Multi-approach methods to predict cryptic carbapenem resistance mechanisms in Klebsiella pneumoniae detected in Central Italy.

The rapid emergence of carbapenem-resistant Klebsiella pneumoniae (Kp) strains in diverse environmental niches, even outside of the clinical setting, poses a challenge for the detection and the real-time monitoring of novel antimicrobial resistance trends using molecular and whole genome sequencing-based methods. The aim of our study was to understand cryptic resistance determinants responsible for the phenotypic carbapenem resistance observed in strains circulating in Italy by using a combined approach involving whole genome sequencing (WGS) and genome-wide association study (GWAS). In this study, we collected 303 Kp strains from inside and outside clinical settings between 2018-2022 in the Abruzzo region of Italy. The antimicrobial resistance profile of all isolates was assessed using both phenotypic and bioinformatic methods. We identified 11 strains resistant to carbapenems, which did not carry any known genetic determinants explaining their phenotype. The GWAS results showed that incongruent carbapenem-resistant phenotype was associated specifically with strains with two capsular types, KL13 and KL116 including genes involved in the capsule synthesis, encoding proteins involved in the assembly of the capsule biosynthesis apparatus, capsule-specific sugar synthesis, processing and export, polysaccharide pyruvyl transferase, and lipopolysaccharide biosynthesis protein. These preliminary results confirmed the potential of GWAS in identifying genetic variants present in KL13 and KL116 that could be associated with carbapenem resistance traits in Kp. The implementation of advanced methods, such as GWAS with increased antimicrobial resistance surveillance will potentially improve Kp infection treatment and patient outcomes.

3D CT-Inclusive Deep-Learning Model to Predict Mortality, ICU Admittance, and Intubation in COVID-19 Patients.

Chest CT is a useful initial exam in patients with coronavirus disease 2019 (COVID-19) for assessing lung damage. AI-powered predictive models could be useful to better allocate resources in the midst of the pandemic. Our aim was to build a deep-learning (DL) model for COVID-19 outcome prediction inclusive of 3D chest CT images acquired at hospital admission. This retrospective multicentric study included 1051 patients (mean age 69, SD = 15) who presented to the emergency department of three different institutions between 20th March 2020 and 20th January 2021 with COVID-19 confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Chest CT at hospital admission were evaluated by a 3D residual neural network algorithm. Training, internal validation, and external validation groups included 608, 153, and 290 patients, respectively. Images, clinical, and laboratory data were fed into different customizations of a dense neural network to choose the best performing architecture for the prediction of mortality, intubation, and intensive care unit (ICU) admission. The AI model tested on CT and clinical features displayed accuracy, sensitivity, specificity, and ROC-AUC, respectively, of 91.7%, 90.5%, 92.4%, and 95% for the prediction of patient's mortality; 91.3%, 91.5%, 89.8%, and 95% for intubation; and 89.6%, 90.2%, 86.5%, and 94% for ICU admission (internal validation) in the testing cohort. The performance was lower in the validation cohort for mortality (71.7%, 55.6%, 74.8%, 72%), intubation (72.6%, 74.7%, 45.7%, 64%), and ICU admission (74.7%, 77%, 46%, 70%) prediction. The addition of the available laboratory data led to an increase in sensitivity for patient's mortality (66%) and specificity for intubation and ICU admission (50%, 52%, respectively), while the other metrics maintained similar performance results. We present a deep-learning model to predict mortality, ICU admittance, and intubation in COVID-19 patients. KEY POINTS: • 3D CT-based deep learning model predicted the internal validation set with high accuracy, sensibility and specificity (> 90%) mortality, ICU admittance, and intubation in COVID-19 patients. • The model slightly increased prediction results when laboratory data were added to the analysis, despite data imbalance. However, the model accuracy dropped when CT images were not considered in the analysis, implying an important role of CT in predicting outcomes.

Sex differences in cardiovascular mortality in diabetics and nondiabetic subjects: a population-based study (Italy).

