Phosphorylation of insulin receptor substrates (IRS-1 and IRS-2) is attenuated following cecal ligation and puncture in mice.

BACKGROUND: Sepsis is characterized as an insulin resistant state. However, the effects of sepsis on insulin's signal transduction pathway are unknown. The molecular activity driving insulin signaling is controlled by tyrosine phosphorylation of the insulin receptor ß-subunit (IRß) and of insulin receptor substrate molecules (IRS) -1 and IRS-2. HYPOTHESIS: Cecal ligation and puncture (CLP) attenuates IRß, IRS-1 and IRS-2 phosphorylation. METHODS: IACUC-approved studies conformed to ARRIVE guidelines. CLP was performed on C57BL/6 mice; separate cohorts received intraperitoneal insulin at baseline (T0) or at 23 or 47 h. post-CLP, 1 h before mice were euthanized. We measured levels of (1) glucose and insulin in serum, (2) IRß, IRS-1 and IRS-2 in skeletal muscle and liver homogenate and (3) phospho-Irß (pIRß) in liver and skeletal muscle, phospho-IRS-1 (pIRS-1) in skeletal muscle and pIRS-2 in liver. Statistical significance was determined using ANOVA with Sidak's post-hoc correction. RESULTS: CLP did not affect the concentrations of IRß, IRS-1or IRS-2 in muscle or liver homogenate or of IRS-1 in liver. Muscle IRS-1 concentration at 48 h. post-CLP was higher than at T0. Post-CLP pIRS-1 levels in muscle and pIRß and pIRS-2 levels in liver were indistinguishable from T0 levels. At 48 h. post-CLP pIRß levels in muscle were higher than at T0. Following insulin administration, the relative abundance of pIRß in muscle and liver at T0 and at both post-CLP time points was significantly higher than abundance in untreated controls. In T0 controls, the relative abundance of pIRS-1 in muscle and of pIRS-2 in liver following insulin administration was higher than in untreated mice. However, at both post-CLP time points, the relative abundance of pIRS-1 in muscle and of pIRS-2 in liver following insulin administration was not distinguishable from the abundance in untreated mice at the same time point. Serum glucose concentration was significantly lower than T0 at 24 h., but not 48 h., post-CLP. Glucose concentration was lower following insulin administration to T0 mice but not in post-CLP animals. Serum insulin levels were significantly higher than baseline at both post-CLP time points. CONCLUSIONS: CLP impaired insulin-induced tyrosine phosphorylation of both IRS-1 in muscle and IRS-2 in liver. These findings suggest that the molecular mechanism underlying CLP-induced insulin resistance involves impaired IRS-1/IRS-2 phosphorylation.

Evaluation of Different Agitation Techniques on Smear Layer Formation and Dentine Erosions- An In Vitro Study.

OBJECTIVE: This study was performed to assess smear layer formation and erosion after final irrigation protocols with metal and non-metal tips in the apical third of root canals. METHODS: Forty mandibular premolars were instrumented with ProTaper Gold files up to F3 and embedded in a closed silicone flask system. The teeth were subsequently cleaved and 4 sequential indentations (1 to 4 mm from the apical foramen) were prepared on the buccal root canal walls to standardize sites for environmental scanning electron microscopy (ESEM) imaging. The samples were cleaned in an ultrasonic bath and observed under ESEM (controls), reassembled and divided into four groups (n=10 each) and subjected to different final irrigation protocols; XPF Group (XP-endo Finisher) and PUI Group (passive ultrasonic irrigation) with metal tips; EA Group (EndoActivator) and MDA Group (Manual dynamic agitation) with non-metal tips. The smear layer formation and dentine erosion were evaluated using ESEM. The data were analyzed with Kruskal-Wallis test with Bonferroni correction. RESULTS: In comparison to the control groups, XPF group had significantly increased smear layer formation at 1 and 2 mm (P<0.05). PUI group had significantly higher smear layer (P<0.05) formation at 3mm while EA and MDA groups did not present with significantly higher smear layer at all levels. Erosion was significantly higher (P<0.05) in MDA, XPF and PUI groups at all levels when compared to controls while EA group presented with significantly more erosion only at 2 and 3 mm. CONCLUSION: Final irrigation protocol using EA and MDA with non-metal tips did not result in significant smear layer formation. Dentine erosion was observed after all experimental irrigation protocols. (EEJ-2021-12-194).

