Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy

Plasma ACTH precursors in cats with pituitary-dependent hyperadrenocorticism

Background: Diagnosis of pituitary-dependent hyperadrenocorticism (PDH) in cats is challenging because there is no specific diagnostic test. Hypothesis/Objective: The determination of plasma ACTH precursor (POMC and pro-ACTH) concentration might facilitate the diagnosis of PDH in cats. The aim of the study was to evaluate prospectively the plasma concentrations of ACTH precursors in a small cohort of cats with PDH and to estimate the value of this approach for diagnosis. Animals: Four groups of cats were included: group 1 (cats with PDH), group 2 (cats with diabetes mellitus but not hyperadrenocorticism (HAC)), group 3 (cats with diabetes mellitus and confirmed acromegaly but not HAC), and group 4 (healthy cats). Methods: PDH diagnosis was based on clinical data, low-dose dexamethasone suppression test (LDDST), and adrenal and pituitary gland computed tomography (CT) scan. For groups 2, 3, and 4, hyperadrenocorticism was excluded by LDDST or urine cortisol:creatinine ratio (UCCR). An immunoluminometric assay was used to determine plasma concentrations of ACTH precursors in the 4 groups of cats. Results: Group 1 contained 9 cats (enlarged pituitary gland in 7/9). Plasma ACTH precursor concentrations ranged from <53 to >1010 pmol/L with 8/9 concentrations 229 pmol/L. Groups 2, 3, and 4 included 13, 7, and 13 cats, respectively. Plasma ACTH precursor concentrations ranged from <53 to 96 pmol/L in group 2, <53 to 72 pmol/L in group 3, and <53 to 99 pmol/L in group 4. Conclusion and Clinical Importance: High plasma concentration of ACTH precursors in cats (>100 pmol/L) is highly suggestive of PD