Search

Abstract Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the Pcsk9 gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.

Krabbe disease is a sphingolipidosis characterized by the genetic deficiency of the acid hydrolase β-galactosylceramidase (GALC). Most of the studies concerning the biological role of GALC performed on Krabbe patients and Galc-deficient twitcher mice (an authentic animal model of the disease) indicate that the pathogenesis of this disorder is the consequence of the accumulation of the neurotoxic GALC substrate β-galactosylsphingosine (psychosine), ignoring the possibility that this enzyme may exert a wider biological impact. Indeed, limited information is available about the effect of GALC downregulation on the cell lipidome in adult and developing organisms. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model human genetic diseases, including sphingolipidoses, and two GALC co-orthologs have been identified in zebrafish (galca and galcb). Here, we investigated the effect of the competitive and irreversible GALC inhibitor β-galactose-cyclophellitol (GCP) on the lipid profile of zebrafish embryos. Molecular modelling indicates that GCP can be sequestered in the catalytic site of the enzyme and covalently binds human GALC, and the zebrafish Galca and Galcb proteins in a similar manner. Accordingly, GCP inhibits the β-galactosylceramide hydrolase activity of zebrafish in vitro and in vivo, leading to significant alterations of the lipidome of zebrafish embryos. These results indicate that the lack of GALC activity deeply affects the lipidome during the early stages of embryonic development, and thereby provide insights into the pathogenesis of Krabbe disease.

Abstract By connecting old and recent notions, different spatial scales, and research domains, we introduce a novel framework on the consequences of brain injury focusing on a key role of slow waves. We argue that the long-standing finding of EEG slow waves after brain injury reflects the intrusion of sleep-like cortical dynamics during wakefulness; we illustrate how these dynamics are generated and how they can lead to functional network disruption and behavioral impairment. Finally, we outline a scenario whereby post-injury slow waves can be modulated to reawaken parts of the brain that have fallen asleep to optimize rehabilitation strategies and promote recovery.

Abstract Purpose The most harmful atmospheric pollutant for human health is particulate matter (PM). We analyzed the correlation between short-term lag exposure to PM10 and PM2.5, salivary cortisol and TNF-α level, and methylation levels of the TNF-α promoter. Methods A pilot study including 20 subjects. Eight salivary samples for each subject at various times of the day were collected for comparing cortisol levels and TNFα detection. TNFα promoter methylation levels on salivary DNA were analyzed. Regression analyses were performed using generalized linear mixed models between the different outcomes and 4, 3, 2 and 1 day’s lag values of PM10/PM2.5.Generalized additive mixed model (GAMM) was used to evaluate any potential deviation from linearity. Results Area under the curve with respect to the ground (AUCg) showed a statistically positive association with 4-, 3-, 2-, and 1-day lag of exposure to PM10. Area under the curve with respect to the increase (AUCi) showed a statistically negative association with 4-, 3- and 1-day lag of exposure to PM10. TNFα showed statistically significant association with both exposures, PM10 and PM2.5, at 4-, 3-, 2-, and 1-day lag. Conclusions Regarding cortisol levels there is an increase of overall hormone levels but a less dynamism of the system to answer to external stressors. Increase of TNF-α may reflect increased levels of oxidative stress and inflammation due to pollution exposure.

Abstract Objective To investigate the Italian experience on the surgical and radiosurgical treatment of drug‐resistant epilepsy due to hypothalamic hamartoma (HH) in the period 2011–2021 in six Italian epilepsy surgery centers, and to compare safety and efficacy profiles of the different techniques. Methods We collected pseudo‐anonymized patient's data with at least 12 months of follow‐up. Surgical outcome was defined according to Engel classification of seizure outcome. Univariate analysis was performed to assess the risk of post‐operative seizures, categorized in dichotomous variable as favorable and unfavorable; explanatory variables were considered. Mann–Whitney or Chi‐squared test were used to assess the presence of an association between variables (p

Carbopol® is a typical jelly agent belonging to the family of cross-linked polyacrylic acid copolymers. It is largely used in antibacterial gels due to its self-wetting properties. In its pristine physical form, Carbopol® falls under the definition of microplastics, though significant changes could occur once added to hydroalcoholic solvents of the liquid formulations. To date, no life-cycle data regarding the physical state are available for this substance or for other similar polymers of the same chemical class. The aim of the present study was the investigation of the fate of Carbopol®-derived microplastics used in the formulation of typical hand sanitizer gels available in the Italian market, such as Amuchina® X-Germ, along the product life cycle. An experimental model was designed to detect the presence of Carbopol® microparticles from product manufacturing to the final use. FTIR and µ-FTIR were used to detect and characterize solid particles after the optimization of the sample preparation of different experimental matrices. While Carbopol® as such can be classified as a microplastic, in the commercial product, Carbopol® particles were not detected. Ten volunteers used the product according to the instructions reported on the label, and finally they rinsed their hands. Carbopol®-based particles were not detected in the water rinse, indicating that, after usage, the original form of the Carbopol microparticles was not retrieved. The study proposes, for the first time, a simple and comprehensive experimental approach to identify and characterize microplastics in finished products and along the life cycle by simulating their real-life usage. This approach could be also useful to evaluate the release of chemical components into the environment through the use of dermal products.

The Ralstonia species (RB) and Burkholderia species (BB) are bacteria responsible for nosocomial infections in frail patients such as hemodialyzed (HD) patients. Here, we report how we managed an outbreak caused by RB and BB that occurred in a dialysis unit. From the 7th to the 16th of April 2021, an infection due to RB and BB occurred in 7 out of 39 (17.9%) HD patients with central venous catheter (CVC). Disinfectants, CVC-lock therapy solutions, water by reverse osmosis unit (ROW) and dialysis concentrates were cultured, including the biofilm from the loading plastic tubes (LPTs) that connect the hemodialysis consoles (HCs) to the ROW delivery line. The antibiotic treatment was successful for all patients. RB and BB were isolated in the biofilm of 11/37 LPTs. Three out of 11 positive LPTs were associated with the infected patients. The ROW delivery line was modified to provide a whole disinfection with the HCs connected, avoiding the risk of new contamination of the LPTs. A filtration module of 0.01 mm was added prior to the ROW delivery line. Our experience suggests that outbreaks sustained by unusual bacteria such as RB and BB should be promptly investigated to treat the infected patients with the appropriate therapy and to identify the possible source of infection, making the needful changes to achieve a safer dialysis unit.

