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Abstract Background Geroscience focuses on interventions to mitigate molecular changes associated with aging. Lifestyle modifications, medications, and social factors influence the aging process, yet the complex molecular mechanisms require an in-depth exploration of the epigenetic landscape. The specific epigenetic clock and predictor effects of a vegan diet, compared to an omnivorous diet, remain underexplored despite potential impacts on aging-related outcomes. Methods This study examined the impact of an entirely plant-based or healthy omnivorous diet over 8 weeks on blood DNA methylation in paired twins. Various measures of epigenetic age acceleration (PC GrimAge, PC PhenoAge, DunedinPACE) were assessed, along with system-specific effects (Inflammation, Heart, Hormone, Liver, and Metabolic). Methylation surrogates of clinical, metabolite, and protein markers were analyzed to observe diet-specific shifts. Results Distinct responses were observed, with the vegan cohort exhibiting significant decreases in overall epigenetic age acceleration, aligning with anti-aging effects of plant-based diets. Diet-specific shifts were noted in the analysis of methylation surrogates, demonstrating the influence of diet on complex trait prediction through DNA methylation markers. An epigenome-wide analysis revealed differentially methylated loci specific to each diet, providing insights into the affected pathways. Conclusions This study suggests that a short-term vegan diet is associated with epigenetic age benefits and reduced calorie intake. The use of epigenetic biomarker proxies (EBPs) highlights their potential for assessing dietary impacts and facilitating personalized nutrition strategies for healthy aging. Future research should explore the long-term effects of vegan diets on epigenetic health and overall well-being, considering the importance of proper nutrient supplementation. Trial registration Clinicaltrials.gov identifier: NCT05297825

Abstract The classic ketogenic diet is an effective treatment option for drug-resistant epilepsy, but its high fat content challenges patient compliance. Optimizing liver ketone production guided by a method comparing substrates for their ketogenic potential may help to reduce the fat content of the diet without loss in ketosis induction. Here, we present a liver cell assay measuring the β-hydroxybutyrate (βHB) yield from fatty acid substrates. Even chain albumin-conjugated fatty acids comprising between 4 and 18 carbon atoms showed a sigmoidal concentration-βHB response curve (CRC) whereas acetate and omega-3 PUFAs produced no CRC. While CRCs were not distinguished by their half-maximal effective concentration (EC50), they differed by maximum response, which related inversely to the carbon chain length and was highest for butyrate. The assay also suitably assessed the βHB yield from fatty acid blends detecting shifts in maximum response from exchanging medium chain fatty acids for long chain fatty acids. The assay further detected a dual role for butyrate and hexanoic acid as ketogenic substrate at high concentration and ketogenic enhancer at low concentration, augmenting the βHB yield from oleic acid and a fatty acid blend. The assay also found propionate to inhibit ketogenesis from oleic acid and a fatty acid blend at low physiological concentration. Although the in vitro assay shows promise as a tool to optimize the ketogenic yield of a fat blend, its predictive value requires human validation.

New onset refractory status epilepticus (NORSE), including its subtype with a preceding febrile illness known as febrile infection-related epilepsy syndrome (FIRES), is one of the most severe forms of status epilepticus. The exact causes of NORSE are currently unknown, and there is so far no disease-specific therapy. Identifying the underlying pathophysiology and discovering specific biomarkers, whether immunologic, infectious, genetic, or other, may help physicians in the management of patients with NORSE. A broad spectrum of biomarkers has been proposed for status epilepticus patients, some of which were evaluated for patients with NORSE. Nonetheless, none has been validated, due to significant variabilities in study cohorts, collected biospecimens, applied analytical methods, and defined outcome endpoints, and to small sample sizes. The NORSE Institute established an open NORSE/FIRES biorepository for health-related data and biological samples allowing the collection of biospecimens worldwide, promoting multicenter research and sharing of data and specimens. Here, we suggest standard operating procedures for biospecimen collection and biobanking in this rare condition. We also propose criteria for the appropriate use of previously collected biospecimens. We predict that the widespread use of standardized procedures will reduce heterogeneity, facilitate the future identification of validated biomarkers for NORSE, and provide a better understanding of the pathophysiology and best clinical management for these patients.