Sleep Pattern Changes and the Level of Fatigue Reported in a Community Sample of Adults During COVID-19 Pandemic.

Purpose: The study aimed to (a) assess the sleep pattern changes and the level of fatigue among COVID positive adults (b) determine the association of sociodemographic and lifestyle factors (age, gender, marital status, occupation, income, exercise, nap, diet, and comorbidities) on sleep pattern and level of fatigue c) examine the relationship between sleep and fatigue, and between sleep problems, sleep quality and fatigue, among a community sample of COVID-19 affected adults. Methods: A non-experimental, descriptive, cross-sectional survey design was used. Participants were adults, between 18 and 63 years (n = 782), who tested positive for COVID-19 infection using RT-PCR or Antigen test, confined to home quarantine/under observation, and without any complications. Data was collected using the socio-demographic-sleep and related activity questionnaire, Fatigue Assessment Scale, and Sleep Quality Scale. Results: A majority of the participants reported either mild to moderate sleep quality problems (97.31%) and 377 of them (48.21%) reported fatigue levels. A significant association between sleep quality and fatigue with gender, and lifestyle factors such as sleep duration, food intake, napping, exercise pattern, and influence of COVID-19 on livelihood after being affected with COVID-19, and time of experiencing sleep problems after COVID-19 infection (all, p ˂ 0.01) were observed, as well as age with sleep quality. Poor sleep quality and fatigue were significantly correlated with each other, and also with sleep problems before being affected with COVID-19 (p = 0.000). Conclusions: The study has shown that COVID-19 has an effect on an individual's demographic factors and a multitude of lifestyle factors, and highlights the need for post-COVID-19 monitoring even after recovery from the disease.

Circadian rhythm disruption results in visual dysfunction.

Artificial light has been increasingly in use for the past 70 years. The aberrant light exposure and round-the-clock nature of work lead to the disruption of biological clock. Circadian rhythm disruption (CRD) contributes to multiple metabolic and neurodegenerative diseases. However, its effect on vision is not understood. Moreover, the mammalian retina possesses an autonomous clock that could be reset with light exposure. We evaluated the impact of CRD on retinal morphology, physiology, and vision after housing mice in a disruption inducing shorter light/dark cycle (L10:D10). Interestingly, the mice under L10:D10 exhibited three different entrainment behaviors; "entrained," "free-running," and "zigzagging." These behavior groups under CRD exhibited reduced visual acuity, retinal thinning, and a decrease in the number of photoreceptors. Intriguingly, the electroretinogram response was decreased only in the mice exhibiting "entrained" behavior. The retinal proteome showed distinct changes with each entrainment behavior, and there was a dysfunctional oxidative stress-antioxidant mechanism. These results demonstrate that CRD alters entrainment behavior and leads to visual dysfunction in mice. Our studies uniquely show the effect of entrainment behavior on retinal physiology. Our data have broader implications in understanding and mitigating the impact of CRD on vision and its potential role in the etiology of retinal diseases.

Dapagliflozin protects neural and vascular dysfunction of the retina in diabetes.

