A protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors.

Sensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose- (or other carbohydrate-) biotin-polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose-biotin-PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.

Synthesis and Evaluation of Natural and Unnatural Tetrahydrocannabiorcol for Its Potential Use in Neuropathologies.

(-)-trans-Δ9-Tetrahydrocannabinol (trans-(-)-Δ9-THC) has shown neuroprotective potential, but its medicinal benefits are not fully exploited due to the limitations of psychoactive properties. The lower homologues are non-psychoactive in nature but lack comprehensive scientific validation regarding neuroprotective potential. The present study describes the synthesis of non-psychoactive lower homologues of THC-type compounds and their neuroprotective potential. Both natural tetrahydro-cannabiorcol (trans-(-)-Δ9-THCO) and unnatural Δ9-tetrahydrocannabiorcol (trans-(+)-Δ9-THCO) were successfully synthesized starting from R-limonene and S-limonene, respectively, and investigated for neuroprotective potential in cellular models. The structures of both enantiomers were confirmed by NMR, HMBC, HQSC, NOESY, and COSY experiments. Results indicated that both enantiomers were nontoxic to the cells treated up to 50 µM. Neuroprotective properties of the enantiomers showed that treatments could significantly reverse the corticosterone-induced toxicity in SH-SY5Y cells and simultaneously cause elevated expression of brain-derived neurotrophic factor (BDNF). It was also observed that unnatural trans-(+)-Δ9-THCO displayed better activity than the natural enantiomer and can be further explored for its potential use in neuropathological ailments.

Assessment of insulin resistance and metabolic syndrome in young reproductive aged women with polycystic ovarian syndrome: analogy of surrogate indices.

BACKGROUND: Polycystic ovarian syndrome has emerged as a cardiometabolic disorder and aim of this study was to evaluate various surrogate indices and their diagnostic potential to determine the most convenient and cost-effective marker of IR, CVD, and MetS in these women. MATERIALS AND METHODS: Ninety-five PCOS women and 45 age matched healthy women were enrolled. Measures included anthropometric and biochemical parameters, BMI, WHR, WHtR, BAI, VAI, LAP, HOMA-IR, and lipid profile. RESULTS: LAP has highest AUC value 0.781 with cut-off value = 39.73 (sensitivity = 75% and specificity = 79.5%) for predicting IR and AUC value 0.83 with cut-off value = 35.63 (sensitivity = 94.4% and specificity = 77.3%) for predicting MetS in women with PCOS. LAP had statistically strong positive correlation with WC, BMI, WHR, fasting glucose, fasting insulin, HOMA-IR, TC, TG, and SBP. CONCLUSIONS: LAP is a powerful and reliable marker for assessment of IR, CVD, and MetS risk in young Indian women with PCOS.

Proteomic sift through serum and endometrium profiles unraveled signature proteins associated with subdued fertility and dampened endometrial receptivity in women with polycystic ovary syndrome.

The objective of this study is to discern the proteomic differences responsible for hampering the receptivity of endometrium and subduing the fertility of females with polycystic ovary syndrome in analogy to healthy fertile females. This study was designed in collaboration with Hakeem Abdul Hameed Centenary Hospital affiliated to Jamia Hamdard, New Delhi, India. Serum samples were taken from infertile PCOS subjects (n = 6) and fertile control subjects (n = 6) whereas endometrial tissue samples were recruited from ovulatory PCOS (n = 4), anovulatory PCOS (n = 4) and normal healthy fertile control subjects (n = 4) for proteomic studies. Additionally, endometrial biopsies from healthy fertile control (n = 8), PCOS with infertility (n = 6), unexplained infertility (n = 3) and endometrial hyperplasia (n = 3) were taken for validation studies. Anthropometric, biochemical and hormonal evaluation was done for all the subjects enrolled in this study. Protein profiles were generated through 2D-PAGE and differential proteins analyzed with PD-QUEST software followed by identification with MALDI-TOF MS protein mass fingerprinting. Validation of identified proteins was done through RT-PCR relative expression analysis. Protein profiling of serum revealed differential expression of proteins involved in transcriptional regulation, embryogenesis, DNA repair, decidual cell ploidy, immunomodulation, intracellular trafficking and degradation processes. Proteins involved in cell cycle regulation, cellular transport and signaling, DNA repair, apoptotic processes and mitochondrial metabolism were found to be differentially expressed in endometrium. The findings of this study revealed proteins that hold strong candidature as potential drug targets to regulate the cellular processes implicating infertility and reduced receptivity of endometrium in women with polycystic ovary syndrome.

Differential Expression of MARK4 Protein and Related Perturbations in Females with Ovulatory PCOS.

