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1.
Cell Mol Life Sci ; 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193609

RESUMO

Biological sex influences inflammatory response, as there is a greater incidence of acute inflammation in men and chronic inflammation in women. Here, we report that acute inflammation is attenuated by X-inactive specific transcript (Xist), a female cell-specific nuclear long noncoding RNA crucial for X-chromosome inactivation. Lipopolysaccharide-mediated acute inflammation increased Xist levels in the cytoplasm of female mouse J774A.1 macrophages and human AML193 monocytes. In both cell types, cytoplasmic Xist colocalizes with the p65 subunit of NF-κB. This interaction was associated with reduced NF-κB nuclear migration, suggesting a novel mechanism to suppress acute inflammation. Further supporting this hypothesis, expression of 5' XIST in male cells significantly reduced IL-6 and NF-κB activity. Adoptive transfer of male splenocytes expressing Xist reduced acute paw swelling in male mice indicating that Xist can have a protective anti-inflammatory effect. These findings show that XIST has functions beyond X chromosome inactivation and suggest that XIST can contribute to sex-specific differences underlying inflammatory response by attenuating acute inflammation in women.

2.
J Am Chem Soc ; 142(5): 2572-2578, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31935080

RESUMO

Single fluoride substitution in trifluoromethylarenes is an ongoing synthetic challenge that often leads to "over-reaction", where multiple fluorides are replaced. Development of this reaction would allow simple access to a vast range of difluoromethyl derivatives of current interest to pharmaceutical, agrochemistry, and materials sciences. Using a catalytic frustrated Lewis pair approach, we have developed a generic protocol that allows a single substitution of one fluoride in trifluoromethyl groups with neutral phosphine and pyridine bases. The resulting phosphonium and pyridinium salts can be further functionalized via nucleophilic substitution, photoredox coupling, and electrophilic transfer reactions allowing the generation of a vast array of difluoromethyl products.

3.
Mol Cell Biochem ; 464(1-2): 205-219, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31792650

RESUMO

Catestatin (CST) is a catecholamine release-inhibitory peptide secreted from the adrenergic neurons and the adrenal glands. It regulates the cardiovascular functions and it is associated with cardiovascular diseases. Though its mechanisms of actions are not known, there are evidences of cross-talk between the adrenergic and CST signaling. We hypothesized that CST moderates the adrenergic overdrive and studied its effects on norepinephrine-mediated hypertrophic responses in H9c2 cardiac myoblasts. CST alone regulated the expression of a number of fetal genes that are induced during hypertrophy. When cells were pre-treated CST, it blunted the modulation of those genes by norepinephrine. Norepinephrine (2 µM) treatment also increased cell size and enhanced the level of Troponin T in the sarcomere. These effects were attenuated by the treatment with CST. CST attenuated the immediate generation of ROS and the increase in glutathione peroxidase activity induced by norepinephrine treatment. Expression of fosB and AP-1 promoter-reporter constructs was used as the endpoint readout for the interaction between the CST and adrenergic signals at the gene level. It showed that CST largely attenuates the stimulatory effects of norepinephrine and other mitogenic signals through the modulation of the gene regulatory modules in a characteristic manner. Depending upon the dose, the signaling by CST appears to be disparate, and at 10-25 nM doses, it primarily moderated the signaling by the ß1/2-adrenoceptors. This study, for the first time, provides insights into the modulation of adrenergic signaling in the heart by CST.

4.
Cell Biol Int ; 44(2): 637-650, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31773824

RESUMO

SG2NA, a WD40 repeat protein of the Striatin subfamily, has four splicing and one messenger RNA edit variants. It is fast emerging as a scaffold for multimeric signaling complexes with roles in tissue development and disease. The green fluorescent protein (GFP)-tagged variants of SG2NA were ectopically expressed in NIH3T3 cells and their modulation by serum and GSK3ß-ERK signaling were monitored. The 87, 78, and 35 kDa variants showed a biphasic modulation by serum till 24 h but the 52 kDa variant remained largely unresponsive. Inhibition of phosphatases by okadaic acid increased the levels of the endogenous 78 kDa and the ectopically expressed GFP-tagged 87 and 78 kDa SG2NAs. Contrastingly, okadaic acid treatment reduced the level of GFP-tagged 35 kDa SG2NA, suggesting differential modes of their stability through phosphorylation-dephosphorylation. The inhibition of GSK3ß by LiCl showed a gradual decrease in the levels of 78 kDa. In the case of the other variants viz, GFP-tagged 35, 52, and 87 kDa, inhibition of GSK3ß caused an initial increase followed by a decrease with a subtle difference in kinetics and intensities. Similar results were also seen upon inhibition of GSK3ß by small interfering RNA. All the variants showed an increase followed by a decrease upon inhibition of extracellular-signal-regulated-kinase (ERK). These variants are localized in the plasma membrane, endoplasmic reticulum, mitochondria, and the nucleus with different propensities and no discernable subcellular distribution was seen upon stimulation by serum and the inhibition of phosphatases, GSK3ß, and ERK. Taken together, the variants of SG2NA are modulated by the kinase-phosphatase network in a similar but characteristic manner.

