CROSSalive: A web server for predicting the in vivo structure of RNA molecules

Motivation: RNA structure is difficult to predict in vivo due to interactions with enzymes and other molecules. Here we introduce CROSSalive, an algorithm to predict the single-and double-stranded regions of RNAs in vivo using predictions of protein interactions. Results: Trained on icSHAPE data in presence (m6a+) and absence of N6 methyladenosine modification (m6a-), CROSSalive achieves cross-validation accuracies between 0.70 and 0.88 in identifying high-confidence single-and double-stranded regions. The algorithm was applied to the long non-coding RNA Xist (17 900 nt, not present in the training) and shows an Area under the ROC curve of 0.83 in predicting structured regions

Zooming in on protein–RNA interactions: a multilevel workflow to identify interaction partners

Interactions between proteins and RNA are at the base of numerous cellular regulatory and functional phenomena. The investigation of the biological relevance of non-coding RNAs has led to the identification of numerous novel RNA-binding proteins (RBPs). However, defining the RNA sequences and structures that are selectively recognised by an RBP remains challenging, since these interactions can be transient and highly dynamic, and may be mediated by unstructured regions in the protein, as in the case of many non-canonical RBPs. Numerous experimental and computational methodologies have been developed to predict, identify and verify the binding between a given RBP and potential RNA partners, but navigating across the vast ocean of data can be frustrating and misleading. In this mini-review, we propose a workflow for the identification of the RNA binding partners of putative, newly identified RBPs. The large pool of potential binders selected by in-cell experiments can be enriched by in silico tools such as catRAPID, which is able to predict the RNA sequences more likely to interact with specific RBP regions with high accuracy. The RNA candidates with the highest potential can then be analysed in vitro to determine the binding strength and to precisely identify the binding sites. The results thus obtained can furthermore validate the computational predictions, offering an all-round solution to the issue of finding the most likely RNA binding partners for a newly identified potential RBP

Geoscience studies in the Maltese Islands: a gateway to the Central Mediterranean region

The location of the Maltese Islands, at the centre of the Mediterranean Sea, places them as a reference for regional climatic, tectonic and marine processes that have affected the Mediterranean basin. Our review work targeted the entire geoscientific literature produced about the Maltese Islands, covering the period from the 19th century to 2023. We identified ca. 390 papers, by using the main literature databases, and classified them according to the main scientific fields. Geomorphology, stratigraphy and sedimentology represent the main research topics, followed by palaeontology and structural geology. We analysed the temporal distribution of this scientific production, highlighting an overall increasing trend in the number of papers over time. This reveals a growing interest in the geoscientific research related to the Maltese Islands. The review emphasises collaborative efforts between scientists from different fields, illustrating the interdisciplinary nature of geoscientific research in Malta. Our results show that the scientific collaboration between authors, and affiliated institutions, is also based on the shared history and linguistic ties, among the Maltese, Italian and British cultures. The outputs of our review aim to be a comprehensive reference for those undertaking geoscientific research on the Islands

In-hospital and out-of-hospital stroke in patients with COVID-19: two different diseases?

Background: Ischemic stroke is a known complication of COVID-19. It may have a different pathogenesis and worse outcome compared to stroke in patients without COVID-19. Furthermore, patients with COVID-19 and out-of-hospital stroke onset might have different characteristics compared to patients with COVID-19 and in-hospital stroke onset. The aim of our study was to analyze the characteristics of patients with stroke with and without COVID-19 and of patients with COVID-19 with in-hospital and out-of-hospital stroke. Methods: We performed a retrospective study of all consecutive patients admitted to our hospital with ischemic stroke between October 2020 and February 2021. We compared functional outcome, lab test, demographic, and clinical characteristics between patients with or without COVID-19. We performed a sub-analysis comparing patients with COVID-19 and in-hospital and out-of-hospital stroke onset. Results: We included in the final analysis 137 patients of whom 26 with COVID-19. Half (13) had out-of-hospital stroke and half in-hospital stroke onset. Overall, patients with COVID-19 had higher mortality compared to the control group (27% vs 9%, p: 0.02), and non-significantly lower rate of good functional outcome (50% vs 63%, p: 0.22). Patients with COVID-19 and out-of-hospital stroke had higher rate of good functional outcome (69% vs 39%, p: 0.05), higher lymphocyte count, and lower D-dimer compared with patients with in-hospital stroke onset. Conclusions: Patients with stroke and COVID-19 had higher mortality compared to patients without COVID-19. Among patients with COVID-19 those with out-of-hospital stroke had better outcome and fewer blood test abnormalities compared to patients with in-hospital stroke

New development: Directly elected mayors in Italy: creating a strong leader doesn’t mean creating strong leadership

More than 20 years after their introduction, directly elected mayors are key players in Italian urban governance. This article explains the main effects of this reform on local government systems and provides lessons for other countries considering directly elected mayors

