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The Kluyveromyces lactis superoxide dismutase 1(KlSOD1) and K. lactis CCS1 (KlCCS1) genes are isolated and the effect of their increased dosage on the secretion of heterologous proteins is evaluated. The results have shown that the production of some heterologous proteins is improved by manipulating genes involved in general stress responses.

Background Hailey–Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, histologically characterized by suprabasal acantholysis. HHD has been linked to mutations in ATP2C1 , the gene encoding the human adenosine triphosphate (ATP)-powered calcium channel pump. Methods In this work, the genetically tractable yeast Kluyveromyces lactis has been used to study the molecular basis of Hailey-Hailey disease. The K. lactis strain depleted of PMR1 , the orthologue of the human ATP2C1 gene, was used to screen a Madin-Darby canine kidney (MDCK) cDNA library to identify genetic interactors able to suppress the oxidative stress occurring in those cells. Results We have identified the Glutathione S-transferase ϴ-subunit (GST), an important detoxifying enzyme, which restores many of the defects associated with the pmr1Δ mutant. GST overexpression in those cells suppressed the sensitivity to calcium chelating agents and partially re-established calcium (Ca 2 + ) homeostasis by decreasing the high cytosolic Ca 2 + levels in pmr1Δ strain. Moreover, we found that in the K. lactis mutant the mitochondrial dysfunction was suppressed by GST overexpression independently from calcineurin. In agreement with yeast results, a decreased expression of the human GST counterpart ( GSTT1/M1 ) was observed in lesion-derived keratinocytes from HHD patients. Conclusions These data highlighted the Glutathione S-transferase as a candidate gene associated with Hailey-Hailey disease. General significance Kluyveromyces lactis can be considered a good model to study the molecular basis of this pathology. [ABSTRACT FROM AUTHOR]

Abstract: Background: In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag− phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing. Methods: A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses. Results: Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced. Conclusions: The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)+/NAD(P)H redox balance state. General significance: The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response. [Copyright &y& Elsevier]

Tirzepatide (TZP) is a new anti-obesity drug, and little is currently known about its effect on body composition (BC) in people with overweight or obesity. The aim of this study is to conduct a systematic review on the impact of TZP on BC compartments in this population during weight loss programs. Literature searches, study selection, method development, and quality appraisal were performed. The data were synthesized using a narrative approach, in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. Of the 1379 papers retrieved, 6 randomized controlled trials met the inclusion criteria and were reviewed, revealing the following findings. Firstly, TZP was shown to result in a significant reduction in total fat mass (FM), visceral adipose tissue (VAT) and waist circumference (WC) between baseline and short as well as intermediate follow-ups. Compared to other anti-obesity medications (e.g., dulaglutide and semaglutide) taken over the same duration, TZP showed a superior decrease in body fat compartments (i.e., total FM, VAT and WC). Finally, the effect of TZP on fat-free mass (FFM) is still uncertain because the findings remain inconclusive. In conclusion, TZP appears to be an effective strategy for achieving significant improvements in body fat and its distribution, but additional investigations are still needed to determine the impact of TZP on lean mass in this population.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels 1 are essential for pacemaking activity and neural signalling 2,3 . Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain 4 and epileptic seizures 5 . The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor 6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs.  7,8 ). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Phospholipid scramblases mediate the rapid movement of lipids between membrane leaflets, a key step in establishing and maintaining membrane homeostasis of the membranes of all eukaryotic cells and their organelles. Thus, impairment of lipid scrambling can lead to a variety of pathologies. How scramblases catalyzed the transbilayer movement of lipids remains poorly understood. Despite the availability of direct structural information on three unrelated families of scramblases, the TMEM16s, the Xkrs, and ATG-9, a unifying mechanism has failed to emerge thus far. Among these, the most extensively studied and best understood are the Ca 2+ activated TMEM16s, which comprise ion channels and/or scramblases. Early work supported the view that these proteins provided a hydrophilic, membrane-exposed groove through which the lipid headgroups could permeate. However, structural, and functional experiments have since challenged this mechanism, leading to the proposal that the TMEM16s distort and thin the membrane near the groove to facilitate lipid scrambling. Here, we review our understanding of the structural and mechanistic underpinnings of lipid scrambling by the TMEM16s and discuss how the different proposals account for the various experimental observations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alessio Accardi reports financial support was provided by National Institutes of Health. Omar E. Alvarenga reports financial support was provided by National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: Many cases of subacute thyroiditis (SAT) have been described related to SARS-CoV-2 infection, but no prospective data about follow-up are known. This prospective, longitudinal, 3-year, multicentre study aims to explore the clinical peculiarities and outcome of SAT in relation to SARS-CoV-2 infection, ascertained with antibody dosage., Methods: All patients receiving SAT diagnosis from November 2020 to May 2022 were enrolled. Data on anamnesis, physical examination, blood tests (TSH, freeT4, freeT3, thyroglobulin, anti-thyroid antibodies, C-reactive protein, erythrocyte sedimentation rate, complete blood count), and thyroid ultrasound were collected. At baseline, the presence of IgG against the SARS-CoV-2 spike protein or nucleocapsid was investigated. Patients were evaluated after 1, 3, 6, and 12 months., Results: Sixty-six subjects were enrolled. At baseline, 54 presented with pain, 36 (67%) for at least 15 days. Serum SARS-CoV-2 IgG measurements documented that 7 out of 52 subjects (13.5%) had infection before SAT diagnosis (COVID+). No significant differences between the COVID+ and COVID- groups were found at baseline, except for respiratory symptoms and fever, which were more common in COVID+ (P = 0.039 and P = 0.021, respectively). Among the 41 subjects who completed follow-up, COVID+ and COVID- did not differ for therapeutic approach to SAT or outcome, all having an improvement in neck pain, inflammation parameters, and ultrasound features., Conclusion: This is the first prospective study investigating any difference both at diagnosis and at follow-up between SAT presentation in patients with previous SARS-CoV-2 infection and those without. Our data demonstrate that SARS-CoV-2 does not impact on SAT onset, evolution, and outcome.

