An integrative epigenome-based strategy for unbiased functional profiling of clinical kinase inhibitors.

More than 500 kinases are implicated in the control of most cellular process in mammals, and deregulation of their activity is linked to cancer and inflammatory disorders. 80 clinical kinase inhibitors (CKIs) have been approved for clinical use and hundreds are in various stages of development. However, CKIs inhibit other kinases in addition to the intended target(s), causing both enhanced clinical effects and undesired side effects that are only partially predictable based on in vitro selectivity profiling. Here, we report an integrative approach grounded on the use of chromatin modifications as unbiased, information-rich readouts of the functional effects of CKIs on macrophage activation. This approach exceeded the performance of transcriptome-based approaches and allowed us to identify similarities and differences among CKIs with identical intended targets, to recognize novel CKI specificities and to pinpoint CKIs that may be repurposed to control inflammation, thus supporting the utility of this strategy to improve selection and use of CKIs in clinical settings.

Worldwide prevalence of obsessive-compulsive symptoms during the COVID-19 pandemic: A systematic review and meta-analysis.

During the COVID-19 pandemic governments worldwide implemented contagion-containing measures (i.e., physical distancing, hand sanitizing, mask wearing and quarantine). The similarities between these measures and obsessive-compulsive phenomenology (e.g., contamination concerns and repetitive washing and/or checking) led to inquiries about the frequency with which obsessive-compulsive symptoms (OCS) were encountered during the COVID-19 pandemic. We conducted a systematic review and meta-analysis to ascertain the prevalence of OCS in individuals of any age during the pandemic (i.e., any obsessive-compulsive symptoms that are clinically significant as shown by a score above the cut-off score of a scale, without necessarily fulfilling the diagnostic threshold for a diagnosis of OCD). A systematic search of relevant databases identified 35 studies, which were included in the systematic review following our inclusion and exclusion criteria. Most of the studies were conducted in adults from the general population and adopted an online assessment method, with 32 studies being eligible for meta-analysis. The meta-analysis resulted in a 20% average prevalence of OCS during the pandemic, with very high heterogeneity among the included studies (I2 99.6%). The highest prevalence of OCS was found in pregnant women (36%, n = 5), followed by individuals diagnosed with COVID-19 (22%, n = 4) and general population (22%, n = 19), undergraduates (21%, n = 5), and healthcare workers (5%, n = 5). The prevalence rates of OCS were higher in Asia (26%, n = 17) and North America (25%, n = 3) than in Europe (13%, n = 12) and Africa (7%, n = 4). Among the studies included, rates appeared higher in certain countries, though this difference did not reach statistical significance and was limited by very few studies conducted in certain countries. When compared to pre-pandemic rates, there seemed to be higher rates of OCS during the COVID-19 pandemic in Asia, Europe, and pregnant women. These findings are discussed considering the impact of the pandemic and contagion-containing measures on the perception and reporting of OCS, and susceptibility of the vulnerable population groups to experiencing OCS during the pandemic.

Perspectives on the impact of vortioxetine on the treatment armamentarium of major depressive disorder.

INTRODUCTION: Major Depressive Disorder (MDD) is a mental health issue that significantly affects patients' quality of life and functioning. Despite available treatments, many patients continue to suffer due to incomplete symptom resolution and side effects. AREAS COVERED: This manuscript examines Vortioxetine's role in Major Depressive Disorder (MDD) treatment, highlighting its potential to reshape therapeutic strategies due to its unique Multimodal action and proven broad-spectrum efficacy in multiple depressive domains. A detailed examination of Vortioxetine's pharmacological aspects, including indications, dosage, pharmacodynamics, and pharmacokinetics, is provided, emphasizing its safety and effectiveness. The discussion extends to Vortioxetine's role in acute-phase treatment and maintenance of MDD and its profound impact on specialized depression domains. EXPERT OPINION: Vortioxetine is distinguished for its novel multimodal serotonin modulation mechanism, showcasing significant promise as an innovative treatment for MDD. Its efficacy, which is dose-dependent, along with a commendable tolerability profile, positions it as a potential leading option for initial treatment strategies. The discourse on dosage titration, particularly the strategy of initiating treatment at lower doses followed by gradual escalation, underscores the approach toward minimizing initial adverse effects while optimizing therapeutic outcomes, aligning with the principles of personalized medicine in psychiatric care.

