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Anti-granulocyte-macrophage colony-stimulating factor autoantibodies (anti-GM-CSF Abs) are a predisposing factor for pulmonary alveolar proteinosis (PAP) and Cryptococcus gattii cryptococcosis. This study aimed to investigate clinical manifestations in anti-GM-CSF Ab-positive patients with C. gattii cryptococcosis and analyze the properties of anti-GM-CSF Abs derived from these patients and patients with PAP.Thirty-nine patients diagnosed with cryptococcosis (caused by C. neoformans or C. gattii) and 6 with PAP were enrolled in the present study. Clinical information was obtained from medical records. Blood samples were collected for analysis of autoantibody properties. We also explored the National Health Insurance Research Database (NHIRD) of Taiwan to investigate the epidemiology of cryptococcosis and PAP.High titers of neutralizing anti-GM-CSF Abs were identified in 15 patients with cryptococcosis (15/39, 38.5%). Most anti-GM-CSF Ab-positive cryptococcosis cases had central nervous system (CNS) involvement (14/15, 93.3%). Eleven out of 14 (78.6%) anti-GM-CSF Ab-positive CNS cryptococcosis patients were confirmed to be infected with C. gattii, and PAP did not occur synchronously or metachronously in a single patient from our cohort. Exploration of an association between HLA and anti-GM-CSF Ab positivity or differential properties of autoantibodies from cryptococcosis patients and PAP yielded no significant results.Anti-GM-CSF Abs can cause two diseases, C. gattii cryptococcosis and PAP, which seldom occur in the same subject. Current biological evidence regarding the properties of anti-GM-CSF Abs cannot provide clues regarding decisive mechanisms. Further analysis, including more extensive cohort studies and investigations into detailed properties, is mandatory to better understand the pathogenesis of anti-GM-CSF Abs.

Clostridioides difficile infection (CDI) is a major enteric disease associated with antibiotic use and a leading cause of hospital-acquired infections worldwide. This is the first guideline for treatment of CDI in Taiwan, aiming to optimize medical care for patients with CDI. The target audience of this document includes all healthcare personnel who are involved in the medical care of patients with CDI. The 2018 Guidelines Recommendations for Evidence-based Antimicrobial agents use in Taiwan (GREAT) working group was formed, comprising of infectious disease specialists from 13 medical centers in Taiwan, to review the evidence and draft recommendations using the grading of recommendations assessment, development, and evaluation (GRADE) methodology. A nationwide expert panel reviewed the recommendations during a consensus meeting in March 2019. The recommendation is endorsed by the Infectious Diseases Society of Taiwan (IDST). This guideline describes the epidemiology and risk factors of CDI, and provides recommendations for treatment of CDI in both adults and children. Recommendations for treatment of the first episode of CDI, first recurrence, second and subsequent recurrences of CDI, severe CDI, fulminant CDI, and pediatric CDI are provided. Keywords: Clostridium difficile, Diarrhea, CDI, Infectious diarrhea

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia – histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism., Pannexin 1 (PANX1) is a membrane channel mediating release of signaling molecules to the extracellular space. PANX1 can be activated by GPCRs. Here, the authors elucidate a non-canonical channel activation pathway by α1-adrenergic receptor that involves HDAC6- mediated lysine deacetylation of PANX1.

New-Delhi metallo-β-lactamase1 (NDM-1) Enterobacteriaceae are increasing worldwide. Herein, we describe a single patient who carried three unusual NDM-1 carbapenem-resistant Enterobacteriaceae - Enterobacter cloacae (E. cloacae) yielded from a urine specimen and Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) from stool specimens. For E. cloacae, its bla NDM-1-encoding plasmid was pKP04NDM with a size of ~54 kb replicons with an IncN backbone. For K. pneumoniae, its bla NDM-1-encoding plasmid was pNDM-BTR with a size of ~59.6 kb and belonged to IncN. For E. coli, its main bla NDM-1-encoding plasmid was pIMP-HK1500, and the NDM-1 gene was obtained from a part of pNDM-BTR (8439 bp). These three clinical strains are reported for the first time and are assumed to be imported from mainland China to Taiwan. The three different plasmids were never reported in K. pneumoniae, E. coli, and Citrobacter spp before. Owing to their associated multidrug resistance, appropriate measures of periodic, targeted surveillance, and development of new antimicrobial agents are urgently needed.

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.

SUMMARYPannexin 1 (Panx1) channels are widely expressed and play important roles in apoptotic cell clearance, inflammation, blood pressure regulation, neurological disorders, opiate withdrawal, and cancer progression and metastasis. We performed (1) physicochemical analysis on a constitutively closed Panx1 channel (designated fPanx1ΔC) to examine the entire population of particles to detect multiple oligomeric states and (2) cryoEM in the membrane mimetics amphipol A8-35 and lipid bilayer nanodiscs. Our results reveal that the dominant if not exclusive oligomeric state of fPanx1ΔC is a heptamer, in solution and by cryoEM. The Panx1 heptamer provides further structural diversity within the family of large-pore channels, including hexameric LRRC8 (SWELL1) channels and connexin hemichannels, octameric CALHM1 channels and innexin hemichannels, and undecameric CALHM2 channels. Conserved structural themes are a large cytoplasmic vestibule with a diameter that corresponds roughly with the oligomeric state and a 4-helix bundle protomer, albeit with noncanonical helical packing for CALHM1 and CALHM2.In BriefThe 4-helix bundle protomer of a constitutively closed pannexin1 channel assembles as a heptamer in solution and by cryoEM.

The serine protease Factor XI (FXI) is a prominent drug target as it holds promise to deliver efficacious anti-coagulation without an enhanced risk of major bleeds. Several efforts have been described targeting the active form of the enzyme, FXIa. Herein we disclose our efforts to identify potent, selective, and orally bioavailable inhibitors of FXIa. Compound 1, identified from a diverse library of internal serine protease inhibitors, was originally designed as a complement Factor D inhibitor and exhibited sub-micromolar FXIa activity and an encouraging ADME profile while being devoid of peptidomimetic architecture. Optimization of interactions in the S1, S1β, and S1` pockets of FXIa through a combination of structure-based drug design and traditional medicinal chemistry led to the discovery of compound 23 with sub-nanomolar potency on FXIa, enhanced selectivity over other coagulation proteases, and a pre-clinical PK profile consistent with bid dosing in patients.

Rationale: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. Objective: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. Methods and Results: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. Conclusions: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.

Background/purpose The aim of this study is to investigate the role of tigecycline in Vibrio vulnificus infection. Methods Eight randomly selected clinical V. vulnificus isolates were studied to obtain the minimal inhibitory concentrations (MICs) of minocycline, cefotaxime, and tigecycline, and the time–kill curves of tigecycline alone or in combination with other drugs. A peritonitis mouse model was used for the evaluation of the therapeutic efficacy of tigecycline alone or cefotaxime in combination with minocycline or tigecycline. Results The MIC of minocycline, cefotaxime, and tigecycline for eight clinical V. vulnificus isolates was 0.06–0.12 μg/mL, 0.03–0.06 μg/mL, and 0.03–0.06 μg/mL, respectively. In time–killing studies, at the concentration of 1 × MIC, the inhibitory effect of tigecycline persisted for 24 hours in five of eight isolates. With 2 × MIC and trough level, the inhibitory effect was noted in all isolates for 24 hours. With the combination of minocycline plus cefotaxime and tigecycline plus cefotaxime at 1/2 × MIC, the bactericidal effect was noted in 25% and 62.5% of eight isolates and synergism in 50% and 75% of isolates. With a low (1.25 × 10 5 CFU/mL) inoculum, all infected mice survived with tigecycline alone, tigecycline plus cefotaxime, or minocycline plus cefotaxime on the 14 th day. At the inoculum of 1.25 × 10 6 CFU, the survival rate was 33.3% on the 14 th day in the tigecycline plus cefotaxime-treated group, but none of the mice treated by tigecycline alone or minocycline plus cefotaxime survived (33.3% vs. 0%, p = 0.01 by Fisher's exact test). Conclusion Our in vitro combination and animal studies indicate that tigecycline could be an option for the treatment of invasive V. vulnificus infections.

Chiu and colleagues review the primary evidence for divergent activation mechanisms and unitary properties of Pannexin 1 channels., Pannexin 1 (Panx1) forms plasma membrane ion channels that are widely expressed throughout the body. Panx1 activation results in the release of nucleotides such as adenosine triphosphate and uridine triphosphate. Thus, these channels have been implicated in diverse physiological and pathological functions associated with purinergic signaling, such as apoptotic cell clearance, blood pressure regulation, neuropathic pain, and excitotoxicity. In light of this, substantial attention has been directed to understanding the mechanisms that regulate Panx1 channel expression and activation. Here we review accumulated evidence for the various activation mechanisms described for Panx1 channels and, where possible, the unitary channel properties associated with those forms of activation. We also emphasize current limitations in studying Panx1 channel function and propose potential directions to clarify the exciting and expanding roles of Panx1 channels.

