Hemin-Induced Transient Senescence Via DNA Damage Response: A Neuroprotective Mechanism Against Ferroptosis in Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) poses acute fatality and long-term neurological risks due to hemin and iron accumulation from hemoglobin breakdown. Our observation that hemin induces DNA double-strand breaks (DSBs), prompting a senescence-like phenotype in neurons, necessitating deeper exploration of cellular responses. Using experimental ICH models and human ICH patient tissue, we elucidate hemin-mediated DNA damage response (DDR) inducing transient senescence and delayed expression of heme oxygenase (HO-1). HO-1 co-localizes with senescence-associated ß-Galactosidase (SA-ß-Gal) in ICH patient tissues, emphasizing clinical relevance of inducible HO-1 expression in senescent cells. We reveal a reversible senescence state protective against acute cell death by hemin, while repeat exposure leads to long-lasting senescence. Inhibiting early senescence expression increases cell death, supporting the protective role of senescence against hemin toxicity. Hemin-induced senescence is attenuated by a pleiotropic carbon nanoparticle that is a catalytic mimic of superoxide dismutase, but this treatment increased lipid peroxidation, consistent with ferroptosis from hemin breakdown released iron. When coupled with iron chelator deferoxamine (DEF), the nanoparticle reduces hemin-induced senescence and upregulates factors protecting against ferroptosis. Our study suggests transient senescence induced by DDR as an early potential neuroprotective mechanism in ICH, but the risk or iron-related toxicity supports a multi-pronged therapeutic approach.

Challenges in diagnosis of calcaneal fractures: an examination using the WIDI SIM platform.

INTRODUCTION: The calcaneus is the most commonly fractured tarsal bone. Diagnosis is often challenging due to subtle radiographic changes and requires timely identification to prevent complications, including subtalar arthritis, neurovascular injury, malunion, osteomyelitis, and compartment syndrome. Treatment varies based on fracture type, with non-surgical methods for non-displaced stress fractures and surgical interventions for displaced or intra-articular fractures. METHODS: This study utilized the Wisdom in Diagnostic Imaging Simulation (WIDI SIM) platform, an emergency imaging simulation designed to assess radiology resident preparedness for independent call. During an 8-hour simulation, residents were tested on 65 cases across various imaging modalities of varying complexity, including normal studies. A single, unique case of calcaneal fracture was included within the simulation in four separate years of testing. Cases were assessed using a standardized grading rubric by subspecialty radiology faculty, with errors subsequently classified by type. RESULTS: A total of 1279 residents were tested in five separate years on the findings of calcaneal fractures of 5 different patients. Analysis revealed a consistent pattern of missed diagnoses across all training years, primarily attributed to observational errors. There was limited improvement with training progression as all training years exhibited similar average performance levels. CONCLUSIONS: Calcaneal fractures pose a diagnostic challenge due to their frequent subtle radiographic findings, especially in stress fractures. Simulation-based evaluations using WIDI SIM highlighted challenges in radiology residents' proficiency in diagnosing calcaneal fractures. Addressing these challenges through targeted education and exposure to diverse cases is essential to improve diagnostic accuracy and reduce complications with calcaneal fractures.

Incidental multifocal granular cell tumor in the setting of chronic gastritis discovered following gastric sleeve operation: a case report and brief review of the literature.

Granular cell tumors are uncommon neoplasms of Schwannian origin that can present nearly anywhere in the body, but are most commonly seen in the head and neck region. While the majority of these tumors are benign, a subset can behave aggressively and even have metastatic potential. We are reporting a case of a 35-year-old female with a history morbid obesity presenting for bariatric surgery (sleeve gastrectomy). Upon receiving the specimen in pathology, nodular vaguely defined lesions were identified in the gastric wall. Representative sections were submitted for microscopic examination, which revealed the incidental finding of a multifocal granular cell tumor with a background of chronic gastritis. The patient has not had any regional recurrences or metastasis in the past 2 years following the procedure.

Oxidized Activated Charcoal Nanozymes: Synthesis, and Optimization for In Vitro and In Vivo Bioactivity for Traumatic Brain Injury.

