Folic acid: The key to a healthy pregnancy - A prospective study on fetomaternal outcome.

Objectives: The objective of the study is to study the fetomaternal outcome associated with folic acid deficiency in pregnancy. Materials and Methods: This hospital-based observational study was conducted in the Department of Obstetrics and Gynaecology at Base Hospital, Delhi Cantt, and a total of 351 participants were enrolled who were fulfilling the inclusion criteria. The plasma folic acid level of the selected patients was measured in the booking visit by automated chemiluminescence assay. The cutoff levels of folic acid were taken at 8.6 ng/mL. Based on these values, the study population was divided into two groups, one with folic acid values <8.6 ng/mL and the other with values ≥8.6 ng/mL. Plasma Vitamin B12 levels were measured to check for any concurrent deficiencies. Obstetric outcomes included first- and second-trimester miscarriages, development of anemia, gestational hypertension/preeclampsia, gestational diabetes mellitus, hypothyroidism, placental abruption, and intrauterine fetal growth restriction (FGR). Furthermore, the period of gestation at delivery, fetal weights, APGAR scores at 5 min were documented. The study also considered fetal neural tube defects, intrauterine fetal demise for data collection. Collected data were analyzed statistically to find the association of the above-mentioned outcomes with levels of folic acid. Results: The rate of preterm deliveries was significantly higher in the folic acid group with levels <8.6 ng/mL (16.94%). The incidence of small for gestational age/FGR was higher in the folic acid group with levels <8.6 ng/mL (27.11%) compared to the high folic acid group with levels ≥8.6 ng/mL (13.38%). The differences in the incidence of anemia, gestational hypertension, gestational diabetes, and preeclampsia between the two groups were not statistically significant and no cases of intrauterine fetal demise or placental abruption were observed in either group. Moreover, there was no significant difference in the relative risk of low Apgar scores at 5 min between the two groups. Conclusion: The present study suggests that low folic acid levels during pregnancy are associated with a higher risk of adverse pregnancy outcomes such as anemia, miscarriages, preterm delivery, and FGR. Therefore, adherence to nutritional recommendation of folic acid supplementation during pregnancy is essential to prevent these adverse outcomes.

Correlation between placental histopathology and perinatal outcome in COVID-19.

Objectives: An alarming rate of adverse perinatal outcomes as well as maternal deaths has been reported worldwide during this pandemic. It would be prudent to start thinking on the lines of acute or chronic intrauterine fetal hypoxia due to placental microvascular pathology or villitis caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. Autopsy studies of deceased patients with severe COVID-19 have revealed the presence of diffuse pulmonary alveolar damage, thrombosis, and microvascular injuries. It is expected that similar pathological features such as microvascular injuries could be found in the placenta of infected pregnant women. Materials and Methods: Placentas of singleton pregnancies from 42 SARS-CoV-2 positive mothers delivered at term were submitted for histopathological examination. Those with multifetal gestation, hypertensive disorder, fetal growth restriction, structural or chromosomal anomalies in the fetus, thrombophilia, prolonged prelabor rupture of membranes, and placenta accreta spectrum were excluded from the study. Histopathological examination was done by two pathologists independently and only those results concurred by both were reported. Histopathological features and corresponding neonatal outcome were analyzed. Results: Reports of 42 placentas from patients with SARS-CoV-2, delivered at term (37-40 weeks) were analyzed in our study. Features of maternal vascular malperfusions (MVM) were present in 45% (n = 19) cases. Features of fetal vascular malperfusions (FVM) were present in 23.8% (n = 10) cases. There were 47.6% (n = 20) cases showing at least one feature of acute inflammatory pathology (AIP) and 42.8% (n = 18) showing features of chronic inflammatory pathology (CIP). Neonatal respiratory distress syndrome was found in 19% (n = 8) of the neonates. Correspondingly, nearly all placentas (n = 7) of these neonates showed features of MVM, FVM, AIP and CIP. There was no maternal or neonatal mortality in our study group. Conclusion: The main findings of our study include maternal as well as fetal vascular malperfusions and placental inflammatory pathology. These findings provide an outline for better understanding of etiological factors and pathogenesis of adverse perinatal outcomes in SARS-CoV-2 infection.

