Identifying the drivers of multidrug-resistant Klebsiella pneumoniae at a European level

Beta-lactam- and in particular carbapenem-resistant Enterobacteriaceae represent a major public health threat. Despite strong variation of resistance across geographical settings, there is limited understanding of the underlying drivers. To assess these drivers, we developed a transmission model of cephalosporin- and carbapenem-resistant Klebsiella pneumoniae. The model is parameterized using antibiotic consumption and demographic data from eleven European countries and fitted to the resistance rates for Klebsiella pneumoniae for these settings. The impact of potential drivers of resistance is then assessed in counterfactual analyses. Based on reported consumption data, the model could simultaneously fit the prevalence of extended-spectrum beta-lactamase-producing and carbapenem-resistant Klebsiella pneumoniae (ESBL and CRK) across eleven European countries over eleven years. The fit could explain the large between-country variability of resistance in terms of consumption patterns and fitted differences in hospital transmission rates. Based on this fit, a counterfactual analysis found that reducing nosocomial transmission and antibiotic consumption in the hospital had the strongest impact on ESBL and CRK prevalence. Antibiotic consumption in the community also affected ESBL prevalence but its relative impact was weaker than inpatient consumption. Finally, we used the model to estimate a moderate fitness cost of CRK and ESBL at the population level. This work highlights the disproportionate role of antibiotic consumption in the hospital and of nosocomial transmission for resistance in gram-negative bacteria at a European level. This indicates that infection control and antibiotic stewardship measures should play a major role in limiting resistance even at the national or regional level.     

Structure-activity relationship (SAR) investigations of substituted imidazole analogs as TRPV1 antagonists

A novel series of 4,5-biarylimidazoles as TRPV1 antagonists were designed based on the previously reported 4,6-disubstituted benzimidazole series. The analogs were evaluated for their ability to block capsaicin- or acid-induced calcium influx in TRPV1-expressing CHO cells. These studies led to the identification of a highly potent and orally bioavailable TRPV1 antagonist, imidazole 33.     

Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs

The seven-transmembrane-spanning receptors of the FZD_1–10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD₂, FZD₄, or FZD₅, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD_2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.     

Signature of an early genetic bottleneck in a population of Moroccan sardines (Sardina pilchardus)

Fishery assessment models meant to determine sustainability of commercial marine fish failed to predict recent stock collapses due to overexploitation. One flaw of assessment models is that they strongly rely on catch and age-composition statistics, but largely ignore the genetic background of the studied populations. We examined population genetic structure of sardine (Sardina pilchardus) in the centraleastern and northeastern Atlantic Ocean and Mediterranean Sea to aid fishery management of this heavily fished small pelagic species. We found that sardine has a striking mitochondrial control region, and sequenced a fragment of 387 bp of its 5'-end in 261 individuals collected off the coasts of Morocco (Dakhla, Tantan, Safi, Larache, and Nador), Portugal (Quarteira), Spain (Pasajes, Barcelona), and Greece (Kavala). High levels of haplotypic diversity rendered a rather unresolved NJ phylogeny. The recovered tree had no phylogeographic structuring except for the clustering of 13 individuals of Safi. In contrast, individuals grouped together according to the presence or absence of a 13-bp insertion in the sequence. Phi(ST) pairwise comparisons and molecular variance analyses supported genetic differentiation between the population of Pasajes (Bay of Biscay), and those of the Mediterranean Sea and Moroccan coast, with a contact zone around the Strait of Gibraltar. This result confirms the existence of two subspecies, S. pilchardus pilchardus and S. pilchardus sardina that were previously identified based on meristics and morphometry. Mismatch distribution analysis showed that sardine populations are expanding since the Pleistocene. Surprisingly, the population of Safi showed strong and statistically significant levels of genetic differentiation that could be related with isolation and genetic drift. Comparative analysis of the Safi population versus the rest including mismatch distributions, and a Bayesian skyline plot suggest that the Safi population likely underwent an early genetic bottleneck. The genetic singularity of the Safi population could have been responsible for the historical collapse of this sardine stock in the 1970s.     

Alterations in the Expression of the Anti-Apoptotic Factor HAX-1 upon Seizures-Induced Hippocampal Injury in the Neonatal Rat Brain

HS1-associated protein X1 (HAX-1) is a mitochondrial protein which interacts with a diverse group of molecules such as inflammatory cytokines; interleukin-1, hematopoietic lineage specific protein-1 and vimentin. It has been reported that HAX-1 may act as antiapoptotic protein in HeLa- and Jurkat cells after Fas-treatment, irradiation or serum deprivation. This underlines the evidence that HAX-1 might be involved in both receptor- and mitochondria-mediated apoptosis pathways. However, the role of HAX-1 in neuronal death induced by status epilepticus in the immature brain has not been reported. In this study, we performed a status epilepticus in rats and investigated the dynamic changes of HAX-1 expression, HtrA2 distribution and caspase-3 activation in the hippocampus. Western blot and immunohistochemistry analysis revealed that HAX-1 was expressed at very low levels in the hippocampus. Status epilepticus in the immature brain significantly induced increased cytosolic accumulation of HAX-1 in a biphasic manner, induced an upregulation of HtrA2 and enhanced caspase-3 activity in the selectively vulnerable hippocampal CA1-subfield. Taken together, these results suggested that HAX-1 is probably involved in the pathophysiology of cell death induced by epilepsy.     

Identification of a Novel Pathway of Transforming Growth Factor-β1 Regulation by Extracellular NAD+ in Mouse Macrophages: IN VITRO AND IN SILICO STUDIES

Extracellular β-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-β1.     

Ischemia-reperfusion injury and pregnancy initiate time-dependent and robust signs of up-regulation of cardiac progenitor cells

To explore how cardiac regeneration and cell turnover adapts to disease, different forms of stress were studied for their effects on the cardiac progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5, and mast cells. Adult female rats were examined during pregnancy, after myocardial infarction and ischemia-reperfusion injury with/out insulin like growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac sub-domains were analyzed at one and two weeks post-intervention, both at the mRNA and protein levels. While pregnancy and myocardial infarction up-regulated Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion injury induced the strongest up-regulation which occurred globally throughout the entire heart and not just around the site of injury. This response seems to be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary to c-Kit, Isl1 was not up-regulated by pregnancy or myocardial infarction while ischemia-reperfusion injury induced not a global but a focal up-regulation in the outflow tract and also in the peri-ischemic region, correlating with the up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1. c-Kit expression was not further influenced by the exogenous growth factors. This indicates that there is a spatial mismatch between on one hand c-Kit and Nkx2.5 expression and on the other hand Isl1 expression. In conclusion, ischemia-reperfusion injury was the strongest stimulus with both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregnancy induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes throughout the heart. Utilization of these pathways could provide new strategies for the treatment of cardiac disease.