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Summary: Saliva is a complex bodily fluid composed of secretions by major and minor salivary glands. Salivary glands and their secretions are known to be unevenly distributed in the human oral cavity. Moreover, saliva flow rate and composition vary across locations and time of the day. This remarkable heterogeneity of salivary secretions suggests that different subtypes of saliva fulfill different functions. By coupling a non-invasive and facile collection method with comprehensive metabolomic profiling, we investigated the spatial and temporal distributions of salivary components. We identified location-specific metabolite profiles, novel oscillating metabolites, and location-specific diurnal patterns. In summary, our study paves the way for a deeper and more comprehensive understanding of the complex dynamics and functionalities of the salivary metabolome and its integration in multi-omics studies related to oral and systemic (patho-)physiology.

Summary: 3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L−1 following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CLpro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay.For complete details on the use and execution of this protocol, please refer to Bafna et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

A timely detection of visual hemifield deficits (VHFDs; hemianopias or quadrantanopias) is critical for both the diagnosis and treatment of stroke patients. The present study determined the sensitivity and specificity of four qualitative visual field tests, including face description, confrontation tests (finger wiggle), and kinetic boundary perimetry, to screen large and dense VHFDs in right-brain-damaged (RBD) stroke patients. Previously, the accuracy of qualitative visual field tests was examined in unselected samples of patients with heterogeneous aetiology, in which stroke patients represented a very small fraction. Building upon existing tests, we introduced some procedural ameliorations (incl. a novel procedure for kinetic boundary perimetry) and provided a scoresheet to facilitate the grading. The qualitative visual field tests’ outcome of 67 consecutive RBD stroke patients was compared with the standard automated perimetry (SAP; i.e., reference standard) outcome to calculate sensitivity and specificity, as well as positive and negative predictive values (PPV and NPV), both for each individual test and their combinations. The face description test scored the lowest sensitivity and NPV, while the kinetic boundary perimetry scored the highest. No test returned false positives. Combining the monocular static finger wiggle test (by quadrants) and the kinetic boundary perimetry returned the highest sensitivity and specificity, in line with previous studies, but with higher accuracy (100% sensitivity and specificity). These findings indicate that the combination of these two tests is a valid approach with RBD stroke patients, prompting referral for a formal visual field examination, and representing a quick, easy-to-perform, and inexpensive tool for improving their care and prognosis.

Stroke survivors with right-brain damage (RBD) often present with attentional deficits such as left unilateral spatial neglect. Some patients also present with contralesional visual hemi-field deficits. A late detection of visual hemi-field deficits (VHFD) contributes to hampering neurorehabilitation and functional outcome of patients with neglect. The Brentano Illusion Test (BRIT) may be used for an early detection of VHFD during the neuropsychological assessment. In the present study, we determined the sensitivity and specificity of the BRIT for screening VHFD in patients with neglect. Sixty-four consecutive RBD patients were examined. Forty-five presented with neglect. Of these, 23 presented with VHFD (hemianopia or quadrantanopia) as detected by the Humphrey automated static visual field testing (reference standard). Consecutive patients also included 19 participants without neglect, who did not have any VHFD. The sensitivity and specificity of the BRIT for neglect patients were 78.3% (95% CI: 61.4–95.1) and 90.9 (95% CI: 78.9–100.0), respectively. Positive predictive value (PPV) was 89.6% (95% CI: 76.4–100.0); negative predictive value (NPV) 80.7% (95% CI: 65.2–96.2). No false positives in the group without neglect were identified. We conclude that the BRIT is an effective tool for clinical neuropsychologists to screen for possible VHFD in neglect patients during the neuropsychological assessment, allowing the refinement of the clinical picture in the neuropsychological report. An early detection of VHFD also allows referring the patient to standard diagnostics for a formal visual field examination, right from the first neuropsychological assessment.

The interest in edible insects is continuously increasing due to their environmental, nutritional, and productive features. The aim of this study was to evaluate the effects and survival of Listeria monocytogenes in Zophobas morio rearing, using two different bacterial loads (2 and 6 log CFU/g). We also considered the effect of washing, fasting, and cooking treatments on the larvae. During the experimental trial, no mortality was observed among the larvae. The L. monocytogenes loads decreased over time, and it was below the detection limit for crates inoculated with 2 log CFU/g, while the substrate inoculated with 6 log CFU/g reached loads of 4.26 (without larvae) and 2.83 log CFU/g (with larvae). Due to the absence of L. monocytogenes in the larvae on day seven, it was not possible to assess the fasting treatment or the washing and the cooking. However, when looking at the total microbial count, significant effects were revealed for all treatments. The unfasted larvae showed no effect of washing, while the total microbial counts decreased after washing in the fasted larvae. This proves that fasting is a good treatment in terms of hygiene assurance for the consumer.

