Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

TREM2 on microglia cell surface binds to and forms functional binary complexes with heparan sulfate modified with 6-O-sulfation and iduronic acid.

The triggering receptor expressed on myeloid cells-2 (TREM2), a pivotal innate immune receptor, orchestrates functions such as inflammatory responses, phagocytosis, cell survival, and neuroprotection. TREM2 variants R47H and R62H have been associated with Alzheimer's disease, yet the underlying mechanisms remain elusive. Our previous research established that TREM2 binds to heparan sulfate (HS) and variants R47H and R62H exhibit reduced affinity for HS. Building upon this groundwork, our current study delves into the interplay between TREM2 and HS and its impact on microglial function. We confirm TREM2's binding to cell surface HS and demonstrate that TREM2 interacts with HS, forming HS-TREM2 binary complexes on microglia cell surfaces. Employing various biochemical techniques, including surface plasmon resonance, low molecular weight HS microarray screening, and serial HS mutant cell surface binding assays, we demonstrate TREM2's robust affinity for HS, and the effective binding requires a minimum HS size of approximately 10 saccharide units. Notably, TREM2 selectively binds specific HS structures, with 6-O-sulfation and, to a lesser extent, the iduronic acid residue playing crucial roles. N-sulfation and 2-O-sulfation are dispensable for this interaction. Furthermore, we reveal that 6-O-sulfation is essential for HS-TREM2 ternary complex formation on the microglial cell surface, and HS and its 6-O-sulfation are necessary for TREM2-mediated ApoE3 uptake in microglia. By delineating the interaction between HS and TREM2 on the microglial cell surface and demonstrating its role in facilitating TREM2-mediated ApoE uptake by microglia, our findings provide valuable insights that can inform targeted interventions for modulating microglial functions in Alzheimer's disease.

Detection of antibodies against the huntingtin protein in human plasma.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder resulting from a CAG expansion in the huntingtin (HTT) gene, which leads to the production and accumulation of mutant huntingtin (mHTT). While primarily considered a disorder of the central nervous system, multiple changes have been described to occur throughout the body, including activation of the immune system. In other neurodegenerative disorders, activation of the immune system has been shown to include the production of antibodies against disease-associated pathological proteins. However, the existence of mHTT-targeted antibodies has never been reported. In this study, we assessed the presence and titer of antibodies recognizing HTT/mHTT in patients with HD (n = 66) and age- and gender-matched healthy controls (n = 66) using a combination of Western blotting and ELISA. Together, these analyses revealed that antibodies capable of recognizing HTT/mHTT were detectable in the plasma samples of all participants, including healthy controls. When antibody levels were monitored at different disease stages, it was observed that antibodies against full-length mHTT were highest in patients with severe disease while antibodies against HTTExon1 were elevated in patients with mild disease. Combined, these results suggest that antibodies detecting different forms of mHTT peak at different disease stages.

Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation.

Glioblastomas (GBs) are the most aggressive and common primary malignant brain tumors. Steroid hormone progesterone (P4) and its neuroactive metabolites, such as allopregnanolone (3α-THP) are synthesized by neural, glial, and malignant GB cells. P4 promotes cellular proliferation, migration, and invasion of human GB cells at physiological concentrations. It has been reported that 3α-THP promotes GB cell proliferation. Here we investigated the effects of 3α-THP on GB cell migration and invasion, the participation of the enzymes involved in its metabolism (AKR1C1-4), and the role of the c-Src kinase in 3α-THP effects in GBs. 3α-THP 100 nM promoted migration and invasion of U251, U87, and LN229 human-derived GB cell lines. We observed that U251, LN229, and T98G cell lines exhibited a higher protein content of AKR1C1-4 than normal human astrocytes. AKR1C1-4 silencing did not modify 3α-THP effects on migration and invasion. 3α-THP activated c-Src protein at 10 min (U251 cells) and 15 min (U87 and LN229 cells). Interestingly, the pharmacological inhibition of c-Src decreases the promoting effects of 3α-THP on cell migration and invasion. Together, these data indicate that 3α-THP promotes GB migration and invasion through c-Src activation.

Effect of support groups on caregiver's quality of life.

