Uncoupling histone modification crosstalk by engineering lysine demethylase LSD1.

Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation, but its functional significance in cells has been difficult to discern. Previous enzymatic studies revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine-specific demethylase 1 (LSD1). In the present study, we engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation. K562 cells with the Y391K LSD1 CRISPR knockin show decreased expression of a set of genes associated with cellular adhesion and myeloid leukocyte activation. Chromatin profiling revealed that the cis-regulatory regions of these silenced genes display a higher level of H3 Lys14 acetylation, and edited K562 cells show diminished H3 mono-methyl Lys4 near these silenced genes, consistent with a role for enhanced LSD1 demethylase activity. These findings illuminate the functional consequences of disconnecting histone modification crosstalk for a key epigenetic enzyme.

Evolution, structure, and drug-metabolizing activity of mammalian prenylcysteine oxidases.

Prenylcysteine oxidases (PCYOXs) metabolize prenylated cysteines produced by protein degradation. They utilize oxygen as a co-substrate to produce free cysteine, an aldehyde, and hydrogen peroxide through the unusual oxidation of a thioether bond. In this study, we explore the evolution, structure, and mechanism of the two mammalian PCYOXs. A gene duplication event in jawed vertebrates originated in these two paralogs. Both enzymes are active on farnesyl- and geranylgeranylcysteine, but inactive on molecules with shorter prenyl groups. Kinetics experiments outline a mechanism where flavin reduction and re-oxidation occur rapidly without any detectable intermediates, with the overall reaction rate limited by product release. The experimentally determined three-dimensional structure of PCYOX1 reveals long and wide tunnels leading from the surface to the flavin. They allow the isoprene substrate to curl up within the protein and position its reactive cysteine group close to the flavin. A hydrophobic patch on the surface mediates membrane association, enabling direct substrate and product exchange with the lipid bilayer. Leveraging established knowledge of flavoenzyme inhibition, we designed sub-micromolar PCYOX inhibitors. Additionally, we discovered that PCYOXs bind and slowly degrade salisirab, an anti-RAS compound. This activity suggests potential and previously unknown roles of PCYOXs in drug metabolism.

Asymmetric Engagement of Dimeric CRL3 KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes.

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

Computationally driven discovery of SARS-CoV-2 Mpro inhibitors: from design to experimental validation.

We report a fast-track computationally driven discovery of new SARS-CoV-2 main protease (Mpro) inhibitors whose potency ranges from mM for the initial non-covalent ligands to sub-µM for the final covalent compound (IC50 = 830 ± 50 nM). The project extensively relied on high-resolution all-atom molecular dynamics simulations and absolute binding free energy calculations performed using the polarizable AMOEBA force field. The study is complemented by extensive adaptive sampling simulations that are used to rationalize the different ligand binding poses through the explicit reconstruction of the ligand-protein conformation space. Machine learning predictions are also performed to predict selected compound properties. While simulations extensively use high performance computing to strongly reduce the time-to-solution, they were systematically coupled to nuclear magnetic resonance experiments to drive synthesis and for in vitro characterization of compounds. Such a study highlights the power of in silico strategies that rely on structure-based approaches for drug design and allows the protein conformational multiplicity problem to be addressed. The proposed fluorinated tetrahydroquinolines open routes for further optimization of Mpro inhibitors towards low nM affinities.

A Review into the Effectiveness of Ozone Technology for Improving the Safety and Preserving the Quality of Fresh-Cut Fruits and Vegetables.

In recent years, consumers have become increasingly aware of the nutritional benefits brought by the regular consumption of fresh fruits and vegetables, which reduces the risk of health problems and disease. High-quality raw materials are essential since minimally processed produce is highly perishable and susceptible to quality deterioration. The cutting, peeling, cleaning and packaging processes as well as the biochemical, sensorial and microbial changes that occur on plant tissue surfaces may accelerate produce deterioration. In this regard, biological contamination can be primary, which occurs when the infectious organisms directly contaminate raw materials, and/or by cross-contamination, which occurs during food preparation processes such as washing. Among the many technologies available to extend the shelf life of fresh-cut products, ozone technology has proven to be a highly effective sterilization technique. In this paper, we examine the main studies that have focused on the effects of gaseous ozone and ozonated water treatments on microbial growth and quality retention of fresh-cut fruit and vegetables. The purpose of this scientific literature review is to broaden our knowledge of eco-friendly technologies, such as ozone technology, which extends the shelf life and maintains the quality of fresh produce without emitting hazardous chemicals that negatively affect plant material and the environment.