Summary: Here, we present a spatially resolved sampling protocol for the oral human cavity aimed at untargeted metabolomics. We describe the spatial collection of salivary biospecimens, their preparation, and subsequent mass-spectrometry-based untargeted metabolomics analysis. Our protocol avoids complex procedures generally required for gland-specific saliva collection. For the human oral cavity, we provide an easy, flexible, and reproducible solution to comprehensively map the highly heterogeneous environment and elucidate the functionality of salivary components.For complete details on the use and execution of this protocol, please refer to Ciurli et al. (2021).

Nickel exposure is associated with tumors of the respiratory tract such as lung and nasal cancers, acting through still-uncharacterized mechanisms. Understanding the molecular basis of nickel-induced carcinogenesis requires unraveling the mode and the effects of Ni(II) binding to its intracellular targets. A possible Ni(II)-binding protein and a potential focus for cancer treatment is hNDRG1, a protein induced by Ni(II) through the hypoxia response pathway, whose expression correlates with higher cancer aggressiveness and resistance to chemotherapy in lung tissue. The protein sequence contains a unique C-terminal sequence of 83 residues (hNDRG1*C), featuring a three-times-repeated decapeptide, involved in metal binding, lipid interaction and post-translational phosphorylation. In the present work, the biochemical and biophysical characterization of unmodified hNDRG1*C was performed. Bioinformatic analysis assigned it to the family of the intrinsically disordered regions and the absence of secondary and tertiary structure was experimentally proven by circular dichroism and NMR. Isothermal titration calorimetry revealed the occurrence of a Ni(II)-binding event with micromolar affinity. Detailed information on the Ni(II)-binding site and on the residues involved was obtained in an extensive NMR study, revealing an octahedral paramagnetic metal coordination that does not cause any major change of the protein backbone, which is coherent with CD analysis. hNDRG1*C was found in a monomeric form by light-scattering experiments, while the full-length hNDRG1 monomer was found in equilibrium between the dimer and tetramer, both in solution and in human cell lines. The results are the first essential step for understanding the cellular function of hNDRG1*C at the molecular level, with potential future applications to clarify its role and the role of Ni(II) in cancer development.

Summary: Saliva is a complex bodily fluid composed of metabolites secreted by major and minor glands, as well as by-products of host oral cells, oral bacteria, gingival crevicular fluid, and exogenous compounds. Major salivary glands include the paired parotid, submandibular, and sublingual glands. The secreted fluids of the salivary glands vary in composition, flow rate, site of release, and clearance suggesting that different types of saliva fulfill different functions and therefore can provide unique biological information. Consequently, for the comprehension of the functionality of the salivary components, spatially resolved investigations are warranted. To understand and comprehensively map the highly heterogeneous environment of the oral cavity, advanced spatial sampling techniques for metabolomics analysis are needed. Here, we present a systematic evaluation of collection devices for spatially resolved sampling aimed at untargeted metabolomics and propose a comprehensive and reproducible collection and analysis protocol for the spatially resolved analysis of the human oral metabolome.

Aquatic ecosystems represent one of the largest reservoirs of phytoplankton accounting for most of the primary production of the Earth. The Lake Massaciuccoli located in Tuscany (Italy) is one of the largest swamps that in ancient times entirely covered the Versilia coastal plain. Despite its peculiar features, especially the eutrophic characteristics, its native microalgal consortia have never been explored up to now. In this work, we isolated and described four autochthonous microalgal strains from different sites in the lake (FB, Idr, CL_Sc, and CL_Ch); the four microalgal strains were identified within the Chlorella sorokiniana clade. We exposed them to ten of the most common or emerging environmental contaminants in order to describe their preliminary response to the tested substances: five metals (As, Fe, Ni, Cu, and Zn), two herbicides (Metolachlor and Sethoxydim), two antibiotics (Ciprofloxacin and Benzylpenicillin) and a non-steroidal anti-inflammatory drug (Ibuprofen). Physiological response of the strains highlighted intraspecific differences; strain CL_Sc was the most tolerant in presence of metals while strain Idr was the most sensitive. All strains were sensitive to sethoxydim and tolerant to metolachlor at all the tested concentrations. Strains FB and Idr were the most sensitive in presence of Ibuprofen while strain CL_Ch was the most sensitive to the highest Benzylpenicillin concentration. Resistance pattern of strain Idr somehow reflects both the phylogenetic and the geographic “isolation” from all other three strains. Finally, optical microscope observation confirmed some differences also in the microalgae morphological aspect. Overall, all the strains showed interesting responses in presence of high concentrations of the tested substances, representing putative interesting candidates for water remediation in wastewater treatment plants.