Carbon-based superoxide dismutase (SOD) mimetic nanozymes have recently been employed as promising antioxidant nanotherapeutics due to their distinct properties. The structural features responsible for the efficacy of these nanomaterials as antioxidants are, however, poorly understood. Here, the process-structure-property-performance properties of coconut-derived oxidized activated charcoal (cOAC) nano-SOD mimetics are studied by analyzing how modifications to the nanomaterial's synthesis impact the size, as well as the elemental and electrochemical properties of the particles. These properties are then correlated to the in vitro antioxidant bioactivity of poly(ethylene glycol)-functionalized cOACs (PEG-cOAC). Chemical oxidative treatment methods that afford smaller, more homogeneous cOAC nanoparticles with higher levels of quinone functionalization show enhanced protection against oxidative damage in bEnd.3 murine endothelioma cells. In an in vivo rat model of mild traumatic brain injury (mTBI) and oxidative vascular injury, PEG-cOACs restore cerebral perfusion rapidly to the same extent as the former nanotube-derived PEG-hydrophilic carbon clusters (PEG-HCCs) with a single intravenous injection. These findings provide a deeper understanding of how carbon nanozyme syntheses can be tailored for improved antioxidant bioactivity, and set the stage for translation of medical applications.

Oxidation of Hydrogen Sulfide to Polysulfide and Thiosulfate by a Carbon Nanozyme: Therapeutic Implications with an Emphasis on Down Syndrome.

Hydrogen sulfide (H2 S) is a noxious, potentially poisonous, but necessary gas produced from sulfur metabolism in humans. In Down Syndrome (DS), the production of H2 S is elevated and associated with degraded mitochondrial function. Therefore, removing H2 S from the body as a stable oxide could be an approach to reducing the deleterious effects of H2 S in DS. In this report we describe the catalytic oxidation of hydrogen sulfide (H2 S) to polysulfides (HS2+n - ) and thiosulfate (S2 O3 2- ) by poly(ethylene glycol) hydrophilic carbon clusters (PEG-HCCs) and poly(ethylene glycol) oxidized activated charcoal (PEG-OACs), examples of oxidized carbon nanozymes (OCNs). We show that OCNs oxidize H2 S to polysulfides and S2 O3 2- in a dose-dependent manner. The reaction is dependent on O2 and the presence of quinone groups on the OCNs. In DS donor lymphocytes we found that OCNs increased polysulfide production, proliferation, and afforded protection against additional toxic levels of H2 S compared to untreated DS lymphocytes. Finally, in Dp16 and Ts65DN murine models of DS, we found that OCNs restored osteoclast differentiation. This new action suggests potential facile translation into the clinic for conditions involving excess H2 S exemplified by DS.

A Case of Unilateral Sixth Cranial Nerve (CN VI) Avulsion in the Prepontine Cistern After Severe Head Trauma.

The patient was a 46-year-old woman who presented with right sixth cranial nerve (CN VI) palsy following severe head trauma secondary to a motor vehicle collision one month prior. In this case report, we aim to add to the literature an additional example of unilateral CN VI avulsion as visualized by MRI secondary to head trauma. 3D T2 MRI was used to visualize the CN VI avulsion. CT was also used in the evaluation of head trauma. In our view, the force trajectory of the patient's impact with the vehicle dashboard, as evidenced by the right occipital lobe fracture, explains the etiology of the unilateral right CN VI avulsion. The combination of clinical and imaging findings was central to the analysis of this case.

Deep cerebral venous abnormalities in premature babies with GMH-IVH: a single-centre retrospective study.

PURPOSE: Germinal matrix haemorrhage/intraventricular haemorrhage (GMH-IVH) is a multifactorial injury with both anatomic and haemodynamic involvement. Normal variants in preterm deep cerebral venous anatomy associated with GMH-IVH have been previously described using MRI susceptibility weighted imaging (SWI). The aims of this study were to use SWI to compare the deep venous systems of a cohort of preterm neonates with various grades of GMH-IVH to a group of age-matched controls without GMH-IVH and to present novel retrospective SWI imaging findings. METHODS: A neuroradiologist retrospectively evaluated 3T MRI SWI and phase imaging of 56 preterm neonates with GMH-IVH (14 of each grade) and 27 controls without GMH-IVH, scoring the venous irregularities according to three variables: decreased venous patency, increased lumen susceptibility and the presence of collaterals. Eight different venous locations, including indicated bilateral components, were evaluated: straight sinus, vein of galen, internal cerebral, direct lateral, thalamostriate, atrial and the anterior septal veins. Variables were analysed for statistical significance. Inter-rater reliability was determined via subset evaluation by a second paediatric radiologist. RESULTS: Deep venous abnormalities were significantly more common in patients with GMH-IVH, with Wilcoxon Rank Sum Test demonstrating significant increase with GMH-IVH for total decreased venous patency (W=0, p<0.0001), increased lumen susceptibility and collateral formation. Venous abnormalities were also positively correlated with an increase in GMH-IVH grade from I to IV (patency, ρ=0.782, p<0.01) (increased lumen susceptibility, ρ=0.739, p<0.01) (collaterals, ρ=0.649, p<0.01), not just GMH-IVH alone. CONCLUSION: Deep venous abnormalities are significantly correlated with GMH-IVH alone and an increase in GMH-IVH grade. Further study is needed to determine cause and effect.

