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SALL2- a member of the Spalt gene family- is a poorly characterized transcription factor found deregulated in various cancers, which suggests it plays a role in the disease. We previously identified SALL2 as a novel interacting protein of neurotrophin rece

Marcelo J Martins,1 Caroline Paiva Matos Oliveira Martins,2 Lucas J Castro-Alves,3 Gabriel Nascimento Jesus,4 Guilherme Oliveira Campos,5 Breno Barbosa Cerqueira Sacramento,4 Leonardo Ferrari Borges,6 Carlos Augusto Bastos Mello,6 Rodrigo Leal Alves,5 Norma Sueli Pinheiro Módolo7 1Department of Anesthesiology, São Paulo State University (UNESP), São Paulo, Brazil; 2Department of Anesthesiology at Federal University of Bahia, Bahia, Brazil; 3Department of Anesthesiology, Santo Antonio Hospital, Salvador, Bahia, Brazil; 4Department of Anesthesia, Bahia University of Medicine and Public Health, Bahia, Brazil, 5Department of Anesthesiology, Hospital Sao Rafael, Salvador, Bahia, Brazil; 6Department of Surgery, Hospital Tereza de Lisieux, Salvador, Bahia, Brazil; 7Department of Anaesthesiology, São Paulo State University (UNESP), Botucatu, Brazil Purpose: Obesity has been considered as a major public health problem in developed countries for which bariatric surgery has become an important treatment strategy. Postoperative pain, however, is a frequent problem in postoperative management. Pregabalin blocks the development of hyperalgesia and central pain sensitization. The objective of this randomized, placebo-controlled, double-blinded trial was to evaluate the effect of a single dose of preoperative pregabalin vs placebo on the quality of postoperative recovery in patients undergoing bariatric surgery.Patients and methods: A total of 70 patients undergoing abdominal gastroplasty were randomly assigned to receive oral pregabalin (75 mg) or an identical placebo 1 hour before surgery. The primary outcome was Quality of Recovery-40 (QoR-40) score at 24 hours. Secondary outcomes included opioid consumption and postoperative pain scores. P

Cell and Gene therapy are referred to as advanced therapies that represent overlapping fields of regenerative medicine. They have similar therapeutic goals such as to modify cellular identity, improve cell function, or fight a disease. These two therapeutic avenues, however, possess major differences. While cell therapy involves introduction of new cells, gene therapy entails introduction or modification of genes. Furthermore, the aim of cell therapy is often to replace, or repair damaged tissue, whereas gene therapy is used typically as a preventive approach. Diabetes mellitus severely affects the quality of life of afflicted individuals and has various side effects including cardiovascular, ophthalmic disorders, and neuropathy while putting enormous economic pressure on both the healthcare system and the patient. In recent years, great effort has been made to develop cutting-edge therapeutic interventions for diabetes treatment, among which cell and gene therapies stand out. This review aims to highlight various cell- and gene-based therapeutic approaches leading to the generation of new insulin-producing cells as a topmost "panacea" for treating diabetes, while deliberately avoiding a detailed molecular description of these approaches. By doing so, we aim to target readers who are new to the field and wish to get a broad helicopter overview of the historical and current trends of cell- and gene-based approaches in β-cell regeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Rohban, Martins and Esni.)

Introduction: Primary health care is a key element in the structuring and coordination of health systems, contributing to overall coverage and performance. PHC financing is therefore central in this context, with variations in sufficiency and regularity depending on the "political dimension" of health systems. Research that systematically examines the political factors and arrangements influencing PHC financing is justified from a global and multidisciplinary perspective. The scoping review proposed here aims to systematically map the evidence on this topic in the current literature, identifying groups, institutions, priorities and gaps in the research., Methods and Analysis: A scoping review will be conducted following the method proposed by Arksey and O'Malley to answer the following question: What is known from the literature about political factors and arrangements and their influence on and repercussions for primary health care financing and resource allocation models? The review will include peer-reviewed papers in Portuguese, English or Spanish published between 1978 and 2023. Searches will be performed of the following databases: Medline (PubMed), Embase, BVS Salud, Web of Science, Scopus and Science Direct. The review will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews checklist. Inclusion and exclusion criteria will be used for literature screening and mapping. Screening and data charting will be conducted by a team of four reviewers., Registration: This protocol is registered on the Open Science Framework (OSF) platform, available at https://doi.org/10.17605/OSF.IO/Q9W3P., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dias et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This cross-sectional study aims to identify the perspective of healthcare professionals and residents regarding the challenges faced in providing care to Indigenous users in a reference hospital in Mato Grosso do Sul, a Brazilian state with the second-largest Indigenous population in the country. The study used a semi-structured online questionnaire emailed to each worker between June and August 2020. The discrete variables were summarized as mean and standard deviation and median and interquartile range (5% significance level). Two hundred thirty healthcare professionals and 29 residents participated in the study. Among the findings, only 14.7% of participants knew the ethnicities served, and 60.2% had never witnessed traditional practices in the hospital, indicating low articulation between biomedical and Indigenous forms of care. When comparing responses from residents and professionals, residents were noted to have a more positive view of approaching the Indigenous context, suggesting that they consider it essential to improve this articulation. When comparing professional categories, some differences of opinion among the medical category stand out concerning assistance. In addition, professionals and residents demonstrated some level of difficulty in caring for the Indigenous population. The results highlight the centrality of the biomedical model, the professionals' lack of knowledge about the context of the communities served, and the devaluation of their practices. The findings contribute to discussions about healthcare policies at different levels of care and management and the qualification of hospital care for Indigenous people.

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8 + T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8 + T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity., (© 2024. The Author(s).)