INTRODUCTION: Dapagliflozin, a sodium-glucose transporter inhibitor, effectively reduces blood glucose and is indicated for individuals with kidney diseases and cardiovascular disorders. In this study, we further expand the therapeutic benefit of dapagliflozin in the neural and vascular retina, with the potential to effectively manage diabetic retinopathy (DR), the most common complication of diabetes. RESEARCH DESIGN AND METHODS: Db/db mice, an animal model of type 2 diabetes, were treated with dapagliflozin orally, and the electroretinogram (ERG) response and acellular capillary numbers were assessed. Messenger RNA levels of inflammatory cytokines were studied using real-time quantitative (q)PCR. We assessed endothelial cell migration in a scratch wound assay and retinal glucose uptake using human retinal endothelial cells. RESULTS: The dapagliflozin treatment improved the ERG b-wave amplitude and decreased acellular capillary numbers. The scratch wound assay demonstrated a reduction in wound closure after dapagliflozin treatment. Retinal glucose uptake reduced after dapagliflozin treatment compared with the respective controls. CONCLUSIONS: Our studies suggest that dapagliflozin treatment effectively corrects neural and vascular dysfunction of the retina in diabetes. This effect is mediated by a decrease in inflammation and improved glycemic control. In addition, dapagliflozin exhibits decreased wound healing and glucose uptake, which could benefit the retina. Thus, dapagliflozin could be helpful in the management of DR, with multimodal therapeutic effects.

Impact of Covid-19 lockdown on the emotional health of schoolchildren in an urban Indian setting.

Background Public health measures taken to prevent the spread of the Covid-19 pandemic can potentially impact the mental health of children. We assessed the prevalence and risk factors for childhood depression during the Covid-19 lockdown. Methods After 100 days of lockdown, we sent a survey questionnaire by WhatsApp to parents of school-aged children (5-16 years) in Chennai. The Short Mood and Feelings questionnaire was used as an objective screening tool to assess depression, with a score of 12 as the cut-off. Results There were 874 responses. The prevalence of childhood depression was 13.7%. Girls were more likely to be depressed than boys; 11-16-year-olds were more likely to be depressed than 5-10-year-old children. Children who had more than 4 hours online education had a higher likelihood of depression. Those who used a cell phone for online classes had a higher likelihood of depression compared to other devices, such as tabs or laptops. Children who slept less than 8 hours a day had a higher likelihood of depression while those who either did not sleep in the afternoon or slept less than 1 hour had a lower likelihood of depression. Children who were interacting with family over 1 hour per day had a lower likelihood of depression. Conclusion Overzealous online education, lack of adequate sleep and failure to spend quality time with the family can negatively impact the mental health of children. The impact of Covid-19 on the emotional health of children should be addressed by public health policy-makers and healthcare professionals.

Retinal Phenotyping of Ferrochelatase Mutant Mice Reveals Protoporphyrin Accumulation and Reduced Neovascular Response.

Purpose: Heme depletion, through inhibition of ferrochelatase (FECH), blocks retinal and choroidal neovascularization. Both pharmacologic FECH inhibition and a partial loss-of-function Fech mutation (Fechm1Pas) are associated with decreased neovascularization. However, the ocular physiology of Fechm1Pas mice under basal conditions has not been characterized. Here, we aimed to characterize the retinal phenotype of Fechm1Pas mice. Methods: We monitored retinal vasculature at postnatal day 17, 2 months, and 6 months in Fechm1Pas homozygotes, heterozygotes, and their wild-type littermates. We characterized Fech substrate protoporphyrin (PPIX) fluorescence in the eye (excitation = 403 nm, emission = 628 nm), retinal function by electroretinogram, visual acuity by optomotor reflex, and retinal morphology by optical coherence tomography and histology. We stained vasculature using isolectin B4 and fluorescein angiography. We determined endothelial sprouting of retinal and choroidal tissue ex vivo and bioenergetics of retinal punches using a Seahorse flux analyzer. Results: Fundi, retinal vasculature, venous width, and arterial tortuosity showed no aberrations. However, VEGF-induced retinal and choroidal sprouting was decreased in Fechm1Pas mutants. Homozygous Fechm1Pas mice had pronounced buildup of PPIX in the posterior eye with no damage to visual function, bioenergetics, and integrity of retinal layers. Conclusions: Even with a buildup of PPIX in the retinal vessels in Fechm1Pas homozygotes, the vasculature remains normal. Notably, stimulus-induced ex vivo angiogenesis was decreased in Fechm1Pas mutants, consistent with reduced pathologic angiogenesis seen previously in neovascular animal models. Our findings indicate that Fechm1Pas mice are a useful model for studying the effects of heme deficiency on neovascularization due to Fech blockade.