Induction of the alternative lengthening of telomeres pathway by trapping of proteins on DNA.

Telomere maintenance is a hallmark of malignant cells and allows cancers to divide indefinitely. In some cancers, this is achieved through the alternative lengthening of telomeres (ALT) pathway. Whilst loss of ATRX is a near universal feature of ALT-cancers, it is insufficient in isolation. As such, other cellular events must be necessary - but the exact nature of the secondary events has remained elusive. Here, we report that trapping of proteins (such as TOP1, TOP2A and PARP1) on DNA leads to ALT induction in cells lacking ATRX. We demonstrate that protein-trapping chemotherapeutic agents, such as etoposide, camptothecin and talazoparib, induce ALT markers specifically in ATRX-null cells. Further, we show that treatment with G4-stabilising drugs cause an increase in trapped TOP2A levels which leads to ALT induction in ATRX-null cells. This process is MUS81-endonuclease and break-induced replication dependent, suggesting that protein trapping leads to replication fork stalling, with these forks being aberrantly processed in the absence of ATRX. Finally, we show ALT-positive cells harbour a higher load of genome-wide trapped proteins, such as TOP1, and knockdown of TOP1 reduced ALT activity. Taken together, these findings suggest that protein trapping is a fundamental driving force behind ALT-biology in ATRX-deficient malignancies.

A key feature of all cancer cells is their ability to divide indefinitely, and this is dependent on circumvention of telomere shortening through induction of a telomere maintenance mechanism, such as the telomerase-independent, Alternative Lengthening of Telomeres (ALT) pathway. The ALT pathway is characterised by loss of the ATRX chromatin remodeler. The current study provides evidence that, in the absence of ATRX, increased trapping of proteins on DNA leads to replication fork stalling and collapse. At telomeres, this leads to ALT pathway activity. These results help to better understand ALT tumours and might, eventually, be instrumental in developing new therapeutic strategies.

The Effects of Antioxidant Nutraceuticals on Cellular Sulfur Metabolism and Signaling.

Significance: Nutraceuticals are ingested for health benefits, in addition to their general nutritional value. These dietary supplements have become increasingly popular since the late 20th century and they are a rapidly expanding global industry approaching a half-trillion U.S. dollars annually. Many nutraceuticals are promulgated as potent antioxidants. Recent Advances: Experimental support for the efficacy of nutraceuticals has lagged behind anecdotal exuberance. However, accumulating epidemiological evidence and recent, well-controlled clinical trials are beginning to support earlier animal and in vitro studies. Although still somewhat limited, encouraging results have been suggested in essentially all organ systems and against a wide range of pathophysiological conditions. Critical Issues: Health benefits of "antioxidant" nutraceuticals are largely attributed to their ability to scavenge oxidants. This has been criticized based on several factors, including limited bioavailability, short tissue retention time, and the preponderance of endogenous antioxidants. Recent attention has turned to nutraceutical activation of downstream antioxidant systems, especially the Keap1/Nrf2 (Kelch like ECH associated protein 1/nuclear factor erythroid 2-related factor 2) axis. The question now becomes, how do nutraceuticals activate this axis? Future Directions: Reactive sulfur species (RSS), including hydrogen sulfide (H2S) and its metabolites, are potent activators of the Keap1/Nrf2 axis and avid scavengers of reactive oxygen species. Evidence is beginning to accumulate that a variety of nutraceuticals increase cellular RSS by directly providing RSS in the diet, or through a number of catalytic mechanisms that increase endogenous RSS production. We propose that nutraceutical-specific targeting of RSS metabolism will lead to the design and development of even more efficacious antioxidant therapeutic strategies. Antioxid. Redox Signal. 38, 68-94.