The Mediterranean diet has, among its cornerstones, the use of olive oil for its nutraceutical and organoleptic properties. Despite the numerous merits, olive-oil mill wastewater (OMWW), which is generated by the olive-oil extraction process, is one of the most serious environmental pollutants in the Mediterranean countries. The polluting potential of OMWW is due to its high content of tannins, polyphenols, polyalcohols, pectins and lipids. In order to close the recovery cycle of a fortified citrus olive oils previously developed, we tested the ability of five microalgae of the Chlorella group (SEC_LI_ChL_1, CL_Sc, CL_Ch, FB and Idr) in lowering the percentage of total phenolic compounds in vegetation water. This was obtained with three different extraction processes (conventional, and lemon and orange peels) at three concentrations each (10%, 25% and 50%). The results showed that strains Idr, FB and CL_Sc from the Lake Massaciuccoli can tolerate vegetation water from conventional and lemon peel extractions up to 25%; these strains can also reduce the phenolic compounds within the tests. The application of microalgae for OMWW treatment represents an interesting opportunity as well as an eco-friendly low-cost solution to be developed within companies as a full-scale approach, which could be applied to obtain a fortified microalgal biomass to be employed in nutraceutical fields.

Self-awareness (SA) is frequently impaired after severe acquired brain injury (sABI) and may lead to reduced subject’s compliance to treatment, worse functional outcome, and high caregiver distress. Considering the multifaceted nature of SA, a specific and effective assessment is crucial to address treatment of impairment of SA (ISA). Many tools can currently assess ISA; however, they have some important limits. In the present study, we proposed the Self-Awareness Multilevel Assessment Scale (SAMAS), a new scale for assessment of SA at different levels (i.e., declarative, emergent, and anticipatory) across all domains of functioning. The SAMAS has been designed to be administered by the cognitive/behavioral therapist with the involvement of a patient’s relative. Findings showed that the SAMAS allowed specifically assessing SA at a declarative level and on all possible functional domains. More interestingly, it seems also able to assess both emergent and anticipatory SA, thus overcoming some important limits of other current assessment methods. Our findings are consistent with a holistic perspective of the patient with sABI because thanks to the combined use of assessing tools, the SAMAS can provide an accurate diagnosis of ISA, thus better addressing the neurorehabilitation treatment and, accordingly, reducing the possible occurrence of its primary and secondary implications.

The increasing global population and the simultaneous growing attention to natural, sustainable, and healthier products are driving the food industry towards research on alternative food sources. In this scenario, microalgae are gaining worldwide attention as “functional feedstocks” for foods, feeds, supplements, and nutraceutical formulations, being a source of high-value metabolites including polyphenols and other antioxidant compounds. In this work, eleven microalgal strains from freshwater environments were evaluated for their nutraceutical properties, focusing on photosynthetic pigments, total polyphenols, and flavonoid content, as well as in vitro antioxidant activities. Data helped to select those strains showing the most promising features for simultaneous massive growth and bioactive compound production. Results highlighted that the microalgae have variable values for both biochemical parameters and antioxidant activities, mainly depending on the solvents and applied treatment rather than on the isolation sources or the phylogenetic attribution. According to our results, the putative best candidates for massive cultivation under laboratory conditions for the simultaneous extraction of different molecules with nutraceutical potential are strains F1 (Scenedesmaceae), F3 (Chlamydomonas debariana), R1 (Chlorella sorokiniana), and C2 (Chlorella-like).