The objective of this study was to evaluate the effect that participating in support groups for caregivers has on the quality of life and psychotropic drug use of family caregivers of adults with limitations in activities of daily living. A controlled quasi-experimental longitudinal design was used with 134 caregivers (64 in the experimental group and 70 in the control group). The outcomes were health-related quality of life (EuroQol 5D3L test) and psychotropic drug use (no/yes). The analyses were performed using SPSS and R statistical software. An interaction was observed between the condition and the level of limitations in activities of daily living of the care receiver, having an effect on the caregiver's psychotropic drug use (p = 0.003), with this use being lower among caregivers who attend support groups when their relatives present fewer limitations in activities of daily living. Moreover, the quality of life was higher in the post-test in the experimental group (B = 8.66, p = 0.015). In conclusion, support groups could improve the caregiver's quality of life and decrease psychotropic drug use when the care receiver has low limitations in activities of daily living.

El objetivo de este estudio es evaluar el efecto que tiene la participación en grupos de apoyo para cuidadores en la calidad de vida y el consumo de fármacos psicotrópicos de cuidadores familiares de adultos con limitaciones en las actividades de la vida diaria. Se usó un diseño longitudinal cuasiexperimental controlado con 134 cuidadores (64 en el grupo experimental y 70 en el grupo de referencia). Los criterios de evaluación fueron la calidad de vida relacionada con la salud (test EuroQol 5D3L) y el consumo de fármacos psicotrópicos (sí / no). Los análisis se realizaron usando los programas estadísticos SPSS y R. Se observó una interacción entre la enfermedad y el nivel de limitaciones en las actividades de la vida diaria del receptor del cuidado y el efecto que tuvo en el consumo de fármacos psicotrópicos del cuidador (p=0.003). Este consumo fue menor entre los cuidadores que asisten a grupos de apoyo cuando sus familiares presentan menos limitaciones en las actividades de la vida diaria. Además, la calidad de vida fue mayor en el grupo experimental después del test (B=8.66, p=0.015). En resumen, los grupos de apoyo podrían mejorar la calidad de vida del cuidador y disminuir el consumo de fármacos psicotrópicos cuando el receptor del cuidado tiene pocas limitaciones en las actividades de la vida diaria.

Design and Implementation of the Puerto Rico Observational Study of Psychosocial, Environmental, and Chronic Disease Trends (PROSPECT).

The Puerto Rico Observational Study of Psychosocial, Environmental, and Chronic Disease Trends (PROSPECT) is a prospective cohort study in Puerto Rico (PR) aiming to identify trends and longitudinal associations in risk factors for cardiovascular disease (CVD). In 2019, PROSPECT investigators started recruiting a sample of 2,000 adults aged 30-75 years in PR using multistage probabilistic sampling of households and community approaches. Culturally sensitive trained research assistants assess participants, at baseline and at 2-year follow-up, in private rooms at a network of partner clinics. The study collects comprehensive data on demographic factors, socioeconomic and environmental factors, medical history, health conditions, lifestyle behaviors, psychosocial status, and biomarkers of CVD and stress. PROSPECT will estimate the prevalence and incidence of psychosocial, lifestyle, and biological CVD risk factors, describe variations in risk factors by urbanicity (urban areas vs. rural areas) and exposure (before and after) to natural disasters, and determine predictors of longitudinal changes in CVD risk factors. The study has 4 coordinated operational strategies: 1) research productivity (including synergy with existing epidemiologic cohorts of Hispanics/Latinos for comparison); 2) research infrastructure (biorepository, ancillary studies, and clinical research network); 3) capacity-building, education, and training; and 4) community outreach, dissemination, and policy. PROSPECT will inform public health priorities to help reduce CVD in PR.

Headache during SARS-CoV-2 infection as an early symptom associated with a more benign course of disease: a case-control study.