It has been shown that many miRNAs, including miR-193b-3p, are differentially expressed in Parkinson's disease (PD). Dysregulation of miR-193b-3p/PGC-1α axis may alter homeostasis in cells and can induce an inflammatory response commonly accompanied by metabolic disturbances. The aim of the present study is to investigate if dysregulation of the miR-193-3p/PGC-1α axis may contribute to the pathological changes observed in the PD brain. Brain tissue were obtained from middle frontal gyrus of non-demented controls and individuals with a PD diagnosis. RT-qPCR was used to determine the expression of miR-193b-3p and in situ hybridization (ISH) and immunological analysis were employed to establish the cellular distribution of miR-193b-3p. Functional assays were performed using SH-SY5Y cells, including transfection and knock-down of miR-193b-3p. We found significantly lower expression of miR-193b-3p in the early stages of PD (PD4) which increased throughout disease progression. Furthermore, altered expression of PGC-1α suggested a direct inhibitory effect of miR-193b-3p in the brain of individuals with PD. Moreover, we observed changes in expression of insulin after transfection of SH-SY5Y cells with miR-193b-3p, which led to dysregulation in the expression of several pro- or anti - inflammatory genes. Our findings indicate that the miR-193b-3p/PGC-1α axis is involved in the regulation of insulin signaling. This regulation is crucial, since insulin induced inflammatory response may serve as a protective mechanism during acute situations but potentially evolve into a pathological process in chronic conditions. This novel regulatory mechanism may represent an interesting therapeutic target with potential benefits for various neurodegenerative diseases.

Fourier-transform near-infrared spectroscopy (FT-NIRS) has recently been used to reduce the processing time for estimates of annual age in several fish species. The present study represents the first application of this technique to marine organisms from the European waters. Atlantic horse mackerel (Trachurus trachurus) from the central Mediterranean Sea was selected for its ecological role, its socioeconomic value, and because its age is regularly estimated by otolith reading under a stereomicroscope for stock assessment purposes. Absorption spectra of the whole otoliths were acquired by FT-NIRS across a multiyear dataset, obtained during acoustic surveys carried out in different regions of the central Mediterranean Sea. The acquired spectra were processed to optimize calibration models to predict age. The best linear models obtained by the optimizing procedure predicted age successfully with a coefficient of determination of 0.95–0.96, mean squared error of 0.5 years, and bias

Abstract Engineering asymmetric transmission between left-handed and right-handed circularly polarized light in planar Fabry–Pérot (FP) microcavities would enable a variety of chiral light-matter phenomena, with applications in spintronics, polaritonics, and chiral lasing. Such symmetry breaking, however, generally requires Faraday rotators or nanofabricated polarization-preserving mirrors. We present a simple solution requiring no nanofabrication to induce asymmetric transmission in FP microcavities, preserving low mode volumes by embedding organic thin films exhibiting apparent circular dichroism (ACD); an optical phenomenon based on 2D chirality. Importantly, ACD interactions are opposite for counter-propagating light. Consequently, we demonstrated asymmetric transmission of cavity modes over an order of magnitude larger than that of the isolated thin film. Through circular dichroism spectroscopy, Mueller matrix ellipsometry, and simulation using theoretical scattering matrix methods, we characterize the spatial, spectral, and angular chiroptical responses of this 2D chiral microcavity.

Abstract Epilepsy is a chronic and heterogenous disease characterized by recurrent unprovoked seizures, that are commonly resistant to antiseizure medications. This study applies a transcriptome network-based approach across epilepsies aiming to improve understanding of molecular disease pathobiology, recognize affected biological mechanisms and apply causal reasoning to identify therapeutic hypotheses. This study included the most common drug-resistant epilepsies (DREs), such as temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), and mTOR pathway-related malformations of cortical development (mTORopathies). This systematic comparison characterized the global molecular signature of epilepsies, elucidating the key underlying mechanisms of disease pathology including neurotransmission and synaptic plasticity, brain extracellular matrix and energy metabolism. In addition, specific dysregulations in neuroinflammation and oligodendrocyte function were observed in TLE-HS and mTORopathies, respectively. The aforementioned mechanisms are proposed as molecular hallmarks of DRE with the identified upstream regulators offering opportunities for drug-target discovery and development.

[This corrects the article DOI: 10.3389/fnut.2023.1220020.].

