Drosophila RET contains an active tyrosine kinase and elicits neurotrophic activities in mammalian cells

AbstractThe RET receptor tyrosine kinase controls kidney organogenesis and development of subpopulations of enteric and sensory neurons in different vertebrate species, including humans, rodents, chicken and zebrafish. RET is activated by binding to a ligand complex formed by a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors bound to its cognate GFRα GPI-linked co-receptor. Despite the absence of GDNF or GFRα molecules in the Drosophila genome, a RET orthologue (dRET) has recently been described in this organism and shown to be expressed in subpopulations of cells of the excretory, digestive and nervous systems, thus resembling the expression pattern of RET in vertebrates. In this study, we report on the initial biochemical and functional characterization of the dRET protein in cell culture systems. Full-length dRET could be produced in mammalian and insect cells. Similar to its human counterpart (hRET), overexpression of dRET resulted in its ligand-independent tyrosine phosphorylation, indicating that it bears an active tyrosine kinase. Unlike hRET, however, the extracellular domain of dRET was unable to interact with mammalian GDNF and GFRα1. Self association between dRET molecules could neither be detected, indicating that dRET is incapable of mediating cell adhesion by homophilic interactions. A chimeric molecule comprising the extracellular domain of hRET and the kinase domain of dRET was constructed and used to probe ligand-mediated downstream activities of the dRET kinase in PC12 cells. GDNF stimulation of cells transfected with the hRET/dRET chimera resulted in neurite outgrowth comparable to that obtained after transfection of wild-type hRET. These results indicate significant conservation between the biological effects elicited by the human and Drosophila RET kinases, and suggest functions for dRET in neuronal differentiation in the fly

Membrane-containing virus particles exhibit the mechanics of a composite material for genome protection

The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport

Haptoglobin from Psoriatic Patients Exhibits Decreased Activity in Binding Hemoglobin and Inhibiting LCAT Activity.

Objective: The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of Haptoglobin (Hpt). Background: Hpt is a plasma acute phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free hemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT) Study design: Hpt was isolated from patients with psoriasis vulgaris, and its activity in hemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. Methods: Two affinity chromatography steps, the first using resin-coupled hemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by ELISA with stationary hemoglobin or ApoA-I, Hpt in solution, and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. Results: Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by hemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both hemoglobin binding and LCAT inhibition. Conclusions: In psoriasis, Hpt displays some structure modification(s) which might be associated with the protein function in the disease