Background: Acute cerebral ischemia triggers a number of cellular mechanisms not only leading to excitotoxic cell death but also to enhanced neuroplasticity, facilitating neuronal reorganization and functional recovery. Objective: Transferring these cellular mechanisms to neurophysiological correlates adaptable to patients is crucial to promote recovery post-stroke. The combination of TMS and EEG constitutes a promising readout of neuronal network activity in stroke patients. Methods: We used the combination of TMS and EEG to investigate the development of local signal processing and global network alterations in 40 stroke patients with motor deficits alongside neural reorganization from the acute to the chronic phase. Results: We show that the TMS-EEG response reflects information about reorganization and signal alterations associated with persistent motor deficits throughout the entire post-stroke period. In the early post-stroke phase and in a subgroup of patients with severe motor deficits, TMS applied to the lesioned motor cortex evoked a sleep-like slow wave response associated with a cortical off-period, a manifestation of cortical bistability, as well as a rapid disruption of the TMS-induced formation of causal network effects. Mechanistically, these phenomena were linked to lesions affecting ascending activating brainstem fibers. Of note, slow waves invariably vanished in the chronic phase, but were highly indicative of a poor functional outcome. Conclusion: In summary, we found evidence that transient effects of sleep-like slow waves and cortical bistability within ipsilesional M1 resulting in excessive inhibition may interfere with functional reorganization, leading to a less favorable functional outcome post-stroke, pointing to a new therapeutic target to improve recovery of function.

Abstract Background Over the last two decades, tumor-derived RNA expression signatures have been developed for the two most commonly diagnosed tumors worldwide, namely prostate and breast tumors, in order to improve both outcome prediction and treatment decision-making. In this context, molecular signatures gained by main components of the tumor microenvironment, such as cancer-associated fibroblasts (CAFs), have been explored as prognostic and therapeutic tools. Nevertheless, a deeper understanding of the significance of CAFs-related gene signatures in breast and prostate cancers still remains to be disclosed. Methods RNA sequencing technology (RNA-seq) was employed to profile and compare the transcriptome of CAFs isolated from patients affected by breast and prostate tumors. The differentially expressed genes (DEGs) characterizing breast and prostate CAFs were intersected with data from public datasets derived from bulk RNA-seq profiles of breast and prostate tumor patients. Pathway enrichment analyses allowed us to appreciate the biological significance of the DEGs. K-means clustering was applied to construct CAFs-related gene signatures specific for breast and prostate cancer and to stratify independent cohorts of patients into high and low gene expression clusters. Kaplan-Meier survival curves and log-rank tests were employed to predict differences in the outcome parameters of the clusters of patients. Decision-tree analysis was used to validate the clustering results and boosting calculations were then employed to improve the results obtained by the decision-tree algorithm. Results Data obtained in breast CAFs allowed us to assess a signature that includes 8 genes (ITGA11, THBS1, FN1, EMP1, ITGA2, FYN, SPP1, and EMP2) belonging to pro-metastatic signaling routes, such as the focal adhesion pathway. Survival analyses indicated that the cluster of breast cancer patients showing a high expression of the aforementioned genes displays worse clinical outcomes. Next, we identified a prostate CAFs-related signature that includes 11 genes (IL13RA2, GDF7, IL33, CXCL1, TNFRSF19, CXCL6, LIFR, CXCL5, IL7, TSLP, and TNFSF15) associated with immune responses. A low expression of these genes was predictive of poor survival rates in prostate cancer patients. The results obtained were significantly validated through a two-step approach, based on unsupervised (clustering) and supervised (classification) learning techniques, showing a high prediction accuracy (≥ 90%) in independent RNA-seq cohorts. Conclusion We identified a huge heterogeneity in the transcriptional profile of CAFs derived from breast and prostate tumors. Of note, the two novel CAFs-related gene signatures might be considered as reliable prognostic indicators and valuable biomarkers for a better management of breast and prostate cancer patients.

Abstract Background The cyclin D1-cyclin dependent kinases (CDK)4/6 inhibitor palbociclib in combination with endocrine therapy shows remarkable efficacy in the management of estrogen receptor (ER)-positive and HER2-negative advanced breast cancer (BC). Nevertheless, resistance to palbociclib frequently arises, highlighting the need to identify new targets toward more comprehensive therapeutic strategies in BC patients. Methods BC cell lines resistant to palbociclib were generated and used as a model system. Gene silencing techniques and overexpression experiments, real-time PCR, immunoblotting and chromatin immunoprecipitation studies as well as cell viability, colony and 3D spheroid formation assays served to evaluate the involvement of the G protein-coupled estrogen receptor (GPER) in the resistance to palbociclib in BC cells. Molecular docking simulations were also performed to investigate the potential interaction of palbociclib with GPER. Furthermore, BC cells co-cultured with cancer-associated fibroblasts (CAFs) isolated from mammary carcinoma, were used to investigate whether GPER signaling may contribute to functional cell interactions within the tumor microenvironment toward palbociclib resistance. Finally, by bioinformatics analyses and k-means clustering on clinical and expression data of large cohorts of BC patients, the clinical significance of novel mediators of palbociclib resistance was explored. Results Dissecting the molecular events that characterize ER-positive BC cells resistant to palbociclib, the down-regulation of ERα along with the up-regulation of GPER were found. To evaluate the molecular events involved in the up-regulation of GPER, we determined that the epidermal growth factor receptor (EGFR) interacts with the promoter region of GPER and stimulates its expression toward BC cells resistance to palbociclib treatment. Adding further cues to these data, we ascertained that palbociclib does induce pro-inflammatory transcriptional events via GPER signaling in CAFs. Of note, by performing co-culture assays we demonstrated that GPER contributes to the reduced sensitivity to palbociclib also facilitating the functional interaction between BC cells and main components of the tumor microenvironment named CAFs. Conclusions Overall, our results provide novel insights on the molecular events through which GPER may contribute to palbociclib resistance in BC cells. Additional investigations are warranted in order to assess whether targeting the GPER-mediated interactions between BC cells and CAFs may be useful in more comprehensive therapeutic approaches of BC resistant to palbociclib.

Lupin seeds are recognized for their nutritional value and potential health benefits, but they contain also a significant amount of alkaloids, an anti-nutritive class of compounds, which vary significantly in composition within and between species due to environmental factors. In this study, we developed a predictive multi-experiment approach using ultra-high performance liquid chromatography coupled with triple quadrupole with linear ionic trap tandem mass spectrometry (UHPLC-QqQ-LIT-MS/MS) for comprehensive alkaloid profiling and geographical classification of Lupinus albus L. samples originating from four different Italian regions. Six targeted quinolizidine alkaloids were detected and 21 other alkaloids were putatively identified. Hierarchical Cluster Analysis (HCA) and partial least squares discriminant analysis (PLS-DA) were applied to explore the data structure and successfully classify samples according to their geographical origin. The data demonstrate the efficacy of the developed approach in providing valuable insights in alkaloid profiles of lupin seeds and their potential as markers for geographical traceability.

