Sensory processing during sleep in Drosophila melanogaster.

During sleep, most animal species enter a state of reduced consciousness characterized by a marked sensory disconnect. Yet some processing of the external world must remain intact, given that a sleeping animal can be awoken by intense stimuli (for example, a loud noise or a bright light) or by soft but qualitatively salient stimuli (for example, the sound of a baby cooing or hearing one's own name1-3). How does a sleeping brain retain the ability to process the quality of sensory information? Here we present a paradigm to study the functional underpinnings of sensory discrimination during sleep in Drosophila melanogaster. We show that sleeping vinegar flies, like humans, discern the quality of sensory stimuli and are more likely to wake up in response to salient stimuli. We also show that the salience of a stimulus during sleep can be modulated by internal states. We offer a prototypical blueprint detailing a circuit involved in this process and its modulation as evidence that the system can be used to explore the cellular underpinnings of how a sleeping brain experiences the world.

Mitochondrial metabolism in neural stem cells and implications for neurodevelopmental and neurodegenerative diseases.

Mitochondria are cytoplasmic organelles having a fundamental role in the regulation of neural stem cell (NSC) fate during neural development and maintenance.During embryonic and adult neurogenesis, NSCs undergo a metabolic switch from glycolytic to oxidative phosphorylation with a rise in mitochondrial DNA (mtDNA) content, changes in mitochondria shape and size, and a physiological augmentation of mitochondrial reactive oxygen species which together drive NSCs to proliferate and differentiate. Genetic and epigenetic modifications of proteins involved in cellular differentiation (Mechanistic Target of Rapamycin), proliferation (Wingless-type), and hypoxia (Mitogen-activated protein kinase)-and all connected by the common key regulatory factor Hypoxia Inducible Factor-1A-are deemed to be responsible for the metabolic shift and, consequently, NSC fate in physiological and pathological conditions.Both primary mitochondrial dysfunction due to mutations in nuclear DNA or mtDNA or secondary mitochondrial dysfunction in oxidative phosphorylation (OXPHOS) metabolism, mitochondrial dynamics, and organelle interplay pathways can contribute to the development of neurodevelopmental or progressive neurodegenerative disorders.This review analyses the physiology and pathology of neural development starting from the available in vitro and in vivo models and highlights the current knowledge concerning key mitochondrial pathways involved in this process.

Infection increases activity via Toll dependent and independent mechanisms in Drosophila melanogaster.

Host behavioural changes are among the most apparent effects of infection. 'Sickness behaviour' can involve a variety of symptoms, including anorexia, depression, and changed activity levels. Here, using a real-time tracking and behavioural profiling platform, we show that in Drosophila melanogaster, several systemic bacterial infections cause significant increases in physical activity, and that the extent of this activity increase is a predictor of survival time in some lethal infections. Using multiple bacteria and D. melanogaster immune and activity mutants, we show that increased activity is driven by at least two different mechanisms. Increased activity after infection with Micrococcus luteus, a Gram-positive bacterium rapidly cleared by the immune response, strictly requires the Toll ligand spätzle. In contrast, increased activity after infection with Francisella novicida, a Gram-negative bacterium that cannot be cleared by the immune response, is entirely independent of both Toll and the parallel IMD pathway. The existence of multiple signalling mechanisms by which bacterial infections drive increases in physical activity implies that this effect may be an important aspect of the host response.

Endothelial cells require functional FLVCR1a during developmental and adult angiogenesis.