Pull-down of Biotinylated RNA and Associated Proteins.

Mapping networks of RNA-protein interactions in cells is essential for understanding the inner workings of many biological processes, including RNA processing, trafficking, and translation. Current in vivo methods for studying protein-RNA interactions rely mostly on purification of poly(A) transcripts, which represent only ~2-3% of total RNAs (Figure 1). Alternate robust methods for tagging RNA molecules with an RNA aptamer (e.g., MS2-, U1A- and biotin-RNA aptamer) and capturing the RNA-protein complex by the respective aptamer-specific partner are not extensively studied. Here, we describe a protocol (Figure 2) in which a biotin-RNA aptamer, referred to as the RNA mimic of biotin (RMB), was conjugated separately to two small RNA secondary structures that contribute to trafficking and translating HAC1 mRNA in the budding yeast Saccharomyces cerevisiae. The RMB-tagged RNA was expressed in yeast cells from a constitutive promoter. The biotinylated RNA bound to proteins was pulled down from the cell lysate by streptavidin agarose beads. RNA was detected by RT-PCR (Figure 3) and associated proteins by mass spectrometry (Figure 4). Our findings show that an RNA aptamer tag to RNA molecule is an effective method to explore the functional roles of RNA-protein networks in vivo.

The cap-proximal RNA secondary structure inhibits preinitiation complex formation on HAC1 mRNA.

Translation of HAC1 mRNA in the budding yeast Saccharomyces cerevisiae is derepressed when RNase Ire1 removes its intron via nonconventional cytosolic splicing in response to accumulation of unfolded proteins inside the endoplasmic reticulum. The spliced HAC1 mRNA is translated into a transcription factor that changes the cellular gene expression patterns to increase the protein folding capacity of cells. Previously, we showed that a segment of the intronic sequence interacts with the 5'-UTR of the unspliced mRNA, resulting in repression of HAC1 translation at the initiation stage. However, the exact mechanism of translational derepression is not clear. Here, we show that at least 11-base-pairing interactions between the 5'-UTR and intron (UI) are sufficient to repress HAC1 translation. We also show that overexpression of the helicase eukaryotic initiation factor 4A derepressed translation of an unspliced HAC1 mRNA containing only 11-bp interactions between the 5'-UTR and intronic sequences. In addition, our genetic screen identifies that single mutations in the UI interaction site could derepress translation of the unspliced HAC1 mRNA. Furthermore, we show that the addition of 24 RNA bases between the mRNA 5'-cap and the UI interaction site derepressed translation of the unspliced HAC1 mRNA. Together, our data provide a mechanistic explanation for why the cap-proximal UI-RNA duplex inhibits the recruitment of translating ribosomes to HAC1 mRNA, thus keeping mRNA translationally repressed.

Detection of HAC1 mRNA Splicing by RT-PCR in Saccharomyces cerevisiae.

HAC1 mRNA remains translationally repressed in the cytoplasm of the budding yeast Saccharomyces cerevisiae. Under conditions of cellular stress, a dual kinase RNase IRE1 (Inositol Requiring Enzyme-1) cleaves out an intervening sequence from the HAC1 mRNA. Cleaved mRNAs are then ligated by tRNA ligase, thus generating a spliced mRNA that translates an active transcription factor. This unconventional splicing of HAC1 mRNA in the cytoplasm is a molecular marker for various cellular stresses including oxidative stress and endoplasmic reticulum (ER) stress. This article describes a PCR-based protocol to detect the HAC1 mRNA splicing.

RNA-binding protein FXR1 drives cMYC translation by recruiting eIF4F complex to the translation start site.

Fragile X-related protein-1 (FXR1) gene is highly amplified in patients with ovarian cancer, and this amplification is associated with increased expression of both FXR1 mRNA and protein. FXR1 expression directly associates with the survival and proliferation of cancer cells. Surface sensing of translation (SUnSET) assay demonstrates that FXR1 enhances the overall translation in cancer cells. Reverse-phase protein array (RPPA) reveals that cMYC is the key target of FXR1. Mechanistically, FXR1 binds to the AU-rich elements (ARE) present within the 3' untranslated region (3'UTR) of cMYC and stabilizes its expression. In addition, the RGG domain in FXR1 interacts with eIF4A1 and eIF4E proteins. These two interactions of FXR1 result in the circularization of cMYC mRNA and facilitate the recruitment of eukaryotic translation initiation factors to the translation start site. In brief, we uncover a mechanism by which FXR1 promotes cMYC levels in cancer cells.