BackgroundThe DIETFITS trial reported no significant difference in 12-month weight loss between a healthy low-fat and healthy low-carbohydrate diet. Participants were instructed to restrict fat or carbohydrates to levels consistent with a ketogenic or ultra low-fat diet for 2  months and to subsequently increase intakes until they achieved a comfortable maintenance level.ObjectiveTo compare 3- and 12-month changes in body weight and cardiometabolic risk factors between a subsample of participants who reported 3-month fat or carbohydrates intakes consistent with either a ketogenic-like diet (KLD) or ultra low-fat diet (ULF).Design3-month and 12-month weight and risk factor outcomes were compared between KLD (n = 18) and ULF (n = 21) sub-groups of DIETFITS participants (selected from n = 609, healthy overweight/obese, aged 18-50  years).ResultsLess than 10% of DIETFITS participants met KLD or ULF criteria at 3-months. Both groups achieved similar weight loss and insulin resistance improvements at 3-months and maintained them at 12- months. Significant differences at 3-months included a transient ~12% increase in LDL cholesterol (LDL-C) for KLD with a concomitant greater reduction in log(TG/HDL), a measure of LDL-C's atherogenic potential. The latter was maintained at 12-months, despite substantial diet recidivism for both groups, whereas LDL-C levels were similar for ULF at baseline and 12-months. KLD participants achieved and maintained the greatest reductions in added sugars and refined grains at 3- months and 12-months, whereas ULF participants reported a 50% increase in refined grains intake from baseline to 12-months.ConclusionAmong the ~10% of study participants that achieved the most extreme restriction of dietary fat vs. carbohydrate after 3  months, weight loss and improvement in insulin sensitivity were substantial and similar between groups. At 12  months, after considerable dietary recidivism, the few significant differences in diet quality and blood lipid parameters tended to favor KLD over ULF.

Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.

BackgroundDigital approaches may be helpful in augmenting care to address unmet mental health needs, particularly for schizophrenia and severe mental illness (SMI). ObjectiveAn international multidisciplinary group was convened to reach a consensus on the challenges and potential solutions regarding collecting data, delivering treatment, and the ethical challenges in digital mental health approaches for schizophrenia and SMI. MethodsThe consensus development panel method was used, with an in-person meeting of 2 groups: the expert group and the panel. Membership was multidisciplinary including those with lived experience, with equal participation at all stages and coproduction of the consensus outputs and summary. Relevant literature was shared in advance of the meeting, and a systematic search of the recent literature on digital mental health interventions for schizophrenia and psychosis was completed to ensure that the panel was informed before the meeting with the expert group. ResultsFour broad areas of challenge and proposed solutions were identified: (1) user involvement for real coproduction; (2) new approaches to methodology in digital mental health, including agreed standards, data sharing, measuring harms, prevention strategies, and mechanistic research; (3) regulation and funding issues; and (4) implementation in real-world settings (including multidisciplinary collaboration, training, augmenting existing service provision, and social and population-focused approaches). Examples are provided with more detail on human-centered research design, lived experience perspectives, and biomedical ethics in digital mental health approaches for SMI. ConclusionsThe group agreed by consensus on a number of recommendations: (1) a new and improved approach to digital mental health research (with agreed reporting standards, data sharing, and shared protocols), (2) equal emphasis on social and population research as well as biological and psychological approaches, (3) meaningful collaborations across varied disciplines that have previously not worked closely together, (4) increased focus on the business model and product with planning and new funding structures across the whole development pathway, (5) increased focus and reporting on ethical issues and potential harms, and (6) organizational changes to allow for true communication and coproduction with those with lived experience of SMI. This study approach, combining an international expert meeting with patient and public involvement and engagement throughout the process, consensus methodology, discussion, and publication, is a helpful way to identify directions for future research and clinical implementation in rapidly evolving areas and can be combined with measurements of real-world clinical impact over time. Similar initiatives will be helpful in other areas of digital mental health and similarly fast-evolving fields to focus research and organizational change and effect improved real-world clinical implementation.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the L2HGDH gene. L-2-HGA patients have a significantly increased lifetime risk of central nervous system (CNS) tumors. Here, we present a 16-year-old girl with L-2-HGA who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. Histologically, the tumor had a high-grade diffuse glioma phenotype. DNA sequencing revealed the inactivating homozygous germline L2HGDH mutation as well as inactivating mutations in TP53, BCOR and NF1. Genome-wide DNA-methylation analysis was unable to classify the tumor with high confidence. More detailed analysis revealed that this tumor clustered amongst IDH-wildtype gliomas by methylation profiling and did not show the glioma CpG island methylator phenotype (G-CIMP) in contrast to IDH-mutant diffuse gliomas with accumulated levels of D-2-HG, the stereoisomer of L-2-HD. These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis of the tumor was diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Due to rapid tumor progression the patient died nine months after initial diagnosis. In this manuscript, we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of L-2-HGA-associated CNS tumors.

