High-resolution analysis of the human placental DNA methylome in early gestation.

BACKGROUND/OBJECTIVE: We communicate high-read-depth bisulfite sequencing analysis of the chorionic villus (CV) DNA methylome from samples obtained between 11 and 13 weeks gestation and samples of gestationally age-matched maternal blood cells (MBC). METHODS: This was achieved through solution-phase targeted region capture (84 Mb) of bisulfite converted human DNA. RESULTS: We identified biphasic distribution of methylation in CV and MBC genomes. We found greater numbers of intermediate methylated sites (20%-80% methylated) in CV and greater number of high methylation sites in MBC and investigated distributions of these in promoters, introns, exons, CpG islands, CpG islands shores, and enhancers. We identified differentially methylated sites distinguishing CV and MBC. These are less likely to occur in CpG islands (CGIs), particularly those that exist outside promoters, exons, and introns. We found that gene promoter and gene body methylation patterns are associated with mRNA transcriptional profiles in CV. Despite the relative hypomethylation of CV genomes, we found that these contain DM regions that are more likely to be hypermethylated in CV relative to MBC. CONCLUSIONS: Our data provide novel insight into the structure and organization of the CV epigenome, which may inform future studies of placental biology and noninvasive prenatal phenotyping.

The CD8 antiviral factor (CAF) can suppress HIV-1 transcription from the long terminal repeat (LTR) promoter in the absence of elements upstream of the CATATAA box.

BACKGROUND: The CD8 Antiviral Factor (CAF) suppresses viral transcription from the HIV-1 Long Terminal Repeat (LTR) promoter in a non-cytolytic manner. However, the region on the LTR upon which CAF acts is unknown. Our objective was to determine the region on the LTR upon which CAF acts to suppress HIV-1 transcription. METHODS: Serial deletions of the LTR from the 5' end and inactivating point mutations were made. RESULTS: Serial deletions of the LTR from the 5' end indicated the importance of a short ~120 bp segment, containing the 3 SpI sites, CATA box (used by HIV-1 instead of the TATA box) and TAR region, in the suppressive process. Introduction of deletions or inactivating point mutations in the SpI sites or deletion of the TAR region did not abolish CAF-mediated transcriptional suppression. Yet, CAF-mediated transcriptional suppression was still retained in the HIV-1 CATA-TAR segment. CONCLUSION: CAF is able to suppress transcription from the LTR lacking all the elements upstream of the CATA box. Our results suggest that the HIV-1 CATA box may be responsible for CAF-mediated suppression of transcription from the HIV-1 LTR.

Building health system resilience and pandemic preparedness using wastewater-based epidemiology from SARS-CoV-2 monitoring in Bengaluru, India.

The COVID-19 pandemic was a watershed event for wastewater-based epidemiology (WBE). It highlighted the inability of existing disease surveillance systems to provide sufficient forewarning to governments on the existing stage and scale of disease spread and underscored the need for an effective early warning signaling system. Recognizing the potentiality of environmental surveillance (ES), in May 2021, COVIDActionCollaborative launched the Precision Health platform. The idea was to leverage ES for equitable mapping of the disease spread in Bengaluru, India and provide early information regarding any inflection in the epidemiological curve of COVID-19. By sampling both networked and non-networked sewage systems in the city, the platform used ES for both equitable and comprehensive surveillance of the population to derive precise information on the existing stage of disease maturity across communities and estimate the scale of the approaching threat. This was in contrast to clinical surveillance, which during the peak of the COVID-19 pandemic in Bengaluru excluded a significant proportion of poor and vulnerable communities from its ambit of representation. The article presents the findings of a sense-making tool which the platform developed for interpreting emerging signals from wastewater data to map disease progression and identifying the inflection points in the epidemiological curve. Thus, the platform accurately generated early warning signals on disease escalation and disseminated it to the government and the general public. This information enabled concerned audiences to implement preventive measures in advance and effectively plan their next steps for improved disease management.

Surveillance of SARS-CoV-2 RNA in open-water sewage canals contaminated with untreated wastewater in resource-constrained regions.

Sewage-based surveillance for COVID-19 has been described in multiple countries and multiple settings. However, nearly all are based on testing sewage treatment plant inflows and outflows using structured sewage networks and treatment systems. Many resource-limited countries worldwide have open canals, lakes and other such waste-contaminated water bodies that act as a means of sewage effluent discharge. These could serve as hyperlocal testing points for detecting COVID-19 incidence using the effluents from nearby communities. However, a sensitive, robust and economical method of SARS-CoV-2 RNA detection from open waste contaminated water bodies in resource-constrained regions is currently lacking. A protocol employed in Bangalore, India, where SARS-CoV-2 RNA levels were evaluated using two open canal systems during the first and second waves in the present study. SARS-CoV-2 RNA was measured using two strategies: a modified TrueNATTM microchip-based rapid method and traditional real-time reverse transcription-PCR (rRT-PCR), which were compared for analytical sensitivity, cost and relative ease of use. SARS-CoV-2 RNA levels were detected at lower levels during the earlier half compared to the later half of the first wave in 2020. The opposite trend was seen in the second wave in 2021. Interestingly, the change in RNA levels corresponded with the community burden of COVID-19 at both sites. The modified TrueNATTM method yielded concordant results to traditional rRT-PCR in sensitivity and specificity and cost. It provides a simple, cost-effective method for detecting and estimating SARS-CoV-2 viral RNA from open-water sewage canals contaminated with human excreta and industrial waste that can be adopted in regions or countries that lack structured sewage systems.

