A Novel Host-Proteome Signature for Distinguishing between Acute Bacterial and Viral Infections

Bacterial and viral infections are often clinically indistinguishable, leading to inappropriate patient management and antibiotic misuse. Bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogens. Our aim was to identify novel viral-induced host proteins that can complement bacterial-induced proteins to increase diagnostic accuracy. Initially, we conducted a bioinformatic screen to identify putative circulating host immune response proteins. The resulting 600 candidates were then quantitatively screened for diagnostic potential using blood samples from 1002 prospectively recruited patients with suspected acute infectious disease and controls with no apparent infection. For each patient, three independent physicians assigned a diagnosis based on comprehensive clinical and laboratory investigation including PCR for 21 pathogens yielding 319 bacterial, 334 viral, 112 control and 98 indeterminate diagnoses; 139 patients were excluded based on predetermined criteria. The best performing host-protein was TNF-related apoptosis-inducing ligand (TRAIL) (area under the curve [AUC] of 0.89; 95% confidence interval [CI], 0.86 to 0.91), which was consistently up-regulated in viral infected patients. We further developed a multi-protein signature using logistic-regression on half of the patients and validated it on the remaining half. The signature with the highest precision included both viral- and bacterial-induced proteins: TRAIL, Interferon gamma-induced protein-10, and CRP (AUC of 0.94; 95% CI, 0.92 to 0.96). The signature was superior to any of the individual proteins (P<0.001), as well as routinely used clinical parameters and their combinations (P<0.001). It remained robust across different physiological systems, times from symptom onset, and pathogens (AUCs 0.87-1.0). The accurate differential diagnosis provided by this novel combination of viral- and bacterial-induced proteins has the potential to improve management of patients with acute infections and reduce antibiotic misuse.     

Estimate of within population incremental selection through branch imbalance in lineage trees

Incremental selection within a population, defined as limited fitness changes following mutation, is an important aspect of many evolutionary processes. Strongly advantageous or deleterious mutations are detected using the synonymous to non-synonymous mutations ratio. However, there are currently no precise methods to estimate incremental selection. We here provide for the first time such a detailed method and show its precision in multiple cases of micro-evolution. The proposed method is a novel mixed lineage tree/sequence based method to detect within population selection as defined by the effect of mutations on the average number of offspring. Specifically, we propose to measure the log of the ratio between the number of leaves in lineage trees branches following synonymous and non-synonymous mutations. The method requires a high enough number of sequences, and a large enough number of independent mutations. It assumes that all mutations are independent events. It does not require of a baseline model and is practically not affected by sampling biases. We show the method''s wide applicability by testing it on multiple cases of micro-evolution. We show that it can detect genes and inter-genic regions using the selection rate and detect selection pressures in viral proteins and in the immune response to pathogens.     

Haptoglobin Phenotype,Preeclampsia Risk and the Efficacy of Vitamin C and E Supplementation to Prevent Preeclampsia in a Racially Diverse Population

Haptoglobin’s (Hp) antioxidant and pro-angiogenic properties differ between the 1-1, 2-1, and 2-2 phenotypes. Hp phenotype affects cardiovascular disease risk and treatment response to antioxidant vitamins in some non-pregnant populations. We previously demonstrated that preeclampsia risk was doubled in white Hp 2-1 women, compared to Hp 1-1 women. Our objectives were to determine whether we could reproduce this finding in a larger cohort, and to determine whether Hp phenotype influences lack of efficacy of antioxidant vitamins in preventing preeclampsia and serious complications of pregnancy-associated hypertension (PAH). This is a secondary analysis of a randomized controlled trial in which 10,154 low-risk women received daily vitamin C and E, or placebo, from 9-16 weeks gestation until delivery. Hp phenotype was determined in the study prediction cohort (n = 2,393) and a case-control cohort (703 cases, 1,406 controls). The primary outcome was severe PAH, or mild or severe PAH with elevated liver enzymes, elevated serum creatinine, thrombocytopenia, eclampsia, fetal growth restriction, medically indicated preterm birth or perinatal death. Preeclampsia was a secondary outcome. Odds ratios were estimated by logistic regression. Sampling weights were used to reduce bias from an overrepresentation of women with preeclampsia or the primary outcome. There was no relationship between Hp phenotype and the primary outcome or preeclampsia in Hispanic, white/other or black women. Vitamin supplementation did not reduce the risk of the primary outcome or preeclampsia in women of any phenotype. Supplementation increased preeclampsia risk (odds ratio 3.30; 95% confidence interval 1.61–6.82, p<0.01) in Hispanic Hp 2-2 women. Hp phenotype does not influence preeclampsia risk, or identify a subset of women who may benefit from vitamin C and E supplementation to prevent preeclampsia.     

Natural history,physiology and energetic strategies of Asellia tridens (Chiroptera)

We used radio-telemetry, observations and physiological measurements to study the basic biology and energetic strategies of Asellia tridens in northern Israel from 2009 to 2010. Between late May and early November, the bats occupied abandoned man-made structures in this area. Parturition occurred between late June and mid-July, and juveniles were independent by late August. A. tridens foraged near the roost in a vegetation-rich, cluttered background environment, catching insects flying close to vegetation. Its diet was diverse, with Coleoptera, Heteroptera, Diptera and Lepidoptera being the main diet components. During summer, males and females differed in their foraging patterns and energetic strategies: Lactating females departed for more frequent foraging bouts than males, and maintained euthermy throughout the day, while males became torpid on a daily basis.     

