Comparison between Standard Expository Cognitive Behavioral Therapy (CBT-E) and Immersive Virtual Reality CBT (CBT-VR) for Rehabilitation of Patients Affected by Occupational Stress Disorders: Study Protocol.

Work-related stress presents a significant impact on work performance and physical health. It has been associated with the onset of a multitude of symptoms that can lead to occupational stress diseases, namely Adjustment Disorder and Post-Traumatic Stress Disorder. The literature has evidenced that "exposure therapy" of cognitive-behavioral training (CBT-E) seems to be the most effective technique to manage stress symptoms, including work stress diseases, and several studies have considered Virtual Reality (VR) as an adjuvant tool to exposure-based psychotherapy (CBT-VR) for the treatment of multiple psychiatric disorders. The aim of this study is to evaluate the effectiveness of CBT with exposure to stressful work scenarios in imaginative (CBT-E) and in immersive virtual reality (CBT-VR) scenarios in a group of workers affected by work-related stress disorders and compare the clinical and physiological outcomes between the two exposure techniques. A long-term goal would be to develop an evidence-based rehabilitation program as a treatment for the reintegration into work of patients affected by these psychiatric disorders.

A synthetic genetic array screen for interactions with the RNA helicase DED1 during cell stress in budding yeast.

During cellular stress it is essential for cells to alter their gene expression to adapt and survive. Gene expression is regulated at multiple levels, but translation regulation is both a method for rapid changes to the proteome and, as one of the most energy-intensive cellular processes, a way to efficiently redirect cellular resources during stress conditions. Despite this ideal positioning, many of the specifics of how translation is regulated, positively or negatively, during various types of cellular stress remain poorly understood. To further assess this regulation, we examined the essential translation factor Ded1, an RNA helicase that has been previously shown to play important roles in the translational response to cellular stress. In particular, ded1 mutants display an increased resistance to growth inhibition and translation repression induced by the TOR pathway inhibitor, rapamycin, suggesting that normal stress responses are partially defective in these mutants. To gain further insight into Ded1 translational regulation during stress, synthetic genetic array analysis was conducted in the presence of rapamycin with a ded1 mutant and a library of nonessential genes in Saccharomyces cerevisiae to identify positive and negative genetic interactions in an unbiased manner. Here, we report the results of this screen and subsequent network mapping and Gene Ontology-term analysis. Hundreds of candidate interactions were identified, which fell into expected categories, such as ribosomal proteins and amino acid biosynthesis, as well as unexpected ones, including membrane trafficking, sporulation, and protein glycosylation. Therefore, these results provide several specific directions for further comprehensive studies.

TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle.

Recent studies have revealed that the growth rate of budding yeast and mammalian cells varies during the cell cycle. By linking a multitude of signals to cell growth, the highly conserved target of rapamycin complex 1 (TORC1) and protein kinase A (PKA) pathways are prime candidates for mediating the dynamic coupling between growth and division. However, measurements of TORC1 and PKA activity during the cell cycle are still lacking. By following the localization dynamics of two TORC1 and PKA targets via time-lapse microscopy in hundreds of yeast (Saccharomyces cerevisiae) cells, we found that the activity of these pathways towards ribosome biogenesis fluctuates in synchrony with the cell cycle even under constant external conditions. Analysis of the effects of mutations of upstream TORC1 and PKA regulators suggests that internal metabolic signals partially mediate these activity changes. Our study reveals a new aspect of TORC1 and PKA signaling, which will be important for understanding growth regulation during the cell cycle.

Systematic In Vivo Characterization of Fluorescent Protein Maturation in Budding Yeast.

Fluorescent protein (FP) maturation can limit the accuracy with which dynamic intracellular processes are captured and reduce the in vivo brightness of a given FP in fast-dividing cells. The knowledge of maturation timescales can therefore help users determine the appropriate FP for each application. However, in vivo maturation rates can greatly deviate from in vitro estimates that are mostly available. In this work, we present the first systematic study of in vivo maturation for 12 FPs in budding yeast. To overcome the technical limitations of translation inhibitors commonly used to study FP maturation, we implemented a new approach based on the optogenetic stimulations of FP expression in cells grown under constant nutrient conditions. Combining the rapid and orthogonal induction of FP transcription with a mathematical model of expression and maturation allowed us to accurately estimate maturation rates from microscopy data in a minimally invasive manner. Besides providing a useful resource for the budding yeast community, we present a new joint experimental and computational approach for characterizing FP maturation, which is applicable to a wide range of organisms.

A convolutional neural network for segmentation of yeast cells without manual training annotations.

