Chemical Synthesis of Δ-4,5 Unsaturated Heparan Sulfate Oligosaccharides for Biomarker Discovery.

A methodology is described that can provide heparan sulfate oligosaccharides having a Δ4,5-double bond, which are needed as analytical standards and biomarkers for mucopolysaccharidoses. It is based on chemical oligosaccharide synthesis followed by modification of the C-4 hydroxyl of the terminal uronic acid moiety as methanesulfonate. This leaving group is stable under conditions used to remove temporary protecting groups, O-sulfation, and hydrogenolysis. Treatment with NaOH results in elimination of the methanesulfonate and formation of a Δ4,5-double bond.

Analysis of Drugs Prescribed to Elderly Patients in a Tertiary Health Care Center in Raipur, Central India: An Observational Study.

Background Most elderly patients suffer from multiple diseases and are on multiple drugs for treatment. Polypharmacy in the elderly, physiological changes with old age, changes in the pharmacokinetics and pharmacodynamic effects of many drugs, and newer drug prescription trends for diseases like diabetes and cardiovascular disease make drug prescribing in the elderly more difficult. There are many chances of drug-drug interactions with easily available over-the-counter (OTC) medications. To prevent the irrational use of drugs in the elderly, there is a need for prescription analysis studies. Prescription analysis studies will help in finding errors in prescriptions and also change trends in the use of medication among the elderly. Methodology This cross-sectional observation study was conducted on 234 elderly patients to investigate medicine use patterns among the geriatric patients attending the Medicine Outpatient Department in a tertiary care teaching hospital. Drug data were collected from the study participants after obtaining written informed consent and analysed, including demographic details, personal history, disease history, and details of the drug, including the generic name of the drug, dose and duration of therapy, and prescription pattern. The proportions of drugs prescribed for different diseases were analysed. Also, the drugs were analysed as per their pharmacological profiles. Results and interpretation A total of 1298 drug prescriptions were analysed in this study. Of the total participants, 60.26% were male, 35% were unemployed, 53% were retired, and 27% were taking OTC herbal medications. Most of the patients included in this study were suffering from diabetes, hypertension, and other comorbidities. Telmisartan and telmisartan in combination with other drugs were the most commonly encountered prescription drugs, i.e., 24% among the cardiovascular drugs. Aspirin and statins alone or in combination were the most commonly encountered prescriptions, i.e., 27.88% of the drugs used for prophylaxis of cardiovascular diseases. Conclusion This study showed a prescription pattern for the elderly and highlighted precautions to be taken with some of the prescribed drugs. As polypharmacy is observed with elderly prescriptions, possible drug interactions must be taken into account. Regular prescription analysis of drugs prescribed to the elderly will help in the appropriate and rational use of drugs.

Religion, Fetal Protection, and Fasting during Pregnancy in Three Subcultures.

Fasting during pregnancy is an enigma: why would a woman restrict her food intake during a period of increased nutritional need? Relative to the costs to healthy individuals who are not pregnant, the physiological costs of fasting in pregnancy are amplified, with intrauterine death being one possible outcome. Given these physiological costs, the question arises as to the socioecological factors that give rise to fasting during pregnancy. There has been little formal research regarding the emic perceptions and socioecological factors associated with such fasting. This study therefore took an emic approach and investigated the types of fasts that are common in pregnancy, women's perceptions of the consequences of fasting, and the socioecological models of pregnancy fasting in three Indian communities. This cross-sectional study took place in Bhubaneshwar, Odisha state, and Mysore, Karnataka state, among two populations of Hindu women and one population of Muslim women (N = 85). In total, 64% of women fasted in prior pregnancies. Findings revealed variation in the number and types of fasts that are common in pregnancy across the three communities. Each community reported differences in positive and negative consequences of fasting, with varied emphasis on reproductive health, religiosity, and general health and well-being. Finally, quantitative analyses indicated that the best-fitting model for fasting during pregnancy was religiosity, and the poorest-fitting models were resource scarcity and general health. This study provides insight into motivations for such fasting and highlights the need to investigate the relationship between supernatural beliefs and maternal-fetal protection further, as well as social functions of pregnancy fasting within the family and community.

Dissecting structure-function of 3-O-sulfated heparin and engineered heparan sulfates.