Allergic airway inflammation affects millions of people world-wide. Among the many factors affecting airway inflammation, type 2 inflammatory responses coordinated by CD4+ T cells are important in disease progression. Pannexins are plasma membrane channels that mediate the release of nucleotides and metabolites into the extracellular milieu to regulate physiological responses. Here, we uncover a novel immunoregulatory role for Panx1 in facilitating the crosstalk between T cells during airway inflammation. Based on an inverse correlation between Panx1 expression and the severity of allergic asthma in children, our analysis in three mouse models revealed the necessity, specificity, and sufficiency of Panx1 expression in T cells to restrict severity of airway disease. Global Panx1KO mice had exacerbated type 2 airway inflammation toward allergens, and T-cell specific Panx1 deletion phenocopied the global Panx1KO mice. A transgenic mouse line specifically designed to re-express Panx1 in T cells reversed the disease severity in Panx1KO mice. Mechanistically, Panx1 channel activation occurred in both pro- inflammatory effector (Teff) and inhibitory (Treg) T cells. While the loss of Panx1 did not intrinsically affect Teff or Treg function, Panx1 was required for extracellular nucleotide based Treg -Teff crosstalk, and optimal Treg -mediated suppression of Teff cell proliferation in vitro and in vivo. Further mechanistic studies identified a novel Panx1::Salt inducible kinase (SIK) interaction, wherein SIK- dependent phosphorylation of Panx1 at serine 205 (S205) led to Panx1 activation. A knock-in mouse strain engineered to express a non-phosphorylatable Panx1S205A channel phenocopied the exacerbated allergic airway inflammation similar to Panx1KO mice. Collectively, these data identify Panx1-dependent Treg:Teff communication, and the functional relevance of this crosstalk in restricting the severity of airway inflammation.

Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels., Pannexins are oligomeric plasma membrane channels that allow permeation of ions and large molecules. Here the authors show that human Pannexin 1 activation is a multistep event, where modification of each monomer opens the channel to a unique conductance state and fine tunes its activity.

Background This study aims to investigate the antimicrobial ability and mechanism analysis of Lactobacillus species against carbapenemase-producing Enterobacteriaceae (CPE). Methods Five Lactobacillus spp. strains and 18 CPE clinical isolates were collected. Their anti-CPE effects were assessed by agar well diffusion and broth microdilution assay, as well as time-kill test. Finally, the specific anti-CPE mechanism, especially for the effect of organic acids was determined using broth microdilution method. Results All of five Lactobacilli isolates displayed the potent activity against most CPE isolates with mean zones of inhibition ranging 10.2 to 21.1 mm. The anti-CPE activity was not affected by heating, catalase, and proteinase treatment. Under the concentration of 50% LUC0180 cell-free supernatant (CFS), lactic acid, and mix acid could totally inhibit the growth of carbapenem-resistant Klebsiella pneumoniae (CPE0011), and acetic acid could inhibit 67.8%. In contrast, succinic acid and citric acid could not inhibit the growth of CPE0011. While we decreased the concentration to 25%, only lactic acid and mix acid displayed 100% inhibition. In contrast, succinic acid, citric acid and acetic acid did not show any inhibitory effect. Conclusions Lactobacillus strains exhibit potent anti-CPE activity, and lactic acid produced by Lactobacillus strains is the major antimicrobial mechanism.

