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It is widely accepted that the pH of insulin granules is acidic, and that its active regulation during granule maturation plays a role in the process of insulin secretion by β-cells. Yet, a calibrated measurement of the absolute granule pH with organelle specificity is still lacking. To tackle this issue, we use the genetically encoded E 1 GFP pH reporter inserted into the C-peptide of proinsulin and expressed in Insulinoma 1E cells. Following verification of correct targeting of the E 1 GFP reporter in the insulin granules, phasor-based Fluorescence Lifetime Imaging Microscopy (FLIM) is applied to obtain a calibrated and probe-concentration-independent measurement of insulin-granule pH. Our results confirm the acidic nature of insulin granules under maintenance cell culture conditions, with an average luminal pH of ~5.8, and show that acidity is actively maintained, as evidenced by its near-neutralization upon treatment with the vacuolar H + -ATPase inhibitor concanamycin. Additionally, by exploiting the intrinsic spatial resolution of FLIM, we highlight that granules which are proximal to the plasma membrane are slightly more acidic than those which are distal, a difference preserved even during the early phase of glucose-induced insulin secretion. This study lays the foundations for future investigations of granule pH in physiology and disease., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: We aimed to describe neuroimaging features, clinical profiles and long-term outcomes in patients with iatrogenic cerebral amyloid angiopathy (iCAA)., Methods: We performed a systematic literature search for case series of iCAA and included individual patients and their longitudinal clinical and neuroimaging data in this pooled cohort study. Patients meeting a modified version of the Queen Square criteria for iCAA were included. Baseline and follow-up MRIs were centrally analysed for markers of CAA using validated rating scales., Results: We included 51 patients (68.6% male, median age at presentation 48 years), 51.0% with probable and 49.0% with possible iCAA. We evaluated 219 MRIs acquired over a median follow-up time of 3.7 years (IQR 1.8-6.4). There were 43 symptomatic intracerebral haemorrhages (ICH) in 24 patients during follow-up, a rate of 16.7 per 100 patient-years.Patients with previous supratentorial brain surgery had an ipsilateral-dominant distribution and spread of haemorrhagic markers on MRI. 14/51 (27.5%) patients had transient inflammatory changes (cortical or parenchymal oedema, sulcal hyperintensities). Haemorrhagic markers progressed during follow-up. In addition to 43 symptomatic ICH, 36 asymptomatic ICH (mostly smaller intragyral haemorrhages) were detected on follow-up scans. Besides numerous lobar microbleeds (median 16 at baseline, 53 at last follow-up), deep microbleeds were present in 19.6% of patients at baseline and 44.4% at follow-up. Severe perivascular spaces in centrum semiovale were common at baseline (64.7%) and follow-up (95.6%)., Conclusions: Patients with iCAA appear to have distinctive MRI characteristics, which might differentiate iCAA from other CAA subtypes and provide new insights into underlying disease mechanisms., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2025. No commercial re-use. See rights and permissions. Published by BMJ Group.)

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, characterized by recurrent strokes, cognitive decline, and psychiatric symptoms. This report presents a novel NOTCH3 c.1564 T > A (p.Cys522Ser) mutation associated with early-onset parkinsonism and significant white matter lesions. We describe a patient who presented with early-onset parkinsonism, characterized by bradykinesia and rigidity, alongside extensive white matter lesions observed through neuroimaging. Genetic testing revealed a novel c.1564 T > A (p.Cys522Ser) mutation in the NOTCH3 gene, contributing to the clinical diagnosis of CADASIL. This case underscores the phenotypic variability of CADASIL and the potential for atypical presentations, including parkinsonism. Early identification of genetic mutations can facilitate appropriate management and counseling for affected individuals and their families. Further research is warranted to explore the mechanisms underlying the association between NOTCH3 mutations and parkinsonism. Our findings contribute to the understanding of CADASIL, suggesting that clinicians should consider CADASIL in differential diagnoses of early-onset parkinsonism, especially in patients with concurrent white matter lesions., Competing Interests: The authors declare that the article was written without any external funding or financial support. The authors have no financial relationships or competing interests to disclose that could influence the content or interpretation of the work., (© 2025 The Authors. Published by Elsevier Ltd.)

