Antagonistic growth regulation by Dpp and Fat drives uniform cell proliferation

We use the Dpp morphogen gradient in the Drosophila wing disc as a model to address the fundamental question of how a gradient of a growth factor can produce uniform growth. We first show that proper expression and subcellular localization of components in the Fat tumor-suppressor pathway, which have been argued to depend on Dpp activity differences, are not reliant on the Dpp gradient. We next analyzed cell proliferation in discs with uniformly high Dpp or uniformly low Fat signaling activity and found that these pathways regulate growth in?a complementary manner. While the Dpp mediator Brinker inhibits growth in the primordium primarily in the lateral regions, Fat represses growth mostly in the medial region. Together, our results indicate that the activities of both signaling pathways are regulated in a parallel rather than sequential manner and that uniform proliferation is achieved by their complementary action on growth.     

The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions.     

Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model

Background

Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV.

Results

Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1∶1024 and 1∶464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations.

Conclusions

Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.     

Podoconiosis and Soil-Transmitted Helminths (STHs): Double Burden of Neglected Tropical Diseases in Wolaita Zone,Rural Southern Ethiopia

Background

Both podoconiosis and soil-transmitted helminth (STH) infections occur among barefoot people in areas of extreme poverty; however, their co-morbidity has not previously been investigated. We explored the overlap of STH infection and podoconiosis in Southern Ethiopia and quantified their separate and combined effects on prevalent anemia and hemoglobin levels in podoconiosis patients and health controls from the same area.

Methods and Principal Findings

A two-part comparative cross-sectional study was conducted in Wolaita zone, southern Ethiopia. Data were collected from adult patients presenting with clinically confirmed podoconiosis, and unmatched adult neighborhood controls living in the same administrative area. Information on demographic and selected lifestyle factors was collected using interviewer-administered questionnaires. Stool samples were collected and examined qualitatively using the modified formalin-ether sedimentation method. Hemoglobin level was determined using two different methods: hemoglobinometer and automated hematology analyzer. A total of 913 study subjects (677 podoconiosis patients and 236 controls) participated. The prevalence of any STH infection was 47.6% among patients and 33.1% among controls (p<0.001). The prevalence of both hookworm and Trichuris trichiura infections was significantly higher in podoconiosis patients than in controls (AOR 1.74, 95% CI 1.25 to2.42, AOR 6.53, 95% CI 2.34 to 18.22, respectively). Not wearing shoes and being a farmer remained significant independent predictors of infection with any STH. There was a significant interaction between STH infection and podoconiosis on reduction of hemoglobin level (interaction p value = 0.002).

Conclusions

Prevalence of any STH and hookworm infection was higher among podoconiosis patients than among controls. A significant reduction in hemoglobin level was observed among podoconiosis patients co-infected with hookworm and ‘non-hookworm STH’. Promotion of consistent shoe-wearing practices may have double advantages in controlling both podoconiosis and hookworm infection in the study area.     

High Prevalence of Diarrhoegenic Intestinal Parasite Infections among Non-ART HIV Patients in Fitche Hospital,Ethiopia

Background

HIV infection has been modifying both the epidemiology and outcome of parasite infections. Hence, this study was undertaken to determine the prevalence of Cryptosporidium and other intestinal parasite infections among HIV positives with and without Antiretroviral Treatment(ART) and its association with CD4+ T-cell count.

Methods

A cross-sectional study was conducted at Fitche hospital focusing on HIV positives who came to hospital for follow-ups. A total of 378 HIV positive persons with and without ART participated in the study. Data on socio-demographic factors and diarrhoea status were obtained by interviewing all 214 with ART and 164 without ART. Stool samples were collected from all patients and examined for intestinal parasites using direct, formol-ether and modified acid-fast staining techniques.

Results

The prevalence of intestinal parasite infections in this study was significantly higher among HIV positive persons not on ART. Specifically, the rate of infection with Cryptosporidium species, Blastocystis spp., Giardia lamblia, and Entamoeba histolytica/E. dispar were higher, particularly in those with CD4+ T-cell counts less than 200 cells/µL. Fifty seven percent of the study participants were on ART. Out of these 164/378 (43%) of the non-ART study participants were infected with at least one intestinal parasite species. Significant association was observed between lower CD4+ T-cell count (<200 cells/µL) and the prevalence of Cryptosporidium spp. and Blastocystis spp. The two parasites were significantly more prevalent in HIV positive non-ART patients.

Conclusion

HIV infection increased the risk of having Cryptosporidium and other intestinal parasites and diarrhoea. Therefore, raising HIV positive’s immune status and screening for intestinal parasites is important. This study showed that patients who are taking ART had a lower prevalence of diarrhoea causing parasites and Cryptosporidium suggesting that ART through improvement of immune status of the patients may have contributed to controlling diarrhoea-causing parasites in HIV positive patients.     