Heparan sulfate (HS) polysaccharides are master regulators of diverse biological processes via sulfated motifs that can recruit specific proteins. 3-O-sulfation of HS/heparin is crucial for anticoagulant activity, but despite emerging evidence for roles in many other functions, a lack of tools for deciphering structure-function relationships has hampered advances. Here, we describe an approach integrating synthesis of 3-O-sulfated standards, comprehensive HS disaccharide profiling, and cell engineering to address this deficiency. Its application revealed previously unseen differences in 3-O-sulfated profiles of clinical heparins and 3-O-sulfotransferase (HS3ST)­specific variations in cell surface HS profiles. The latter correlated with functional differences in anticoagulant activity and binding to platelet factor 4 (PF4), which underlies heparin-induced thrombocytopenia, a known side effect of heparin. Unexpectedly, cells expressing the HS3ST4 isoenzyme generated HS with potent anticoagulant activity but weak PF4 binding. The data provide new insights into 3-O-sulfate structure-function and demonstrate proof of concept for tailored cell-based synthesis of next-generation heparins.

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation.

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Exposure to Gestational Diabetes Enriches Immune-Related Pathways in the Transcriptome and Methylome of Human Amniocytes.

CONTEXT: Gestational diabetes (GDM) has profound effects on the intrauterine metabolic milieu and is linked to obesity and diabetes in offspring, but the mechanisms driving these effects remain largely unknown. Alterations in DNA methylation and gene expression in amniocytes exposed to GDM in utero represent a potential mechanism leading to metabolic dysfunction later in life. OBJECTIVE: To profile changes in genome-wide DNA methylation and expression in human amniocytes exposed to GDM. DESIGN: A nested case-control study (n = 14 pairs) was performed in amniocytes matched for offspring sex, maternal race/ethnicity, maternal age, gestational age at amniocentesis, and gestational age at birth. Sex-specific genome-wide DNA methylation analysis and RNA-sequencing were completed and differentially methylated regions (DMRs) and gene expression changes were identified. Ingenuity pathway analysis identified biologically relevant pathways enriched after GDM exposure. In silico high-throughput chromosome conformation capture (Hi-C) analysis identified potential chromatin interactions with DMRs. RESULTS: Expression of interferon-stimulated genes was increased in GDM amniocytes, accounting for 6 of the top 10 altered genes (q < 0.05). Enriched biological pathways in GDM amniocytes included pathways involving inflammation, the interferon response, fatty liver disease, monogenic diabetes, and atherosclerosis. Forty-two DMRs were identified in male GDM-exposed amniocytes and 20 in female amniocyte analysis (q < 0.05). Hi-C analysis identified interactions between DMRs and 11 genes with significant expression changes in male amniocytes and 9 in female amniocytes (P < .05). CONCLUSION: In a unique repository of human amniocytes exposed to GDM in utero, transcriptome analysis identified enrichment of inflammation and interferon-related pathways and novel DMRs with potential distal regulatory functions.

In utero Bisphenol A Exposure Is Linked with Sex Specific Changes in the Transcriptome and Methylome of Human Amniocytes.

CONTEXT: Prenatal exposure to bisphenol A (BPA) is linked to obesity and diabetes but the molecular mechanisms driving these phenomena are not known. Alterations in deoxyribonucleic acid (DNA) methylation in amniocytes exposed to BPA in utero represent a potential mechanism leading to metabolic dysfunction later in life. OBJECTIVE: To profile changes in genome-wide DNA methylation and expression in second trimester human amniocytes exposed to BPA in utero. DESIGN: A nested case-control study was performed in amniocytes matched for offspring sex, maternal race/ethnicity, maternal age, gestational age at amniocentesis, and gestational age at birth. Cases had amniotic fluid BPA measuring 0.251 to 23.74 ng/mL. Sex-specific genome-wide DNA methylation analysis and RNA-sequencing (RNA-seq) were performed to determine differentially methylated regions (DMRs) and gene expression changes associated with BPA exposure. Ingenuity pathway analysis was performed to identify biologically relevant pathways enriched after BPA exposure. In silico Hi-C analysis identified potential chromatin interactions with DMRs. RESULTS: There were 101 genes with altered expression in male amniocytes exposed to BPA (q < 0.05) in utero, with enrichment of pathways critical to hepatic dysfunction, collagen signaling and adipogenesis. Thirty-six DMRs were identified in male BPA-exposed amniocytes and 14 in female amniocyte analysis (q < 0.05). Hi-C analysis identified interactions between DMRs and 24 genes with expression changes in male amniocytes and 12 in female amniocytes (P < 0.05). CONCLUSION: In a unique repository of human amniocytes exposed to BPA in utero, sex-specific analyses identified gene expression changes in pathways associated with metabolic disease and novel DMRs with potential distal regulatory functions.

