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NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

The Coma Recovery Scale-Revised (CRS-R) is the recommended tool to assess consciousness in patients with prolonged Disorders of Consciousness (pDoC). However, the time needed to administer it may limit its use. A shorter tool has been validated: the Simplified Evaluation of CONsciousness Disorders (SECONDs). This multicentre study aimed to develop and validate a cross-cultural adaptation of the SECONDs into Italian. An interdisciplinary expert team, from both Fondazione Don Carlo Gnocchi and Istituto Neurologico Carlo Besta, led the translation processes. Independent certified translators were also involved in a blinded modality. Patients diagnosed with Unresponsive Wakefulness Syndrome (UWS) or Minimally Conscious State (MCS) admitted to 3 Italian rehabilitation units were enrolled. The CRS-R and SECONDs were administered in 5 sessions over two weeks by 3 blinded examiners at each center (3 times, with 2 sessions conducted by the same examiner). Weighted Fleiss' kappa and Spearman correlation coefficients were used to assess intrarater and interrater reliability and concurrent validity. Sixty adults with pDoC were assessed: 23 women; median age: 64 years; 14 trauma, median post-onset time: 2 months. Intrarater and interrater reliability showed almost perfect agreement (kappa coefficients 0.968 and 0.935, respectively; p<0.001). The comparison of CRS-R vs. SECONDs on the same day or the best out of 5 SECONDs/CRS-R led to a substantial to almost perfect agreement both for the total score of the CRS-R and the SECONDs' Additional Index (ρ = 0.772-1.000; p<0.001) and for the consciousness diagnosis (k = 0.784-0.935; p<0.001). The disagreement rate between the overall best diagnosis of the SECONDs and the best CRS-R diagnosis was 6.7%. The Italian version of the SECONDs has been cross-culturally adapted to serve as a shorter assessment tool for the diagnosis of pDoC. Our study shows its excellent reliability and concurrent validity when compared to the CRS-R.

ObjectivesCaregivers of patients diagnosed with disorders of consciousness (DoCs) play a pivotal role in the care pathway of these patients. Due to the high costs of care, among other symptoms and disorders previously described in the literature, they can manifest also mood and stress-related disorders which greatly impact their life and increase their burden. It is noteworthy to identify which factors are better related to the manifestation of mood and stress-related disorders to care for the caregivers in time. However, no studies have explored which factors are related to the manifestation of these disorders within this population yet.Materials and methodsWe explored with different questionnaires whether patient-, caregiver-, and caregiving environment-related factors are associated with mood and stress-related disorders on 114 caregivers of patients with DoCs.ResultsOur results showed that female caregivers manifested higher levels of both depression and prolonged grief disorder; furthermore, the presence of economic problems increased the levels of depression. Moreover, different levels of caregivers’ depression, anxiety, anger expression, and prolonged grief disorder were closely linked to the degree of behavioural responsiveness of the patients.ConclusionsOur results highlighted the need to consider also caregivers’ mood and stress-related disorders when defining the care pathway of patients with DoCs; indeed, caregivers constitute the main environment of DoC patients and they need tailored interventions aimed at reducing their burden due to caregiving.

There are [almost equal to]350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermostable mutants of a GPCR, the β1-adrenergic receptor. The most stable mutant receptor, βAR-m23, contained six point mutations that led to an apparent Tm 21°C higher than the native protein, and, in the presence of bound antagonist, βAR-m23 was as stable as bovine rhodopsin. In addition, βAR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand.

Magnani et al . describe a protocol to generate thermostable membrane proteins for structural analysis. This approach combines mutagenesis with a rapid, radioligand-based thermostability assay to screen and identify mutants with optimal stability. The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6–12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an 125 I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML. [Display omitted] In the context of CAR T cell immunotherapy, a proper balance between safety and efficacy should be evaluated before reaching the clinic. Arcangeli et al. demonstrated how context-dependent variables, i.e., CAR binding affinity, CAR expression, and target antigen density, modulate CAR T cell functionality in the context of AML targeting by an anti-CD123 CAR.