Background/Purpose This study was conducted to compare the mutation rates of different rpoB sites and rifampin minimum inhibitory concentration (MIC) changes prior to and after rifampin therapy for biofilm-embedded methicillin-resistant Staphylococcus aureus (MRSA) isolates. Methods The screening of rifampin-resistant MRSA isolates, from the biofilm at Day 5 with or without exposure to the susceptible breakpoint concentration of rifampin recommended by the Clinical and Laboratory Standards Institute (1 mg/L), was conducted using agar plates containing rifampin. A partial fragment of RNA polymerase B subunit gene ( rpoB ), including clusters I and II, was amplified and sequenced. The rifampin MIC values and mutation frequencies at different sites of rpoB were measured and evaluated in rifampicin-resistant isolates. Results Rifampin-resistant mutants could be selected from all of 39 randomly selected rifampin-susceptible MRSA isolates in the biofilm model. The spontaneous mutation frequency ranged from 1.00 × 10 −4 to 3.85 × 10 −7 . Mutation at codon 481 was most commonly found at 35 (89.7%) of 39 MRSA isolates. Without rifampin induction, the MIC ranged between 0.125 mg/L and1024 mg/L and mutation sites included cluster I 464, 466, 468, 471, 474, 477, 481, 484, 486 and cluster II 519, 527, 529 with the percentage of 471 (35.9%), 477 (33.3%), 481 (53.8%), and 484 (35.9%). Conversely, with the induction of rifampin, the MIC value ranged ∼256–1024 mg/L. The mutation sites that were more concentrated included 468 (17.9%), 477 (30.8%), 481 (89.7%), 484 (17.9%), and 486 (33.3%). Conclusion We documented high rifampin resistance induction activity when MRSA was engaged in biofilm with rifampin exposure. Monotherapy seems to be inadequate for MRSA in biofilm. There is an urgent need for developing effective combination therapies with less rifampin resistance-inducing activities for treating MRSA in biofilms.

A large unmet medical need exists for safer antithrombotic drugs because all currently approved anticoagulant agents interfere with hemostasis, leading to an increased risk of bleeding. Genetic and pharmacologic evidence in humans and animals suggests that reducing factor XI (FXI) levels has the potential to effectively prevent and treat thrombosis with a minimal risk of bleeding. We generated a fully human antibody (MAA868) that binds the catalytic domain of both FXI (zymogen) and activated FXI. Our structural studies show that MAA868 traps FXI and activated FXI in an inactive, zymogen-like conformation, explaining its equally high binding affinity for both forms of the enzyme. This binding mode allows the enzyme to be neutralized before entering the coagulation process, revealing a particularly attractive anticoagulant profile of the antibody. MAA868 exhibited favorable anticoagulant activity in mice with a dose-dependent protection from carotid occlusion in a ferric chloride–induced thrombosis model. MAA868 also caused robust and sustained anticoagulant activity in cynomolgus monkeys as assessed by activated partial thromboplastin time without any evidence of bleeding. Based on these preclinical findings, we conducted a first-in-human study in healthy subjects and showed that single subcutaneous doses of MAA868 were safe and well tolerated. MAA868 resulted in dose- and time-dependent robust and sustained prolongation of activated partial thromboplastin time and FXI suppression for up to 4 weeks or longer, supporting further clinical investigation as a potential once-monthly subcutaneous anticoagulant therapy.

In our recent publication, we reported the discovery that spironolactone—an antihypertensive—is also a new Panx1 (pannexin 1) inhibitor. In addition, we found that spironolactone interferes with α1AR (α1 adrenoceptor)-mediated vasoconstriction of resistance vessels and acutely lowers blood pressure in mice; importantly, these effects require Panx1 channel expression in vascular smooth muscle cells but are independent of the MR (mineralocorticoid receptor)—the traditional target of spironolactone. Based on the accumulated evidence, we proposed that Panx1 is a novel target of spironolactone that, in combination with MR-dependent actions, may contribute to the beneficial blood pressure-lowering effects of spironolactone that are especially relevant for treatment of resistant hypertensive patients. We are pleased that Drs Wright and Angus recognize that this novel action adds to the well-accepted MR-dependent antihypertensive effects of spironolactone and that this unveils a potentially important mechanism with translational clinical implications. In addition, however, these authors raise some points of contention with our studies that can be summarized in 2 general arguments: (1) based on perceived differences with their own pharmacological reports, they insist that additional pharmacological studies are necessary to support the fundamental underlying mechanism (ie, that Panx1-mediated ATP release contributes to α1AR-mediated vasoconstriction) and (2) that spironolactone acts less potently at Panx1 than at MR and thus may require concentrations not achieved clinically. Below, we address both of these points. Despite the narrow focus of their argument on work involving a single Panx1 inhibitor (mefloquine)1 and a single P2X1 blocker (NF449),2 there is now substantial pharmacological and, importantly, genetic evidence supporting a role for Panx1-mediated ATP release in α1AR-mediated vasoconstriction. For Panx1, this …

Objective Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis. Methods We assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients. Results Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance. Conclusions We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis., Graphical abstract, Highlights • Adipocytes express Pannexin 1 channels that can be activated to release ATP. • Inhibition of Pannexin 1 decreased insulin-induced glucose uptake in adipocytes. • Adipocyte Pannexin 1 knockout mice are more insulin resistant on high fat diet. • We identify insulin as a novel activator of Pannexin 1 channels. • Pannexin 1 expression in human adipose tissue correlates with insulin resistance.