The Feline Leukemia Virus Subgroup C Receptor 1a (FLVCR1a) is a transmembrane heme exporter essential for embryonic vascular development. However, the exact role of FLVCR1a during blood vessel development remains largely undefined. Here, we show that FLVCR1a is highly expressed in angiogenic endothelial cells (ECs) compared to quiescent ECs. Consistently, ECs lacking FLVCR1a give rise to structurally and functionally abnormal vascular networks in multiple models of developmental and pathologic angiogenesis. Firstly, zebrafish embryos without FLVCR1a displayed defective intersegmental vessels formation. Furthermore, endothelial-specific Flvcr1a targeting in mice led to a reduced radial expansion of the retinal vasculature associated to decreased EC proliferation. Moreover, Flvcr1a null retinas showed defective vascular organization and loose attachment of pericytes. Finally, adult neo-angiogenesis is severely affected in murine models of tumor angiogenesis. Tumor blood vessels lacking Flvcr1a were disorganized and dysfunctional. Collectively, our results demonstrate the critical role of FLVCR1a as a regulator of developmental and pathological angiogenesis identifying FLVCR1a as a potential therapeutic target in human diseases characterized by aberrant neovascularization.

Ethoscopes: An open platform for high-throughput ethomics.

Here, we present the use of ethoscopes, which are machines for high-throughput analysis of behavior in Drosophila and other animals. Ethoscopes provide a software and hardware solution that is reproducible and easily scalable. They perform, in real-time, tracking and profiling of behavior by using a supervised machine learning algorithm, are able to deliver behaviorally triggered stimuli to flies in a feedback-loop mode, and are highly customizable and open source. Ethoscopes can be built easily by using 3D printing technology and rely on Raspberry Pi microcomputers and Arduino boards to provide affordable and flexible hardware. All software and construction specifications are available at http://lab.gilest.ro/ethoscope.

Regulation of apoptosis is impaired in atrophic gastritis associated with gastric cancer.

BACKGROUND: Gastric premalignant conditions, atrophic gastritis (AG) and intestinal metaplasia (IM) are characterized by an increase of proliferation and a reduction of apoptosis in epithelial cells. The epithelial cell kinetics in AG and IM in gastric mucosa adjacent to gastric cancer is still unclear. The aim of this study was to evaluate the epithelial cell turnover and expression of proliferation and apoptosis-related genes in gastric cancer (GC) and adjacent mucosa with atrophic gastritis or intestinal metaplasia (AG/IM GC+), as well as in atrophic gastritis or intestinal metaplasia mucosa of patients without GC (AG/IM GC-) and in control biopsy samples of non-transformed gastric mucosa (Control). METHODS: We selected 58 patients (M: F = 34:24; age range 20-84 years, median 61.06 years) with 4 well defined histological conditions: 20 controls with histological finding of non-transformed gastric mucosa, 20 patients with AG or IM (AG/IM GC-), and 18 patients with intestinal type gastric adenocarcinoma (GC) and AG or IM in the adjacent mucosa (3 cm from the macroscopic tumour margin, AG/IM GC+). We performed an immunohistochemical staining of Ki67 and TUNEL and quantitative RT-PCR to determine the expression of PCNA and Bax/Bcl-2. RESULTS: The immunohistochemical expression of Ki67 and TUNEL in AG/IM GC- was significantly increased compared to not transformed gastric mucosa (p < 0.0001) but not compared to AG/IM in gastric mucosa adjacent to GC. Levels of Bcl-2 were reduced in GC and AG/IM GC- compared to controls as well as in AG/IM GC- compared to AG/IM in mucosa adjacent to GC+ (p < 0.05). Proliferation and apoptosis markers did not correlate with H.pylori status in our study population. CONCLUSIONS: In AG/IM associated with GC, no significant changes in the epithelial cell turnover were detected. Decreased Bcl-2 gene expression signified atrophic gastritis and IM in presence of cancer, as well as intestinal type gastric adenocarcinoma.

Sequential vs. prolonged 14-day triple therapy for Helicobacter pylori eradication: the meta-analysis may be influenced by 'geographical weighting'.

BACKGROUND: Sequential therapy is a first-line regimen obtaining satisfactory Helicobacter pylori eradication. Triple therapy prolongation improves the success rate even if a recent meta-analysis showed satisfying results only for the 14-day regimen. Studies from Africa and North America were unavailable in previous meta-analyses. AIM: To perform a meta-analysis comparing sequential vs. prolonged 14-day triple therapy with regard to 'geographic weighting' by considering subgroups analysis according to metronidazole/clarithromycin low and high resistance areas. METHODS: Based on PRISMA recommendations, we considered all first-line clinical studies from 2003 to November 2014. Randomised clinical trials (RCTs) were included by a search on PubMed, MEDLINE, Science Direct, EMBASE. Data on eradication rates were expressed as ITT. Risk ratio (RR), pooled RR and 95% confidence intervals were calculated by the Mantel-Haenszel method. Data were entered into RevMan 5.2 software (Nordic Cochrane Centre) using a random-effects model. RESULTS: Databases identified 194 studies; seven met the inclusion criteria. Overall results showed a similar effectiveness of the two regimens considered (RR = 0.99; 95% CI = 0.94-1.05; p = 0.75). In areas with high resistance to clarithromycin, sequential was superior to 14-day triple therapy (RR = 0.95; 95% CI = 0.90-1.00; p = 0.03). In areas with high metronidazole resistance, sequential and 14-day triple therapy were equivalent (RR = 0.99; 95% CI = 0.91-1.08; p = 0.82). CONCLUSIONS: 'Geographic weighting' could be the main factor affecting the lack of differences between sequential and 14-day triple therapy outcomes.

Expression of basic fibroblastic growth factor, syndecan 1 and tumour necrosis factor α in resected acute colonic diverticulitis.

AIM: Inflammation and fibrosis are present in both colonic diverticulitis and Crohn's disease (CD). The molecular pattern of basic fibroblastic growth factor (bFGF) and syndecan 1 (SD1) expression is altered in stenosing CD, but their expression in resected complicated colonic diverticulitis (ACD) is unknown. METHOD: The expression of bFGF, SD1 and tumour necrosis factor α (TNF-α) in 20 patients after resection of ACD was compared with 15 patients having a resection for CD. Analysis was conducted using real-time reverse transcriptase polymerase chain reaction in biopsy samples. RESULTS: Lymphocytic and neutrophil inflammation scores were similar in both groups (P = 0.771 and P = 0.562). TNF-α and bFGF expression was significantly higher in ACD than in CD (P < 0.0001 and P = 0.009). SD1 expression was similar in both groups (P = 0.841). CONCLUSION: TNF-α and bFGF are significantly overexpressed in ACD with respect to CD, whilst SD1 levels do not differ. The findings confirm that inflammation and its association with altered molecular patterns of mucosal healing may play an important role in the phenotype of the diseases.

Divergent evolution of sleep in Drosophila species.

Living organisms synchronize their biological activities with the earth's rotation through the circadian clock, a molecular mechanism that regulates biology and behavior daily. This synchronization factually maximizes positive activities (e.g., social interactions, feeding) during safe periods, and minimizes exposure to dangers (e.g., predation, darkness) typically at night. Beyond basic circadian regulation, some behaviors like sleep have an additional layer of homeostatic control, ensuring those essential activities are fulfilled. While sleep is predominantly governed by the circadian clock, a secondary homeostatic regulator, though not well-understood, ensures adherence to necessary sleep amounts and hints at a fundamental biological function of sleep beyond simple energy conservation and safety. Here we explore sleep regulation across seven Drosophila species with diverse ecological niches, revealing that while circadian-driven sleep aspects are consistent, homeostatic regulation varies significantly. The findings suggest that in Drosophilids, sleep evolved primarily for circadian purposes. The more complex, homeostatically regulated functions of sleep appear to have evolved independently in a species-specific manner, and are not universally conserved. This laboratory model may reproduce and recapitulate primordial sleep evolution.

Ethoscopy and ethoscope-lab: a framework for behavioural analysis to lower entrance barrier and aid reproducibility.

Summary: High-throughput analysis of behaviour is a pivotal instrument in modern neuroscience, allowing researchers to combine modern genetics breakthrough to unbiased, objective, reproducible experimental approaches. To this extent, we recently created an open-source hardware platform (ethoscope; Geissmann Q, Garcia Rodriguez L, Beckwith EJ et al. Rethomics: an R framework to analyse high-throughput behavioural data. PLoS One 2019;14:e0209331) that allows for inexpensive, accessible, high-throughput analysis of behaviour in Drosophila or other animal models. Here we equip ethoscopes with a Python framework for data analysis, ethoscopy, designed to be a user-friendly yet powerful platform, meeting the requirements of researchers with limited coding expertise as well as experienced data scientists. Availability and implementation: Ethoscopy is best consumed in a prebaked Jupyter-based docker container, ethoscope-lab, to improve accessibility and to encourage the use of notebooks as a natural platform to share post-publication data analysis. Ethoscopy is a Python package available on GitHub and PyPi. Ethoscope-lab is a docker container available on DockerHub. A landing page aggregating all the code and documentation is available at https://lab.gilest.ro/ethoscopy.

A reductionist paradigm for high-throughput behavioural fingerprinting in Drosophila melanogaster.

Understanding how the brain encodes behaviour is the ultimate goal of neuroscience and the ability to objectively and reproducibly describe and quantify behaviour is a necessary milestone on this path. Recent technological progresses in machine learning and computational power have boosted the development and adoption of systems leveraging on high-resolution video recording to track an animal pose and describe behaviour in all four dimensions. However, the high temporal and spatial resolution that these systems offer must come as a compromise with their throughput and accessibility. Here, we describe coccinella, an open-source reductionist framework combining high-throughput analysis of behaviour using real-time tracking on a distributed mesh of microcomputers (ethoscopes) with resource-lean statistical learning (HCTSA/Catch22). Coccinella is a reductionist system, yet outperforms state-of-the-art alternatives when exploring the pharmacobehaviour in Drosophila melanogaster.

Long-term follow-up after bronchoscopic lung volume reduction in patients with emphysema.

Bronchoscopic lung volume reduction (BLVR) is a novel emphysema therapy. We evaluated long-term outcome in patients with heterogeneous emphysema undergoing BLVR with one-way valves. 40 patients undergoing unilateral BLVR entered our study. Pre-operative mean forced expiratory volume in 1 s (FEV(1)) was 0.88 L · s(-1) (23%), total lung capacity was 7.45 L (121%), intrathoracic gas volume was 6 L (174%), residual volume (RV) was 5.2 L (232%), and the 6-min walk test (6MWT) was 286 m. All patients required supplemental oxygen; the Medical Research Council (MRC) dyspnoea score was 3.9. High-resolution computed tomography (HRCT) results were reviewed to assess the presence of interlobar fissures. 33 patients had a follow-up of >12 months (median 32 months). 37.5% of the patients had visible interlobar fissures. 40% of the patients died during follow-up. Three patients were transplanted and one underwent lung volume reduction surgery. Supplemental oxygen, FEV(1), RV, 6MWT and MRC score showed a statistically significant improvement (p ≤ 0.0001, p = 0.004, p = 0.03, p = 0.003 and p<0.0001, respectively). Patients with visible fissures had a functional advantage. BLVR is feasible and safe. Long-term sustained improvements can be achieved. HRCT-visible interlobar fissures are a favourable prognostic factor.

Haemophagocytic syndrome associated with mucormycosis infection.

Several clinical forms of mucormycosis are recognized. The tendency of mucoraceous zygomycetes to invade the blood vessels often produces a disseminated infection. A case of disseminate mucormycosis complicated by a haemophagocytic syndrome (HS) in a 32-year-old Caucasian male is reported in this article. Few cases of infection-associated HS (IAHS), involving infections caused by fungi, have been reported. In all the recorded cases, the fungal infection coexists with malignant lymphoma, immunodeficiency and a long-term steroid therapy for renal transplant or Crohn's disease. This is the second described case of the HS due to mucormycosis.

Musosal tumour necrosis factor α in diverticular disease of the colon is overexpressed with disease severity.

