A sensitive high-throughput assay for evaluating host-pathogen interactions in Cryptococcus neoformans infection

Background

Cryptococcus neoformans causes serious disease in immunocompromised individuals, leading to over 600,000 deaths per year worldwide. Part of this impact is due to the organism''s ability to thwart what should be the mammalian hosts'' first line of defense against cryptococcal infection: internalization by macrophages. Even when C. neoformans is engulfed by host phagocytes, it can survive and replicate within them rather than being destroyed; this ability is central in cryptococcal virulence. It is therefore critical to elucidate the interactions of this facultative intracellular pathogen with phagocytic cells of its mammalian host.

Methodology/Principal Findings

To accurately assess initial interactions between human phagocytic cells and fungi, we have developed a method using high-throughput microscopy to efficiently distinguish adherent and engulfed cryptococci and quantitate each population. This method offers significant advantages over currently available means of assaying host-fungal cell interactions, and remains statistically robust when implemented in an automated fashion appropriate for screening. It was used to demonstrate the sensitivity of human phagocytes to subtle changes in the cryptococcal capsule, a major virulence factor of this pathogen.

Conclusions/Significance

Our high-throughput method for characterizing interactions between C. neoformans and mammalian phagocytic cells offers a powerful tool for elucidating the relationship between these cell types during pathogenesis. This approach will be useful for screens of this organism and has potentially broad applications for investigating host-pathogen interactions.     

Enhanced susceptibility of Ago1/3 double-null mice to influenza A virus infection

RNA interference (RNAi) is a critical component of many cellular antiviral responses in plants, invertebrates, and mammals. However, its in vivo role in host protection from the negative-sense RNA virus influenza virus type A (flu) is unclear. Here we have examined the role of RNAi in host defense to flu by analyzing Argonaute 1 and 3 double-knockout mice deficient in components of the RNA-induced silencing complex. Compared to littermate controls, flu-infected double-knockout mice exhibited increased mortality, consistent with more severe alveolitis and pneumonitis. These data indicate that optimal resistance to flu requires Argonaute 1 and/or 3. Enhanced mortality of double-knockout mice was not associated either with increased viral replication or with differential pulmonary recruitment or function of innate and adaptive immune cells. Given the absence of detectable immune defects, our results support the notion that the enhanced flu susceptibility of double-knockout mice arises from an intrinsic impairment in the ability of lung cells to tolerate flu-elicited inflammation.     

Modulation of recombinant, α2*, α3* or α4*-nicotinic acetylcholine receptor (nAChR) function by nAChR β3 subunits

The nicotinic acetylcholine receptor (nAChR) β3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When β3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing α2, α3 or α4 plus either β2 or β4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type β3 subunits generally (except for α3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20-50% without having dramatic effects (≤ 2-fold) on agonist potencies. By contrast, co-expression with mutant β3(V9'S) subunits generally (except for α4β2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any α subunit plus β4 and β3(V9'S) subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that β3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with β3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes.     

Detection of Rotavirus from Hospitalized Diarrheic Children in Uttar Pradesh, India

In the present study 220 stool samples collected from diarrheic children admitted to different hospitals and nursing homes of Uttar Pradesh and Uttarakhand were screened for rotavirus. Of 220 diarrheic samples screened 46 samples were found to be positive for rotavirus by RNA PAGE. All the isolates exhibited 4-2-3-2 migration pattern suggesting group A rotavirus. Both long and short electropherotypes were prevalent in these regions. Six different electropherotypes were detected in this study period. Male diarrheic children were found to be more susceptible to rotavirus infection (22.96 %) than that of the female ones (17.64 %). Viral RNA isolated from stool samples again subjected to VP4 gene amplification by RT-PCR using con2 and con3 primer which resulted 876 bp product suggesting group A rotavirus. Besides virus isolation was successfully done using MA104 cell line.     