Efficacy and safety of ketamine and esketamine for unipolar and bipolar depression: an overview of systematic reviews with meta-analysis.

Background: Unipolar and bipolar depression present treatment challenges, with patients sometimes showing limited or no response to standard medications. Ketamine and its enantiomer, esketamine, offer promising alternative treatments that can quickly relieve suicidal thoughts. This Overview of Reviews (OoR) analyzed and synthesized systematic reviews (SRs) with meta-analysis on randomized clinical trials (RCTs) involving ketamine in various formulations (intravenous, intramuscular, intranasal, subcutaneous) for patients with unipolar or bipolar depression. We evaluated the efficacy and safety of ketamine and esketamine in treating major depressive episodes across various forms, including unipolar, bipolar, treatment-resistant, and non-resistant depression, in patient populations with and without suicidal ideation, aiming to comprehensively assess their therapeutic potential and safety profile. Methods: Following PRIOR guidelines, this OoR's protocol was registered on Implasy (ID:202150049). Searches in PubMed, Scopus, Cochrane Library, and Epistemonikos focused on English-language meta-analyses of RCTs of ketamine or esketamine, as monotherapy or add-on, evaluating outcomes like suicide risk, depressive symptoms, relapse, response rates, and side effects. We included studies involving both suicidal and non-suicidal patients; all routes and formulations of administration (intravenous, intramuscular, intranasal) were considered, as well as all available comparisons with control interventions. We excluded meta-analysis in which the intervention was used as anesthesia for electroconvulsive therapy or with a randomized ascending dose design. The selection, data extraction, and quality assessment of studies were carried out by pairs of reviewers in a blinded manner. Data on efficacy, acceptability, and tolerability were extracted. Results: Our analysis included 26 SRs and 44 RCTs, with 3,316 subjects. The intervention is effective and well-tolerated, although the quality of the included SRs and original studies is poor, resulting in low certainty of evidence. Limitations: This study is limited by poor-quality SRs and original studies, resulting in low certainty of the evidence. Additionally, insufficient available data prevents differentiation between the effects of ketamine and esketamine in unipolar and bipolar depression. Conclusion: While ketamine and esketamine show promising therapeutic potential, the current evidence suffers from low study quality. Enhanced methodological rigor in future research will allow for a more informed application of these interventions within the treatment guidelines for unipolar and bipolar depression. Systematic review registration: [https://inplasy.com/inplasy-2021-5-0049/], identifier (INPLASY202150049).

Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide.

Histone-modifying enzymes depend on the availability of cofactors, with acetyl-coenzyme A (CoA) being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA-producing enzymes translocate to the nucleus suggests that high concentrations of locally synthesized metabolites may impact acylation of histones and other nuclear substrates, thereby controlling gene expression. Here, we show that 2-ketoacid dehydrogenases are stably associated with the Mediator complex, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in lipopolysaccharide-stimulated macrophages, inhibited the activity of Mediator-associated 2-ketoacid dehydrogenases. Elevation of NO levels and the disruption of Mediator complex integrity both affected de novo histone acetylation within a shared set of genomic regions. Our findings indicate that the local supply of acetyl-CoA generated by 2-ketoacid dehydrogenases bound to Mediator is required to maximize acetylation of histone tails at sites of elevated HAT activity.

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription.

Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements. The Restrictor complex, composed of the Pol II-interacting protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription at thousands of extragenic sites in mammalian genomes. Restrictor-driven termination does not involve nascent RNA cleavage, and its interplay with other termination machineries is unclear. Here we show that efficient termination at Restrictor-controlled extragenic transcription units involves the recruitment of the protein phosphatase 1 (PP1) regulatory subunit PNUTS, a negative regulator of the SPT5 elongation factor, and Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent and phosphatase-dependent termination of noncoding RNAs in yeast. PNUTS and Symplekin act synergistically with, but independently from, Restrictor to dampen processive extragenic transcription. Moreover, the presence of limiting nuclear levels of Symplekin imposes a competition for its recruitment among multiple transcription termination machineries, resulting in mutual regulatory interactions. Hence, by synergizing with Restrictor, Symplekin and PNUTS enable efficient termination of processive, long-range extragenic transcription.

The impact of COVID-19 pandemic in patients with a diagnosis of Oral Lichen Planus: Pain perception and psychological profile analysis.

OBJECTIVES: To assess the impact of COVID-19 in patients affected by OLP, in terms of level of pain, stress, depression and anxiety and their impact on the clinical manifestation of the disease. MATERIAL AND METHODS: A longitudinal design was employed. Psychometric evaluations of anxiety, stress, and depression were conducted using the DASS21 scale, while pain levels were measured using the VAS scale. Clinical diagnosis and phenotype evaluation were performed. RESULTS: The study included 24 patients with an average age of 62.9 years, with 70.8% presenting erosive OLP. Results revealed a significant worsening of anxiety, stress, and depression scores during the pandemic. Pain level (1.5 ± 1.2 pre-pandemic VS 3.8 ± 1.1 during the pandemic, p < 0.0001) was also negatively affected. CONCLUSIONS: These findings highlight the potential interplay between psychological stress and oral health conditions, emphasizing the need for a comprehensive understanding of OLP's complex etiology and its response to external stressors. CLINICAL RELEVANCE: Multidisciplinary care strategies to address both physical and psychological aspects of OLP patients is recommended following the present findings. Further research is warranted to confirm these observations in larger multicenter studies and to guide tailored guidance approaches for OLP patients during challenging times.

Is intravenous valproate more efficacious than oral valproate for inpatients with bipolar I disorder with a manic or depressive episode and concomitant symptoms of opposite polarity?

INTRO: Valproic acid (VPA) is a commonly prescribed mood stabilizer, available in both oral (OS) and intravenous (IV) formulations. However, few studies have compared their safety and efficacy. This retrospective study aimed to investigate the safety and efficacy of and IV-VPA in patients with Bipolar Disorder. METHODS: Fifty patients with Bipolar Disorder experiencing a manic or depressive episode, with concomitant symptoms of opposite polarity, admitted to our inpatient unit and treated with IV-VPA were included in a retrospective, single-centre, non-randomized, open-label, parallel-group comparative study. Fifty patients experiencing a manic or depressive episode, with concomitant symptoms of opposite polarity, treated with oral-VPA and selected among those who were admitted to the inpatient unit prior to the introduction of IV-VPA in our clinical practice, were included as the control group (matched based on age, gender and clinical scales score at baseline). The Clinical Global Impression (CGI), Young Mania Rating Scale (YMRS), Montgomery-Asberg Depression Rating Scale (MADRS) and Hamilton Anxiety Rating Scale (HAM-A) scores were recorded at baseline, after 3 days of treatment and discharge from the inpatient unit. Patients were asked to respond on the basis of the symptoms present on the day the scale was administered. Response rate and the presence of adverse effects were also recorded. RESULTS: Both patients treated with oral and IV-VPA demonstrated significant improvements in all psychometric scales (p < 0.001). However, the IV group exhibited superior efficacy, with significantly lower scores on the CGI, YMRS, MADRS and HAM-A scales on Day 3 and at discharge from the inpatient unit. The IV-VPA treatment showed higher response rates on all psychometric scales, and no adverse effects were reported in either group. CONCLUSION: This retrospective study supports the use of IV-VPA as a more efficacious treatment option for patients with Bipolar Disorder, particularly in acute settings where rapid symptom improvement is crucial. Both oral and IV-VPA were found to be safe and well-tolerated.