Background: The understanding of human postural control has advanced with the introduction of optimization process modeling. These models, however, only provide control parameters, rather than analytical descriptors of optimization processes. Here, we use a newly developed direct (pattern) search algorithm to investigate changes in postural optimization process in poststroke individuals., Objective: This cross-sectional study investigated the optimization properties of postural stability during upright standing in poststroke individuals., Methods: Twenty-nine poststroke and 15 healthy age-matched individuals underwent posturography with a force platform while standing for 60 s for acquisition of center-of-pressure data. Poststroke individuals were grouped depending on their weight-bearing (WB) pattern and their balance capability assessed through Berg Balance Scale (BBS). The optimization properties of postural stability were computed assuming the minimization of postural sway as cost function., Results: The asymmetric WB poststroke group showed larger convergence rate toward the local minimum of postural sway than the symmetric WB group. Additionally, the low-balance capability group exhibited smaller values for averaged local minima and global minimum of postural sway coordinates compared with high-balance capability group. Significant correlations were found for BBS and the local minima and global minimum (Pearson's r ranged 0.378-0.424, P < 0.05)., Conclusions: In summary, the optimization properties describing postural dynamic stability, steadiness, and global reference are altered in poststroke individuals with asymmetric WB pattern and low-balance capability.

Shrimp antilipopolysaccharide factors (ALFs) form a multifunctional and diverse family of antimicrobial host defense peptides (AMPs) composed of seven members (groups A to G), which differ in terms of their primary structure and biochemical properties. They are amphipathic peptides with two conserved cysteine residues stabilizing a central β-hairpin that is understood to be the core region for their biological activities. In this study, we synthetized three linear (cysteine-free) peptides based on the amino acid sequence of the central β-hairpin of the newly identified shrimp ( Litopenaeus vannamei ) ALFs from groups E to G. Unlike whole mature ALFs, the ALF-derived peptides exhibited an α-helix secondary structure. In vitro assays revealed that the synthetic peptides display a broad spectrum of activity against both Gram-positive and Gram-negative bacteria and fungi but not against the protozoan parasites Trypanosoma cruzi and Leishmania ( L .) infantum . Remarkably, they displayed synergistic effects and showed the ability to permeabilize bacterial membranes, a mechanism of action of classical AMPs. Having shown low cytotoxicity to THP-1 human cells and being active against clinical multiresistant bacterial isolates, these nature-inspired peptides represent an interesting class of bioactive molecules with biotechnological potential for the development of novel therapeutics in medical sciences.

AZD4625 is a potent, selective, and orally bioavailable inhibitor of oncogenic KRASG12C as demonstrated in cellular assays and in vivo in preclinical cell line-derived and patient-derived xenograft models. In vitro and cellular assays have shown selective binding and inhibition of the KRASG12C mutant isoform, which carries a glycine to cysteine mutation at residue 12, with no binding and inhibition of wild-type RAS or isoforms carrying non-KRASG12C mutations. The pharmacology of AZD4625 shows that it has the potential to provide therapeutic benefit to patients with KRASG12C mutant cancer as either a monotherapy treatment or in combination with other targeted drug agents., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Postural instability with an excessive body sway is a disabling manifestation in spinocerebellar ataxia type 3. Whether the larger body sway reflects distinct movement strategies for postural control remains uncertain. This study compared the control of postural stability of people with spinocerebellar ataxia type 3 with healthy subjects using body sway and movement strategy analyses derived from bi- and three-dimensional posturography., Methods: Twenty-three patients (7 men, 16 women, 47 ± 11 years) and 102 healthy participants (34 men, 68 women; 44 ± 22 years) underwent posturography while standing with eyes open/closed tasks. Postural stability was assessed using elliptical area and average velocity of body sway. Spatial patterns (single-, double-, or multi-centered) were derived from the number of high-density regions in the three-dimensional statokinesigram., Findings: Repeated measures two-way analysis-of-variance showed a vision-by-group interaction effect for area (F 1,122  = 28.831, P < 0.001, η 2  = 0.037) and velocity (F 1,123  = 59.367, P < 0.001, η 2  = 0.073); sway area and velocity were higher in spinocerebellar ataxia type 3 and increased under eyes-closed condition, with a higher increase in the spinocerebellar ataxia type 3. A main effect for group (F 1,123  = 11.702, P < 0.001, η 2  = 0.061) but not vision (F 1,123  = 2.257, P = 0.136, η 2  = 0.005) was found for the number of high-density regions. Spatial patterns were different between groups under trials with eyes closed (χ 2 2,125  = 7.46, P = 0.023) but not open (χ 2 2,125  = 2.026, P = 0.363), with a shift from single- to double- or multi-centered spatial patterns., Interpretation: Compared to healthy subjects, a larger body displacement and velocity in spinocerebellar ataxia type 3, mainly under visual constraints, are not related to the predominance of either ankle or hip movement strategies., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Since distinguishing pulmonary (PTB) from latent tuberculosis (LTBI) in pediatric patients remains a challenge, we aimed to investigate the efficacy of immune mediators in diagnosing PTB and LTBI in this population. In this cross-sectional study performed with children and adolescents, serum levels of 20 biomarkers were assessed and data were analyzed according to age groups. We included 65 participants (PTB, n = 28 and LTBI, n = 37). Overall, levels of TNF-α, IL-1Ra, IL-6, IL-17A, VEGF, MMP-1, and procalcitonin were significantly higher (P < 0.05) in adolescents and children <10 years-old with PTB. Also, principal component analysis (PCA) showed that immune mediators were able to distinguish PTB from LTBI. VEGF and IL-1Ra presented the highest area under the curve (AUC) values, both separately (AUC 0.890 and 0.785) and combined (AUC 0.99). Taken together, we showed that VEGF and IL-1Ra are promising biomarkers to distinguish PTB from LTBI in pediatric patients, especially in children <5 years-old., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Importance: A widely cited meta-analysis of randomized clinical trials has claimed ivermectin as an effective treatment for prevention of mortality in COVID-19. However, an unrecognized interaction variable with the relative risk (RR) of mortality may substantially change the appropriate interpretation of this analysis., Objective: To evaluate the association between regional prevalence of strongyloidiasis and ivermectin trial results for the outcome of mortality by testing the hypothesis that strongyloidiasis prevalence interacts with the RR of mortality., Data Sources: Original meta-analysis as well as a manual review of all references in a dedicated ivermectin trial database (c19ivermectin) from January 1, 2019, to November 6, 2021., Study Selection: Randomized clinical trials using ivermectin as a treatment for COVID-19 and reporting the outcome of mortality. Studies were excluded in the event of publications revealing suspected trial fraud and/or randomization failure., Data Extraction and Synthesis: Study characteristics and RR estimates were extracted from each source. Estimates were pooled using random-effects meta-analysis. Differences by strongyloidiasis prevalence were estimated using subgroup meta-analysis and meta-regression. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline was followed., Main Outcomes and Measures: Relative risk of mortality in ivermectin trials in regions of high vs low strongyloidiasis prevalence and correlation coefficient of meta-regression analysis between RR of mortality and regional prevalence of strongyloidiasis., Results: A total of 12 trials comprising 3901 patients were included in the analysis. Four trials (33%) took place in regions of high strongyloidiasis prevalence and 8 (67%) trials took place in regions of low strongyloidiasis prevalence. Ivermectin trials that took place in areas of low regional strongyloidiasis prevalence were not associated with a statistically significant decreased risk of mortality (RR, 0.84 [95% CI, 0.60-1.18]; P = .31). By contrast, ivermectin trials that took place in areas of high regional strongyloidiasis prevalence were associated with a significantly decreased risk of mortality (RR, 0.25 [95% CI, 0.09-0.70]; P = .008). Testing for subgroup differences revealed a significant difference between the results of groups with low and high strongyloidiasis prevalence (χ21 = 4.79; P = .03). The estimate for τ2 (the variance of the study effect sizes) was 0 (95% CI, 0.0000-0.2786), and the estimate for I2 (percentage of variability that is explained by between-study heterogeneity) was 0 (95% CI, 0-43.7%). The meta-regression analysis revealed an RR decrease of 38.83% (95% CI, 0.87%-62.25%) for each 5% increase in strongyloidiasis prevalence., Conclusions and Relevance: In this meta-analysis of 12 trials including 3901 patients, strongyloidiasis prevalence was found to interact with the RR of mortality for ivermectin as a treatment for COVID-19. No evidence was found to suggest ivermectin has any role in preventing mortality among patients with COVID-19 in regions where strongyloidiasis was not endemic.

