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Recent evidence indicates that the human gut microbiome plays a significant role in health and disease. Dysbiosis, defined as a pathological imbalance in a microbial community, is becoming increasingly appreciated as a 'central environmental factor' that is both associated with complex phenotypes and affected by host genetics, diet and antibiotic use. More recently, a link has been established between the dysmetabolism of bile acids (BAs) in the gut to dysbiosis.BAs, which are transformed by the gut microbiota, have been shown to regulate intestinal homeostasis and are recognized as signaling molecules in a wide range of metabolic processes. This review will examine the connection between BA metabolism as it relates to the gut microbiome and its implication in health and disease.A disrupted gut microbiome, including a reduction of bile salt hydrolase (BSH)-active bacteria, can significantly impair the metabolism of BAs and may result in an inability to maintain glucose homeostasis as well as normal cholesterol breakdown and excretion. To better understand the link between dysbiosis, BA dysmetabolism and chronic degenerative disease, large-scale metagenomic sequencing studies, metatranscriptomics, metaproteomics and metabolomics should continue to catalog functional diversity in the gastrointestinal tract of both healthy and diseased populations. Further, BSH-active probiotics should continue to be explored as treatment options to help restore metabolic levels.

The treatment of chronic wounds poses a significant challenge for clinicians and patients alike. Here we report design and preclinical efficacy of a novel nitric oxide gas (gNO)-producing probiotic patch for wound healing. Specifically, a wound healing patch using lactic acid bacteria in an adhesive gas permeable membrane has been designed and investigated for treating ischaemic and infected full-thickness dermal wounds in a New Zealand white rabbit model for ischaemic wound healing. Kaplan-Meier survival curves showed increased wound closure with gNO-producing patch-treated wounds over 21 days of therapy (log-rank P = 0·0225 and Wilcoxon P = 0·0113). Cox proportional hazard regression showed that gNO-producing patch-treated wounds were 2·52 times more likely to close compared with control patches (hazard P = 0·0375, score P = 0·032 and likelihood ratio P = 0·0355), and histological analysis showed improved wound healing in gNO-producing patch-treated animals. This study may provide an effective, safe and less costly alternative for treating chronic wounds.

This review describes the antimicrobial properties of nitric oxide (NO) and its application as an antimicrobial agent in different formulations and medical devices. We depict the eukaryotic biosynthesis of NO and its physiologic functions as a cell messenger and as an antimicrobial agent of the cell-mediated immune response. We analyze the antimicrobial activity of NO and the eukaryotic protective mechanisms against NO for the purpose of delineating the therapeutic NO dosage range required for an efficacious and safe antimicrobial activity. We also examine the role of NO produced by virulent bacteria in lessening the efficacy of traditional antimicrobials. In addition, we discuss the efficacy of NO in the healing of infected wounds, describing different NO-producing devices by category, analyzing therapeutic levels, duration of NO production, as well as commercial considerations. Finally, we provide current and future prospects for the design and use of NO-producing devices.

Microbial and fungal infections are a significant consideration in the etiology of all wounds. Numerous antimicrobial and antifungal formulations have been developed with varying degrees of efficacy and stability. Here, we report a nitric oxide producing probiotic adhesive patch device and investigate its antimicrobial and antifungal efficacy in vitro. This probiotic patch utilizes the metabolic activity of immobilized lactic acid bacteria, glucose, and nitrite salts for the production of gaseous nitric oxide (gNO), which is used as an antimicrobial agent against bacterial and fungal pathogens. Results show that application of gNO-producing probiotic patches to cultures of E. coli, S. aureus, P. aeruginosa, MRSA, T. mentagrophytes, and T. rubrum resulted in complete cell death at between 4 and 8 h, and application to cultures of A. baumannii, resulted in fewer than ten colonies detected per milliliter at 6 h. These results demonstrate that a gNO-producing probiotic patch device containing bacteria, glucose, and nitrite salts can produce sufficient levels of gNO over a therapeutically relevant duration to kill common bacterial and fungal wound pathogens in humans.

SummaryIncreased risk of thrombosis, with propitious conditions for fibrin deposition, along with upregulation of inflammation, are important factors that enhance plaque formation in atherosclerosis. Evidence supporting the role of anticoagulant protein C (PC) as an inflammatory agent has emerged, supplementing its wellknown function as an anticoagulant. Thus, we sought to examine whethera PC deficiency would lead to an enhanced response to an acute arterial hyperplasic challenge. The presentation of early arterial inflammation was studied using a copper/silicone arterial cuff model of accelerated focal neointimal remodeling in mice with a heterozygous total deficiency of PC (PC+/−). Increased inflammation, cell proliferation, cell migration, fibrin elevation, and tissue necrosis were observed in the treated arteries of PC+/−mice, as compared to arteries of equally challenged age- and gender-matched WT mice. These results indicate that PC+/−mice subjected to this challenge displayed enhanced focal arterial inflammation and thrombosis, leading to larger neointimas and subsequent localized occlusion, as compared to their WT counterparts.