Focal brain injuries, such as stroke, cause local structural damage as well as alteration of neuronal activity in distant brain regions. Experimental evidence suggests that one of these changes is the appearance of sleep-like slow waves in the otherwise awake individual. This pattern is prominent in areas surrounding the damaged region and can extend to connected brain regions in a way consistent with the individual's specific long-range connectivity patterns. In this paper we present a generative whole-brain model based on (f)MRI data that, in combination with the disconnection mask associated with a given patient, explains the effects of the sleep-like slow waves originated in the vicinity of the lesion area on the distant brain activity. Our model reveals new aspects of their interaction, being able to reproduce functional connectivity patterns of stroke patients and offering a detailed, causal understanding of how stroke-related effects, in particular slow waves, spread throughout the brain. The presented findings demonstrate that the model effectively captures the links between stroke occurrences, sleep-like slow waves, and their subsequent spread across the human brain.

ObjectiveThis longitudinal study aimed to quantitatively document and evaluate the implementation and outcomes of the Open Dialogue (OD) approach within Italian Mental Health Departments (MHDs), focusing on the ratings of OD-network meetings by patients and their families and assessing the clinical outcomes over a span of 12 months.ResultsOver the course of the study, 58 patients participated in 517 OD-network meetings, demonstrating a high level of satisfaction with the care received, as evidenced by the Session Rating Scale (SRS) and Outcome Rating Scale (ORS). Clinically, significant improvements were observed in the Global Assessment of Functioning (GAF), Clinical Outcomes in Routine Evaluation (CORE-OM), and the Lubben Social Network Scale (LSNS), indicating enhanced psychological and social functioning. The SRS scores showed that satisfaction with the meetings increased over time, while the ORS indicated that both patients and their social networks perceived gradual improvements throughout the therapy.ConclusionThe OD approach within Italian MHDs was successfully implemented and well-received by patients and their social networks, yielding significant clinical improvements. These findings suggest the feasibility and effectiveness of integrating the OD model into the Italian public mental health system, supporting its potential for broader application in diverse healthcare settings. The study highlights the importance of continuous engagement and evaluation to maintain high standards of practice and suggests that OD can be a valuable addition to existing mental health care practices, promoting recovery through inclusive, dialogue-based interventions.

The first measurement of two-particle angular correlations for charged particles with LEP-II data is presented. The study is performed using archived hadronic e+e− data collected by ALEPH at center-of-mass energies up to 209 GeV, above the W+W− production threshold, which provide access to unprecedented charged-particle multiplicities and more complex color-string configurations if compared to previous measurements at LEP-I energies. An intriguing long-range near-side excess is observed in the correlation function measured with respect to the thrust axis in the highest multiplicity interval (Ntrk≥50). Such a structure is not predicted by the Monte-Carlo simulation. The harmonic anisotropy coefficients vn, which result from the Fourier expansion of the two-particle correlation functions, were also measured for the first time in e+e− data, and compared to pythia 6 predictions and to the results obtained in proton-proton collisions. The results presented in the Letter provide novel experimental constraints on the formation of collective phenomena in point-like e+e− collisions.

Abstract Introduction Bcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2. Methods We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts. Results We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation. Conclusions We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

Abstract Background Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERβ, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells. Methods The potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients. Results After the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients. Conclusions Overall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

Background: In last years the diffusion of carbapenem resistance in Gram-negative bacteria (CR-GNB) is increasing worldwide, mainly due to the expression of carbapenemases. Cefiderocol has molecular characteristics that ideally confers activity against all CR-GNB, but resistant strains have already been identified. We describe cefiderocol susceptibility profile among multi-drug resistant Gram-negative isolated from pediatric patients. Methods: Prospective, single pediatric center study, 1st January 2020–15th June 2023. All GNB carbapenemases producers or phenotypically carbapenem-resistant isolated in the study period were tested for cefiderocol susceptibility. Clinical and microbiological data were collected. A descriptive analysis was performed, comparing the groups of cefiderocol-resistant vs. cefiderocol-susceptible Enterobacterales and non-fermenting Gram-negative bacteria (NF-GNB). Results: Forty-seven GNB were tested for cefiderocol susceptibility; 38% were cefiderocol-resistant: 16/30 (52%) among Enterobacterales and 2/17 (12%) among NF-GNB. None of the patients were previously exposed to cefiderocol. Looking at Enterobacterales, resistance to ceftazidime/avibactam was higher among cefiderocol-resistant vs. cefiderocol-susceptible strains (62% vs 36%, respectively), as MBL expression (67% vs. 36%, respectively). Too few NF-GNB were cefiderocol-resistance to draw any conclusion. No difference in ICU admission and mortality was identified comparing cefiderocol-resistant vs. susceptible strains. Patients colonized/infected by cefiderocol-resistant strains had been previously hospitalized more frequently. Conclusion: In our cohort cefiderocol resistance was mostly registered among Enterobacterales, and especially among MBL producers’ strains (that were alongside resistant to ceftazidime/avibactam). This could be explained by the known possible cross resistance mechanism among ceftazidime/avibactam and cefiderocol. Also, correlation of cefiderocol-resistance with previous hospitalization could be associated with horizontal resistance transmission. Looking at our data, we believe that cefiderocol should be use cautiously, especially empirically and in monotherapy, due to the high resistance rate.

O objetivo deste trabalho foi analisar o comportamento da cinética de secagem em estufa de circulação de ar forçada da alga Sargassum sp. em temperaturas de 60 a 80 °C e seu potencial como adsorvente na remoção do corante azul de metileno em um efluente têxtil sintético (ETS). Foi aplicado o planejamento fatorial 23 para o processo de adsorção com as seguintes variáveis independentes: massa de adsorvente, concentração de corante e tempo de contato, sendo avaliadas as respostas de redução na concentração de corante (% R) e de quantidade adsorvida (qt). O percentual de umidade da massa da alga Sargassum sp. foi de 84,47%. Para a temperatura de secagem de 60 °C, o modelo que obteve o melhor ajuste aos dados experimentais foi o modelo de Midili, e o modelo que obteve o melhor ajuste para os dados da temperatura de secagem de 80 °C foi o logarítmico. O melhor resultado em relação à variável qt foi de 231,90 mg g−1, e o melhor resultado para a variável resposta % R obteve um valor de 93,31%. O modelo de pseudo-segunda ordem obteve o melhor ajuste aos dados experimentais de cinética, com R2 de 0,99. O adsorvente produzido a partir da alga Sargassum sp. teve resultados satisfatórios no estudo do planejamento experimental e da cinética de adsorção, apresentando eficiência na redução do corante azul de metileno em ETS.

