Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice

Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.     

Parasitism of the “Fuller’s rose weevil” Naupactus cervinus by Microctonus sp. in Argentina

We report the occurrence of an unidentified species of the wasp Microctonus Wesmael (Hymenoptera: Braconidae) parasitizing adults of the Fuller’s rose weevil Naupactus cervinus (Boheman) (Coleoptera: Curculionidae), a widespread pest of economically important crops included in the South American tribe Naupactini. Cytochrome c oxidase subunit I-based phylogenetic analysis indicates that the parasitoid is closely related to Microctonus hyperodae Loan. Their first instar larvae show slight morphological differences with this species. Superparasitism by first instar larvae occurred at low frequency. Some teratocytes were observed. Microctonus sp. and its host were infected with different strains of the reproductive parasite Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae), although the bacterial lineage harbored by the wasp coincides with that infecting most parthenogenetic Naupactini. This multipartite association (weevil bearing both Wolbachia and Microctonus, and Microctonus bearing Wolbachia) opens challenging perspectives for future research on biological interactions and biological control.     

Diagnostic performance of multi-organ ultrasound with pocket-sized device in the management of acute dyspnea

Background

The availability of ultra-miniaturized pocket ultrasound devices (PUD) adds diagnostic power to the clinical examination. Information on accuracy of ultrasound with handheld units in immediate differential diagnosis in emergency department (ED) is poor. The aim of this study is to test the usefulness and accuracy of lung ultrasound (LUS) alone or combined with ultrasound of the heart and inferior vena cava (IVC) using a PUD for the differential diagnosis of acute dyspnea (AD).

Methods

We included 68 patients presenting to the ED of “Maurizio Bufalini” Hospital in Cesena (Italy) for AD. All patients underwent integrated ultrasound examination (IUE) of lung-heart-IVC, using PUD. The series was divided into patients with dyspnea of cardiac or non-cardiac origin. We used 2 × 2 contingency tables to analyze sensitivity, specificity, positive predictive value and negative predictive value of the three ultrasonic methods and their various combinations for the diagnosis of cardiogenic dyspnea (CD), comparing with the final diagnosis made by an independent emergency physician.

Results

LUS alone exhibited a good sensitivity (92.6%) and specificity (80.5%). The highest accuracy (90%) for the diagnosis of CD was obtained with the combination of LUS and one of the other two methods (heart or IVC).

Conclusions

The IUE with PUD is a useful extension of the clinical examination, can be readily available at the bedside or in ambulance, requires few minutes and has a reliable diagnostic discriminant ability in the setting of AD.

     

Targeted screening strategies to detect Trypanosoma cruzi infection in children

Background

Millions of people are infected with Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Anti-trypanosomal drug therapy can cure infected individuals, but treatment efficacy is highest early in infection. Vector control campaigns disrupt transmission of T. cruzi, but without timely diagnosis, children infected prior to vector control often miss the window of opportunity for effective chemotherapy.

Methods and Findings

We performed a serological survey in children 2–18 years old living in a peri-urban community of Arequipa, Peru, and linked the results to entomologic, spatial and census data gathered during a vector control campaign. 23 of 433 (5.3% [95% CI 3.4–7.9]) children were confirmed seropositive for T. cruzi infection by two methods. Spatial analysis revealed that households with infected children were very tightly clustered within looser clusters of households with parasite-infected vectors. Bayesian hierarchical mixed models, which controlled for clustering of infection, showed that a child''s risk of being seropositive increased by 20% per year of age and 4% per vector captured within the child''s house. Receiver operator characteristic (ROC) plots of best-fit models suggest that more than 83% of infected children could be identified while testing only 22% of eligible children.

Conclusions

We found evidence of spatially-focal vector-borne T. cruzi transmission in peri-urban Arequipa. Ongoing vector control campaigns, in addition to preventing further parasite transmission, facilitate the collection of data essential to identifying children at high risk of T. cruzi infection. Targeted screening strategies could make integration of diagnosis and treatment of children into Chagas disease control programs feasible in lower-resource settings.     

