Search

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Objectives: Remdesivir is an antiviral agent, which was shown to be safe and effective in treating early COVID-19, but its favourable impact in hospitalised patients with non-critical disease is still under investigation. The present study aimed to assess the effectiveness and safety of remdesivir as a treatment for hospitalised patients with COVID-19 by a propensity score analysis of observational data., Methods: In this monocentric retrospective cohort study, the effectiveness and safety of a 5-day course of remdesivir (200 mg intravenously at Day 1, then 100 mg from Days 2-5) in association with the standard of care were assessed in comparison with the standard of care only. The primary endpoint was the proportion of recovery on Day 14., Results: Of 3662 eligible inpatients who tested positive for the severe acute respiratory syndrome coronavirus 2 genome by nasopharyngeal swab at admission, 861 (24%) non-critical patients were included in a propensity score analysis and 281 (33%) were exposed to remdesivir. In total, 242/281 (86.1%) and 435/580 (75.0%) patients recovered in exposed and non-exposed, respectively, with a relative improvement of 11.1% (95% CI + 5.8 to 16.5%; unadjusted odds ratio: 2.07, 95% CI 1.40-3.05, p = 0.0001; after adjustment by propensity score weighting, odds ratio: 1.92, 95% CI 1.30-2.83, p = 0.001). In treated patients, 1 (0.03%) anaphylactic reaction and 1 (0.03%) acute reaction during drug injection were reported, and 24 (8.5%) patients stopped the treatment due to adverse reactions. No significant differences were found with respect to the secondary efficacy endpoints (in-hospital all-cause death, need for intensive care treatments, clinical improvement score at Day 28) and safety endpoints (any and serious adverse reactions)., Conclusion: A 5-day course of remdesivir in association with the standard of care effectively promoted recovery from COVID-19 among non-critical in-hospital patients and had an acceptable safety profile., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The heart depends on a functional vasculature for oxygenation and transport of nutrients, and it is of interest to learn how primary impairment of the vasculature can indirectly affect cardiac function and heart morphology. Notch3-deficiency causes vascular smooth muscle cell (VSMC) loss in the vasculature but the consequences for the heart remain largely elusive. Here, we demonstrate that Notch3 -/- mice have enlarged hearts with left ventricular hypertrophy and mild fibrosis. Cardiomyocytes were hypertrophic but not hyperproliferative, and the expression of several cardiomyocyte markers, including Tnt2, Myh6, Myh7 and Actn2, was altered. Furthermore, expression of genes regulating the metabolic status of the heart was affected: both Pdk4 and Cd36 were downregulated, indicating a metabolic switch from fatty acid oxidation to glucose consumption. Notch3 -/- mice furthermore showed lower liver lipid content. Notch3 was expressed in heart VSMC and pericytes but not in cardiomyocytes, suggesting that a perturbation of Notch signalling in VSMC and pericytes indirectly impairs the cardiomyocytes. In keeping with this, Pdgfb ret/ret mice, characterized by reduced numbers of VSMC and pericytes, showed left ventricular and cardiomyocyte hypertrophy. In conclusion, we demonstrate that reduced Notch3 or PDGFB signalling in vascular mural cells leads to cardiomyocyte dysfunction., (© 2023. Springer Nature Limited.)

Rheumatic disease patients are at greater risk of infection due to their disease, comorbidities, and immunosuppressive therapy. COVID-19 outcomes in this patient setting appeared to be similar to those of the general population. However, data on this topic were mainly related to small studies on a limited number of patients. Consequently, to date, this field remains poorly explored, particularly in the pre-vaccine era. This monocentric study aimed to describe the intrahospital mortality in rheumatic patients with SARS-CoV-2 consecutively hospitalized from 21 February to 31 December 2020, before anti-SARS-CoV-2 vaccine administration spread, compared with non-rheumatic patients. Of 2491 included patients, 65 [3%, median (interquartile range) age 75 (64.76-82.239 years, 65% women] were suffering from rheumatic diseases. A total of 20 deaths were reported [case fatality rate 31%, 95% confidence interval (CI): 19-42] compared with 433 deaths (19%, 95% CI: 17-20) in patients without rheumatic diseases (p=0.024). However, the rheumatic disease was not associated with a significant increase in univariate mortality hazards (hazard ratio 1.374, 95% CI: 0.876-2.154), and after adjustment (hazard ratio 1.199, 95% CI: 0.759-1.894) by age, sex and Charlson comorbidity index. The incidence of intensive care unit admission, death, and discharge in the case-control study was comparable between rheumatic and non-rheumatic patients. The presence of rheumatic diseases in SARS-CoV-2-hospitalized patients did not represent an independent risk factor for severe disease or mortality.

Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain diseases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivascular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation protocol suitable for different experimental modalities, such as single-cell analyses, western blotting, and primary cell culture., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease; no preventive or curative therapy is available. CADASIL is caused by mutations in the NOTCH3 gene, resulting in a mutated NOTCH3 receptor, with aggregation of the NOTCH3 extracellular domain (ECD) around vascular smooth muscle cells. In this study, we have developed a novel active immunization therapy specifically targeting CADASIL-like aggregated NOTCH3 ECD. Immunizing CADASIL TgN3R182C 150 mice with aggregates composed of CADASIL-R133C mutated and wild-type EGF 1-5 repeats for a total of 4 months resulted in a marked reduction (38-48%) in NOTCH3 deposition around brain capillaries, increased microglia activation and lowered serum levels of NOTCH3 ECD. Active immunization did not impact body weight, general behavior, the number and integrity of vascular smooth muscle cells in the retina, neuronal survival, or inflammation or the renal system, suggesting that the therapy is tolerable. This is the first therapeutic study reporting a successful reduction of NOTCH3 accumulation in a CADASIL mouse model supporting further development towards clinical application for the benefit of CADASIL patients., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs 1 ) such as meningeal and perivascular macrophages 2-7 -are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma 2,8-10 . It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors 11-15 . However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Notch signalling is an evolutionarily highly conserved signalling mechanism governing differentiation and regulating homeostasis in many tissues. In this review, we discuss recent advances in our understanding of the roles that Notch signalling plays in the vasculature. We describe how Notch signalling regulates different steps during the genesis and remodelling of blood vessels (vasculogenesis and angiogenesis), including critical roles in assigning arterial and venous identities to the emerging blood vessels and regulation of their branching. We then proceed to discuss how experimental perturbation of Notch signalling in the vasculature later in development affects vascular homeostasis. In this review, we also describe how dysregulated Notch signalling, as a consequence of direct mutations of genes in the Notch pathway or aberrant Notch signalling output, contributes to various types of vascular disease, including CADASIL, Snedden syndrome and pulmonary arterial hypertension. Finally, we point out some of the current knowledge gaps and identify remaining challenges in understanding the role of Notch in the vasculature, which need to be addressed to pave the way for Notch-based therapies to cure or ameliorate vascular disease.

Background: Robust data on case fatality rate (CFR) among inpatients with COVID-19 are still lacking, and the role of patient characteristics in in-hospital deaths remains under-investigated. This study quantified the overall CFR and described its trend in a cohort of hospitalized patients with SARS-CoV-2 in Italy. Admission to ICU, death, or discharge were the secondary outcomes., Methods: This retrospective study is based on administrative health data and electronic case records of inpatients consecutively admitted to Niguarda Hospital between 21 February and 8 November 2020., Results: An overall CFR of 18% was observed. CFR was significantly reduced during the second wave of contagion (1 June to 30 September, 16%) compared with the first wave (21 February to 31 May, 21% p  = 0.015). Such reduction was mainly observed among male inpatients between 40 and 80 years with limited comorbidities. Admission to ICU was associated with a high risk of mortality in both waves. The incidence of severe disease and the need for ICU admission were lower in the second wave., Conclusion: CFR in SARS-CoV-2 inpatients was demonstrated to decrease over time. This reduction may partly reflect the changes in hospital strategy and clinical practice. The reasons for this improvement should be further investigated to plan an exit strategy in case of future outbreaks., Key Messages: What is already known on this topic Before the advent of anti-COVID-19 vaccines, a multi-wave pattern of contagion was observed, and this trend conditioned the inpatient case fatality rate (CFR), which varied over time accordingly to the waves of contagion.Only preliminary results on the in-hospital mortality trend are available, along with a partial analysis of its determinants. Consequently, robust data on CFR among inpatients with SARS-CoV-2 infection are still lacking, and the role of patient characteristics in in-hospital deaths remains under-investigated. What this study adds This study shows that the in-hospital mortality in patients with SARS-CoV-2 infection decreases over time.Such reduction was mainly observed among male inpatients between 40 and 80 years with limited comorbidities. Admission to ICU was invariably associated with a high risk of mortality during the whole study period (21 February to 8 November 2020), but the incidence of severe disease and the need for ICU admission were lower in the second wave of contagions (1 October to 8 November 2020). This reduction may partly reflect the impact of changes in hospital strategy and clinical practice. The reasons for this improvement should be further investigated to inform the response to future outbreaks and to plan exit strategy by prioritizing high-risk populations., Supplementary Information: The online version contains supplementary material available at 10.1007/s10389-021-01675-y., Competing Interests: Conflicts of interestNone of the authors declared conflict of interests., (© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021.)

