Minimum number of oligonucleotide-based stable monomeric branched DNA nanostructure: Biochemical and biophysical study.

DNA has been employed as building blocks of nanomaterial construction due to their programmability and wide range applications. The functional branched DNA (bDNA) nanostructure is largely dependent on the sequence and structural symmetry. Despite the discovery of different structures, the synthesis of bDNA nanostructures from optimal number of oligonucleotides is yet to be explored. In the current study, for the first time we demonstrate the designing of stable monomeric bDNA structures using two or three oligonucleotides. Furthermore, the stability of bDNA nanostructures was thoroughly investigated in presence of different pH, cations, fetal bovine serum and DNaseI. The thermodynamic parameters indicate that hydrogen bonding and van der Waals interaction played a major role during binding of bDNA oligonucleotides. From the gel retardation assay, we confirmed the binding of complementary oligonucleotides to the bDNA nanostructures, thus can be explored for target specific transcript regulation. In conclusion, the self-assembled DNA structures developed from optimal oligonucleotides are stable in physiological environment and can be used for biomedical applications.

Sex-specific differences in presenting symptoms of obstructive sleep apnea.

OBJECTIVES: Scant data from India are available on the gender differences in presenting features of Obstructive Sleep Apnea (OSA) in India. This study aims to compare male and female patients with OSA for general characteristics and presenting symptoms. METHODOLOGY: Retrospective study was done in OSA patients diagnosed in our sleep lab. History, biochemical reports, and polysomnography variables were retrieved from the sleep registry and were compared between males and females. RESULTS: Out of 514 patients of OSA (367 males; 147 females). Females were older (55.97 ± 9.73 v/s 50.2 + 12.70 years, P<0.001) and more obese (BMI 35.26 ± 7.17 v/s 29.58 ± 5.49 Kg/m2; P<0.001). Waist and hip circumference were significantly higher in the female patients (P = 0.009 and <0.001 respectively). Morning headache, nocturia, fatigability (P < 0.001), and depression (P = 0.005) was more common in females (P = 0.036). Hypersomnia was more commonly seen in males (P < 0.001). Mean diastolic blood pressure was significantly higher in males, although no difference was seen in Systolic BP. Females had higher mean Fasting Blood glucose (FBS) (P = 0.02). Apnea hypopnea index was significantly higher in females {P = 0.01}. CONCLUSION: Women with OSA are more obese, elderly, and with higher fasting blood glucose than males at the time of diagnosis. Females have a higher prevalence of symptoms like fatigability, depression, nocturia and early morning headache and had more severe AHI than males.

Peptide nanostructures-based delivery of DNA nanomaterial therapeutics for regulating gene expression.

Self-assembled branched DNA (bDNA) nanomaterials have exhibited their functionality in various biomedical and diagnostic applications. However, the anionic cellular membrane has restricted the movement of bDNA nanostructures. Recently, amphiphilic peptides have been investigated as cationic delivery agents for nucleic acids. Herein, we demonstrate a strategy for delivering functional bDNA nanomaterials into mammalian cells using self-assembled linear peptides. In this study, antisense oligonucleotides of vascular endothelial growth factor (VEGF) were inserted in the overhangs of bDNAs. Novel linear peptides have been synthesized and the peptide-bound bDNA complex formation was examined using various biophysical experiments. Interestingly, the W4R4-bound bDNAs were found to be exceptionally stable against DNase I compared to other complexes. The delivery of fluorescent-labeled bDNAs into the mammalian cells confirmed the potential of peptide transporters. Furthermore, the functional efficacy of the peptide-bound bDNAs has been examined through RT-PCR and western blot analysis. The observed results revealed that W4R4 peptides exhibited excellent internalization of antisense bDNAs and significantly suppressed (3- to 4-fold) the transcripts and translated product of VEGF compared to the control. In summary, the results highlight the potential use of peptide-based nanocarrier for delivering bDNA nanostructures to regulate the gene expression in cell lines.

Self-assembled DNA nanostructure containing oncogenic miRNA-mediated cell proliferation by downregulation of FOXO1 expression.

FOXO1 transcription factor not only limits the cell cycle progression but also promotes cell death as a tumor suppressor protein. Though the expression of FOXO1 is largely examined in breast cancer, the regulation of FOXO1 by miRNA is yet to be explored. In the current study, self-assembled branched DNA (bDNA) nanostructures containing oncogenic miRNAs were designed and transfected to the MCF7 cell line to decipher the FOXO1 expression. bDNA containing oncogenic miRNAs 27a, 96, and 182 synergistically downregulate the expression of FOXO1 in MCF7 cells. The down-regulation is evident both in mRNA and protein levels suggesting that bDNA having miRNA sequences can selectively bind to mRNA and inhibit translation. Secondly, the downstream gene expression of p21 and p27 was also significantly downregulated in presence of miR-bDNA nanostructures. The cell proliferation activity was progressively increased in presence of miR-bDNA nanostructures which confirms the reduced tumor suppression activity of FOXO1 and the downstream gene expression. This finding can be explored to design novel bDNA structures which can downregulate the tumor suppressor proteins in normal cells and induce cell proliferation activity to identify early-phase markers of cancer.

