Discovery of novel isothiazole inhibitors of the TrkA kinase: structure-activity relationship, computer modeling, optimization, and identification of highly potent antagonists

The design, synthesis, and biological evaluation of potent inhibitors of the TrkA kinase is presented. A homology model is created to aid in the enhancement of potency and selectivity of isothiazole inhibitors found during a high-throughput screen. Three different syntheses are utilized to make diverse analogs within this series. Aminoheterocycles are found to be good urea surrogates, whereas bicyclic substituents on the C3 thio group were found to be extremely potent TrkA inhibitors in kinase and cell assays.     

Synthesis of unique 17beta-estradiol homo-dimers, estrogen receptors binding affinity evaluation and cytocidal activity on breast, intestinal and skin cancer cell lines

A rapid and efficient synthesis of a series of C2-symmetric 17beta-estradiol homo-dimers is described. The new molecules are linked at position 17alpha of the steroid nucleus with either an alkyl chain or a polyethylene glycol chain. They are made from estrone in only five chemical steps with an overall yield exceeding 30%. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER+ and ER-) human breast tumor cell lines: MCF-7 and MDA-MB-231. Some of the dimers present selective cytotoxic activity against the ER+ cell line. However, they are not very cytotoxic when compared to the antiestrogen tamoxifen. Unfortunately, they show only weak affinity for the estrogen receptor alpha (ERalpha) and no affinity for the estrogen receptor beta (ERbeta). The new compounds were also tested on human intestinal (HT-29) cancer and on murine skin cancer (B16-F10) cell lines for further biological assessment. Interestingly, the dimers were found to be cytotoxic to the murine skin cancer cell line but were inactive towards the intestinal cancer cell line.     

Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α₂-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.     

Elastin peptides signaling relies on neuraminidase-1-dependent lactosylceramide generation

The sialidase activity of neuraminidase-1 (Neu-1) is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex. In this work, we demonstrate that the receptor and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains as well as their depletion in glycolipids blocks the receptor signaling. Following elastin peptide treatment, the cellular GM(3) level decreases while lactosylceramide (LacCer) content increases consistently with a GM(3)/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is responsible for this lipid conversion. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation. Further, the use of a monoclonal anti-GM(3) blocking antibody shows that GM(3) is required for signaling. In conclusion, our data strongly suggest that Neu-1-dependent GM(3)/LacCer conversion is the key event leading to signaling by the elastin receptor complex. As a consequence, we propose that LacCer is an early messenger for this receptor.     

Potentiation of Polarized Intestinal Caco-2 Cell Responsiveness to Probiotics Complexed with Secretory IgA

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.     

Association of maternal and nutrient supply line factors with DNA methylation at the imprinted IGF2/H19 locus in multiple tissues of newborn twins

Epigenetic events are crucial for early development, but can be influenced by environmental factors, potentially programming the genome for later adverse health outcomes. The insulin-like growth factor 2 (IGF2)/H19 locus is crucial for prenatal growth and the epigenetic state at this locus is environmentally labile. Recent studies have implicated maternal factors, including folate intake and smoking, in the regulation of DNA methylation at this locus, although data are often conflicting in the direction and magnitude of effect. Most studies have focused on single tissues and on one or two differentially-methylated regions (DMRs) regulating IGF2/H19 expression. In this study, we investigated the relationship between multiple shared and non-shared gestational/maternal factors and DNA methylation at four IGF2/H19 DMRs in five newborn cell types from 67 pairs of monozygotic and 49 pairs of dizygotic twins. Data on maternal and non-shared supply line factors were collected during the second and third trimesters of pregnancy and DNA methylation was measured via mass spectrometry using Sequenom MassArray EpiTyper analysis. Our exploratory approach showed that the site of umbilical cord insertion into the placenta in monochorionic twins has the strongest positive association with methylation in all IGF2/H19 DMRs (p < 0.05). Further, evidence for tissue- and locus-specific effects were observed, emphasizing that responsiveness to environmental exposures in utero cannot be generalized across genes and tissues, potentially accounting for the lack of consistency in previous findings. Such complexity in responsiveness to environmental exposures in utero has implications for all epigenetic studies investigating the developmental origins of health and disease.     