Recent studies suggest that COVID-19 survivors may experience long-term health consequences: in particular, neurological and mental health symptoms might be associated with long-term negative outcomes. This study is a secondary analysis of a larger cohort study and aims to determine the extent to which neurological and mental health sequelae are associated with survivors’ disability. Participants include COVID-19 survivors, with no pre-morbid brain conditions, who were discharged from the COVID-19 Unit of the ASST Spedali Civili Hospital between February and April 2020. At an average of 3.5 months after discharge, they were submitted to a neurological examination and completed the WHO Disability Assessment Schedule (WHODAS-12), the Hospital Anxiety and Depression Score, the Pittsburgh Sleep Quality Index and the Montreal Cognitive Assessment. Multivariable regression analysis was carried out to analyze variables that explain WHODAS-12 variation. In total, 83 patients (63 males, average age 66.9, 95% CI: 64.2–69.7) were enrolled; average WHODAS-12 was 13.2 (95% CI: 9.7–16.6). Cognitive dysfunction, anxiety, fatigue, and hyposmia/hypogeusia explained 28.8% of WHODAS-12 variation. These findings underline the importance and need for longitudinal follow-up assessments after recovery from COVID-19 and suggest the need for early rehabilitation of residual symptoms to enhance patients’ functioning.

Structural studies on mammalian integral membrane proteins have long been hampered by their instability in detergent. This is particularly true for the agonist conformation of G protein-coupled receptors (GPCRs), where it is thought that the movement of helices that occurs upon agonist binding results in a looser and less stable packing in the protein. Here, we show that mutagenesis coupled to a specific selection strategy can be used to stabilize the agonist and antagonist conformations of the adenosine A₂a receptor. Of the 27 mutations identified that improve the thermostability of the agonist conformation, only three are also present in the 17 mutations identified that improve the thermostability of the antagonist conformation, suggesting that the selection strategies used were specific for each conformation. Combination of the stabilizing mutations for the antagonist- or agonist-binding conformations resulted in mutants that are more stable at higher temperatures than the wild-type receptor by 17°C and 9°C, respectively. The mutant receptors both showed markedly improved stability in short-chain alkyl-glucoside detergents compared with the wild-type receptor, which will facilitate their structural analysis.

Sleep disorders are among the main comorbidities in patients with a Disorder of Consciousness (DOC). Given the key role of sleep in neural and cognitive functioning, detecting and treating sleep disorders in DOCs might be an effective therapeutic strategy to boost consciousness recovery and levels of awareness. To date, no systematic reviews have been conducted that explore the effect of sleep treatments in DOCs; thus, we systematically reviewed the existing studies on both pharmacological and non-pharmacological treatments for sleep disorders in DOCs. Among 2267 assessed articles, only 7 were included in the systematic review. The studies focused on two sleep disorder categories (sleep-related breathing disorders and circadian rhythm dysregulation) treated with both pharmacological (Modafinil and Intrathecal Baclofen) and non-pharmacological (positive airway pressure, bright light stimulation, and central thalamic deep brain stimulation) interventions. Although the limited number of studies and their heterogeneity do not allow generalized conclusions, all the studies highlighted the effectiveness of treatments on both sleep disorders and levels of awareness. For this reason, clinical and diagnostic evaluations able to detect sleep disorders in DOC patients should be adopted in the clinical routine for the purpose of intervening promptly with the most appropriate treatment.

The amount of knowledge on human consciousness has created a multitude of viewpoints and it is difficult to compare and synthesize all the recent scientific perspectives. Indeed, there are many definitions of consciousness and multiple approaches to study the neural correlates of consciousness (NCC). Therefore, the main aim of this article is to collect data on the various theories of consciousness published between 2007–2017 and to synthesize them to provide a general overview of this topic. To describe each theory, we developed a thematic grid called the dimensional model, which qualitatively and quantitatively analyzes how each article, related to one specific theory, debates/analyzes a specific issue. Among the 1130 articles assessed, 85 full texts were included in the prefinal step. Finally, this scoping review analyzed 68 articles that described 29 theories of consciousness. We found heterogeneous perspectives in the theories analyzed. Those with the highest grade of variability are as follows: subjectivity, NCC, and the consciousness/cognitive function. Among sub-cortical structures, thalamus, basal ganglia, and the hippocampus were the most indicated, whereas the cingulate, prefrontal, and temporal areas were the most reported for cortical ones also including the thalamo-cortical system. Moreover, we found several definitions of consciousness and 21 new sub-classifications.