Poor Outcomes in a Cohort of HIV-Infected Adolescents Undergoing Treatment for Multidrug-Resistant Tuberculosis in Mumbai,India

Background

Little is known about the treatment of multidrug-resistant tuberculosis (MDR-TB) in HIV-co-infected adolescents. This study aimed to present the intermediate outcomes of HIV-infected adolescents aged 10–19 years receiving second-line anti-TB treatment in a Médecins Sans Frontières (MSF) project in Mumbai, India.

Methods

A retrospective review of medical records of 11 adolescents enrolled between July 2007 and January 2013 was undertaken. Patients were initiated on either empirical or individualized second-line ambulatory anti-TB treatment under direct observation.

Results

The median age was 16 (IQR 14–18) years and 54% were female. Five (46%) adolescents had pulmonary TB (PTB), two (18%) extrapulmonary disease (EPTB) and four (36%) had both. Median CD4 count at the time of MDR-TB diagnosis was 162.7 cells/µl (IQR: 84.8–250.5). By January 2013, eight patients had final and 3 had interim outcomes. Favourable results were seen in four (36.5%) patients: one was cured and three were still on treatment with negative culture results. Seven patients (64%) had poor outcomes: four (36.5%) died and three (27%) defaulted. Three of the patients who died never started on antiretroviral and/or TB treatment and one died 16 days after treatment initiation. Two of the defaulted died soon after default. All patients (100%) on-treatment experienced adverse events (AEs): two required permanent discontinuation of the culprit drug and two were hospitalized due to AEs. No patient required permanent discontinuation of the entire second-line TB or antiretroviral regimens.

Conclusions

Early mortality and mortality after default were the most common reasons for poor outcomes in this study. Early mortality suggests the need for rapid diagnosis and prompt treatment initiation, and adolescents might benefit from active contact-tracing and immediate referral. Default occurred at different times, suggesting the need for continuous, intensified and individualized psychosocial support for co-infected adolescents. Operational research among co-infected adolescents will be especially important in designing effective interventions for this vulnerable group.     

Ca2+ influx through reverse mode Na+/Ca2+ exchange is critical for vascular endothelial growth factor-mediated extracellular signal-regulated kinase (ERK) 1/2 activation and angiogenic functions of human endothelial cells

VEGF is a key angiogenic cytokine and a major target in anti-angiogenic therapeutic strategies. In endothelial cells (ECs), VEGF binds VEGF receptors and activates ERK1/2 through the phospholipase γ (PLCγ)-PKCα-B-Raf pathway. Our previous work suggested that influx of extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation, and we hypothesized that this could occur through reverse mode (Ca(2+) in and Na(+) out) Na(+)-Ca(2+) exchange (NCX). However, the role of NCX activity in VEGF signaling and angiogenic functions of ECs had not previously been described. Here, using human umbilical vein ECs (HUVECs), we report that extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation and that release of Ca(2+) from intracellular stores alone, in the absence of extracellular Ca(2+), is not sufficient to activate ERK1/2. Furthermore, inhibitors of reverse mode NCX suppressed the VEGF-induced activation of ERK1/2 in a time- and dose-dependent manner and attenuated VEGF-induced Ca(2+) transients. Knockdown of NCX1 (the main NCX isoform in HUVECs) by siRNA confirmed the pharmacological data. A panel of NCX inhibitors also significantly reduced VEGF-induced B-Raf activity and inhibited PKCα translocation to the plasma membrane and total PKC activity in situ. Finally, NCX inhibitors reduced VEGF-induced HUVEC proliferation, migration, and tubular differentiation in surrogate angiogenesis functional assays in vitro. We propose that Ca(2+) influx through reverse mode NCX is required for the activation and the targeting of PKCα to the plasma membrane, an essential step for VEGF-induced ERK1/2 phosphorylation and downstream EC functions in angiogenesis.     

Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APC(Cdh1)

Mammalian oocytes are arrested in prophase of the first meiotic division. Progression into the first meiotic division is driven by an increase in the activity of maturation-promoting factor (MPF). In mouse oocytes, we find that early mitotic inhibitor 1 (Emi1), an inhibitor of the anaphase-promoting complex (APC) that is responsible for cyclin B destruction and inactivation of MPF, is present at prophase I and undergoes Skp1-Cul1-F-box/betaTrCP-mediated destruction immediately after germinal vesicle breakdown (GVBD). Exogenous Emi1 or the inhibition of Emi1 destruction in prophase-arrested oocytes leads to a stabilization of cyclin B1-GFP that is sufficient to trigger GVBD. In contrast, the depletion of Emi1 using morpholino oligonucleotides increases cyclin B1-GFP destruction, resulting in an attenuation of MPF activation and a delay of entry into the first meiotic division. Finally, we show that Emi1-dependent effects on meiosis I require the presence of Cdh1. These observations reveal a novel mechanism for the control of entry into the first meiotic division: an Emi1-dependent inhibition of APC(Cdh1).