Assessment of materno-foetal transmission of SARS-CoV-2: A prospective pilot study.

BACKGROUND: The issue of vertical transmission of SARS-CoV-2 infection to the foetus has not yet been resolved. Its main reason is lack of a bigger study to analyse this question. The evidence of the affection of the foetus during antenatal or intrapartum period is limited to some anecdotal reports. To look for the possibility of vertical transmission of Severe Acute Respiratory Syndrome - Corona Virus-2 (SARS-CoV-2) infection to the foetus, this prospective pilot study was conducted at a tertiary health care COVID-19 designated centre of Armed Forces. METHODS: This study was conducted during 01 June 2020 and 15 October 2020 and included 54 covid-positive pregnant mothers. During delivery, amniotic fluid and cord blood samples were collected in a sterile manner. Amniotic fluid samples were not collected during vaginal deliveries as chances of contamination was very high. These samples were tested for the presence of SARS-CoV-2 gene by Reverse Transcriptasee Polymerase Chain Reaction (RT-PCR) test, and the results were analysed. Newborns were allowed to room in with mother, and they underwent throat and nasal swab RT-PCR testing of covid within 24-48 h of delivery. RESULTS: A total of 1520 pregnant mothers underwent RT-PCR test during the study period. Total positivity rate among our pregnant women was 2.8%. Out of 54 covid-positive women during the study period, amniotic fluid RT-PCR tests were carried out for 43 women, and cord blood was tested for 45 women. CONCLUSION: RT-PCR test of amniotic fluid, cord blood and nasal and throat swab of all newborns delivered by SARS-CoV-2-positive pregnant women were negative. Based on our study, the possibility of intrauterine vertical transmission of SARS-CoV-2 infection appears to be unlikely.

Phosphorylation of Pal2 by the protein kinases Kin1 and Kin2 modulates HAC1 mRNA splicing in the unfolded protein response in yeast.

During cellular stress in the budding yeast Saccharomyces cerevisiae, an endoplasmic reticulum (ER)-resident dual kinase and RNase Ire1 splices an intron from HAC1 mRNA in the cytosol, thereby releasing its translational block. Hac1 protein then activates an adaptive cellular stress response called the unfolded protein response (UPR) that maintains ER homeostasis. The polarity-inducing protein kinases Kin1 and Kin2 contribute to HAC1 mRNA processing. Here, we showed that an RNA-protein complex that included the endocytic proteins Pal1 and Pal2 mediated HAC1 mRNA splicing downstream of Kin1 and Kin2. We found that Pal1 and Pal2 bound to the 3' untranslated region (3'UTR) of HAC1 mRNA, and a yeast strain lacking both Pal1 and Pal2 was deficient in HAC1 mRNA processing. We also showed that Kin1 and Kin2 directly phosphorylated Pal2, and that a nonphosphorylatable Pal2 mutant could not rescue the UPR defect in a pal1Δ pal2Δ strain. Thus, our work uncovers a Kin1/2-Pal2 signaling pathway that coordinates HAC1 mRNA processing and ER homeostasis.

Vps34 and TOR Kinases Coordinate HAC1 mRNA Translation in the Presence or Absence of Ire1-Dependent Splicing.

In the budding yeast Saccharomyces cerevisiae, an mRNA, called HAC1, exists in a translationally repressed form in the cytoplasm. Under conditions of cellular stress, such as when unfolded proteins accumulate inside the endoplasmic reticulum (ER), an RNase Ire1 removes an intervening sequence (intron) from the HAC1 mRNA by nonconventional cytosolic splicing. Removal of the intron results in translational derepression of HAC1 mRNA and production of a transcription factor that activates expression of many enzymes and chaperones to increase the protein-folding capacity of the cell. Here, we show that Ire1-mediated RNA cleavage requires Watson-Crick base pairs in two RNA hairpins, which are located at the HAC1 mRNA exon-intron junctions. Then, we show that the translational derepression of HAC1 mRNA can occur independent of cytosolic splicing. These results are obtained from HAC1 variants that translated an active Hac1 protein from the unspliced mRNA. Additionally, we show that the phosphatidylinositol-3-kinase Vps34 and the nutrient-sensing kinases TOR and GCN2 are key regulators of HAC1 mRNA translation and consequently the ER stress responses. Collectively, our data suggest that the cytosolic splicing and the translational derepression of HAC1 mRNA are coordinated by unique and parallel networks of signaling pathways.