BACKGROUND AND PURPOSE: Headache is an important manifestation during SARS-CoV-2 infection. In this study, the aim was to identify factors associated with headache in COVID-19 and headache characteristics. METHODS: This case-control study includes COVID-19 hospitalized patients with pneumonia during March 2020. Controls comprise COVID-19 patients without headache and the cases are COVID-19 patients with headache. Demographic, clinical and laboratory data were obtained from the medical records. Headache characteristics were evaluated by semi-structured telephonic interview after discharge. RESULTS: Of a total of 379 COVID-19 patients, 48 (13%) developed headache. Amongst these, 30 (62%) were men and the median age was 57.9 (47-73) years. Headache was associated with younger age, fewer comorbidities and reduced mortality, as well as with low levels of C-reactive protein, mild acute respiratory distress syndrome and oropharyngeal symptoms. A logistic multiple regression model revealed that headache was directly associated with D-dimer and creatinine levels, the use of high flow nasal cannula and arthromyalgia, whilst urea levels, beta-lactamic treatment and hypertension were negatively associated with headache. COVID-19-associated headache characteristics were available for 23/48 (48%) patients. Headache was the onset symptom in 8/20 (40%) patients, of mild or moderate intensity in 17/20 (85%) patients, with oppressive characteristics in 17/18 (94%) and of holocranial 8/19 (42%) or temporal 7/19 (37%) localization. CONCLUSIONS: Our results show that headache is associated with a more benign SARS-CoV-2 infection. COVID-19-associated headache appears as an early symptom and as a novel headache with characteristics of headache attributed to systemic viral infection. Further research addressing the underlying mechanisms to confirm these findings is warranted.

Delays in diagnosing pulmonary tuberculosis within a context of medium incidence, Medellín, Colombia, 2017: an operational research.

BACKGROUND: Delay in tuberculosis (TB) diagnosis is one of the first obstacles for controlling the disease. Delays generate greater deterioration of the health of the patients and increase the possibilities of transmission and infection at home and in the community. The aim of the study was to identify profiles and individual variables associated with patient delays and health care system delays in patients with pulmonary tuberculosis (PTB) in Medellín, Colombia, a city that notifies 1400 new cases per year. METHODS: A retrospective cohort study in adults with PTB was conducted from May to September of 2017. Sociodemographic, health care-seeking behaviour, and clinical variables were measured. The outcomes were patient delay and health care system delay. The data were obtained from records of the local TB program, and a questionnaire was applied by the health care team that performs routine field visits. Simple correspondence analysis was used to identify groups (profiles), and their characteristics. Cox's proportional hazards model was carried out to identify the variables associated with the delays. RESULTS: The study included 183 patients. The total delay median was 101 days (IQR: 64-163). Patient delay was of 35 days (IQR: 14-84), the profile with greater delay belonged to consumers of psychoactive substances. The health care system delay was of 27 days (IQR: 7-89), the attributes of the profile with greater delay were being a female, having more than two consultations before the diagnosis, and having prescribed antibiotics. Basic-medium educational level [HRa = 0.69; 95% CI (0.49-0.97)] and having a TB home contact [HRa = 0.68; 95% CI (0.48-0.96)] were associated with greater patient delay. Having negative acid-fast bacilli (AFB) smear [HRa = 0.64; 95% CI (0.45-0.92)] and more than two consultations before the diagnosis [HRa = 0.33; 95% CI (0.22-0.49)] was associated with greater health care system delay. CONCLUSIONS: Data from epidemiological surveillance allowed locating risk groups with delays in TB diagnosis which requires the prioritisation of the local TB control program to promote early detection and prevention of adverse outcomes.

Patients with chronic mild or moderate traumatic brain injury have abnormal brain enlargement.

Introduction: Much less is known about brain volume abnormalities in patients with chronic mild or moderate traumatic brain injury (TBI) compared with patients with more severe injury. Commercially available software methods including NeuroQuant® are being used increasingly to assess MRI brain volume in patients with TBI.Methods: 50 patients with mild or moderate TBI were compared to the NeuroQuant® normal control database (n = thousands) with respect to MRI brain volume.Results: The patients had many areas of abnormal enlargement and fewer areas of atrophy, including abnormally small cerebral white matter (CWM) limited to the first 10 months after injury. Examination of correlations within the patient group between CWM volume and volumes of the abnormally enlarged regions showed multiple significant negative correlations, indicating that CWM atrophy correlated with enlargement of the other regions.Discussion: The finding of many regions of abnormal brain enlargement was relatively new, although a couple of previous studies of patients with mild TBI found similar but more limited findings. The cause of the abnormal enlargement was unknown, but possibilities included: (1) hyperactivity and hypertrophy; or (2) chronic neuro-inflammation and edema.Abbreviations: ADNI: Alzheimer's Disease Neuroimaging Initiative; CWM: cerebral white matter; GM: cerebral cortical gray matter; ICC: intraclass correlations coefficient; IFT: infratentorial; MRI: magnetic resonance imaging; mTBI: mild TBI; NQ: NeuroQuant®; SCN: subcortical nuclei; t0: time of injury; t1: time of first NeuroQuanted MRI scan after injury; t2: time of second NeuroQuanted MRI scan after injury; TBI: traumatic brain injury; VBR: ventricle-to-brain ratio; WBP: whole-brain parenchyma.

Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.

Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires' Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases) when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM) to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the 'open' conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.

Enhancement of corneal epithelium cell survival, proliferation and migration by red light: Relevance to corneal wound healing.

The aim of the present study was to analyse how short wave blue and long wave red light differentially affect corneal epithelial (HCE-2) cells in culture. The corneal epithelium in situ is exposed to more blue light than in the past because of Light Emitting Diodes (LEDs) used for indoor lighting and computer, television and phone screens. Compared with cultures maintained in the dark, low intensity blue light, such as that emitted from computer screens, reduced the proliferation rate of HCE-2 cells and caused cell death at greater intensities in a dose-dependent manner. In contrast, red light at high intensity slightly enhanced the proliferation rates of HCE-2 cells and importantly blunted the negative influence of blue light on cell survival when delivered after the insult. The toxic influence of blue light on HCE-2 cells involves mitochondrial dysfunction and the activation of AIF, p38-MAPK and HO-1. Importantly, red light blocks the effects caused by blue light and enhances mitochondrial function when delivered independently. The mechanism of action of red light is to directly stimulate mitochondrial function, suggested by staining with JC-1, which results in the activation of multiple biochemical mechanisms and the ability to blunt a variety of death pathways. As a consequence, even sodium azide-induced toxicity to HCE-2 cells in culture is blunted by red light. We interpret our studies on HCE-2 cell cultures to suggest that red light can be used prophylactically to protect the corneal epithelial in situ and is also able to counteract a variety of potential environmental insults to the tissue that includes blue light. This might be of particular significance when the cornea is already affected as, for example, in dry eye.

Changes in maxillary incisor inclination and position after traction of unilateral vs bilateral maxillary impacted canines in nonextraction treatment: A cone-beam computed tomography study.

INTRODUCTION: The aim of this study was to compare the inclination and position changes of maxillary incisors after traction of unilateral vs bilateral maxillary impacted canines in nonextraction orthodontic treatment. METHODS: This longitudinal and retrospective study evaluated 24 patients with impacted maxillary canines; 12 with unilateral impaction and 12 with bilateral impaction. All subjects had Angle Class I malocclusion and were orthodontically treated with a standardized traction protocol that did not include premolar extractions. Cone-beam computed tomographies were obtained before and after canine traction and the inclination and position of both maxillary central incisors were measured. Furthermore, dental arch, skeletal, and canine impaction characteristics were evaluated. Paired and independent t tests were used for intra and inter group comparisons, respectively. Multiple linear regressions were also used. RESULTS: After canine traction, a significant incisor labial inclination was observed in the bilateral group (10.41° right side, P = 0.008 and 12.79° left side, P = 0.001), while in the unilateral group, this was observed only on the nonaffected side (6.67°, P = 0.008). Furthermore, a significant protrusion of incisors was observed in the bilateral group (2.66 mm right side, P = 0.006, and 3.15 mm left side, P = 0.001) and in the nonaffected side of the unilateral group (1.74 mm, P = 0.022). Intergroup comparisons showed greater values of incisor labial inclination for the bilateral group when compared with the unilateral group, independently of the sides. CONCLUSIONS: Traction of maxillary impacted canines, in nonextraction treatment, produces greater labial inclination of maxillary incisors in bilateral cases and similar protrusion in both unilateral and bilateral cases. Unilateral impaction cases showed significant incisor inclination and protrusion in the nonaffected side. These treatment effects should be considered by clinicians.

Agreement between jaw-muscle activity measurement with portable single-channel electromyography and polysomnography in children.

