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Vγ9Vδ2 T cells are potent but elusive cytotoxic effectors. Butyrophilin subfamily 2 member A1 (BTN2A1) is a surface protein that has recently been shown to bind the Vγ9 chain of the γδ T-cell receptor, but its precise role in modulating Vγ9Vδ2 T-cell functions remains unknown. Here, we show that 107G3B5, a monoclonal BTN2A1 agonist antibody, was able to significantly enhance Vγ9Vδ2 T-cell functions against hematologic or solid cell lines and against primary cells from patients with adult acute lymphoblastic leukemia. New computer vision strategies applied to holotomographic microscopy videos showed that 107G3B5 enhanced the interaction between Vγ9Vδ2 T cells and target cells in a quantitative and qualitative manner. In addition, we found that Vγ9Vδ2 T cells activated by 107G3B5 induced caspase 3/7 activation in tumor cells, thereby triggering tumor cell death by pyroptosis. Together, these data demonstrate that targeting BTN2A1 with 107G3B5 enhances the Vγ9Vδ2 T-cell antitumor response by triggering pyroptosis-induced immunogenic cell death. These new pyroptosis-based therapies have great potential to stimulate the immune system to fight cancer, especially "cold" tumors. See related Spotlight by Kabelit, p. 1662., (©2024 American Association for Cancer Research.)

One commercial production run of Provolone del Monaco - a long-ripened pasta filata cheese - was followed up to the end of ripening for a total of 20 samples. 371 LAB isolates were subject to genetic characterization followed by 16S rRNA gene sequencing. The dominant species were Lacticaseibacillus casei/paracasei (19.4 %), Streptococcus macedonicus (19.1 %) and Enterococcus faecalis (13.2 %). Strains were screened for features of technological interest or safety relevance. Tyramine-producing cultures were quite common, above all within enterococci. By MALDI TOF Mass Spectrometry, one Lactococcus lactis and one Enterococcus faecium strain proved to be bacteriocin producers. Four further cheese wheels from the same production run at 623 days of ripening were evaluated for volatile organic compounds, biogenic amines, and bacterial community by metagenomic sequencing. Three individual wheel samples shared a rather similar microbiome with Lactobacillus delbrueckii and Streptococcus thermophilus as the most represented species, while the fourth wheel appeared wholly different being dominated by Lentilactobacillus buchneri and St. infantarius. Additionally, this sample had the greatest content of biogenic amines and a different VOCs composition. Given the variance seen among cheese wheels processed and ripened under the same conditions, the search for adjunct cultures in the production of this cheese seems to be of utmost importance., 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 © 2024 Elsevier Ltd. All rights reserved.)

This study explores Universal Design for Learning (UDL) and Game-Based Learning (GBL) to promote the inclusion of secondary school students with intellectual disabilities. Through qualitative methods, including participant observation and post-workshop interviews, the impact of these methodologies was assessed on 31 students, including 5 with intellectual disabilities. The findings highlight how these methodologies enhance accessibility, motivation, and learning outcomes, developing narrative skills and fostering creativity. Challenges in curriculum integration and active student participation are also addressed. This study underscores the transformative potential of UDL and GBL in inclusive education, significantly improving the educational experience for students of varying abilities., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

Background Multiple studies have emphasized the increased use of physician rating websites by patients when searching for surgeons to perform elective procedures. This study aimed to analyze the comments associated with online five-star patient reviews for orthopedic foot and ankle surgeons. Methods A retrospective analysis of five-star online reviews and corresponding comments using Vitals.com in 2024 was completed. Surgeons were included if they could be found on Vitals.com, were within a 10-mile radius of one of the top 10 largest cities in the United States, and if they had at least one review with one corresponding comment. Comments were further stratified into the following categories: good outcomes, well-controlled pain, correct diagnosis, clear plan, bedside manner/patient experience, staff compliment, wait time, nice facility, and offering a nonsurgical option. Results In a sampling of 2,425 orthopedic foot and ankle surgeons, 148 physicians (6.1%) had at least one review with one comment. Ultimately, 1,833 five-star reviews comprising 3,215 comments were included in the final analysis. Comments stratified by category revealed the most common comments being related to good outcomes (n = 940; 29.2%) and bedside manner/patient experience (n = 921; 28.6%). From the comments related to bedside manner/patient experience (p < 0.001) and presence of a clear plan (p < 0.001), a significantly higher proportion of comments in the nonoperative group was found relative to the operative group. Conversely, from the comments related to well-controlled pain (p < 0.001), a significantly higher proportion of comments in the operative group was found relative to the nonoperative group. Conclusions The most common reasons behind five-star patient comments for orthopedic foot and ankle surgeons were related to good outcomes and bedside manner/patient experience. Comments from surgical patients were most likely to include mention of well-controlled pain, whereas comments from patients who underwent nonoperative care were more likely to center on bedside manner/patient experience and presence of a clear plan., Competing Interests: Human subjects: All authors have confirmed that this study did not involve human participants or tissue. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: Hanselman declare(s) personal fees from Arthrex. Consultant/Advisory Role. Hanselman declare(s) personal fees from Trilliant Surgical. Consultant/Advisory Role. Hanselman declare(s) personal fees from Medline Industries. Consultant/Advisory Role. Adams declare(s) personal fees from Restor3d. Consultant/Advisory Role. Adams declare(s) personal fees from in2bones. Consultant/Advisory Role. Adams declare(s) personal fees from Enovis. Consultant/Advisory Role. Adams declare(s) personal fees from Conventus. Consultant/Advisory Role. Hanselman declare(s) personal fees from Bioventus. Consultant/Advisory Role. Hanselman declare(s) personal fees from Tricoast Surgical Solutions. Consultant/Advisory Role. Hanselman, Adams declare(s) personal fees from Stryker. Consultant/Advisory Role. Hanselman declare(s) personal fees from MedShape. Consultant/Advisory Role. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Anastasio et al.)

Background: Historically, ankle fractures have been treated with open reduction and internal fixation (ORIF) procedures, which are considered safe and effective. Patient characteristics may contribute to postoperative difficulties thereby increasing risk of hospital readmission. The objective of this study was to determine the frequency of and reasons for 30-day readmission and postoperative complications following ORIF for ankle fractures., Methods: A retrospective review of the National Surgical Quality Improvement Program (NSQIP) database from 2015 to 2021 identified patients undergoing ORIF for ankle fractures. Patient demographics, complication incidence, and reasons for unplanned hospital readmission were collected. Multivariable analyses identified patient risk factors for any adverse event (AAE) and readmission within 30-days of surgery., Results: The 29,905 patients queried who underwent ORIF procedures for ankle fractures between 2015 and 2021 were 49.6 ± 18.40 years of age, 30.9 ± 7.10 kg/m 2 , and 40.81% male. Of this cohort, 981 (3.30%) experienced 30-day postoperative adverse events, with surgical site infections (SSI; 1.25%) the most common. Unplanned readmission was observed in 2.08% of patients after a mean of 14.64 days. Surgical site related readmissions were 20.55% (n = 128) of reported readmissions with the most common being superficial incisional SSI. Notable risk factors for adverse events included ASA class (OR = 1.579, P < .001), COPD (OR = 1.522, P < .001), bleeding disorders (OR = 1.489, P = .001), diabetes (OR = 1.254, P = .008), and current smoking status (OR = 1.295, P = .002). Risk factors for readmission were ASA class (OR = 1.762, P < .001), COPD (OR = 1.599, P < .001), bleeding disorder (OR = 1.711, P < .001), diabetes (OR = 1.494, P < .001), end stage renal disease (OR = 3.304, P < .001), steroid use (OR = 2.144, P < .001), and current smoking status (OR = 1.667, P < .001)., Conclusion: Despite a low adverse event rate, 2% of patients required unplanned readmission after ORIF for ankle fractures. Surgical site complications account for almost half of reported readmissions. ASA class and various medical comorbidities were found to significantly increase the risk of postoperative adverse events and hospital readmission., (© 2024. The Author(s).)

