Additional file 1 of Comparative pangenomic analysis of predominant human vaginal lactobacilli strains towards population-specific adaptation: understanding the role in sustaining a balanced and healthy vaginal microenvironment

Supplementary Material

Additional file 3 of Comparative pangenomic analysis of predominant human vaginal lactobacilli strains towards population-specific adaptation: understanding the role in sustaining a balanced and healthy vaginal microenvironment

Supplementary Material

Additional file 2 of Comparative pangenomic analysis of predominant human vaginal lactobacilli strains towards population-specific adaptation: understanding the role in sustaining a balanced and healthy vaginal microenvironment

Supplementary Material

ELISA to determine relative anti-rK39 titers in serum sample.

<p>Serum samples were diluted at 1∶6400 dilution. The 2 circled samples in blood-negative, serum-positive samples were re-evaluated and found to be positive of the rK39 immunochromatographic RDT. Compared to Blood (−)/Serum (−), the difference with Blood (+)/Serum (+) was p = 2.3×10⁻²⁹; the difference with Blood (−)/Serum (+) was p = 0.017; the difference Blood (+)/Seum (−) was p = 0.31.</p

Relation of breast feeding status with Rotavirus infection and related outcome.

<p>(A) Breast-feeding wise distribution of Rotavirus positivity among all patients. (B, D-E) Age stratified data among breastfed and non-breastfed infants for Rotavirus positivity (B), Rotavirus antigenemia (D), and Rotavirus RNAemia (E). (C) Percentage positivity for Rotavirus antigenemia and Rotavirus RNAemia among breastfed and non-breastfed infants of the study.</p

Percentage distribution of Rotavirus-positivity among hospitalalized infants <2 yrs age suffering from severe diarrhea in Patna.

<p>(A-B) Temporal distribution of Rotavirus positivity: data of 2 consecutive years (A) and age wise stratified data among all patients (B). (C) Breast-feeding wise distribution of Rotavirus positivity among all patients.</p

Correlation of Rotavirus antigenemia and RNAemia with stool viral load.

<p>(A-D) Correlation of stool viral load with Rotavirus antigenemia (A,B) and Rotavirus RNAemia (C,D) in acute-phase (A,C) and convalescent phase (B,D) serum of infants suffering from acute gastroenteritis and diarrhea. Paired acute-phase and convalescent phase sera were tested for (i) Rotavirus antigenemia levels, that is denoted by levels of Rotavirus antigen (optical density/O.D.) measured by RV-EIA at 450 nm X 1000 units and (ii) Rotavirus RNAemia levels, denoted by levels of Rotavirus RNA in form of VP7 gene in reverse-transcriptase PCR calculated by band scanning software, as mentioned in the text. Stool viral load is represented by C(t) values in real-time RT-PCR.</p

Rotavirus antigenemia levels among the study groups.

<p>(A-C) Data demonstrates level of Rotavirus antigenemia in acute-phase (A) and convalescent phase (B) sera in infants suffering from severe diarrhea in comparison with the healthy control group. Paired acute-phase and convalescent phase sera were tested for Rotavirus antigenemia levels, that is denoted by levels of Rotavirus antigen (optical density/O.D.) measured by RV-EIA at 450 nm X 1000 units, as mentioned in the text. Broken lines denote the cut-off value.</p

Data_Sheet_1.pdf

<p>Micro RNAs (miRNAs) have emerged as a critical regulator of several biological processes in both animals and plants. They have also been associated with regulation of immune responses in many human diseases during recent years. Visceral leishmaniasis (VL) is the most severe form of leishmaniasis, which is characterized by impairment of both innate and adaptive immune responses. In the present study, we observed that Leishmania establishes hypoxic environment in host macrophages that induces the expression of hypoxia inducible factor-1α (HIF-1α) and miRNA-210. Further, the expression of miRNA-210 was found to be dependent on activation of HIF-1α expression. The HIF-1α silencing by siRNA resulted in significantly (p < 0.001) decreased expression of miR-210 in parasites infected macrophages. We also observed that in siHIF-1α or antagomir-210 treated L. donovani infected macrophages, the parasitic load and percentage infectivity were significantly (p < 0.001) decreased. Furthermore, we found that inhibition of miR-210 leads to activation of NF-κB subunit p50, and it forms heterodimer with p65 and translocates into the nucleus from the cytoplasm. This significantly (p < 0.05) induced the transcription of pro-inflammatory cytokines genes such as TNF-α and IL-12 in miRNA-210 inhibited macrophages compared to uninhibited macrophages whereas the level of IL-10, an anti-inflammatory cytokine, was found to be significantly decreased (p < 0.001). These findings suggested that L. donovani infection induces hypoxic environment inside the macrophages that activates HIF-1α. Further, HIF-1α upregulates miR-210, which eventually establishes a suitable environment for the survival of parasite inside the host macrophages by downregulating NF-κB mediated pro-inflammatory immune responses.</p

<i>Leishmania donovani</i> induced Unfolded Protein Response delays host cell apoptosis in PERK dependent manner

<div><p>Background</p><p>Endoplasmic reticulum (ER) stress generated unfolded stress response (UPR) is a basic survival mechanism which protects cell under unfavourable conditions. Leishmania parasite modulates host macrophages in various ways to ensure its survival. Modulation of PI3K-Akt pathway in delayed apoptotic induction of host; enables parasite to stabilize the infection for further propagation.</p><p>Methodology</p><p>Infected RAW macrophages were exposed to campothecin or thagsigargin and phosphorylation status of PERK, Akt, BAD and Cyt-C was determined through western blotting using phospho specific antibody. Expression at transcriptional level for cIAP1 &2, ATF4, CHOP, ATF3, HO-1 and sXBP1 was determined using real time PCR. For inhibition studies, RAW macrophages were pre-treated with PERK inhibitor GSK2606414 before infection.</p><p>Findings</p><p>Our studies in RAW macrophages showed that induction of host UPR against <i>L</i>.<i>donovani</i> infection activates Akt mediated pathway which delays apoptotic induction of the host. Moreover, Leishmania infection results in phosphorylation and activation of host PERK enzyme and increased transcription of genes of inhibitor of apoptosis gene family (cIAP) mRNA. In our inhibition studies, we found that inhibition of infection induced PERK phosphorylation under apoptotic inducers reduces the Akt phosphorylation and fails to activate further downstream molecules involved in protection against apoptosis. Also, inhibition of PERK phosphorylation under oxidative exposure leads to increased Nitric Oxide production. Simultaneously, decreased transcription of cIAP mRNA upon PERK phosphorylation fates the host cell towards apoptosis hence decreased infection rate.</p><p>Conclusion</p><p>Overall the findings from the study suggests that Leishmania modulated host UPR and PERK phosphorylation delays apoptotic induction in host macrophage, hence supports parasite invasion at early stages of infection.</p></div