Mutations in Human αA-Crystallin/sHSP Affect Subunit Exchange Interaction with αB-Crystallin

BACKGROUND: Mutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique. METHODOLOGY/PRINCIPAL FINDINGS: In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data. CONCLUSIONS/SIGNIFICANCE: Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract

Knowledge, attitude, and practice towards face mask use among residents of Greater Chennai Corporation, India, March 2021

BackgroundWearing a mask is one of the simplest ways to reduce the spread of COVID-19. Studies reported poor mask compliance in Greater Chennai Corporation, India. Hence, we described the knowledge, attitude, and practice regarding mask use among adults (≥18 years) in Greater Chennai Corporation, Tamil Nadu, India.MethodsWe conducted a cross-sectional survey among residents of Greater Chennai Corporation in March 2021. We estimated the sample size to be 203 per strata (slum and non-slum). We used a simple random sampling technique to select 20 locations using a digital map in the slum and non-slum areas. After reaching the location chosen, we selected 10 consecutive households and one adult (≥18 years of age) from each household. We used a validated, semi-structured questionnaire for collecting data regarding knowledge, attitudes, and practices for mask use. We estimated proportions and 95% CI for key variables and compared the variables between slums and non-slums.ResultsOf 430 participants included in the study, 51.4% were males. The mean (S.D.) age of the participants is 41.1 (14.6) years. The majority (86.7%) of the participants felt that wearing a mask helped in reducing the spread of coronavirus and the knowledge differed (p-value < 0.05) between the slum (81.4%) and non-slum (92.3%). Nearly half (46.5%) of the participants did not like being forced to wear the mask. About 63.9% of the participants reported the practice of mask use while going out which was similar across slums and non-slums.ConclusionAlthough the knowledge regarding mask use was good among the public, the attitude was unfavorable. We suggest continuous reinforcement by spreading awareness and educating the community on the appropriate use of the mask

Contribution of Cystine-Glutamate Antiporters to the Psychotomimetic Effects of Phencyclidine

Altered glutamate signaling contributes to a myriad of neural disorders, including schizophrenia. While synaptic levels are intensely studied, nonvesicular release mechanisms, including cystine–glutamate exchange, maintain high steady-state glutamate levels in the extrasynaptic space. The existence of extrasynaptic receptors, including metabotropic group II glutamate receptors (mGluR), pose nonvesicular release mechanisms as unrecognized targets capable of contributing to pathological glutamate signaling. We tested the hypothesis that activation of cystine–glutamate antiporters using the cysteine prodrug N-acetylcysteine would blunt psychotomimetic effects in the rodent phencyclidine (PCP) model of schizophrenia. First, we demonstrate that PCP elevates extracellular glutamate in the prefrontal cortex, an effect that is blocked by N-acetylcysteine pretreatment. To determine the relevance of the above finding, we assessed social interaction and found that N-acetylcysteine reverses social withdrawal produced by repeated PCP. In a separate paradigm, acute PCP resulted in working memory deficits assessed using a discrete trial t-maze task, and this effect was also reversed by N-acetylcysteine pretreatment. The capacity of N-acetylcysteine to restore working memory was blocked by infusion of the cystine–glutamate antiporter inhibitor (S)-4-carboxyphenylglycine into the prefrontal cortex or systemic administration of the group II mGluR antagonist LY341495 indicating that the effects of N-acetylcysteine requires cystine–glutamate exchange and group II mGluR activation. Finally, protein levels from postmortem tissue obtained from schizophrenic patients revealed significant changes in the level of xCT, the active subunit for cystine–glutamate exchange, in the dorsolateral prefrontal cortex. These data advance cystine–glutamate antiporters as novel targets capable of reversing the psychotomimetic effects of PCP

Cápsula endoscópica e estudos imagiológicos contrastados: diferentes perspectivas para uma imagem mais completa do intestino delgado

Small bowel evaluation is a challenging task and has been revolutionized by high-quality contrasted sectional imaging (CT enterography - CTE) and magnetic resonance enterography (MRE) as well as by small bowel capsule endoscopy (SBCE).The decision of which technique to employ during the investigation of small bowel diseases is not always simple or straightforward. Moreover, contraindications may preclude the use of these techniques in some patients, and although they are noninvasive procedures, may present with various complications. SBCE plays a crucial role in the investigation of both obscure gastrointestinal bleeding and Crohn's disease, but it is also useful for surveillance of patients with Peutz-Jeghers syndrome, while CTE is very accurate in small bowel tumours and in established Crohn's Disease, and its use in patients presenting with gastrointestinal bleeding is increasing. MRE, an expensive and not widely available technique, is essential for the study of patients with Crohn's Disease, and presents an attractive alternative to SBCE in Peutz-Jeghers syndrome surveillance.These diagnostic modalities are often not competitive but synergistic techniques. Knowing their characteristics, strengths and limitations, indications, contraindications and potential complications, as well as the adaptation to local availability and expertise, is essential to better select which procedures to perform in each patient, both safely and effectively, in order to optimize management and improve patient outcomes.A investigação do intestino delgado, previamente difícil e limitada, sofreu uma revolução com o aparecimento de técnicas imagiológicas contrastadas de elevada qualidade, como a enterografia por tomografia axial computadorizada (enteroTC) e a enterografia por ressonância magnética (enteroRM), assim como pela enteroscopia por cápsula (EC). A decisão na escolha da técnica a utilizar nas diferentes patologias do intestino delgado não é na maioria das vezes simples ou óbvia. Adicionalmente, a presença de contraindicações pode restringir o uso destas técnicas em alguns doentes, e apesar de não serem consideradas técnicas invasivas, não são isentas de riscos e complicações. A EC tem um papel crucial na investigação da hemorragia digestiva de causa obscura e da doença de Crohn, mas tem-se revestido também de utilidade na vigilância de doentes com síndrome de Peutz-Jeghers; a enteroTC revelou uma elevada capacidade diagnóstica para neoplasias do intestino delgado e na doença de Crohn estabelecida, e a sua utilização na hemorragia digestiva de causa obscura tem vindo a expandir. A enteroRM, apesar de dispendiosa e de disponibilidade limitada, tem uma elevada eficácia no estudo da doença de Crohn, e é uma alternativa válida à EC no síndrome de Peutz-Jeghers. Estas técnicas diagnósticas são frequentemente singergísticas e complementares, ao invés de competitivas. O reconhecimento das suas características, das suas capacidades e limitações, assim como das indicações, contraindicações e potenciais complicações, e aliado à adaptação à disponibilidade e competências locais, é essencial na correcta escolha de procedimentos seguros e eficazes para cada doente, de forma a optimizar a abordagem e o prognóstico.(undefined)info:eu-repo/semantics/publishedVersio