The signal transducer and activator of transcription 3 (STAT3) is known to be involved in hypertrophy and fibrosis in cardiac dysfunction. The activation of STAT3 via the phosphorylation of STAT3 is required for the production of functional activity. It has been established that lipopolysaccharide (LPS)‑induced phosphorylation of STAT3 in cardiomyocytes primarily occurs through a direct receptor‑mediated action. This effect is demonstrated to be produced rapidly. STAT3 in cardiac fibrosis of diabetes is induced by high glucose through promotion of the STAT3‑associated signaling pathway. However, the time schedule for STAT3 activation between LPS and high glucose appears to be different. Therefore, the difference in STAT3 activation between LPS and hyperglycemia in cardiomyocytes requires elucidation. The present study investigated the phosphorylation of STAT3 induced by LPS and hyperglycemia in the rat cardiac cell line H9c2. Additionally, phosphorylation of STAT3 induced by erythropoietin (EPO) via receptor activation was compared. Then, the downstream signals for fibrosis, including the connective tissue growth factor (CTGF) and matrix metalloproteinase (MMP)‑9, were determined using western blotting, while the mRNA levels were quantified. LPS induced a rapid elevation of STAT3 phosphorylation in H9c2 cells within 30 min, similar to that produced by EPO. However, LPS or EPO failed to modify the mRNA level of STAT3, and/or the downstream signals for fibrosis. High glucose increased STAT3 phosphorylation to be stable after a long period of incubation. Glucose incubation for 24 h may augment the STAT3 expression in a dose‑dependent manner. Consequently, fibrosis‑associated signals, including CTGF and MMP‑9 protein, were raised in parallel. In the presence of tiron, an antioxidant, these changes by hyperglycemia were markedly reduced, demonstrating the mediation of oxidative stress. Therefore, LPS‑ or EPO‑induced STAT3 phosphorylation is different compared with that caused by high glucose in H9c2 cells. Sustained activation of STAT3 by hyperglycemia may promote the expression of fibrosis‑associated signals, including CTGF and MMP‑9, in H9c2 cells. Therefore, regarding the cardiac dysfunctions associated with diabetes and/or hyperglycemia, the identification of nuclear STAT3 may be more reliable compared with the assay of phosphorylated STAT3 in cardiac cells.

Neuropathic pain symptoms respond poorly to available therapeutics, with most treated patients reporting unrelieved pain and significant impairment in daily life. Here, we show that Pannexin 1 (Panx1) in hematopoietic cells is required for pain-like responses following nerve injury in mice, and a potential therapeutic target. Panx1 knockout mice (Panx1−/−) were protected from hypersensitivity in two sciatic nerve injury models. Bone marrow transplantation studies show that expression of functional Panx1 in hematopoietic cells is necessary for mechanical hypersensitivity following nerve injury. Reconstitution of irradiated Panx1 knockout mice with hematopoietic Panx1−/− cells engineered to re-express Panx1 was sufficient to recover hypersensitivity after nerve injury; this rescue required expression of a Panx1 variant that can be activated by G protein-coupled receptors (GPCRs). Finally, chemically distinct Panx1 inhibitors blocked development of nerve injury-induced hypersensitivity and partially relieved this hypersensitivity after it was established. These studies indicate that Panx1 expressed in immune cells is critical for pain-like effects following nerve injury in mice, perhaps via a GPCR-mediated activation mechanism, and suggest that inhibition of Panx1 may be useful in treating neuropathic pain.

The innate immune system deploys a variety of sensors to detect signs of infection. Nucleic acids represent a major class of pathogen signatures that can trigger robust immune responses. The presence of DNA in the cytoplasm of mammalian cells is a danger signal that activates innate immune responses; however, how cytosolic DNA triggers these responses remained unclear until recently. In this review, we focus on the mechanism of DNA sensing by the newly discovered cGAS-cGAMP-STING pathway and highlight recent progress in dissecting the in vivo functions of this pathway in immune defense as well as autoimmunity.

