recognized by DNA sensors. For instance, the free cytoplasmic DNA can be recognized, by DNA-sensing molecules like cGAS-STING (cyclic GMP-AMP synthase linked to a stimulator of interferon genes), triggering transcriptional factors involved in the secretion of pro-inflammatory mediators. However, the contribution of this pathway to the aging immune system remains largely unknown. Here, we highlight recent advances in understanding the biology of the cGAS-STING pathway, its influence on the senescence-associated secretory phenotype (SASP), and its modulation of the immune system during sterile inflammation. We propose that this important stress sensor of DNA damage is also a trigger of immunosenescence and inflammaging.

Prospective insight into the role of benzyl propylene glycoside as a modulator of the cGAS-STING signaling pathway in the management of nonalcoholic fatty pancreas animal model.

BACKGROUND: Nonalcoholic fatty pancreatitis (NAFP) is one of the metabolic syndrome manifestations that need further studies to determine its molecular determinants and find effective medications. We aimed to investigate the potential effect of benzyl propylene glycoside on NAFP management via targeting the pancreatic cGAS-STING pathway-related genes (DDX58, NFκB1 & CHUK) and their upstream regulator miRNA (miR-1976) that were retrieved from bioinformatics analysis. METHODS: The rats were fed either normal chow or a high-fat high-sucrose diet (HFHS), as a nutritional model for NAFP. After 8 weeks, the HFHS-fed rats were subdivided randomly into 4 groups; untreated HFHS group (NAFP model group) and three treated groups which received 3 doses of benzyl propylene glycoside (10, 20, and 30 mg/kg) daily for 4 weeks, parallel with HFHS feeding. RESULTS: The molecular analysis revealed that benzyl propylene glycoside could modulate the expression of the pancreatic cGAS-STING pathway-related through the downregulation of the expression of DDX58, NFκB1, and CHUK mRNAs and upregulation of miR-1976 expression. Moreover, the applied treatment reversed insulin resistance, inflammation, and fibrosis observed in the untreated NAFP group, as evidenced by improved lipid panel, decreased body weight and the serum level of lipase and amylase, reduced protein levels of NFκB1 and caspase-3 with a significant reduction in area % of collagen fibers in the pancreatic sections of treated animals. CONCLUSION: benzyl propylene glycoside showed a potential ability to attenuate NAFP development, inhibit pancreatic inflammation and fibrosis and reduce the pathological and metabolic disturbances monitored in the applied NAFP animal model. The detected effect was correlated with modulation of the expression of pancreatic (DDX58, NFκB1, and CHUK mRNAs and miR-1976) panel.

Prospective insight into the role of benzyl propylene glycoside as a modulator of the cGAS-STING signaling pathway in the management of nonalcoholic fatty pancreas animal model

BACKGROUND: Nonalcoholic fatty pancreatitis (NAFP) is one of the metabolic syndrome manifestations that need further studies to determine its molecular determinants and find effective medications. We aimed to investigate the potential effect of benzyl propylene glycoside on NAFP management via targeting the pancreatic cGAS-STING pathway-related genes (DDX58, NFκB1 & CHUK) and their upstream regulator miRNA (miR-1976) that were retrieved from bioinformatics analysis. METHODS: The rats were fed either normal chow or a high-fat high-sucrose diet (HFHS), as a nutritional model for NAFP. After 8 weeks, the HFHS-fed rats were subdivided randomly into 4 groups; untreated HFHS group (NAFP model group) and three treated groups which received 3 doses of benzyl propylene glycoside (10, 20, and 30 mg/kg) daily for 4 weeks, parallel with HFHS feeding. RESULTS: The molecular analysis revealed that benzyl propylene glycoside could modulate the expression of the pancreatic cGAS-STING pathway-related through the downregulation of the expression of DDX58, NFκB1, and CHUK mRNAs and upregulation of miR-1976 expression. Moreover, the applied treatment reversed insulin resistance, inflammation, and fibrosis observed in the untreated NAFP group, as evidenced by improved lipid panel, decreased body weight and the serum level of lipase and amylase, reduced protein levels of NFκB1 and caspase-3 with a significant reduction in area % of collagen fibers in the pancreatic sections of treated animals. CONCLUSION: benzyl propylene glycoside showed a potential ability to attenuate NAFP development, inhibit pancreatic inflammation and fibrosis and reduce the pathological and metabolic disturbances monitored in the applied NAFP animal model. The detected effect was correlated with modulation of the expression of pancreatic (DDX58, NFκB1, and CHUK mRNAs and miR-1976) panel.