Background: This retrospective cohort study compared short-term complication rates following total ankle arthroplasty (TAA), alone or with concomitant procedures. Secondary independent risk factors were also examined as they related to postoperative outcomes., Methods: The American College of Surgeons (ACS) National Surgical Quality Improvement Program (NSQIP) database was queried using Current Procedural Terminology ( CPT ) codes to identify patients who underwent TAA (27702) between 2010 to 2021. Patients were divided into cohorts based on the presence or absence of ancillary procedures. Propensity score matching was employed to account for demographic differences, and statistical analyses were performed to compare short-term complication rates between matched cohorts., Results: A total of 2225 patients were identified, with 1432 (64.4%) receiving TAA alone and 793 (35.6%) with ancillary procedure(s). After matching, 793 patients were included in each cohort. The ancillary cohort had longer operative times ( P  < .001) and length of hospital stay (LOS) ( P  < 0.001). Rates for extended LOS were significantly higher in the ancillary cohort than in the simple cohort ( P  = .01). No other complications varied significantly between cohorts, including the incidence of any adverse event (AAE). American Society of Anesthesiologists classification of 4 was found to be an independent risk factor for development of AAE (odds ratio [OR] = 1.091, P  = .04). Matched subgroup analysis excluding tendon lengthening as a concomitant procedure found that the ancillary cohort still had longer operative time ( P  < .001) and LOS ( P  < .05) than patients undergoing simple TAA., Conclusion: Without significant difference in rates of AAE other than extended LOS, the relative safety of ancillary TAA appears similar to that of TAA alone. Such knowledge can help inform surgical decision-making and assuage safety concerns for patients requiring additional corrective procedures at the time of TAA., Level of Evidence: Level III, retrospective comparative study., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Disclosure forms for all authors are available online., (© The Author(s) 2024.)

The ubiquity of perfluoroalkyl substances has raised concerns about the unintended consequences of PFAS exposure on human health. In the present study, an eco-friendly ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of 17 PFAS in human serum and semen samples. QuEChERS salts MgSO 4 :NaCl 4:1 (w/w) were used for the extraction. The separation of analytes was performed on an ACQUITY BEH C 18 column (100 × 2.1 mm, 1.7 μm), using water:methanol 95:5 and methanol as mobile phases A and B, respectively, both containing 2 mM ammonium acetate. Multiple reaction monitoring (MRM) in negative ion mode was used, selecting two transitions for each analyte, except for perfluorobutanoic acid (PFBA) and perfluoropentanoic acid (PFPeA). The analytical method was validated according to the Organization of Scientific Area Committees (OSAC) for Forensic Sciences guidelines and AGREE approach software was used to evaluate the greenness of the method. The developed procedure was applied to the analysis of 10 paired human serum and semen samples, proving the suitability in high throughput laboratories due to the easy preparation and the reduced volume of toxic solvents. Moreover, it allows to perform further investigation on the correlation between serum and semen PFAS concentration, focusing on male reproductive system correlated pathologies, such as male infertility., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Intrauterine fetal death and perinatal death represent one of the most relevant medical scientific problems since, in many cases, even after extensive investigation, the causes remain unknown. The considerable increase in medical legal litigation in the obstetrical field that has witnessed in recent years, especially in cases of stillborn births, has simultaneously involved the figure of the forensic pathologist in scientific research aimed at clarifying the pathophysiological processes underlying stillbirth., Methods: our study aims to analyze cases of sudden intrauterine unexplained death syndrome (SIUD) to evaluate the role of oxidative stress in the complex pathogenetic process of stillbirth. In particular, the immunohistochemical expression of specific oxidative stress markers (NOX2, NT, iNOS, 8-HODG, IL-6) was evaluated in tissue samples of placentas of SIUDs belonging to the extensive case series (20 cases), collected from autopsy cases of the University of Ferrara and Politecnica delle Marche between 2017 and 2023., Results: The study demonstrated the involvement of oxidative stress in intrauterine fetal deaths in the placenta of the cases examined. In SIUD, the most expressed oxidative stress markers were NOX2 and 8-HODG., Conclusions: The study contributes to investigating the role of oxidative stress in modulating different pathways in unexplained intrauterine fetal death (SIUD) tissues.

Recently, hexahydrocannabinol (HHC) was posed under strict control in Europe due to the increasing HHC-containing material seizures. The lack of analytical methods in clinical laboratories to detect HHC and its metabolites in biological matrices may result in related intoxication underreporting. We developed and validated a comprehensive GC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC, 9αOH-HHC, 9βOH-HHC, 8(R)OH-9(R)-HHC, 8(S)OH-9(S)HHC, 11OH-9(R)HHC, 11OH-9(S)HHC, 11nor-carboxy-9(R)-HHC, and 11nor-carboxy-9(S)-HHC in whole blood, urine, and oral fluid. A novel QuEChERS extraction protocol was optimized selecting the best extraction conditions suitable for all the three matrices. Urine and blood were incubated with β-glucuronidase at 60 °C for 2 h. QuEChERS extraction was developed assessing different ratios of Na 2 SO 4 :NaCl (4:1, 2:1, 1:1, w / w ) to be added to 200 µL of any matrix added with acetonitrile. The chromatographic separation was achieved on a 7890B GC with an HP-5ms column, (30 m, 0.25 mm × 0.25 µm) in 12.50 min. The analytes were detected with a triple-quadrupole mass spectrometer in the MRM mode. The method was fully validated following OSAC guidelines. The method showed good validation parameters in all the matrices. The method was applied to ten real samples of whole blood (n = 4), urine (n = 3), and oral fluid (n = 3). 9(R)-HHC was the prevalent epimer in all the samples (9(R)/9(S) = 2.26). As reported, hydroxylated metabolites are proposed as urinary biomarkers, while carboxylated metabolites are hematic biomarkers. Furthermore, 8(R)OH-9(R)HHC was confirmed as the most abundant metabolite in all urine samples.

Ostarine (enobasarm) is a selective androgen receptor modulator with great therapeutic potential. However, it is also used by athletes to promote muscle growth and enhance performances without the typical adverse effects of anabolic steroids. Ostarine popularity increased in recent years, and it is currently the most abused "other anabolic agent" (subclass S1.2. of the "anabolic agents" class S1) from the World Anti-Doping Agency's (WADA) prohibited list. Several cases of liver toxicity were recently reported in regular users. Detecting ostarine or markers of intake in biological matrices is essential to document ostarine use in doping. Therefore, we sought to investigate ostarine metabolism to identify optimal markers of consumption. The substance was incubated with human hepatocytes, and urine samples from six ostarine-positive cases were screened. Analyses were performed via liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) and software-assisted data mining, with in silico metabolite predictions. Ten metabolites were identified with hydroxylation, ether cleavage, dealkylation, O -glucuronidation, and/or sulfation. The production of cyanophenol-sulfate might participate in the mechanism of ostarine liver toxicity. We suggest ostarine-glucuronide (C 25 H 22 O 9 N 3 F 3 , diagnostic fragments at m / z 118, 185, and 269) and hydroxybenzonitrile-ostarine-glucuronide (C 25 H 22 O 10 N 3 F 3 , diagnostic fragments at m / z 134, 185, and 269) in non-hydrolyzed urine and ostarine and hydroxybenzonitrile-ostarine (C 19 H 14 O 4 N 3 F 3 , diagnostic fragments at m / z 134, 185, and 269) in hydrolyzed urine as markers to document ostarine intake in doping.