Multiple Aggregates and Aggresomes of C-Terminal Truncated Human αA-Crystallins in Mammalian Cells and Protection by αB-Crystallin

Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM).YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes.αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes

Congenital cataract causing mutants of αA-crystallin/sHSP form aggregates and aggresomes degraded through ubiquitin-proteasome pathway.

BACKGROUND: Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy. METHODOLOGY/PRINCIPAL FINDINGS: YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control. CONCLUSIONS/SIGNIFICANCE: Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells

FoxO3a Serves as a Biomarker of Oxidative Stress in Human Lens Epithelial Cells under Conditions of Hyperglycemia.

BACKGROUNDForkhead box 'O' transcription factors (FoxOs) are implicated in the pathogenesis of type2 diabetes and other metabolic diseases. Abnormal activity of FoxOs was reported in the glucose and insulin metabolism. Expression of FoxO proteins was reported in ocular tissues; however their function under hyperglycemic conditions was not examined.METHODSHuman lens epithelial cell line was used to study the function of FoxO proteins. Immunofluorescence, flow cytometry and Western blotting were employed to detect the FoxO proteins under the conditions of hyperglycemia.RESULTSIn this study we examined the role of FoxO3a in hyperglycemia-induced oxidative stress in human lens epithelial cells. FoxO3a protein expression was elevated in a dose- and time-dependent fashion after high glucose treatment. Anti-oxidant defense mechanisms of the lens epithelial cells were diminished as evidenced from loss of mitochondrial membrane integrity and lowered MnSOD after 72 h treatment with high glucose. Taken together, FoxO3a acts as a sensitive indicator of oxidative stress and cell homeostasis in human lens epithelial cells during diabetic conditions.CONCLUSIONFoxO3a is an early stress response protein to glucose toxicity in diabetic conditions

Overexpression of αA-Crystallin mutants showed increased ubiquitination.

<p><i>A: Western blot analysis:</i> After 48 h transfection, cells were lysed and 5 µg of total protein was subjected to immunoblot probed with an anti-ubiquitin (FK2) monoclonal antibody. The pattern of polyubiquitinated species were dramatically increased in the mutants, R21W, R116C and R116H compared to other mutants and wild-type. * indicates a non-specific band. The same blot was stripped and re-probed for αA-crystallin and β-actin for loading controls. The blot shown here is a representative blot of three independent experiments. <i>B: Quantitative data for the western blot:</i> Using NIH Image J software, densitometric measurements were normalized against β-actin. The mutants, R21L, R21W, R116C and R116H showed increased ubiquitination as obtained in three independent experiments and plotted. The results were expressed as mean ± SD. The p value for αA-wt Vs αA-R12C is not significant, for αA-wt Vs αA-R21L is < 0.05; for αA-wt Vs αA-R21W is < 0.01; for αA-wt Vs αA-R49C is not significant; for αA-wt Vs αA-R54C is not significant; for αA-wt Vs αA-R116C is < 0.003 and for αA-wt Vs αA-R116H is < 0.001.</p

YFP-αA-wt and the mutant constructs, R12C, R21L, R21W, R49C, R54C, R116C and R116H were individually expressed in HeLa cells.

<p><i>A: LSM Images were captured after 48 hours transfection.</i> HeLa cells were individually transfected with 2 µg of YFP-tagged αA-wt and mutants of αA-crystallin. A homogenous expression of αA-crystallin was evident in αA-wt transfected cells. Cytoplasmic aggregates were evident in αA- crystallin mutants transfected cells. The YFP signal was excited at 514 nm and the images were collected by BP 530-600 nm filter. The images represent one of the four similar images obtained in three independent experiments. <i>B: Graph represents per cent of cells with aggregates.</i> The results obtained after 48 hours transfection for the individually expressed αA-wt or its mutants in HeLa cells. Cells containing aggregates were counted in 10 random fields each field with 30 cells. The mutant, R116C showed a high per cent (∼47) of cells having aggregates and the mutant R21L showed least per cent (∼10) of cells containing aggregates. The results were presented as means ± SD obtained in three independent experiments. All the mutants were statistically significant, p < 0.01.</p