Drug-drug interactions between COVID-19 therapeutics and psychotropic medications.

INTRODUCTION: The coronavirus (COVID-19) pandemic has led to as well as exacerbated mental health disorders, leading to increased use of psychotropic medications. Co-administration of COVID-19 and psychotropic medications may result in drug-drug interactions (DDIs), that may compromise both the safety and efficacy of both medications. AREAS COVERED: This review provides an update of the current evidence on DDIs between COVID-19 and psychotropic medications. The interactions are categorized into pharmacokinetic, pharmacodynamic, and other relevant types. A thorough literature search was conducted using electronic databases to identify relevant studies, and extract data to highlight potential DDIs, clinical implications, and management strategies. EXPERT OPINION: Understanding and managing potential DDIs between COVID-19 and psychotropic medications is paramount to ensuring safe and effective treatment of patients with COVID-19 and mental illness. Awareness of the diverse spectrum of DDIs, vigilant monitoring, and judicious dose modifications, while choosing pharmacotherapeutic options with low risk of interaction whenever possible, are necessary. Ongoing and future investigations should continue to review the dynamic landscape of COVID-19 therapeutic modalities and their interactions with psychotropic medications.

GTF2I dosage regulates neuronal differentiation and social behavior in 7q11.23 neurodevelopmental disorders.

Copy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations. Deletion causes Williams-Beuren syndrome featuring hypersociability, while duplication causes 7q11.23 microduplication syndrome (7Dup), frequently exhibiting autism spectrum disorder (ASD). Converging evidence indicates GTF2I as key mediator of the cognitive-behavioral phenotypes, yet its role in cortical development and behavioral hallmarks remains largely unknown. We integrated proteomic and transcriptomic profiling of patient-derived cortical organoids, including longitudinally at single-cell resolution, to dissect 7q11.23 dosage-dependent and GTF2I-specific disease mechanisms. We observed dosage-dependent impaired dynamics of neural progenitor proliferation, transcriptional imbalances, and highly specific alterations in neuronal output, leading to precocious excitatory neuron production in 7Dup, which was rescued by restoring physiological GTF2I levels. Transgenic mice with Gtf2i duplication recapitulated progenitor proliferation and neuronal differentiation defects alongside ASD-like behaviors. Consistently, inhibition of lysine demethylase 1 (LSD1), a GTF2I effector, was sufficient to rescue ASD-like phenotypes in transgenic mice, establishing GTF2I-LSD1 axis as a molecular pathway amenable to therapeutic intervention in ASD.

Autistic symptoms in unaffected first-degree relatives of people with schizophrenia: results from the Italian Network for Research on Psychoses multicenter study.

BACKGROUND: Autistic symptoms represent a frequent feature in schizophrenia spectrum disorders (SSD). However, the prevalence and the cognitive and functional correlates of autistic symptoms in unaffected first-degree relatives of people with SSD remain to be assessed. METHODS: A total of 342 unaffected first-degree relatives related to 247 outpatients with schizophrenia were recruited as part of the multicenter study of the Italian Network for Research on Psychoses (NIRP). Autistic features were measured with the PANSS Autism Severity Scale. Three groups of participants, defined on the presence and severity of autistic symptoms, were compared on a wide array of cognitive and functional measures. RESULTS: Of the total sample, 44.9% presented autistic symptoms; 22.8% showed moderate levels of autistic symptoms, which can be observed in the majority of people with SSD. Participants with higher levels of autistic symptoms showed worse performance on Working Memory (p = 0.014) and Social Cognition (p = 0.025) domains and in the Global Cognition composite score (p = 0.008), as well as worse on functional capacity (p = 0.001), global psychosocial functioning (p < 0.001), real-world interpersonal relationships (p < 0.001), participation in community activities (p = 0.017), and work skills (p = 0.006). CONCLUSIONS: A high prevalence of autistic symptoms was observed in first-degree relatives of people with SSD. Autistic symptoms severity showed a negative correlation with cognitive performance and functional outcomes also in this population and may represent a diagnostic and treatment target of considerable scientific and clinical interest in both patients and their first-degree relatives.

Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells.

Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases.

Vulnerabilities in Mental Health due to Covid-19 Pandemic: The Response of the Italian Physicians.

COVID-19 pandemic has exacerbated the pre-existing vulnerabilities and inequalities in societies. In this paper we analyse the categories that have suffered more than others from the pandemic and the restrictions on social life in terms of mental health. We rely on the Serendipity project based on a survey administered between November 2021 and February 2022 to a sample of Italian physicians (n = 1281). The survey aimed to assess the perception of general practitioners, paediatricians, geriatricians, and mental health specialists (psychiatrists, neurologists, child neuropsychiatrists), about changes in the mental health of the population as an effect of the COVID-19 pandemic and the lockdown. The strategies implemented by the doctors interviewed in terms of the intensity of the prevention, emergence, and treatment of mental health interventions, and their association with physicians' characteristics and their opinions on patient vulnerability have been illustrated by means of a multiple correspondence analysis. An overall result of the survey is the consensus of doctors on the worsening of mental health in general population, especially among their patients, due to the pandemic and on the onset of new discomforts. The most exposed individuals to the risk of onset or worsening of mental disorders include women, young people, and patients with psychiatric comorbidity. The paper also illustrates the interventions put in place by the physicians and deemed necessary from a public heath response perspective, that include providing psychoeducation to the general population, improving telehealth services, and increasing financial and human resources for community-based care.

FAM46C Is an Interferon-Stimulated Gene That Inhibits Lentiviral Particle Production by Modulating Autophagy.

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment.

Exploratory study of ultraviolet B (UVB) radiation and age of onset of bipolar disorder.

BACKGROUND: Sunlight contains ultraviolet B (UVB) radiation that triggers the production of vitamin D by skin. Vitamin D has widespread effects on brain function in both developing and adult brains. However, many people live at latitudes (about > 40 N or S) that do not receive enough UVB in winter to produce vitamin D. This exploratory study investigated the association between the age of onset of bipolar I disorder and the threshold for UVB sufficient for vitamin D production in a large global sample. METHODS: Data for 6972 patients with bipolar I disorder were obtained at 75 collection sites in 41 countries in both hemispheres. The best model to assess the relation between the threshold for UVB sufficient for vitamin D production and age of onset included 1 or more months below the threshold, family history of mood disorders, and birth cohort. All coefficients estimated at P ≤ 0.001. RESULTS: The 6972 patients had an onset in 582 locations in 70 countries, with a mean age of onset of 25.6 years. Of the onset locations, 34.0% had at least 1 month below the threshold for UVB sufficient for vitamin D production. The age of onset at locations with 1 or more months of less than or equal to the threshold for UVB was 1.66 years younger. CONCLUSION: UVB and vitamin D may have an important influence on the development of bipolar disorder. Study limitations included a lack of data on patient vitamin D levels, lifestyles, or supplement use. More study of the impacts of UVB and vitamin D in bipolar disorder is needed to evaluate this supposition.

Employment and Social Security/Insurance among patients affected by mental disorders in Italy: A descriptive multi-center study.