Metformin Corrects Abnormal Circadian Rhythm and Kir4.1 Channels in Diabetes.

Purpose: Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5' adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice. Methods: Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel. Results: The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression. Conclusions: Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR.

Diabetes Alters Diurnal Rhythm of Electroretinogram in db/db Mice.

Diabetic retinopathy (DR) is the most common complications of diabetes and a leading cause of blindness in the United States. The retinal neuronal changes precede the vascular dysfunction observed in DR. The electroretinogram (ERG) determines the electrical activity of retinal neural and non-neuronal cells. The retinal ERG amplitude is reduced gradually on the progression of DR to a more severe form. Circadian rhythms play an important role in the physiological function of the body. While ERG is known to exhibit a diurnal rhythm, it is not known whether a progressive increase in the duration of diabetes affects the physiological rhythm of retinal ERG. To study this, we determined the ERG rhythm of db/db mice, an animal model of type 2 diabetes at 2, 4, and 6 months of diabetes under a regular light-dark cycle and constant dark. Our studies demonstrate that the diurnal rhythm of ERG amplitude for retinal a-wave and b-wave was altered in diabetes. The implicit time was increased in db/db mice while the oscillatory potential was reduced. Moreover, there was a progressive decline in an intrinsic rhythm of ERG upon an increase in the duration of diabetes. In conclusion, our studies provide novel insights into the pathogenic mechanism of DR by showing an altered circadian rhythm of the ERG.

Anti-N-methyl-d-aspartate Receptor Encephalitis: A Case Series and Review of the Literature.

Anti-NMDAR (N-methyl-d-aspartate receptor) encephalitis is a potentially severe form of encephalitis associated with antibodies against NR1 and NR2 subunits of the NMDAR. Anti-NMDAR encephalitis is a treatable cause of encephalitis. An underlying tumor should be actively looked for as this is also considered to be a paraneoplastic syndrome. We report two children with anti-NMDAR encephalitis with a literature review of current evidence in diagnosing and managing this rare condition. Resection of the tumor, glucocorticoids, intravenous immunoglobulin, and plasma exchange often result in improvement, usually within four weeks. Outcome corresponds with the rapidity of commencing appropriate treatment.

Impaired Thermogenesis and a Molecular Signature for Brown Adipose Tissue in Id2 Null Mice.

Inhibitor of DNA binding 2 (ID2) is a helix-loop-helix transcriptional repressor rhythmically expressed in many adult tissues. Our previous studies have demonstrated that Id2 null mice have sex-specific elevated glucose uptake in brown adipose tissue (BAT). Here we further explored the role of Id2 in the regulation of core body temperature over the circadian cycle and the impact of Id2 deficiency on genes involved in insulin signaling and adipogenesis in BAT. We discovered a reduced core body temperature in Id2-/- mice. Moreover, in Id2-/- BAT, 30 genes including Irs1, PPARs, and PGC-1s were identified as differentially expressed in a sex-specific pattern. These data provide valuable insights into the impact of Id2 deficiency on energy homeostasis of mice in a sex-specific manner.

Engaging with a Wiki related to knowledge translation: a survey of WhatisKT Wiki users.

