Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases. © 2013 Neal et al

Early pyloric stenosis: a case control study

Pyloric stenosis (PS) is rare in the first 2 weeks of life, often leading to delays in diagnosis and treatment. We conducted a case control study to delineate the characteristics of patients with early PS (EPS). In addition, we tested the hypothesis that patients with EPS present with a smaller pylorus than older patients. A database of all patients presenting with PS to a children’s hospital over a 5-year period (2002–2006) was obtained. Each patient admitted during the first 2 weeks of life (subject) was matched to a patient admitted after 4 weeks of age (control), with the same gender, electrolyte status, and treating surgeon. A single pediatric radiologist, blinded to patient age, reviewed all available ultrasounds retrospectively. Demographic, clinical, diagnostic, therapeutic, and outcome data were compared. During the study period, 278 pyloromyotomies were performed for PS. Sixteen patients (5.8%) presented with EPS between 2 and 14 days of life. EPS patients had a higher prevalence of positive family history (31 vs. 0%, P = 0.043), and breast milk feeding (75 vs. 31%, P = 0.045). Sonographic measurements showed a pylorus that was of significantly less length (17.1 ± 0.6 vs. 20.5 ± 0.9 mm, P = 0.006) and muscle thickness (3.5 ± 0.2 vs. 4.9 ± 0.2 mm, P < 0.001) in patients with EPS. Hospital stay was significantly longer for EPS patients (4.3 ± 0.9 vs. 2.0 ± 0.1 days, P = 0.19) Babies presenting with EPS are more likely to be breast fed and to have a positive family history. EPS is associated with a longer hospital stay. Use of sonographic diagnostic measurements specific to this age group may prevent delays in diagnosis and treatment, and improve outcomes

Generation and Validation of a Shewanella oneidensis MR-1 Clone Set for Protein Expression and Phage Display

A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST- tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro

Gram Negative Bacteria Are Associated with the Early Stages of Necrotizing Enterocolitis

Introduction: Necrotizing enterocolitis (NEC) affects 5–10 % of infants born weighing less than 1500 g. Most models of NEC recapitulate late-stage disease with gut necrosis and elevated inflammatory mediators. Evaluation of NEC at earlier, less lethal stages of disease will allow investigation of initial disease triggers and may advance our understanding of temporal relationships between factors implicated in NEC pathogenesis. In this manuscript, we describe our investigation of early NEC and test the hypothesis that bacteria and inflammatory mediators differ between animals with early NEC and disease fre

Global survey of the roles, satisfaction, and barriers of home healthcare nurses on the provision of palliative care

Background: the World Health Assembly urges members to build palliative care (PC) capacity as an ethical imperative. Nurses provide PC services in a variety of settings, including the home and may be the only health care professional able to access some disparate populations. Identifying current nursing services, resources, and satisfaction and barriers to nursing practice are essential to build global PC capacity. Objective: to globally examine home health care nurses' practice, satisfaction, and barriers, regarding existing palliative home care provision. Design: needs assessment survey. Setting/Subjects: five hundred thirty-two home health care nurses in 29 countries. Measurements: a needs assessment, developed through literature review and cognitive interviewing. Results: nurses from developing countries performed more duties compared with those from high-income countries, suggesting a lack of resources in developing countries. Significant barriers to providing home care exist: personnel shortages, lack of funding and policies, poor access to end-of-life or hospice services, and decreased community awareness of services provided. Respondents identified lack of time, funding, and coverages as primary educational barriers. In-person local meetings and online courses were suggested as strategies to promote learning. Conclusions: it is imperative that home health care nurses have adequate resources to build PC capacity globally, which is so desperately needed. Nurses must be up to date on current evidence and practice within an evidence-based PC framework. Health care policy to increase necessary resources and the development of a multifaceted intervention to facilitate education about PC is indicated to build global capacity

Trauma Hemorrhagic Shock-Induced Lung Injury Involves a Gut-Lymph-Induced TLR4 Pathway in Mice

Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation.The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan's blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice.Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation

On the Relationship between Sialomucin and Sulfomucin Expression and Hydrogenotrophic Microbes in the Human Colonic Mucosa

The colonic mucus layer is comprised primarily of acidomucins, which provide viscous properties and can be broadly classified into sialomucins or sulfomucins based on the presence of terminating sialic acid or sulfate groups. Differences in acidomucin chemotypes have been observed in diseases such as colorectal cancer and inflammatory bowel disease, and variation in sialo- and sulfomucin content may influence microbial colonization. For example, sulfate derived from sulfomucin degradation may promote the colonization of sulfate-reducing bacteria (SRB), which through sulfate respiration generate the genotoxic gas hydrogen sulfide. Here, paired biopsies from right colon, left colon, and rectum of 20 subjects undergoing routine screening colonoscopies were collected to enable parallel histochemical and microbiological studies. Goblet cell sialo- and sulfomucins in each biopsy were distinguished histochemically and quantified. Quantitative PCR and multivariate analyses were used to examine the abundance of hydrogenotrophic microbial groups and SRB genera relative to acidomucin profiles. Regional variation was observed in sialomucins and sulfomucins with the greatest abundance of each found in the rectum. Mucin composition did not appear to influence the abundance of SRB or other hydrogenotrophic microbiota but correlated with the composition of different SRB genera. A higher sulfomucin proportion correlated with higher quantities of Desulfobacter, Desulfobulbus and Desulfotomaculum, relative to the predominant Desulfovibrio genus. Thus, acidomucin composition may influence bacterial sulfate respiration in the human colon, which may in turn impact mucosal homeostasis. These results stress the need to consider mucus characteristics in the context of studies of the microbiome that target intestinal diseases

Environmental Adaptation: Genomic Analysis of the Piezotolerant and Psychrotolerant Deep-Sea Iron Reducing Bacterium Shewanella piezotolerans WP3

Shewanella species are widespread in various environments. Here, the genome sequence of Shewanella piezotolerans WP3, a piezotolerant and psychrotolerant iron reducing bacterium from deep-sea sediment was determined with related functional analysis to study its environmental adaptation mechanisms. The genome of WP3 consists of 5,396,476 base pairs (bp) with 4,944 open reading frames (ORFs). It possesses numerous genes or gene clusters which help it to cope with extreme living conditions such as genes for two sets of flagellum systems, structural RNA modification, eicosapentaenoic acid (EPA) biosynthesis and osmolyte transport and synthesis. And WP3 contains 55 open reading frames encoding putative c-type cytochromes which are substantial to its wide environmental adaptation ability. The mtr-omc gene cluster involved in the insoluble metal reduction in the Shewanella genus was identified and compared. The two sets of flagellum systems were found to be differentially regulated under low temperature and high pressure; the lateral flagellum system was found essential for its motility and living at low temperature