Objectives: Recently, a subset of patients affected by cerebral amyloid angiopathy (CAA) distinguished by atypical juvenile onset and a hypothesized iatrogenic origin (iatrogenic CAA, iCAA) has emerged. β-Amyloid (Aβ) accumulation evidenced by amyloid PET positivity or CSF Aβ decrease was included in the iCAA diagnostic criteria. Conversely, diagnostic criteria for sporadic CAA (sCAA) do not involve biomarker analysis. The aim of this study was to assess CSF and plasma levels of Aβ and tau in iCAA and sCAA cohorts., Methods: Patients affected by probable or possible CAA according to established criteria (Boston 2.0) were prospectively recruited at Fondazione IRCCS Carlo Besta and San Gerardo dei Tintori from May 2021 to January 2024. Patients with probable and possible iCAA or sCAA with available plasma and/or CSF samples were included. Clinical and neurologic data were collected, and levels of Aβ40, Aβ42, total tau, and phospho-tau (p-tau) were assessed in CSF and plasma by SiMoA and Lumipulse., Results: 21 patients with iCAA (72% male, mean age at symptom onset 50 years [36-74]) and 32 patients with sCAA (44% male, mean age at symptom onset 68 years [52-80]) were identified. Cognitive impairment and cardiovascular risk factors in the sCAA cohort were more common compared with the iCAA cohort. Patients with sCAA and iCAA showed similar CSF levels for Aβ40 ( p = 0.5 [sCAA, 95% CI 2,604-4,228; iCAA, 95% CI 1,958-3,736]), Aβ42 ( p = 0.7 [sCAA, 95% CI 88-157; iCAA, 95% CI 83-155]), and total tau ( p = 0.08 [sCAA, 95% CI 80-134; iCAA, 95% CI 37-99]). Plasma levels of Aβ40 ( p = 0.08, 95% CI 181-222), Aβ42 ( p = 0.3, 95% CI 6-8), and total tau ( p = 0.4, 95% CI 3-6) were not statistically different in patients with sCAA compared with iCAA ones (Aβ40, 95% CI 153-193; Aβ42, 95% CI 6-7 and total tau, 95% CI 2-4)., Discussion: Despite presenting with a younger age at onset, fewer cardiovascular risk factors, and lower cognitive impairment, patients with iCAA demonstrated Aβ and tau levels comparable with elderly patients with sCAA, supporting a common molecular paradigm between the 2 CAA forms.

Despite the growing interest in gender medicine, the influence of sex and gender on human diseases, including stroke, continues to be underestimated and understudied. The COVID-19 pandemic has overall impacted not only the occurrence and management of stroke but has also exacerbated sex and gender disparities among both patients and healthcare providers. This paper aims to provide an updated overview on the influence of sex and gender in stroke pathophysiology and care during COVID-19 pandemic, through biological, clinical, psychosocial and research perspectives. Gender equity and awareness of the importance of sexual differences are sorely needed, especially in times of health crisis but have not yet been achieved to date. To this purpose, the sudden yet worldwide diffusion of COVID-19 represents a unique learning experience that highlights critical unmet needs also in gender medicine. The failures of this recent past should be kept as food for thought to inspire proper strategies reducing inequalities and to address women's health and wellbeing issues, particularly in case of future pandemics., (© 2024. Fondazione Società Italiana di Neurologia.)