What influences adherence among HIV patients presenting with first-line antiretroviral therapy failure (ART failure)? A retrospective, cross-sectional study from a private clinic in Nagpur, India.

BACKGROUND: Multiple reports show increasing occurrences of ART failure in India. Despite the fact that a significant volume of outpatient and on-going healthcare occurs in private clinics, there are very few studies on adherence from private clinics in India. OBJECTIVE: To evaluate the factors influencing adherence to ART in patients with first-line ART failure. MATERIALS AND METHODS: Data were collected from a convenience sample of 139 individuals diagnosed with clinical, immunological or virologic failure from a private HIV clinic in Nagpur, India. A retrospective cross-sectional study was undertaken and data were statistically analysed. RESULTS: Of the 139 patients, 118 (84.9%) were male and 21 (15.1%) were female. 64 (46%) had received pre-treatment and adherence counselling. 81 (58.3%) were not told about the side effects of ART medications and 65 (46.8%) avoided friends and family. Most common reasons for suboptimal adherence by stopping treatment were high cost, alcoholism, choosing non-allopathic medications and depression. Reasons cited for suboptimal adherence due to missed doses included feeling healthy, depression, forgetfulness and busy schedule. A significant association was found between pre-treatment counselling, adherence counselling and being told the importance of lifelong treatment and decreased occurrence of complete stoppage of treatment. CONCLUSION: This study brings to light some of the predictors of ART failure. Counselling, having a strong support system as well as early identification and tackling of reasons for suboptimal adherence plays an important role in preventing ART failure.

Reasons for obstetric referrals from community facilities to a tertiary obstetric facility: A study from Southern Karnataka.

CONTEXT: Pregnancy-related preventable morbidity and mortality remain high in India. Safe delivery services should focus on improving neonatal and maternal outcomes while also enabling a positive childbirth experience. However, high rates of intrapartum obstetric referrals are common. OBJECTIVE: To describe the timing and the reasons for obstetric referrals to a public tertiary care hospital in Bangalore and characteristics of the referring facilities. METHODS: We interviewed 320 women who delivered at the tertiary care hospital within a one-month time frame prior to the interview and who originally planned to deliver elsewhere. RESULTS: Ninety four percent of women in the study reported that the decision to transfer to the tertiary hospital was made after the onset of labour. Referrals were made for medical as well as non-medical reasons. About a third (35%) had to take loans to cover the expenses of childbirth. CONCLUSIONS: Referrals frequently occurred after the onset of labour. Our data imply that improving obstetric referral protocols will improve the birth experience and reduce the burden on tertiary care facilities and on the women themselves.

Comprehensive analysis of preeclampsia-associated DNA methylation in the placenta.

BACKGROUND: A small number of recent reports have suggested that altered placental DNA methylation may be associated with early onset preeclampsia. It is important that further studies be undertaken to confirm and develop these findings. We therefore undertook a systematic analysis of DNA methylation patterns in placental tissue from 24 women with preeclampsia and 24 with uncomplicated pregnancy outcome. METHODS: We analyzed the DNA methylation status of approximately 27,000 CpG sites in placental tissues in a massively parallel fashion using an oligonucleotide microarray. Follow up analysis of DNA methylation at specific CpG loci was performed using the Epityper MassArray approach and high-throughput bisulfite sequencing. RESULTS: Preeclampsia-specific DNA methylation changes were identified in placental tissue samples irrespective of gestational age of delivery. In addition, we identified a group of CpG sites within specific gene sequences that were only altered in early onset-preeclampsia (EOPET) although these DNA methylation changes did not correlate with altered mRNA transcription. We found evidence that fetal gender influences DNA methylation at autosomal loci but could find no clear association between DNA methylation and gestational age. CONCLUSION: Preeclampsia is associated with altered placental DNA methylation. Fetal gender should be carefully considered during the design of future studies in which placental DNA is analyzed at the level of DNA methylation. Further large-scale analyses of preeclampsia-associated DNA methylation are necessary.

Loss of HGF/c-Met signaling in pancreatic ß-cells leads to incomplete maternal ß-cell adaptation and gestational diabetes mellitus.

Hepatocyte growth factor (HGF) is a mitogen and insulinotropic agent for the ß-cell. However, whether HGF/c-Met has a role in maternal ß-cell adaptation during pregnancy is unknown. To address this issue, we characterized glucose and ß-cell homeostasis in pregnant mice lacking c-Met in the pancreas (PancMet KO mice). Circulating HGF and islet c-Met and HGF expression were increased in pregnant mice. Importantly, PancMet KO mice displayed decreased ß-cell replication and increased ß-cell apoptosis at gestational day (GD)15. The decreased ß-cell replication was associated with reductions in islet prolactin receptor levels, STAT5 nuclear localization and forkhead box M1 mRNA, and upregulation of p27. Furthermore, PancMet KO mouse ß-cells were more sensitive to dexamethasone-induced cytotoxicity, whereas HGF protected human ß-cells against dexamethasone in vitro. These detrimental alterations in ß-cell proliferation and death led to incomplete maternal ß-cell mass expansion in PancMet KO mice at GD19 and early postpartum periods. The decreased ß-cell mass was accompanied by increased blood glucose, decreased plasma insulin, and impaired glucose tolerance. PancMet KO mouse islets failed to upregulate GLUT2 and pancreatic duodenal homeobox-1 mRNA, insulin content, and glucose-stimulated insulin secretion during gestation. These studies indicate that HGF/c-Met signaling is essential for maternal ß-cell adaptation during pregnancy and that its absence/attenuation leads to gestational diabetes mellitus.