A case of clinically aggressive EBV-negative ENKTL in a non-Asian female patient.

BACKGROUND: The patient was a 65-year-old White woman who presented to dermatology with a painless, rapidly growing exophytic nodule on her left upper cheek. AIMS/PURPOSE: In this case report, we aim to demonstrate the difficulty of diagnosing Epstein-Barr virus-negative extranodal NK cell lymphomas given the broad differential of NK cell lymphomas and the rarity of EBV-negative extranodal NK cell lymphoma. METHODS: Immunohistochemical studies confirmed the diagnosis of cutaneous, extranodal NK cell lymphoma. Interestingly, Epstein-Barr virus in situ hybridization was negative, which is unusual for most NK cell lymphomas. RESULTS/CONCLUSIONS: In our view, a combination of immunohistochemistry, clonality assessment, sequencing, and flow cytometric studies is required.

Naphthoquinones Oxidize H2S to Polysulfides and Thiosulfate, Implications for Therapeutic Applications.

1,4-Napththoquinones (NQs) are clinically relevant therapeutics that affect cell function through production of reactive oxygen species (ROS) and formation of adducts with regulatory protein thiols. Reactive sulfur species (RSS) are chemically and biologically similar to ROS and here we examine RSS production by NQ oxidation of hydrogen sulfide (H2S) using RSS-specific fluorophores, liquid chromatography-mass spectrometry, UV-Vis absorption spectrometry, oxygen-sensitive optodes, thiosulfate-specific nanoparticles, HPLC-monobromobimane derivatization, and ion chromatographic assays. We show that NQs, catalytically oxidize H2S to per- and polysulfides (H2Sn, n = 2−6), thiosulfate, sulfite and sulfate in reactions that consume oxygen and are accelerated by superoxide dismutase (SOD) and inhibited by catalase. The approximate efficacy of NQs (in decreasing order) is, 1,4-NQ ≈ juglone ≈ plumbagin > 2-methoxy-1,4-NQ ≈ menadione >> phylloquinone ≈ anthraquinone ≈ menaquinone ≈ lawsone. We propose that the most probable reactions are an initial two-electron oxidation of H2S to S0 and reduction of NQ to NQH2. S0 may react with H2S or elongate H2Sn in variety of reactions. Reoxidation of NQH2 likely involves a semiquinone radical (NQ·−) intermediate via several mechanisms involving oxygen and comproportionation to produce NQ and superoxide. Dismutation of the latter forms hydrogen peroxide which then further oxidizes RSS to sulfoxides. These findings provide the chemical background for novel sulfur-based approaches to naphthoquinone-directed therapies.

Using Mobile Video-Teleconferencing to Deliver Secondary Stroke Prevention Interventions: A Pilot Study.

Objectives: Patient self-management support (SMS) interventions help stroke survivors control stroke risk factors and assist with secondary prevention. We examined utility and preliminary effectiveness of mobile video-teleconferencing (VT) to deliver SMS to stroke survivors in rural and low-income urban Texas communities. Methods: We applied a within-subjects design to assess improvement in self-management behaviors and stroke risk factors among stroke survivors receiving SMS intervention through mobile VT. Adults with stroke and two or more uncontrolled stroke risk factors were eligible. The SMS program, Video-teleconference-Self-management TO Prevent stroke (V-STOP) was delivered over 6 weeks by trained health coaches through VT. We applied Generalized Estimating Equations with site and time in intervention as covariates to evaluate psychological, social, physiological outcomes, self-management behaviors, and quality of life. Results: Mean age of 106 participants was 59.3 (±10.9); most were White, Hispanic men, living with someone, with low income. Approximately 69% completed all measures at 6 weeks. Median number of sessions attended was 5 (interquartile range 3) potentially avoiding 210 km of travel per person. Satisfaction with V-STOP and VT delivery was high, at (4.8 [±0.5]) and (4.7 [±0.5]), respectively. Stroke knowledge was improved from 8.8 (±1.0) at baseline to 9.6 (±0.7) at 12 weeks, (p < 0.0001). Improvements were observed in self-efficacy, exercise behaviors, depression and anxiety, disability, and quality of life. Conclusion: Implementation of SMS is feasible and shows good utility and preliminary effectiveness of using mobile VT to provide stroke follow-up care to stroke survivors. Participants improved self-management behaviors and stroke risk factors.