The human gastrointestinal tract hosts a large number of microbial cells which exceed their mammalian counterparts by approximately 3-fold. The genes expressed by these microorganisms constitute the gut microbiome and may participate in diverse functions that are essential to the host, including digestion, regulation of energy metabolism, and modulation of inflammation and immunity. The gut microbiome can be modulated by dietary changes, antibiotic use, or disease. Different ailments have distinct associated microbiomes in which certain species or genes are present in different relative quantities. Thus, identifying specific disease-associated signatures in the microbiome as well as the factors that alter microbial populations and gene expression will lead to the development of new products such as prebiotics, probiotics, antimicrobials, live biotherapeutic products, or more traditional drugs to treat these disorders. Gained knowledge on the microbiome may result in molecular lab tests that may serve as personalized tools to guide the use of the aforementioned products and monitor interventional progress.

Activated protein C functions directly as an anticoagulant and indirectly as a profibrinolytic enzyme. To determine whether the fibrin deposition previously observed in PC −/− murine embryos and neonates was mediated through the FXI pathway, PC +/− /FXI −/− mice were generated and crossbred to produce double-deficient progeny ( PC −/− /FXI −/− ). PC −/− /FXI −/− mice survived the early lethality observed in the PC −/− /FXI +/+ neonates, with the oldest PC −/− /FXI −/− animal living to 3 months of age. However, the majority of these animals was sedentary and significantly growth-retarded. On sacrifice or natural death, all of these PC −/− /FXI −/− mice demonstrated massive systemic fibrin deposition with concomitant hemorrhage and fibrosis, as confirmed through histological analyses. Several of these animals also presented with enlarged lymph nodes and extensive lymphatic fluid in the thoracic cavity. Thus, although a number of the PC −/− /FXI −/− mice survived the lethal perinatal coagulopathy seen in the PC −/− neonates, they nonetheless succumbed to overwhelming thrombotic disease in later life. This combined deficiency state provided the first clear indication that the course of a severe thrombotic disorder could be manipulated by blocking the intrinsic pathway and provided the first opportunity to study a total protein C deficiency in an adult animal.

Gastrointestinal (GI) symptoms are conditions that are frequently observed in clinical practice. A post-hoc analysis has been undertaken to evaluate the effect of bile salt hydrolase-active L. reuteri NCIMB 30242 on GI health status based on Rome III questionnaire response in otherwise healthy hypercholesterolemic subjects.A total of 127 subjects received either L. reuteri NCIMB 30242 or placebo capsules over a 9-week intervention in a randomized, double-blind, placebo-controlled, parallel-arm, multicenter study. Subjects were asked to complete the Rome III diagnostic GI questionnaire prior to the baseline and end point visits of the clinical study.GI health status was evaluated, per questionnaire, by assessing all questions with 5- or 7-point response scales for symptoms of the stomach and intestines.Subjects receiving L. reuteri NCIMB 30242 reported significant improvements in general GI health status (p = 0.029) and in symptoms related to diarrhea (p = 0.018) as compared to placebo over the intervention period. Further, a greater proportion of L. reuteri-treated subjects showed improved general GI health status (p = 0.042) and improved diarrhea symptoms (p = 0.03).L. reuteri NCIMB 30242 capsules appear to be well tolerated and potentially beneficial for GI health status. Further clinical investigation is warranted for the treatment of functional GI disorders.

The densities of aqueous solutions resulting from the partial neutralization of boric acid with sodium hydroxide were measured as a function of concentration, between 375 K and 523 K at pressures close to saturation. The speciation in this system is complex, di- and triborate ions are present in addition to the monoborate ion. The concentration dependence of the apparent partial molar volume of the mixture can be described using the Pitzer equation only if the formation of the polyborate species in the concentrated solutions is taken into account. The partial molar volumes at infinite dilution for the diborate and triborate anions, obtained by assuming ideal mixing, are reported in the range of temperature studied.