INTRODUCTION: Many mental disorders especially chronic serious ones such as schizophrenia-spectrum disorders, are disabling syndromes and impact on patients' social and cognitive functioning, including work activity. Thus, affected patients may show a particular socio-economic vulnerability and need specific social security as well as rehabilitation interventions, including pensions or job-placements. In Italy, the Working Group named 'Employment and Social Security/Insurance in Mental Health (ESSIMH)' was founded in 2020 in order to collect research evidence on mental illness, employment, social security, and rehabilitation. METHODS: A descriptive, observational and multi-center study has been conducted in eleven Departments of Mental Health in Italy (Foggia, Brindisi, Putignano, Rome, Bologna, Siena, Pavia, Mantova, Genova, Brescia, and Torino) and involved 737 patients affected by major mental illness and classified in five diagnostic categories: psychoses, mood disorders, personality disorders, anxiety disorders, and others. The data collection was performed in 2020 among patients aged 18 to 70 years old. RESULTS: The rate of employment in our sample was 35.8% (n = 264). Occupational disability in our sample was recognized in 58.0% of patients with a mean percentage of severity 51.7 ± 43.1; patients with psychoses (73%) reported higher disability followed by personality (60%) and mood disorders (47.3%) ones. In a logistic multivariate modeling, factors significantly associated with diagnosis were (a) higher level of occupational disability in psychoses; (b) higher number of job- placement programs among psychoses patients; (c) lower level of employment in psychoses; (d) more psychotherapy in personality disorder patients; and (e) more years of MHC program in psychoses patients; factors associated with sex were: (a) higher number of drive licenses among males; (b) more physical activity among males; and (c) higher number of job-placement programs among males. CONCLUSION: patients affected by psychoses were more likely to be unemployed, reported higher occupational disability as well as received more incentives and rehabilitation interventions. These findings confirmed that schizophrenia-spectrum disorders are disabling and patients need psychosocial support and interventions in the framework of a recovery-oriented treatment.

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed. To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Methylation of CENP-A/Cse4 on arginine 143 and lysine 131 regulates kinetochore stability in yeast.

Post-translational modifications on histones are well known to regulate chromatin structure and function, but much less information is available on modifications of the centromeric histone H3 variant and their effect at the kinetochore. Here, we report two modifications on the centromeric histone H3 variant CENP-A/Cse4 in the yeast Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me), that affect centromere stability and kinetochore function. Both R143me and K131me lie in the core region of the centromeric nucleosome, near the entry/exit sites of the DNA from the nucleosome. Unexpectedly, mutation of Cse4-R143 (cse4-R143A) exacerbated the kinetochore defect of mutations in components of the NDC80 complex of the outer kinetochore (spc25-1) and the MIND complex (dsn1-7). The analysis of suppressor mutations of the spc25-1 cse4-R143A growth defect highlighted residues in Spc24, Ndc80, and Spc25 that localize to the tetramerization domain of the NDC80 complex and the Spc24-Spc25 stalk, suggesting that the mutations enhance interactions among NDC80 complex components and thus stabilize the complex. Furthermore, the Set2 histone methyltransferase inhibited kinetochore function in spc25-1 cse4-R143A cells, possibly by methylating Cse4-K131. Taken together, our data suggest that Cse4-R143 methylation and Cse4-K131 methylation affect the stability of the centromeric nucleosome, which is detrimental in the context of defective NDC80 tetramerization and can be compensated for by strengthening interactions among NDC80 complex components.

Serum proteomics profiling identifies a preliminary signature for the diagnosis of early-stage lung cancer.

PURPOSE: Lung cancer is the most common cause of death from cancer worldwide, largely due to late diagnosis. Thus, there is an urgent need to develop new approaches to improve the detection of early-stage lung cancer, which would greatly improve patient survival. EXPERIMENTAL DESIGN: The quantitative protein expression profiles of microvesicles isolated from the sera from 46 lung cancer patients and 41 high-risk non-cancer subjects were obtained using a mass spectrometry method based on a peptide library matching approach. RESULTS: We identified 33 differentially expressed proteins that allow discriminating the two groups. We also built a machine learning model based on serum protein expression profiles that can correctly classify the majority of lung cancer cases and that highlighted a decrease in the levels of Arysulfatase A (ARSA) as the most discriminating factor found in tumors. CONCLUSIONS AND CLINICAL RELEVANCE: Our study identified a preliminary, non-invasive protein signature able to discriminate with high specificity and selectivity early-stage lung cancer patients from high-risk healthy subjects. These results provide the basis for future validation studies for the development of a non-invasive diagnostic tool for lung cancer.