Objective: This study aimed to improve the post-marketing surveillance on mRNA anti-SARS-CoV-2 vaccines, characterizing the adverse events (AEs) after the first dose of mRNA BNT162b vaccine. The associations between the AEs and individuals' characteristics were explored., Patients and Methods: All adult healthcare workers at Niguarda Hospital (Milan, Italy) who were referred for the first dose of vaccine were offered to participate in a cross-sectional survey during the second-dose administration, between 18 January and 7 February 2021. All participants completed a questionnaire about age, gender, weight, height, medical history, concurrent therapies, employment status, previous diagnosis/testing for SARS-CoV-2 infection, and a list of 24 AEs (solicited AEs). The development of at least one solicited AEs was the main outcome. AEs were stratified by the presence of injection-site symptoms, systemic symptoms or both, and the differences between strata were assessed as a secondary outcome. Biometric data and reports of a previous diagnosis of SARS-CoV-2 infection were also explored, as predictors of the main outcome., Results: 7,014 healthcare workers were included. An incidence of 3 per 10.000 persons for serious AEs following the first administration of the mRNA BNT162b vaccine was found. An association between the development of non-serious AEs with young age, female gender, low body mass index, and previous history of SARS-CoV-2 was described., Conclusions: This real-life study supported data on the safety profile of the BNT162b2 mRNA vaccine. Our findings on the associations between the development of non-serious AEs with some individual characteristics may help physicians and patients make educated and informed medical decisions towards anti-COVID-19 vaccination.

Objective: Anti-COVID-19 vaccines were mainly associated with non-serious adverse events (AEs), whose prevalence was reported to be up to 70% in healthcare workers (HCWs). This may lead to sick leave requests, but this impact has never been quantified. This study aimed to investigate the absence from work among HCWs following anti-COVID-19 vaccination. Its association with age and previous COVID-19 infection was also assessed., Patients and Methods: This is a retrospective observational cross-sectional study on administrative data about sick leave requests after anti-COVID-19 vaccination. All the HCWs employed at the Niguarda Hospital (Milan, Italy) who received the vaccine from December 27, 2020 to February 28, 2021 were included., Results: In total, 4,088 HCWs received the first dose of the vaccine and 4,043 completed the vaccination cycle. After the first injection, 1.6% of HCWs requested sick leave, while after the second injection, the number of requests significantly increased (+6.1%, p<0.001). A significant increase in sick leave was detected for those who have had SARS-CoV-2 infection after the first injection (+2.3%, p<0.001). After the second dose, a significant increase in sick leave was observed in the 20-30-year-old group compared to >30 years (+3.6%, p=0.017), if HCWs without a history of SARS-CoV-2 infection were considered., Conclusions: The requests for sick leave among HCWs following the anti-COVID-19 vaccine were limited and higher after the second injection. This may help the management of the human resources when the large-scale administration of the anti-COVID-19 vaccines will involve other categories of workers.