The use of percutaneous medical devices often results in nosocomial infections. Attachment of microorganisms to the surfaces of these medical devices triggers biofilm formation, which presents significant complications to the health of a patient and may lead to septicemia, thromboembolism, or endocarditis if not correctly treated. Although several antimicrobials are commonly used for prevention of biofilm formation, they have limited efficacy against formed biofilms. In this study, we report the use of an enzymatic, gaseous nitric oxide (gNO)-releasing dressing for the prevention and treatment of Acinetobacter baumannii , methicillin-resistant Staphylococcus aureus , and Pseudomonas aeruginosa biofilms. Results show that the bactericidal activity against biofilms of the test strains was dependent on time and rate of gNO release from the dressing. Following 6 h of treatment, gNO-releasing dressings significantly inhibited the growth of test strains relative to vehicle control dressings, demonstrating eradication of bacterial concentrations of up to 10 5 CFU/cm 2 . Complete cell death was observed for both prevention of biofilm formation and treatment of 24-h-grown biofilms after 6 h of treatment with the gNO-releasing dressings. Further, gNO-releasing dressings were more efficient against formed biofilms than other antimicrobial agents currently used. These results demonstrate that the gNO-releasing dressing can produce sufficient levels of gNO over a therapeutically relevant duration for maximal bactericidal effects against virulent bacterial strains known to cause nosocomial infections.

BACKGROUND The leading causes of death for trauma patients in civilian and combat settings are traumatic brain injury and uncontrolled hemorrhage, respectively. This study examines the hemostatic activity of an ideal amphipathic peptide (IAP) attached to a biocompatible surface. METHODS Procoagulant properties of IAP attached to a surface were first tested, in vitro, using factor Xa and thrombin generation assays and thromboelastography. Rabbits and swine were used for in vivo studies. Injuries were performed using scalpel blade 11, and free bleeding was allowed for five seconds. While bleeding, IAP coupled to a hydrogel or QuikClot were applied to the wound and the time was recorded until bleeding stopped. RESULTS Results show that when IAP is attached to a surface, both factor IXa and factor Xa activities are promoted. Thromboelastography shows that surface-attached IAP results in earlier onset and stronger clot formation. In rabbits, the incorporation of IAP onto a biocompatible hydrogel reduces bleeding times by 40% (p < 0.03). In pigs, bleeding times are reduced 30% to 50% (p < 0.02) by surface-coupled IAP. Finally, using a rabbit liver laceration model, the properties of surface-coupled IAP are less damaging when compared with QuikClot, a currently used material in external hemorrhagic injuries. CONCLUSIONS This study provides a relevant proof of concept for the development of IAP coupled to a biocompatible surface as a hemostatic agent, that is potentially safer than the commercially available QuikClot.

On the basis of previous evidence that amphipathic helical peptides accelerate Factor IXa activation of Factor X [Blostein, Rigby, Furie, Furie and Gilbert (2000) Biochemistry 39, 12000–12006], the present study was designed to assess the procoagulant activity of an IAP (ideal amphipathic peptide) of Lys7Leu15 composition. The results show that IAP accelerates Factor X activation by Factor IXa in a concentration-dependent manner and accelerates thrombin generation by Factor Xa with a comparable peptide- and substrate-concentration-dependence. A scrambled helical peptide with the same amino acid composition as IAP, but with its amphipathicity abolished, eliminated most of the aforementioned effects. The Gla (γ-carboxyglutamic acid)-rich domain of Factor X is required for IAP activity, suggesting that this peptide behaves as a phospholipid membrane. This hypothesis was confirmed, using fluorescence spectroscopy, by demonstrating direct binding between IAP and the Gla-rich domain of Factor X. In addition, the catalytic efficiencies of the tenase and prothrombinase enzymatic complexes, containing cofactors Factor VIIIa and Factor Va respectively, are enhanced by IAP. Finally, we show that IAP delays clot lysis in vitro. In summary, these observations demonstrate that IAP not only enhances essential procoagulant reactions required for fibrin generation, but also inhibits fibrinolysis, suggesting a potential role for IAP as a haemostatic agent.

During the systemic inflammatory state induced by sepsis, the potential for coagulopathy exists because of up-regulation of natural procoagulants and anti-fibrinolytics, and down-regulation of natural anti-coagulants, with protein C (PC) being a critical example of the latter case. PC functions as an anti-coagulant, profibrinolytic, and anti-inflammatory agent, and, thus, its administration or deficiency may affect the course and outcome of sepsis in patients. In this study, a cecal ligation and puncture model of septic peritonitis was applied to wild-type mice and littermates with a targeted heterozygous deficiency of PC (PC(+/-)) to characterize the importance of a PC-deficiency on polymicrobial sepsis. An enhanced mortality rate was found to accompany a PC deficiency. Plasma cytokines, as well as organ-specific expression of cytokine transcripts, were elevated in PC(+/-) mice. No signs of severe disseminated intravascular coagulation (DIC) were observed in wild-type or PC(+/-) mice, as indicated by an increase in fibrinogen levels and the invariability of platelet counts after cecal ligation and puncture. Consumption of coagulation factors was similar in both genotypes and a decrease in the PC mRNA and protein levels was more prominent in PC(+/-) mice. Renal and organ muscle damage was enhanced in PC(+/-) mice, as shown by increases in plasma blood urea nitrogen, creatinine, and creatinine kinase. Hypotension and bradycardia were more enhanced in PC(+/-) mice than in wild-type mice, thus provoking a more severe septic shock response. Thus, the hemodynamic role of PC during sepsis is of critical importance to the outcome of the disease.