Membrane asymmetry is important for cellular physiology and established by energy-dependent unidirectional lipid translocases, which have diverse physiological functions in plants. By contrast, the role of phospholipid scrambling (PLS), the passive bidirectional lipid transfer leading to the break-down of membrane asymmetry, is currently still unexplored. The Arabidopsis thaliana genome contains a single gene (At1g73020) with homology to the eukaryotic TMEM16 family of Ca 2+ -activated phospholipid scramblases. Here, we investigated the protein function of this Arabidopsis homolog. Fluorescent AtTMEM16 fusions localized to the ER both in transiently expressing Arabidopsis protoplasts and HEK293 cells. A putative scrambling domain (SCRD) was identified on the basis of sequence conservation and conferred PLS to transfected HEK293 cells, when grafted into the backbone of the non-scrambling plasma membrane-localized TMEM16A chloride channel. Finally, AtTMEM16 'gain-of-function' variants gave rise to cellular phenotypes typical of aberrant scramblase activity, which were reversed by the additional introduction of a 'loss-of-function' mutation into the SCRD. In conclusion, our data suggest AtTMEM16 works as an ER-resident lipid scramblase in Arabidopsis., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

ClC-7 is a chloride-proton antiporter of the CLC protein family. In complex with its accessory protein Ostm-1, ClC-7 localizes to lysosomes and to the osteoclasts' ruffled border, where it plays a critical role in acidifying the resorption lacuna during bone resorption. Gene inactivation in mice causes severe osteopetrosis, neurodegeneration, and lysosomal storage disease. Mutations in the human CLCN7 gene are associated with diverse forms of osteopetrosis. The functional evaluation of ClC-7 variants might be informative with respect to their pathogenicity, but the cellular localization of the protein hampers this analysis. Here we investigated the functional effects of 13 CLCN7 mutations identified in 13 new patients with severe or mild osteopetrosis and a known ADO2 mutation. We mapped the mutated amino acid residues in the homology model of ClC-7 protein, assessed the lysosomal colocalization of ClC-7 mutants and Ostm1 through confocal microscopy, and performed patch-clamp recordings on plasma-membrane-targeted mutant ClC-7. Finally, we analyzed these results together with the patients' clinical features and suggested a correlation between the lack of ClC-7/Ostm1 in lysosomes and severe neurodegeneration. © 2020 American Society for Bone and Mineral Research (ASBMR)., (© 2020 American Society for Bone and Mineral Research (ASBMR).)