5.
Indian J Ophthalmol ; 68(1): 209, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31856522
6.
Indian J Ophthalmol ; 68(1): 234-236, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31856536

RESUMO

We describe a case of 34-year-old male with post penetrating keratoplasty glaucoma, post trabeculectomy with aphakia in the only seeing eye, in which a modified surgical technique of inserting Ahmed glaucoma valve (AGV) tube in vitreous cavity was done to reduce the risks associated with pars plana incision during pars plana vitrectomy (PPV). A hybrid 20-25 gauge PPV was done concurrently, implant fixed to sclera, and tube inserted through the 25 gauge sclerotomy port in supero-temporal quadrant. Visual acuity and intraocular pressure remained stable during 1-year follow-up.

7.
J Biol Chem ; 294(51): 19486-19497, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31690623

RESUMO

Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαß) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gtα) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαß in its nonactivated state. Following reconstitution of PDE6 and activated Gtα with liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα-binding sites per holoenzyme. Each Gtα interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.

8.
Indian J Ophthalmol ; 67(11): 1843-1849, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31638046

RESUMO

Purpose: To evaluate diagnostic ability of macular ganglion cell layer-inner plexiform layer (GCL-IPL) for detection of preperimetric glaucoma (PPG) and perimetric glaucoma and comparison with peripapillary RNFL. Methods: Three hundred and thirty seven eyes of 190 patients were enrolled (127 normals, 70 PPG, 140 perimetric glaucoma). Each patient underwent detailed ocular evaluation, standard automated perimetry, and spectral domain optical coherence tomography. Diagnostic abilities of GCL-IPL and RNFL parameters were determined. Data were compared using one-way analysis of variance, Pearson's Chi-square test, and area under the curve (AUC). Results: After adjusting for age, gender, and signal strength, all GCL-IPL and RNFL parameters except mean thickness and disc area differed significantly. Among GCL-IPL thicknesses, inferotemporal had the highest AUC (0.865) for classifying perimetric glaucoma from normals, inferior (0.746) for PPG from normals, and inferotemporal (0.750) for perimetric glaucoma from PPG. When using RNFL, inferior thickness had the highest AUC (0.922) in discriminating POAG from normal, while the same parameter had lower AUC (0.813) in discriminating PPG from normal. The average thickness had maximum AUC (0.775) for discriminating POAG from PPG. For discriminating perimetric glaucoma and normals, inferotemporal GCL-IPL had the highest strength (sensitivity 81.43% and specificity 77.96%), slightly lower than inferior RNFL thickness (sensitivity 87.85% and specificity 84.26%). The same parameters were sensitive in discriminating perimetric glaucoma from PPG (87.14% and 92.85%, respectively). However, their specificities were poor (56.43% both). Conclusion: RNFL had better diagnostic ability, when compared with GCL-IPL for detecting PPG and perimetric glaucoma. However, difference was small and may not be clinically relevant.

9.
Sci Rep ; 9(1): 13990, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570736

RESUMO

Memory loss is one of the most tragic symptoms of Alzheimer's disease. Our laboratory has recently demonstrated that 'i-Extract' of Ashwagandha (Withania somnifera) restores memory loss in scopolamine (SC)-induced mice. The prime target of i-Extract is obscure. We hypothesize that i-Extract may primarily target muscarinic subtype acetylcholine receptors that regulate memory processes. The present study elucidates key target(s) of i-Extract via cellular, biochemical, and molecular techniques in a relevant amnesia mouse model and primary hippocampal neuronal cultures. Wild type Swiss albino mice were fed i-Extract, and hippocampal cells from naïve mice were treated with i-Extract, followed by muscarinic antagonist (dicyclomine) and agonist (pilocarpine) treatments. We measured dendritic formation and growth by immunocytochemistry, kallikrein 8 (KLK8) mRNA by reverse transcription polymerase chain reaction (RT-PCR), and levels of KLK8 and microtubule-associated protein 2, c isoform (MAP2c) proteins by western blotting. We performed muscarinic receptor radioligand binding. i-Extract stimulated an increase in dendrite growth markers, KLK8 and MAP2. Scopolamine-mediated reduction was significantly reversed by i-Extract in mouse cerebral cortex and hippocampus. Our study identified muscarinic receptor as a key target of i-Extract, providing mechanistic evidence for its clinical application in neurodegenerative cognitive disorders.