Fibromyalgia (FM) is a complex pathology described as persistent hyperalgesia including somatic and mood dysfunctions, depression and anxiety. Although the etiology of FM is still unknown, a significant decrease in biogenic amines is a common characteristic in its pathogenesis. Here, our main objective was to investigate the role of dopamine D3/D2 receptor during the reserpine-induced pain in mice. Our results showed that pramipexole (PPX) - a dopaminergic D3/D2 receptor agonist - inhibited mechanical allodynia and thermal sensitivity induced by reserpine. Relevantly, PPX treatment decreased immobility time and increased the number of grooming in the forced swimming test and splash test, respectively. Animals that received PPX remained longer in the open arms than the reserpine group using elevated plus-maze apparatus. The repeated PPX administration, given daily for 4 days, significantly blocked the mechanical and thermal allodynia during FM model, similarly to pregabalin, although it failed to affect the reserpine-induced thermal nociception. Reserpine administration induced significant downregulation of dopamine concentration in the central nervous system, and repeated treatment with PPX restored dopamine levels in the frontal cortex and spinal cord tissues. Moreover, PPX treatment inhibited oxidants production such as DCFH (2',7'-dichlorodihydrofluorescein) and nitrite, also decreased oxidative damage (carbonyl), and upregulated the activity of superoxide dismutase in the spinal cord. Together, our findings demonstrated the ability of dopamine D3/D2 receptor-preferring agonist in reducing pain and mood dysfunction allied to FM in mice. All experimental protocols were approved by the Universidade Federal de Santa Catarina (UFSC) Ethics Committee (approval No. 2572210218) on May 10, 2018., Competing Interests: None

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019, has caused millions of deaths worldwide. The virus is transmitted by inhalation of infectious particles suspended in the air, direct deposition on mucous membranes and indirect contact via contaminated surfaces. Disinfection methods that can halt such transmission are important in this pandemic and in future viral infections., Aim: To highlight the efficacy of several disinfection methods against SARS-CoV-2 based on up-to-date evidence found in the literature., Methods: Two databases were searched to identify studies that assessed disinfection methods used against SARS-CoV-2. In total, 1229 studies were identified and 60 of these were included in this review. Quality assessment was evaluated by the Office of Health Assessment and Translation's risk-of-bias tool., Findings: Twenty-eight studies investigated disinfection methods on environmental surfaces, 16 studies investigated disinfection methods on biological surfaces, four studies investigated disinfection methods for airborne coronavirus, and 16 studies investigated methods used to recondition personal protective equipment (PPE)., Conclusions: Several household and hospital disinfection agents and ultraviolet-C (UV-C) irradiation were effective for inactivation of SARS-CoV-2 on environmental surfaces. Formulations containing povidone-iodine can provide virucidal action on the skin and mucous membranes. In the case of hand hygiene, typical soap bars and alcohols can inactivate SARS-CoV-2. Air filtration systems incorporated with materials that possess catalytic properties, UV-C devices and heating systems can reduce airborne viral particles effectively. The decontamination of PPE can be conducted safely by heat and ozone treatment., (Copyright © 2021 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Objective: The use of resin cements in clinical practice entails photopolymerization through prosthetic devices, which precludes light penetration. The objective of this study was to modify experimental resin cements (ERCs) with diphenyliodonium hexafluorophosphate (DPI) in an attempt to improve chemical and mechanical properties of materials cured with reduced irradiance and final radiant exposure., Methods: A co-monomer base containing a 1:1 mass ratio of 2.2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (bis-GMA) and triethyleneglycol dimethacrylate (TEGDMA) was prepared, with 1mol% of camphorquinone and 2mol% of ethyl 4-(dimethylamino)benzoate as initiator system. The resin was divided into 4 fractions according to the DPI concentrations (0, 0.5, 1 and 2mol%). The challenging polymerization condition was simulated performing the light activation (12, 23 and 46s) through a ceramic block (3mm thick). The irradiance was assessed with a calibrated spectrometer (1320mW/cm 2 ), resulting in three levels of radiant exposure (0.58, 1.1 and 2.2J/cm 2 ). The polymerization kinetics was evaluated in real-time using a spectrometer (Near-IR). Water sorption and solubility was analyzed and the cohesive strength of resins obtained through the microtensile test. Polymerization stress was assessed by Bioman method., Results: Resins containing DPI had higher degree of conversion and rate of polymerization than the control (without DPI). The use of DPI reduced water sorption and solubility, and led to higher cohesive strength compared to resins without the iodonium salt. However, the stress of polymerization was higher for experimental resins with DPI., Significance: Even under remarkably reduced irradiance, cements containing a ternary initiating system with an iodonium salt can present an optimal degree of conversion and chemical/mechanical properties., (Copyright © 2021. Published by Elsevier Inc.)