Reemergence of Strongyloidiasis, Northern Italy

Strongyloidiasis is a helminth infection caused by Strongyloides stercoralis, a nematode ubiquitous in tropical and subtropical countries and occasionally reported in temperate countries, including Italy (1). Sources of infection are filariform strongyloid larvae present in soil contaminated by infected feces; the larvae penetrate through the skin of a human host. After the first life cycle, a process of autoinfection begins, which persists indefinitely in the host if the infection is not effectively treated. The infection can remain totally asymptomatic for many years or forever or cause cutaneous (itching and rash), abdominal (epigastric pain, pseudoappendicitis, diarrhea), respiratory (cough, recurrent asthma), and systemic (weight loss, cachexia) symptoms that can be enervating. More importantly, when host immunity is impaired because of a concurrent disease or immunosuppressive therapy (including corticosteroids, sometimes used to treat symptoms of the unrecognized infection or the concurrent eosinophilia), disseminated strongyloidiasis may occur (2\u20134), causing a massive and almost invariably fatal invasion of virtually all organs and tissues by filariform larvae and even adult worms (Figure), often combined with bacterial superinfection. This complication is believed to be rare but is probably underestimated because of the extreme variability of the clinical presentation. Although strongyloidiasis can be suspected in the presence of symptoms or eosinophilia (which is frequent but not mandatory), the low sensitivity of direct diagnostic methods often lets the disease go unrecognized (5\u20137). By far the most sensitive diagnostic tools are serologic tests: sensitivity and specificity of indirect fluorescent antibody test (IFAT) (in-house produced IFAT) are 97.4% and 97.9%, respectively, at a dilution >1/20, and 70.5% and 99.8% at a dilution >1/80 (6). A suspected case is defined by a positive antibody titer >20 (IFAT); a case is confirmed by a positive direct test result (culture in agar being the most sensitive direct technique) or by a positive antibody titer >80 (6). Despite some anecdotal reports on the presence of strongyloidiasis in Italy (1,6), reliable information about the real prevalence of the infection is lacking. After seeing several patients affected by the disease, 1 of whom died because of dissemination (Z. Bisoffi, unpub. data), we decided to carry out a preliminary rapid assessment of the extent of the problem in elderly patients with eosinophilia. During a 4-month period, from February through May 2008, every patient born in 1940 or earlier who came to the clinical laboratories of 2 contiguous health districts in northern Italy (Mantova, Lombardy Region, and Legnago, Veneto Region) for a diagnostic blood test (hematocrit and leukocyte count/formula) for whatever reason and having a eosinophil count >500 cells/\u3bcL was asked to join the study. This study was the pilot phase of a larger, multicentered study, which obtained formal approval from the Ethical Committee of Sacro Cuore Hospital of Negrar, Verona. Informed consent was required of each patient. Of the 132 patients eligible for inclusion (mean age 76.4 years, range 68\u201390 years, male:female ratio 1.6), none refused to give informed consent. Serum specimens were subjected to the IFAT for S. stercoralis at the Sacro Cuore Hospital Centre for Tropical Diseases. Unexpectedly, we found that 37 (28%) of 132 patients were positive, with titers ranging between 20 and >320 (and >80 in most cases). However, caution should be exercised in interpreting the results because the patients may not be representative of the general population. Moreover, our results are based on an indirect (although highly sensitive and specific) test. Because the reported cases involve only a few patients every year (of whom some are anecdotally reported as dying from the infection, usually unpublished), we suspect that most strongyloidiasis cases remain undetected. If relevant transmission still exists in the area, it is unknown but is unlikely because of the improvement of hygienic conditions in the past 5 decades. Reports of the infection in children or young adults with no travel history outside Italy are lacking. Strongyloidiasis in the elderly is therefore most likely to result from an infection that occurred much earlier in life, either in infancy or at a young age, while walking or working barefoot in agricultural fields. The long persistence is the consequence of the autoinfection cycle typical of this parasite as described above. The result is an important and unrecognized public health problem affecting the geriatric population of northern Italy. These preliminary results confirm the need for the already planned, multicentered study involving a larger sample and a wider geographic area

Epidemiology of Strongyloides stercoralis in northern Italy: Results of a multicentre case-control study, February 2013 to July 2014

Strongyloides stercoralis is a soil-transmitted helminth widely diffused in tropical and subtropical regions of the world. Autochthonous cases have been also diagnosed sporadically in areas of temperate climate. We aimed at defining the epidemiology of strongyloidiasis in immigrants and Italians living in three northern Italian Regions. Screening for S. stercoralis infection was done with serology, confirmation tests were a second serological method or stool agar culture. A case-control approach was adopted and patients with a peripheral eosinophil count 65 500/mcL were classified as cases. Of 2,701 individuals enrolled here 1,351 were cases and 1,350 controls; 86% were Italians, 48% women. Italians testing positive were in 8% (97/1,137) cases and 1% (13/1,178) controls (adjusted odds ratio (aOR) 8.2; 95% confidence interval (CI): 4.5-14.8), while positive immigrants were in 17% (36/214) cases and in 2% (3/172) controls (aOR 9.6; 95% CI: 2.9-32.4). Factors associated with a higher risk of infection for all study participants were eosinophilia (p < 0.001) and immigration (p = 0.001). Overall, strongyloidiasis was nine-times more frequent in individuals with eosinophilia than in those with normal eosinophil count

Impact of antiretroviral dosing and daily pill burden on viral rebound rates in naive patients receiving a tenofovir-based regimen