Recently we published in this journal an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 3,4-methylenedioxymethamphetamine (MDMA) and its major phase-1 metabolites, 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA) in human plasma, sweat, oral fluid and urine. Since we did not achieve simultaneous enantioseparation of all 4 compounds with a single chiral column, two amylose-based chiral columns were used alternatively. Further optimization of the mobile phase in the present study enabled baseline separation of all four pairs of enantiomers on a single Lux AMP column. In addition, by optimization of the column dimension and applied flow-rate it became possible to complete the separation within 6 minutes. These new methods were applied to the analysis of human plasma, oral fluid and urine. While results on the concentration of MDMA and its metabolites in various biological fluids were reported in our recent publication, in the present study an attempt was made to hydrolyze glucuronides in urine samples by using alternatively, hydrochloric acid or glucuronidase and to evaluate the effect of hydrolysis on the concentration and enantiomeric distribution of hydroxy metabolites of MDMA such as HMA and HMMA., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Achilles tendon (AT) pathologies are common musculoskeletal conditions that can significantly impair function. Despite various traditional treatments, recovery is often slow and may not restore full functionality. The use of extracellular vesicles (EVs) has emerged as a promising therapeutic option due to their role in cell signaling and tissue regeneration. This systematic review aims to consolidate current in vivo animal study findings on the therapeutic effects of EVs on AT injuries. An extensive literature search was conducted using the PubMed, Scopus, and Embase databases for in vivo animal studies examining the effects of EVs on AT pathologies. The extracted variables included but were not limited to the study design, type of EVs used, administration methods, efficacy of treatment, and proposed therapeutic mechanisms. After screening, 18 studies comprising 800 subjects were included. All but one study reported that EVs augmented wound healing processes in the AT. The most proposed mechanisms through which this occurred were gene regulation of the extracellular matrix (ECM), the enhancement of macrophage polarization, and the delivery of therapeutic microRNAs to the injury site. Further research is warranted to not only explore the therapeutic potential of EVs in the context of AT pathologies, but also to establish protocols for their clinical application.

Background: Approximately 30 million people worldwide consume new psychoactive substances (NPS), creating a serious public health issue due to their toxicity and potency. Drug-induced liver injury is the leading cause of liver disease, responsible for 4% of global deaths each year., Content: A systematic literature search revealed 64 case reports, in vitro and in vivo studies on NPS hepatotoxicity. Maximum elevated concentrations of aspartate aminotransferase (136 to 15 632 U/L), alanine transaminase (121.5 to 9162 U/L), total bilirubin (0.7 to 702 mg/dL; 0.04 to 39.03 mmol/L), direct (0.2-15.1 mg/dL; 0.01-0.84 mmol/L) and indirect (5.3 mg/dL; 0.29 mmol/L) bilirubin, alkaline phosphatase (79-260 U/L), and gamma-glutamyltransferase (260 U/L) were observed as biochemical markers of liver damage, with acute and fulminant liver failure the major toxic effects described in the NPS case reports. In vitro laboratory studies and subsequent in vivo NPS exposure studies on rats and mice provide data on potential mechanisms of toxicity. Oxidative stress, plasma membrane stability, and cellular energy changes led to apoptosis and cell death. Experimental studies of human liver microsome incubation with synthetic NPS, with and without specific cytochrome P450 inhibitors, highlighted specific enzyme inhibitions and potential drug-drug interactions leading to hepatotoxicity., Summary: Mild to severe hepatotoxic effects following synthetic NPS exposure were described in case reports. In diagnosing the etiology of liver damage, synthetic NPS exposure should be considered as part of the differential diagnosis. Identification of NPS toxicity is important for educating patients on the dangers of NPS consumption and to suggest promising treatments for observed hepatotoxicity., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Ankle arthritis differs from arthritis of the hip and knee in that 80% is posttraumatic and thus often occurs in a younger patient population. The literature supporting total ankle arthroplasty (TAA) in younger patients has increased over recent years and has bolstered the argument that in the short term, TAA in younger patients has successful outcomes that are comparable to older, lower-demand patients.The purpose of our study was to evaluate patient-reported outcomes (PROs) and implant survivorship at midterm after primary TAA in patients ≤ 50 years of age at the time of surgery., Methods: A retrospective chart review was conducted of patients ≤ 50 years of age who underwent primary TAA at a single institution from 2000 to 2017. Patient demographics, outcome measures, and complications were recorded. All patients had a minimum clinical follow-up of 5 years. PRO measures were evaluated at preoperative, 1-year postoperative, and final follow-up visits. Paired t tests were performed to compare individual patient changes in PROs from preoperative. Implant survivorship was evaluated based on need for revision of either the tibial or talar component. The need for additional surgery related to the TAA was also evaluated., Results: A total of 58 patients were included. The average age at the time of the index surgery was 43.3 years (range 22-50 years). All patients had a minimum follow-up of 5 years with a mean follow-up of 8.8 years. A total of 11 patients required additional surgery related to their TAA. Six patients (10.3%) required bone grafting of peri-implant cysts, 3 patients (5.2 %) required gutter debridement, and 1 patient underwent complete revision of metal components. Mean visual analog scale, 36-item Short Form Health Survey, Short Musculoskeletal Function Assessment, and American Orthopaedic Foot & Ankle Society hindfoot scores significantly improved from preoperative to 1-year postoperative and final postoperative follow-up., Conclusion: The patients aged ≤50 years treated with a TAA whom we have been able to observe for a minimum of 5 years showed generally maintained improvement in functional scores and thus far have had a relatively low rate of secondary surgeries.Level of Evidence:Level III, retrospective cohort study., Competing Interests: Ethical ApprovalEthical approval for this study was obtained from Institutional Review Board ID # Pro00103431. Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: James K. DeOrio, MD, reports support for the present manuscript (eg, funding, provision of study materials, medical writing, article processing charges, etc); royalties or license; consulting fees; and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Exactech. Mark E. Easley, MD, reports grants or contracts from any entity; royalties or licenses; consulting fees; and patents planned, issued, or pending from Exactech. James A. Nunley, MD, reports royalties or licenses; consulting fees; and patents planned, issued, or pending from Exactech. Disclosure forms for all authors are available online.

A sensitive LC-MS/MS method for the simultaneous quantification of the (9 R)- and (9 S)- hexahydrocannabinols (HHCs), and their metabolites, in human urine, oral fluid (OF) and blood samples were developed, validated and used to the biological samples of volunteers. The analytes were extracted from 100 μL human samples. An isocratic elution mode with methanol was used for chromatographic separation of (9 R)- and (9 S)-HHC on an immobilized amylose tris(3-chloro-5-methylphenylcarbamate)-based chiral column Lux i-Amylose-3. The flow-rate of the mobile phase was 0.5 mL/min. An isocratic elution mode of methanol and water (80/20, v/v) was used for chromatographic separation of metabolites of (9 R)- and (9 S)-HHC on a Lux AMP chiral column (with a proprietary chiral selector) at a flow rate of 0.5 mL/min. MS/MS analysis was performed in positive ionization mode for HHC epimers, while in negative ionization mode was used for metabolites of HHCs. The calibration curves for HHCs and their metabolites in human samples ranged from 0.25- 240 ng mL -1 and 1 - 100 ng mL -1 , respectively, with determination coefficients (r 2 ) of ≥ 0.99. All analytes were stable at room temperature, 4 °C, in the autosampler (+10 °C) and -20 °C for 24 h, after three freeze/thaw cycles, and when stored at -20 °C up to one week after quality control (QC) sample preparation (concentration differences less than 20% with respect to time zero response), in blood, urine and OF., 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 © 2024 Elsevier B.V. All rights reserved.)

The valorization of milk whey appears to be a promising strategy for managing by-products from dairy food industries, which incur demanding economic costs for treatment and/or disposal. Thanks to its numerous bioactive components, whey is expected to be increasingly incorporated into foods in the future. We investigated the safety of ovine milk whey through in vitro experiments on human primary gingival fibroblast (HGF-1) proliferation and wound healing. Fibroblasts play a crucial role in the repair processes from the late inflammatory phase until the final stages. Cells treated with varying concentrations of ovine whey (0.01%, 0.1%, 1%, and 10%) were able to close wounds more rapidly than vehicle-treated cells. Time- and dose-dependent responses were observed in cell populations exposed to ovine whey. Specifically, wounds treated with 0.1% and 10% milk whey showed better migratory capabilities compared to those treated with 0.01% and 1% milk whey after 24 and 48 h. In addition, ovine milk whey stimulates extracellular matrix deposition, as evidenced by the increasing levels of CD44 antigen density evaluated through FACS analysis, as well as COL1A1 expression measured both via RT-qPCR and immunofluorescence. This phenomenon was particularly evident at concentrations of 0.01% and 10%. Ensuring quality and safety has become a major concern for health authorities in the food industry. Our findings suggest that ovine milk whey is safe and possesses regenerative properties. It facilitates tissue re-establishment following exposure to environmental stress, particularly accelerating gingival wound closure.