The objective of this study is to assess the impact of diabetes on cardiovascular mortality, focusing on sex differences. The inhabitants of Reggio Emilia province on December 31, 2009, aged 20-84 were followed up for three years for mortality. The exposure was determined using Reggio Emilia diabetes register. The age-adjusted death rates were estimated as well as the incidence rate ratios using Poisson regression model. Interaction terms for diabetes and sex were tested by the Wald test. People with diabetes had an excess of mortality, compared with nondiabetic subjects (all cause: IRR = 1.68; 95%CI 1.60-1.78; CVD: IRR = 1.61; 95%CI 1.47-1.76; AMI: IRR = 1.59; 95%CI 1.27-1.99; renal causes: IRR = 1.71; 95%CI 1.22-2.38). The impact of diabetes is greater in females than males for all causes (P = 0.0321) and for CVD, IMA, and renal causes. Further studies are needed to investigate whether the difference in cardiovascular risk profile or in the quality of care delivered justifies the higher excess of mortality in females with diabetes compared to males.

p66(ShcA): linking mammalian longevity with obesity-induced insulin resistance.

Traditionally linked to an accumulation of cell and tissue oxidative damage, the aging process is currently viewed as the result of a chronic metabolic imbalance and deranged body response to nutrients. This is in keeping with the pivotal role of the insulin/IGF pathway in longevity determination from model organisms to primates, with the association between obesity and age-related disorders, and with the dramatic acceleration of senescence by diabetes. Importantly, metabolic and oxidative damages concomitantly occur in aging as consequences of tissue hyperstimulation by insulin. With its double identity of generator of mitochondrial oxidant species and of signaling adaptor in the insulin receptor cascade, the 66Kd Shc protein (p66Shc) has drawn major attention as a negative determinant of life span and healthy longevity in mammals. We have demonstrated that these effects are related, at least in part, to p66Shc effect on the mTOR/S6K cascade that promotes obesity and insulin resistance and shortens life span.

Monitoring nutrient signaling through the longevity protein p66(SHC¹).

Nutrient availability and nutrient-dependent biochemical signals represent major determinants of cellular senescence and organismal aging. The present chapter describes simple procedures to reliably evaluate the response of cultured cell to nutrients through the longevity protein p66(SHC1) and the mTOR/S6K cascade, which might be used to study cellular senescence and its chemical modulation by pharmaceutical agents in vitro.

A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction.

Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD(+)-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-γ coactivator-1α and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes.

Nutrient withdrawal rescues growth factor-deprived cells from mTOR-dependent damage.

Deregulated nutrient signaling plays pivotal roles in body ageing and in diabetic complications; biochemical cascades linking energy dysmetabolism to cell damage and loss are still incompletely clarified, and novel molecular paradigms and pharmacological targets critically needed. We provide evidence that in the retrovirus-packaging cell line HEK293-T Phoenix, massive cell death in serum-free medium is remarkably prevented or attenuated by either glucose or aminoacid withdrawal, and by the glycolysis inhibitor 2-deoxy-glucose. A similar protection was also elicited by interference with mitochondrial function, clearly suggesting involvement of energy metabolism in increased cell survival. Oxidative stress did not account for nutrient toxicity on serum-starved cells. Instead, nutrient restriction was associated with reduced activity of the mTOR/S6 Kinase cascade. Moreover, pharmacological and genetic manipulation of the mTOR pathway modulated in an opposite fashion signaling to S6K/S6 and cell viability in nutrient-repleted medium. Additionally, stimulation of the AMP-activated Protein Kinase concomitantly inhibited mTOR signaling and cell death, while neither event was affected by overexpression of the NAD+ dependent deacetylase Sirt-1, another cellular sensor of nutrient scarcity. Finally, blockade of the mTOR cascade reduced hyperglycemic damage also in a more pathophysiologically relevant model, i.e. in human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia. Taken together these findings point to a key role of the mTOR/S6K cascade in cell damage by excess nutrients and scarcity of growth-factors, a condition shared by diabetes and other ageing-related pathologies.

Mammalian life-span determinant p66shcA mediates obesity-induced insulin resistance.