BackgroundConsensus has not been reached on what constitutes an optimal diet in individuals with prediabetes and type 2 diabetes mellitus (T2DM), especially between low-carbohydrate options.ObjectivesWe compared 2 low-carbohydrate diets with 3 key similarities (incorporating nonstarchy vegetables and avoiding added sugars and refined grains) and 3 key differences (incorporating compared with avoiding legumes, fruits, and whole, intact grains) for their effects on glucose control and cardiometabolic risk factors in individuals with prediabetes and T2DM.MethodsKeto-Med was a randomized, crossover, interventional trial. Forty participants aged ≥18 years with prediabetes or T2DM followed the well-formulated ketogenic diet (WFKD) and the Mediterranean-plus diet (Med-Plus) for 12 weeks each, in random order. The diets shared the 3 key similarities noted above. The Med-Plus incorporated legumes, fruits, and whole, intact grains, while the WFKD avoided them. The primary outcome was the percentage change in glycated hemoglobin (HbA1c) after 12 weeks on each diet. Secondary and exploratory outcomes included percentage changes in body weight, fasting insulin, glucose, and blood lipids; average glucose from continuous glucose monitor (CGM), and nutrient intake.ResultsThe primary analysis was of 33 participants with complete data. The HbA1c values did not differ between diets at 12 weeks. Triglycerides decreased more for the WFKD [percentage changes, -16% (SEM, 4%) compared with -5% (SEM, 6%) for the Med-Plus; P = 0.02] and LDL cholesterol was higher for the WFKD [percentage changes, +10% (SEM, 4%) compared with -5% (SEM, 5%) for the Med-Plus; P = 0.01]. Weight decreased 8% (SEM, 1%) compared with 7% (SEM, 1%) and HDL cholesterol increased 11% (SEM, 2%) compared with 7% (SEM, 3%) for the WFKD compared with the Med-Plus, respectively; however, there was a significant interaction of diet × order for both. Participants had lower intakes of fiber and 3 nutrients on the WFKD compared with the Med-Plus. Twelve-week follow-up data suggest the Med-Plus is more sustainable.ConclusionsHbA1c values were not different between diet phases after 12 weeks, but improved from baseline on both diets, likely due to several shared dietary aspects. The WFKD led to a greater decrease in triglycerides, but also had potential untoward risks from elevated LDL cholesterol and lower nutrient intakes from avoiding legumes, fruits, and whole, intact grains, as well as being less sustainable. This trial was registered at clinicaltrials.gov as NCT03810378.

Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43 kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43 kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43 kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43 kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43 kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43 kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies.

Abstract A subgroup of patients infected with SARS-CoV-2 remain symptomatic over three months after infection. A distinctive symptom of patients with long COVID is post-exertional malaise, which is associated with a worsening of fatigue- and pain-related symptoms after acute mental or physical exercise, but its underlying pathophysiology is unclear. With this longitudinal case-control study (NCT05225688), we provide new insights into the pathophysiology of post-exertional malaise in patients with long COVID. We show that skeletal muscle structure is associated with a lower exercise capacity in patients, and local and systemic metabolic disturbances, severe exercise-induced myopathy and tissue infiltration of amyloid-containing deposits in skeletal muscles of patients with long COVID worsen after induction of post-exertional malaise. This study highlights novel pathways that help to understand the pathophysiology of post-exertional malaise in patients suffering from long COVID and other post-infectious diseases.