In 2023, hexahydrocannabinol (HHC) attracted the attention of international agencies due to its rapid spread in the illegal market. Although it was discovered in 1940, less is known about the pharmacology of its two naturally occurring epimers, 9(R)-HHC and 9(S)-HHC. Thus, we aimed to investigate the disposition of hexahydrocannabinol epimers and their metabolites in whole blood, urine and oral fluid following a single controlled administration of a 50:50 mixture of 9(R)-HHC and 9(S)-HHC smoked with tobacco. To this end, six non-user volunteers smoked 25 mg of the HHC mixture in 500 mg of tobacco. Blood and oral fluid were sampled at different time points up to 3 h after the intake, while urine was collected between 0 and 2 h and between 2 and 6 h. The samples were analyzed with a validated HPLC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC and eight metabolites. 9(R)-HHC showed the highest C max and AUC 0-3h in all the investigated matrices, with an average concentration 3-fold higher than that of 9(S)-HHC. In oral fluid, no metabolites were detected, while they were observed as glucuronides in urine and blood, but with different profiles. Indeed, 11nor-9(R)-HHC was the most abundant metabolite in blood, while 8(R)OH-9(R) HHC was the most prevalent in urine. Interestingly, 11nor 9(S) COOH HHC was detected only in blood, whereas 8(S)OH-9(S) HHC was detected only in urine.

Objectives: N-piperidinyl etonitazene (etonitazepipne) is a newly synthesized opioid related to the 2-benzylbenzimidazole analog class. Etonitazepipne has been formally notified and placed under intensive monitoring in Europe in January 2022. Nitazenes have high affinity at µ-opioid receptor (MOR). Etonitazepipne, specifically shows a EC 50 of 2.49 nM, suggesting about 50 times higher potency combined with higher efficacy compared to morphine. Antinociceptive potency l ('hot plate test' with rats) was 192-fold greater than that of morphine., Methods: Here we report on a post-mortem case involving etonitazepipne and its quantification using a standard addition method (SAM) through liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, characterization and identification of phase I human metabolites using in vitro assay based on pooled human liver microsomes (pHLM) was performed along with the analysis of authentic urine samples by means of high-performance liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS)., Results: The concentration of etonitazepipne in post-mortem blood and urine was 8.3 and 11 ng/mL, respectively. SAM was validated by assessing the following parameters: intraday and interday repeatability, matrix effect and recovery rate in post-mortem blood. A total of 20 and 14 metabolites were identified after pHLM incubation and urine analysis, respectively. Most pronounced in vitro and in vivo transformations were O-deethylation, hydroxylation, ketone reduction, and combinations thereof., Conclusions: Considering small traces of the parent drug often found in real cases, the identification of metabolic biomarkers is crucial to identify exposure to this drug. O-deethylated, oxidated metabolites, and combination thereof are proposed as urinary biomarkers along with the parent compound., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

IOX2 is a potent inhibitor of prolyl hydroxylase 2, a key enzyme in the regulation of hypoxia-inducible factor (HIF) and oxygen homeostasis. As such, it can be used to enhance athletic performance and is currently banned by the World Anti-Doping Agency (WADA). Detection of metabolites is critical to demonstrate drug use in doping. However, there is currently little data on IOX2 human metabolism. Our aim was to identify relevant biomarkers of IOX2 use in humans. For this purpose, IOX2 was incubated with 10-donor-pooled human hepatocytes for 3 h, incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and LC-HRMS/MS data were screened with Compound Discoverer (Thermo Scientific) for a comprehensive identification of IOX2 metabolites. Additionally, IOX2 human metabolites were predicted with GLORYx open-access software (University of Hamburg, Germany) to assist in the LC-HRMS/MS analysis and data mining. Thirteen metabolites were identified, oxidation at the quinolinyl group, O-glucuronidation, and combinations being predominant biotransformations. The results were consistent with previous animal studies and a single case of oral microdose administration. We suggest hydroxyquinolinyl-IOX2 as major biomarker of IOX2 use in biological samples, glucuronide hydrolysis being critical to increase IOX2 and hydroxyquinolinyl-IOX2 detectability in urine., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Recently we proposed an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the separation and quantitative determination of dextro- (DXM) and levo-methorphan (LVM) and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. This method was based on the polysaccharide-based chiral column Lux AMP, a specialty column characterized with high stability in mobile phases of pH 11.0 and above. The use of a single-source column is a limitation for any analytical method. Therefore, the major goal of the present study was to develop an enantioselective method for the differentiation of dextro- and levo-methorphan, as well as their metabolites dextrorphan and levorphanol, using Lux Cellulose-3 as alternative chiral column with methanol containing 0.1 % diethylamine mobile phase. A newly developed method uses a chiral selector part of HPLC columns available from multiple manufacturers and a fairly common mobile phase. The method was validated and applied to post-mortem blood samples. Out of 50 analyzed new samples, dextromethorphan (DXM) was detected in 17 samples. Of these 17 cases DXM was accompanied with LVM in 7 samples. The proposed analytical method is relatively simple, accurate and fast and can be adopted for routine use in forensic and clinical toxicology laboratories., 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 © 2023. Published by Elsevier B.V.)

This study assesses the potential impacts on human health of volcanic ash emitted during the 2021 Tajogaite eruption (La Palma Island, Spain). Ash samples were physically and chemically characterized and leaching tests (with deionized water and acidic solution) were performed according to the IVHHN protocols to elucidate i) the leachable elements that may affect water quality and represent a potential threat for livestock and humans through drinking water supply; and ii) the bioaccessible fraction of toxicants able to be solubilized from ash surfaces if ashes are incidentally ingested by children. The most abundant readily water-soluble compounds were SO 4 , F, Cl, Na, Ca, Ba, Mg, and Zn. Fluoride and chloride (up to 1085 and 1347 mg/kg) showed higher values in distal ash samples than closer ones. The potential F availability assessed from water leachates may suggest important environmental and health implications. In addition, long-term health hazard due to a long-term weathering of tephra deposits should be possible as confirmed by the greater amount of F extracted by acidic solution. Concentration of other trace elements (e.g., As, V, Mn, Mo, Cr, Fe, Se, Ti, Pb) were low compared to global medians and within the range globally assessed. Indicative calculation of hazard for water supply showed that F concentration may exceed both the recommended value (1 mg/L) for irrigation purpose and the health-based drinking water limits of 1.5 mg/L (for humans) and 2 mg/L (for livestock). If the predicted concentrations in water were compared with the toxicologically dose, F showed a potential health-risk for children through drinking water. The indicative health-risk characterization via accidental ash ingestion showed that the direct exposure does not represent a primary source of F daily intake for children. This important outcome confirmed F as element with the greatest health threat during Tajogaite 2021 eruption., 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 © 2023 Elsevier B.V. All rights reserved.)