SARS-CoV-2 and the central nervous system: Emerging insights into hemorrhage-associated neurological consequences and therapeutic considerations.

Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to impact our lives by causing widespread illness and death and poses a threat due to the possibility of emerging strains. SARS-CoV-2 targets angiotensin-converting enzyme 2 (ACE2) before entering vital organs of the body, including the brain. Studies have shown systemic inflammation, cellular senescence, and viral toxicity-mediated multi-organ failure occur during infectious periods. However, prognostic investigations suggest that both acute and long-term neurological complications, including predisposition to irreversible neurodegenerative diseases, can be a serious concern for COVID-19 survivors, especially the elderly population. As emerging studies reveal sites of SARS-CoV-2 infection in different parts of the brain, potential causes of chronic lesions including cerebral and deep-brain microbleeds and the likelihood of developing stroke-like pathologies increases, with critical long-term consequences, particularly for individuals with neuropathological and/or age-associated comorbid conditions. Our recent studies linking the blood degradation products to genome instability, leading to cellular senescence and ferroptosis, raise the possibility of similar neurovascular events as a result of SARS-CoV-2 infection. In this review, we discuss the neuropathological consequences of SARS-CoV-2 infection in COVID survivors, focusing on possible hemorrhagic damage in brain cells, its association to aging, and the future directions in developing mechanism-guided therapeutic strategies.

Thrombolysis Experience in Costa Rica Compared Against Individual Patient Data from two Randomized Controlled Trials.

OBJECTIVES: We sought to compare thrombolysis outcomes from the Costa Rican Stroke Registry Program (CRSRP) with published individual patient data from NINDS and CLOTBUST-ER trials using matching and outcome modeling from randomized clinical trials (RCTs). MATERIALS AND METHODS: A retrospective observational study matching subjects on baseline characteristics, from the CRSRP, the control arm of CLOTBUST-ER, and the interventional arm of NINDS trials. Day 7-10/discharge modified Rankin Score (mRS), and early mortality was compared between matched subjects. A mortality model derived from RCTs was developed, and outcomes were compared at similar baseline NIHSS scores. CRSRP symptomatic hemorrhage (SICH) rate was compared with an Ibero-American cohort (IAC). RESULTS: Of 540 CRSRP patients, 351 received rt-PA under 3 hours and were matched with NINDS subjects yielding 292 pairs; 161 CRSRP subjects treated within 4.5 hours were matched with CLOTBUST-ER subjects resulting in 151 pairs. The proportion of patients achieving excellent outcomes (mRS 0-1) did not differ between CRSRP and either NINDS or CLOTBUST-ER (CRSRP vs NINDS: 36.6% vs 32.9%, p=0.3; CRSRP vs CLOTBUST-ER: 26.5% vs 24.5%, p=0.8). Mortality was higher for CRSRP vs CLOTBUST-ER (7.3% vs 0.7%, p=0.006), but not vs NINDS (6.5% vs 4.5%, p=0.4). A pooled mortality model derived from 15 RCTs representing 4410 patients (R2=0.39) showed CRSRP and NINDS within expected mortality, while CLOTBUST-ER showed lower than expected mortality. CRSRP SICH rate equaled IAC (5.7% vs 5.7%; p=0.9). CONCLUSIONS: Functional outcomes and SICH of thrombolysed Costa Rican patients compared favorably with published datasets, with a potential increase in early mortality.

Coenzyme Q10 and related quinones oxidize H2S to polysulfides and thiosulfate.