We have previously demonstrated that a 22 amino acid ideal amphipathic peptide (IAP) of K7L15 composition dramatically accelerates both factor IXa and factor Xa activity. In the present work, we investigate the activity of IAP attached to a surface in view of designing a procoagulant surface to reduce hemorrhage. Our results show that IAP maintains its catalytic enhancing properties for factor IXa and factor Xa when attached to a surface. This enhancement is dependent on the presence of the gamma-carboxyglutamic acid domain of factor X, consistent with the hypothesis that IAP behaves as a phospholipid membrane, providing a surface for the assembly of procoagulant enzymes and substrates. To further confirm this hypothesis, we demonstrate direct binding between surface-bound IAP and the Gla domain of factor X using an ELISA-based binding assay. Based on the aforementioned evidence that immobilized IAP enhances procoagulant activity, we conducted in vivo experiments using an ear-bleeding model in rabbits. We incorporated IAP into DuraSeal, a commercially available sealing agent, and found that the addition of IAP decreases the bleeding time in rabbits by 25% (p=0.0065). In conclusion, the above data provide a rationale for designing procoagulant surfaces in vivo. Further evaluation in larger animal models is warranted.

We have previously demonstrated that amphipathic helical peptides can accelerate the turnover of the substrate factor X by the enzyme, factor IXa in a gamma-carboxyglutamic acid (Gla) domain dependent manner (Biochemistry,39:12000Biochemistry,39:12000). Such improvement was due to a remarkable decrease in KM and a mild increase in kcat, mimicking the presence of phospholipid membranes. It is hypothesized that amphipathic helical peptides could exert similar activities in other reactions of the blood coagulation cascade, such as thrombin generation and in whole blood clotting. In the present work, we analyze the activity of a 22-amino acid ideal amphipathic helical peptide (IAP) of K7L15 composition, in factor X activation and thrombin generation. The addition of IAP accelerates factor X activation by factor IXa in a concentration dependent manner. IAP also accelerates thrombin generation by factor Xa with a comparable peptide and substrate concentration dependence. Further, the Gla domain is also required for peptide activity confirming the hypothesis this peptide behaves as a membrane mimetic. Using fluorescence spectroscopy and an ELISA-based binding assay, we demonstrate direct binding between IAP and the Gla domain of factor X. Additionally, when IAP is immobilized to a reaction surface, factor X activation and thrombin generation are dramatically enhanced, as compared to the corresponding reactions on untreated surfaces. Finally, activated partial thromboplastin times (APTTs) of pooled plasma are significantly shortened on surfaces treated with IAP, in comparison with clotting times measured on untreated surfaces. The above results suggest that immobilized IAP may serve as a potential hemostatic agent, as demonstrated in isolated critical reactions of the coagulation cascade and in whole plasma.

Growth Arrest Specific gene product 6 (GAS-6), a γ-carboxylated protein expressed in quiescent fibroblasts and endothelial cells, exerts an anti-apoptotic function by binding to the receptor tyrosine kinase Axl. Recently, our laboratory has demonstrated that gas6-Axl interactions activate PI3-kinase with subsequent Akt phosphorylation during gas6-mediated protection from apoptosis. The current study explores further the mechanism by which this survival mechanism is achieved. FOXO1 is a member of the Forkhead family of transcription factors that plays a role in the expression of pro-apoptototic genes. Phosphorylation of FOXO1 at Thr24, Ser256 and Ser319 results in phospho-FOXO1 translocation from the nucleus to the cytoplasm, with consequent suppression of FOXO1 transcriptional activity and inhibition of apoptosis. In the present study we show, for the first time, that the treatment of serum-starved endothelial cells with 100 ng/ml of GAS-6 induces FOXO1 phosphorylation in a time dependent manner. Phosphorylated FOXO1 is translocated from the nucleus to the cytoplasm as evidenced by Western blot analysis of both nuclear and cytoplasmic extracts. Using fluorescence microscopy, FOXO1 is found predominantly in the nucleus during apoptosis induced by serum starvation. Upon gas6 stimulation, phosphorylated FOXO1 is translocated to the cytoplasm (see Figure 1). It is suggested that anti-apoptotic genes are then released from suppression and are thereby able to mediate cell survival. Both FOXO1 phosphorylation and translocation are suppressed by Wortmannin, a PI3-kinase inhibitor demonstrating that FOXO1 phosphorylation is PI3-kinase dependent. These results provide mechanistic insight of how gas6 rescues endothelial cells from serum-starvation-induced apoptosis. Foxo1 distribution Foxo1 distribution