Seasonal weight loss (SWL), is a major limitation to animal production. In the Canary Islands, there are two dairy goat breeds with different levels of tolerance to SWL: Majorera (tolerant) and Palmera (susceptible). Our team has studied the response of these breeds to SWL using different Omics tools. The objective of this study was to integrate such results in a data driven approach and using dedicated tools, namely the DIABLO method. The outputs of our analysis mainly separate unrestricted from restricted goats. Metabolites behave as "hub" molecules, grouping interactions with several genes and proteins. Unrestricted goats upregulated protein synthesis, along with arginine catabolism and adipogenesis pathways, which are related with higher anabolic rates and a larger proportion of secretory tissue, in agreement with their higher milk production. Contrarily, restricted goats seemingly increased the synthesis of acetyl-CoA through serine and acetate conversion into pyruvate. This may have occurred to increase fatty acid synthesis and/or to use them as an energy source in detriment to glucose, which was more available in the diet of unrestricted goats. Lastly, restricted Palmera upregulated the expression of PEBP4 and GPD1 genes compared to all other groups, which might support their use as putative biomarkers for SWL susceptibility. SIGNIFICANCE: Seasonal weight loss (SWL) is a major issue influencing animal production in the tropics and Mediterranean. By studying its impact on the mammary gland of tolerant and susceptible dairy goat breeds, using Omics, we aim at surveying the tissue for possible biomarkers that reflect these traits. In this study, data integration of three Omics (transcriptomics, proteomics and metabolomics) was performed using bioinformatic tools, to relate putative biomarkers and evaluate all three levels of information; in a novel approach. This information can enhance selection programs, lowering the impact of SWL on food production systems., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study aimed to characterize the colostrum quality in three different local goat breeds of Northern Italy (i.e., Frisa Valtellinese, Orobica, and Lariana) and a cosmopolitan one (i.e., Camosciata delle Alpi) ( n = 30 per breed), reared under traditional semi-extensive and intensive systems, respectively. Lariana showed the highest percentage of fat (10.18 ± 3.14%) and total solids (30.73 ± 4.89%) but the lowest percentage of lactose (1.87 ± 0.82%; p < 0.05); Orobica had the lowest percentage of fat (7.13 ± 2.48%), total solids (24.11 ± 5.48%), and protein (10.77 ± 4.53%) but the highest percentage of lactose (3.16 ± 0.73%; p < 0.05). This suggests that breeds which have a more pronounced meat aptitude (i.e., Frisa and Lariana) have a higher concentration of components than breeds with more dairy aptitude (i.e., Orobica and Camosciata). Uni- and multivariate analyses showed that IgG is the parameter that best differentiates local breeds from cosmopolitan ones ( p < 0.01). Colostrum from Frisa goats showed the highest IgG concentration (100.90 ± 8.11 mg/mL), while the lowest concentration was in the Camosciata breed (74.75 ± 20.16 mg/mL). Finally, the highest lactoferrin concentration was in Frisa (1781.3 ± 892.6 µg/mL) and the lowest in Camosciata and Lariana (763.1 ± 357.9 and 1148.0 ± 858.6 µg/mL, respectively; p < 0.05). Differences between Camosciata and local breeds could be due to the different farming systems, in addition to the genetic characteristics. The higher quality of colostrum produced by some local goats could be an adaptive characteristic that helps the growth and survival of the kids.

To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen-presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTE) system, a recombinant optogenetics-based molecular tool targeting the T cell receptor (TCR). The LiTE system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation. We established that CD4 + T cells respond to intermittent TCR stimulation more efficiently than their CD8 + T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTE system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTE system provides opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with high spatiotemporal control.

We present an apparatus that applies Ramsey's method of separated oscillatory fields to proton spins in water molecules. The setup consists of a water circuit, a spin polarizer, a magnetically shielded interaction region with various radio frequency elements, and a nuclear magnetic resonance system to measure the spin polarization. We show that this apparatus can be used for Rabi resonance measurements and to investigate magnetic and pseudomagnetic field effects in Ramsey-type precision measurements with a sensitivity below 100 pT., 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 © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the present study an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the first time for quantitative determination of the recreational drug of abuse methylone and its major metabolites in oral fluid. The simultaneous chemo- and enantioseparation of methylone and its major metabolites was performed on a polysaccharide-based chiral column based on amylose tris(5-chloro-3-methylphenylcarbamate) as chiral selector (Lux i-Amylose-3) with methanol containing 0.4 % (v/v) aqueous ammonium hydroxide as mobile phase. The time required for enantioselective analysis of methylone and its 2 major metabolites was 15 min. This method was fully validated following the Organization of Scientific Area Committees (OSAC) for Forensic Science guidelines. This method was applied for the enantioselective determination of methylone and its metabolites in oral fluid and enantioselectivity in metabolism and pharmacokinetic of the parent compound and metabolites was observed. While the first enantiomer of methylone was found at higher concentration, both metabolites shown greater concentration for the second enantiomer. The results revealed that MET undergoes an enantioselective biotransformation to its metabolites HMMC and MDC, with S-(-)-MET more rapidly metabolized and eliminated from the body., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This study involves an investigation of the effects of various cooking temperatures, freeze-thaw processes, and food preservatives on the quality and shelf-life of sous vide Mediterranean mussels. Cooking temperatures of 80 °C or above significantly improved the microbiological quality, with bacterial counts remaining within the acceptability range for human consumption even after 21 days of refrigerated storage. Fast freezing followed by slow thawing preserved the highest moisture content, potentially improving texture. Sensory analysis revealed that refrigerated sous vide mussels maintained a comparable taste to freshly cooked samples. Frozen samples reheated via microwaving exhibited more intense flavour than pan-reheated or fresh mussels. Food additives, including citric acid, potassium benzoate, and potassium sorbate, alone or in combination with grape seed oil, significantly reduced total volatile basic nitrogen and thiobarbituric acid-reactive substances during 28 days of storage, indicating decreased spoilage and lipid oxidation. Mussels with a combination of these additives registered a nitrogen content as low as 22 mg of N/100g after 28 days, well below the limit of acceptability (<35 mg of N/100g). Food additives also inhibited bacterial growth, with mesophilic bacteria count below 3.35 Log CFU/g after 28 days, compared with 5.37 Log CFU/g in control samples. This study provides valuable insights for developing optimal cooking and preservation methods for sous vide cooked seafood, underscoring the need for further research on optimal cooking and freeze-thaw protocols for various seafood types.

The cooling applied during the firming and brining processes represents an important production step in mozzarella cheese-making. The temperature fluctuations of the cooling water can negatively affect the hygiene, composition, and quality of mozzarella. Some sustainable cooling systems can minimize this problem by using hot process fluids as heat sources to generate refrigerated energy. This study aimed to evaluate the effects of a new cooling system equipped with a water-ammonia absorption chiller (MA) on the characteristics of buffalo mozzarella through a comparative study with products cooled with a traditional ice water chiller (MT). The buffalo mozzarella cheese manufacture was monitored, and the samples were analyzed for chemical, nutritional, microbiological, and sensory characteristics. The MT samples showed an overall weight loss of 7.4% compared to an average of 2.8% for the MA samples. The MT samples were characterized by greater sapidity than the MA ones, which instead showed a higher moisture content that increased juiciness. The microbiological analysis showed a lower concentration of mesophilic bacterial load in the MA samples than in the MT ones [difference of 1 Log (CFU/g)], which is probably due to the low and constant temperatures that reduced the permanence time of the mozzarella in the vats (firming and brining). This study represents a preliminary positive evaluation of the use of this sustainable cooling system for mozzarella cheese, which is useful for dairy plants with an annual cheese production volume sufficient to justify the operating cost of the plant and the annual energy cost., Competing Interests: Conflict of interest: the authors declare no potential conflict of interest., (©Copyright: the Author(s).)

In the present work an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the separation and quantitative determination of dextro - and levo -methorphan and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. The separation of enantiomers of methorphan and metabolites was performed on the polysaccharide-based chiral column Lux AMP in combination with acetonitrile and 5 mM aqueous ammonium bicarbonate pH 11 in the ratio 50:50 (%, v/v) as mobile phase with the flow rate 1 mL/min. The mass spectrometer was operated in scheduled multiple reaction monitoring (MRM) mode, with four transitions for each dextromethorpan, levomethorphan, dextrorphan and dextromethorphan-d3 and two transitions for each levorphanol, levorphanol-d3 and dextrorphan-d3. Application of this method to human post-mortem blood samples confirmed cases of severe overdosing with dextromethorphan, levomethorphan, and less commonly with both., 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 © 2023 Elsevier B.V. All rights reserved.)