Abstract A growing body of literature on intrinsically disordered proteins (IDPs) led scientists to rethink the structure-function paradigm of protein folding. Enzymes are often considered an exception to the rule of intrinsic disorder (ID), believed to require a unique structure for catalysis. However, recent studies revealed the presence of disorder in several functional native enzymes. In the present work, we address the importance of dynamics for catalysis, by investigating the relationship between folding and activity in Sporosarcina pasteurii UreG (SpUreG), a P-loop GTPase and the first discovered native ID enzyme, involved in the maturation of the nickel-containing urease. The effect of denaturants and osmolytes on protein structure and activity was analyzed using circular dichroism (CD), Site-Directed Spin Labeling (SDSL) coupled to EPR spectroscopy, and enzymatic assays. Our data show that SpUreG needs a “flexibility window” to be catalytically competent, with both too low and too high mobility being detrimental for its activity.

Neuropsychological studies on acquired topographical disorientation have provided useful insights into the contribution of different brain regions to human navigation. However, little is known about the possibility to restore navigational skills after brain damage. Here we describe the case of No Longer Lost (NLL), a 49-year-old man who complained of severe topographical disorientation following traumatic brain injury. Extensive neuropsychological evaluation at baseline revealed selective episodic memory deficits and topographical disorientation. NLL underwent 8-week imagery-based treatment (IBT) inspired by current cognitive models of human spatial navigation. After IBT, NLL improved topographical skills and episodic memory. From a clinical point of view, the present study describes a model-based intervention for topographical disorientation. From a theoretical point of view, it provides new insights into the cognitive models of human spatial navigation and straightforward evidence about common phylogenetic roots of brain mechanisms devoted to spatial navigation and memory.

UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of Helicobacter pylori (Hp) UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution. Here, isothermal titration calorimetry and site-directed spin labeling coupled to electron paramagnetic spectroscopy were successfully combined to investigate HpUreG structural dynamics in solution and the effect of Ni(II) and GTP on protein mobility. The results demonstrate that, although the protein maintains a flexible behavior in the metal and nucleotide bound forms, concomitant addition of Ni(II) and GTP exerts a structural change through the crosstalk of different protein regions.

Urease is a nickel-containing enzyme that is essential for the survival of several and often deadly pathogenic bacterial strains, including Helicobacter pylori. Notwithstanding several attempts, the development of direct urease inhibitors without side effects for the human host remains, to date, elusive. The recently solved X-ray structure of the HpUreDFG accessory complex involved in the activation of urease opens new perspectives for structure-based drug discovery. In particular, the quaternary assembly and the presence of internal tunnels for nickel translocation offer an intriguing possibility to target the HpUreDFG complex in the search of indirect urease inhibitors. In this work, we adopted a theoretical framework to investigate such a hypothesis. Specifically, we searched for putative binding sites located at the protein–protein interfaces on the HpUreDFG complex, and we challenged their druggability through structure-based virtual screening. We show that, by virtue of the presence of tunnels, some protein–protein interfaces on the HpUreDFG complex are intrinsically well suited for hosting small molecules, and, as such, they possess good potential for future drug design endeavors.

The interaction between macromolecules is a fundamental aspect of most biological processes. The computational techniques used to study protein-protein and protein-nucleic acid interactions have evolved in the last few years because of the development of new algorithms that allow the a priori incorporation, in the docking process, of experimentally derived information, together with the possibility of accounting for the flexibility of the interacting molecules. Here we review the results and the evolution of the techniques used to study the interaction between metallo-proteins and DNA operators, all involved in the nickel and iron metabolism of pathogenic bacteria, focusing in particular on Helicobacter pylori (Hp). In the first part of the article we discuss the methods used to calculate the structure of complexes of proteins involved in the activation of the nickel-dependent enzyme urease. In the second part of the article, we concentrate on two applications of protein-DNA docking conducted on the transcription factors HpFur (ferric uptake regulator) and HpNikR (nickel regulator). In both cases we discuss the technical expedients used to take into account the conformational variability of the multi-domain proteins involved in the calculations.

Sweet basil (Ocimum basilicum L.) is one of the most important herbs widely used for its medicinal properties and as food ingredient. The marketing of this product highlights the problem that these plants have long and slender stems, which are easy to break off and thus making difficult their market distribution. In this work, two cultivars of basil (Genovese and Profumo) at the adequate development stage for sale were used. We evaluated the effect of supplemental ultraviolet (UV)-B irradiation (15 W m-2; 3 h day-1) on plant growth and market quality. Both cultivars of basil plants under UV-B irradiation resulted in increased leaf size and biomass, and decreased shoot length in comparison to that of under control growth conditions. These results indicate that the application of UV-B irradiation beneficially influenced plant architecture in basil improving their greenhouse production for fresh market.