Obstructive sleep apnea is highly prevalent in COVID19 related moderate to severe ARDS survivors: findings of level I polysomnography in a tertiary care hospital.

STUDY OBJECTIVES: Studies have found Obstructive Sleep Apnea (OSA) as a risk factor for increased risk for COVID19 Acute respiratory Distress Syndrome (ARDS); but most of the studies were done in already known patients of OSA. This study was done to find prevalence of OSA in patients with COVID-19 related acute respiratory distress syndrome. METHODOLOGY: A hospital based longitudinal study was conducted among COVID 19 Intensive Care Unit (ICU) survivors. All consecutive COVID19 with moderate to severe ARDS were evaluated for OSA by Level I Polysomnography (PSG) after 4-6 weeks of discharge. Prevalence of OSA and PSG variables {Total sleep time, Sleep efficiency, sleep stage percentage, Apnea Hypopnea Index (AHI), T90, nadir oxygen} was estimated. RESULTS: Out of 103 patients discharged from ICU during study period (October 2020 to 15 December 2020), 67 underwent Level I PSG. Mean Age was 52.6 ± 10.9 years and mean Body Mass Index was 27.5 ± 6.2 kg/m2. Total sleep time was 343.2 ± 86 min, sleep efficiency was 75.9 ± 14.2%. OSA (AHI ≥5) was seen in 65/67 patients and 49 patients had moderate to severe OSA (ie AHI ≥15). CONCLUSION: Moderate-severe OSA was highly prevalent (73%) in COVID19 moderate to severe ARDS survivors. Role of OSA in pathophysiology of COVID19 ARDS needs further evaluation.

Sleep EEG Signatures in COVID-19 Survivors.

Study Objectives: Effect of COVID-19 on sleep architecture is not known. This study was done to find out EEG changes seen during sleep in COVID-19 survivors. Methodology: In this prospective single centre study, consecutive patients diagnosed with RTPCR confirmed COVID 19 were included after 4-6 weeks of discharge from hospital. All patients underwent level I PSG. EEG was analysed for presence of abnormal EEG pattern. Results: Total 189 patients were contacted telephonically for participation in this study. Finally 81 patients (55 males, 26 females) underwent Level I PSG. Total sleep time was 345.1 ± 85.1 min. Sleep efficiency was 76.0 ± 14.2%. Mean time (%) during N1, N2, N3 and Rapid Eye movement (REM) was 16.4%, 59.2%, 7.9% and 18.4% percentage, respectively. Mean AHI was 28.7 ± 22.8 per hour and arousal index was 23.9 ± 13.3. Alpha intrusion was the most common EEG finding (78%), followed by cyclical alternating pattern (59%). REM density was significantly increased in 38% of patients. REM alpha bursts and increased spindles were also seen in 27% and 16%. Conclusion: Abnormal EEG waves are very commonly seen in COVID-19 survivors. Presence of these abnormal PSG-EEG waves hints that COVID-19 might have similar effects as depression, insomnia on these subjects, at least in short run. Whether these changes are temporary or permanent needs to be evaluated by performing serial polysomnographies in patients with COVID-19 ARDS.

Assessment of Obstructive Sleep Apnea in Association with Severity of COVID-19: A Prospective Observational Study.

INTRODUCTION: OSA has been postulated to be associated with mortality in COVID-19, but studies are lacking thereof. This study was done to estimate the prevalence of OSA in patients with COVID-19 using various screening questionnaires and to assess effect of OSA on outcome of disease. METHODOLOGY: In this prospective observational study, consecutive patients with RT-PCR confirmed COVID-19 were screened for OSA by different questionnaires (STOPBANG, Berlin Questionnaire, NoSAS, and Epworth Scale). Association between OSA, outcome (mortality) and requirement for respiratory support was assessed. RESULTS: In study of 213 patients; screening questionnaires for OSA [STOPBANG, Berlin Questionnaire (BQ), NoSAS] were more likely to be positive in patients who died compared to patients who survived. On binary logistic regression analysis, age ≥ 55 and STOPBANG score ≥ 5 were found to have small positive but independent effect on mortality even after adjusting for other variables. Proportion of patients who were classified as high risk for OSA by various OSA screening tools significantly increased with increasing respiratory support (p < 0.001 for STOPBANG, BQ, ESS and p = 0.004 for NoSAS). CONCLUSION: This is one of the first prospective studies of sequentially hospitalized patients with confirmed COVID-19 status who were screened for possible OSA could be an independent risk factor for poor outcome in patients with COVID-19.