Benign prostate hyperplasia (BPH) is a frequent condition in aging men, which affects life quality, causing principally lower urinary tract symptoms. Epidemiologic studies suggest that BPH may raise the risk of developing prostate cancer (PCa), most likely promoting a chronic inflammatory environment. Studies aiming at elucidating the link and risk factors that connect BPH and PCa are urgently needed to develop prevention strategies. The BPH microenvironment, similar to the PCa one, increases immune infiltration of the prostate, but, in contrast to PCa, immunosuppression may not be established yet. In this study, we found that prostate-infiltrating lymphocytes (PILs) expanded from hyperplastic prostate tissue recognized tumor-associated antigens (TAA) and autologous tissue, regardless of the presence of tumor cells. PILs expanded from BPH samples of patients with PCa, however, seem to respond more strongly to autologous tissue. Phenotypic characterization of the infiltrating PILs revealed a trend towards better expanding CD4 + T cells in infiltrates derived from PCa, but no significant differences were found. These findings suggest that T cell tolerance is compromised in BPH-affected prostates, likely due to qualitative or quantitative alterations of the antigenic landscape. Our data support the hypothesis that BPH increases the risk of PCa and may pave the way for new personalized preventive vaccine strategies for these patients.

Nowadays, the search for food products that promote consumers' health has gained interest, and dairy by-products, due to their biological quality, could have a prominent position among products with health benefits. However, little is known about their activity on cancer cells. This study aimed to provide evidence about the effect of ovine colostrum and milk whey on K562 cells, a model of the human chronic myeloid leukemia cell line. The exposure of K562 cells to a single administration of sheep by-products at different concentrations for three days and three treatments for three days was carried out. Using a flow cytometric approach, we found that CD235a expression remained stable in the cells exposed to ovine whey (milk and colostrum) at concentrations ranging from 1 ng/mL to 100 μg/mL, after three days from one or three administrations, respectively. A significant reduction in fluorescent cells was observed in the populations exposed to 1 mg/mL of both milk and colostrum at the same time points. In these conditions, the size and granularity of the leukemic cells also changed, with a substantial reduction in the number of actively dividing cells in the S phase of the cell cycle. This phenomenon was highlighted by the Annexin V/PI cytofluorimetric test, which is able to provide quantitative results regarding the population of cells in early or late apoptosis or necrotic cells after exposure to a single dose or three doses of colostrum or sheep whey for three days, respectively. This report showed that both colostrum and milk whey were able to modify the phenotypic profile and cell cycle of the K562 cell line, inducing apoptosis at the highest concentration.

The aim of this study was to investigate methylone and its metabolites concentration in oral fluid following controlled increasing doses, focusing on the effect of oral fluid pH. Samples were obtained from a clinical trial where twelve healthy volunteers participated after ingestion of 50, 100, 150 and 200 mg of methylone. Concentration of methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone in oral fluid were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters were estimated, and the oral fluid-to-plasma ratio (OF/P) at each time interval was calculated and correlated with the oral fluid pH using data from our previous study in plasma. Methylone was detected at all time intervals after each dose; MDC and HMMC were not detectable after the lowest dose. Oral fluid concentrations of methylone ranged between 88.3-503.8, 85.5-5002.3, 182.8-13,201.8 and 214.6-22,684.6 ng/mL following 50, 100, 150 and 200 mg doses, respectively, peaked between 1.5 and 2.0 h, and were followed by a progressive decrease. Oral fluid pH was demonstrated to be affected by methylone administration. Oral fluid is a valid alternative to plasma for methylone determination for clinical and toxicological studies, allowing for a simple, easy and non-invasive sample collection.

Background: No consensus exists regarding the superiority of operative versus nonoperative management for Achilles tendon ruptures, as multiple randomized controlled trials conducted since the advent of early mobilization protocols have found outcomes for these 2 interventions to be more similar than were previously held., Purpose: To use a large national database to (1) compare reoperation and complication rates between operative and nonoperative treatment of acute Achilles tendon ruptures and (2) evaluate trends in treatment and cost over time., Study Design: Cohort study; Level of evidence, 3., Methods: The MarketScan Commercial Claims and Encounters database was used to identify an unmatched cohort of 31,515 patients who sustained primary Achilles tendon ruptures between 2007 and 2015. Patients were stratified into operative and nonoperative treatment groups, and a propensity score-a matching algorithm-was used to establish a matched cohort of 17,996 patients (n = 8993 per treatment group). Reoperation rates, complications, and aggregate treatment costs were compared between groups with an alpha level of .05. A number needed to harm (NNH) was calculated from the absolute risk difference in complications between cohorts., Results: The operative cohort experienced a significantly larger total number of complications within 30 days of injury (1026 vs 917; P = .0088). The absolute increase in cumulative risk was 1.2% with operative treatment, which resulted in an NNH of 83. Neither 1-year (1.1% [operative] vs 1.3% [nonoperative]; P = .1201) nor 2-year reoperation rates (1.9% [operative] vs 2% [nonoperative]; P = .2810) were significantly different. Operative care was more expensive than nonoperative care at 9 months and 2 years after injury; however, there was no difference in cost between treatments at 5 years. Before matching, the rate of surgical repair for Achilles tendon rupture remained stable, from 69.7% to 71.7% between 2007 and 2015, indicating little change in practice in the United States., Conclusion: Results indicated no differences in reoperation rates between operative and nonoperative management of Achilles tendon ruptures. Operative management was associated with an increased risk of complications and higher initial costs, which dissipated over time. Between 2007 and 2015 the proportion of Achilles tendon ruptures managed operatively remained similar despite increasing evidence that nonoperative management of Achilles tendon rupture may provide equivalent outcomes., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: B.C.L. has received research support from Arthrex, DJO, and Zimmer Biomet; education payments from Arthrex and Smith & Nephew; and hospitality payments from Crossroads Extremity Systems, Stryker, and Wright Medical. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2023.)

The buffalo dairy sector is extending its boundaries to include new buffalo cheese productions beyond mozzarella, overcoming some barriers that make cheeses expensive and unsustainable. This study aimed to evaluate the effects of both the inclusion of green feed in the diet of Italian Mediterranean buffaloes and an innovative ripening system on buffalo cheese quality, providing solutions capable of guaranteeing the production of nutritionally competitive and sustainable products. For this purpose, chemical, rheological, and microbiological analyses were carried out on cheeses. Buffaloes were fed with or without the inclusion of green forage. Their milk was used to produce dry ricotta and semi-hard cheeses, ripened according to both respective traditional (MT) and innovative methods (MI); these are based on automatic adjustments of climatic recipe guided by the continuous control of pH. Green feed enhances the nutritional profile of the final products (high content of MUFAs and PUFAs). As far as the ripening method is concerned, to our knowledge, this is the first study that tests aging chambers, commonly used for meat, for the maturing of buffalo cheeses. Results pointed out the MI validity also in this field of application, as it shortens the ripening period without negatively compromising any of desirable physicochemical properties and the safety and hygiene of the final products. Conclusively, this research highlights the benefits of diets rich in green forage on productions and provides support for the ripening optimization of buffalo semi-hard cheeses.

Competing Interests: Competing interests: None declared.