Abstract Oligomeric clusters of amyloid-β (Aβ) are one of the major biomarkers for Alzheimer’s disease (AD). However, proficient methods to detect Aβ-oligomers in brain tissue are lacking. Here we show that synthetic M13 bacteriophages displaying Aβ-derived peptides on their surface preferentially interact with Aβ-oligomers. When exposed to brain tissue isolated from APP/PS1-transgenic mice, these bacteriophages detect small-sized Aβ-aggregates in hippocampus at an early age, prior to the occurrence of Aβ-plaques. Similarly, the bacteriophages reveal the presence of such small Aβ-aggregates in post-mortem hippocampus tissue of AD-patients. These results advocate bacteriophages displaying Aβ-peptides as a convenient and low-cost tool to identify Aβ-oligomers in post-mortem brain tissue of AD-model mice and AD-patients.

Abstract The human hypothalamus modulates mental health by balancing interactions between hormonal fluctuations and stress responses. Stress-induced progesterone release activates progesterone receptors (PR) in the human brain and triggers alterations in neuropeptides/neurotransmitters. As recent epidemiological studies have associated peripheral progesterone levels with suicide risks in humans, we mapped PR distribution in the human hypothalamus in relation to age and sex and characterized its (co-) expression in specific cell types. The infundibular nucleus (INF) appeared to be the primary hypothalamic structure via which progesterone modulates stress-related neural circuitry. An elevation of the number of pro-opiomelanocortin+ (POMC, an endogenous opioid precursor) neurons in the INF, which was due to a high proportion of POMC+ neurons that co-expressed PR, was related to suicide in patients with mood disorders (MD). MD donors who died of legal euthanasia were for the first time enrolled in a postmortem study to investigate the molecular signatures related to fatal suicidal ideations. They had a higher proportion of PR co-expressing POMC+ neurons than MD patients who died naturally. This indicates that the onset of endogenous opioid activation in MD with suicide tendency may be progesterone-associated. Our findings may have implications for users of progesterone-enriched contraceptives who also have MD and suicidal tendencies.

Abstract Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these ‘design rules’ to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.

In this work, we study a Mediterranean cyclone, Helios, which took place during 9–11 February 2023 in the southeastern part of Sicily and Malta, by a multiparametric approach combining microseism results with sea state and meteorological data provided by wavemeter buoy, HF radar, hindcast maps and satellite SEVIRI images. The sub-tropical system Helios caused heavy rainfall, strong wind gusts and violent storm surges with significant wave heights greater than 5 m. We deal with the relationships between such a system and the features of microseism (the most continuous and ubiquitous seismic signal on Earth) in terms of spectral content, space–time variation of the amplitude and source locations tracked by means of two methods (amplitude-based grid search and array techniques). By comparing the location of the microseism sources and the area affected by significant storm surges derived from sea state data, we note that the microseism location results are in agreement with the real position of the storm surges. In addition, we are able to obtain the seismic signature of Helios using a method that exploits the coherence of continuous seismic noise. Hence, we show how an innovative monitoring system of the Mediterranean cyclones can be designed by integrating microseism information with other techniques routinely used to study meteorological phenomena.

ABSTRACTIn line with internationally defined goals of sustainable development, European agricultural policies today have a far-sighted vision for rural areas. Using a case study approach, this paper explores how receptive rural farms in Sicily are to a long-term vision of development. The study focuses on three key factors of a long-term vision, that is, digitalisation, innovation and sustainability, to examine not only whether farms have invested in these areas but also how they perceive their role in the post-pandemic era. Empirical results provide insights into the concentration of farms in the central inland areas of Sicily without any real long-term vision of development. Nevertheless, the analysis also shows that some of them do have a positive attitude to change.

Abstract We present a comprehensive multi-omic analysis of the EPISTOP prospective clinical trial of early intervention with vigabatrin for pre-symptomatic epilepsy treatment in Tuberous Sclerosis Complex (TSC), in which 93 infants with TSC were followed from birth to age 2 years, seeking biomarkers of epilepsy development. Vigabatrin had profound effects on many metabolites, increasing serum deoxycytidine monophosphate (dCMP) levels 52-fold. Most serum proteins and metabolites, and blood RNA species showed significant change with age. Thirty-nine proteins, metabolites, and genes showed significant differences between age-matched control and TSC infants. Six also showed a progressive difference in expression between control, TSC without epilepsy, and TSC with epilepsy groups. A multivariate approach using enrollment samples identified multiple 3-variable predictors of epilepsy, with the best having a positive predictive value of 0.987. This rich dataset will enable further discovery and analysis of developmental effects, and associations with seizure development in TSC.