The Relationship between Reactive Oxygen Species and the cGAS/STING Signaling Pathway in the Inflammaging Process.

During Inflammaging, a dysregulation of the immune cell functions is generated, and these cells acquire a senescent phenotype with an increase in pro-inflammatory cytokines and ROS. This increase in pro-inflammatory molecules contributes to the chronic inflammation and oxidative damage of biomolecules, classically observed in the Inflammaging process. One of the most critical oxidative damages is generated to the host DNA. Damaged DNA is located out of the natural compartments, such as the nucleus and mitochondria, and is present in the cell's cytoplasm. This DNA localization activates some DNA sensors, such as the cGAS/STING signaling pathway, that induce transcriptional factors involved in increasing inflammatory molecules. Some of the targets of this signaling pathway are the SASPs. SASPs are secreted pro-inflammatory molecules characteristic of the senescent cells and inducers of ROS production. It has been suggested that oxidative damage to nuclear and mitochondrial DNA generates activation of the cGAS/STING pathway, increasing ROS levels induced by SASPs. These additional ROS increase oxidative DNA damage, causing a loop during the Inflammaging. However, the relationship between the cGAS/STING pathway and the increase in ROS during Inflammaging has not been clarified. This review attempt to describe the potential connection between the cGAS/STING pathway and ROS during the Inflammaging process, based on the current literature, as a contribution to the knowledge of the molecular mechanisms that occur and contribute to the development of the considered adaptative Inflammaging process during aging.

Efficacy and safety of methylphenidate and behavioural parent training for children aged 3-5 years with attention-deficit hyperactivity disorder: a randomised, double-blind, placebo-controlled, and sham behavioural parent training-controlled trial.

BACKGROUND: There is insufficient evidence to support treatment recommendations for preschool children aged 3-5 years with attention-deficit hyperactivity disorder (ADHD). We aimed to investigate the efficacy and safety of methylphenidate and behavioural parent training in reducing the frequency and severity of symptoms and improving global functioning in preschool children with ADHD. METHODS: We did an 8-week, randomised, double-blind, placebo-controlled and sham behavioural parent training-controlled clinical trial (the MAPPA Study) in children aged 3-5 years with moderate-to-severe ADHD. The trial was conducted at the Institute of Psychiatry, Hospital das Clinicas, University of São Paulo Medical School, São Paulo, Brazil. Participants were randomly assigned (1:1:1) to receive immediate-release methylphenidate plus educational intervention (sham behavioural parent training), placebo medication plus behavioural parent training, or placebo medication plus educational intervention. Randomisation was done by an independent research manager by use of a permuted block randomisation procedure. Parents, teachers, study staff, and evaluators remained masked to group allocation. Methylphenidate and placebo were titrated to a maximum dose of 1·25 mg/kg per day administered orally twice daily, and behavioural parent training and the educational intervention were delivered weekly through 90 min sessions with both the child and parent, conducted by two psychologists or learning therapists. The primary outcomes were parents' and teachers' composite scores of the Swanson, Nolan, and Pelham-IV scale (SNAP-IV-P/T), the Clinical Global Impressions Severity (CGI-S) scale, and the Children's Global Assessment Scale (CGAS). This trial is registered with ClinicalTrials.gov, NCT02807870, and is now complete. All participants were invited to participate in an open observational follow-up, which is ongoing. FINDINGS: Between Aug 21, 2016, and Oct 21, 2019, 153 children were randomly assigned to receive methylphenidate plus the educational intervention (n=51), placebo plus behavioural parent training (n=51), or placebo plus the educational intervention (n=51). Nine (6%) children discontinued treatment. All participants were included in the intention-to-treat analysis. Children in the methylphenidate plus educational intervention group showed greater reductions in the SNAP-IV-P/T (endpoint mean difference -3·93 [95% CI -7·14 to -0·73], p=0·049; effect size -0·55 [95% CI -0·99 to -0·10]) and CGI-S scores (endpoint mean difference -0·49 [-0·82 to -0·17], p=0·0088; effect size -0·70 [-1·16 to -0·24]) and a greater increase in CGAS scores (endpoint mean difference 5·25 [95% CI 2·09 to 8·40], p=0·0036; effect size 0·80 [95% CI 0·32 to 1·28]) than children in the placebo plus educational intervention group. Children in the placebo plus behavioural parent training group did not have significantly different SNAP-IV-P/T scores (endpoint mean difference -3·18 [95% CI -6·38 to 0·02], p=0·077; effect size -0·44 [95% CI -0·89 to 0·003]) or CGI-S scores (endpoint mean difference -0·35 [-0·68 to -0·03], p=0·052; effect size -0·50 [-0·96 to -0·04]) compared to children in the placebo plus educational intervention group, but they had a greater increase in CGAS scores compared to the placebo plus educational intervention group (endpoint mean difference 3·69 [0·53 to 6·85], p=0·033; effect size 0·56 [0·08 to 1·04]). Children in the methylphenidate plus educational intervention versus placebo plus behavioural parent training group did not have statistically or clinically significant differences in primary outcomes. Children in the methylphenidate plus educational intervention group had more mild adverse events than the other two groups, and there were no between-group differences for moderate or severe adverse events. INTERPRETATION: Methylphenidate was effective in reducing ADHD symptoms and improving functionality, and behavioural parent training was effective in improving functionality for preschool children with ADHD after 8 weeks of treatment. FUNDING: São Paulo Research Foundation and Brazilian National Council for Scientific and Technological Development.