"Light cannabis" is a product legally sold in Europe with Δ9-tetrahydrocannabinol (THC) concentration <0.2% and variable cannabidiol (CBD) content. In this study, we aimed to assess the time courses of THC and metabolites (11-nor-9-carboxy-THC and 11-hydroxy-THC) and CBD and metabolites (CBD-7-oic acid, 7-hydroxy-CBD, 6α-hydroxy-CBD and 6β-hydroxy-CBD) in whole blood of 10 healthy participants after smoking one or four light cannabis cigarettes (0.16% THC and 5.8% CBD). Blood samples were collected 0.5-4 h after administration. Blood analysis was performed by reversed-phase ultra-performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode after glucuronide hydrolysis and liquid-liquid extraction in basic and acidic conditions. The method was validated following the most recent guidelines in toxicology: the method was linear, accurate, precise and sensitive (lower limits of quantification ranged from 0.005 to 0.01 ng/mL); carryover, matrix effect, recovery, process efficiency and dilution integrity were also assessed. As previously reported, the main metabolites of THC were THC-COOH and then 11-OH-THC, and the main metabolites of CBD were 7-OH-CBD and then 7-COOH-CBD. The time of the first collection, which likely occurred after the maximal concentration of most of the analytes, and the short monitoring time, up to 4 h after smoking, limited the evaluation of the pharmacokinetic parameters., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As a result of the increase of game meat intended for human consumption through Europe, a plethora of food-borne diseases, including trichinellosis, may occur in consumers, posing a relevant public health threat. Thus, this study aims to a citizen science approach to monitor the occurrence of Trichinella spp. in wild boar meat intended for human consumption, evaluating the risk of infection for consumers. Following the European Regulation 2015/1375 ( laying down specific rules on official controls for Trichinella in meat ), from 2015 to 2021, hunters ( n  = 478) were involved to collect diaphragm pillar samples of wild boars from mainland southern Italy, which were tested for Trichinella spp. L 1 larvae via HCl-pepsin digestion and Multiplex PCR. Overall, 139,160 animals were collected (average of 19,880 per year), being 14 (i.e., 0.01%) tested positive to Trichinella britovi by the combined biochemical and molecular approach. An average larval burden of 28.4 L 1 per gram of meat was found (minimum 3.2 - maximum 132.6). A statistically significant difference was found in the prevalence according to hunting seasons ( p  < 0.01, with higher values in 2016 and 2021) and regions of the study area ( p  < 0.01). No statistically significant decrease in the prevalence of T. britovi throughout the study period was found ( p  = 0.51), except in Apulia region ( p  < 0.01). These findings revealed a stable prevalence of T. britovi in wild boar meat intended for human consumption, suggesting a risk of infection for consumers, especially hunters and local markets users. Citizen science surveillance models could be promoted to improve trichinellosis control and prevention in a One Health perspective., Competing Interests: None., (© 2023 The Authors. Published by Elsevier B.V.)

Foodborne infections cause illness and death every year worldwide. The aim of this study was to describe trends in 2013-2019 in the occurrence of human cases of salmonellosis, campylobacteriosis, listeriosis, and hepatitis A in the Campania region. Human case data were provided by the National Surveillance System of disease and were grouped by year, province, age group, and sex. Moreover, the number of people hospitalized was recorded. In the Campania region, the total number of confirmed human cases for the diseases investigated was 1924, with Hepatitis A and the Salmonellosis as the first most reported (1009 and 825 cases, respectively). The incidence rates of gastroenteritis under study were lower than those in Italy and European Union in the same period, with the exception of Hepatitis A whose incidence was higher than that recorded in Italy. Data on hospitalizations pointed out the onset of severe forms of infection also for listeriosis and campylobacteriosis, whose incidence was very low (27 and 63 cases, respectively). Unfortunately, no information on the foods implicated is available. Although probably underestimated, gastroenteritis due to foodborne agents still represents a burden in Campania, and continuous monitoring and implementation of the currently available regional surveillance system is required.

Purpose: Outpatient classified total hip arthroplasty (THA) is a safe option for a select group of patients. An analysis of a national database was conducted to understand the risk factors for unplanned discharge to a skilled nursing facility (SNF) or acute rehabilitation (rehab) after outpatient classified THA., Materials and Methods: A query of the National Surgical Quality Improvement Program (NSQIP) database for THA (Current Procedural Terminology [CPT] 27130) performed from 2015 to 2018 was conducted. Patient demographics, American Society of Anesthesiologists (ASA) classification, functional status, NSQIP morbidity probability, operative time, length of stay (LOS), 30-day reoperation rate, readmission rate, and associated complications were collected., Results: A total of 2,896 patients underwent outpatient classified THA. The mean age of patients was 61.2 years. The mean body mass index (BMI) was 29.6 kg/m 2 with median ASA 2. The results of univariate comparison of SNF/rehab versus home discharge showed that a significantly higher percentage of females (58.7% vs. 46.8%), age >70 years (49.3% vs. 20.9%), ASA ≥3 (58.0% vs. 25.8%), BMI >35 kg/m 2 (23.3% vs. 16.2%), and hypoalbuminemia (8.0% vs. 1.5%) ( P <0.0001) were discharged to SNF/rehab. The results of multivariable logistic regression showed that female sex (odds ratio [OR] 1.47; P =0.03), age >70 years (OR 3.08; P =0.001), ASA ≥3 (OR 2.56; P =0.001), and preoperative hypoalbuminemia (<3.5 g/dL) (OR 3.76; P =0.001) were independent risk factors for SNF/rehab discharge., Conclusion: Risk factors associated with discharge to a SNF/rehab after outpatient classified THA were identified. Surgeons will be able to perform better risk stratification for patients who may require additional postoperative intervention., Competing Interests: CONFLICT OF INTEREST: The authors declare that there is no potential conflict of interest relevant to this article., (Copyright © 2022 by Korean Hip Society.)

Background: 4-Hydroxy- N,N -methylpropyltryptamine (4-OH-MPT) is a psychedelic tryptamine whose use is regulated in several countries. Due to unspecific effects, consumption can be ascertained only through toxicological analyses. However, the trace amounts of tryptamines are usually challenging to detect in biological samples. 4-OH-MPT metabolism was characterized to identify optimal metabolite markers of intake in clinical/forensic toxicology., Research Design and Methods: 4-OH-MPT was incubated with 10-donor-pooled human hepatocytes to simulate in vivo conditions; samples were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and data were processed with Compound Discoverer from Thermo Scientific. LC-HRMS/MS and data mining were supported by in silico metabolite predictions (GLORYx)., Results: Three phase I and four phase II metabolites were identified, including N -oxidation and N -demethylation at the alkylamine chain, and O -glucuronidation and sulfation at the hydroxylindole core., Conclusions: 4-OH-MPT metabolic fate was consistent with the human metabolism of tryptamine analogues: we suggest 4-OH-MPT- N -oxide and 4-hydroxy- N,N -propyltryptamine (4-OH-PT) as metabolite biomarkers of 4-OH-MPT consumption after glucuronide/sulfate hydrolysis in biological samples to improve detection of 4-OH-MPT and phase I metabolites; 4-OH-MPT-glucuronide is suggested as an additional biomarker when hydrolysis is not performed. Further research on the metabolism of structural analogues is necessary to evaluate the specificity of 4-OH-MPT metabolite biomarkers.

In this study new compounds consisting of activated carbon cloths (ACC) modified with calcium silicate (CaSiO 3 ) were prepared for hydroxyapatite (HAP) generation. ACC samples were oxidized with 8 M HNO 3 at different times (15 min and 2 h), to increase oxygenated functional groups. The CaSiO 3 fine powders were prepared by chemical coprecipitation using Ca(NO 3 ) 2 ∙4H 2 O and Si(OC 2 H 5 ) 4 , and 5 M NaOH was used as precipitant. The resulting powders were mixed with ethanol by ultrasound stirring and the previously oxidized activated carbon fibers were placed leaving under stirring for 30 min to allow particle dispersion. Once the formed compounds were dried, the samples were immersed in a simulated body fluid (SBF) solution for 21 days in conical tubes at 36.5 °C to allow the HAP formation on the ACC/CaSiO 3 composite surface. The results indicated that the increase in oxidation time improves HAP formation on the surface from ACC/CaSiO 3 compounds and this bioactive composite may be a potential material for bone regeneration., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

We report on a search for dark matter axionlike particles (ALPs) using a Ramsey-type apparatus for cold neutrons. A hypothetical ALP-gluon coupling would manifest in a neutron electric dipole moment signal oscillating in time. Twenty-four hours of data have been analyzed in a frequency range from 23  μHz to 1 kHz, and no significant oscillating signal has been found. The usage of present dark-matter models allows one to constrain the coupling of ALPs to gluons in the mass range from 10^{-19} to 4×10^{-12}  eV. The best limit of C&#95;{G}/f&#95;{a}m&#95;{a}=2.7×10^{13}  GeV^{-2} (95% C.L.) is reached in the mass range from 2×10^{-17} to 2×10^{-14}  eV.

