Aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family, is well characterized to regulate the biochemical and toxic effects of environmental chemicals. More recently, AhR ...activation has been shown to regulate the differentiation of Foxp3(+) Tregs as well as Th17 cells. However, the precise mechanisms are unclear. In the current study, we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand, on epigenetic regulation leading to altered Treg/Th17 differentiation, and consequent suppression of colitis.
Dextran sodium sulphate (DSS) administration induced acute colitis in C57BL/6 mice, as shown by significant weight loss, shortening of colon, mucosal ulceration, and increased presence of CXCR3(+) T cells as well as inflammatory cytokines. Interestingly, a single dose of TCDD (25 µg/kg body weight) was able to attenuate all of the clinical and inflammatory markers of colitis. Analysis of T cells in the lamina propria (LP) and mesenteric lymph nodes (MLN), during colitis, revealed decreased presence of Tregs and increased induction of Th17 cells, which was reversed following TCDD treatment. Activation of T cells from AhR(+/+) but not AhR (-/-) mice, in the presence of TCDD, promoted increased differentiation of Tregs while inhibiting Th17 cells. Analysis of MLN or LP cells during colitis revealed increased methylation of CpG islands of Foxp3 and demethylation of IL-17 promoters, which was reversed following TCDD treatment.
These studies demonstrate for the first time that AhR activation promotes epigenetic regulation thereby influencing reciprocal differentiation of Tregs and Th17 cells, and amelioration of inflammation.
Background & objectives: In developed countries, efforts have been made to restrict episiotomy practice. However, in developing countries the episiotomy rates continue to be high. This study was ...conducted to evaluate the pattern of episiotomy use and its immediate complications among women delivering at tertiary level public hospitals in India.
Methods: Prospective data of all women undergoing vaginal delivery including instrumental delivery were collected daily from the labour room registers of the 18 tertiary care hospitals on a structured proforma. Weekly data from all sites were sent to a central unit for compilation and analysis. Odds ratio was used to compare the proportion of genital trauma among women with and without episiotomy both in nulliparous and multiparous women.
Results: Among 1,20,243 vaginal deliveries, episiotomy was performed in 63.4 per cent (n=76,305) cases. Nulliparaous women were 8.8 times more likely to undergo episiotomy than multiparous women. The various genital tract injuries reported were first degree perineal tear (n=4805, 3.9%), second degree perineal tear (n=1082, 0.9%), third and fourth degree perineal tear (n=186, 0.2%), anterior vaginal trauma requiring suturing (n=490, 0.4%), extension of episiotomy/vaginal laceration/excessive bleeding from episiotomy or tear (n=177, 0.15%), vulval/vaginal haematoma (n=70, 0.06%) and cervical tear (n=108, 0.08%). The combined rate of third and fourth degree perineal tears was observed to be significantly lower (p<0.001) among nullipara who received episiotomy (0.13%) compared to those who delivered without episiotomy (0.62%).
Interpretations & conclusions: Significantly lower rates of third or fourth degree perineal tear were seen among nulliparous women undergoing episiotomy. The risk and benefit of episiotomy and its complications need to be evaluated through randomized clinical trials in the Indian context.
In low-transmission settings, accurate estimates of malaria transmission are needed to inform elimination targets. Detection of antimalarial antibodies provides exposure history, but previous studies ...have mainly relied on species-specific antigens. The use of chimeric antigens that include epitopes from multiple species of malaria parasites in population-based serological surveys could provide data for exposure to multiple Plasmodium species circulating in an area. Here, the utility of P. vivax/P. falciparum chimeric antigen for assessing serological responses was evaluated in Ethiopia, an endemic country for all four human malarias, and Costa Rica, where P. falciparum has been eliminated with reports of sporadic P. vivax cases.
A multiplex bead-based assay was used to determine the seroprevalence of IgG antibodies against a chimeric malaria antigen (PvRMC-MSP1) from blood samples collected from household surveys in Ethiopia in 2015 (n = 7,077) and Costa Rica in 2015 (n = 851). Targets specific for P. falciparum (PfMSP1) and P. vivax (PvMSP1) were also included in the serological panel. Seroprevalence in the population and seroconversion rates were compared among the three IgG targets.
Seroprevalence in Costa Rica was 3.6% for PfMSP1, 41.5% for PvMSP1 and 46.7% for PvRMC-MSP1. In Ethiopia, seroprevalence was 27.6% for PfMSP1, 21.4% for PvMSP1, and 32.6% for PvRMC-MSP1. IgG levels in seropositive individuals were consistently higher for PvRMC-MSP1 when compared to PvMSP1 in both studies. Seroconversion rates were 0.023 for PvMSP1 and 0.03 for PvRMC-MSP1 in Costa Rica. In Ethiopia, seroconversion rates were 0.050 for PfMSP1, 0.044 for PvMSP1 and 0.106 for PvRMC-MSP1.
Our data indicate that chimeric antigen PvRMC-MSP1 is able to capture antibodies to multiple epitopes from both prior P. falciparum and P. vivax infections, and suitable chimeric antigens can be considered for use in serosurveys with appropriate validation.
Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA ...countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.
As malaria incidence and transmission in a region decreases, it becomes increasingly difficult to identify areas of active transmission. Improved methods for identifying and monitoring foci of active ...malaria transmission are needed in areas of low parasite prevalence in order to achieve malaria elimination. Serological assays can provide population-level infection history to inform elimination campaigns.
A bead-based multiplex antibody detection assay was used to evaluate a chimeric Plasmodium vivax MSP1 protein (PvRMC-MSP1), designed to be broadly immunogenic for use in vaccine studies, to act as a pan-malaria serological tool based on its ability to capture IgG in plasma samples obtained from naturally exposed individuals. Samples from 236 US travellers with PCR confirmed infection status from all four major Plasmodium species infecting humans, Plasmodium falciparum (n = 181), Plasmodium vivax (n = 38), Plasmodium malariae (n = 4), and Plasmodium ovale (n = 13) were tested for IgG capture using PvRMC-MSP1 as well as the four recombinant MSP1-19 kD isoforms representative of these Plasmodium species.