The most common arrhythmia associated with COronaVIrus-related Disease (COVID) infection is sinus tachycardia. It is not known if high Heart Rate (HR) in COVID is simply a marker of higher systemic response to sepsis or if its persistence could be related to a long-term autonomic dysfunction. The aim of our work is to assess the prevalence of elevated HR at discharge in patients hospitalized for COVID-19 and to evaluate the variables associated with it. We enrolled 697 cases of SARS-CoV2 infection admitted in our hospital after February 21 and discharged within 23 July 2020. We collected data on clinical history, vital signs, laboratory tests and pharmacological treatment. Severe disease was defined as the need for Intensive Care Unit (ICU) admission and/or mechanical ventilation. Median age was 59 years (first-third quartile 49, 74), and male was the prevalent gender (60.1%). 84.6% of the subjects showed a SARS-CoV-2 related pneumonia, and 13.2% resulted in a severe disease. Mean HR at admission was 90 ± 18 bpm with a mean decrease of 10 bpm to discharge. Only 5.5% of subjects presented HR > 100 bpm at discharge. Significant predictors of discharge HR at multiple linear model were admission HR (mean increase = β = 0.17 per bpm, 95% CI 0.11; 0.22, p < 0.001), haemoglobin (β = -0.64 per g/dL, 95% CI -1.19; -0.09, p = 0.023) and severe disease (β = 8.42, 95% CI 5.39; 11.45, p < 0.001). High HR at discharge in COVID-19 patients is not such a frequent consequence, but when it occurs it seems strongly related to a severe course of the disease.

Objective: To conduct a clinical study of a family with neurologic symptoms and findings carrying a novel NOTCH3 mutation and to analyze the molecular consequences of the mutation., Methods: We analyzed a family with complex neurologic symptoms by MRI and neurologic examinations. Exome sequencing of the NOTCH3 locus was conducted, and whole-genome sequencing was performed to identify COL4A1 , COL4A2 , and HTRA1 mutations. Cell lines expressing the normal or NOTCH3 A1604T receptors were analyzed to assess proteolytic processing, cell morphology, receptor routing, and receptor signaling., Results: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary form of cerebral small vessel disease (SVD) and caused by mutations in the NOTCH3 gene. Most CADASIL mutations alter the number of cysteine residues in the extracellular domain of the NOTCH3 receptor, but in this article, we describe a family in which some members carry a novel cysteine-sparing NOTCH3 mutation (c.4810 G>A, p.Ala1604Thr). Two of 3 siblings heterozygous for the NOTCH3 A1604T mutation presented with migraine and white matter lesions (WMLs), the latter of a type related to but distinct from what is normally observed in CADASIL. Two other members instead carried a novel COL4A1 missense mutation (c.4795 G>A; p.(Ala1599Thr)). The NOTCH3 A1604T receptor was aberrantly processed, showed reduced presence at the cell surface, and less efficiently activated Notch downstream target genes., Conclusions: We identify a family with migraine and WML in which some members carry a cysteine-sparing hypomorphic NOTCH3 mutation. Although a causal relationship is not established, we believe that the observations contribute to the discussion on dysregulated Notch signaling in cerebral SVDs., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

This study aims to develop techniques for ankle joint kinematics analysis using motion capture based on stereophotogrammetry. The scope is to design marker attachments on the skin for a most reliable identification of the instantaneous helical axis, to be targeted for the fabrication of customized hinged ankle-foot orthoses. These attachments should limit the effects of the experimental artifacts, in particular the soft-tissue motion artifact, which affect largely the accuracy of any in vivo ankle kinematics analysis. Motion analyses were carried out on two healthy subjects wearing customized rigid shells that were designed through 3D scans of the subjects' lower limbs and fabricated by additive manufacturing. Starting from stereophotogrammetry data collected during walking and dorsi-plantarflexion motor tasks, the instantaneous and mean helical axes of ankle joint were calculated. The customized shells matched accurately the anatomy of the subjects and allowed for the definition of rigid marker clusters that improved the accuracy of in vivo kinematic analyses. The proposed methodology was able to differentiate between subjects and between the motor tasks analyzed. The observed position and dispersion of the axes were consistent with those reported in the literature. This methodology represents an effective tool for supporting the customization of hinged ankle-foot orthoses or other devices interacting with human joints functionality.

Infantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal dominant forms of IMF are caused by mutations in the PDGFRB gene, but a family carrying a L1519P mutation in the NOTCH3 gene has also recently been identified. In this report, we address the molecular consequences of the NOTCH3 L1519P mutation and the relationship between the NOTCH and PDGFRB signaling in IMF. The NOTCH3 L1519P receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3 L1519P receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3 L1519P receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3 L1519P extracellular domain and decreases signaling. Finally, NOTCH3 L1519P upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3-PDGFRB axis in IMF, where an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function., (© 2021. Published by The Company of Biologists Ltd.)