In contrast to the skin and the gut, where somatic stem cells and their niche are well characterized, a definitive pancreatic multipotent cell population in the adult pancreas has yet to be revealed. Of particular interest is whether such cells may be endogenous in patients with diabetes, and if so, can they be used for therapeutic purposes? In the current study, we used two separate reporter lines to target Cre-recombinase expression to the Lgr5- or glucagon-expressing cells in the pancreas. We provide evidence for the existence of a population of cells within and in the proximity of the ducts that transiently express the stem-cell marker Lgr5 during late gestational stages. Careful timing of tamoxifen treatment in Lgr5 EGFP-IRES-CreERT2 ;R26 Tomato mice allowed us to show that these Lgr5 -expressing progenitor cells can differentiate into α-cells during pregnancy. Furthermore, we report on a spontaneous lineage conversion of α- to β-cells specifically after parturition. The contribution of Lgr5 progeny to the β-cell compartment through an α-cell intermediate phase early after pregnancy appears to be part of a novel mechanism that would counterbalance against excessive β-cell mass reduction during β-cell involution., (© 2021 by the American Diabetes Association.)

In Type 1 Diabetes (T1D), CD4 + T cells initiate autoimmune attack of pancreatic islet β cells. Importantly, bioenergetic programs dictate T cell function, with specific pathways required for progression through the T cell lifecycle. During activation, CD4 + T cells undergo metabolic reprogramming to the less efficient aerobic glycolysis, similarly to highly proliferative cancer cells. In an effort to limit tumor growth in cancer, use of glycolytic inhibitors have been successfully employed in preclinical and clinical studies. This strategy has also been utilized to suppress T cell responses in autoimmune diseases like Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), and Rheumatoid Arthritis (RA). However, modulating T cell metabolism in the context of T1D has remained an understudied therapeutic opportunity. In this study, we utilized the small molecule PFK15, a competitive inhibitor of the rate limiting glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3 (PFKFB3). Our results confirmed PFK15 inhibited glycolysis utilization by diabetogenic CD4 + T cells and reduced T cell responses to β cell antigen in vitro . In an adoptive transfer model of T1D, PFK15 treatment delayed diabetes onset, with 57% of animals remaining euglycemic at the end of the study period. Protection was due to induction of a hyporesponsive T cell phenotype, characterized by increased and sustained expression of the checkpoint molecules PD-1 and LAG-3 and downstream functional and metabolic exhaustion. Glycolysis inhibition terminally exhausted diabetogenic CD4 + T cells, which was irreversible through restimulation or checkpoint blockade in vitro and in vivo . In sum, our results demonstrate a novel therapeutic strategy to control aberrant T cell responses by exploiting the metabolic reprogramming of these cells during T1D. Moreover, the data presented here highlight a key role for nutrient availability in fueling T cell function and has implications in our understanding of T cell biology in chronic infection, cancer, and autoimmunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martins, New, O’Connor, Previte, Cargill, Tse, Sims- Lucas and Piganelli.)

Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid β, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and β-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid β as the protagonist and potential therapeutic target.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

Mollusk shells can provide important information regarding environmental parameters. It is known that shell morphology is affected by both natural and anthropogenic factors. However, few studies have investigated alterations in shell morphology over a historical perspective and considering chemical contamination and climate changes. The present study assessed shell form (shape and size) variations of limpet (Lottia subrugosa) shells sampled from 1950 to 1981 (past) in comparison with organisms obtained in 2018 (present). Differences between shells from the past and present (2018) were detected, being shell weight and height the two most important affected parameters. The differences observed were attributed to the possible increase in contamination over the years due to human population growth and to climate change. Additionally, when shells from the past were evaluated according to the decade they were sampled, results indicate that it was necessary an interval of 40 years to shell form be altered within populations., Competing Interests: Declaration of competing interest The authors 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 © 2020 Elsevier Ltd. All rights reserved.)

Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence-associated lysosomal β-galactosidase (SA-β-gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose-encapsulated nanoparticles within these cells. Here, we show that galacto-conjugation of the BCL-2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav-Gal), that can be preferentially activated by SA-β-gal activity in a wide range of cell types. Nav-Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav-Gal enhances the cytotoxicity of standard senescence-inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav-Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto-conjugation reduces Navitoclax-induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

The present study evaluated the association among traditional biochemical biomarkers with biometric, morphometric, and elemental composition of Lottia subrugosa (patelliform gastropod) shells from three multi-impacted coastal areas in Brazil. The study was carried out in Todos os Santos Bay (TSB), Santos/São Vicente Estuarine System (SESS) and Paranaguá Estuarine Complex (CEP), using three sampling sites to seek contamination gradients in each area. Results showed that all biomarkers evaluated responded to environmental contamination, regardless the presence (SESS and CEP) or absence (TSB) of a gradient of contamination. The responses found using biometric and morphometric parameters were consistent with the traditional biomarkers of exposure and effects (lipid peroxidation and DNA damage). Indeed, changes in elemental composition of L. subrugosa shells suggest that exposure to contaminated environments is probably responsible for the alterations detected. Despite the simplicity and lower cost of biometric and morphometric analyzes, these parameters are influenced by natural environmental conditions from which biases may arise. Therefore, these tools should be evaluated through experimental studies before it can be used in future assessments. However, the findings from the present study were observed in three aquatic systems distributed over a wide range of latitudes, which indicates that gastropod shells reflect effects resulting from environmental contamination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Cardiac implantable electronic device infections are associated with substantial morbidity and mortality. There are varied recommendations in the literature about treatment of the wound after extraction of all hardware, but only conservative, time-consuming approaches such as open packing and negative-pressure therapy along with a long interval before reimplanting any hardware have generally been recommended for the treatment. 1-4 ., Methods: A retrospective review was performed of 42 patients treated at Jersey Shore University Medical Center for implantable cardioverter defibrillator and permanent pacemaker infections between July 2010 and April 2018 with an aggressive, multidisciplinary approach utilizing an invasive cardiologist and a plastic surgeon. Clinical and demographic data were collected, and a descriptive analysis was conducted., Results: A total of 42 patients, with a median age of 76 years, were selected for our treatment of pacemaker pocket infection. Patients underwent removal of all hardware followed by debridement and flap closure of the wound soon after extraction. Reimplantation was performed when indicated typically within a week after initial extraction and typically on the contralateral side. There were no reports of reinfection and no mortality in all 42 patients treated., Conclusion: We found that the aggressive removal of all hardware and excisional debridement of the entire capsule followed by flap coverage and closure of the wound allowed for a shortened interval to reimplantation with no ipsilateral or contralateral infections during the follow-up period.

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4 + T cells and tempers their homeostatic expansion. Because CD4 + T cell proliferation is tightly coupled to bioenergetics, we investigate the role of LAG-3 in modulating naive CD4 + T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4 + T cells by elevating levels of mitochondrial biogenesis. In vivo, LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4 + T cells to levels of Lag3 -/- CD4 + T cells, solidifying that LAG-3 controls these processes. Lag3 -/- CD4 + T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation, enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally, enhancement of STAT5 activation, as a result of LAG-3 deficiency, contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4 + T cell responses., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Osmotic regulation is a vital homoeostatic process in all cells and tissues. Cells initially respond to osmotic stresses by activating transmembrane transport proteins to move osmotically active ions. Disruption of ion and water transport is frequently observed in cellular transformations such as cancer. We report that genes involved in membrane transport are significantly deregulated in many cancers, and that their expression can distinguish cancer cells from normal cells with a high degree of accuracy. We present an executable model of osmotic regulation and membrane transport in mammalian cells, providing a mechanistic explanation for phenotype change in varied disease states, and accurately predicting behaviour from single cell expression data. We also predict key proteins involved in cellular transformation, SLC4A3 (AE3), and SLC9A1 (NHE1). Furthermore, we predict and verify a synergistic drug combination in vitro, of sodium and chloride channel inhibitors, which target the osmoregulatory network to reduce cancer-associated phenotypes in fibroblasts.

KRAS is the most frequently mutated driver oncogene in human adenocarcinoma of the lung. There are presently no clinically proven strategies for treatment of KRAS-driven lung cancer. Activating mutations in KRAS are thought to confer independence from upstream signaling; however, recent data suggest that this independence may not be absolute. We show that initiation and progression of KRAS-driven lung tumors require input from ERBB family receptor tyrosine kinases (RTKs): Multiple ERBB RTKs are expressed and active from the earliest stages of KRAS-driven lung tumor development, and treatment with a multi-ERBB inhibitor suppresses formation of KRAS G12D -driven lung tumors. We present evidence that ERBB activity amplifies signaling through the core RAS pathway, supporting proliferation of KRAS-mutant tumor cells in culture and progression to invasive disease in vivo. Brief pharmacological inhibition of the ERBB network enhances the therapeutic benefit of MEK (mitogen-activated protein kinase kinase) inhibition in an autochthonous tumor setting. Our data suggest that lung cancer patients with KRAS-driven disease may benefit from inclusion of multi-ERBB inhibitors in rationally designed treatment strategies., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.

Tumours have developed strategies to interfere with most steps required for anti-tumour immune responses. Although many populations contribute to anti-tumour responses, tumour-infiltrating cytotoxic T cells dominate, hence, many suppressive strategies act to inhibit these. Tumour-associated T cells are frequently restricted to stromal zones rather than tumour islands, raising the possibility that the tumour microenvironment, where crosstalk between malignant and "normal" stromal cells exists, may be critical for T cell suppression. We provide evidence of direct interactions between stroma and T cells driving suppression, showing that cancer-associated fibroblasts (CAFs) sample, process and cross-present antigen, killing CD8 + T cells in an antigen-specific, antigen-dependent manner via PD-L2 and FASL. Inhibitory ligand expression is observed in CAFs from human tumours, and neutralisation of PD-L2 or FASL reactivates T cell cytotoxic capacity in vitro and in vivo. Thus, CAFs support T cell suppression within the tumour microenvironment by a mechanism dependent on immune checkpoint activation.