COVID Eyes: REM in COVID-19 Survivors.

Increased REM density and alpha intrusion are routinely seen in COVID-19 patient. These findings may correlate with unstable sleep pattern in COVID-19 survivors and therefore, sleep hygiene and proper counselling should be emphasized upon. Clinicians and technicians should be aware of these EEG changes with reference to COVID-19 survivors in interpreting polysomnography.

miRNA-mediated alteration of sulfatase modifying factor 1 expression using self-assembled branched DNA nanostructures.

Sulfatase enzymes catalyze sulfate ester hydrolysis, thus deficiencies of sulfatases lead to the accumulation of biomolecules resulting in several disorders. One of the important sulfatases is estrone sulfatase that converts inactive estrone sulfate to active estradiol. Posttranslational modification of highly conserved cysteine residue leads to unique formylglycine in the active site of sulfatases being critical for its catalytic activity. The essential factor responsible for this modification of sulfatase is Sulfatase-Modifying Factor 1 (SUMF1). The role of estrone sulfatase is well evident in breast cancer progression. However, the function and regulation of SUMF1 in cancer are not studied. In the present study, for the first time, we have assessed the expression of SUMF1 in breast cancer and report the oncogenic behavior upon overexpression of SUMF1. Although increased expression or activity of SUMF1 is anticipated based on its function, the expression of SUMF1 was found to be reduced in breast cancer cells at both mRNA and protein levels. An estrogen receptor (ER) dependent expression of SUMF1 was observed and higher SUMF1 expression is associated with improved breast cancer patient survival in ER-positive cases. However, high SUMF1 expression leads to reduced median survival in ER-negative breast cancer patients. Putative binding sites for miRNAs-106b-5p, 128-3p and 148b-3p were found at 3'-UTR of SUMF1. Since self-assembled branched DNA (bDNA) structures have emerged as a highly efficient strategy for targeting multiple miRNAs simultaneously, we studied the alteration in SUMF1 expression using bDNA nanostructures with a complementary sequence to miRNAs. The findings suggest the involvement of co-regulators and repressors in miRNA-mediated SUMF1 expression in breast cancer cells and reveal the therapeutic potential of SUMF1 in endocrine-related malignancies.

"Necklace in the lung:" Multilocularis hydatid cyst mimicking left-sided massive pleural effusion.

Massive pleural effusions are commonly caused by malignancy, parapneumonic effusion, and tuberculosis. Parasitic infections are rare causes of massive pleural effusion. Echinococcosis or hydatid disease is a major public health problem in poor hygienic environments. The liver and lungs are the most frequently involved organs. Pulmonary disease appears to be more common in younger individuals. Echinococcus multilocularis causes alveolar echinococcosis, which accounts for <5% of all cases of hydatid liver disease and less frequently lung disease. Here, we present an unusual case of multilocular pulmonary hydatid cysts mimicking massive pleural effusion in a 25-year-old young male.

A meta-analysis of TLR4 and TLR9 SNPs implicated in severe malaria.

Toll-like receptors (TLRs) are critical mediators of the inflammatory response to malarial infection, and gene polymorphisms affecting TLR function may be partially responsible for inter-individual variation in disease manifestation. However, there are inconsistencies in the associations of common genetic variants of TLR4 (D299G) and TLR9 (T-1237C and T-1486C) with malaria outcome. A comprehensive search was conducted to identify relevant and independent Plasmodium falciparum-infected case-control studies, and meta-analysis including six studies for each SNP was performed to obtain more precise estimates of the pooled effects of these variants. The results showed significant associations of the -1486C allele with the risk of severe malaria in allele contrast (T vs. C, p = 0.004, OR = 1.26) and homozygous (TT vs. CC, p = 0.03, OR = 1.51) genetic models. There was no association between the D299G or T-1237C variants and uncomplicated or severe malaria using any of the genetic models tested. However, in stratified analysis, -1237C was associated with the risk of severe malaria in Indian adults (TT vs. TC, p = 0.06, OR = 2.13; TT vs. TC+CC, p <0.00001, OR = 2.65), suggesting that our results must be considered preliminary. The robustness of -1486C as a risk factor warrants investigation into its functionality in malaria pathogenesis. Further, the lack of an association with the T-1237C variant was weak, and future studies examining more detailed individual data from different ethnic groups are essential for confirmation of its genetic contribution to malaria.