BACKGROUND: Polysomnography (PSG) is the gold standard for sleep bruxism (SB) assessment. High economical costs, complex technical equipment, and unfamiliar laboratory setting limit its use in children. AIM: To determine the night-to-night variability of electromyography (EMG) episodes during a five-night recording with the GrindCare Measure (GCM), and the agreement in the assessment of masticatory muscle activity (MMA) between GCM and PSG in children. DESIGN: Forty-seven children from clinics of Universidad CES participated. Each participant was assessed with GCM for five consecutive nights. The last night, children underwent a single-night PSG study, together with the GCM. Spearman correlation coefficients were used to analyze data. RESULTS: The frequency of SB occurrence was 'sometimes' in 12 (25.5%) and 'usually' in 19 (40.4%) children. Simultaneous measurements with GCM and PSG obtained during the fifth night of measurement were not significantly correlated. Correlation between GCM total EMG episodes and EMG episodes/h and PSG total SB episodes, SB episodes/h, total bursts and burst/h measured with PSG was also not significant. CONCLUSION: EMG measurement with GCM was not accurate to detect PSG/SB in children. There was not advantage of multiple assessment for five nights with GCM, reducing the impact of night-to-night EMG episodes' variability on the GCM/PSG correlation.

[Non reported spa types in our country in Staphylococcus aureus from adult patients of a school hospital, Santa Fe - Argentina]. / Tipos de spa no reportados en nuestro país en Staphylococcus aureus de pacientes adultos de un hospital escuela, Santa Fe, Argentina.

Staphylococcus aureus is a pathogen associated a different kind of infection. Molecular markers are useful tools to study microbial epidemiology. Twenty two methicillin-resistant S. aureus (MRSA) and 23 methicillin-susceptible S. aureus (MSSA) were studied by mecA gene, SCCmec cassette, Panton Valentine leucocidin (PVL) and spa polymorphism. The clinical data patients were analyzed. MSSA was prevalent in samples different from skin and soft tissue (SST) and in hospitalized patients, whereas MRSA in SST. SCCmec type IV was predominant, followed by type I. Low presence of PVL was found. In MRSA 11 different types of spa were detected, t019 was the most frequent and associated with outpatient, 17 types were found in MSSA and t189 was prevalent. spa t002 was present in MSSA and MRSA. We found 11 types of spa not reported in our country.

Hyperinsulinism-Causing Mutations Cause Multiple Molecular Defects in SUR1 NBD1.

The sulfonylurea receptor 1 (SUR1) protein forms the regulatory subunit in ATP sensitive K+ (KATP) channels in the pancreas. SUR proteins are members of the ATP binding cassette (ABC) superfamily of proteins. Binding and hydrolysis of MgATP at the SUR nucleotide binding domains (NBDs) lead to channel opening. Pancreatic KATP channels play an important role in insulin secretion. SUR1 mutations that result in increased levels of channel opening ultimately inhibit insulin secretion and lead to neonatal diabetes. In contrast, SUR1 mutations that disrupt trafficking and/or decrease gating of KATP channels cause congenital hyperinsulinism, where oversecretion of insulin occurs even in the presence of low glucose levels. Here, we present data on the effects of specific congenital hyperinsulinism-causing mutations (G716V, R842G, and K890T) located in different regions of the first nucleotide binding domain (NBD1). Nuclear magnetic resonance (NMR) and fluorescence data indicate that the K890T mutation affects residues throughout NBD1, including residues that bind MgATP, NBD2, and coupling helices. The mutations also decrease the MgATP binding affinity of NBD1. Size exclusion and NMR data indicate that the G716V and R842G mutations cause aggregation of NBD1 in vitro, possibly because of destabilization of the domain. These data describe structural characterization of SUR1 NBD1 and shed light on the underlying molecular basis of mutations that cause congenital hyperinsulinism.

Near infra-red light attenuates corneal endothelial cell dysfunction in situ and in vitro.