Exercise modulation of the host-tumor interaction in an orthotopic model of murine prostate cancer

The purpose of this study is to investigate the effects of exercise on cancer progression, metastasis, and underlying mechanisms in an orthotopic model of murine prostate cancer. C57BL/6 male mice (6-8 wk of age) were orthotopically injected with transgenic adenocarcinoma of mouse prostate C-1 cells (5 × 10(5)) and randomly assigned to exercise (n = 28) or a non-intervention control (n = 31) groups. The exercise group was given voluntary access to a wheel 24 h/day for the duration of the study. Four mice per group were serially killed on days 14, 31, and 36; the remaining 38 mice (exercise, n = 18; control, n = 20) were killed on day 53. Before death, MRI was performed to assess tumor blood perfusion. Primary tumor growth rate was comparable between groups, but expression of prometastatic genes was significantly modulated in exercising animals with a shift toward reduced metastasis. Exercise was associated with increased activity of protein kinases within the MEK/MAPK and PI3K/mTOR signaling cascades with subsequent increased intratumoral protein levels of HIF-1α and VEGF. This was associated with improved tumor vascularization. Multiplex ELISAs revealed distinct reductions in plasma concentrations of several angiogenic cytokines in the exercise group, which was associated with increased expression of angiogenic and metabolic genes in the skeletal muscle. Exercise-induced stabilization of HIF-1α and subsequent upregulation of VEGF was associated with "productive" tumor vascularization with a shift toward suppressed metastasis in an orthotopic model of prostate cancer.     

Do ribosomal RNAs act merely as scaffold for ribosomal proteins?

Investigations that are being carried out in various laboratories including ours clearly provide the answer which is in the negative. Only the direct evidences obtained in this laboratory will be presented and discussed. It has been unequivocally shown that the interaction between 16S and 23S RNAs plays the primary role in the association of ribosomal subunits. Further, 23S RNA is responsible for the Binding of 5S RNA to 16S.23S RNA complex with the help of three ribosomal proteins, L5, L18, L15/L25. The 16S.23S RNA complex is also capable of carrying out the following ribosomal functions, although to small but significant extents, with the help of a very limited number of ribosomal proteins and the factors involved in protein synthesis: (a) poly U-Binding, (B) poly U-dependent Binding of phenylalanyl tRNA, (c) EF-G-dependent GTPase activity, (d) initiation complex formation, (e) peptidyl transferase activity (puromycin reaction) and (f) polyphenylalanine synthesis. These results clearly indicate the direct involvement of rRNAs in the various steps of protein synthesis. Very recently it has Been demonstrated that the conformational change of 23S RNA is responsible for the translocation of peptidyl tRNA from the aminoacyl (A) site to the peptidyl (P) site. A model has Been proposed for translocation on the Basis of direct experimental evidences. The new concept that ribosomal RNAs are the functional components in ribosomes and proteins act as control switches may eventually turn out to Be noncontroversial.     

Insecticide resistance status of three vectors of Japanese encephalitis in east central India

Japanese encephalitis (JE) has been reported in different districts of Odisha state (east central India) since 1992. During 2016, a major outbreak of JE and acute encephalitis syndrome (AES) occurred in the Malkangiri district of Odisha, causing 103 deaths in children, of which 37 were caused by JE and 66 by AES. Information on insecticide resistance in JE vectors is important for the selection of appropriate insecticides for use in vector control. The present study was designed to determine the resistance status of three important vectors of JE, Culex vishnui, Culex tritaeniorhynchus and Culex bitaeniorhynchus (Diptera: Culicidae), against dichlorodiphenyltrichloroethane (DDT), malathion and deltamethrin in three districts of Odisha state affected by JE. Female adult mosquitoes were collected using mouth aspirators both indoors and outdoors in JE‐affected villages and used in susceptibility bioassays following World Health Organization guidelines. Knock‐downs were recorded every 10 min up to 1 h and mortality rates were recorded at 24 h post‐exposure. Culex vishnui and Cx. tritaeniorhynchus showed resistance to DDT, malathion and deltamethrin, whereas Cx. bitaeniorhynchus was susceptible in all study districts. The information generated by this study will be highly useful in the planning and implementing of appropriate vector control operations for the prevention and control of JE in east central India.