MOTIVATION: Single-cell time-lapse microscopy is a ubiquitous tool for studying the dynamics of complex cellular processes. While imaging can be automated to generate very large volumes of data, the processing of the resulting movies to extract high-quality single-cell information remains a challenging task. The development of software tools that automatically identify and track cells is essential for realizing the full potential of time-lapse microscopy data. Convolutional neural networks (CNNs) are ideally suited for such applications, but require great amounts of manually annotated data for training, a time-consuming and tedious process. RESULTS: We developed a new approach to CNN training for yeast cell segmentation based on synthetic data and present (i) a software tool for the generation of synthetic images mimicking brightfield images of budding yeast cells and (ii) a convolutional neural network (Mask R-CNN) for yeast segmentation that was trained on a fully synthetic dataset. The Mask R-CNN performed excellently on segmenting actual microscopy images of budding yeast cells, and a density-based spatial clustering algorithm (DBSCAN) was able to track the detected cells across the frames of microscopy movies. Our synthetic data creation tool completely bypassed the laborious generation of manually annotated training datasets, and can be easily adjusted to produce images with many different features. The incorporation of synthetic data creation into the development pipeline of CNN-based tools for budding yeast microscopy is a critical step toward the generation of more powerful, widely applicable and user-friendly image processing tools for this microorganism. AVAILABILITY AND IMPLEMENTATION: The synthetic data generation code can be found at https://github.com/prhbrt/synthetic-yeast-cells. The Mask R-CNN as well as the tuning and benchmarking scripts can be found at https://github.com/ymzayek/yeastcells-detection-maskrcnn. We also provide Google Colab scripts that reproduce all the results of this work. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Vacuolar Localization via the N-terminal Domain of Sch9 is Required for TORC1-dependent Phosphorylation and Downstream Signal Transduction.

The budding yeast Sch9 kinase (functional orthologue of the mammalian S6 kinase) is a major effector of the Target of Rapamycin Complex 1 (TORC1) complex in the regulation of cell growth in response to nutrient availability and stress. Sch9 is partially localized at the vacuolar surface, where it is phosphorylated by TORC1. The recruitment of Sch9 on the vacuole is mediated by direct interaction between phospholipids of the vacuolar membrane and the region of Sch9 encompassing amino acid residues 1-390, which contains a C2 domain. Since many C2 domains mediate phospholipid binding, it had been suggested that the C2 domain of Sch9 mediates its vacuolar recruitment. However, the in vivo requirement of the C2 domain for Sch9 localization had not been demonstrated, and the phenotypic consequences of Sch9 delocalization remained unknown. Here, by examining cellular localization, phosphorylation state and growth phenotypes of Sch9 truncation mutants, we show that deletion of the N-terminal domain of Sch9 (aa 1-182), but not the C2 domain (aa 183-399), impairs vacuolar localization and TORC1-dependent phosphorylation of Sch9, while causing growth defects similar to those observed in Sch9Δ cells. These defects can be reversed either via artificial tethering of the protein to the vacuole, or by introducing phosphomimetic mutations at the TORC1 target sites, suggesting that Sch9 localization on the vacuole is needed for the TORC1-dependent activation of the kinase. Our study uncovers a key role for the N-terminal domain of Sch9 and provides new mechanistic insight into the regulation of a major TORC1 signaling branch.

Medulloblastoma-associated mutations in the DEAD-box RNA helicase DDX3X/DED1 cause specific defects in translation.

Medulloblastoma is the most common pediatric brain cancer, and sequencing studies identified frequent mutations in DDX3X, a DEAD-box RNA helicase primarily implicated in translation. Forty-two different sites were identified, suggesting that the functional effects of the mutations are complex. To investigate how these mutations are affecting DDX3X cellular function, we constructed a full set of equivalent mutant alleles in DED1, the Saccharomyces cerevisiae ortholog of DDX3X, and characterized their effects in vivo and in vitro. Most of the medulloblastoma-associated mutants in DDX3X/DED1 (ded1-mam) showed substantial growth defects, indicating that functional effects are conserved in yeast. Further, while translation was affected in some mutants, translation defects affecting bulk mRNA were neither consistent nor correlated with the growth phenotypes. Likewise, increased formation of stress granules in ded1-mam mutants was common but did not correspond to the severity of the mutants' growth defects. In contrast, defects in translating mRNAs containing secondary structure in their 5' untranslated regions (UTRs) were found in almost all ded1-mam mutants and correlated well with growth phenotypes. We thus conclude that these specific translation defects, rather than generalized effects on translation, are responsible for the observed cellular phenotypes and likely contribute to DDX3X-mutant medulloblastoma. Examination of ATPase activity and RNA binding of recombinant mutant proteins also did not reveal a consistent defect, indicating that the translation defects are derived from multiple enzymatic deficiencies. This work suggests that future studies into medulloblastoma pathology should focus on this specific translation defect, while taking into account the wide spectrum of DDX3X mutations.