Objectives: Cerebral amyloid angiopathy (CAA)-related features on neuroimaging often coexist with signs of arteriolosclerosis-small vessel disease on neuroimaging in people with intracranial hemorrhage (ICH). This study aimed at defining the value of amyloid pathology detected by 18F flutemetamol PET in reclassification and stratification of risk of bleeding in people with mixed CAA-arteriolosclerosis features., Methods: We included consecutive patients admitted to 2 institutions (2018-2023) with spontaneous symptomatic ICH, subarachnoid hemorrhage (SAH), transient focal neurologic episodes (TFNE), or cognitive impairment and MRI showing CAA hallmarks. All patients underwent brain magnetic resonance imaging (MRI) with susceptibility weighted imaging and 18F flutemetamol PET imaging and were followed up for at least 1 year. We compared cases with CAA and arteriolosclerosis + CAA features and defined long-term outcomes (composite outcome including death, ICH, ischemic stroke, SAH, TFNE) depending on PET status (CAA/amyloid pathology vs arteriolosclerosis-predominant groups)., Results: Among 47 patients, according to PET and MRI imaging, 38 patients were reclassified in the CAA/amyloid pathology group and 9 in the arteriolosclerosis-predominant group, with similar cardiovascular risk factors but a significantly higher lobar microbleed burden for the former group. The CAA/amyloid pathology group had higher rates of composite outcome (43.9 vs 11.1 events per 100 patient-year; p = 0.039) and ICH (36.5 vs 5.6 events per 100 patient-years; p = 0.04) compared with the arteriolosclerosis-predominant group., Discussion: 18F Flutemetamol PET imaging can help in reclassification of mixed arteriolosclerosis + CAA into CAA/amyloid pathology and arteriolosclerosis-predominant, with implications on long-term risk of recurrent events., Classification of Evidence: This study provides Class IV evidence that 18F flutemetamol PET can distinguish between CAA + arteriolosclerosis and arteriolosclerosis-predominant pathology.

Aim: To review the current data on cognitive and psychological characteristics of patients with CAA and on the instruments used for their evaluation., Methods: A systematic search was performed in Embase, Scopus and PubMed with terms related to "cerebral amyloid angiopathy", "neuropsychological measures" and "patient-reported outcome measures" from January 2001 to December 2021., Results: Out of 2851 records, 18 articles were selected. The cognitive evaluation was present in all of which, while the psychological one only in five articles. The MMSE (Mini Mental State Examination), TMT (Trail Making Test), fluency test, verbal learning test, digit span, digit symbol and Rey figure tests were the most used cognitive tests, while executive function, memory, processing speed, visuospatial function, attention and language were the most frequent impaired cognitive functions. Depression was the most considered psychological factor usually measured with BDI (Beck Depression Inventory) and GDS (Geriatric Depression Scale)., Conclusions: The results of this study might be used in clinical practice as a guide to choose cognitive and psychological instruments and integrate them in the clinical evaluation. The results might also be used in the research field for studies investigating the impact of cognitive and psychological variables on the disease course and for consensus studies aimed at define a standardized evaluation of these aspects., (© 2024. Fondazione Società Italiana di Neurologia.)