Pannexin 1 (Panx1) channels are generally represented as non-selective, large-pore channels that release ATP. Emerging roles have been described for Panx1 in mediating purinergic signaling in the normal nervous, cardiovascular, and immune systems, where they may be activated by mechanical stress, ionotropic and metabotropic receptor signaling, and via proteolytic cleavage of the Panx1 C-terminus. Panx1 channels are widely expressed in various cell types, and it is now thought that targeting these channels therapeutically may be beneficial in a number of pathophysiological contexts, such as asthma, atherosclerosis, hypertension, and ischemic-induced seizures. Even as interest in Panx1 channels is burgeoning, some of their basic properties, mechanisms of modulation, and proposed functions remain controversial, with recent reports challenging some long-held views regarding Panx1 channels. In this brief review, we summarize some well-established features of Panx1 channels; we then address some current confounding issues surrounding Panx1 channels, especially with respect to intrinsic channel properties, in order to raise awareness of these unsettled issues for future research.

SummaryObjectiveTo determine whether acid aspiration provokes the development of multidrug-resistant Acinetobacter baumannii (MDRAB) pneumonia in its host.MethodsGroups of mice were inoculated intratracheally (IT) with 50μl of 0.1N HCl and 1×108 colony-forming units (CFU) Ab396 (A+Ab group), or 50μl of 0.1N HCl and 20μl of 0.9% saline (A+S group), or 20μl of 0.9% saline and 1×108 CFU of Ab396 (S+Ab group), or 50μl of 0.9% saline and 20μl of 0.9% saline (S+S group). Cytokines, bacterial loads in the bronchoalveolar lavage fluid (BALF), lung permeability, histopathology of the lungs, and survival rates were evaluated.ResultsOnly the A+Ab mice developed extensive Ab396 pneumonia and had significantly elevated bacterial loads, increased lung leakage, and lower levels of tumor necrosis factor alpha (TNF-α) compared with the other three groups (p

Background/Purpose: In vitro studies of the combination of an aminoglycoside with tigecycline or doxycycline against Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates are rarely published. The goal of this study was to evaluate the antibacterial activity of the combination regimens. Methods: Thirteen genetically different KPC-producing K. pneumoniae isolates were randomly selected. Drug concentrations of amikacin, gentamicin, tigecycline, and doxycycline were adjusted to 1-, 1/2-, and 1/4-fold of respective minimum inhibitory concentrations (MICs). Each drug alone or the combinations of amikacin or gentamicin with tigecycline or doxycycline were tested by combination studies. Results: Treatment with the 1× MIC concentration in combinations of amikacin or gentamicin and tigecycline or doxycycline for 24 hours resulted in bactericidal activity of 84–100% in the isolates. Treatment with 1/2× MIC combinations resulted in synergism of 69–100% in the isolates. Notably, doxycycline plus gentamicin or amikacin was synergistic for all tested isolates. However, bactericidal or synergistic effect was barely evident following 1/4× MIC combinations. There was no antagonism in any of the combination regimens. Conclusion: Enhanced activity was noted following treatment with doxycycline combined with gentamicin or amikacin against KPC-producing K. pneumoniae isolates, warranting further in vitro and animal investigations before clinical application. Keywords: aminoglycoside, doxycycline, KPC, synergism, tigecycline

Using a series of immunoprecipitation (IP) – tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

The emergence of multidrug-resistant Salmonella isolates has created the need for new therapeutic agents. We evaluated the intracellular activity of four carbapenem compounds against clinical nontyphoid Salmonella (NTS) isolates in vitro and ex vivo . Subsequently, the efficacy of carbapenem treatment against selected Salmonella isolates in vivo was assessed using a murine peritonitis model. The MIC 50 and MIC 90 for doripenem, ertapenem, imipenem, and meropenem against 126 NTS isolates were found to be 0.062 and 0.062, 0.015 and 0.015, 0.5 and 1, and 0.031 and 0.031 μg/ml, respectively. The intracellular killing effect of ertapenem was sustained for 24 h and was superior to that of imipenem, meropenem, and doripenem; its effect was comparable to that of ceftriaxone. Ertapenem demonstrated an excellent pharmacokinetic profile with a percent time above the MIC of 75.5% and an area under the concentration-time curve/MIC ratio of 20,733. When peritoneal exudate cells were examined directly ex vivo from mice with Salmonella -induced peritonitis, cells from mice treated with ertapenem and ceftriaxone had intracellular and extracellular bacterial counts reduced 10 2 - to 10 4 -fold and exhibited killing effects similar to each other. The survival rates of mice inoculated with 1 × 10 5 and 10 6 CFU of a ceftriaxone-susceptible Salmonella isolate that were subsequently treated with ertapenem or ceftriaxone were 100% and 90%, respectively. When mice were inoculated with 5 × 10 4 and 10 5 CFU of a ceftriaxone-resistant and ciprofloxacin-resistant Salmonella isolate, mice treated with ertapenem had a higher survival rate than mice treated with ceftriaxone (70% versus 0% and 50% versus 0%, respectively; P < 0.001). Our results suggest that ertapenem is at least as effective as ceftriaxone in treating murine Salmonella infections and show that further clinical investigations on the potential use of ertapenem in treatment of human Salmonella infections are warranted.