Cannabidiol (CBD) exhibits anti-inflammatory, anxiolytic, antiseizure, and neuroprotective proprieties without addictive or psychotropic side effects, as opposed to Δ 9 -tetrahydrocannabinol (THC). While recreational cannabis contains higher THC and lower CBD concentrations, medical cannabis contains THC and CBD in different ratios, along with minor phytocannabinoids, terpenes, flavonoids and other chemicals. A volumetric absorptive microsampling (VAMS) method combined with ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem for quantification of CBD, THC and their respective metabolites: cannabidiol-7-oic acid (7-COOH-CBD); 7-hydroxy-cannabidiol (7-OH-CBD); 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD); and 6-beta-hydroxycannabidiol (6-β-OH-CBD); 11- Hydroxy-Δ 9 -tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THCCOOH). After overnight enzymatic glucuronide hydrolysis at 37°C, samples underwent acidic along with basic liquid-liquid extraction with hexane: ethyl acetate (9:1, v/v). Chromatographic separation was carried out on a C18 column, with the mass spectrometer operated in multiple reaction monitoring mode and negative electrospray ionization. Seven patients with intractable epilepsy were dosed with various CBD-containing formulations and blood collected just before their daily morning administration. The method was validated following international guidelines in toxicology. Linear ranges were (ng/ml) 0.5-25 THC, 11-OH-THC, THCCOOH, 6-α-OH-CBD and 6-β-OH-CBD; 10-500 CBD and 7-OH-CBD; and 20-5000 7-COOH-CBD. 7-COOH-CBD was present in the highest concentrations, followed by 7-OH-CBD and CBD. This analytical method is useful for investigating CBD, THC and their major metabolites in epilepsy patients treated with CBD preparations employing a minimally invasive microsampling technique requiring only 30 µL blood., 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 © 2022 Pigliasco, Malaca, Lo Faro, Tini, Cangemi, Cafaro, Barco, Riva, Pisati, Amadori, Striano, Tagliabracci, Huestis and Busardò.)

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella . Here, the mechanism of action of 1018-K6 was explored further to understand the peptide-membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium 2GN , Escherichia coli 3GN . The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.Staphylococcus aureus 3GP . The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.

Carbonic anhydrase inhibitors (CAIs) are prescription drugs also used in doping to dilute urine samples and tamper with urinalyses. Dorzolamide, brinzolamide, and acetazolamide are prohibited by the World Anti-Doping Agency. Detecting CAIs and their metabolites in biological samples is crucial to documenting misuse in doping. We quantified dorzolamide, brinzolamide, acetazolamide, and their metabolites in the urine and hair of 88 patients under treatment for ocular hypertension or glaucoma. Samples of the patients' relatives were analyzed to assess potential for accidental exposure. After washing, 25 mg hair was incubated with an acidic buffer at 100 °C for 1 h. After cooling and centrifugation, the supernatant was analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Urine (100 μL) was diluted and centrifuged before UHPLC-MS/MS analysis. Run time was 8 min through a reverse-phase column with a mobile phase gradient. MS/MS analysis was performed in a multiple-reaction monitoring mode after positive electrospray ionization. Median urinary concentration was 245 ng/mL (IQR: 116.2-501 ng/mL) for dorzolamide, 81.1 ng/mL (IQR: 35.9-125.3 ng/mL) for N -deethyl-dorzolamide, 0.77 ng/mL (IQR: 0.64 ng/mL-0.84 ng/mL) for N -acetyl-dorzolamide, 38.9 ng/mL (IQR: 20.4-79.2 ng/mL) for brinzolamide, and 72.8 ng/mL (IQR: 20.7-437.3 ng/mL) for acetazolamide. Median hair concentration was 0.48 ng/mg (IQR: 0.1-0.98 ng/mg) for dorzolamide, 0.07 ng/mg (IQR: 0.06-0.08 ng/mg) for N -deethyl-dorzolamide, 0.40 ng/mL (IQR: 0.13-1.95 ng/mL) for brinzolamide. Acetazolamide was detected in only one hair sample. Dorzolamide and brinzolamide were detected in the urine of three and one relatives, respectively. Cutoff concentrations of urinary dorzolamide and brinzolamide are necessary to preclude false positives due to contamination or passive exposure. We reported the first concentrations of brinzolamide in hair.

Superoxide dismutase (SOD) is a fundamental antioxidant enzyme that neutralises superoxide ions, one of the main reactive oxygen species (ROS). Extremophile organisms possess enzymes that offer high stability and catalytic performances under a wide range of conditions, thus representing an exceptional source of biocatalysts useful for industrial processes. In this study, SODs from the thermo-halophilic Aeropyrum pernix (SOD Ap ) and the thermo-acidophilic Saccharolobus solfataricus (SOD Ss ) were heterologously expressed in transgenic tomato cell cultures. Cell extracts enriched with SOD Ap and SOD Ss showed a remarkable resistance to salt and low pHs, respectively, together with optimal activity at high temperatures. Moreover, the treatment of tuna fillets with SOD Ap -extracts induced an extension of the shelf-life of this product without resorting to the use of illicit substances. The results suggested that the recombinant plant extracts enriched with the extremozymes could find potential applications as dietary supplements in the nutrition sector or as additives in the food preservation area, representing a more natural and appealing alternative to chemical preservatives for the market.

Background : Since 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic (COVID-19) has caused millions of deaths worldwide and is the second most serious pandemic after the Spanish flu. Despite SARS-CoV-2 infection having a dominant effect on morbidity and life-threatening outcomes, the role of bacterial co-infection in patients with COVID-19 is poorly understood. The present study aimed to verify the existence of bacterial co-infections and their possible role as cofactors worsening COVID-19-related clinical manifestations. Methods : All patients with suspected SARS-CoV-infection, hospitalised in COVID-19 wards at the Sant'Anna University Hospital of Ferrara, were retrospectively included in this single-centre study and their specific bacterial serologies were assessed. Univariate and logistic regression analyses were performed. Results : A total of 1204 individual records were retrieved. Among them, 959 were excluded because of a negative nasopharyngeal swab or missing data; of the eligible 245 patients, 51 were co-infected. Compared to patients with SARS-CoV-2 infection alone, those with Chlamydia pneumoniae or Mycoplasma pneumoniae co-infections had worse respiratory/radiological features and more intensive care unit admissions. However, the co-infection did not result in a higher mortality rate. Conclusions : The present study, comparing clinical, laboratory and radiological findings between patients with COVID-19 vs. those with co-infections ( C. pneumoniae or M. pneumoniae ) showed that, on admission, these features were worse in co-infected patients, although the mortality rate did not differ between the two groups.

Fresh fishery products are highly perishable foods mainly due to their high-water content and high level of pH which act as promoters of spoilage processes. In these matrices, the deterioration phenomena are the result of the action of oxidative, and enzymatic processes due in part to the presence of specific microorganisms. Indeed, the microbial communities responsible for spoilage are a small fraction of the flora detectable in the fish and are known as specific spoilage organisms (SSOs). In the last decades, the scientific community has worked to achieve the ambitious goal of reducing the impact of microbial deterioration on food losses through innovative solutions, including antimicrobial packaging. The goal of this study was to evaluate the efficacy of an active polypropylene (PP)- based packaging functionalized with the antimicrobial peptide 1018K6 to extend the shelf life of dolphinfish burgers ( Coryphaena hippurus ) by evaluating its effect on sensorial and microbiological profile. The microbiological results showed an evident antimicrobial activity of the active packaging against hygiene indicator microorganisms and SSOs, recording a reduction of about 1 Log (CFU/g) of their concentrations compared to those of the control groups. Furthermore, a significant influence of functionalized packaging on the organoleptic characteristics was noted, accentuating the differences in freshness between the two experimental groups. This work confirmed the hypothesis of considering antimicrobial packaging as a potential tool capable of slowing down surface microbial replication and, therefore, extending the shelf-life and improving the health and hygiene aspect of fresh fish products., Competing Interests: Conflict of interest: The authors declare no potential conflict of interests., (©Copyright: the Author(s).)