Cryosectioning-enabled super-resolution microscopy for studying nuclear architecture at the single protein level.

DNA-PAINT combined with total Internal Reflection Fluorescence (TIRF) microscopy enables the highest localization precisions, down to single nanometers in thin biological samples, due to TIRF's unique method for optical sectioning and attaining high contrast. However, most cellular targets elude the accessible TIRF range close to the cover glass and thus require alternative imaging conditions, affecting resolution and image quality. Here, we address this limitation by applying ultrathin physical cryosectioning in combination with DNA-PAINT. With "tomographic & kinetically-enhanced" DNA-PAINT (tokPAINT), we demonstrate the imaging of nuclear proteins with sub-3 nanometer localization precision, advancing the quantitative study of nuclear organization within fixed cells and mouse tissues at the level of single antibodies. We believe that ultrathin sectioning combined with the versatility and multiplexing capabilities of DNA-PAINT will be a powerful addition to the toolbox of quantitative DNA-based super-resolution microscopy in intracellular structural analyses of proteins, RNA and DNA in situ.

PRECOGx: exploring GPCR signaling mechanisms with deep protein representations.

In this study we show that protein language models can encode structural and functional information of GPCR sequences that can be used to predict their signaling and functional repertoire. We used the ESM1b protein embeddings as features and the binding information known from publicly available studies to develop PRECOGx, a machine learning predictor to explore GPCR interactions with G protein and ß-arrestin, which we made available through a new webserver (https://precogx.bioinfolab.sns.it/). PRECOGx outperformed its predecessor (e.g. PRECOG) in predicting GPCR-transducer couplings, being also able to consider all GPCR classes. The webserver also provides new functionalities, such as the projection of input sequences on a low-dimensional space describing essential features of the human GPCRome, which is used as a reference to track GPCR variants. Additionally, it allows inspection of the sequence and structural determinants responsible for coupling via the analysis of the most important attention maps used by the models as well as through predicted intramolecular contacts. We demonstrate applications of PRECOGx by predicting the impact of disease variants (ClinVar) and alternative splice forms from healthy tissues (GTEX) of human GPCRs, revealing the power to dissect system biasing mechanisms in both health and disease.

Down-regulation of adenosine A1 and A2A receptors in peripheral cells from idiopathic normal-pressure hydrocephalus patients.

Idiopathic normal-pressure hydrocephalus (iNPH) is a neurological disease that usually develops in the elderly. Natural history of iNPH is still unknown. It has been hypothesized that cerebrovascular diseases could have a role in etiology of chronic hydrocephalus and studies show an increased prevalence of cardiovascular diseases in iNPH patients. Moreover, evidences show a possible alteration of immune system in iNPH patients. Adenosine (Ado) is a metabolite produced in response to metabolic stress and injury. Adenosine and its receptors play an important role in vascular protection and in the modulation of inflammatory reactions and neuroinflammation. Our aim is to evaluate gene and protein expression of A1R and A2AR in the peripheral blood mononuclear cells (PBMCs) from iNPH patients compared to control subjects. We investigate if Ado system, that plays an important role in central nervous system, in vascular system, and also in inflammation, is involved in pathophysiology of iNPH disease. Our analysis showed that A1R mRNA levels and A1R density in PBMCs from iNPH patients were significantly lower than CT subjects (0.84 ± 0.12 and 2.42 ± 0.42, p<0.001 and 0.31 ± 0.02 and 0.42 ± 0.04, p=0.043; respectively). About A2AR, the gene expression in PBMCs was significantly lower in iNPH than CT (0.65 ± 0.09 and 1.5 ± 0.14, p<0.001) as well as there was a trend in protein expression: iNPH and CT (0.51 ± 0.05 and 0.62 ± 0.03; p=0.172). This preliminary study underlines the involvement of Ado system in iNPH disease whose pathophysiology is still unclear.

Analysis of propofol/remifentanil infusion protocol for tumor surgery with intraoperative brain mapping.

BACKGROUND: There is no general consensus about the best anesthesiologic approach to use during craniotomies with intraoperative brain mapping, and large prospective studies evaluating the complications associated with different approaches are lacking. Objective of this study was to prospectively collect and evaluate data about a large series of consecutive asleep-awake and asleep-asleep craniotomies. METHODS: We analyzed 238 consecutive procedures from January 2005 to December 2008. During asleep-awake procedures, patients were initially ventilated through a laryngeal mask which was removed to allow language testing. During asleep-asleep procedures, patients remained sedated and intubated to permit motor testing. RESULTS: In asleep-awake craniotomies [n=135, age 42 y (range: 16 to 72 y), American Society of Anesthesiologists classification (ASA) 1 (1 to 3), and body mass index 24.2+/-3.7 kg/m], 43% of the procedures were free of complications. Most common complications were hypertension (27%) and brief clinical seizures (16%), but also hypotension (10%), vomiting (7%), brief periods of apnea (4%), and agitation (6%) were observed. In 7% of the procedures, seizures required pharmacologic treatment. Fifty-nine percent of the asleep-asleep procedures [n=103, age 51 y (range: 21 to 76 y), ASA 1 (1 to 3), body mass index 25.4+/-3.9 kg/m, P<0.05 vs. asleep-awake] were free of complications. Clinical seizures were observed in 31% of the cases. The administration of boluses of hypnotics was rarely necessary (6%) and safer because of secured airways. CONCLUSIONS: With this study, we demonstrated the feasibility and safety of our protocols on large prospective case series. Asleep-awake protocol can be safely used when intraoperative language mapping is planned, whereas an asleep-asleep protocol with secured airway might be preferred when motor testing only is required.