Molecular Cloning, Expression Pattern, and Chromosomal Localization of the Human Na–Cl Thiazide-Sensitive Cotransporter (SLC12A3)

Electrolyte homeostasis is maintained by several ion transport systems. Na–(K)–Cl cotransporters promote the electrically silent movement of chloride across the membrane in absorptive and secretory epithelia. Two kidney-specific Na–(K)–Cl cotransporter isoforms are known, so far, according to their sensitivity to specific inhibitors. We have cloned the human cDNA coding for the renal Na–Cl cotransporter selectively inhibited by the thiazide class of diuretic agents. The predicted protein sequence of 1021 amino acids (112 kDa) shows a structure common to the other members of the Na–(K)–Cl cotransporter family: a central region harboring 12 transmembrane domains and the 2 intracellular hydrophilic amino and carboxyl termini. The ex- pression pattern of the human Na–Cl thiazide-sensitive cotransporter (hTSC, HGMW-approved symbol SLC12A3) confirms the kidney specificity. hTSC has been mapped to human chromosome 16q13 by fluorescencein situhybridization. The cloning and characterization of hTSC now render it possible to study the involvement of this cotransport system in the pathogenesis of tubulopathies such as Gitelman syndrome.     

Visual and Olfactory Female-Borne Cues Evoke Male Courtship in the Aphid Parasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae)

We investigated the courtship and mating behavior of the pan-tropical polyphagous endoparasitoid Aphidius colemani Viereck. The courtship and mating displays, the magnitude of male-male sexual approaches and the role of female-borne cues evoking male courtship behavior were quantified. The sequence of events leading to copulation in this parasitoid did not differ from that found for other braconids. Females refused to copulate more than once. Same-sex courtships were observed among males and their possible role in an adaptive context is discussed. Olfactory female-borne cues played a key role in eliciting the courtship responses of males. Males were attracted by freshly dead females, but not by dead females soaked in hexane, nor by visual cues from females alone. Intense male wing fanning behavior was elicited by crushed abdomens of virgin females, suggesting that the female abdomen is the source of a short-distance pheromone crucial in evoking male courtship. Further studies are required to clarify the exact nature of the chemicals involved.     

The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 ( AFR1 ) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1- overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1 Δ mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1 -overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans –microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.     

IFN-α Regulates Blimp-1 Expression via miR-23a and miR-125b in Both Monocytes-Derived DC and pDC

Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and in vitro IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.     

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.     

Evaluation of Cervical Cancer Screening Programs in C?te d’Ivoire,Guyana, and Tanzania: Effect of HIV Status

Background

HIV infection increases a woman’s risk for cervical cancer, and cervical cancer incidence and mortality rates are higher in countries with high HIV prevalence and limited resources for screening. Visual inspection with acetic acid (VIA) allows screening and treatment of cervical lesions in a single-visit approach (SVA), but data on its performance in HIV-infected women are limited. This study’s objective was to examine cervical cancer screening using VIA/SVA in programs serving HIV-infected women.

Methods

A VIA/SVA program with cryotherapy for VIA-positive lesions was implemented in Côte d’Ivoire, Guyana, and Tanzania from 2009 to 2012. The effect of HIV status on VIA positivity and on presence of cryotherapy-eligible lesions was examined using a cross-sectional study design, with Chi-square tests for comparisons and constructed multivariate logistic regression models. A P-value of < 0.05 was significant.

Findings

VIA was performed on 34,921 women, 10% (3,580) were VIA positive; 2,508 (85%) eligible women received cryotherapy during the same visit; only 234 (52%) of those who postponed returned for treatment; 622 (17%) VIA-positive women had lesions too large to be treated with cryotherapy and were referred for excisional treatment. In multivariate analysis—controlling for HIV status, location of the screening clinic, facility location, facility type, and country—compared to HIV-uninfected/unknown women, HIV-infected women had higher odds of being VIA positive (OR 1.95, 95% CI 1.76, 2.16, P<0.0001) and of having large lesions requiring referral (OR 1.93, 95% CI 1.49, 2.51, P< 0.0001). Minor treatment complications occurred in 19 of 3,032 (0.63%) women; none required further intervention.

Conclusions

This study found that compared to HIV-uninfected/unknown women, HIV-infected women had nearly twice the odds of being VIA-positive and to require referral for large lesions. SVA was safe and resulted in significant reductions in loss to follow-up. There is increased need for excisional treatment in countries with high HIV prevalence.     

The Impact of Heat on an Emergency Department in Italy: Attributable Visits among Children,Adults, and the Elderly during the Warm Season

Introduction

Recent studies suggest that heat is associated with an increase in the number of ambulance calls and emergency department visits. We investigated the association between heat and daily number of emergency department visits at the University Hospital of Verona during the warm seasons 2011–2012 and we assessed the magnitude of the impact in terms of attributable events, focusing on the role of age and triage codification.

Materials and methods

We used a Poisson model to analyse the association between daily number of visits and daily mean apparent temperature, accounting for air pollution level and seasonality. The analyses were stratified by age group and were performed both on the total number of emergency department visits and on the subsample of high-priority visits. Impact estimates were obtained only for this subsample, using a Monte Carlo approach to account for sampling variability. Number of attributable events and attributable community rate were calculated.

Results

We found a positive and immediate association between event occurrence and mean apparent temperatures exceeding a threshold located around 28–29°C. The estimated percent change in the total number of visits per 1°C increase of exposure above the threshold was equal to 3.75 (90% CI: 3.01; 4.49). Focusing only on high-priority visits, the estimated percent change was larger and the greatest effect was among children. We estimated that apparent temperatures above the threshold were responsible for 1177 high-priority visits during the study period. Due to the record high temperatures observed in 2012 in Italy and in Europe, the impact in 2012 was much larger than in 2011, and consisted in 34 high-priority visits every 10000 children, 30 every 10000 people aged 15–64, and 38 every 10000 people aged 65 and over.

Discussion

Our results indicate that heat affects not only the elderly, but also children and non-elderly adults, stressing the need for developing public health preparedness plans for the entire community.     

Mutations in DOCK7 in Individuals with Epileptic Encephalopathy and Cortical Blindness

Epileptic encephalopathies are increasingly thought to be of genetic origin, although the exact etiology remains uncertain in many cases. We describe here three girls from two nonconsanguineous families affected by a clinical entity characterized by dysmorphic features, early-onset intractable epilepsy, intellectual disability, and cortical blindness. In individuals from each family, brain imaging also showed specific changes, including an abnormally marked pontobulbar sulcus and abnormal signals (T2 hyperintensities) and atrophy in the occipital lobe. Exome sequencing performed in the first family did not reveal any gene with rare homozygous variants shared by both affected siblings. It did, however, show one gene, DOCK7, with two rare heterozygous variants (c.2510delA [p.Asp837Alafs^∗48] and c.3709C>T [p.Arg1237^∗]) found in both affected sisters. Exome sequencing performed in the proband of the second family also showed the presence of two rare heterozygous variants (c.983C>G [p.Ser328^∗] and c.6232G>T [p.Glu2078^∗]) in DOCK7. Sanger sequencing confirmed that all three individuals are compound heterozygotes for these truncating mutations in DOCK7. These mutations have not been observed in public SNP databases and are predicted to abolish domains critical for DOCK7 function. DOCK7 codes for a Rac guanine nucleotide exchange factor that has been implicated in the genesis and polarization of newborn pyramidal neurons and in the morphological differentiation of GABAergic interneurons in the developing cortex. All together, these observations suggest that loss of DOCK7 function causes a syndromic form of epileptic encephalopathy by affecting multiple neuronal processes.