Knock-down of plasminogen-activator inhibitor-1 enhances expression of E-cadherin and promotes epithelial differentiation of human pancreatic adenocarcinoma cells

High levels of plasminogen activator inhibitor-1 (PAI-1), which is produced by stromal, endothelial, and cancer cells and has multiple complex effects on cancers, correlate with poor cancer prognosis. To more definitively study the role of endogenously produced PAI-1 in human pancreatic adenocarcinoma (PAC) PANC-1 cell line biology, we used anti-PAI-1 shRNA to create stable PAI-1 deficient cells (PD-PANC-1s). PD-PANC-1s exhibited a heterogeneous morphology. While the majority of cells exhibited a cuboidal shape similar to the parental PANC-1 or the vector-infected control cells, numerous large cells with long filopodia and a neuronal-like appearance were observed. Although both Vector-control cells and PD-PANC-1s expressed mRNAs that are characteristic of mesenchymal, neural, and epithelial phenotypes, epithelial marker RNAs were up-regulated (e.g., E-cadherin, 32-fold) whereas mesenchymal marker RNAs were down-regulated (e.g., Thy1, ninefold) in PD-PANC-1s, suggesting mesenchymal-to-epithelial transition. Neural markers exhibited both up- and down-regulation. Immunocytochemistry indicated that epithelial-like PD-PANC-1s expressed E-cadherin and β-catenin in significantly more cells, while neural-like cells exhibited robust expression of organized β-3-tubulin. PAI-1 and E-cadherin were rarely co-expressed in the same cells. Indeed, examination of PAI-1 and E-cadherin mRNAs expression in additional cell lines yielded clear inverse correlation. Indeed, infection of Colo357 PAC cells (that exhibit high expression of E-cadherin) with PAI-1-expressing adenovirus led to a marked decrease in E-cadherin expression and to enhanced migration of cells from clusters. Our results suggest that endogenous PAI-1 suppresses expression of E-cadherin and differentiation in PAC cells in vitro, supporting its negative impact on tumor prognosis.     

Targeting of nanoparticles to the clathrin-mediated endocytic pathway

Nanoparticles (NPs) are considered attractive carriers for gene therapy and drug delivery owing to their minor toxic effect and their ability to associate and internalize into mammalian cells. In this study, we compared the endocytosis into HeLa cells of NPs exposing either a negative or positive charge on their surface. The exposed charge significantly affected their ability to internalize as well as the cellular endocytosis mechanism utilized. Negatively charged NPs show an inferior rate of endocytosis and do not utilize the clathrin-mediated endocytosis pathway. On the other hand, positively charged NPs internalize rapidly via the clathrin-mediated pathway. When this pathway is blocked, NPs activate a compensatory endocytosis pathway that results in even higher accumulation of NPs. Overall, the addition of a positive charge to NPs may improve their potential as nanoparticulate carriers for drug delivery.     

Alteration in the regulation of plasma membrane glycoproteins of the hepatocyte during ontogeny

The expression of four integral membrane glycoproteins was examined in detail utilizing monospecific antibodies during liver development. These included asialoglycoprotein receptor, a hepatocyte glycoprotein residing in the sinusoidal domain, and three bile canalicular glycoproteins, leucine aminopeptidase, dipeptidyl peptidase IV, and a Mr 110,000 glycoprotein denoted GP 110. It was observed that asialoglycoprotein receptor, GP 110, and dipeptidyl peptidase IV were present in low amounts in fetal liver and reached adult levels between 1 to 3 weeks. In contrast, leucine aminopeptidase was present in nearly adult amounts in 18-day-old fetal livers. These observations were qualitatively confirmed by indirect immunofluorescent staining of frozen thin liver sections obtained from fetal and adult rats. Further, in fetal livers it was found that leucine aminopeptidase was not localized to typical bile canalicular areas. Immunoprecipitation studies performed in the presence of proteolytic inhibitors using detergent-solubilized extracts of metabolically labeled liver minces revealed that GP 110 was present in low amounts as Mr 110,000 and Mr 105,000 polypeptides in 17-day fetal livers but by 21 days of gestation the larger polypeptide was the major synthesis product. Conversely, the apparent molecular weights of leucine aminopeptidase and dipeptidyl peptidase IV were not altered during development. Experiments determining relative rates of synthesis using excess amounts of antibodies showed that the concentrations of the three bile canalicular glycoproteins in liver during ontogeny reflect their rates of synthesis. These results underscore that plasma membrane constituents of the hepatocyte undergo dramatic changes in expression and localization as the liver changes its physiological role at birth.     

Insulin action in intact mouse diaphragm II.

Summary Incubation of intact mouse diaphragms with insulin in the absence of glucose resulted in a rapid inhibition of the subsequent cell-free phosphorylation of endogenous protein substrates in tissue extracts. The phosphorylation of added histone was inhibited to a lesser extent. The inhibition was observed both in the absence and in the presence of added cyclic 35 adenosine monophosphate. Acrylamide gel electrophoresis of phosphorylation products revealed a number of major phosphorylated polypeptides. The phosphorylation of several polypeptides was inhibited following short treatment with insulin. These results represent a novel experimental approach to the elucidation of the mechanism of the action of insulin and are consistent with our hypothesis that the inhibition of protein kinase activities in the tissue may be the first step in this mechanism.Abbreviations Tricine N-tris(hydroxymethyl)methyl glycine - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-amino ethyl ether) N,N¹-tetraacetic acid - Mes 2(N-morpholino)ethane sulfonic acid J. Larner is an established investigator of the American Diabetes Association.