Abstract Dynamic contrast optical coherence tomography (DyC-OCT), an emerging imaging method, utilizes fluctuation patterns in OCT signals to enhance contrast, thereby enabling non-invasive label-free volumetric live cell imaging. In this mini review, we explain the core concepts behind DyC-OCT image formation and its system configurations, serving as practical guidance for future DyC-OCT users. Subsequently, we explore its applications in delivering high-quality, contrast-enhanced images of cellular morphology, as well as in monitoring changes in cellular activity/viability assay experiments.

Abstract Background and study aim The development of a new cholangioscope, the SpyGlass™ Discover (Boston Scientific), has allowed the laparoscopic transcystic common bile duct exploration and stone clearance. The possibility of simultaneous treatment of choledocholithiasis during early laparoscopic cholecystectomy offers the opportunity to enormously reduce the time between acute cholecystitis diagnosis and the execution of cholecystectomy with better outcomes for patients. Furthermore, an altered anatomy of the gastrointestinal tract is not an obstacle to this technique. The aim of the study was to determine whether this new procedure is feasible, safe, and effective. Patients and methods The investigation employs a retrospective case series study including all consecutive patients with a diagnosis of common bile duct stones undergoing cholecystectomy and intraoperative laparoscopic common bile duct clearance using SpyGlass™ Discover at IRCCS Policlinico San Matteo in Pavia (Italy). Eighteen patients were included from May 2022 to May 2023. Results A complete clearance of the common bile duct was obtained in 88.9% of patients. The mean postoperative length of stay was 3 days. No major complications occurred. After a median follow-up of 8 months, no recurrence of biliary events or readmissions occurred. Conclusion This procedure has proven to be feasible, safe, and effective.

Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.

Abstract Background Guidelines about febrile neutropenia in paediatric patients are not homogeneous; the best empiric treatment of this condition should be driven by local epidemiology. The Weighted-Incidence Syndromic Combination Antibiogram (WISCA) addresses the need for disease-specific local susceptibility evidence that could guide empiric antibiotic prescriptions based on outcome estimates of treatment regimens obtained as a weighted average of pathogen susceptibilities. This study developed a WISCA model to inform empirical antibiotic regimen selection for febrile neutropenia (FN) episodes in onco-haematological paediatric patients treated at two Italian paediatric tertiary centres. Methods We included blood cultures from patients with a bloodstream infection and neutropenia admitted to the Paediatric Haematology-Oncology wards in Padua and Genoa Hospitals from 2016 to 2021. WISCAs were developed by estimating the coverage of 20 antibiotics as monotherapy and of 21 combined regimens with a Bayesian probability distribution. Results We collected 350 blood cultures, including 196 g-negative and 154 g-positive bacteria. Considering the most used antibiotic combinations, such as piperacillin–tazobactam plus amikacin, the median coverage for the pool of bacteria collected in the study was 78%. When adding a glycopeptide, the median coverage increased to 89%, while the replacement of piperacillin–tazobactam with meropenem did not provide benefits. The developed WISCAs showed that no monotherapy offered an adequate coverage rate for the identified pathogens. Conclusions The application of WISCA offers the possibility of maximizing the clinical utility of microbiological surveillance data derived from large hospitals to inform the choice of the best empiric treatment while contributing to spare broad-spectrum antibiotics.

Abstract Background First identified in Drosophila melanogaster, the Hippo pathway is considered a major regulatory cascade controlling tissue homeostasis and organ development. Hippo signaling components include kinases whose activity regulates YAP and TAZ final effectors. In response to upstream stimuli, YAP and TAZ control transcriptional programs involved in cell proliferation, cytoskeletal reorganization and stemness. Main text While fine tuning of Hippo cascade components is essential for maintaining the balance between proliferative and non-proliferative signals, pathway signaling is frequently dysregulated in gastrointestinal cancers. Also, YAP/TAZ aberrant activation has been described in conditions characterized by chronic inflammation that precede cancer development, suggesting a role of Hippo effectors in triggering carcinogenesis. In this review, we summarize the architecture of the Hippo pathway and discuss the involvement of signaling cascade unbalances in premalignant lesions of the gastrointestinal tract, providing a focus on the underlying molecular mechanisms. Conclusions The biology of premalignant Hippo signaling dysregulation needs further investigation in order to elucidate the evolutionary trajectories triggering cancer inititation and develop effective early therapeutic strategies targeting the Hippo/YAP pathway.

This work is concerned with the study of a necessary and sufficient condition for the existence of solutions with a given regularity to a system of linear equations with coefficients of given regularity. First, to properly contextualize the subject matter and to introduce crucial analytical solving tools, we go through results by C.Fefferman- J.Kollár and by C.Fefferman - G.K.Luli. Then we prove our result to determine a necessary and sufficient condition for the existence of continuous (C0) semialgebraic solutions in case of a system of linear equations with continuous semialgebraic coefficients.

Background: Combined oral contraceptives (COCs) work mostly by preventing the pre-ovulatory gonadotropin surge, but the action of COCs on spontaneous episodic and GnRH (gonadotropin-releasing hormone)-induced LH (luteinizing hormone) release has been poorly evaluated. Oral contraceptives are known to act on the spontaneous hypothalamic–pituitary functions reducing both GnRH and gonadotropin release and blocking ovulation. Aim: To evaluate spontaneous and GnRH-induced LH release during both phases of the menstrual cycle or under the use of the contraceptive pill. Methods: A group of 12 women, subdivided into two groups, volunteered for the study. Group A (n = 6, controls) received no treatments, while Group B (n = 6) received a 21 + 7 combination of ethinyl-estradiol (EE) 30 µg + drospirenone (DRSP) 3 mg. Both groups were evaluated twice: Group A during follicular and luteal phases, Group B during pill assumption and during the suspension interval, performing a pulsatility test, GnRH stimulation test, and hormonal parameters evaluation. Spontaneous and GnRH-induced secretory pulses were evaluated, as well as the instantaneous secretory rate (ISR). Results: COC treatment lowered LH and FSH (follicle stimulating hormone) levels significantly if compared to the follicular phase of spontaneous cycles. During the suspension interval, hormone levels rapidly rose and became comparable to those of the follicular phase of the control group. The LH pulse frequency under COC administration during the suspension interval was similar to that observed during the follicular phase (2.6 ± 0.3 pulses/180 min and 2.3 ± 0.2 pulses/180 min, respectively). The GnRH-induced LH peaks were greater in amplitude and duration than those observed after ISR computation in both groups. The GnRH-induced LH release during the luteal phase of the control subjects was higher than in the follicular phase (51.2 ± 12.3 mIU/mL and 14.9 ± 1.8 mIU/mL, respectively). Conversely, subjects under COC showed a GnRH-induced LH response similar during COC and during the suspension interval. Conclusions: Our data support that the EE + DRSP preparation acts on both spontaneous pulsatile release and GnRH-induced LH release during the withdrawal period of the treatment, and that after 5–7 days from the treatment suspension, steroidal secretion from the ovary is resumed, such as that of androgens. This suggests that in hyperandrogenic patients, a suspension interval as short as 4 days might be clinically better.