In the canonical pathway for mitochondrial H2S oxidation electrons are transferred from sulfide:quinone oxidoreductase (SQR) to complex III via ubiquinone (CoQ10). We previously observed that a number of quinones directly oxidize H2S and we hypothesize that CoQ10 may have similar properties. Here we examine H2S oxidation by CoQ10 and more hydrophilic, truncated forms, CoQ1 and CoQ0, in buffer using H2S and polysulfide fluorophores (AzMC and SSP4), silver nanoparticles to measure thiosulfate (H2S2O3), mass spectrometry to identify polysulfides and O2-sensitive optodes to measure O2 consumption. We show that all three quinones concentration-dependently catalyze the oxidization of H2S to polysulfides and thiosulfate in buffer with the potency CoQ0>CoQ1>CoQ10 and that CoQ0 specifically oxidizes H2S to per-polysulfides, H2S2,3,4. These reactions consume and require oxygen and are augmented by addition of SOD suggesting that the quinones, not superoxide, oxidize H2S. Related quinones, MitoQ, menadione and idebenone, oxidize H2S in similar reactions. Exogenous CoQ0 decreases cellular H2S and increases polysulfides and thiosulfate production and this is also O2-dependent, suggesting that the quinone has similar effects on sulfur metabolism in cells. Collectively, these results suggest an additional endogenous mechanism for H2S metabolism and a potential therapeutic approach in H2S-related metabolic disorders.

Alternative Lengthening of Telomeres: Lessons to Be Learned from Telomeric DNA Double-Strand Break Repair.

The study of the molecular pathways underlying cancer has given us important insights into how breaks in our DNA are repaired and the dire consequences that can occur when these processes are perturbed. Extensive research over the past 20 years has shown that the key molecular event underpinning a subset of cancers involves the deregulated repair of DNA double-strand breaks (DSBs) at telomeres, which in turn leads to telomere lengthening and the potential for replicative immortality. Here we discuss, in-depth, recent major breakthroughs in our understanding of the mechanisms underpinning this pathway known as the alternative lengthening of telomeres (ALT). We explore how this gives us important insights into how DSB repair at telomeres is regulated, with relevance to the cell-cycle-dependent regulation of repair, repair of stalled replication forks and the spatial regulation of DSB repair.

Sonothrombolysis in Patients With Acute Ischemic Stroke With Large Vessel Occlusion: An Individual Patient Data Meta-Analysis.

BACKGROUND AND PURPOSE: Evidence about the utility of ultrasound-enhanced thrombolysis (sonothrombolysis) in patients with acute ischemic stroke (AIS) is conflicting. We aimed to evaluate the safety and efficacy of sonothrombolysis in patients with AIS with large vessel occlusion, by analyzing individual patient data of available randomized-controlled clinical trials. METHODS: We included all available randomized-controlled clinical trials comparing sonothrombolysis with or without addition of microspheres (treatment group) to intravenous thrombolysis alone (control group) in patients with AIS with large vessel occlusion. The primary outcome measure was the rate of complete recanalization at 1 to 36 hours following intravenous thrombolysis initiation. We present crude odds ratios (ORs) and ORs adjusted for the predefined variables of age, sex, baseline stroke severity, systolic blood pressure, and onset-to-treatment time. RESULTS: We included 7 randomized controlled clinical trials that enrolled 1102 patients with AIS. A total of 138 and 134 confirmed large vessel occlusion patients were randomized to treatment and control groups respectively. Patients randomized to sonothrombolysis had increased odds of complete recanalization compared with patients receiving intravenous thrombolysis alone (40.3% versus 22.4%; OR, 2.17 [95% CI, 1.03-4.54]; adjusted OR, 2.33 [95% CI, 1.02-5.34]). The likelihood of symptomatic intracranial hemorrhage was not significantly different between the 2 groups (7.3% versus 3.7%; OR, 2.03 [95% CI, 0.68-6.11]; adjusted OR, 2.55 [95% CI, 0.76-8.52]). No differences in the likelihood of asymptomatic intracranial hemorrhage, 3-month favorable functional and 3-month functional independence were documented. CONCLUSIONS: Sonothrombolysis was associated with a nearly 2-fold increase in the odds of complete recanalization compared with intravenous thrombolysis alone in patients with AIS with large vessel occlusions. Further study of the safety and efficacy of sonothrombolysis is warranted.

Effects of the Hydrophilic N-Terminal Region on Aß-Mediated Membrane Disruption.