BACKGROUND: In 2008, WhatisKT wiki was launched as a collaborative platform for knowledge translation (KT) researchers and stakeholders to debate the use and definitions of KT-related terms. The wiki has definitions for over 110 terms from disciplines including health care, information technology, education, accounting, and business. WhatisKT wiki has over 115 registered users. Approximately 73,000 unique visitors have visited the wiki since 2008. Despite annual increases in visitors and regular maintenance of the wiki, no visitors have contributed content or started a discussion. OBJECTIVE: We surveyed wiki users to gain an understanding of the perceived value of the website, reasons for not engaging in the wiki, and suggestions to facilitate collaboration and improve the usability of the wiki. METHODS: We surveyed three cohorts: KT Canada members who were previously invited to join the wiki, registered wiki members, and unregistered visitors. The first two cohorts completed a Web-based survey that included the System Usability Scale (SUS) questionnaire to assess usability; additionally 3 participants were interviewed. Unregistered wiki visitors were surveyed with polls posted on the wiki. The study received ethics approval from the McMaster University Faculty of Health Sciences Research Ethics Board. RESULTS: Twenty-three participants completed the Web-based and SUS surveys; 15 participants indicated that they would collaborate on the wiki. The mean SUS score of 67 (95% CI 56-77) indicated that the wiki could be considered for design improvements. Study participants indicated that the wiki could be improved by email notification regarding new terms, better grouping of terms, user friendly interface, and training for users interested in editing content. CONCLUSIONS: The findings from this survey will be used to enhance the design and content of WhatisKT wiki. Further feedback from participants will be used to make the wiki an ideal collaboration platform for KT researchers interested in terminology.

Ablation of the ID2 gene results in altered circadian feeding behavior, and sex-specific enhancement of insulin sensitivity and elevated glucose uptake in skeletal muscle and brown adipose tissue.

Inhibitor of DNA binding 2 (ID2) is a helix-loop-helix transcriptional repressor rhythmically expressed in many adult tissues. Our earlier studies have demonstrated a role for ID2 in the input pathway, core clock function and output pathways of the mouse circadian system. We have also reported that Id2 null (Id2-/-) mice are lean with low gonadal white adipose tissue deposits and lower lipid content in the liver. These results coincided with altered or disrupted circadian expression profiles of liver genes including those involved in lipid metabolism. In the present phenotypic study we intended to decipher, on a sex-specific basis, the role of ID2 in glucose metabolism and in the circadian regulation of activity, important components of energy balance. We find that Id2-/- mice exhibited altered daily and circadian rhythms of feeding and locomotor activity; activity profiles extended further into the late night/dark phase of the 24-hr cycle, despite mice showing reduced total locomotor activity. Also, male Id2-/- mice consumed a greater amount of food relative to body mass, and displayed less weight gain. Id2-/- females had smaller adipocytes, suggesting sexual-dimorphic programing of adipogenesis. We observed increased glucose tolerance and insulin sensitivity in male Id2-/- mice, which was exacerbated in older animals. FDG-PET analysis revealed increased glucose uptake by skeletal muscle and brown adipose tissue of male Id2-/- mice, suggesting increased glucose metabolism and thermogenesis in these tissues. Reductions in intramuscular triacylglycerol and diacylglycerol were detected in male Id2-/- mice, highlighting its possible mechanistic role in enhanced insulin sensitivity in these mice. Our findings indicate a role for ID2 as a regulator of glucose and lipid metabolism, and in the circadian control of feeding/locomotor behavior; and contribute to the understanding of the development of obesity and diabetes, particularly in shift work personnel among whom incidence of such metabolic disorders is elevated.

Neonatal testicular torsion--a lost cause?

UNLABELLED: Twenty-four neonates presented with signs of testicular ischaemia over a 13-year period. They had a mean birth weight of 3.706 kg. The right testicle was affected in 13, the left in 9 and there was bilateral torsion in 2 babies. Two babies had no twist in the cord, but the testicles were nonviable macroscopically and microscopically. Twenty-one babies had primary exploration revealing necrotic testes in all patients and they underwent orchidectomies. The other three babies had conservative management and the affected testes had atrophied on follow-up. Sixteen babies had contralateral orchidopexy. Doppler ultrasound scans were reported as normal in 2 of 13 babies who had scans. No testes were salvaged following surgery. CONCLUSION: The incidence of testicular torsion in the neonatal period was calculated as 6.1 per 100,000 live births. No testis was salvaged following surgery in our series of 24 patients. This dismal outcome underlines that immediate surgical exploration, although commonly performed, rarely saves torted testes.