AbstractObjective Diagnosis of cancer is challenging in primary care due to the low incidence of cancer cases in primary care practice. A prolonged diagnostic interval may be due to doctor, patient or system factors, or may be due to the characteristics of the cancer itself. The objective of this study was to learn from Primary Care Physicians’ (PCP) experiences of incidents when they had failed to think of, or act on, a cancer diagnosis.Design A qualitative, online survey eliciting PCP narratives. Thematic analysis was used to analyse the data.Setting and subjects A primary care study, with narratives from 159 PCPs in 23 European countries.Main outcome measures PCPs’ narratives on the question ‘If you saw this patient with cancer presenting in the same way today, what would you do differently?Results The main themes identified were: thinking broadly; improvement in communication and clinical management; use of other available resources and ‘I wouldn’t do anything differently’.Conclusion (Implications) To achieve more timely cancer diagnosis, PCPs need to provide a long-term, holistic and active approach with effective communication, and to ensure shared decision-making, follow-up and continuing re-assessment of the patients’ clinical conditions.

Therapeutic drug monitoring (TDM) is a critical clinical tool used to optimize the safety and effectiveness of drugs by measuring their concentration in biological fluids. These fluids are primarily plasma or blood. TDM, together with real-time dosage adjustment, contributes highly to the successful management of glycopeptide antimicrobial therapies. Understanding pharmacokinetic/pharmacodynamic (PK/PD) properties is vital for optimizing antimicrobial therapies, as the efficacy of these therapies depends on both the exposure of the patient to the drug (PK) and pharmacodynamic (PD) parameters such as the in vitro estimated minimum drug concentration that inhibits bacterial growth (MIC). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is widely recognized as the gold standard for measuring small molecules, such as antibiotics. This review provides a comprehensive overview of LC-MS/MS methods available for TDM of glycopeptide antibiotics, including vancomycin, teicoplanin, dalbavancin, oritavancin, and telavancin.

Abstract Background Amyloid A (AA) amyloidosis is a protein misfolding disease arising from serum amyloid A (SAA). Systemic AA amyloidosis recently was shown to have a high prevalence in shelter cats in Italy and was associated with azotemia and proteinuria. Objectives Investigate urine protein profiles and diagnostic biomarkers in cats with renal AA amyloidosis. Animals Twenty‐nine shelter cats. Methods Case‐control study. Cats with renal proteinuria that died or were euthanized between 2018 and 2021 with available necropsy kidney, liver and spleen samples, and with surplus urine collected within 30 days before death, were included. Histology was used to characterize renal damage and amyloid amount and distribution; immunohistochemistry was used to confirm AA amyloidosis. Urine protein‐to‐creatinine (UPC) and urine amyloid A‐to‐creatinine (UAAC) ratios were calculated, and sodium dodecyl sulfate‐agarose gel electrophoresis (SDS‐AGE) and liquid chromatography‐mass spectrometry (LC‐MS) of proteins were performed. Results Twenty‐nine cats were included. Nineteen had AA amyloidosis with renal involvement. Cats with AA amyloidosis had a higher UPC (median, 3.9; range, 0.6‐12.7 vs 1.5; 0.6‐3.1; P = .03) and UAAC ratios (median, 7.18 × 10−3; range, 23 × 10−3‐21.29 × 10−3 vs 1.26 × 10−3; 0.21 × 10−3‐6.33 × 10−3; P = .04) than unaffected cats. The SDS‐AGE identified mixed‐type proteinuria in 89.4% of cats with AA amyloidosis and in 55.6% without AA amyloidosis (P = .57). The LC‐MS identified 63 potential biomarkers associated with AA amyloidosis (P

Abstract Background Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. Conclusions Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Abstract Background Several socioeconomic conditions may influence subjects’ adherence to healthy eating habits. Food consumption may be influenced by external stress during crisis periods; however, the effects of these events on food habits are difficult to predict. Also, a pandemic crisis like the recent COVID-19 pandemic may have influenced dietary habits and food consumption. The objective of this study was to compare the dietary habits of Italian people before the COVID-19 pandemic with those belonging to the year 2020 in a nationwide population sample. Materials and methods Information on dietary habits has been obtained from the multi-purpose survey on families’ “Aspects of daily life”, carried out in Italy by the Italian National Statistics Institute (ISTAT). We analyzed data coming from 2016 and 2020 editions of the survey (43,000 subjects each year). We used population attributable fraction (PAF) adjusted for age, defined as the proportional reduction in unhealthy diet that would occur if all participants had had a higher education, assuming higher educated individuals as more socially advantaged. Prevalence association for each dietary exposition has been calculated through logistic regression. Results Looking at aggregated data from 2016 and 2020 both men and women showed a high prevalence of unhealthy dietary habits. Regarding men, excessive consumption of eggs, pork meat, and bovine meat was characterized by a PAF attributable to socioeconomic conditions to an extent greater than 30%. Women showed the same trend. Focusing on different years of investigation, in 2020, during the COVID-19 pandemic, men and women increased their consumption of eggs, cooked fats, snacks, and sweets, and reduced consumption of fruits and vegetables. Additionally, women increased the assumption of dietary products and meat. Both sexes registered an increase in overweight and obese subjects in 2020. Conclusions To our knowledge, this study was the first in our country to use a yearly, nationwide sample to analyze dietary habits by examining specific types of various foods on a nationwide scale and establishing a correlation between these habits and the COVID-19 pandemic. Our results showed unbalanced dietary habits of the Italian population with an excess of consumption of several foods like eggs, cooked fats snacks, and sweets with low consumption of fruits and vegetables. Socioeconomic differences influence food choices but in a complex way since they seemed to affect some wrong dietary habits but not others, especially regarding fruits and vegetables assumption where differences were less evident among social classes. Outside stressors like a crisis period such as the COVID-19 pandemic seem to have an important role in both men and women regarding the assumption of so-called “junk food”.

IntroductionAutoimmune cytopenias (AICs) are a group of disorders characterized by immune-mediated destruction of blood cells. In children, they are often secondary to immune dysregulation that may require long-lasting immunosuppression. Mycophenolate mofetil and sirolimus represent two well-tolerated options to treat these disorders, often as a steroid-sparing option. However, no data are available on the infection risk for patients undergoing long-lasting treatments.Patients and methodsThe rate of severe infective events was calculated in episodes per 100 persons/months at risk (p/m/r) documented by the analysis of hospitalization charts between January 2015 and July 2023 of patients treated with mycophenolate mofetil or sirolimus given for isolated AIC or AICs associated with autoimmune lymphoproliferative syndrome (ALPS)/ALPS-like syndromes in two large Italian pediatric hematology units.ResultsFrom January 2015 to July 2023, 13 out of 96 patients treated with mycophenolate mofetil or sirolimus developed 16 severe infectious events requiring hospitalization. No patients died. Overall infection rate was 0.24 person/*100 months/risk (95% CI 0.09–0.3). Serious infectious events incidence was higher in patients with ALPS-like compared to others (0.42 versus 0.09; p = 0.006) and lower in patients who underwent mycophenolate treatment alone compared to those who started sirolimus after mycophenolate failure (0.04 versus 0.29, p = 0.03). Considering only patients who started treatment at the beginning of study period, overall cumulative hazard was 18.6% at 60 months (95% CI 3.4–31.4) with higher risk of infectious events after 5 years in ALPS-like patients (26.1%; 95% CI 3.2–43.5) compared to other AICs (4%; 95% CI 0–11.4; p = 0.041).DiscussionTo the best of our knowledge, this is the first study to describe the infectious risk related to mycophenolate and sirolimus chronic treatment in patients with AICs and immune dysregulation. Our data highlight that infection rate is very low and mainly related to the underlying hematological condition.ConclusionsMycophenolate and sirolimus represent a safe immunosuppressive therapy in AICs and immune dysregulation syndromes.

The impact of common and rare variants in COVID-19 host genetics has been widely studied. In particular, in Fallerini et al. (Human genetics, 2022, 141, 147–173), common and rare variants were used to define an interpretable machine learning model for predicting COVID-19 severity. First, variants were converted into sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. After that, the Boolean features, selected by these logistic models, were combined into an Integrated PolyGenic Score (IPGS), which offers a very simple description of the contribution of host genetics in COVID-19 severity.. IPGS leads to an accuracy of 55%–60% on different cohorts, and, after a logistic regression with both IPGS and age as inputs, it leads to an accuracy of 75%. The goal of this paper is to improve the previous results, using not only the most informative Boolean features with respect to the genetic bases of severity but also the information on host organs involved in the disease. In this study, we generalize the IPGS adding a statistical weight for each organ, through the transformation of Boolean features into “Boolean quantum features,” inspired by quantum mechanics. The organ coefficients were set via the application of the genetic algorithm PyGAD, and, after that, we defined two new integrated polygenic scores (IPGSph1 and IPGSph2). By applying a logistic regression with both IPGS, (IPGSph2 (or indifferently IPGSph1) and age as inputs, we reached an accuracy of 84%–86%, thus improving the results previously shown in Fallerini et al. (Human genetics, 2022, 141, 147–173) by a factor of 10%.

In all reported cases of foreign body migration or impaction in the biliary tree, there is no mention of the presence of hypereosinophilia among the laboratory findings. This could possibly be attributed to the local tissue reaction caused by the impacted foreign body. Here, we present our experience with the removal of a screw from a surgical retractor that became lodged in the common bile duct (CBD) and migrated in a patient who had previously undergone a left lateral hepatectomy for hepatic hydatidosis. The imaging was not sufficient to make a diagnosis, and the interpretation of hypereosinophilia in such a case could pose a challenge.

Abstract Emerging evidence highlights the potential prognostic relevance of circulating lipids in metastatic castration-resistant prostate cancer (mCRPC), with a proposed 3-lipid signature. This study aims to analyze the lipidomic profiles of individuals with mCRPC to identify lipid species that could serve as predictive indicators of prognosis and therapeutic response. Plasma samples were collected from mCRPC patients initiating first-line treatment (1 L) (n = 29) and those previously treated with at least two lines of therapy (> 2 L) (n = 19), including an androgen-receptor signaling inhibitor and a taxane. Employing an untargeted lipidomic approach, lipids were extracted from the plasma samples and subjected to analysis. A comprehensive identification and quantification of 789 plasma lipids was achieved. Notably, 75 species displayed significant dysregulation in > 2 L patients in comparison to the 1 L group. Among these, 63 species exhibited elevated levels, while 12 were reduced. Patients included in > 2 L cohort showed elevated levels of acylcarnitines (CAR), diacylglycerols (DG), phosphatidylethanolamines (PE), triacylglycerols (TG), and ceramides (Cer). Notably, some upregulated lipids, including CAR 14:0, CAR 24:1, Cer d18:1/16:0, Cer d18:1/18:0 (C18 Cer), Cer d18:2/18:0, Cer d18:1/24:1, and Cer d20:1/24:1, showed significant associations with overall survival (OS) in univariate models. Specifically, increased levels of C18 Cer remained significantly associated with poorer OS in the multivariate model, even after adjusting for treatment line and PSA levels (Hazard Ratio: 3.59 [95% Confidence Interval 1.51–8.52], p = 0.004). Employing quantitative mass spectrometry, our findings underscore the independent prognostic significance of C18 Cer in individuals with mCRPC. This discovery opens avenues for further studies within this field.

Abstract Background Molecular Tumor Boards (MTB) operating in real-world have generated limited consensus on good practices for accrual, actionable alteration mapping, and outcome metrics. These topics are addressed herein in 124 MTB patients, all real-world accrued at progression, and lacking approved therapy options. Methods Actionable genomic alterations identified by tumor DNA (tDNA) and circulating tumor DNA (ctDNA) profiling were mapped by customized OncoKB criteria to reflect diagnostic/therapeutic indications as approved in Europe. Alterations were considered non-SoC when mapped at either OncoKB level 3, regardless of tDNA/ctDNA origin, or at OncoKB levels 1/2, provided they were undetectable in matched tDNA, and had not been exploited in previous therapy lines. Results Altogether, actionable alterations were detected in 54/124 (43.5%) MTB patients, but only in 39 cases (31%) were these alterations (25 from tDNA, 14 from ctDNA) actionable/unexploited, e.g. they had not resulted in the assignment of pre-MTB treatments. Interestingly, actionable and actionable/unexploited alterations both decreased (37.5% and 22.7% respectively) in a subset of 88 MTB patients profiled by tDNA-only, but increased considerably (77.7% and 66.7%) in 18 distinct patients undergoing combined tDNA/ctDNA testing, approaching the potential treatment opportunities (76.9%) in 147 treatment-naïve patients undergoing routine tDNA profiling for the first time. Non-SoC therapy was MTB-recommended to all 39 patients with actionable/unexploited alterations, but only 22 (56%) accessed the applicable drug, mainly due to clinical deterioration, lengthy drug-gathering procedures, and geographical distance from recruiting clinical trials. Partial response and stable disease were recorded in 8 and 7 of 19 evaluable patients, respectively. The time to progression (TTP) ratio (MTB-recommended treatment vs last pre-MTB treatment) exceeded the conventional Von Hoff 1.3 cut-off in 9/19 cases, high absolute TTP and Von Hoff values coinciding in 3 cases. Retrospectively, 8 patients receiving post-MTB treatment(s) as per physician’s choice were noted to have a much longer overall survival from MTB accrual than 11 patients who had received no further treatment (35.09 vs 6.67 months, p = 0.006). Conclusions MTB-recommended/non-SoC treatments are effective, including those assigned by ctDNA-only alterations. However, real-world MTBs may inadvertently recruit patients electively susceptible to diverse and/or multiple treatments.