10.
J Thorac Oncol ; 14(12): 2084-2096, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31605795

RESUMO

INTRODUCTION: CKLF like MARVEL transmembrane domain containing 6 (CMTM6) has been described as a programmed death ligand 1 (PD-L1) regulator at the protein level by modulating stability through ubiquitination. In this study, we describe the patterns of CMTM6 expression and assess its association with response to programmed cell death 1 pathway blockade in NSCLC. METHODS: We used multiplexed quantitative immunofluorescence to determine the expression of CMTM6 and PD-L1 in 438 NSCLCs represented in tissue microarrays, including in two independent retrospective cohorts of immunotherapy-treated (n = 69) and non-immunotherapy-treated (n = 258) patients and a third collection of EGFR- and KRAS-genotyped tumors (n = 111). RESULTS: Tumor and stromal CMTM6 expression was detected in approximately 70% of NSCLCs. CMTM6 expression was not associated with clinical features or EGFR/KRAS mutational status and showed a modest correlation with T-cell infiltration (R2 < 0.40). We found a significant correlation between CMTM6 and PD-L1, which was higher in the stroma (R2 = 0.51) than in tumor cells (R2 = 0.35). In our retrospective NSCLC cohort, neither CMTM6 nor PD-L1 expression alone significantly predicted immunotherapy outcomes. However, high CMTM6 and PD-L1 coexpression in the stromal and CD68 compartments (adjusted hazard ratio = 0.38, p = 0.03), but not in tumor cells (p = 0.15), was significantly associated with longer overall survival in treated patients but was not observed in the absence of immunotherapy. CONCLUSION: This study supports the mechanistic role for CMTM6 in stabilization of PD-L1 in patient tumors and suggests that high coexpression of CMTM6 and PD-L1, particularly in stromal immune cells (macrophages), might identify the greatest benefit from programmed cell death 1 axis blockade in NSCLC.

11.
Indian J Ophthalmol ; 67(10): 1560-1563, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31546480

RESUMO

Purpose: To validate the smartphone photography as a screening tool for amblyogenic conditions in children. Methods: Children between 5 to 8 years attending eye out patient department (OPD) were photographed (by an optometrist) with a smartphone to capture their pupillary red reflexes followed by clinical examination by the principal investigator (PI). The PI on the basis of clinical examination identified children with significant amblyogenic conditions and, subsequently, two ophthalmologists independently categorized the photographs on the basis of color, symmetry, and shape of the pupillary reflex into normal or abnormal. The identification of amblyogenic conditions on clinical examination was compared to that on photography. Refractive errors <3D and anisometropia <2D were excluded. Sensitivity, specificity, positive predictive value, and negative predictive value of smartphone photography screening were determined. Results: In all, 250 children were screened. Clinically 23.6% were harboring amblyogenic conditions. The mean sensitivity and specificity of screening by smartphone were 94% and 91%, respectively. Conclusion: Smartphone photography is a reliable tool for detection of amblyogenic conditions in children.

12.
Indian J Ophthalmol ; 67(10): 1657-1662, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31546503

RESUMO

Purpose: To study macular ganglion cell layer--inner plexiform layer complex (GCL + IPL) in relation to peripapillary retinal nerve fiber layer (RNFL) in glaucomatous eyes with superior or inferior hemifield defects (HD) and to study structural configuration in normal hemifield. Methods: This was an observational cross-sectional study. Data from consecutive 45 superior HD (SHD) and 50 inferior HD (IHD) eyes were analyzed. Each patient underwent detailed ocular examination, standard automated perimetry, and spectral domain optical coherence tomography (SD-OCT). After adjusting for age, gender, and signal strength, area under receiver operating characteristic curve (AUC) was calculated to determine diagnostic ability of GCL + IPL and peripapillary RNFL. Apparently normal hemifield was compared with true normal hemifield. Data were analyzed with SPSS, analysis of variance, t-test, Chi-square test, and receiver operating curve. Results: In the SHD glaucoma group, best parameters for discriminating normal eyes from glaucomatous eyes were inferotemporal GCL + IPL thickness (0.935) and inferior quadrant RNFL thickness (0.971). For IHD glaucoma, average GCL + IPL thickness (0.877) and average RNFL thickness (0.950) had best AUC values. When evaluating apparently normal hemifield in both groups, statistically significant difference was found in inferior GCL + IPL sector (0.865) and inferior quadrant RNFL (0.883) in IHD and superonasal GCL + IPL (0.725) and superior quadrant RNFL (0.842) in SHD groups. Conclusion: SD-OCT may be a useful ancillary diagnostic tool for evaluation of early macular and circumpapillary structural changes in glaucomatous eyes with localized visual field defects. Apparently normal hemifields show structural damage and should be considered in management of glaucoma.