Photon bursts with a wavelength smaller than the plasma interparticle distance can drive plasma wakes via Compton scattering. We investigate this fundamental process analytically and numerically for different photon frequencies, photon flux, and plasma magnetization. Our results show that Langmuir and extraordinary modes are driven efficiently when the photon energy density lies above a certain threshold. The interaction of photon bursts with magnetized plasmas is of distinguished interest as the generated extraordinary modes can convert into pure electromagnetic waves at the plasma-vacuum boundary. This could possibly be a mechanism for the generation of radio waves in astrophysical scenarios in the presence of intense sources of high energy photons.

A positive prognosis of triple-negative breast cancer can be considered as one of the major challenges in clinical studies; accordingly, scientific research has the mission to find out novel chemotherapeutics to make it curable. In recent times, a good potential of dietary bioactive natural substances, called nutraceuticals, in suppressing cancer cell proliferation via gene expression regulation has been discovered: this effect and the lack of toxicity make nutraceuticals potentially effective agents against cancers. Monacolin K from red rice, a FDA-approved and well-tolerated compound generally employed to treat hypercholesterolemia, has been proved to have anti-proliferative and apoptotic effects in a wide panel of triple-negative breast cancers. Thus, an unbiased analysis of monacolin K-induced MDA-MB-231 cellular pathway alterations has been carried out by quantitative proteomics exploiting isobaric tags. Despite the positive modulation of some proteins already reported in the literature, an increased concentration of the tissue-type plasminogen activator PLAT has interestingly been found. This is a marker of good prognosis in mammary cancer, suggesting the anti-metastatic properties of this molecule as strongly associated with the alterations in the cytoskeleton organization and the consequent modulation of adhesion, motility and proteolysis. In accordance, some of the found monacolin K-induced phosphoproteome alterations have a tight connection to cell migration mechanisms. In this setting, the over-phosphorylation of Lamin A and of melanophilin induced by monacolin K has been very attractive. Moreover, monacolin K exerts its effect on the over-expression of the tissue inhibitor metalloproteinase-2 (TIMP-2), an endogenous metalloproteinase inhibitor. This protein modulates growth, migration and invasion of tumor cells and inhibits tumor angiogenesis.

We demonstrate the experimental feasibility of probing the fully nonperturbative regime of quantum electrodynamics with a 100 GeV-class particle collider. By using tightly compressed and focused electron beams, beamstrahlung radiation losses can be mitigated, allowing the particles to experience extreme electromagnetic fields. Three-dimensional particle-in-cell simulations confirm the viability of this approach. The experimental forefront envisaged has the potential to establish a novel research field and to stimulate the development of a new theoretical methodology for this yet unexplored regime of strong-field quantum electrodynamics.

Recent findings have shown that Farnesoid X Receptor (FXR) antagonists might be useful in the treatment of cholestasis and related metabolic disorders. In this paper, we report the discovery of a new chemotype of FXR antagonists featured by a 3,5-disubstituted oxadiazole core. In total, 35 new derivatives were designed and synthesized, and notably, compounds 3f and 13 , containing a piperidine ring, displayed the best antagonistic activity against FXR with promising cellular potency (IC 50 = 0.58 ± 0.27 and 0.127 ± 0.02 μM, respectively). The excellent pharmacokinetic properties make compound 3f the most promising lead identified in this study., Competing Interests: The authors declare no competing financial interest.

Acetaminophen misuse is a leading cause of acute liver failure and liver transplantation for which therapy is poorly effective. FXR ligands have shown effective in reducing liver injury in several experimental and clinical settings. In this Letter, we have elaborated on the structure of GW4064, the first nonsteroidal agonist for FXR, to identify novel isoxazoles endowed with FXR agonistic activity and improved ADME properties. The pharmacological characterization and molecular docking studies for the structure-activity rationalization allowed the identification of several FXR agonists with nanomolar potency in transactivation and SRC-1 recruitment assays. This characterization resulted in the identification of a potent FXR agonist, compound 20 that was orally active, and rescued mice from acute liver failure caused by acetaminophen overdose in a FXR-dependent manner., Competing Interests: The authors declare no competing financial interest.

The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.

Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub)types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.

In Fig. 1b of this Article, 'Csf1r' was misspelt 'Csfr1'. In addition, in Extended Data Fig. 11b, owing to an error during figure formatting, the genes listed in the first column shifted down three rows below the first gene on the list, causing a mismatch between the gene names and their characteristics. These errors have been corrected online, and the original Extended Data Fig. 11b is provided as Supplementary Information to the accompanying Amendment.

The quinoline-5,8 dicarboxylic acid scaffold has been identified by a fragment-based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, 3-(2,4-dimethoxypyrimidin-5-yl)quinoline-5,8-dicarboxylic acid (compound 3) shows low micromolar inhibitory activity against Jumonji domain-containing protein 3 (JMJD3). The experimental evaluation of inhibitory activity against seven related isoforms of JMJD3 highlighted an unprecedented selectivity toward the biological target of interest., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.

Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Extracts of Ruscus aculeatus are a rich source of bioactive steroidal glycosides, such as ruscogenins which are reported to act against chronic venous disorders. Nowadays, several preparations of its roots, commonly used in traditional medicine, are on the market as food supplements for health care and maintenance. Although spirostanol deglucoruscin is one of the main metabolites in these extracts, literature reports about its pharmacological profile are scarce. In this paper, a multi-disciplinary approach, based on chemical proteomics, molecular modelling and bio-organic assays, has been used to disclose the whole interactome of deglucoruscin and the F0-F1 ATP synthase complex has been found as its main target.

The Boswellia gum resin extracts have been used in traditional medicines because of their remarkable anti-inflammatory properties. Nowadays, these extracts are on the market as food supplements. β-Boswellic acid (βBA) is one of the main pentacyclic triterpene components, among the family of BAs, of the Boswellia gum resins. BAs have been broadly studied and are well known for their wide anti-inflammatory and potential anticancer properties. In this paper, a mass spectrometry-based chemoproteomic approach has been applied to characterize the whole βBA interacting profile. Among the large numbers of proteins fished out, proteasome, 14-3-3 and some ribosomal proteins were considered the most interesting targets strictly connected to the modulation of the cancer progression. In particular, because of their recent assessment as innovative chemotherapeutic targets, the ribosomal proteins were considered the most attractive βBA partners, and the biological role of their interaction with the natural compound has been evaluated. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Purpose: Systematic family-centered cancer care is needed. We conducted a randomized controlled trial of family therapy, delivered to families identified by screening to be at risk from dysfunctional relationships when one of their relatives has advanced cancer., Patients and Methods: Eligible patients with advanced cancer and their family members screened above the cut-off on the Family Relationships Index. After screening 1,488 patients or relatives at Memorial Sloan Kettering Cancer Center or three related community hospice programs, 620 patients (42%) were recruited, which represented 170 families. Families were stratified by three levels of family dysfunction (low communicating, low involvement, and high conflict) and randomly assigned to one of three arms: standard care or 6 or 10 sessions of a manualized family intervention. Primary outcomes were the Complicated Grief Inventory-Abbreviated (CGI) and Beck Depression Inventory-II (BDI-II). Generalized estimating equations allowed for clustered data in an intention-to-treat analysis., Results: On the CGI, a significant treatment effect (Wald χ(2) = 6.88; df = 2; P = .032) and treatment by family-type interaction was found (Wald χ(2) = 20.64; df = 4; P < .001), and better outcomes resulted from 10 sessions compared with standard care for low-communicating and high-conflict groups compared with low-involvement families. Low-communicating families improved by 6 months of bereavement. In the standard care arm, 15.5% of the bereaved developed a prolonged grief disorder at 13 months of bereavement compared with 3.3% of those who received 10 sessions of intervention (Wald χ(2) = 8.31; df = 2; P =.048). No significant treatment effects were found on the BDI-II., Conclusion: Family-focused therapy delivered to high-risk families during palliative care and continued into bereavement reduced the severity of complicated grief and the development of prolonged grief disorder., (© 2016 by American Society of Clinical Oncology.)

Bile acids are the endogenous modulators of the nuclear receptor FXR and the membrane receptor GPBAR1. FXR represents a promising pharmacological target for the treatment of cholestatic liver disorders. Currently available semisynthetic bile acid derivatives cover the same chemical space of bile acids and therefore they are poorly selective toward BA receptors, increasing patient risk for adverse side effects. In this report, we have investigated around the structure of CDCA describing the synthesis and the in vitro and in vivo pharmacological characterization of a novel family of compounds modified on the steroidal tetracyclic core and on the side chain. Pharmacological characterization resulted in the identification of several potent and selective FXR agonists. These novel agents might add utility in the treatment of cholestatic disorders by potentially mitigating side effects linked to unwanted activation of GPBAR1.