Proteome-wide observation of the phenomenon of life on the edge of solubility

To function effectively proteins must avoid aberrant aggregation, and hence they are expected to be expressed at concentrations safely below their solubility limits. By analyzing proteome-wide mass spectrometry data of Caenorhabditis elegans, however, we show that the levels of about three-quarters of the nearly 4, 000 proteins analyzed in adult animals are close to their intrinsic solubility limits, indeed exceeding them by about 10% on average. We next asked how aging and functional self-assembly influence these solubility limits. We found that despite the fact that the total quantity of proteins within the cellular environment remains approximately constant during aging, protein aggregation sharply increases between days 6 and 12 of adulthood, after the worms have reproduced, as individual proteins lose their stoichiometric balances and the cellular machinery that maintains solubility undergoes functional decline. These findings reveal that these proteins are highly prone to undergoing concentration-dependent phase separation, which on aging is rationalized in a decrease of their effective solubilities, in particular for proteins associated with translation, growth, reproduction, and the chaperone system

Enzyme stability in nanoparticle preparations part 1: Bovine serum albumin improves enzyme function

Enzymes have gained attention for their role in numerous disease states, calling for research for their efficient delivery. Loading enzymes into polymeric nanoparticles to improve biodistribution, stability, and targeting in vivo has led the field with promising results, but these enzymes still suffer from a degradation effect during the formulation process that leads to lower kinetics and specific activity leading to a loss of therapeutic potential. Stabilizers, such as bovine serum albumin (BSA), can be beneficial, but the knowledge and understanding of their interaction with enzymes are not fully elucidated. To this end, the interaction of BSA with a model enzyme B-Glu, part of the hydrolase class and linked to Gaucher disease, was analyzed. To quantify the natural interaction of beta-glucosidase (B-Glu,) and BSA in solution, isothermal titration calorimetry (ITC) analysis was performed. Afterwards, polymeric nanoparticles encapsulating these complexes were fully characterized, and the encapsulation efficiency, activity of the encapsulated enzyme, and release kinetics of the enzyme were compared. ITC results showed that a natural binding of 1:1 was seen between B-Glu and BSA. Complex concentrations did not affect nanoparticle characteristics which maintained a size between 250 and 350 nm, but increased loading capacity (from 6% to 30%), enzyme activity, and extended-release kinetics (from less than one day to six days) were observed for particles containing higher B-Glu:BSA ratios. These results highlight the importance of understanding enzyme:stabilizer interactions in various nanoparticle systems to improve not only enzyme activity but also biodistribution and release kinetics for improved therapeutic effects. These results will be critical to fully characterize and compare the effect of stabilizers, such as BSA with other, more relevant therapeutic enzymes for central nervous system (CNS) disease treatments

Middle cerebral artery ischemic stroke and COVID-19: a case report

We present a clinical case of a patient with SARS-CoV-2 infection and respiratory symptoms, complicated with a pro-thrombotic state involving multiple vascular territories and concomitant interleukin-6 increase. This case underlines the possibility to develop a COVID-19-related coagulopathy

Why Me? To Be an Ultra-Responder to Antiplatelet Therapy: A Case Report

Background: Platelet function testing is a valid tool to investigate the clinical response to antiplatelet therapy in different clinical settings; in particular, it might supply helpful information in patients with cerebrovascular disease. Oral antiplatelet treatment, such as Aspirin (ASA) and Clopidogrel, is the gold standard in secondary stroke prevention of non-cardiogenic ischemic stroke; conversely, its application as a primary prevention therapy is not routinely recommended in patients with vascular risk factors. Multiple electrode platelet aggregometry (MEA) impedance aggregometer is a validated device to test platelet inhibition induced by ASA or Clopidogrel. Case Report: We report the case of a 78-year-old patient without relevant clinical history, taking ASA as primary prevention strategy, who was admitted for sudden onset of dysarthria and left facial hyposthenia during physical effort. Brain CT revealed two small subcortical bilateral spontaneous intracranial hemorrhages. Platelet aggregometry with MEA performed upon admission revealed a very strong platelet inhibition induced by ASA (result of the ASPI Test was 5 U, consistent with an ultra-responsiveness to ASA, and the cutoff value of correct responsiveness is <40 U). MRI at longitudinal follow-up revealed the presence of two small cavernous angioma underlying hemorrhagic spots. Conclusion: The evaluation of platelet reactivity in stroke patients undergoing antiplatelet therapies, not commonly performed in clinical practice, could be useful to optimize prevention strategies; the verification of the biological effectiveness of ASA or Clopidogrel could be a valid tool in the definition of each patient's risk profile, particularly in patients with cerebrovascular disease known to be at increased risk for both hemorrhagic and thrombotic complications

Strategies for Improved pDNA Loading and Protection Using Cationic and Neutral LNPs with Industrial Scalability Potential Using Microfluidic Technology

Purpose: In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity. Methods: The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical “post-formulation” DNA addition vs “pre” addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake. Results: Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 μg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 μg/mL of pDNA with the “post” method, while maintaining their characteristics up to 100 μg/mL of pDNA with the “pre” addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 μg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes. Conclusion: In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy