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Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Together, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Murine adenosine deaminase (mADA) is a prototypic system for studying the thermal activation of active site chemistry within the TIM barrel family of enzyme reactions. Previous temperature-dependent hydrogen deuterium exchange studies under various conditions have identified interconnected thermal networks for heat transfer from opposing protein-solvent interfaces to active site residues in mADA. One of these interfaces contains a solvent exposed helix-loop-helix moiety that presents the hydrophobic face of its long α-helix to the backside of bound substrate. Herein we pursue the time and temperature dependence of solvation dynamics at the surface of mADA, for comparison to established kinetic parameters that represent active site chemistry. We first created a modified protein devoid of native tryptophans with close to native kinetic behavior. Single site-specific tryptophan mutants were back inserted into each of the four positions where native tryptophans reside. Measurements of nanosecond fluorescence relaxation lifetimes and Stokes shift decays, that reflect time dependent environmental reoroganization around the photo-excited state of Trp*, display minimal temperature dependences. These regions serve as controls for the behavior of a new single tryptophan inserted into a solvent exposed region near the helix-loop-helix moiety located behind the bound substrate, Lys54Trp. This installed Trp displays a significantly elevated value for Ea(kStokesshift) ; further, when Phe61 within the long helix positioned behind bound substrate is replaced by a series of aliphatic hydrophobic side chains, the trends in Ea(kStokesshift) mirror the earlier reported impact of the same series of function-altering hydrophobic side chains on the activation energy of catalysis, Ea(kcat) .The reported experimental findings implicate a solvent initiated and rapid (>ns) protein restructuring that contributes to the enthalpic activation barrier to catalysis in mADA.

Vincenzo Monda,1,* Marco La Marra,1,* Raffaella Perrella,2 Giorgio Caviglia,2 Alessandro Iavarone,3 Sergio Chieffi,1 Giovanni Messina,4 Marco Carotenuto,5 Marcellino Monda,1 Antonietta Messina1 1Department of Experimental Medicine, Section of Human Physiology, University of Campania Luigi Vanvitelli, Naples, Italy; 2Neurological and Stroke Unit, CTO Hospital, AORN ‘‘Ospedali dei Colli’’, Naples, Italy; 3Department of Psychology, University of Campania Luigi Vanvitelli, Naples, Italy; 4Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy; 5Clinic of Child and Adolescent Neuropsychiatry, Department of Mental Health, Physical and Preventive Medicine, University of Campania Luigi Vanvitelli, Naples, Italy *These authors contributed equally to this work Abstract: Recent findings showed that obesity represents an additional risk factor to developing brain illness such as cognitive impairments and psychopathological disorders. However, some benefits of overweight in the elderly have been identified and an “obesity paradox” has been shown. Currently, it is still unknown how obesity and brain functioning could be linked, and the process by which body fat independently injures cognitive abilities and psychological well-being remains unclear. To establish the independent role of obesity on cognitive abilities and mental health, clarifying the role played by several factors and understanding their interaction is essential. In this review, we discuss the relationship between obesity and brain illness and underline the role played by confounders and other covariates to determine this link. Keywords: obesity, cognitive decline, mood disorders, obesity paradox

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein-protein and protein-peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosylmethionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes.

Abstract Sore throat represents one of the main causes of antibiotic overprescription in children. Its management is still a matter of debate, with countries considering streptococcal pharyngotonsillitis a benign and self-limiting condition and others advocating for its antibiotic treatment to prevent suppurative complications and acute rheumatic fever. Italian paediatricians frequently prescribe antibiotics on a clinical basis regardless of microbiological results. Moreover, broad-spectrum antibiotics are inappropriately prescribed for this condition. In this regard, an intersociety consensus conference was issued to promote the judicious use of antibiotic therapy in paediatric outpatient settings. A systematic review of the literature was performed, and updated recommendations were developed according to the GRADE methodology. Antibiotic treatment with amoxicillin (50 mg/kg/day) for 10 days is recommended in all children with proven streptococcal pharyngitis. Benzathine-penicillin could be prescribed in children with impaired intestinal absorption or inability to tolerate enteral intake and in those at high risk of suppurative complications with low compliance to oral therapy. In children with suspected amoxicillin allergy, third-generation cefalosporins for five days are recommended in low-risk patients, and macrolides are recommended in high-risk ones. Candidates for tonsillectomy due to recurrent pharyngitis could be treated with amoxicillin-clavulanic acid, clindamycin, or combined therapy with amoxicillin plus rifampicin for four days, in an attempt to avoid surgery.

Abstract The purpose of the analysis was to evaluate if 10 mg naloxone, administered intramuscularly, could reverse or prevent opioid‐induced respiratory depression (OIRD), including OIRD associated with the administration of lethal doses of high‐potency opioids. A naloxone population pharmacokinetic (PK) model was generated using data from two naloxone auto‐injector (NAI) clinical PK studies. Mechanistic OIRD PK‐pharmacodynamic (PD) models were constructed using published data for buprenorphine, morphine, and fentanyl. Due to the lack of published carfentanil data in humans, interspecies allometric scaling methods were used to predict carfentanil PK parameters in humans. A PD model of a combined effect‐compartment and receptor kinetics model with a linear relationship between ventilation and carbon dioxide was used to predict the respiratory depression induced by carfentanil. Model‐based simulations were performed using the naloxone population PK model and the constructed mechanistic OIRD PK–PD models. Changes in ventilation were assessed after opioid exposure and treatment with 2 mg naloxone or one or two doses of 10 mg naloxone. A higher percentage of subjects recovered back to the rescue ventilation thresholds and/or had a faster recovery to 40% or 70% of baseline ventilation with 10 mg compared with 2 mg naloxone. A second dose of 10 mg naloxone, administered 60 min post‐opioid exposure, expedited recovery to 85% of baseline ventilation and delayed time to renarcotization compared with a single dose. In addition, when 10 mg naloxone was administered at 5, 15, 30, or 60 min before fentanyl or carfentanil exposure, rapid and profound OIRD was prevented.

Alessandro Iavarone,1,2 Antonio Rosario Ziello,3,4 Francesca Pastore,3 Angiola Maria Fasanaro,3 Carla Poderico5 1Neurological and Stroke Unit, CTO Hospital, 2Italian Association on Alzheimer's Disease (AIMA), 3Memory Clinic, Neurological Unit, AORN Cardarelli Hospital, Naples, Italy; 4Clinical Research, Telemedicine and Telepharmacy Centre, University of Camerino, Camerino, Italy; 5Department of Psychology, Second University of Naples, Caserta, Italy Background: Alzheimer’s disease (AD) causes considerable distress in caregivers who are continuously required to deal with requests from patients. Coping strategies play a fundamental role in modulating the psychologic impact of the disease, although their role is still debated. The present study aims to evaluate the burden and anxiety experienced by caregivers, the effectiveness of adopted coping strategies, and their relationships with burden and anxiety. Methods: Eighty-six caregivers received the Caregiver Burden Inventory (CBI) and the ­State-Trait Anxiety Inventory (STAI Y-1 and Y-2). The coping strategies were assessed by means of the Coping Inventory for Stressful Situations (CISS), according to the model proposed by Endler and Parker in 1990.Results: The CBI scores (overall and single sections) were extremely high and correlated with dementia severity. Women, as well as older caregivers, showed higher scores. The trait anxiety (STAI-Y-2) correlated with the CBI overall score. The CISS showed that caregivers mainly adopted task-focused strategies. Women mainly adopted emotion-focused strategies and this style was related to a higher level of distress. Conclusion: AD is associated with high distress among caregivers. The burden strongly correlates with dementia severity and is higher in women and in elderly subjects. Chronic anxiety affects caregivers who mainly rely on emotion-oriented coping strategies. The findings suggest providing support to families of patients with AD through tailored strategies aimed to reshape the dysfunctional coping styles. Keywords: Alzheimer’s disease, distress, caregiver burden, stress, coping strategies

Abstract Recently, in Hepatocellular carcinoma (HCC) setting, the use of metformin has been associated to a trend toward worse response rate, overall survival and progression free survival in patients who received immunotherapy. The study population included individuals from both Eastern and Western regions with a confirmed diagnosis of HCC and receiving first line treatment with Atezolizumab plus bevacizumab or Lenvatinib. Univariate and multivariate analyses were performed by Cox proportional. For the analysis, patients were stratified based on their use of concomitant medication or not. At the time of database lock, 319 deaths were observed: 209 in the Lenvatinib cohort, 110 in the Atezolizumab plus bevacizumab cohort. In the Atezolizumab plus Bevacizumab arm, 50 (16.5%) patients were on chronic metformin use. At the univariate analysis for OS, patients who used metformin showed significantly shorter OS compared to patients who did not use metformin (HR 1.9, 95% CI 1.1–3.2). Multivariate analysis confirmed that patients in metformin group had significantly shorter OS compared to patients in no-metformin group (HR 1.9; 95% CI 1.1–3.1). At the univariate analysis for PFS, patients in metformin group had significantly shorter PFS compared to patients in no-metformin group (HR 1.6, 95% CI 1.0–2.6). Multivariate analysis confirmed that patients in metformin group had significantly shorter PFS compared to patients in no-metformin group (HR 1.7; 95% CI 1.1–2.7; p = 0.0147). No differences were reported in terms of ORR and DCR between patients in metformin group and those in no-metformin group. In the Lenvatinib cohort, 65 (15%) patients were recorded to chronically use metformin. No statistically significant differences in terms of both OS and PFS were found between patients in metformin group and patients in no-metformin group. This analysis unveils a negative prognostic role associated with metformin use specifically within the Atezolizumab plus Bevacizumab group.

Abstract Chronic liver disease and cancer are global health challenges. The role of the circadian clock as a regulator of liver physiology and disease is well established in rodents, however, the identity and epigenetic regulation of rhythmically expressed genes in human disease is less well studied. Here we unravel the rhythmic transcriptome and epigenome of human hepatocytes using male human liver chimeric mice. We identify a large number of rhythmically expressed protein coding genes in human hepatocytes of male chimeric mice, which includes key transcription factors, chromatin modifiers, and critical enzymes. We show that hepatitis C virus (HCV) infection, a major cause of liver disease and cancer, perturbs the transcriptome by altering the rhythmicity of the expression of more than 1000 genes, and affects the epigenome, leading to an activation of critical pathways mediating metabolic alterations, fibrosis, and cancer. HCV-perturbed rhythmic pathways remain dysregulated in patients with advanced liver disease. Collectively, these data support a role for virus-induced perturbation of the hepatic rhythmic transcriptome and pathways in cancer development and may provide opportunities for cancer prevention and biomarkers to predict HCC risk.

Natural products provide an unparalleled diversity of small molecules to fuel drug screening efforts, but deconvoluting the pharmacological activity of natural product mixtures to identify key bioactive compounds remains a vexing and labor-intensive process. Therefore, we have developed a new platform to probe the non-specific pharmacological potential of compounds present in common dietary supplements via shotgun derivatization with isotopically labeled propanoic acid, a live cell affinity assay, which was used to selectively recognize the population of compounds which bind tightly to HeLa cells in culture, and a computational LC-MS data analysis of isotopically labeled compounds from cell lysate. The data analysis showed that hundreds of compounds were successfully derivatized in each extract, and dozens of those compounds showed high affinity for HeLa cells. In total, over a thousand isotopically labeled compounds were screened for cell affinity across three separate experiments, resulting in the identification of several known bioactive compounds with specific protein targets and six previously unreported structures. The new natural products include three tulsinol compounds which were isolated from Ocimum tenuiflorum and three valeraninium alkaloids from Valeriana officinalis. The valeraninium alkaloids constitute a distinct new family of alkaloids from valerian, which may have previously undescribed bioactivity. These results collectively demonstrate the tag and snag workflows viability as a drug discovery method.

Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.

BackgroundOvulatory dysfunction is more common in women with obesity. Body fat distribution is also crucial because anovulatory women have a greater waist circumference and more abdominal fat than ovulatory women of similar BMI. The primary aim of the present study is to determine whether there is a relationship between BMI and reproductive characteristics, including hormonal values, antral follicle count (AFC), endometrial assessment at transvaginal ultrasound evaluation (TVUS) during controlled ovarian stimulation (COS), and oocyte retrieval after Ovum Pick-Up (OPU).MethodsData from a cohort of 183 patients were analyzed and divided into three groups based on weight status: normal weight, overweight, and obesity. Evaluated reproductive characteristics included: age, basal values of follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17-beta-estradiol (E2), thyroid stimulating hormone (TSH), anti-müllerian hormone (AMH), antral-follicle-count (AFC), duration of COS, E2, and progesterone at the last monitoring, TVUS endometrial thickness at the last monitoring before OPU, FOI after OPU. Additionally, the number of meiosis II oocytes retrieved (MII), the total dose of FSH administered, the ratio between MII and total FSH administered, and OSI were registered.ResultsAMH levels were significantly lower in obese patients compared to normal weight and overweight women (1.05 IQR 1.20, 1.58 IQR 2.16, 1.32 IQR 1.38, respectively, p-value = 0.032). When looking at the MII/FSH ratio, the normal weight group showed a median value of 3.3 with an IQR of 4.0, the overweight group showed a median value of 2.3 with an IQR of 1.9, and the obese group had a median value of 2.6 with an IQR of 2.8. Those data were statistically significant (p-value = 0.049).ConclusionThese results emphasize the importance of considering weight status in fertility assessment and treatment planning.

Scramjet engines offer a promising option for powering hypersonic vehicles, although the unique characteristics of supersonic combustion present numerous challenges. Notably, turbulence-chemistry interactions are significant in supersonic flows, demanding specific combustion formulations in numerical simulations due to their dissimilarity from traditional combustion systems. This work evaluates the performance of the Partially Stirred Reactor (PaSR) closure of the mean chemical source term by performing Reynolds-Averaged Navier-Stokes (RANS) simulations of the German Aerospace Centre (DLR) scramjet. The PaSR approach requires defining a chemical timescale, and this study assesses several formulations. Among them, the Fastest Formation Rate (FFR) does not provide stable combustion, while the Slowest Formation Rate (SFR) achieve fairly good predictions. Moreover, the sensitivity to species selection in calculating the chemical timescale is investigated, as well as the impact of two PaSR residence time formulations. A newly proposed Batchelor-based mixing timescale performs well with both the SFR and FFR formulations and guarantees a coupling between chemical and mixing times. It also accounts for species diffusivity, which can have important effects on hydrogen-based fuel combustion.

The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen-deuterium exchange experiments to define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein-solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.

A variety of electron transfer mechanisms link bacterial cytosolic electron pools with functionally diverse redox activities in the cell envelope and extracellular space. In Listeria monocytogenes, the ApbE-like enzyme FmnB catalyzes extracytosolic protein flavinylation, covalently linking a flavin cofactor to proteins that transfer electrons to extracellular acceptors. L. monocytogenes uses an energy-coupling factor (ECF) transporter complex that contains distinct substrate-binding, transmembrane, ATPase A, and ATPase A' subunits (RibU, EcfT, EcfA, and EcfA') to import environmental flavins, but the basis of extracytosolic flavin trafficking for FmnB flavinylation remains poorly defined. In this study, we show that the EetB and FmnA proteins are related to ECF transporter substrate-binding and transmembrane subunits, respectively, and are essential for exporting flavins from the cytosol for flavinylation. Comparisons of the flavin import versus export capabilities of L. monocytogenes strains lacking different ECF transporter subunits demonstrate a strict directionality of substrate-binding subunit transport but partial functional redundancy of transmembrane and ATPase subunits. Based on these results, we propose that ECF transporter complexes with different subunit compositions execute directional flavin import/export through a broadly conserved mechanism. Finally, we present genomic context analyses that show that related ECF exporter genes are distributed across members of the phylum Firmicutes and frequently colocalize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes. IMPORTANCE Bacteria import vitamins and other essential compounds from their surroundings but also traffic related compounds from the cytosol to the cell envelope where they serve various functions. Studying the foodborne pathogen Listeria monocytogenes, we find that the modular use of subunits from a prominent class of bacterial transporters enables the import of environmental vitamin B2 cofactors and the extracytosolic trafficking of a vitamin B2-derived cofactor that facilitates redox reactions in the cell envelope. These studies clarify the basis of bidirectional small-molecule transport across the cytoplasmic membrane and the assembly of redox-active proteins within the cell envelope and extracellular space.

Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology.

IntroductionCommunities affected by large scale and long lasting industrial contamination are often keen to understand whether their health has been impaired by such contamination. This requires answers that integrate environmental public health and environmental justice perspectives. At these sites, exposure scenarios from environmental contamination over time by multiple chemicals, often involving different environmental matrices, are complex and challenging to reconstruct.MethodsAn approach for describing the health of such communities in association with environmental contamination is presented, with the methods applied across the three domains of environmental contamination, population exposure and toxicology, environmental and social epidemiology, and environmental public health communication. The approach is described with examples from its application to the case study of Porto Torres, a town with a substantial industrially conditioned evolution.ResultsActivities in the field of environmental contamination, population exposure and toxicology focus on the collection and systematization of available contamination data, the identification of priority pollutants based on their toxicological profiles, the qualitative assessment of the likelihood of exposure for the population to priority pollutants and their known health effects. Environmental and social epidemiology methods are applied to describe the health profiles and socioeconomic conditions of the local population, taking into account multiple health outcomes from local information systems and considering specific diseases based on exposure and toxicological assessments. The environmental public health communication methods are directed to produce a communication plan and for its implementation through interaction with local institutional and social actors. The interpretation of health profiles benefits from a transdisciplinary analysis of the results.DiscussionThe proposed approach combines the needs of environmental public health and environmental justice allowing the integration of multidisciplinary knowledge to define recommendations for reducing and/or preventing hazardous environmental exposures and adverse health effects, stimulating the interactions between stakeholders, and making the study results more accessible to citizens.

COVID-19 has shaped and changed our normalities, and, with the discovery of new variants, the long-COVID syndrome, and stress disorders, the end of the pandemic seems distant. The current scenario is impacting all aspects of our lives. In particular, many studies reported that the pandemic resulted in increased psychiatric disorders and grief-related symptoms in adolescents. The project developed between Italy and Slovenia investigated students' experiences during the lockdown and, in particular, reported the perception of distanced learning, producing a transparent qualitative analysis that can inform future research and open to discussions on learning strategies. A survey was conducted with secondary school students in Southern Italy between 1 April and 31 May 2020 and in Central Slovenia between 16 March and 28 February 2021. Qualitative and quantitative data were collected based on students' perception of distanced learning, via an online survey platform. The results focused on three main aspects: learning experience, relationships with peers and teachers, and anxiety levels. The data showed similarities and differences in the two cohorts and suggested strategies to improve education (e.g., with blended approaches) to prepare students, teachers, and tutors for the challenges of returning to classes. In particular, data showed that it is recommended to foster collaboration between EU countries and to work to prevent students' social isolation.

The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend temperature-dependent hydrogen deuterium exchange from apo-enzyme structures to protein-ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different hydrogen deuterium exchange-mass spectrometry properties for the two ligands, as expected from their 106-fold differences in strength of binding. By contrast, analogous patterns for temperature-dependent hydrogen deuterium exchange mass spectrometry emerge in the presence of both 1-deaza-adenosine and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended temperature-dependent hydrogen deuterium exchange to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility versus site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient catalysis.

Cellular iron homeostasis is vital and maintained through tight regulation of iron import, efflux, storage and detoxification1-3. The most common modes of iron storage use proteinaceous compartments, such as ferritins and related proteins4,5. Although lipid-bounded iron compartments have also been described, the basis for their formation and function remains unknown6,7. Here we focus on one such compartment, herein named the ferrosome, that was previously observed in the anaerobic bacterium Desulfovibrio magneticus6. Using a proteomic approach, we identify three ferrosome-associated (Fez) proteins that are responsible for forming ferrosomes in D. magneticus. Fez proteins are encoded in a putative operon and include FezB, a P1B-6-ATPase found in phylogenetically and metabolically diverse species of bacteria and archaea. We show that two other bacterial species, Rhodopseudomonas palustris and Shewanella putrefaciens, make ferrosomes through the action of their six-gene fez operon. Additionally, we find that fez operons are sufficient for ferrosome formation in foreign hosts. Using S. putrefaciens as a model, we show that ferrosomes probably have a role in the anaerobic adaptation to iron starvation. Overall, this work establishes ferrosomes as a new class of iron storage organelles and sets the stage for studying their formation and structure in diverse microorganisms.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

The civil education approach uses sports practise as a preventive intervention to increase self-awareness and to help modulate emotion in adolescents. Indeed, sports participation results in a better quality of life, more favourable cardio-metabolic and sleeping profiles and a healthier body composition. Adolescents involved in sport activities also report a higher level of social and emotional skills, and reduced mental health distress. However, the Italian school's program provides only two hours weekly of physical education, with lack of options and participation. In alignment with the civil educational approach, the research team aims to develop a predictive sport, movement, eating habits, relationships and technologies (S.M.A.R.T.) questionnaire to identify the youths that present a higher risk of delinquency. The following research aims to explore preliminary data from 501 adolescents completing the S.M.A.R.T. The results show that the female group (n = 260) was healthier than the male group (n = 241); particularly, the female group demonstrated a higher level of self-awareness in the use of technologies (p < 0.05). Our results produce the first data set on a cohort of young adolescents in Italy using the S.M.A.R.T. questionnaire. The questionnaire possesses a high response rate and should now be implemented towards validation.

The first case of pneumonia was reported on 8 December 2019 and identified on 7 January 2020 as COVID-19. On 9 March 2020, to stop the spread of COVID-19 cases, the Italian government declared a health emergency, forcing all citizens to go into lockdown. Suddenly, schools were constrained to using distance learning strategies with little or limited experience on the topic. Particularly, in the southern regions of Italy, approximately 20% of the students did not have access to any devices and were excluded from learning, producing a direct risk of increased adolescent delinquency. This research team intended to report the results of an observational study that focused on the perceptions of distance learning in adolescents from secondary school in Naples (Italy) between April and May 2020. The questionnaire comprised 11 questions focused on the perceptions of distance learning in comparison to live classrooms, relationships with peers and teachers, and levels of anxiety. The study is amongst the first to report the effect of the pandemic from a student-centred perspective and hopes to produce information to develop future research on asynchronous learning.