Abstract The G protein-coupled estrogen receptor (GPER) mediates estrogen action in different pathophysiological conditions, including cancer. GPER expression and signaling have been found to join in the progression of triple-negative breast cancer (TNBC), even though controversial data have been reported. In present study, we aimed at providing new mechanistic and biological discoveries knocking out (KO) GPER expression by CRISPR/Cas9 technology in MDA-MB-231 TNBC cells. GPER KO whole transcriptome respect to wild type (WT) MDA-MB-231 cells was determined through total RNA sequencing (RNA-Seq) and gene ontology (GO) enrichment analysis. We ascertained that anti-proliferative and pro-apoptotic gene signatures characterize GPER KO MDA-MB-231 cells. Thereafter, we determined that these cells exhibit a reduced proliferative, clonogenic and self-renewal potential along with an increased mitochondria-dependent apoptosis phenotype. In addition, we recognized that decreased cAMP levels trigger the JNK/c-Jun/p53/Noxa axis, which in turn orchestrates the pro-apoptotic effects observed in GPER KO cells. In accordance with these data, survival analyses in TNBC patients of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset indicated that high Noxa expression correlates with improved outcomes in TNBC patients. Furthermore, we demonstrated that GPER KO in TNBC cells impairs the expression and secretion of the well-acknowledged GPER target gene named CTGF, thus resulting in the inhibition of migratory effects in cancer-associated fibroblasts (CAFs). Overall, the present study provides novel mechanistic and biological insights on GPER KO in TNBC cells suggesting that GPER may be considered as a valuable target in comprehensive therapeutic approaches halting TNBC progression.

Abstract Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.

The relationship between serum 25-hydroxyvitamin D (25(OH)D) levels, genomic response to vitamin D (Vit.D), and positivity to SARS-CoV-2 remains understudied. In this pilot study, during the follow-up of patients with Inflammatory Bowel Disease (IBD) and COVID-19, we investigated this issue by analyzing the molecular contents of serum extracellular vesicles (EVs) from six groups of IBD patients (n = 32), classified according to anti-SARS-CoV-2 status, 25(OH)D level, and Vit.D supplementation, by small RNA-seq. This analysis revealed differentially expressed miRNAs, PIWI-RNA, transfer RNA, small nucleolar RNAs, and protein-coding RNAs in the EVs obtained from these cohorts of IBD patients. Experimental validation evidenced a statistically significant increase in miR30d-5p, miR150-5p, Let-7f-5p, and Let-7a-5p in the anti-SARS-CoV-2-positive and low 25(OH)D and Vit.D supplemented groups with respect to the non-Vit.D supplemented group, indicating their responsiveness to Vit.D treatment. Bioinformatics analysis highlighted the regulation of these validated miRNAs by oxidative stress and inflammation, hallmarks of IBD and COVID-19. Our study reports an unprecedented panel of circulating EV-enclosed inflammation- and oxidative stress-related miRNAs, the potentiality of which, as biomarkers for Vit.D responsivity in IBD patients, needs to be explored in future studies on larger cohorts in order to allow clinicians to optimize current treatment strategies upon viral infection.

The carbon source/sink nature and the water balance of a drip-irrigated and mulched watermelon cultivated under a semi-arid climate were investigated. Biodegradable films, plants and some fruits were left on the soil as green manure. The study spanned from watermelon planting to the subsequent crop (June–November 2023). The eddy covariance technique was employed to monitor water vapor (H2O) and carbon dioxide (CO2) fluxes, which were partitioned into transpiration, evaporation, photosynthesis and respiration, respectively, using the flux variance similarity method.This method utilizesthe Monin–Obukhov similarity theory to separate stomatal (photosynthesis and transpiration) from non-stomatal (respiration and evaporation) processes. The results indicate that mulching films contribute to carbon sequestration in the soil (+19.3 g C m−2). However, the mulched watermelon crop presented in this study functions as a net carbon source, with a net biome exchange, representing the net rate of C accumulation in or loss from ecosystems, equal to +230 g C m−2. This is primarily due to the substantial amount of carbon exported through marketable fruits. Fixed water scheduling led to water waste through deep percolation (approximately 1/6 of the water supplied), which also contributed to the loss of organic carbon via leaching (−4.3 g C m−2). These findings recommend further research to enhance the sustainability of this crop in terms of both water and carbon balances.

The transcription factor ΔNp63 plays a pivotal role in maintaining the integrity of stratified epithelial tissues by regulating the expression of distinct target genes involved in lineage specification, cell stemness, cell proliferation and differentiation. Here, we identified the ABC transporter subfamily member ABCC1 as a novel ΔNp63 target gene. We found that in immortalized human keratinocytes and in squamous cell carcinoma (SCC) cells, ∆Np63 induces the expression of ABCC1 by physically occupying a p63-binding site (p63 BS) located in the first intron of the ABCC1 gene locus. In cutaneous SCC and during the activation of the keratinocyte differentiation program, ∆Np63 and ABCC1 levels are positively correlated raising the possibility that ABCC1 might be involved in the regulation of the proliferative/differentiative capabilities of squamous tissue. However, we did not find any gross alteration in the structure and morphology of the epidermis in humanized hABCC1 knock-out mice. Conversely, we found that the genetic ablation of ABCC1 led to a marked reduction in inflammation-mediated proliferation of keratinocytes, suggesting that ABCC1 might be involved in the regulation of keratinocyte proliferation upon inflammatory/proliferative signals. In line with these observations, we found a significant increase in ABCC1 expression in squamous cell carcinomas (SCCs), a tumor type characterized by keratinocyte hyper-proliferation and a pro-inflammatory tumor microenvironment. Collectively, these data uncover ABCC1 as an additional ∆Np63 target gene potentially involved in those skin diseases characterized by dysregulation of proliferation/differentiation balance.

Background: Heart failure (HF) with preserved ejection fraction (HFpEF) represents a major comorbidity in the elderly and is associated with cognitive impairment (CoI) and type 2 diabetes mellitus (T2DM). In this context, there is an increase in oxidative stress and platelet activation biomarkers. The aim of this study was to evaluate the effects of 6 months’ treatment with SGLT2i on functional, mood-related, and cognitive aspects, assessed by performing a comprehensive geriatric assessment (CGA), and on oxidative stress and platelet activation biomarkers, in a cohort of HFpEF elderly patients with T2DM. We recruited 150 elderly outpatients (mean age 75.8 ± 7.4 years). Results: At six-month follow-up, there was a significant improvement in MMSE (p < 0.0001), MoCA (p < 0.0001), GDS score (p < 0.0001), and SPPB (p < 0.0001). Moreover, we observed a significant reduction in Nox-2 (p < 0.0001), 8-Isoprostane (p < 0.0001), Sp-Selectin (p < 0.0001), and Gp-VI (p < 0.0001). Considering ΔMMSE as the dependent variable, ΔE/e’, ΔNox-2, ΔHOMA, Δ8-Isoprostane, and ΔUricemia were associated for 59.6% with ΔMMSE. When ΔMoCA was considered as the dependent variable, ΔHOMA, ΔE/e’, Δ8-Isoprostane, ΔNox-2 and ΔUricemia were associated for 59.2%. Considering ΔGDS as the dependent variable, ΔHOMA, ΔNox-2, Δ8-Isoprostane, and ΔUricemia were associated with 41.6% of ΔGDS variation. Finally, ΔHOMA was the main predictor of ΔSPPB, which was associated with 21.3% with ΔSPPB, Δ8-Isoprostane, ΔNox-2, ΔE/e’, and ΔUricemia added another 24.1%. Conclusion: The use of SGLT2i in elderly patients with T2DM and HFpEF significantly contributes to improving CGA scales and biomarkers of OS and PA.

Knowledge of epidemiology is essential for guiding correct antibiotic prescription, reducing bacteremia-associated mortality, and implementing targeted infection control programs. However, only a few studies have reported on the epidemiology of bloodstream infections (BSIs) in pediatrics. We performed a retrospective analysis of all BSIs (excluding those caused by common skin contaminants) diagnosed from 2006 to 2022 in patients younger than 18 years who were treated at an Italian pediatric tertiary care hospital. Overall, 2395 BSIs were recorded, including 2207 (92.15%) due to bacteria and 188 (7.85%) due to fungi. The incidence rate (BSIs/10,000 hospital discharges, IR) of bacterial BSIs significantly increased during the study period. In particular, BSIs caused by S. aureus (including MRSA), Enterobacterales (including ESBL and AmpC producers), Enterococcus spp., and P. aeruginosa became more common. The frequency of carbapenem-resistant strains was S. pneumoniae. The BSIs were stratified by patient age, and S. aureus was the most frequent cause of BSIs in all age groups, while E. coli was the most frequent in the Enterobacterales family. S. agalactiae was the third most frequent cause of neonatal early-onset BSIs. The prevalence of Enterococcus spp. increased in the subgroups from 8 days to 5 years of age, while P. aeruginosa became more prevalent in children over 5 years of age. S. aureus was also the most frequent isolate in both community- and hospital-onset BSIs, followed by E. coli. The prevalence of multidrug-resistant (MDR) pathogens was very low. It was C. albicans was the most frequently isolated strain, followed by C. parapsilosis. Notably, the IR of fungemia did not change significantly during the study period, in spite of an increase in the absolute number of events. The continuous monitoring of local epidemiology is essential to identify changes in the IRs of pathogens and antibiotic susceptibility and to guide antibiotic treatments, especially in the phase when antibiograms are not yet available.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder lacking reliable biomarkers for early diagnosis and disease progression monitoring. This study aimed to identify the novel biomarkers in plasmatic extracellular vesicles (EVs) isolated from ALS patients and healthy controls (HCs). A total of 61 ALS patients and 30 age-matched HCs were enrolled in the study and the protein content of circulating EVs was analyzed by shotgun proteomics. The study was divided into a discovery phase (involving 12 ALS and 12 HC patients) and a validation one (involving 49 ALS and 20 HC patients). In the discovery phase, more than 300 proteins were identified, with 32 proteins showing differential regulation in ALS patients compared to HCs. In the validation phase, over 400 proteins were identified, with 20 demonstrating differential regulation in ALS patients compared to HCs. Notably, seven proteins were found to be common to both phases, all of which were significantly upregulated in EVs from ALS patients. Most of them have previously been linked to ALS since they have been detected in the serum or cerebrospinal fluid of ALS patients. Among them, proteoglycan (PRG)-4, also known as lubricin, was of particular interest since it was significantly increased in ALS patients with normal cognitive and motor functions. This study highlights the significance of EVs as a promising avenue for biomarker discovery in ALS. Moreover, it sheds light on the unexpected role of PRG-4 in relation to cognitive status in ALS patients.

Background: The efficacy of endoscopic ultrasound-guided liver biopsy (EUS-LB) compared to percutaneous liver biopsy (PC-LB) remains uncertain. Methods: Our data consist of randomized controlled trials (RCTs) comparing EUS-LB to PC-LB, found through a literature search via PubMed/Medline and Embase. The primary outcome was sample adequacy, whereas secondary outcomes were longest and total lengths of tissue specimens, diagnostic accuracy, and number of complete portal tracts (CPTs). Results: Sample adequacy did not significantly differ between EUS-LB and PC-LB (risk ratio [RR] 1.18; 95% confidence interval [CI] 0.58–2.38; p = 0.65), with very low evidence quality and inadequate sample size as per trial sequential analysis (TSA). The two techniques were equivalent with respect to diagnostic accuracy (RR: 1; CI: 0.95–1.05; p = 0.88), mean number of complete portal tracts (mean difference: 2.29, −4.08 to 8.66; p = 0.48), and total specimen length (mean difference: −0.51, −20.92 to 19.9; p = 0.96). The mean maximum specimen length was significantly longer in the PC-LB group (mean difference: −3.11, −5.51 to −0.71; p = 0.01), and TSA showed that the required information size was reached. Conclusion: EUS-LB and PC-LB are comparable in terms of diagnostic performance although PC-LB provides longer non-fragmented specimens.

Hepatitis E virus (HEV) infection is typically a self-limiting, acute illness that spreads through the gastrointestinal tract but replicates in the liver. However, chronic infections are possible in immunocompromised individuals. The HEV virion has two shapes: exosome-like membrane-associated quasi-enveloped virions (eHEV) found in circulating blood or in the supernatant of infected cell cultures and non-enveloped virions (“naked”) found in infected hosts’ feces and bile to mediate inter-host transmission. Although HEV is mainly spread via enteric routes, it is unclear how it penetrates the gut wall to reach the portal bloodstream. Both virion types are infectious, but they infect cells in different ways. To develop personalized treatment/prevention strategies and reduce HEV impact on public health, it is necessary to decipher the entry mechanism for both virion types using robust cell culture and animal models. The contemporary knowledge of the cell entry mechanism for these two HEV virions as possible therapeutic target candidates is summarized in this narrative review.