Regardless of infecting Plasmodium species, a large proportion of plasma samples from infected US travellers provided a high assay signal to the PvRMC-MSP1 chimeric protein, with 115 high responders out of 236 samples assessed (48.7%). When grouped by active infection, 38.7% P. falciparum-, 92.1% of P. vivax-, 75.0% P. malariae-, and 53.4% of P. ovale-infected individuals displayed high assay signals in response to PvRMC-MSP1. It was also determined that plasma from P. vivax-infected individuals produced increased assay signals in response to the PvRMC-MSP1 chimera as compared to the recombinant PvMSP1 for 89.5% (34 out of 38) of individuals. PvRMC-MSP1 also showed improved ability to capture IgG antibodies from P. falciparum-infected individuals when compared to the capture by recombinant PvMSP1, with high assay signals observed for 38.7% of P. falciparum-infected travellers in response to PvRMC-MSP1 IgG capture compared to just 1.1% who were high responders to capture by the recombinant PvMSP1 protein.
These results support further study of designed antigens as an approach for increasing sensitivity or broadening binding capacity to improve existing serological tools for determining population-level exposure to Plasmodium species. Including both broad-reacting and Plasmodium species-specific antigen-coated beads in an assay panel could provide a nuanced view of population-level exposure histories, an extensive IgG profile, and detailed seroestimates. A more sensitive serological tool for detection of P. vivax exposure would aid malaria elimination campaigns in co-endemic areas and regions where P. vivax is the dominant parasite.
Zero Budget Natural Farming (ZBNF), utilizing natural resources, multiple cropping systems, and cow-dung- and urine-based products to improve soil biology, has been practiced by thousands of farmers ...in India. However, without any scientific proof, this traditional and ancient technique is mocked as a bugged theory in the scientific community. In the current study, we have investigated the effect of Jeevamrit—cow-dung- and urine-based formulation—on soil chemical and microbial properties of the ZBNF field coupled with metagenomic analysis and the economics of ZBNF. The percentage increase in soil properties, such as organic carbon, available phosphorus, and available potassium, was recorded up to 46%, 439%, and 142%, respectively, while micronutrients, such as Zn, Fe, Cu, and Mn, also increased up to 98%, 23%, 62%, and 55%, respectively, from 2017 to 2019. Whole genome metagenomic analysis revealed that Proteobacteria were dominantly present, and bacterial phyla including Bacillus, Pseudomonas, Rhizobium, and Panibacillus. On the other hand, Ascomycota was the dominating fungal phyla present in the soil sample. Further, functional analysis showed a high representation of genes/enzymes involved in amino acids and carbohydrate metabolism contributing to soil fertility, plant growth, defense, and development. Additionally, the cost–benefit ratio of ZBNF was double the farmer’s practice when tested with the rice and wheat cropping system. The results from this study provide a new proof of concept and understanding of the potential of the ZBNF component, i.e., Jeevamrit, in improving soil properties.
Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs ...used in the field.
This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay.
Purified nHRP2 was identified by SDS-PAGE and western blot as a - 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL.
Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.
•PvMSp9E795-A808 synthetic constructs were immunogenic in mice.•IgG1, IgG2a and IgG2b were prevalent in humoral response induced by synthetic peptides.•T-cell epitopes enhanced the humoral ...response.•Anti-PvMSP9E795-A808 antibodies were able to recognize the native PvMSP9 by immunofluorescence assay.
Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.
In the age of industrialization, numerous non-biodegradable pollutants like plastics, HMs, polychlorinated biphenyls, and various agrochemicals are a serious concern. These harmful toxic compounds ...pose a serious threat to food security because they enter the food chain through agricultural land and water. Physical and chemical techniques are used to remove HMs from contaminated soil. Microbial-metal interaction, a novel but underutilized strategy, might be used to lessen the stress caused by metals on plants. For reclaiming areas with high levels of heavy metal contamination, bioremediation is effective and environmentally friendly. In this study, the mechanism of action of endophytic bacteria that promote plant growth and survival in polluted soils-known as heavy metal-tolerant plant growth-promoting (HMT-PGP) microorganisms-and their function in the control of plant metal stress are examined. Numerous bacterial species, such as Arthrobacter, Bacillus, Burkholderia, Pseudomonas, and Stenotrophomonas, as well as a few fungi, such as Mucor, Talaromyces, Trichoderma, and Archaea, such as Natrialba and Haloferax, have also been identified as potent bioresources for biological clean-up. In this study, we additionally emphasize the role of plant growth-promoting bacteria (PGPB) in supporting the economical and environmentally friendly bioremediation of heavy hazardous metals. This study also emphasizes future potential and constraints, integrated metabolomics approaches, and the use of nanoparticles in microbial bioremediation for HMs.
Summary
Plasmodium vivax and P. cynomolgi produce numerous caveola–vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron‐dense ...knob protrusions expressed at the surface of Plasmodium falciparum‐infected erythrocytes. Here we investigate the three‐dimensional (3‐D) structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography – tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC‐8195 and PcyPHIST/CVC‐8195, analysed their structural features, including a PEXEL motif, repeated sequences and a C‐terminal PHIST domain, and show that PHIST/CVC‐8195 is most highly expressed in trophozoites. We generated images of CVCs in 3‐D using electron tomography (ET), and used immuno‐ET to show PHIST/CVC‐8195 localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc‐8195 gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC‐8195 is essential for survival of these malaria parasites.
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