AIM: Inflammation occurs in diverticular disease (DD), but there is little information on inflammatory cytokines such as tumour necrosis factor α (TNF-α). The aim of this study was to assess TNF-α expression in DD and to see whether it is related to the severity of the disease. METHOD: Twenty-four patients with symptomatic DD were divided into those with acute uncomplicated diverticulitis (AUD) (12 patients) and those with symptomatic uncomplicated diverticular disease (SUDD) (12 patients). Twelve further patients with asymptomatic diverticulosis (AD), six with segmental colitis associated with diverticulosis (SCAD), with ulcerative colitis (UC) and six healthy individuals (HC) were enrolled as controls. TNF-α expression in the colonic mucosa was assessed by the amount of mRNA codifying for the synthesis of TNF-α. RESULTS: TNF-α expression was significantly higher in AUD than in HC (P=0.0007), in AD (P=0.0001) and in SUDD (P=0.0179). It was significantly higher also in SUDD than in HC (P=0.0007) and in AD (P=0.0001). TNF-α expression in AUD did not differ significantly from that in UC (P=0.0678) and SCAD (P=0.0610). It was significantly higher in UC, SCAD and AUD than in SUDD (P=0.0007, P=0.0001, P=0.0179). CONCLUSION: TNF-α expression in DD seems to be related to the severity of the disease. In particular, it appears to be overexpressed in DD with inflammation (AUD and SUDD) compared with DD without (AD).

Pathways and gene expression profiles in hepatocellular carcinoma.

Hepatocarcinogenesis is a process attributed to progressive genomic changes which alter the hepatocellular phenotype producing cellular intermediates evolving into clearly neoplastic cells (hepatocellular carcinoma, HCC). During the preneoplastic phase, the liver is often the site of chronic hepatitis and/or cirrhosis, and this process leads to the production of monoclonal populations of aberrant and dysplastic hepatocytes that develop genetic and chromosomal alterations. At the moment three main molecular pathways of liver carcinogenesis have been described and several attempts of genetic classification of HCC have been proposed. The definition of genomic and molecular changes which occur during the development of HCC should improve the classification and prognostis of liver tumors. The development of sorafenib and other new targeted developing therapies were rendered possible by the discovery and understanding of the molecular and genetic pathogenesis of hepatocellular carcinoma. Besides viruses, such as Hepatitis B virus (HBV) and Hepatitis C virus (HCV), may contribute to cancer development by several ways; however, additional factors, such as host immunity and chronic inflammation and host cellular mutations also play a role in the transformation process. The understanding of these pathways will in the future enable the clinician to focus the treatment patients with HCC and customize single or combination therapy.

COVID-19 pandemic and days of absence from work in workers with flu-like symptoms in the City of Rome, Italy.

ABSTRACT: COVID-19 has dramatically affected working forces. We aim to report our occupational medicine service's experience in managing suspected COVID-19 cases during the pandemic through a retrospec-tive observational study. We compared the number of days employees were absent from work due to flu-like symptoms from March 2020 to February 2021 to the same period the previous year (2019-2020). Two hundred thirty-four patients (+47.2% compared to the previous year) who tested negative for SARS-CoV-2 reported flu-like symp-toms; the number of days of absence from work was 2812 (+190.2% compared to the previous year). On average, employees with flu-like symptoms lost 12.07 working days compared to 6.12 in the previous year (p<0.0001). In conclusion, in our sample COVID-19 has increased the number of working day loss. However, our approach proved to be important, especially during the first months of the pandemic, to limit SARS-CoV-2 spread in workplaces.

Evaluation of dental demands and needs of people experiencing homelessness in the city of Rome, Italy.

OBJECTIVE: Poor oral health and oral diseases are common among people experiencing homelessness. The aim of this study was to evaluate the dental demands and needs of a population of homeless persons in the city of Rome, Italy. PATIENTS AND METHODS: The clinical records of 165 homeless patients admitted between October 2020 and October 2021 to the dental service of the Primary Care Services of the Eleemosynaria Apostolica, Vatican City, were retrospectively reviewed. The service employed dentists to evaluate dental needs and oral conditions in patients experiencing homelessness. The main dental and oral pathological conditions were noted. RESULTS: One hundred and sixty-five records of homeless patients were included in the study. The sample consisted in 138 males (76.97%) and 27 females (23.03%) with a mean age of 46.9 years (range 7-85 years). Acute tooth pain was reported by 132 (80%) patients, 42 (25.45%) had edentulism or missing teeth and 18 (10.91%) patients had oral lesions. Both dental and oral pathologies were intercepted and managed in secondary healthcare facilities. CONCLUSIONS: Given the specific peculiarities of this vulnerable population, it is important to implement strategies that facilitate the access of persons experiencing homelessness to dental evaluation with a preventive and curative perspective.

Sleep: Imaging the Fly Brain Reveals New Paradoxes.

In Drosophila melanogaster, brain imaging with single-cell resolution reveals new physiological correlates of sleep and wakefulness, possibly defining different stages of sleep with functional relevance.

Paracetamol (acetaminophen) rescues cognitive decline, neuroinflammation and cytoskeletal alterations in a model of post-operative cognitive decline (POCD) in middle-aged rats.

Post-operative cognitive dysfunction (POCD) is a debilitating clinical phenomenon in elderly patients. Management of pain in elderly is complicated because analgesic opiates elicit major side effects. In contrast, paracetamol (acetaminophen) has shown analgesic efficacy, no impact on cognition, and its side effects are well tolerated. We investigated the efficacy of paracetamol, compared to the opioid analgesic buprenorphine, in a model of POCD by investigating cognitive decline, allodynia, peripheral and hippocampal cytokines levels, and hippocampal microtubule dynamics as a key modulator of synaptic plasticity. A POCD model was developed in middle-aged (MA) rats by inducing a tibia fracture via orthopaedic surgery. Control MA rats did not undergo any surgery and only received isoflurane anaesthesia. We demonstrated that cognitive decline and increased allodynia following surgery was prevented in paracetamol-treated animals, but not in animals which were exposed to anesthesia alone or underwent the surgery and received buprenorphine. Behavioral alterations were associated with different peripheral cytokine changes between buprenorphine and paracetamol treated animals. Buprenorphine showed no central effects, while paracetamol showed modulatory effects on hippocampal cytokines and markers of microtubule dynamics which were suggestive of neuroprotection. Our data provide the first experimental evidence corroborating the use of paracetamol as first-choice analgesic in POCD.

SARS-CoV-2 infection prevalence in people experiencing homelessness.

OBJECTIVE: People experiencing homelessness have peculiar characteristics that make them more vulnerable to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission and to more serious forms of Coronavirus Disease 19 (COVID-19). The aim of this study was to evaluate the prevalence of SARS-CoV-2 infection in the homeless population assisted by the primary care services of the Eleemosynaria Apostolica, Vatican City. PATIENTS AND METHODS: Persons experiencing homelessness and the volunteers assisting them were tested for COVID-19 through PCR and antigen rapid test between October 1st, 2020, and June 5th, 2021, in the clinical facilities of the Eleemosynaria Apostolica. RESULTS: A total of 1665 subjects from 96 different countries in five continents were included in the study; age range was 1-90 years. Overall, 2315 COVID-19 tests through nasopharyngeal swab were performed; 1052 Polymerase Chain Reaction (PCR) tests and 1263 antigen rapid tests. Nearly 40% of the subjects underwent both tests (n=650, 39.04%), 402 were tested with PCR test only (24.14%) and 613 with antigen test only (36.8%). PCR tests were negative in 966 cases and positive in 86 (8.17%), while antigen tests were negative in 1205 cases and positive in 58 (4.59%). The number of positive cases varied over time, with a drastic increase during the winter months of 2020 and a progressive decrease over 2021. Among positive cases, 24.41% were symptomatic; symptoms included fever, breathing difficulties, anosmia/hyposmia, cough, headache, and diarrhea. CONCLUSIONS: This study reported an overall prevalence of SARS-CoV-2 infection in our sample slightly above 8%. Additional data on viral genome through sequencing of SARS-CoV-2 in positive cases are of utmost importance to help identify variants and implement specific infection control measures.