Following the discovery of the first specific and essential role of nickel in biology in 1975 (the dinuclear active site of the enzyme urease) [...]

The aviation industry stands as a well-known "hard to electrify" sector, currently responsible for the consumption of massive amounts of fossil fuels, considerably contributing to global CO 2 emissions. The need to reduce the aviation carbon footprint to comply with the European Green Deal objectives, has driven intensive research into the so-called bio-jet fuels (BJF) that can be obtained from different lipid-rich substrates, including microalgae among the most promising. Starting from the analysis of existing studies dealing with the selection of the strains more indicated for BJF production, this review examines the most recent breakthrough in microalgae cultivation techniques and lipid accumulation strategies, focusing on the approaches targeting the enhancement of the process environmental sustainability. The main bottlenecks in each phase of the production process are identified and critically reviewed. The most recent solutions are also thoroughly discussed to point out room for improvements in consolidated engineering strategies, as well as areas of further scientific research to advance the state of the art on micro-algal potential for BJF production. [Display omitted] • Microalgae use for the the production of bio-jet fuels is critically reviewed. • Production costs of microalgal BJF should be reduced to make this technology competitive. • Main costs are associated to microalgae cultivation rather than algal oil upgrade. • Different strategies adopted to make the cultivation sustainable are critically discussed. [ABSTRACT FROM AUTHOR]

Oral mucositis is an important side effect of hematopoietic stem cell transplantation (HCST), mainly due to toxicity of conditioning regimens. It produces significant pain and morbidity. The present study reports a prospective, randomized, non-blinded study testing the efficacy of a new mouthwash, called Baxidil Onco® (Sanitas Farmaceutici Srl, Tortona, Italy) in 60 hematologic patients undergoing HCST (28 autologous, 32 allogeneic). Baxidil Onco®, used three times a day from Day -1 to Day +30, in addition to standard prophylactic schedules, was administered to 14 patients undergoing autologous and 14 patients undergoing allogeneic HCST. The remaining 32 patients (14 autologous and 18 HCST) were treated only with standard prophylactic schedules and served as control. In our study, the overall incidence of oral mucositis, measured according to the World Health Organization 0-4 scale, was 50% in the Baxidl Onco® group versus 82% in the control group (P=0.022). In addition, a significant reduction in scale 2-4 oral mucositis was observed in the Baxidil Onco® group (25% vs 56.2%; P=0.0029). The results obtained indicate that incidence, severity and duration of oral mucositis induced by conditioning regi- mens for HCST can be significantly reduced by oral rinsing with Baxidil Onco®, in addition to the standard prophylaxis scheme. Since Camelia Sinensin extract, which is used to produce green tea, is the main agent in this mouthwash, we hypothesize that the anti-oxidative properties of polyphenolic compounds of tea might exert protective effects on oral mucosa.

Objective Impaired self-awareness (ISA) of altered functional capacities is a common sequelae of severe acquired brain injury that can severely hamper neuro-rehabilitation in this clinical population. ISA is frequently associated with anosodiaphoria and/or apathy. Although several scales are available to measure apathy, no tools have been published to specifically assess anosodiaphoria after acquired brain injury. In this paper, we reported an initial effort to develop an anosodiaphoria subscale in a commonly used measure of ISA, that is, the Patient Competency Rating scale-neurorehabilitation form (PCRS-NR). Method A sample of 46 participants with severe acquired brain injury completed a functional, ISA, apathy, and anosodiaphoria assessment. One informal caregiver of each patient participated in the study. Thus, we were able to obtain external data on his/her level of functional competencies, and self-awareness, which allowed separating patients with low self-awareness (LSA) from those with high self-awareness (HSA). Finally, the patients were compared with 44 healthy age–gender–years of formal education matched control participants (HCs). Results Compared to both patients with HSA and HCs, patients with LSA demonstrated greater anosodiapvhoria and lower levels of functioning than both HSA patients and HCs. A stronger relationship emerged between ISA and anosodiaphoria rather than with apathy. Conclusions These initial findings provide support that PCRS scale can be adapted to measure anosodiaphoria as well as ISA. The findings reveal a stronger correlation between this measure of anosodiaphoria and ISA compared with the correlation of apathy to ISA. The present method for measuring anosodiaphoria takes into account the actual levels of patients' functioning. [ABSTRACT FROM AUTHOR]