Numb asymmetrically segregates at mitosis to control cell fate choices during development. Numb inheritance specifies progenitor over differentiated cell fates, and, paradoxically, also promotes neuronal differentiation, thus indicating that the role of Numb may change during development. Here we report that Numb nuclear localization is restricted to early thymocyte precursors, whereas timed appearance of pre-T-cell receptor (pre-TCR) and activation of protein kinase Cθ promote phosphorylation-dependent Numb nuclear exclusion. Notably, nuclear localization of Numb in early thymocyte precursors favors p53 nuclear stabilization, whereas pre-TCR-dependent Numb nuclear exclusion promotes the p53 downmodulation essential for further differentiation. Accordingly, the persistence of Numb in the nucleus impairs the differentiation and promotes precursor cell death. This study reveals a novel regulatory mechanism for Numb function based on its nucleus-cytosol shuttling, coupling the different roles of Numb with different stages of T-cell development.

Background: Clinicians meet a variety of ethnicities among patients and families in hospice programs. This article focuses on Latino families., Methods: Within a controlled trial of family therapy in the context of palliative care, 17 families identified as Hispanic. Five were examined qualitatively herein., Results: A synopsis of each family's narrative is presented here. Patterns of strong family loyalty (Familismo), the gender roles of Machismo and Marianismo, the importance of family tradition, expectations about caregiving, and the place of faith and religion emerged as prominent and able potentially to impact on the therapy., Conclusions: Family therapists need to be thoughtful about cultural issues as they strive to support families.

Background: Supporting the family-as-a-whole presents challenges in palliative care, although family meetings are increasingly used in routine practice. The Family Focused Grief Therapy (FFGT) Model guides clinicians in using a range of intervention strategies., Aim: To examine the therapists' techniques used in assessing 'at risk' families in palliative care to better illuminate what helps and what remains challenging., Method: Recorded sessions 1 and 2 were coded using the FFGT fidelity coding measure, with its glossary of definitions. Inter-rater reliability between three coders was satisfactory at 88%. Frequencies of strategy utilization were computed, with extraction of examples of both successful and problematic approaches., Setting/participants: From within a larger study of family therapy during palliative care at a comprehensive cancer center, the first two sessions (n = 144) delivered to 74 families (299 individuals) by 32 therapists were coded and analyzed., Results: Therapists readily explored the story of illness and families' ways of coping (97%) and assessed communication and cohesiveness in the majority. Exploration of relational patterns occurred in 89% of sessions, use of a genogram in 80%, understanding members' roles in 65% and family values and beliefs in 62%. Less use was made of summaries (39%), family mottos (34%), exploration of family conflict (35%) and the formalization of a comprehensive family treatment plan (20%)., Conclusions: Challenges exist in therapy with difficult families. Therapy in the home brings special issues. Therapists can apply most of the interventions prescribed by the FFGT model.

In order to study effects of different total parenteral nutritional regimens on the recovery of liver function and on plasma amino acid pattern after major hepatectomy, twelve healthy mongrel dogs were submitted to 70% liver resection and randomly divided into three different groups: group I (control group) received only 10% glucose; group II received 10% glucose plus 8.5% Freamine; group III received 10% glucose plus 100% branched chain amino acid (BCAA) solution. Blood samples were analyzed for determining biochemical parameters, including plasma amino acid levels, before surgery and on 3rd and 7th postoperative day. The 'BCAA group', compared to other groups, showed higher values of plasma proteins (p less than 0.01) and prothrombin activity (p less than 0.05) on the 3rd postoperative day. The total concentration of plasma amino acids decreased after hepatectomy. Gluconeogenetic amino acids alanine and glutamine were markedly decreased in groups II and III, likely due to their increased consumption during gluconeogenesis. Aromatic amino acids were increased in all groups, due to the transient liver insufficiency. BCAA markedly increased in group III on 3rd postoperative day and returned to basal values on 7th postoperative day; possible explanations of such alterations are discussed. Our findings suggest that BCAA may represent a first choice substrate in the early phase after hepatectomy.

Some 2-substituted 3-pyridinolethers were synthesized, with one side chain bearing a carboxylic function; among these there are products having structure analogies with the prostaglandins. The results of hypolipidemic and hypocholesterolemic tests on experimental hyperdyslipidemic animals, as well as those on the inhibition of platelets aggregation and on fibrinolytic activity are reported.