Background and Purpose: The motor impairments related to gait and balance have a huge impact on the life of individuals with spinocerebellar ataxia (SCA). Here, the aim was to assess the possibility of retraining gait, improving cardiopulmonary capacity, and challenging balance during gait in SCA using a partial body weight support (BWS) and a treadmill. Also, the effects of this training over functionality and quality of life were investigated., Methods: Eight SCA patients were engaged in the first stage of the study that focused on gait training and cardiovascular conditioning. From those, five took part in a second stage of the study centered on dynamic balance training during gait. The first and second stages lasted 8 and 10 weeks, respectively, both comprising sessions of 50 min (2 times per week)., Results: The results showed that gait training using partial BWS significantly increased gait performance, treadmill inclination, duration of exercise, and cardiopulmonary capacity in individuals with SCA. After the second stage, balance improvements were also found., Conclusion: Combining gait training and challenging tasks to the postural control system in SCA individuals is viable, well tolerated by patients with SCA, and resulted in changes in capacity for walking and balance.

Lung cancer is the leading cause of cancer-related death worldwide, reflecting an unfortunate combination of very high prevalence and low survival rates, as most cases are diagnosed at advanced stages when treatment efficacy is limited. Lung cancer comprises several disease groups with non small cell lung cancer (NSCLC) accounting for ~ 85% of cases and lung adenocarcinoma being its most frequent histological subtype. Mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) affect ~ 30% of lung adenocarcinomas but unlike other commonly altered proteins (EGFR and ALK, affected in ~ 14% and 7% of cases respectively), mutant KRAS remains untargetable. Therapeutic strategies that rely instead on the inhibition of mutant KRAS functional output or the targeting of mutant KRAS cellular dependencies (i.e. synthetic lethality) are an appealing alternative approach. Recent studies focused on the metabolic properties of mutant KRAS lung tumours have uncovered unique metabolic features that can potentially be exploited therapeutically. We review these findings here with a particular focus on in vivo, physiologic, mutant KRAS activity., (© 2017 Federation of European Biochemical Societies.)

Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine Kras G12D - p53 null , - p53 R172H (conformational), and - p53 R270H (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53 R270H -specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant., (© 2017 Turrell et al.; Published by Cold Spring Harbor Laboratory Press.)

Main Conclusion: Overexpression of the citrus CsTIP2;1 improves plant growth and tolerance to salt and drought stresses by enhancing cell expansion, H 2 O 2 detoxification and stomatal conductance. Tonoplast intrinsic proteins (TIPs) are a subfamily of aquaporins, belonging to the major intrinsic protein family. In a previous study, we have shown that a citrus TIP isoform, CsTIP2;1, is highly expressed in leaves and also transcriptionally regulated in leaves and roots by salt and drought stresses and infection by 'Candidatus Liberibacter asiaticus', the causal agent of the Huanglongbing disease, suggesting its involvement in the regulation of the flow of water and nutrients required during both normal growth and stress conditions. Here, we show that the overexpression of CsTIP2;1 in transgenic tobacco increases plant growth under optimal and water- and salt-stress conditions and also significantly improves the leaf water and oxidative status, photosynthetic capacity, transpiration rate and water use efficiency of plants subjected to a progressive soil drying. These results correlated with the enhanced mesophyll cell expansion, midrib aquiferous parenchyma abundance, H 2 O 2 detoxification and stomatal conductance observed in the transgenic plants. Taken together, our results indicate that CsTIP2;1 plays an active role in regulating the water and oxidative status required for plant growth and adaptation to stressful environmental conditions and may be potentially useful for engineering stress tolerance in citrus and other crop plants.

The identification of therapeutic vulnerabilities in mutant KRAS tumors has proven difficult to achieve. Burgess and colleagues recently reported in Cell that mutant/wild-type Kras allelic dosage determines clonal fitness and MEK inhibitor sensitivity in a leukemia model, demonstrating that KRAS allelic imbalance is likely an important and overlooked variable., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The immune system is necessary for protecting against various pathogens. However, under certain circumstances, self-reactive immune cells can drive autoimmunity, like that exhibited in type 1 diabetes (T1D). CD4+ T cells are major contributors to the immunopathology in T1D, and in order to drive optimal T cell activation, third signal reactive oxygen species (ROS) must be present. However, the role ROS play in mediating this process remains to be further understood. Recently, cellular metabolic programs have been shown to dictate the function and fate of immune cells, including CD4+ T cells. During activation, CD4+ T cells must transition metabolically from oxidative phosphorylation to aerobic glycolysis to support proliferation and effector function. As ROS are capable of modulating cellular metabolism in other models, we sought to understand if blocking ROS also regulates CD4+ T cell activation and effector function by modulating T cell metabolism. To do so, we utilized an ROS scavenging and potent antioxidant manganese metalloporphyrin (MnP). Our results demonstrate that redox modulation during activation regulates the mTOR/AMPK axis by maintaining AMPK activation, resulting in diminished mTOR activation and reduced transition to aerobic glycolysis in diabetogenic splenocytes. These results correlated with decreased Myc and Glut1 upregulation, reduced glucose uptake, and diminished lactate production. In an adoptive transfer model of T1D, animals treated with MnP demonstrated delayed diabetes progression, concurrent with reduced CD4+ T cell activation. Our results demonstrate that ROS are required for driving and sustaining T cell activation-induced metabolic reprogramming, and further support ROS as a target to minimize aberrant immune responses in autoimmunity.