Personality-dependent dispersal cancelled under predation risk

Dispersal is a fundamental life-history trait for many ecological processes. Recent studies suggest that dispersers, in comparison to residents, display various phenotypic specializations increasing their dispersal inclination or success. Among them, dispersers are believed to be consistently more bold, exploratory, asocial or aggressive than residents. These links between behavioural types and dispersal should vary with the cause of dispersal. However, with the exception of one study, personality-dependent dispersal has not been studied in contrasting environments. Here, we used mosquitofish (Gambusia affinis) to test whether personality-dependent dispersal varies with predation risk, a factor that should induce boldness or sociability-dependent dispersal. Corroborating previous studies, we found that dispersing mosquitofish are less social than non-dispersing fish when there was no predation risk. However, personality-dependent dispersal is negated under predation risk, dispersers having similar personality types to residents. Our results suggest that adaptive dispersal decisions could commonly depend on interactions between phenotypes and ecological contexts.     

Impaired transient vasodilation and increased vasoconstriction to digital local cooling in primary Raynaud's phenomenon

Raynaud's phenomenon (RP) is defined as episodic ischemia of the extremities in response to cold. Although the structure of skin capillaries is normal in primary RP, some data suggest impairment of microvascular function. We aimed at testing whether digital skin blood flow was lower in RP than in controls while cooling locally. We further evaluated the contribution of sensory nerves in the response. We recruited 21 patients with primary RP and 20 healthy volunteers matched on age and gender. After a 10-min baseline at 33°C, skin temperature was cooled at 15 or 24°C during 30 min on the forearm and the finger while monitoring perfusion with a custom-design laser Doppler flowmetry probe. Perfusion was also assessed after topical anesthesia. Blood flow was expressed as cutaneous vascular conductance (CVC). Data were subsequently expressed as area above the curve (AAC(0-30)) of the percentage decrease from baseline CVC (%BL). CVC on the dorsum of the finger was lower in RP patients compared with controls at 15°C (AAC(0-30) were 106,237.2 and 69,544.3%BL·s, respectively; P = 0.02) and at 24°C (AAC(0-30) were 86,915 and 57,598%BL·s, respectively; P = 0.04) whereas we observed no significant difference on the finger pad and the forearm. Topical anesthesia increased CVC in patients with RP (P = 0.05), whereas it did not affect reactivity in controls (P = 0.86). Our study shows exaggerated skin microvascular vasoconstriction to local cooling on the dorsum of the finger in primary RP compared with controls. Part of this abnormal response in primary RP depends on sensitive nerves.     

Staphylococcal biofilm disassembly

Staphylococcus aureus and Staphylococcus epidermidis are a frequent cause of biofilm-associated infections that are a tremendous burden on our healthcare system. Staphylococcal biofilms exhibit extraordinary resistance to antimicrobial killing, limiting the efficacy of antibiotic therapy, and surgical intervention is often required to remove infected tissues or implanted devices. Recent work has provided new insight into the molecular basis of biofilm development in these opportunistic pathogens. Extracellular bacterial products, environmental conditions, and polymicrobial interactions have all been shown to influence profoundly the ability of these bacteria to colonize and disperse from clinically relevant surfaces. We review new developments in staphylococcal biofilm disassembly and set them in the context of potential strategies to control biofilm infections.     

Viral Etiology of Respiratory Tract Infections in Children at the Pediatric Hospital in Ouagadougou (Burkina Faso)

Background

Acute respiratory infections (ARIs) are a major cause of morbidity and mortality in children in Africa. The circulation of viruses classically implicated in ARIs is poorly known in Burkina Faso. The aim of this study was to identify the respiratory viruses present in children admitted to or consulting at the pediatric hospital in Ouagadougou.

Methods

From July 2010 to July 2011, we tested nasal aspirates of 209 children with upper or lower respiratory infection for main respiratory viruses (respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza viruses 1, 2 and 3, influenza A, B and C, rhinovirus/enterovirus), by immunofluorescence locally in Ouagadougou, and by PCR in France. Bacteria have also been investigated in 97 samples.

Results

153 children (73.2%) carried at least one virus and 175 viruses were detected. Rhinoviruses/enteroviruses were most frequently detected (rhinovirus n = 88; enterovirus n = 38) and were found to circulate throughout the year. An epidemic of RSV infections (n = 25) was identified in September/October, followed by an epidemic of influenza virus (n = 13), mostly H1N1pdm09. This epidemic occurred during the period of the year in which nighttime temperatures and humidity were at their lowest. Other viruses tested were detected only sporadically. Twenty-two viral co-infections were observed. Bacteria were detected in 29/97 samples with 22 viral/bacterial co-infections.

Conclusions

This study, the first of its type in Burkina Faso, warrants further investigation to confirm the seasonality of RSV infection and to improve local diagnosis of influenza. The long-term objective is to optimize therapeutic management of infected children.