Mutations in the human TMEM16E/ANO5 gene are causative for gnathodiaphyseal dysplasia (GDD), a rare bone malformation and fragility disorder, and for two types of muscular dystrophy (MD). Previous studies have demonstrated that TMEM16E/ANO5 is a Ca 2+ -activated phospholipid scramblase and that the mutation c.1538C>T (p.Thr513Ile) causing GDD leads to a gain-of-function phenotype. Here, using established HEK293-based functional assays, we investigated the effects of MD-related and further GDD-related amino acid exchanges on TMEM16E/ANO5 function in the same expression system. These experiments also revealed that the gradual changes in HEK293 cell morphology observed upon expression of TMEM16E/ANO5 GDD mutants are a consequence of aberrant protein activity. Our results collectively demonstrate that, on the level of protein function, MD mutations are associated to loss-of-function and GDD mutations to gain-of-function phenotypes, confirming conjectures made on the basis of inheritance modes., (© 2020 Wiley Periodicals, Inc.)

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Although a number of bacteria can cause UTIs, most cases are due to infection by uropathogenic Escherichia coli (UPEC). UPEC are a genetically heterogeneous group that exhibit several virulence factors associated with colonization and persistence of bacteria in the urinary tract. Caenorhabditis elegans is a tiny, free-living nematode found worldwide. Because many biological pathways are conserved in C. elegans and humans, the nematode has been increasingly used as a model organism to study virulence mechanisms of microbial infections and innate immunity. The virulence of UPEC strains, characterized for antimicrobial resistance, pathogenicity-related genes associated with virulence and phylogenetic group belonging was evaluated by measuring the survival of C. elegans exposed to pure cultures of these strains. Our results showed that urinary strains can kill the nematode and that the clinical isolate ECP110 was able to efficiently colonize the gut and to inhibit the host oxidative response to infection. Our data support that C. elegans, a free-living nematode found worldwide, could serve as an in vivo model to distinguish, among uropathogenic E. coli, different virulence behavior., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca 2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca 2+ concentrations. Contrarily, mutation of the homologous position in the Ca 2+ -activated Cl - channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca 2+ -dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.

Heterozygous mutations in the PHOX2B gene are causative of congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by defective autonomic control of breathing due to the impaired differentiation of neural crest cells. Among PHOX2B mutations, polyalanine (polyAla) expansions are almost exclusively associated with isolated CCHS, whereas frameshift variants, although less frequent, are often more severe than polyAla expansions and identified in syndromic CCHS. This article provides a complete review of all the frameshift mutations identified in cases of isolated and syndromic CCHS reported in the literature as well as those identified by us and not yet published. These were considered in terms of both their structure, whether the underlying indels induced frameshifts of either 1 or 2 steps ("frame 2" and "frame 3" mutations respectively), and clinical associations. Furthermore, we evaluated the structural and functional effects of one "frame 3" mutation identified in a patient with isolated CCHS, and one "frame 2" mutation identified in a patient with syndromic CCHS, also affected with Hirschsprung's disease and neuroblastoma. The data thus obtained confirm that the type of translational frame affects the severity of the transcriptional dysfunction and the predisposition to isolated or syndromic CCHS., (© 2017 The Authors. Human Mutation published byWiley Periodicals, Inc.)

Background: Secondary caries are considered the main cause of dental restoration failure. In this context, anti-biofilm and bactericidal properties are desired in dental materials against pathogens such as Streptococcus mutans. To this purpose, graphene based materials can be used as fillers of polymer dental adhesives. In this work, we investigated the possibility to use as filler of dental adhesives, graphene nanoplatelets (GNP), a non toxic hydrophobic nanomaterial with antimicrobial and anti-biofilm properties., Results: Graphene nanoplatelets have been produced starting from graphite intercalated compounds through a process consisting of thermal expansion and liquid exfoliation. Then, a dental adhesive filled with GNPs at different volume fractions has been produced through a solvent evaporation method. The rheological properties of the new experimental adhesives have been assessed experimentally. The adhesive properties have been tested using microtensile bond strength measurements (µ-TBS). Biocidal activity has been studied using the colony forming units count (CFU) method. The anti-biofilm properties have been demonstrated through FE-SEM imaging of the biofilm development after 3 and 24 h of growth., Conclusions: A significantly lower vitality of S. mutans cells has been demonstrated when in contact with the GNP filled dental adhesives. Biofilm growth on adhesive-covered dentine tissues demonstrated anti-adhesion properties of the produced materials. µ-TBS results demonstrated no significant difference in µ-TBS between the experimental and the control adhesive. The rheology tests highlighted the necessity to avoid low shear rate regimes during adhesive processing and application in clinical protocol, and confirmed that the adhesive containing the 0.2%wt of GNPs possess mechanical properties comparable with the ones of the control adhesive.