Lung cancer is the leading cause of cancer deaths. Lethal pulmonary adenocarcinomas (ADC) present with frequent mutations in the EGFR. Genetically engineered murine models of lung cancer expedited comprehension of the molecular mechanisms driving tumorigenesis and drug response. Here, we systematically analyzed the evolution of tumor heterogeneity in the context of dynamic interactions occurring with the intermingled tumor microenvironment (TME) by high-resolution transcriptomics. Our effort identified vulnerable tumor-specific epithelial cells, as well as their cross-talk with niche components (endothelial cells, fibroblasts, and tumor-infiltrating immune cells), whose symbiotic interface shapes tumor aggressiveness and is almost completely abolished by treatment with Unesbulin, a tubulin binding agent that reduces B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) activity. Simultaneous magnetic resonance imaging (MRI) analysis demonstrated decreased tumor growth, setting the stage for future investigations into the potential of novel therapeutic strategies for EGFR-mutant ADCs., Significance: Targeting the TME is an attractive strategy for treatment of solid tumors. Here we revealed how EGFR-mutant landscapes are affected at the single-cell resolution level during Unesbulin treatment. This novel drug, by targeting cancer cells and their interactions with crucial TME components, could be envisioned for future therapeutic advancements., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Background: The transmission of amyloid β (Aβ) in humans leading to iatrogenic cerebral amyloid angiopathy (iCAA) is a novel concept with analogies to prion diseases. However, the number of published cases is low, and larger international studies are missing., Aims: We aimed to build a large multinational collaboration on iCAA to better understand the clinical spectrum of affected patients., Methods: We collected clinical data on patients with iCAA from Austria, Croatia, Italy, Slovenia, and Spain. Patients were included if they met the proposed Queen Square diagnostic criteria (QSC) for iCAA. In addition, we pooled data on disease onset, latency, and cerebrospinal fluid (CSF) biomarkers from previously published iCAA cases based on a systematic literature review., Results: Twenty-seven patients (22% women) were included in this study. Of these, 19 (70%) met the criteria for probable and 8 (30%) for possible iCAA. Prior neurosurgical procedures were performed in all patients (93% brain surgery, 7% spinal surgery) at median age of 8 (interquartile range (IQR) = 4-18, range = 0-26 years) years. The median symptom latency was 39 years (IQR = 34-41, range = 28-49). The median age at symptom onset was 49 years (IQR = 43-55, range = 32-70). Twenty-one patients (78%) presented with intracranial hemorrhage and 3 (11%) with seizures., Conclusions: Our large international case series of patients with iCAA confirms a wide age boundary for the diagnosis of iCAA. Dissemination of awareness of this rare condition will help to identify more affected patients., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation ( Haptoglobin , MCP-1 , IL-6 , PAI-1 ), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells.

Background and Purpose: Iatrogenic cerebral amyloid angiopathy (iCAA) is a specific type of cerebral amyloid angiopathy which is becoming increasingly diagnosed. It has been hypothesized that iCAA might arise as a late consequence of past neurosurgical interventions involving dural patch grafts. Positron emission tomography (PET) scans with amyloid tracers and the assay of beta-amyloid levels in cerebrospinal fluid (CSF) are auxiliary criteria, however, definite diagnosis remains histopathologically determined., Methods: Case report., Results: We present a 48-year-old patient who suffered multiple lobar cerebral haemorrhages from the age of 47. The patient had undergone surgery for remolval of hemangioblastoma with lyophilized dural graft at the age of 11, in 1987. Brain MRI, amiloid PET and CSF analysis led to a diagnosis of probable iCAA., Conclusion: It is necessary to increase the awareness of iCAA, in order to avoid overlooking the potential causal involvement of surgical procedures which took place far back in time. Moreover, the diagnostic relevance of amyloid PET and beta-amyloid levels in CSF must be emphasised., (© 2023 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Thanks to a more widespread knowledge of the disease, and improved diagnostic techniques, the clinical spectrum of cerebral amyloid angiopathy (CAA) is now broad. Sporadic CAA, hereditary CAA, CAA-related inflammation (CAA-ri) and iatrogenic CAA (iCAA) create a clinical and radiological continuum which is intriguing and only partially discovered. Despite being relatively rare, CAA-ri, an aggressive subtype of CAA with vascular inflammation, has gained growing attention also because of the therapeutic efficacy of anti-inflammatory and immunomodulating drugs. More recently, diagnostic criteria have been proposed for an unusual variant of CAA, probably related to an iatrogenic origin (iCAA), toward which there is mounting scientific interest. These atypical forms of CAA are still poorly known, and their recognition can be challenging and deserve to be pursued in specialized referral centres. The aim of this brief review is to focus current developments in the field of rare forms of CAA, its pathogenesis as well as clinical and biological features in order to increase awareness of these rare forms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Storti, Gabriel, Sennfält, Canavero, Rifino, Gatti and Bersano.)