Large-conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene are often components of large multiprotein complexes in excitable and nonexcitable cells. Here we show that Slo1 proteins interact with Neph1, a member of the immunoglobulin superfamily expressed in slit diaphragm domains of podocytes and in vertebrate and invertebrate nervous systems. This interaction was established by reciprocal coimmunoprecipitation of endogenous proteins from differentiated cells of a podocyte cell line, from parasympathetic neurons of the embryonic chick ciliary ganglion, and from HEK293T cells heterologously expressing both proteins. Neph1 can interact with all three extreme COOH-terminal variants of Slo1 (Slo1VEDEC, Slo1QEERL, and Slo1EMVYR) as ascertained by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation. Neph1 is partially colocalized in intracellular compartments with endogenous Slo1 in podocytes and ciliary ganglion neurons. Coexpression in HEK293T cells of Neph1 with any of the Slo1 extreme COOH-terminal splice variants suppresses their steady-state expression on the cell surface, as assessed by cell surface biotinylation assays, confocal microscopy, and whole cell recordings. Consistent with this, small interfering RNA (siRNA) knockdown of endogenous Neph1 in embryonic day 10 ciliary ganglion neurons causes an increase in steady-state surface expression of Slo1 and an increase in whole cell Ca2+-dependent K+ current. Surprisingly, a comparable Neph1 knockdown in podocytes causes a decrease in surface expression of Slo1 and a decrease in whole cell BKCa currents. In podocytes, Neph1 siRNA also caused a decrease in nephrin, even though the Neph1 siRNA had no sequence homology with nephrin. However, we could not detect nephrin in ciliary ganglion neurons.

1.1. The ubiquitin-proteasome pathway Ubiquitin is a highly conserved, 76-amino acid protein that is ubiquitously expressed in eukaryotic cells 1. This small protein controls almost all aspects of a cell's life and death, through its covalent modification of other cellular proteins in a process known as ubiquitination 2,3. The enzymatic cascade of ubiquitination begins with ubiquitin activation by an E1 (ubiquitin-activating enzyme), followed by transfer of the activated ubiquitin to an E2 (ubiquitin-conjugating enzyme, also known as Ubc), and ends with conjugation of ubiquitin to a target protein through the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and an e-amino group of a lysine residue on the protein substrate. The last step requires a member of a very large family of ubiquitin-protein ligases (E3), which, together with E2s, determine substrate specificity. E3s can be divided into two categories, depending on whether they contain a HECT (homology to E6AP C-terminus) or RING (really interesting new gene) domain. The HECT domain E3s contain an active-site cysteine, which can accept ubiquitin from an E2 and transfer the ubiquitin to a target protein. In contrast, the RING domain E3s do not contain a conventional enzyme active site, but they promote ubiquitination by binding to both protein substrates and E2s, facilitating the conjugation of ubiquitin to specific protein targets. Ubiquitination reactions are reversed by members of a large family of deubiquitination enzymes (DUBs, also known as isopeptidases)4,5. Thus, ubiquitination is a reversible covalent modification, similar to phosphorylation. Ubiquitin has seven lysines, each of which can be conjugated by another ubiquitin to form a polyubiquitin chain 6. The topology of polyubiquitin chains can influence the fate of target proteins. For example, polyubiquitin chains linked through lysine 48 (K48) of ubiquitin normally target a protein for degradation by the proteasome, whereas K63 polyubiquitin chains have functions independent of proteolysis, including protein kinase activation, DNA repair and membrane trafficking. Monoubiquitination usually does not lead to proteasomal degradation; instead, it regulates important cellular functions such as chromatin remodeling and vesicle trafficking.

