Inhibition of Plant-Pathogenic Bacteria by Short Synthetic Cecropin A-Melittin Hybrid Peptides

Short peptides of 11 residues were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Xanthomonas vesicatoria and compared to the previously described peptide Pep3 (WKLFKKILKVL-NH₂). The antimicrobial activity of Pep3 and 22 analogues was evaluated in terms of the MIC and the 50% effective dose (ED₅₀) for growth. Peptide cytotoxicity against human red blood cells and peptide stability toward protease degradation were also determined. Pep3 and several analogues inhibited growth of the three pathogens and had a bactericidal effect at low micromolar concentrations (ED₅₀ of 1.3 to 7.3 μM). One of the analogues consisting of a replacement of both Trp and Val with Lys and Phe, respectively, resulted in a peptide with improved bactericidal activity and minimized cytotoxicity and susceptibility to protease degradation compared to Pep3. The best analogues can be considered as potential lead compounds for the development of new antimicrobial agents for use in plant protection either as components of pesticides or expressed in transgenic plants.     

Identification and Localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae)

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor “Candidatus Portiera aleyrodidarum,” an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.     

The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.     

High-sensitivity cardiac troponin T: risk stratification tool in patients with symptoms of chest discomfort

Background

Recent studies have demonstrated the association between increased concentrations of high-sensitivity cardiac troponin T (hs-cTnT) and the incidence of myocardial infarction, heart failure, and mortality. However, most prognostic studies to date focus on the value of hs-cTnT in the elderly or general population. The value of hs-cTnT in symptomatic patients visiting the outpatient department remains unclear. The aim of this study was to investigate the prognostic value of hs-cTnT as a biomarker in patients with symptoms of chest discomfort suspected for coronary artery disease and to assess its additional value in combination with other risk stratification tools in predicting cardiac events.

Methods

We studied 1,088 patients (follow-up 2.2±0.8 years) with chest discomfort who underwent coronary calcium scoring and coronary CT-angiography. Traditional cardiovascular risk factors and concentrations of hs-cTnT, N-terminal pro-brain-type natriuretic peptide (NT-proBNP) and high-sensitivity C-reactive protein (hsCRP) were assessed. Study endpoint was the occurrence of late coronary revascularization (>90 days), acute coronary syndrome, and cardiac mortality.

Results

Hs-cTnT was a significant predictor for the composite endpoint (highest quartile [Q4]>6.7 ng/L, HR 3.55; 95%CI 1.88–6.70; P<0.001). Survival analysis showed that hs-cTnT had significant predictive value on top of current risk stratification tools (Chi-square change P<0.01). In patients with hs-cTnT in Q4 versus P<0.01). This was not the case for hsCRP and NT-proBNP.

Conclusions

Hs-cTnT is a useful prognostic biomarker in patients with chest discomfort suspected for coronary artery disease. In addition, hs-cTnT was an independent predictor for cardiac events when corrected for cardiovascular risk profiling, calcium score and CT-angiography results.     

Changes in ant communities along an age gradient of cocoa cultivation in the Oumé region,central Côte d'Ivoire

We identified the extent to which ant diversity occurs despite conversion of forests into cocoa plantations by examining the communities across four age classes of plantations (classes I–IV with increasing age from 0–5 to 21–40 years) and in their original forests. An extensive sampling protocol consisting of pitfall trapping, leaf litter sampling, soil sampling and hand sampling was used to characterize ant species richness and composition in three replicates of each age class and in the remaining forest patches. A total of one hundred ant species was found in all habitats combined. While the forest was the richest habitat (73 species), species richness in the different plantation age classes varied as follows (sorted in descending order): class IV (69 species) > class III (57 species) > class I (52 species) > class II (43 species). Age gradient was thus significantly positively correlated with mean species richness and with the relative abundance of some subfamilies. The species composition differed greatly between some plantation age classes and the forest. The two youngest cocoa age classes (I and II) were most dissimilar to the forest. In contrast, forest ants were well represented in the old cocoa age classes (III and IV). Three functional guilds (generalist predators, specialist predators and territorially dominant arboreal species) were in their relative abundance significantly correlated to the age gradient. Overall, cocoa cultivations retaining a floristically diverse and structurally complex forest structure are a suitable management system for the conservation of ant species of the formerly forested habitats.     

TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.     

High Resolution X Chromosome-Specific Array-CGH Detects New CNVs in Infertile Males

Context

The role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far.

Objectives

In order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls.

Results

We identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10⁻⁶) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10⁻⁴).

Conclusions

By the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations.     

Vitamin k-antagonists accelerate atherosclerotic calcification and induce a vulnerable plaque phenotype

Background

Vitamin K-antagonists (VKA) are treatment of choice and standard care for patients with venous thrombosis and thromboembolic risk. In experimental animal models as well as humans, VKA have been shown to promote medial elastocalcinosis. As vascular calcification is considered an independent risk factor for plaque instability, we here investigated the effect of VKA on coronary calcification in patients and on calcification of atherosclerotic plaques in the ApoE^−/− model of atherosclerosis.

Methodology/Principal Findings

A total of 266 patients (133 VKA users and 133 gender and Framingham Risk Score matched non-VKA users) underwent 64-slice MDCT to assess the degree of coronary artery disease (CAD). VKA-users developed significantly more calcified coronary plaques as compared to non-VKA users. ApoE^−/− mice (10 weeks) received a Western type diet (WTD) for 12 weeks, after which mice were fed a WTD supplemented with vitamin K₁ (VK₁, 1.5 mg/g) or vitamin K₁ and warfarin (VK₁&W; 1.5 mg/g & 3.0 mg/g) for 1 or 4 weeks, after which mice were sacrificed. Warfarin significantly increased frequency and extent of vascular calcification. Also, plaque calcification comprised microcalcification of the intimal layer. Furthermore, warfarin treatment decreased plaque expression of calcification regulatory protein carboxylated matrix Gla-protein, increased apoptosis and, surprisingly outward plaque remodeling, without affecting overall plaque burden.

Conclusions/Significance

VKA use is associated with coronary artery plaque calcification in patients with suspected CAD and causes changes in plaque morphology with features of plaque vulnerability in ApoE^−/− mice. Our findings underscore the need for alternative anticoagulants that do not interfere with the vitamin K cycle.