Coffee Chlorogenic Acids Incorporation for Bioactivity Enhancement of Foods: A Review.

The demand of foods with high antioxidant capacity have increased and research on these foods continues to grow. This review is focused on chlorogenic acids (CGAs) from green coffee, which is the most abundant source. The main CGA in coffee is 5-O-caffeoylquinic acid (5-CQA). Coffee extracts are currently the most widely used source to enhance the antioxidant activity of foods. Due to the solubility of CGAs, their extraction is mainly performed with organic solvents. CGAs have been associated with health benefits, such as antioxidant, antiviral, antibacterial, anticancer, and anti-inflammatory activity, and others that reduce the risk of cardiovascular diseases, type 2 diabetes, and Alzheimer's disease. However, the biological activities depend on the stability of CGAs, which are sensitive to pH, temperature, and light. The anti-inflammatory activity of 5-CQA is attributed to reducing the proinflammatory activity of cytokines. 5-CQA can negatively affect colon microbiota. An increase in anthocyanins and antioxidant activity was observed when CGAs extracts were added to different food matrices such as dairy products, coffee drinks, chocolate, and bakery products. The fortification of foods with coffee CGAs has the potential to improve the functionality of foods.

Intravitreal injection of mitochondrial DNA induces cell damage and retinal dysfunction in rats.

BACKGROUND: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. METHODS: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light- or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING pathway and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. RESULTS: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-ß mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-ß were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. CONCLUSIONS: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.

Natterin-Induced Neutrophilia Is Dependent on cGAS/STING Activation via Type I IFN Signaling Pathway.