Amyloidogenesis of amyloid-ß (Aß) peptides is intimately related to pathological neurodegeneration in Alzheimer's disease. Here, we investigated the membrane damage activity of Aß40 and its derivatives that contain mutation at the N-terminal charged residues using a membrane leakage assay. A model 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipid vesicle encapsulating the fluorescent dye carboxyfluorescein was used in the study. Our results show that the mutations of the N-terminal charged residues of Aß40 significantly affect the peptide-induced membrane leakage. The results suggest that favorable electrostatic interactions of the N-terminal charged residues and the phosphatidylcholine membrane surface are crucial in Aß-mediated membrane permeation. The flexible and charge-rich N-terminal region may play a critical role in directing Aß self-association on the membrane surface and in anchoring and stabilizing the peptide aggregates inserted in the phospholipid membrane, which are closely related with membrane disruption activity of Aß. The results provide new mechanistic insight into the Aß-mediated membrane damage process, which may be critical for understanding the mechanism of Aß neurotoxicity in Alzheimer's disease.

Automatable Distributed Regression Analysis of Vertically Partitioned Data Facilitated by PopMedNet: Feasibility and Enhancement Study.

BACKGROUND: In clinical research, important variables may be collected from multiple data sources. Physical pooling of patient-level data from multiple sources often raises several challenges, including proper protection of patient privacy and proprietary interests. We previously developed an SAS-based package to perform distributed regression-a suite of privacy-protecting methods that perform multivariable-adjusted regression analysis using only summary-level information-with horizontally partitioned data, a setting where distinct cohorts of patients are available from different data sources. We integrated the package with PopMedNet, an open-source file transfer software, to facilitate secure file transfer between the analysis center and the data-contributing sites. The feasibility of using PopMedNet to facilitate distributed regression analysis (DRA) with vertically partitioned data, a setting where the data attributes from a cohort of patients are available from different data sources, was unknown. OBJECTIVE: The objective of the study was to describe the feasibility of using PopMedNet and enhancements to PopMedNet to facilitate automatable vertical DRA (vDRA) in real-world settings. METHODS: We gathered the statistical and informatic requirements of using PopMedNet to facilitate automatable vDRA. We enhanced PopMedNet based on these requirements to improve its technical capability to support vDRA. RESULTS: PopMedNet can enable automatable vDRA. We identified and implemented two enhancements to PopMedNet that improved its technical capability to perform automatable vDRA in real-world settings. The first was the ability to simultaneously upload and download multiple files, and the second was the ability to directly transfer summary-level information between the data-contributing sites without a third-party analysis center. CONCLUSIONS: PopMedNet can be used to facilitate automatable vDRA to protect patient privacy and support clinical research in real-world settings.

The Key Regulator of Necroptosis, RIP1 Kinase, Contributes to the Formation of Astrogliosis and Glial Scar in Ischemic Stroke.

Necroptosis initiation relies on the receptor-interacting protein 1 kinase (RIP1K). We recently reported that genetic and pharmacological inhibition of RIP1K produces protection against ischemic stroke-induced astrocytic injury. However, the role of RIP1K in ischemic stroke-induced formation of astrogliosis and glial scar remains unknown. Here, in a transient middle cerebral artery occlusion (tMCAO) rat model and an oxygen and glucose deprivation and reoxygenation (OGD/Re)-induced astrocytic injury model, we show that RIP1K was significantly elevated in the reactive astrocytes. Knockdown of RIP1K or delayed administration of RIP1K inhibitor Nec-1 down-regulated the glial scar markers, improved ischemic stroke-induced necrotic morphology and neurologic deficits, and reduced the volume of brain atrophy. Moreover, knockdown of RIP1K attenuated astrocytic cell death and proliferation and promoted neuronal axonal generation in a neuron and astrocyte co-culture system. Both vascular endothelial growth factor D (VEGF-D) and its receptor VEGFR-3 were elevated in the reactive astrocytes; simultaneously, VEGF-D was increased in the medium of astrocytes exposed to OGD/Re. Knockdown of RIP1K down-regulated VEGF-D gene and protein levels in the reactive astrocytes. Treatment with 400 ng/ml recombinant VEGF-D induced the formation of glial scar; conversely, the inhibitor of VEGFR-3 suppressed OGD/Re-induced glial scar formation. RIP3K and MLKL may be involved in glial scar formation. Taken together, these results suggest that RIP1K participates in the formation of astrogliosis and glial scar via impairment of normal astrocyte responses and enhancing the astrocytic VEGF-D/VEGFR-3 signaling pathways. Inhibition of RIP1K promotes the brain functional recovery partially via suppressing the formation of astrogliosis and glial scar.