Sudden and persistent dysphonia within the framework of COVID-19: The case report of a nurse.

In December 2019, clusters of atypical pneumonia with unknown etiology emerged in the city of Wuhan in China. In early January 2020, the Center for Disease Control in China announced that it was identified a new coronavirus, first tentatively named 2019-nCoV and officially named SARS-CoV-2 by the International Committee on Taxonomy of Viruses. On February 11, 2020 the WHO identified the disease caused by SARS-CoV-2 as COVID-19 (COronaVIrus Disease-19 based on the year of appearance). Although only a few months have passed since the beginning of this pandemic, numerous studies, case reports, reviews by leading international scientific and medical journals have been published. However, given the unpredictability of virus behaviour and the still limited knowledge about it, many aspects of the infection are still little known. A recent epidemiological study has shown the presence of dysphonia in some patients with COVID-19, with a minority reporting aphonia during the clinical course of the disease. This case study draws attention on a 50-year-old female nurse presented with a history of fatigue resulting from minor exertion and persistent dysphonia at the Occupational Health Department of a major University Hospital in central Italy. The patient had a history of COVID-19 infection, which lasted about two months with pulmonary and extrapulmonary symptoms. After two RT-PCR negativities for SARS-CoV-2, dysphonia and fatigue due to minor exertionpersisted. The patient, following the persistence of the symptomatology, was subject to numerous specialist examinations, which showed no organic alterations. Based on her clinical and instrumental history, we hypothesized a psychogenetic dysphonia related to COVID-19. This case report highlights the importance of personalized medicine with long-term follow-up and rubustpsychological support in patients who tested positive for COVID-19 and in particular in the categories at greatest risk of both contagion and adverse physical and mental outcomes like health care workers.

Mental disability management within occupational health surveillance.

INTRODUCTION: The management of workers with mental disability is a current topic of great interest. The aim of the article is to report the experience of managing cases of employees with mental disabilities in an Italian university hospital and to describe the preventive measures adopted and the therapeutic programs carried out. METHODS: A group of 100 workers suffering from psychiatric disorders has been included in a medical surveillance program lasting at least 6 months. The workers were followed up within a protocol that included psychiatric visits, psychotherapeutic interventions, visits by the occupational physician and medico-legal evaluations. Evaluation scales of disease severity and treatment efficacy (CGI) and overall functioning (GAF) were administered at the baseline and after 6 months of follow-up. RESULTS: The sample was mainly composed of nurses (44%) and nursing assistants (24%) and the most commonly diagnosed disorders were mood and anxiety disorders. Participation in the medical surveillance program with the implementation of specific therapeutic strategies and organizational interventions resulted in a statistically significant reduction in the severity of the disease and an improvement in overall functioning and made it possible to keep the job and place of work in almost all cases. DISCUSSION: The results of this experience allow us to affirm that the integration of skills is a valid tool both for the multidisciplinary diagnostic assessment and for the monitoring and management of workers with mental disability under periodic health surveillance.

Differential scaling between G1 protein production and cell size dynamics promotes commitment to the cell division cycle in budding yeast.

In the unicellular eukaryote Saccharomyces cerevisiae, Cln3-cyclin-dependent kinase activity enables Start, the irreversible commitment to the cell division cycle. However, the concentration of Cln3 has been paradoxically considered to remain constant during G1, due to the presumed scaling of its production rate with cell size dynamics. Measuring metabolic and biosynthetic activity during cell cycle progression in single cells, we found that cells exhibit pulses in their protein production rate. Rather than scaling with cell size dynamics, these pulses follow the intrinsic metabolic dynamics, peaking around Start. Using a viral-based bicistronic construct and targeted proteomics to measure Cln3 at the single-cell and population levels, we show that the differential scaling between protein production and cell size leads to a temporal increase in Cln3 concentration, and passage through Start. This differential scaling causes Start in both daughter and mother cells across growth conditions. Thus, uncoupling between two fundamental physiological parameters drives cell cycle commitment.

Work-related asthma in a sample of subjects with established asthma.

OBJECTIVE: To assess the impact of occupational exposure to irritants or sensitizers on the occurrence, recrudescence and worsening of asthma and to identify unrecognized cases of work related asthma (WRA) including Work-Exacerbated Asthma (WEA) and Occupational Asthma (OA), in a general asthma clinic population sample. SETTING, DESIGN AND PARTICIPANTS: The study was a population-based cross sectional survey. 1289 asthmatic subjects (from 15 to 46 yrs old) living in a vast district of Tuscany (Italy) were identified from the Medical Reimbursement Register of the National Health System. 893 subjects agreed to take part in the study. Subjects who were currently working or had worked in past were classified in different categories of occupational risk exposure (No, Low or High) according to the italian standard classification for industries and job titles, associated with the judgment of occupational hygiene experts. RESULTS: 41% of subjects worked in industries and in job titles at risk for exposure to airway irritants and/or sensitizers, 48.6% reported an occupational exposure to gases, dust and fumes, more males than females. Prevalence of WEA and OA was higher in subjects who worked at higher risk exposure; these subjects reported a higher prevalence of markers of asthma severity (asthma control, level of treatment, FEV1) than subjects without WRA. Risk of WEA was significantly associated to female gender, older age, and self-reported exposure, while risk of OA was associated to job title with higher exposure risk to occupational asthmogens. CONCLUSIONS: Our study shows a high prevalence of WRA (especially WEA) associated with employment in industries and job titles at risk for airways sensitizers and/or irritants; data also support a role for occupational exposure in determining a poor asthma control and a higher level of asthma severity.

Oncogenic osteomalacia caused by a phosphaturic mesenchymal tumor of the thoracic spine.

Phosphaturic mesenchymal tumors that cause the paraneoplastic syndrome known as oncogenic osteomalacia are rare. The authors report on the case of a 57-year-old man with a history of osteomalacia and in whom was diagnosed a thoracic spine tumor at the T-4 level. Complete tumor resection was accomplished. The histological diagnosis was phosphaturic mesenchymal tumor (mixed connective tissue variant). After lesion removal, the paraneoplastic syndrome resolved. At the 24-month follow-up, no recurrence of the disease was observed. The clinical presentation, surgical technique, and follow-up in this case were reviewed in detail.

Severely symptomatic craniovertebral junction abnormalities in children: long-term reliability of aggressive management.

OBJECTIVES: The treatment of symptomatic cranio-vertebral junction (CVJ) instability in children affected by CVJ abnormalities is a challenge. A series of severely symptomatic children has been reviewed to understand the controversial long-term effectiveness of the aggressive management of CVJ abnormalities, in terms of clinical improvement, spinal stability and growth. METHODS: Three Down syndrome patients, 1 with mucopolysaccharidosis and 1 with os odontoideum (range 3-6 years old) with a CVJ instability determining spinal cord compression with severe neurological deficits (the patients presented at admission a Ranawat III A/III B neurological condition), were consecutively treated at our institution. Medical records, imaging studies, adopted surgical techniques and long-term results were reviewed. Details of the presenting symptoms, clinical and radiological signs were compared to the signs and symptoms at follow-up. RESULTS: The perioperative use of an halo-orthosis, the operative techniques and the timing of rehabilitation were always tailored to the patient's anatomical features. All the patients showed remarkable neurological improvements, along with construct stability and bone fusion without abnormalities of the developing spine. CONCLUSIONS: Considering the effective long-term results, we recommend, even in severely symptomatic children with CVJ abnormalities, a multidisciplinary aggressive tailored treatment of instability with rigid internal fixation.

Microwave-treated gelatin microspheres as drug delivery system.

The crosslinking process of natural macromolecules with microwave energy should have the potentiality to overcome the problems due to the toxicity of the residuals of chemical crosslinking agents and moreover of the "in vivo" biodegradation products of the chemical crosslinked macromolecule. To evaluate the effective crosslinking of the gelatin forming the microspheres, the water-soluble fraction at 37 degrees C, the water absorption capability, the free amino and free carboxylic acid groups of the gelatin were determined. The structural change in the gelatin microspheres has been detected by the porosity studies. Moreover, both the "in vitro" biodegradability and the biocompatibility of the gelatin microspheres microwave-treated after a subcutaneous injection into female albino guinea pigs were tested. As the results suggest only the gelatin microspheres microwave-treated for 10 min at an inlet temperature of 250 degrees C could have been modified by the crosslink formation among the macromolecular chains. The gelatin microspheres treated with the microwave energy were very well biodegraded as indicated both by the "in vitro" enzymatic degradation studies and mainly by the histopathological examination. This latter study has also demonstrated the biocompatibility of the gelatin microspheres crosslinked with the microwave energy. In order to evaluate the feasibility of the microwave crosslinking process for pharmaceutical applications, both the drug loading and the drug release processes were evaluated using diclofenac as drug model, either as acidic form or as sodium salt. The microspheres were swollen in aqueous solution of diclofenac sodium salt, followed by a washing procedure with cool water to maintain the sodium salt into the microspheres or with pH 1.5 HCl to induce the diclofenac precipitation. To increase the amount of diclofenac acid form in the microspheres, the procedure was repeated three times washing with pH 1.5 HCl after each swelling process. Both the X-ray diffractometry and thermal analysis investigations showed a different physical state of the two drug forms in the microspheres, i.e. the amorphous state of the sodium salt and the crystalline state of the acidic form. According to the experimental results, the drug is released from gelatin microspheres according to the drug loading and the drug solubility.