Diffraction-based automated crystal centering.

A fully automated procedure for detecting and centering protein crystals in the X-ray beam of a macromolecular crystallography beamline has been developed. A cryo-loop centering routine that analyzes video images with an edge detection algorithm is first used to determine the dimensions of the loop holding the sample; then low-dose X-rays are used to record diffraction images in a grid over the edge and face plane of the loop. A three-dimensional profile of the crystal based on the number of diffraction spots in each image is constructed. The derived center of mass is then used to align the crystal to the X-ray beam. Typical samples can be accurately aligned in approximately 2-3 min. Because the procedure is based on the number of ;good' spots as determined by the program Spotfinder, the best diffracting part of the crystal is aligned to the X-ray beam.

Membrane estrogen receptors: genomic actions and post transcriptional regulation.

The primary cellular location of the nuclear estrogen receptor II (nER II) is the plasma membrane. A number of reports that have appeared in the recent past indicate that plasma membrane localized estrogen receptor alpha (ERalpha) also exists. Whether the membrane localized ERalpha represents the receptor that binds to the estrogen responsive element (ERE) remains to be known. The mechanisms that underlie the internalization of nER II (non-activated estrogen receptor, deglycosylated) have been identified to a certain extent. The question remains: is the primary location of the ERalpha also the plasma membrane? If that is the case, it will be a challenging task to identify the molecular events that underlie the plasma membrane-to-nucleus movement of ERalpha. The internalization mechanisms for the two 66kDa plasma membrane ERs, following hormone binding, appear to be distinct and without any overlaps. Interestingly, while the major gene regulatory role for ERalpha appears to be at the level of transcription, the nER II has its major functional role in post transcriptional mechanisms. The endoplasmic reticulum associated anchor protein-55 (ap55) that was recently reported from the author's laboratory needs a closer look. It is a high affinity estrogen binding protein that anchors the estrogen receptor activation factor (E-RAF) in an estrogen-mediated event. It will be interesting to examine whether ap55 bears any structural similarity with either ERalpha or ERbeta.

Possible evidence for shift work schedules in the media workers of the ant species Camponotus compressus.

The locomotor activity rhythm of the media workers of the ant species Camponotus compressus was monitored under constant conditions of the laboratory to understand the role of circadian clocks in social organization. The locomotor activity rhythm of most ants entrained to a 24h light/dark (12:12h; LD) cycle and free-ran under constant darkness (DD) with circadian periodicities. Under entrained conditions about 75% of media workers displayed nocturnal activity patterns, and the rest showed diurnal activity patterns. In free-running conditions these ants displayed three types of activity patterns (turn-around). The free-running period (tau) of the locomotor activity rhythm of some ants (10 out of 21) showed period lengthening, and those of a few (6 out of 21) showed period shortening, whereas the locomotor activity rhythm of the rest of the ants (5 out of 21) underwent large phase shifts. Interestingly, the pre-turn-around tau of those ants that showed nocturnal activity patterns during earlier LD entrainment was shorter than 24 h, which became greater than 24 h after 6-9 days of free-run in DD. On the other hand, the pre-turn-around tau of those ants, which exhibited diurnal patterns during earlier LD entrainment, was greater than 24 h, which became shorter than 24 h after 6-9 days of free-run in DD. The patterns of activity under LD cycles and the turn-around of activity patterns in DD regime suggest that these ants are shift workers in their respective colonies, and they probably use their circadian clocks for this purpose. Circadian plasticity thus appears to be a general strategy of the media workers of the ant species C. compressus to cope with the challenges arising due to their roles in the colony constantly exposed to a fluctuating environment.