Background: Small intestine bacterial overgrowth is a heterogeneous syndrome characterized by an increase in the number and/or the presence of atypical microbiota in the small intestine. The symptoms of small intestine bacterial overgrowth are unspecific, encompassing abdominal pain/distension, diarrhea and flatulence. Due to the increased cost and complexity for carrying out the jejunal aspirate, the gold standard for diagnosis of the syndrome, routinely the hydrogen (H 2 ) breath test has been used, utilizing glucose or lactulose as substrate, which is able to determine, in the exhaled air, the H 2 concentration produced from the intestinal bacterial metabolism. However, due to a number of individuals presenting a methanogenic microbiota, which does not produce H 2 , the testing on devices capable of detecting, concurrently, the concentration of exhaled H 2 and methane (CH 4 ) is justified., Objective: This study aimed to determine the prevalence of small intestine bacterial overgrowth in patients with digestive symptoms, through a comparative analysis of breath tests of H 2 or H 2 and CH 4 associated, using glucose as substrate ., Methods: A total of 200 patients of both sexes without age limitation were evaluated, being directed to a Breath Test Laboratory for performing the H 2 test (100 patients) and of exhaled H 2 and CH 4 (100 patients) due to gastrointestinal complaints, most of them patients with gastrointestinal functional disorders., Results: The results indicated a significant prevalence of small intestine bacterial overgrowth in the H 2 test and in the test of exhaled H 2 and CH 4 (56% and 64% respectively) in patients with gastrointestinal symptoms, and higher prevalence in females. It found further that methane gas was alone responsible for positivity in 18% of patients., Conclusion: The data found in this study is consistent with the findings of the current literature and underscores the need for using devices capable of capturing the two gases (exhaled H 2 and CH 4 ) to improve the sensitivity and hence the accuracy of small intestine bacterial overgrowth diagnosis in daily medical practice.

9-cis-epoxycarotenoid dioxygenase (NCED) encodes a key enzyme in abscisic acid (ABA) biosynthesis. Little is known regarding the regulation of stress response by NCEDs at physiological levels. In the present study, we generated transgenic tobacco overexpressing an NCED3 ortholog from citrus (CsNCED3) and investigated its relevance in the regulation of drought stress tolerance. Wild-type (WT) and transgenic plants were grown under greenhouse conditions and subjected to drought stress for 10 days. Leaf predawn water potential (Ψw leaf ), stomatal conductance (gs), net photosynthetic rate (A), transpiration rate (E), instantaneous (A/E) and intrinsic (A/gs) water use efficiency (WUE), and in situ hydrogen peroxide (H 2 O 2 ) and abscisic acid (ABA) production were determined in leaves of irrigated and drought-stressed plants. The Ψw leaf decreased throughout the drought stress period in both WT and transgenic plants, but was restored after re-watering. No significant differences were observed in gs between WT and transgenic plants under normal conditions. However, the transgenic plants showed a decreased (P ≤ 0.01) gs on the 4th day of drought stress, which remained lower (P ≤ 0.001) than the WT until the end of the drought stress. The A and E levels in the transgenic plants were similar to those in WT; therefore, they exhibited increased A/gs under drought conditions. No significant differences in A, E, and gs values were observed between the WT and transgenic plants after re-watering. The transgenic plants had lower H 2 O 2 and higher ABA than the WT under drought conditions. Our results support the involvement of CsNCED3 in drought avoidance.

Background: Acne in adult women is a frequent hard-to-manage disease with many relapse cases. It mostly interferes with the quality of life of patients, bringing them major metabolic and social losses. As androgenic hormones play a very important role in the acne pathogenesis, the early diagnosis of hyperandrogenic states is very useful for the proper evaluation of each patient and for a better choice of therapeutic management. Defining a pattern for laboratory profile analysis is important for the control of relapses of acne breakouts in adult women, which lately has been the aim of many published studies., Aim: To establish the relation between 3 alpha-diol G levels and acne in female patients with normal androgenic status without menstrual dysfunctions., Patients/methods: The evaluation of serum 3 alpha-androstanediol glucuronide levels through an enzymatic immunoassay method (Androstanediol Glucuronide ELISA Kit) for a direct quantitative measurement in 26 patients with grade II and III acne, ages ranging from 13 to 50., Results: Among the analyzed patients, 83% had grade II acne, and among this total, 60% were aged 14 or over. According to age, 12 studied patients showed serum 3 alpha-diol G levels within normal range and 11 patients had increased levels., Conclusions: A total of 60% of adult women with acne present increased levels of androgens and among those with normal levels and without menstrual dysfunctions, 50% show an increase in 3 alpha-diol G. Therefore, a pharmacological approach with anti-androgenic drugs for acne therapy in most of these patients is advisable., (© 2016 Wiley Periodicals, Inc.)

Oral manifestations of common variable immunodeficiency (CVID) are rare, have rarely been studied and have given controversial results. There are few data about IgA, IgG, and IgM antibody salivary levels in the literature, and there are few papers about the clinical impact of antibody deficiencies and CVID on the oral health of such patients. The aim of this study was to measure serum and salivary IgA, IgG, and IgM levels in CVID participants and controls, and to associate immunoglobulin levels with caries and periodontal disease. This was a case-control study involving 51 CVID individuals and 50 healthy controls. All participants underwent examination for dental caries and periodontal disease. Blood and whole saliva samples were collected on the same day of the oral examination. Serum IgA, IgM, and IgG levels were measured by turbidimetry and salivary IgA, IgM, and IgG titers were assessed by enzyme-linked immunosorbent assay. Incidences of caries and gingivitis were significantly higher in the CVID group than in the control group (p<0.05). Salivary and blood IgA and IgM titers were significantly reduced in the CVID group, but there was no association of salivary immunoglobulin levels with periodontal disease or with caries incidence (p>0.05 for both). Although CVID was associated with increased susceptibility to caries and gingivitis, it was not associated with low salivary levels of IgA and IgM.

Objective: The aim of this study was to investigate the clinical and laboratorial factors associated with serum sodium variation during continuous renal replacement therapy and to assess whether the perfect admixture formula could predict 24-hour sodium variation., Methods: Thirty-six continuous renal replacement therapy sessions of 33 patients, in which the affluent prescription was unchanged during the first 24 hours, were retrieved from a prospective collected database and then analyzed. A mixed linear model was performed to investigate the factors associated with large serum sodium variations (≥ 8mEq/L), and a Bland-Altman plot was generated to assess the agreement between the predicted and observed variations., Results: In continuous renal replacement therapy 24-hour sessions, SAPS 3 (p = 0.022) and baseline hypernatremia (p = 0.023) were statistically significant predictors of serum sodium variations ≥ 8mEq/L in univariate analysis, but only hypernatremia demonstrated an independent association (β = 0.429, p < 0.001). The perfect admixture formula for sodium prediction at 24 hours demonstrated poor agreement with the observed values., Conclusions: Hypernatremia at the time of continuous renal replacement therapy initiation is an important factor associated with clinically significant serum sodium variation. The use of 4% citrate or acid citrate dextrose - formula A 2.2% as anticoagulants was not associated with higher serum sodium variations. A mathematical prediction for the serum sodium concentration after 24 hours was not feasible.