Abstract Cigarette smoking impairs the lung innate immune response making smokers more susceptible to infections and severe symptoms. Dysregulation of cell death is emerging as a key player in chronic inflammatory conditions. We have recently reported that short exposure of human monocyte-derived macrophages (hMDMs) to cigarette smoke extract (CSE) altered the TLR4-dependent response to lipopolysaccharide (LPS). CSE caused inhibition of the MyD88-dependent inflammatory response and activation of TRIF/caspase-8/caspase-1 pathway leading to Gasdermin D (GSDMD) cleavage and increased cell permeability. Herein, we tested the hypothesis that activation of caspase-8 by CSE increased pro-inflammatory cell death of LPS-stimulated macrophages. To this purpose, we measured apoptotic and pyroptotic markers as well as the expression/release of pro-inflammatory mediators in hMDMs exposed to LPS and CSE, alone or in combination, for 6 and 24 h. We show that LPS/CSE-treated hMDMs, but not cells treated with CSE or LPS alone, underwent lytic cell death (LDH release) and displayed apoptotic features (activation of caspase-8 and -3/7, nuclear condensation, and mitochondrial membrane depolarization). Moreover, the negative regulator of caspase-8, coded by CFLAR gene, was downregulated by CSE. Activation of caspase-3 led to Gasdermin E (GSDME) cleavage. Notably, lytic cell death caused the release of the damage-associated molecular patterns (DAMPs) heat shock protein-60 (HSP60) and S100A8/A9. This was accompanied by an impaired inflammatory response resulting in inhibited and delayed release of IL6 and TNF. Of note, increased cleaved caspase-3, higher levels of GSDME and altered expression of cell death-associated genes were found in alveolar macrophages of smoker subjects compared to non-smoking controls. Overall, our findings show that CSE sensitizes human macrophages to cell death by promoting pyroptotic and apoptotic pathways upon encountering LPS. We propose that while the delayed inflammatory response may result in ineffective defenses against infections, the observed cell death associated with DAMP release may contribute to establish chronic inflammation. CS exposure sensitizes human macrophages to pro-inflammatory cell death. Upon exposure to LPS, CS inhibits the TLR4/MyD88 inflammatory response, downregulating the pro-inflammatory genes TNF and IL6 and the anti-apoptotic gene CFLAR, known to counteract caspase-8 activity. CS enhances caspase-8 activation through TLR4/TRIF, with a partial involvement of RIPK1, resulting on the activation of caspase-1/GSDMD axis leading to increased cell permeability and DAMP release through gasdermin pores [19]. At later timepoints caspase-3 becomes strongly activated by caspase-8 triggering apoptotic events which are associated with mitochondrial membrane depolarization, gasdermin E cleavage and secondary necrosis with consequent massive DAMP release.

Inflammatory processes induced by brain injury are important for recovery; however, when uncontrolled, inflammation can be deleterious, likely explaining why most anti-inflammatory treatments have failed to improve neurological outcomes after brain injury in clinical trials. In the thalamus, chronic activation of glial cells, a proxy of inflammation, has been suggested as an indicator of increased seizure risk and cognitive deficits that develop after cortical injury. Furthermore, lesions in the thalamus, more than other brain regions, have been reported in patients with viral infections associated with neurological deficits, such as SARS-CoV-2. However, the extent to which thalamic inflammation is a driver or by-product of neurological deficits remains unknown. Here, we found that thalamic inflammation in mice was sufficient to phenocopy the cellular and circuit hyperexcitability, enhanced seizure risk, and disruptions in cortical rhythms that develop after cortical injury. In our model, down-regulation of the GABA transporter GAT-3 in thalamic astrocytes mediated this neurological dysfunction. In addition, GAT-3 was decreased in regions of thalamic reactive astrocytes in mouse models of cortical injury. Enhancing GAT-3 in thalamic astrocytes prevented seizure risk, restored cortical states, and was protective against severe chemoconvulsant-induced seizures and mortality in a mouse model of traumatic brain injury, emphasizing the potential of therapeutically targeting this pathway. Together, our results identified a potential therapeutic target for reducing negative outcomes after brain injury.