Evaluation of the Prescribed Drugs to Elderly in a Tertiary Healthcare Center for Possible Drug Interactions with Investigational Drugs for COVID-19 Treatment.

OBJECTIVE: Earlier identifying drug interactions may help in risk reduction in elderly patients. METHODS: Drug prescription data of 212 elderly patients of tertiary health care center had been analyzed for possible drug interactions with investigational drugs for COVID-19 treatment. Drug interaction had been checked from Stockley's Drug Interaction 2019 and Martindale the Complete Drug Reference 2017 and standard reference books of Pharmacology. FINDINGS: Different types of drugs prescribed in the elderly were 260 and out of which 68 (26.36%) were in the category of fixed-dose combinations. Around 150 (70.75%) elderly patients were having one or more associated comorbidities. Thirty-five drugs prescribed to elderly had been found to cause drug interaction with investigational drugs for COVID-19. Possible drug interactions are mediated through CYP3A4 (eighteen patients), CYP2D6 (seven patients) isoenzymes, or P glycoproteins transporters (three patients). CONCLUSION: Possible drug interactions predicted in this study suggested need for modification of dose of drug or watchfulness for adverse effects. If these drug interactions are considered beforehand, complications can be prevented on account of these drug interactions in elderly who are suffering from COVID-19.

Gestational diabetes and maternal obesity are associated with sex-specific changes in miRNA and target gene expression in the fetus.

BACKGROUND/OBJECTIVES: Pregnancies complicated by gestational diabetes (GDM) or maternal obesity have been linked to the development of diabetes, obesity, and fatty liver disease later in life with sex-specific manifestations. Alterations in miRNA expression in offspring exposed to GDM and maternal obesity and effects on hepatic development are unknown. Here, we describe how exposure to maternal obesity in utero leads to sex-specific changes in miRNA and target gene expression in human fetal liver. METHODS: Candidate miRNA expression was measured in second trimester amniotic fluid (AF) from women with GDM. Targets of differentially expressed miRNAs were determined and pathway enrichment of target genes was performed. MiRNA and target gene expression were measured in a separate cohort of second trimester primary human fetal hepatocytes (PHFH) exposed to maternal obesity via qPCR and western blot. All studies were IRB approved. RESULTS: GDM-exposed AF had significant increases in miRNAs 199a-3p, 503-5p, and 1268a (fold change (FC) ≥ 1.5, p < 0.05). Female offspring-specific analysis showed enrichment in miRNAs 378a-3p, 885-5p, and 7-1-3p (p < 0.05). MiRNA gene targets were enriched in hepatic pathways. Key genes regulating de novo lipogenesis were upregulated in obesity-exposed PHFH, especially in males. Significantly altered miRNAs in GDM AF were measured in obese-exposed PHFH, with consistent increases in miRNAs 885-5p, 199-3p, 503-5p, 1268a, and 7-1-3p (FC ≥ 1.5, p < 0.05). Female PHFH exposed to maternal obesity had increased expression of miR-885-5p, miR-199-3p, miR-503-5p, miR-1268s, and miR-7-1-3p (p < 0.05), corresponding to decreased target genes expression for ABCA1, PAK4, and INSR. In male PHFHs, no miRNA changes were measured but there was increased expression of ABCA1, PAK4, and INSR (p < 0.05). CONCLUSIONS: Our data suggest sex-specific changes in miRNA and gene expression in PHFH may be one mechanism contributing to the sexual dimorphism of metabolic disease in offspring exposed to GDM and maternal obesity in utero.

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis.

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

Oral treatment with plecanatide or dolcanatide attenuates visceral hypersensitivity via activation of guanylate cyclase-C in rat models.