BACKGROUND: Ovulatory PCOS (OPCOS) is the mildest form of the polycystic ovarian syndrome among all four determined phenotypes. Though the females with OPCOS are ovulating, hyperandrogenism and polycystic ovarian morphology increase the susceptibility of cardiovascular diseases, insulin resistance, hyperlipidemia and metabolic syndrome in these females. OBJECTIVES: The aim of the study was to identify the significance associated with OPCOS phenotype through serum proteomic profiling of OPCOS females and normal age-matched healthy ovulating females. METHODS: One and two-dimensional gel-based proteomic approaches were adopted to fractionate the complex serum proteome. Differential protein profiles generated were analyzed with PD-QUEST Software. Protein spots differing in intensity by >2-fold were selected and identified further by MALDI-TOF MS. Validation of identified protein was carried out by Biolayer Interferometry. RESULTS: One and two-dimensional gel profiles revealed a differential expression pattern of proteins. 10 selected spots were identified as GMP synthase [glutamine hydrolyzing], zinc finger protein 518A, pericentriolar material 1 protein, BCLAF1 and THRAP3 family member 3, MAP/microtubule affinityregulating kinase 4, H/ACA ribonucleoprotein complex subunit 1, Melanoma-associated antigen B3 and Zinc finger protein 658B. Expression of MAP/microtubule affinity-regulating kinase 4 (MARK4) was found to be downregulated in OPCOS females as compared to controls on validation. CONCLUSION: Reduced expression of MARK4 protein in OPCOS increases the associated risk of hyperlipidemia, hyperandrogenism and metabolic syndrome, thus the protein holds strong candidature as a drug target for the syndrome.

Prenatal Ethanol-induced Effect on Cell Count of Pars Distalis of Pituitary Gland in the Rat Pups.

OBJECTIVE: To determine the effects of prenatal administration of ethanol on cell count in pars distalis of pituitary gland in the rat pups. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Anatomy, College of Physicians and Surgeons Pakistan, Regional Centre, Islamabad, Pakistan, from April 2014 to April 2015. METHODOLOGY: Sixteen female rats (Sprague Dawley) were selected by random sampling method. Rats were mated and divided into control group A and experimental group B. From gestational day 10 to 18, mother rats received intraperitoneal injection of ethanol (Group B) and normal saline (Group A). Mother rats were allowed to complete their gestation and deliver spontaneously. When pups were born, only male pups were selected for the study. They were reared till day five. At 5ᵗʰ; day, pituitary glands were taken out and histological study was done in PAS-OG stain. Cell count was made in unit area (10,000 µ2) of pars distalis of pituitary gland. Student t-test was applied for analysing the data of cell counts in unit area. RESULTS: Mean acidophil count was reduced in experimental group (70.19 ± 11.4) as compared to control group (92.65 ± 8.52, p<0.001). Mean basophil count was reduced in experimental group (27.05 ± 3.9) in comparison to control group (34.03 ± 7.9, p<0.001). Mean chromophobe count was increased (131.95 ± 10.7) in experimental group against the control group (104.62 ± 7.62, p < 0.001). CONCLUSION: Pups exposed to ethanol during gestation, showed significant reduction in acidophil and basophil count while increase in chromophobe cell count in pars distalis of pituitary gland.

Association of IL-1ß, IL-1Ra and FABP1 gene polymorphisms with the metabolic features of polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS), a highly prevalent endocrinopathy is currently being designated as chronic low grade inflammatory state. IL-1ß, IL-1Ra and FABP1 are critical mediators of inflammatory processes and are speculated to play a role in the pathogenesis of PCOS. The aim of this study was to study the association of IL-ß, IL-1Ra and FABP1 gene polymorphisms with PCOS and related metabolic features. SUBJECTS: 95 PCOS and 45 age matched healthy control subjects were enrolled in this study. METHODS: Polymorphism in genes IL-1ß, IL-1Ra and FABP1 was studied by PCR, PCR-RFLP and sequencing methods, respectively. Hormonal and lipid profiles were evaluated for all the subjects. RESULTS: Hormonal and lipid profiles showed significant differences between PCOS and control subjects. Allele and genotype frequencies of IL-1ß, IL-1Ra and FABP1 gene polymorphisms did not vary between the control and PCOS group. However, T allele of C[-511]T variant of IL-1ß, allele II in intron 2 of IL-1Ra and A allele of A/G variant of FABP1 (rs2197076) showed significant association with many metabolic features associated with PCOS. CONCLUSIONS: Polymorphism in genes encoding cytokines and proteins involved in lipid metabolism can provide insights into the genetics of the disease and may contribute to assess the associated risk of cardiovascular diseases (CVD), dyslipidemia and metabolic syndrome (MetS) associated with PCOS.

Factors affecting the uptake of cervical cancer screening among nurses in Singapore.

OBJECTIVE: To identify factors other than socioeconomic status that influence participation in cervical cancer screening. METHODS: A prospective, questionnaire-based, cross-sectional study was conducted among all female nurses working at Singapore General Hospital, Singapore, between November 1 and December 15, 2013. Characteristics assessed included age, knowledge score (0-10, on the basis of 10 true-or-false statements), perceived risk of cervical cancer, and health facility use. RESULTS: Among 2000 nurses, 1622 (81.1%) responded. The mean knowledge score was 4.70±1.76. Among 1593 nurses who reported on self-perception of risk, 97 (6.1%) reported high risk, 675 (42.4%) reported low risk, and 821 (51.5%) reported uncertainty. Of the 815 nurses reporting on their history of screening, 344 (42.2%) were screened regularly, 103 (12.6%) underwent opportunistic screening, and 368 (45.2%) had never undergone screening. The likelihood of screening was increased among women aged 35-4years, those who had recent experience of medical screening, those who had recently had a specialist consultation, or those who had recently had a consultation with a gynecologist (P<0.001 for all). Nurses undergoing regular screening reported positive effects of a doctor's recommendation, husband's encouragement, people talking about screening, and people close to the respondent undergoing screening. CONCLUSION: Advocacy and herd signaling positively influenced the cervical cancer screening rate.