Adaptation to Endoplasmic Reticulum Stress Requires Transphosphorylation within the Activation Loop of Protein Kinases Kin1 and Kin2, Orthologs of Human Microtubule Affinity-Regulating Kinase.

Perturbations in endoplasmic reticulum (ER) homeostasis, a condition termed ER stress, activate the unfolded protein response (UPR), an intracellular network of signaling pathways. Recently, we have shown that protein kinase Kin1 and its paralog, Kin2, in the budding yeast Saccharomyces cerevisiae (orthologs of microtubule affinity-regulating kinase in humans) contribute to the UPR function. These Kin kinases contain a conserved kinase domain and an autoinhibitory kinase-associated 1 (KA1) domain separated by a long undefined domain. Here, we show that Kin1 or Kin2 protein requires minimally a kinase domain and an adjacent kinase extension region (KER) for UPR function. We also show that the functional mini-Kin2 protein is predominantly visualized inside the cells and precipitated with the cellular membrane fraction, suggesting its association with the cellular endomembrane system. Furthermore, we show that transphosphorylation of the Kin1 residue T302 and the analogous Kin2 residue T281 within the activation loop are important for full kinase activity. Collectively, our data suggest that, during ER stress, the Kin kinase domain is released from its autoinhibitory KA1 domain and is activated by transphosphorylation.

Phosphorylation of translation initiation factor eIF2α at Ser51 depends on site- and context-specific information.

Protein kinases phosphorylate specific amino acid residues of substrate proteins and regulate many cellular processes. Specificity for phosphorylation depends on the accessibility of these residues, and more importantly, kinases have preferences for certain residues flanking the phospho-acceptor site. Translation initiation factor 2α [eukaryotic translation initiation factor 2α (eIF2α)] kinase phosphorylates serine51 (Ser51) of eIF2α and downregulates cellular protein synthesis. Structural information on eIF2α reveals that Ser51 is located within a flexible loop, referred to as the Ser51 loop. Recently, we have shown that conformational change of the Ser51 loop increases the accessibility of Ser51 to the kinase active site for phosphorylation. Here, we show that the specificity of Ser51 phosphorylation depends largely on its relative position in the Ser51 loop and minimally on the flanking residues.

Outcomes of Multifetal Reduction: A Hospital-Based Study.

BACKGROUND: Higher-order multiple (HOM) pregnancies are associated with increased incidences of pregnancy complications mainly abortions, pre-eclampsia, preterm delivery and fetal death. Multifetal reduction (MFR) during first trimester and subsequent delivery of twins can reduce pregnancy associated morbidities. This study was conducted to evaluate the maternal and fetal outcomes of MFR procedure in patients with HOMs those managed in a tertiary care hospital. METHODS AND MATERIAL: It was a prospective observational study carried out in a tertiary care military hospital, India, and all women with higher-order multiples (triplets or more) conceived spontaneously or after infertility treatment (ovulation induction, intra-uterine insemination, or in vitro fertilization) during the 3-year period from Jan 2014 to Dec 2016 were included for MFR. Demographic and clinical data, and obstetric and neonatal outcomes were tabulated. RESULTS: The study included 32 HOM pregnancies which underwent MFR. 16% patients had pre-eclampsia and 12% patients had gestational diabetes. The study had 2 pregnancy losses before 24 weeks period of gestation (POG). 70% patients underwent cesarean delivery with mean gestational age of 35.5 weeks. Average birth weight of newborn was 1820 gm and 80% of them required NICU admission. CONCLUSION: Favorable pregnancy outcomes can be achieved after multifetal reductions during first trimester in higher-order multiples, but the procedure is not totally safe.

Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress.

High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1ß. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.

Efficacy of Use of Long-Term, Low-Dose Mifepristone for the Treatment of Fibroids.

BACKGROUND: Fibroid or myoma is the commonest reported tumor of uterus, and is one of the important reasons for hysterectomy in our setting. Different drugs are available for medical management of fibroid uterus including mifepristone, a progesterone antagonist. Varying dosage regimen for mifepristone was studied as medical management of fibroid uterus. The aim of the project was to study the effect of mifepristone on the symptoms and size of fibroids, especially using a low-dose regimen for 6 months. In addition, any symptomatic improvement of menorrhagia and dysmenorrhea was also studied. METHODS: The study was designed as an observational prospective "before-after" study. Women diagnosed with uterine fibroids attending OPD of a tertiary care hospital were selected according to the inclusion criteria. A total of 36 patients were enrolled in the study. Sample size was calculated to study changes in various parameters after 6 months treatment with mifepristone 50 mg once a week. Baseline investigations were performed and menstrual blood loss was assessed using pictorial blood assessment charts. Fifty milligrams of mifepristone weekly was used, and patient assessed at 1 and 6 month. They were also further followed up till 3 months after stopping the drug to observe the changes in menstrual pattern, fibroid volume, hemoglobin and liver function tests. Baseline endometrial biopsy and another at 6 month on cessation of drug therapy were done for all patients. RESULTS: Majority of the study population comprised of perimenopausal women, i.e., 41-45 years (44 %). Fifty percent of the patients were Para 2 and belonged to the perimenopausal age-group (18 out of 36). The dominant presenting symptom was menorrhagia associated with dysmenorrhea and pelvic pain. After 6 months of treatment with mifepristone, the mean fibroid volume reduced from 204.33 to 113.16 cm(3) (n = 33); p ≤ 0.001, and the percentage mean volume reduction of the fibroid in the study population was 44.57 % (range 1.10-100 %). Immediate reduction in bleeding PV was observed in 100 %, and 88.89 % (32/36) patients attained amenorrhea. The mean hemoglobin increased from 9.18 to 10.82 g/dl (p = 0.001). There was a transient rise in mean transaminases (AST/ALT) levels at 6 months which reverted to normal at 9 months follow-up. CONCLUSION: To conclude, 6 months therapy with 50 mg of mifepristone given weekly is efficacious and acceptable for the treatment of symptomatic leiomyoma, especially in a select group of patients. Although its use as a primary medical therapy is limited due to recurrence of fibroid after stopping treatment, it is useful for perimenopausal women whose myoma would regress after menopause, and younger infertile patients with small-size deep intramural myomas not easily accessible to either hysteroscopic or laparoscopic surgery. It is also beneficial as a preoperative adjunct, in patients with preoperative severe anemia and large fibroids where surgery is technically difficult. Mode of surgery can be changed to a less-invasive vaginal hysterectomy rather than an abdominal procedure.

Evidence That Base-pairing Interaction between Intron and mRNA Leader Sequences Inhibits Initiation of HAC1 mRNA Translation in Yeast.

The Hac1 transcription factor in yeast up-regulates a collection of genes that control protein homeostasis. Base-pairing interactions between sequences in the intron and the 5'-untranslated region (5' UTR) of the HAC1 mRNA represses Hac1 protein production under basal conditions, whereas cytoplasmic splicing of the intron by the Ire1 kinase-endonuclease, activated under endoplasmic reticulum stress conditions, relieves the inhibition and enables Hac1 synthesis. Using a random mutational screen as well as site-directed mutagenesis, we identify point mutations within the 5' UTR-intron interaction site that derepress translation of the unspliced HAC1 mRNA. We also show that insertion of an in-frame AUG start codon upstream of the interaction site releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhances production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. These results suggest that the major block of translation occurs at the initiation stage. Supporting this interpretation, the point mutations that enhanced Hac1 production resulted in an increased percentage of the HAC1 mRNA associating with polysomes versus free ribosomal subunits. Thus, our results provide evidence that the 5' UTR-intron interaction represses translation initiation on the unspliced HAC1 mRNA.