Analysis of drug residues in urban wastewater could complement epidemiological studies in detecting the use of new psychoactive substances (NPS), a continuously changing group of drugs hard to monitor by classical methods. We initially selected 52 NPS potentially used in Italy based on seizure data and consumption alerts provided by the Antidrug Police Department and the National Early Warning System. Using a linear ion trap-Orbitrap high resolution mass spectrometer, we designed a suspect screening and a target method approach and compared them for the analysis of 24 h wastewater samples collected at the treatment plant influents of four Italian cities. This highlighted the main limitations of these two approaches, so we could propose requirements for future research. A library of MS/MS spectra of 16 synthetic cathinones and 19 synthetic cannabinoids, for which analytical standards were acquired, was built at different collision energies and is available on request. The stability of synthetic cannabinoids was studied in analytical standards and wastewater, identifying the best analytical conditions for future studies. To the best of our knowledge, these are the first stability data on NPS. Few suspects were identified in Italian wastewater samples, in accordance with recent epidemiological data reporting a very low prevalence of use of NPS in Italy. This study outlines an analytical approach for NPS identification and measurement in urban wastewater and for estimating their use in the population.

The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.

This work focuses on the influence of the selected LC-HRMS platform on the final annotated compounds in non-targeted metabolomics. Two platforms that differed in columns, mobile phases, gradients, chromatographs, mass spectrometers (Orbitrap [Platform#1] and Q-TOF [Platform#2]), data processing and marker selection protocols were compared. A total of 42 wines samples from three different protected denomination of origin (PDO) were analyzed. At the feature level, good (O)PLS-DA models were obtained for both platforms (Q(2)[Platform#1]=0.89, 0.83 and 0.72; Q(2)[Platform#2]=0.86, 0.86 and 0.77 for Penedes, Ribera del Duero and Rioja wines respectively) with 100% correctly classified samples in all cases. At the annotated metabolite level, platforms proposed 9 and 8 annotated metabolites respectively which were identified by matching standards or the MS/MS spectra of the compounds. At this stage, there was no coincidence among platforms regarding the suggested metabolites. When screened on the raw data, 6 and 5 of these compounds were detected on the other platform with a similar trend. Some of the detected metabolites showed complimentary information when integrated on biological pathways. Through the use of some examples at the annotated metabolite level, possible explanations of this initial divergence on the results are presented. This work shows the complications that may arise on the comparison of non-targeted metabolomics platforms even when metabolite focused approaches are used in the identification., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: The human T-lymphotropic virus-1 (HTLV-1) is associated with chronic inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic inflammatory disease. Disturbances in lipid metabolism are involved in inflammatory and demyelinating diseases., Methods: Plasma levels of triglycerides, total cholesterol, and fractions of HTLV-1-infected individuals of both sexes with different clinical progressions were determined., Results: Elevated levels of triglyceride and very low-density lipoproteins (VLDL) were exclusively detected in HTLV-1-infected women from asymptomatic and HAM/TSP groups compared with uninfected individuals (p = 0.02)., Conclusions: Elevated triglyceride and VLDL levels in HTLV-1-infected women may be related to the predominance of HAM/TSP in women.

Most human tissues used in research are of post mortem origin. This is the case for all brain samples, and due to the difficulty in obtaining a good number of samples, especially in the case of neurodegenerative diseases, male and female samples are often included in the same experimental group. However, the effects of post mortem interval (PMI) and gender differences in the endpoints being analyzed are not always fully understood, as is the case for DNA repair activities. To investigate these effects, in a controlled genetic background, base excision repair (BER) activities were measured in protein extracts obtained from Wistar rat brains from different genders and defined PMI up to 24 hours, using a novel fluorescent-based in vitro incision assay. Uracil and AP-site incision activity in nuclear and mitochondrial extracts were similar in all groups included in this study. Our results show that gender and PMI up to 24 hours have no influence in the activities of the BER proteins UDG and APE1 in rat brains. These findings demonstrate that these variables do not interfere on the BER activities included in these study, and provide a security window to work with UDG and APE1 proteins in samples of post mortem origin., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The modelling and prediction of reversed-phase chromatographic retention time (tR) under gradient elution conditions for 166 pharmaceuticals in wastewater extracts is presented using artificial neural networks for the first time. Radial basis function, multilayer perceptron and generalised regression neural networks were investigated and a comparison of their predictive ability for model solutions discussed. For real world application, the effect of matrix complexity on tR measurements is presented. Measured tR for some compounds in influent wastewater varied by >1min in comparison to tR in model solutions. Similarly, matrix impact on artificial neural network predictive ability was addressed towards developing a more robust approach for routine screening applications. Overall, the best neural network had a predictive accuracy of <1.3min at the 75th percentile of all measured tR data in wastewater samples (<10% of the total runtime). Coefficients of determination for 30 blind test compounds in wastewater matrices lay at or above R(2)=0.92. Finally, the model was evaluated for application to the semi-targeted identification of pharmaceutical residues during a weeklong wastewater sampling campaign. The model successfully identified native compounds at a rate of 83±4% and 73±5% in influent and effluent extracts, respectively. The use of an HRMS database and the optimised ANN model was also applied to shortlisting of 37 additional compounds in wastewater. Ultimately, this research will potentially enable faster identification of emerging contaminants in the environment through more efficient post-acquisition data mining., (Copyright © 2015 Elsevier B.V. All rights reserved.)