Out of the forest: past and present range expansion of a parthenogenetic weevil pest,or how to colonize the world successfully

Previous research revealed complex diversification patterns in the parthenogenetic weevil Naupactus cervinus. To understand the origin of clonal diversity and successful spreading of this weevil, we investigated its geographic origin and possible dispersal routes and whether parthenogens can persist in habitats under unsuitable environmental conditions. This study is based on samples taken throughout a broad area of the species’ range. We used both mitochondrial and nuclear markers and applied phylogenetic and network analyses to infer possible relationships between haplotypes. Bayesian phylogeographic analyses and ecological niche modeling were used to investigate the processes that shaped genetic diversity and enabled the colonization of new geographic areas. Southeastern Brazil emerges as the original distribution area of N. cervinus. We detected two range expansions, one along natural corridors during the Pleistocene and the other in countries outside South America during recent times. Isolation due to climate shifts during the early Pleistocene led to diversification in two divergent clades, which probably survived in different refugia of the Paranaense Forest and the Paraná River delta. The origin of the clonal diversity was probably a complex process including mutational diversification, hybridization, and secondary colonization. The establishment of N. cervinus in areas outside its native range may indicate adaptation to drier and cooler conditions. Parthenogenesis would be advantageous for the colonization of new environments by preventing the breakup of successful gene combinations. As in other insect pests, the present distribution of N. cervinus results from both its evolutionary history and its recent history related to human activities.     

Grape marcs as unexplored source of new yeasts for future biotechnological applications

In recent years the potential of using microbes as biotechnological sources of industrially relevant enzymes has stimulated a renewed interest in the exploration of new unconventional habitats like trove of natural biodiversity. In this work, grape marcs was selected as extreme environment because of its limited nutrients, exposure to solar radiation, temperature fluctuations and ethanol. One hundred and eighty non-Saccharomyces yeasts and two hundred and twenty Saccharomyces cerevisiae strains were screened for the production of extracellular amylases, cellulases, lipases, pectinases, proteases and xylanases. Two non-Saccharomyces strains were found effective for the hydrolysis of cellulose and starch while eleven S. cerevisiae isolates were described as proficient pectinase producers. For the first time, thirteen S. cerevisiae strains, potentially able to use starch as the sole carbon source, were reported and their potential amylolytic phenotype was found to be related to a non extracellular alpha-amylase. This study encourages the selection of yeasts isolated from grape marcs as sources of unusual and industrially interesting enzymes for future biotechnological applications.     

Circulating Levels of Dimethylarginines,Chronic Kidney Disease and Long-Term Clinical Outcome in Non-ST-Elevation Myocardial Infarction

Background

Mechanisms linking chronic kidney disease (CKD) and adverse outcomes in acute coronary syndromes (ACS) are not fully understood. Among potential key players, reduced nitric oxide (NO) synthesis due to its endogenous inhibitors, asymmetric (ADMA) and symmetric (SDMA) dimethylarginine could be involved. We measured plasma concentration of arginine, ADMA and SDMA and investigated their relationship with CKD and long-term outcome in non-ST-elevation myocardial infarction (NSTEMI).

Methodology/Principal Findings

We prospectively measured arginine, ADMA, and SDMA at hospital admission in 104 NSTEMI patients. CKD was defined as an estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m². We considered a primary end point of combined cardiac death and re-infarction at a median follow-up of 21 months. In CKD (n = 33) and no-CKD (n = 71) patients, arginine and ADMA were similar, whereas SDMA was significantly higher in CKD patients (0.65±0.23 vs. 0.42±0.12 µmol/L; P<0.0001). Twenty-four (23%) patients had an adverse cardiac event during follow-up: 12 (36%) were CKD and 12 (17%) no-CKD patients (P = 0.02). When study population was stratified according to arginine, ADMA and SDMA median values, only SDMA (median 0.46 µmol/L) was associated with the primary end-point (P = 0.0016). In models adjusted for age, hemoglobin and left ventricular ejection fraction, the hazard ratio (HR) for CKD and SDMA were high (HR 2.93, interquartile range [IQR] 1.15–7.53; P = 0.02 and HR 6.80, IQR 2.09–22.2; P = 0.001, respectively) but, after mutual adjustment, only SDMA remained significantly associated with the primary end point (HR 5.73, IQR 1.55–21.2; P = 0.009).

Conclusions/Significance

In NSTEMI patients, elevated SDMA plasma levels are associated with CKD and worse long-term prognosis.