AIM: To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS: Transport of fluorescein isothiocyanate (FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1 (ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid (TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress (PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests. RESULTS: Treatment of T84 and Caco-2 monolayers with lipopolysaccharide (LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model. CONCLUSION: Our results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent anti-nociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.

Negative Electron Transfer Dissociation Sequencing of 3-O-Sulfation-Containing Heparan Sulfate Oligosaccharides.

Among dissociation methods, negative electron transfer dissociation (NETD) has been proven the most useful for glycosaminoglycan (GAG) sequencing because it produces informative fragmentation, a low degree of sulfate losses, high sensitivity, and translatability to multiple instrument types. The challenge, however, is to distinguish positional sulfation. In particular, NETD has been reported to fail to differentiate 4-O- versus 6-O-sulfation in chondroitin sulfate decasaccharide. This raised the concern of whether NETD is able to differentiate the rare 3-O-sulfation from predominant 6-O-sulfation in heparan sulfate (HS) oligosaccharides. Here, we report that NETD generates highly informative spectra that differentiate sites of O-sulfation on glucosamine residues, enabling structural characterizations of synthetic HS isomers containing 3-O-sulfation. Further, lyase-resistant 3-O-sulfated tetrasaccharides from natural sources were successfully sequenced. Notably, for all of the oligosaccharides in this study, the successful sequencing is based on NETD tandem mass spectra of commonly observed deprotonated precursor ions without derivatization or metal cation adduction, simplifying the experimental workflow and data interpretation. These results demonstrate the potential of NETD as a sensitive analytical tool for detailed, high-throughput structural analysis of highly sulfated GAGs. Graphical Abstract.

Plecanatide-mediated activation of guanylate cyclase-C suppresses inflammation-induced colorectal carcinogenesis in Apc+/Min-FCCC mice.

AIM: To evaluate the effect of orally administered plecanatide on colorectal dysplasia in Apc+/Min-FCCC mice with dextran sodium sulfate (DSS)-induced inflammation. METHODS: Inflammation driven colorectal carcinogenesis was induced in Apc+/Min-FCCC mice by administering DSS in their drinking water. Mice were fed a diet supplemented with plecanatide (0-20 ppm) and its effect on the multiplicity of histopathologically confirmed polypoid, flat and indeterminate dysplasia was evaluated. Plecanatide-mediated activation of guanylate cyclase-C (GC-C) signaling was assessed in colon tissues by measuring cyclic guanosine monophosphate (cGMP) by ELISA, protein kinase G-II and vasodilator stimulated phosphoprotein by immunoblotting. Ki-67, c-myc and cyclin D1 were used as markers of proliferation. Cellular levels and localization of ß-catenin in colon tissues were assessed by immunoblotting and immunohistochemistry, respectively. Uroguanylin (UG) and GC-C transcript levels were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). A mouse cytokine array panel was used to detect cytokines in the supernatant of colon explant cultures. RESULTS: Oral treatment of Apc+/MinFCCC mice with plecanatide produced a statistically significant reduction in the formation of inflammation-driven polypoid, flat and indeterminate dysplasias. This anti-carcinogenic activity of plecanatide was accompanied by activation of cGMP/GC-C signaling mediated inhibition of Wnt/ß-catenin signaling and reduced proliferation. Plecanatide also decreased secretion of pro-inflammatory cytokines (IL-6, IL1 TNF), chemokines (MIP-1, IP-10) and growth factors (GCSF and GMCSF) from colon explants derived from mice with acute DSS-induced inflammation. The effect of plecanatide-mediated inhibition of inflammation/dysplasia on endogenous expression of UG and GC-C transcripts was measured in intestinal tissues. Although GC-C expression was not altered appreciably, a statistically significant increase in the level of UG transcripts was detected in the proximal small intestine and colon, potentially due to a reduction in intestinal inflammation and/or neoplasia. Taken together, these results suggest that reductions in endogenous UG, accompanied by dysregulation in GC-C signaling, may be an early event in inflammation-promoted colorectal neoplasia; an event that can potentially be ameliorated by prophylactic intervention with plecanatide. CONCLUSION: This study provides the first evidence that orally administered plecanatide reduces the multiplicity of inflammation-driven colonic dysplasia in mice, demonstrating the utility for developing GC-C agonists as chemopreventive agents.