Subarachnoid hemorrhage (SAH) carries high mortality and disability rates, which are substantially driven by complications. Early brain injury and vasospasm can happen after SAH and are crucial events to prevent and treat to improve prognosis. In recent decades, immunological mechanisms have been implicated in SAH complications, with both innate and adaptive immunity involved in mechanisms of damage after SAH. The purpose of this review is to summarize the immunological profile of vasospasm, highlighting the potential implementation of biomarkers for its prediction and management. Overall, the kinetics of central nervous system (CNS) immune invasion and soluble factors' production critically differs between patients developing vasospasm compared to those not experiencing this complication. In particular, in people developing vasospasm, a neutrophil increase develops in the first minutes to days and pairs with a mild depletion of CD45+ lymphocytes. Cytokine production is boosted early on after SAH, and a steep increase in interleukin-6, metalloproteinase-9 and vascular endothelial growth factor (VEGF) anticipates the development of vasospasm after SAH. We also highlight the role of microglia and the potential influence of genetic polymorphism in the development of vasospasm and SAH-related complications.

The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10-200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

Hyper-/hypoglycemic states are rare but well-established causes of hyperkinetic movements, including chorea and ballismus, usually associated with brain lesions in the basal ganglia. We report a case of hemichorea-hemiballismus (HCHB) syndrome that developed after a severe hypoglycemic episode in a 71-year-old man with poorly controlled type 2 diabetes mellitus. Uncommonly, brain MRI showed contralateral cortical-subcortical T2 and T2-FLAIR-hyperintense frontoparietal lesions, with cingulate gyrus involved, while the basal ganglia were unaffected. In patients with hypoglycemic encephalopathy associated with cortical lesions, the long-term prognosis is usually poor. Nevertheless, in our patient, the dyskinesias and the cerebral lesions progressively regressed by achieving good glycemic control. After four and 12 months, the patient's neurological examination was normal. To our knowledge, this is the first evidence of hypoglycemic etiology of cortical HCHB syndrome, supporting recent theories that cortical circuitries may independently contribute to the pathogenesis of chorea and ballismus. This is also the first report of cingulate gyrus involvement in hypoglycemic encephalopathy. Finally, this case may indicate that a subset of patients with cortical lesions due to hypoglycemia could present a good clinical outcome, likely depending on the size of the lesions and the duration and severity of the hypoglycemic episode., Competing Interests: The authors have no conflicts of interest to declare., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Objective: Cerebral amyloid angiopathy (CAA) is a major cause of spontaneous intracranial hemorrhage in older adults. Epilepsy represents a possible sequela of the disease. To date, studies on epilepsy in CAA are lacking, and the few data available mainly focus on CAA-related inflammation (CAA-ri), the inflammatory form of the disease., Methods: In this retrospective observational study, we consecutively recruited CAA patients observed over a time span of 10 years, collecting demographic, clinical, and instrumental data. Significant baseline characteristics were evaluated as potential risk factors for the development of epilepsy in the CAA population, and in the subgroups of CAA-ri and CAA without inflammatory reaction (CAA-nri). The effect of potential risk factors for epilepsy was measured as odds ratio with 95% confidence interval., Results: Within 96 recruited CAA cases, 33 (34.4%) developed epilepsy during follow-up (median = 13.5 months). The prevalent type of seizure was focal (81.3%); 12.1% of the epileptic patients presented status epilepticus, and 6.1% developed drug-resistant epilepsy. Electroencephalographic traces revealed slow and epileptic discharge activity in the majority of epileptic patients, but also in those without epilepsy. The presence of focal or disseminated cortical superficial siderosis (cSS) was associated with an increased risk of epilepsy in the CAA-nri group, and the association with CAA-ri and epilepsy was present in the overall population., Significance: Epilepsy is a common manifestation during the course of CAA, where CAA-ri and cSS represent predisposing factors for the development of seizures. These data suggest the importance of a deep characterization of CAA patients, to better select those more prone to develop epilepsy., (© 2022 International League Against Epilepsy.)