Obesity and metabolic syndrome result from excess calorie intake and genetic predisposition and are mechanistically linked to type II diabetes and accelerated body aging; abnormal nutrient and insulin signaling participate in this pathologic process, yet the underlying molecular mechanisms are incompletely understood. Mice lacking the p66 kDa isoform of the Shc adaptor molecule live longer and are leaner than wild-type animals, suggesting that this molecule may have a role in metabolic derangement and premature senescence by overnutrition. We found that p66 deficiency exerts a modest but significant protective effect on fat accumulation and premature death in lepOb/Ob mice, an established genetic model of obesity and insulin resistance; strikingly, however, p66 inactivation improved glucose tolerance in these animals, without affecting (hyper)insulinaemia and independent of body weight. Protection from insulin resistance was cell autonomous, because isolated p66KO preadipocytes were relatively resistant to insulin desensitization by free fatty acids in vitro. Biochemical studies revealed that p66shc promotes the signal-inhibitory phosphorylation of the major insulin transducer IRS-1, by bridging IRS-1 and the mTOR effector p70S6 kinase, a molecule previously linked to obesity-induced insulin resistance. Importantly, IRS-1 was strongly up-regulated in the adipose tissue of p66KO lepOb/Ob mice, confirming that effects of p66 on tissue responsiveness to insulin are largely mediated by this molecule. Taken together, these findings identify p66shc as a major mediator of insulin resistance by excess nutrients, and by extension, as a potential molecular target against the spreading epidemic of obesity and type II diabetes.

Role of the life span determinant P66(shcA) in ethanol-induced liver damage.

Mice lacking the 66 kDa isoform of the adapter molecule shcA (p66(shcA)) display increased resistance to oxidative stress and delayed aging. In cultured cell lines, p66 promotes formation of Reactive Oxygen Species (ROS) in mitochondria, and apoptotic cell death in response to a variety of pro-oxidant noxious stimuli. As mitochondrial ROS and oxidative cell damage are clearly involved in alcohol-induced pathology, we hypothesized that p66 may also have a role in ethanol. In vivo, changes observed in p66+/+ mice after 6-week exposure to ethanol in the drinking water, including elevated serum alanine aminotransferase (ALT), liver swelling and evident liver steatosis, were significantly attenuated in p66-/- mutant mice. Biochemical analysis of liver tissues revealed induction of the p66 protein by ethanol, whereas p66-deficient livers responded to alcohol with a significant upregulation of the mitochondrial antioxidant enzyme MnSOD, nearly absent in control mice. Evidence of an inverse correlation between expression level of p66 and protection from alcohol-induced oxidative stress was also confirmed in vitro in primary hepatocytes and in HepG2-E47 cells, an ethanol-responsive hepatoma cell line. In fact, MnSOD upregulation by exposure to ethanol in vitro was much more pronounced in p66KO versus wild-type isolated liver cells, and blunted in HepG2 cells overexpressing p66shc. p66 overexpression also prevented the activation of a luciferase reporter gene controlled by the SOD2 promoter, indicating that p66 repression of MnSOD operates at a transcriptional level. Finally, p66 generated ROS in HepG2 cells and potentiated oxidative stress and mitochondrial depolarization by ethanol. Taken together, the above observations clearly indicate a role for p66 in alcohol-induced cell damage, likely via a cell-autonomous mechanism involving reduced expression of antioxidant defenses and mitochondrial dysfunction.

Bilirubin as an endogenous modulator of neurotrophin redox signaling.

Bilirubin is neurotoxic upon excess accumulation in the brain, but it also plays important physiological roles related to its antioxidant properties. Here we report that exposure of PC12 and primary rat cerebellar granule neurons to bilirubin (0.5-10 microM) drastically decreases nerve growth factor (NGF)/brain-derived neurotrophic factor signaling to Akt and extracellular signal-regulated kinases (ERKs), indicating a direct interference of the molecule with crucial prosurvival signaling pathways. This effect likely involves the scavenging capacity of bilirubin, the latter being able to inhibit, in PC12 cells, accumulation of intracellular reactive oxygen species and phosphorylation of Akt and ERKs in response to extracellular hydrogen peroxide. Interestingly, in the absence of exogenous growth factor, bilirubin elicited the phosphorylation of ERKs and of the cAMP responsive element binding (CREB) transcription factor, a signature of NGF-dependent survival signaling. These growth factor-like signaling effects were paralleled by the induction of the neuronal nitric oxide synthase (nNOS) and generation of nitric oxide (NO). Pharmacological dissection of the signaling cascade triggered by bilirubin revealed that phosphorylation of ERKs requires NO signaling through soluble guanylyl cyclase, and, further upstream, influx of extracellular calcium is necessary for nNOS induction and NO release, likely through calcium-dependent phosphorylation of CREB. Importantly, the cascade elicited by bilirubin through NO and ERK is cytoprotective, as revealed by exacerbated bilirubin toxicity in cultures treated by either NOS or MEK inhibitors. Taken together, these observations indicate an important action of bilirubin on redox signaling by neurotrophins, with either inhibitory or agonistic effects based on growth factor availability.