Methods A total of 480 ART-naive patients were selected from the GNOMO cohort. Incidence rate of viral rebound (VR = first of two consecutive VL>50 cp/ml) was calculated as number of events over PYFU and expressed at univariate and multivariate analysis as incidence rate ratio (IRR). Number of both pills and doses per day were used to define three different types of regimens: twice-a-day regimens (BID regimens); once-a-day regimens with 3 pills (high-pill QD [hp-QD]). Adjusted rates of viral rebound were estimated by Poisson regression using date of first HIV-RNA <50 c/ml as baseline. Follow-up was censored at the date of VR, death, or loss to follow-up

Impact of social determinants on antiretroviral therapy access and outcomes entering the era of universal treatment for people living with HIV in Italy

Background: Social determinants are known to be a driving force of health inequalities, even in high income countries. Aim of our study was to determine if these factors can limit antiretroviral therapy (ART) access, outcome and retention in care of people living with HIV (PLHIV) in Italy. Methods: All ART naïve HIV+ patients (pts) of Italian nationality enrolled in the ICONA Cohort from 2002 to 2016 were included. The association of socio-demographic characteristics (age, sex, risk factor for HIV infection, educational level, occupational status and residency area) with time to: ART initiation (from the first positive anti-HIV test), ART regimen discontinuation, and first HIV-RNA &lt; 50 cp/mL, were evaluated by Cox regression analysis, Kaplan Meier method and log-rank test. Results: A total of 8023 HIV+ pts (82% males, median age at first pos anti-HIV test 36 years, IQR: 29-44) were included: 6214 (77.5%) started ART during the study period. Women, people who inject drugs (PWID) and residents in Southern Italy presented the lowest levels of education and the highest rate of unemployment compared to other groups. Females, pts aged &gt; 50 yrs., unemployed vs employed, and people with lower educational levels presented the lowest CD4 count at ART initiation compared to other groups. The overall median time to ART initiation was 0.6 years (yrs) (IQR 0.1-3.7), with a significant decrease over time [2002-2006 = 3.3 yrs. (0.2-9.4); 2007-2011 = 1.0 yrs. (0.1-3.9); 2012-2016 = 0.2 yrs. (0.1-2.1), p &lt; 0.001]. By multivariate analysis, females (p &lt; 0.01) and PWID (p &lt; 0.001), presented a longer time to ART initiation, while older people (p &lt; 0.001), people with higher educational levels (p &lt; 0.001), unemployed (p = 0.02) and students (p &lt; 0.001) were more likely to initiate ART. Moreover, PWID, unemployed vs stable employed, and pts. with lower educational levels showed a lower 1-year probability of achieving HIV-RNA suppression, while females, older patients, men who have sex with men (MSM), unemployed had higher 1-year risk of first-line ART discontinuation. Conclusions: Despite median time to ART start decreased from 2002 to 2016, socio-demographic factors still contribute to disparities in ART initiation, outcome and durability

Four lectures on secant varieties

This paper is based on the first author's lectures at the 2012 University of Regina Workshop "Connections Between Algebra and Geometry". Its aim is to provide an introduction to the theory of higher secant varieties and their applications. Several references and solved exercises are also included.Comment: Lectures notes to appear in PROMS (Springer Proceedings in Mathematics & Statistics), Springer/Birkhause

Impact of antiretroviral dosing and daily pill burden on viral rebound rates in naive patients receiving a tenofovir-based regimen

Methods A total of 480 ART-naive patients were selected from the GNOMO cohort. Incidence rate of viral rebound (VR = first of two consecutive VL>50 cp/ml) was calculated as number of events over PYFU and expressed at univariate and multivariate analysis as incidence rate ratio (IRR). Number of both pills and doses per day were used to define three different types of regimens: twice-a-day regimens (BID regimens); once-a-day regimens with 3 pills (high-pill QD [hp-QD]). Adjusted rates of viral rebound were estimated by Poisson regression using date of first HIV-RNA <50 c/ml as baseline. Follow-up was censored at the date of VR, death, or loss to follow-up