In the present study mechanical damage to the corneal endothelium was induced by elevation of intraocular pressure (IOP, 140 mmHg, 60 min) to one eye of rats, delivered either in complete darkness or in the presence of red light (16.5 W/m2, 3000 lx, 625-635 nm). IOP raised in the dark revealed the endothelium to be damaged as staining for the gap junction protein ZO-1 was irregular in appearance with some cells displaced in position or lost to leave gaps or holes. This damage was clearly attenuated when red light was focused through the pupil during the insult of raised IOP. Moreover, staining of endothelium with JC-1 dye showed mitochondria to be activated by both elevated IOP and red light but the activation of mitochondria persisted longer for red light. We interpret this finding to suggest that raised IOP causes apoptosis of endothelial cells and that their mitochondria are activated in the initial stages of the process. In contrast, red light activates mitochondria to induce a protective mechanism to counteract the negative influence of raised IOP on endothelial cells. Evidence is provided to support this notion by the finding that red light stimulates mitochondrial cytochrome oxidase IV (COX IV). Moreover, mitochondria in corneal endothelial cell cultures are activated by red light, revealed by staining with JC-1, that results in an increased rate of proliferation and are also able to counteract toxic insults (sodium azide or cobalt chloride) to the cultures. The present studies therefore show that a non-toxic level of red light attenuates damage to the corneal endothelium both in situ and in vitro through action on COX IV located in mitochondria that results in an enhancement of a cell's survival mechanisms. The study provides proof of principle for the non-invasive use of red-light therapy to attenuate any dysfunctions associated with the corneal endothelium and so preserve maximum visual acuity.

Diagnostic accuracy of the use of parental-reported sleep bruxism in a polysomnographic study in children.

BACKGROUND: Polysomnography (PSG) is the gold standard for sleep bruxism (SB) diagnosis. PSG/SB children's criteria are not available; thus, parental-report SB is widely used. AIM: Assessing the diagnostic accuracy of parental report of sleep tooth grinding (STG) with a PSG/SB diagnosis in children, adopting adult criteria. DESIGN: Thirty-seven children from clinics of Universidad CES were included. Parents filled the Children's Sleep Habits Questionnaire (CSHQ) assessing the single-observation report - CSHQ - of STG with a No/Yes answer and five ordinal answers. A 5-day diary reporting the presence/absence of STG (multiple-observation report) was also completed. Each child underwent a single-night PSG study. Sensitivity, specificity, positive predictive value, negative predictive value, accuracy, likelihood ratios, Spearman correlation coefficient, and Cohen's kappa coefficient were used to analyze data. RESULTS: Single observation, using No/Yes answer, showed acceptable specificity and NPV, while low PPV and sensitivity. Accuracy and likelihood ratios were low. When using the five ordinal answers, weak correlation and fair agreement (r = 0.34 and κ = 0.40) with PSG/SB adult criteria were found. Multiple-observation evaluation of STG presented moderate correlation and agreement (r = 0.50 and κ = 0.48). CONCLUSIONS: Although multiple-observation report achieved better agreement than single-observation report, our results failed supporting the validity of report strategies for the diagnosis of SB in children, as an equivalent of PSG/SB adult criteria.

Phosphorylation-dependent changes in nucleotide binding, conformation, and dynamics of the first nucleotide binding domain (NBD1) of the sulfonylurea receptor 2B (SUR2B).

The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels.

Blue Light Action on Mitochondria Leads to Cell Death by Necroptosis.

Blue light impinging on the many mitochondria associated with retinal ganglion cells (RGCs) in situ has the potential of eliciting necroptosis through an action on RIP1/RIP3 to stimulate RGC death in diseases like glaucoma and diabetic retinopathy. Cells in culture die when exposed to blue light. The death process is mitochondria-dependent and is known to involve a decrease in the production of ATP, a generation of ROS, the activation of poly-(ADP-ribose) polymerase, the stimulation of apoptosis-inducing factor (AIF) as well as the up-regulation of heme-oxygenase-1 (HO-1). Our present results show that blue light-induced activation of AIF is not directly linked with the stimulation of RIP1/RIP3. Down-regulation of RIP1/RIP3 did not influence AIF. AIF activation therefore appears to enhance the rate of necroptosis by a direct action on DNA breakdown, the end stage of necroptosis. This implies that silencing of AIF mRNA may provide a degree of protection to blue light insult. Also, necrostatin-1 attenuated an increased turnover of HO-1 mRNA caused by blue light to suggest an indirect inhibition of necroptosis, caused by the action of necrostatin-1 on RIP1/RIP3 to reduce oxidative stress. This is supported by the finding that gene silencing of RIP1 and RIP3 has no effect on HO-1. We therefore conclude that inhibitors of RIP kinase might be more specific than necrostatin-1 as a neuroprotective agent to blunt solely necroptosis caused by blue light.