A meta-analysis of TLR4 and TLR9 SNPs implicated in severe malaria

Abstract Toll-like receptors (TLRs) are critical mediators of the inflammatory response to malarial infection, and gene polymorphisms affecting TLR function may be partially responsible for inter-individual variation in disease manifestation. However, there are inconsistencies in the associations of common genetic variants of TLR4 (D299G) and TLR9 (T-1237C and T-1486C) with malaria outcome. A comprehensive search was conducted to identify relevant and independent Plasmodium falciparum-infected case-control studies, and meta-analysis including six studies for each SNP was performed to obtain more precise estimates of the pooled effects of these variants. The results showed significant associations of the -1486C allele with the risk of severe malaria in allele contrast (T vs. C, p = 0.004, OR = 1.26) and homozygous (TT vs. CC, p = 0.03, OR = 1.51) genetic models. There was no association between the D299G or T-1237C variants and uncomplicated or severe malaria using any of the genetic models tested. However, in stratified analysis, -1237C was associated with the risk of severe malaria in Indian adults (TT vs. TC, p = 0.06, OR = 2.13; TT vs. TC+CC, p <0.00001, OR = 2.65), suggesting that our results must be considered preliminary. The robustness of -1486C as a risk factor warrants investigation into its functionality in malaria pathogenesis. Further, the lack of an association with the T-1237C variant was weak, and future studies examining more detailed individual data from different ethnic groups are essential for confirmation of its genetic contribution to malaria.

Genetic predisposition of variants in TLR2 and its co-receptors to severe malaria in Odisha, India.

Although the role of TLRs signalling in malaria pathogenesis is well established, contribution of individual TLR to clinical outcome of malaria still remains inconclusive. Given the importance of TLR2 and its co-receptors in recognising distinct structural forms of key malaria toxins and mediating innate immune response, it is essential to delineate their genetic contribution. Variants in TLR1 (I602S) and TLR6 (P249S) were genotyped by PCR-RFLP methods, and TLR2 (I/D) was genotyped by PCR in 200 samples each from uncomplicated malaria (UM) and severe malaria (SM). Further, SM was categorised into its sub-clinical groups (CM and NCSM or SOD and MODS) and analysed. The results showed the PP genotype of TLR6 (P249S) to be significantly more common in UM (P < 0.0001), whereas the 'SS' genotype was the risk factor for SM including its sub-clinical categories. The TLR1 (602S) and TLR2 (D) variants were significantly high in patients with CM; however, negative LD was observed between TLR2 and TLR6 in NCSM and MODS. Haplotype analysis showed significantly high frequency of I-I-S haplotype in all forms of subclinical SM and was associated with low parasite load in SM (P = 0.013). The haplotypes I-D-S and S-I-P were significantly high in SOD and CM, respectively. The TLR6 '249S' variant appeared to be the dominant determinant for genetic predisposition to SM and that its association with either TLR2 'D' or TLR1 '602S' modulates for CM development. The present study opens up several new avenues for their exploration and validation in future studies in different global settings for malaria.

Influence of common variants of TLR4 and TLR9 on clinical outcomes of Plasmodium falciparum malaria in Odisha, India.

BACKGROUND: In malaria, the toll-like receptors (TLRs) have recently emerged as major player of innate immunity. However, implication of TLR variants on clinical manifestations of malaria is conflicting. The present study aims to provide relevant information of growing interest in understanding the role of TLR4D299G, TLR9T-1237C and TLR9T-1486C polymorphisms on clinical outcomes of malaria. METHODS: We genotyped TLR4D299G, TLR9T-1237C and TLR9T-1486C polymorphisms by PCR-RFLP methods and subsequently analyzed in 200 uncomplicated patients and 200 severe patients. Further, the severe malaria categorized into sub-clinical groups such as cerebral malaria (CM), non-cerebral severe malaria (NCSM), single organ dysfunction (SOD) and multi-organ dysfunctions (MODS) are analyzed. RESULT: The TLR9-1237CC genotype was observed at significantly low frequency in MODS (p=0.0008), while in heterozygous state (TC) it was proportionately more frequent in SOD (p=0.087) as compared to mild malaria. The TLR9T-1486C heterozygote was more common in all categories of severe malaria. However, pair wise LD analysis revealed significant linkage between T-1237C and T-1486C, whereas haplotype analysis showed significantly low frequency of C-T haplotype in CM (p=0.005, pc=0.02) and high frequency of T-C haplotype in NCSM as compared to mild malaria. CONCLUSION: Although TLR9-1237C could be a risk factor for severe malaria in heterozygous state, negative association of CC genotype with MODS warrants caution of segregating severe malaria into its sub-clinical groups while interpreting data. Further, clinical outcome in malaria was observed to be apparently modulated by LD between TLR9 promoter variants.