13.
J Mol Biol ; 431(19): 3677-3689, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31394113

RESUMO

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαßγ2) is composed of a catalytic heterodimer (Pαß) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pß) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαß, Pαß-cGMP, Pαßγ2, and Pαßγ2-cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαß catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.

14.
J Neurochem ; 151(2): 139-165, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31318452

RESUMO

The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.

15.
J Affect Disord ; 256: 567-577, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31280082

RESUMO

BACKGROUND: There has been growing interest in the potential of emerging internet-delivered psychological treatments for supporting the mental health needs of university students. However, no large-scale prospective effectiveness trials examining their real-world potential have been reported. OBJECTIVE: The aim of the current study was to evaluate the acceptability and effectiveness of a brief, 5-week, internet-delivered and therapist-guided intervention for anxiety and depression, when delivered as part of routine care by a university counselling service. DESIGN: A large, prospective, single-group Phase-IV clinical trial. Students (n = 1326) engaging with the university counselling service were provided the opportunity to receive the intervention based on their preferences and identified needs. Students completed standardised measures of anxiety and depression at pre-treatment, each week of the intervention, post-treatment and 3-month follow-up. RESULTS: Over a 4 year period, 1081 students (10% of those presenting to the counselling service) participated in the intervention. Large clinical reductions in symptoms of both anxiety (% reduction = 41%; Cohen's d = 0.94) and depression (% reduction = 36%; Cohen's d = 0.81) were observed alongside high levels of acceptability. The intervention required relatively little counsellor time (M = 36.28 mins; SD = 20.56) per student, and symptom deterioration was observed in less than 5% of students. CONCLUSION: The findings of the current study are supportive of internet-delivered interventions provided as routine care to university students. Further research is needed to carefully explore whether these interventions could be used with a larger proportion of students presenting to counselling services, paying close attention to acceptability, engagement and clinical outcomes.

16.
Can J Ophthalmol ; 54(4): 473-478, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358146

RESUMO

OBJECTIVE: This study was conducted to identify factors associated with visual outcome in patients with open globe injuries (OGIs). DESIGN: Retrospective case series of OGIs presenting to a tertiary eye care institute in North India from October 2009 to December 2016. METHODS: A total of 157 patients with open globe injury have been included in the study. Multivariate analysis to ascertain the effects of different identified variables on the likelihood of poor visual outcome was done using binomial logistic regression. "Visual survival" (counting fingers or better) versus "minimal/no vision" (hand motion, light perception, and no light perception) was predicted using the classification and regression tree (CART) model. Main outcome measures were visual outcomes, risk factors, and rates of postoperative complications. RESULTS: Univariate analysis determined 9 predictors associated with poor visual outcome. Out of these, presence of relative afferent pupillary defect (RAPD), poor presenting visual acuity, presence of adnexal injuries, and location of injuries were the most significant predictors of vision loss. Absence of RAPD led to 79% chance of vision survival. Sixty-eight percent of patients with RAPD and initial visual acuity (VA) of less than 6/60 resulted in poor vision. CONCLUSION: The CART model is useful in predicting final VA based on some prognostic factors present initially.

19.
Nat Commun ; 10(1): 1207, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30872584

RESUMO

In bacteria, transcription-coupled repair of DNA lesions initiates after the Mfd protein removes RNA polymerases (RNAPs) stalled at the lesions. The bacterial RNA helicase, Rho, is a transcription termination protein that dislodges the elongation complexes. Here, we show that Rho dislodges the stalled RNAPs at DNA lesions. Strains defective in both Rho and Mfd are susceptible to DNA-damaging agents and are inefficient in repairing or propagating UV-damaged DNA. In vitro transcription assays show that Rho dissociates the stalled elongation complexes at the DNA lesions. We conclude that Rho-dependent termination recycles stalled RNAPs, which might facilitate DNA repair and other DNA-dependent processes essential for bacterial cell survival. We surmise that Rho might compete with, or augment, the Mfd function.


Assuntos
Reparo do DNA/fisiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Terminação da Transcrição Genética/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/genética , Mitomicina/farmacologia , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , RNA Bacteriano/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios Ultravioleta/efeitos adversos
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