Both purinergic signaling through nucleotides such as ATP (adenosine 5′-triphosphate) and noradrenergic signaling through molecules such as norepinephrine regulate vascular tone and blood pressure. Pannexin1 (Panx1), which forms large-pore, ATP-releasing channels, is present in vascular smooth muscle cells in peripheral blood vessels and participates in noradrenergic responses. Using pharmacological approaches and mice conditionally lacking Panx1 in smooth muscle cells, we found that Panx1 contributed to vaso-constriction mediated by the α1 adrenoreceptor (α1AR), whereas vasoconstriction in response to serotonin or endothelin-1 was independent of Panx1. Analysis of the Panx1-deficient mice showed that Panx1 contributed to blood pressure regulation especially during the night cycle when sympathetic nervous activity is highest. Using mimetic peptides and site-directed mutagenesis, we identified a specific amino acid sequence in the Panx1 intracellular loop that is essential for activation by α1AR signaling. Collectively, these data describe a specific link between noradrenergic and purinergic signaling in blood pressure homeostasis.

The activation of NF-κB and IKK requires an upstream kinase complex consisting of TAK1 and adaptor proteins such as TAB1, TAB2, or TAB3. TAK1 is in turn activated by TRAF6, a RING domain ubiquitin ligase that facilitates the synthesis of lysine 63-linked polyubiquitin chains. Here we present evidence that TAB2 and TAB3 are receptors that bind preferentially to lysine 63-linked polyubiquitin chains through a highly conserved zinc finger (ZnF) domain. Mutations of the ZnF domain abolish the ability of TAB2 and TAB3 to bind polyubiquitin chains, as well as their ability to activate TAK1 and IKK. Significantly, replacement of the ZnF domain with a heterologous ubiquitin binding domain restored the ability of TAB2 and TAB3 to activate TAK1 and IKK. We also show that TAB2 binds to polyubiquitinated RIP following TNFα stimulation. These results indicate that polyubiquitin binding domains represent a new class of signaling domains that regulate protein kinase activity through a nonproteolytic mechanism.

PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely unknown. Here we show that PARK2 is altered in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitric oxide synthase (eNOS), enhanced levels of reactive oxygen species, and a concomitant increase in oxidized nitric oxide levels, thereby promoting the inhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylation in supporting cell survival and proliferation under conditions of energy deprivation., We thank Rodrick Bronson, the entire HMS Rodent Histopathology Core, and the ICR Pathology Core for technical help with the mouse histopathology and discussions concerning the project. We also thank David Carling for the generous gift of the 991 activator, Nick Leslie for the pHR-SIN-PTEN-WT and Tina Yuan for the pLV430G-oFL-T2A-eGFP vectors, Pier Paolo Pandolfi for giving us access to Pten knockout (KO) mice, and Olga Corti and Alexis Brice for providing Park2 KO mice. We thank Susanne Breitkopf and Min Yuan for help with mass spectrometry. This work was supported by grants from the NIH P01-CA120964 (J.M.A. and L.C.C.) and R01-GM041890. A.V. was funded by the Ministry of Education, Culture and Sport under the Program for Promoting and Hiring of Talent and its Employability (Subprogram for Mobility) of the Spanish Government. G.P. is funded by the ICR. Work in the D.A. lab is supported by MRC grant MC_UP_1202/1. L.C.C. owns equity in, receives compensation from, and serves on the Board of Directors and Scientific Advisory Board of Agios Pharmaceuticals. Agios Pharmaceuticals is identifying metabolic pathways in cancer cells and developing drugs to inhibit such enzymes to disrupt tumor cell growth and survival. Finally, we would like to dedicate this work to the memory of Professor Chris Marshall who was an esteemed colleague and mentor, whose scientific discoveries will continue to inspire us and translate basic science into benefits for cancer patients.

Bromodomain-containing protein 7 (BRD7) is a member of the bromodomain-containing protein family that is known to play a role as tumor suppressors. Here, we show that BRD7 is a component of the unfolded protein response (UPR) signaling through its ability to regulate X-box binding protein 1 (XBP1) nuclear translocation. BRD7 interacts with the regulatory subunits of phosphatidylinositol 3-kinase (PI3K) and increases the nuclear translocation of both p85 alpha and p85 beta and the spliced form of XBP1 (XBP1s). Deficiency of BRD7 blocks the nuclear translocation of XBP1s. Furthermore, our in vivo studies have shown that BRD7 protein levels are reduced in the liver of obese mice, and reinstating BRD7 levels in the liver restores XBP1s nuclear translocation, improves glucose homeostasis, and ultimately reduces the blood glucose levels in the obese and diabetic mouse models.