Natterin is a potent pro-inflammatory fish molecule, inducing local and systemic IL-1ß/IL-1R1-dependent neutrophilia mediated by non-canonical NLRP6 and NLRC4 inflammasome activation in mice, independent of NLRP3. In this work, we investigated whether Natterin activates mitochondrial damage, resulting in self-DNA leaks into the cytosol, and whether the DNA sensor cGAS and STING pathway participate in triggering the innate immune response. Employing a peritonitis mouse model, we found that the deficiency of the tlr2/tlr4, myd88 and trif results in decreased neutrophil influx to peritoneal cavities of mice, indicative that in addition to MyD88, TRIF contributes to neutrophilia triggered by TLR4 engagement by Natterin. Next, we demonstrated that gpcr91 deficiency in mice abolished the neutrophil recruitment after Natterin injection, but mice pre-treated with 2-deoxy-d-glucose that blocks glycolysis presented similar infiltration than WT Natterin-injected mice. In addition, we observed that, compared with the WT Natterin-injected mice, DPI and cyclosporin A treated mice had a lower number of neutrophils in the peritoneal exudate. The levels of dsDNA in the supernatant of the peritoneal exudate and processed IL-33 in the supernatant of the peritoneal exudate or cytoplasmic supernatant of the peritoneal cell lysate of WT Natterin-injected mice were several folds higher than those of the control mice. The recruitment of neutrophils to peritoneal cavity 2 h post-Natterin injection was intensely impaired in ifnar KO mice and partially in il-28r KO mice, but not in ifnγr KO mice. Finally, using cgas KO, sting KO, or irf3 KO mice we found that recruitment of neutrophils to peritoneal cavities was virtually abolished in response to Natterin. These findings reveal cytosolic DNA sensors as critical regulators for Natterin-induced neutrophilia.

An overview of the crosstalk between YAP and cGAS-STING signaling in non-small cell lung cancer: it takes two to tango.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is recognized as a main mediator bridging innate and adaptive immunity, recent advances have expanded its roles to anti-tumor immunity and carcinogenesis. Loss of cGAS-STING signaling in non-small cell lung cancer (NSCLC) leads to enhanced tumorigenicity and decreased cytotoxic T lymphocyte infiltration. Apart from its anticancer response, persistent overreaction of cGAS-STING signaling promotes progression of certain inflammation-aggravated cancers. Activation of the pro-inflammatory nucleic acid sensing pathway can trigger Hippo pathway, which mediates the inactivation of Yes-associated protein 1 (YAP1) and its paralogue transcriptional co-regulators with PDZ-binding motif (TAZ, also known as WWTR1), and subsequent suppression of tumorigenesis. Active YAP acts as a transcriptional driver in bolstering immunosuppressive cytokines to evade immune surveillance and promote occurrence of preneoplasia. It is reasonable that aggressive tumors co-opt these regulators to generate few immunogenic antigens and drive tumorigenic behaviors via a highly cooperative manner. Given their multifaced roles, we profile the molecular biology characteristic and current status underpinning oncogenic YAP, review its crosstalk roles with cGAS/STING pathway in NSCLC, and summarize the major clinical investigations in NSCLC with TCGA database.

Intravitreal injection of mitochondrial DNA induces cell damage and retinal dysfunction in rats

BACKGROUND: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. METHODS: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light-or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING path-way and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. RESULTS: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-ß mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-ß were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. CONCLUSIONS: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.

Design of Novel Phosphopantetheine Adenylyltransferase Inhibitors: A Potential New Approach to Tackle Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) has been a challenging disease worldwide, especially for the neglected poor populations. Presently, there are approximately 2 billion people infected with TB worldwide and 10 million people in the world fell ill with active TB, leading to 1.5 million deaths. INTRODUCTION: The classic treatment is extensive and the drug- and multi-drug resistance of Mycobacterium tuberculosis has been a threat to the efficacy of the drugs currently used. Therefore, the rational design of new anti-TB candidates is urgently needed. METHODS: With the aim of contributing to face this challenge, 78 compounds have been proposed based on SBDD (Structure-Based Drug Design) strategies applied to target the M. tuberculosis phosphopantetheine adenylyltransferase (MtPPAT) enzyme. Ligand-Based Drug Design (LBDD) strategies were also used for establishing Structure-Activity Relationships (SAR) and for optimizing the structures. MtPPAT is important for the biosynthesis of coenzyme A (CoA) and it has been studied recently toward the discovery of new inhibitors. RESULTS: After docking simulations and enthalpy calculations, the interaction of selected compounds with MtPPAT was found to be energetically favorable. The most promising compounds were then synthesized and submitted to anti-M. tuberculosis and MtPPAT inhibition assays. CONCLUSION: One of the compounds synthesized (MCP163), showed the highest activity in both of these assays.

Structure and Sequence Requirements for RNA Capping at the Venezuelan Equine Encephalitis Virus RNA 5' End.

Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5' end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.