Side-to-side esophagojejunostomy during totally laparoscopic total gastrectomy for malignant disease: a multicenter study.

BACKGROUND: Esophagojejunostomy (EJS) represents the most difficult steps during totally laparoscopic total gastrectomy (TLTG). Over the past few years, several techniques have been developed. This study aimed to evaluate the feasibility and surgical outcomes of the laparoscopic intracorporeal side-to-side EJS during TLTG used to treat malignant disease of the stomach. METHODS: This study was conducted from June 2001 to December 2006 at three different institutions. Data were collected from patients' medical notes, and a database was established that recorded gender, age, American Society of Anesthesiology (ASA) classification, tumor site, operative duration, time required for anastomosis, length of hospital stay, morbidity, mortality, tumor node metastasis (TNM) staging, grading, type of procedure performed, type of lymphadenectomy, conversion rate, reason for conversion, histology type, reoperation rate, reason for reoperation, time required for closure of leak, flatus time, time enteral feeding started, morbidity, and mortality. RESULTS: In this study, 56 totally laparoscopic gastrectomies (TLGs) (83.6%) and 11 totally laparoscopic degastrogastrectomies (TLDGs) (16.4%) with intracorporeal side-to-side EJS were performed. The average operating time was 249 min (range, 195-349 min). The average time required for both anastomoses was 44 min (17.7% of the average total time). The conversion rate was 10.4%, and the reoperation rate was 13.4%. The mean hospital stay was 12.4 days (range, 8-45 days). The major complications comprised four anastomotic leakage (6%), five postoperative bleeding (7.5%), and two duodenal stump leakage (3%). Most of the patients (91%) were enteral fed on day 6. The mean time for closure of leaks was 12 days (range, 4-18 days). The minor complications comprised two esophagojejunal anastomotic strictures (3%) subsequently treated by endoscopic dilatation. There was one death (1.5%), which occurred within 45 postoperative days. CONCLUSIONS: Laparoscopic intracorporeal side-to-side EJS is a safe and feasible technique. It represents a valid method for performing a reconstruction of the digestive tract in laparoscopic surgery after TLG, especially in presence of a narrow esophagus.

Laparoscopic ureteroneocystostomy and vesicopsoas hitch for infiltrative ureteral endometriosis.

The aim of the study was to assess the safety and efficacy of laparoscopic treatment of distal infiltrative ureteral endometriosis with segmental ureteral resection, ureteroneocystostomy, and vesicopsoas hitch. We performed a retrospective analysis of perioperative data and looked at follow-up outcomes of patients with deep endometriosis with ureteral involvement treated by laparoscopic vesicopsoas hitch. Six patients were treated for left ureteral endometriosis in the study period. Four of those were diagnosed during previous laparoscopies. A ureteroneocystostomy (Lich-Gregoir reimplantation procedure) with vesicopsoas hitch was fashioned laparoscopically in all cases, and a double-J stent was applied intraoperatively. There were no intraoperative or postoperative complications and no cases of extravasation of contrast at cystogram one week after surgery. The median follow-up time was 38 months (range 12-56). All patients had normal renal ultrasound or intravenous pyelogram results at one year follow-up. This study confirmed that laparoscopic ureteroneocystostomy and vesicopsoas hitch is a safe and effective option in the management of distal ureteral endometriosis. In view of the small size of this series, multicenter studies are needed to confirm these conclusions.

Association between Work Related Stress and Health Related Quality of Life: The Impact of Socio-Demographic Variables. A Cross Sectional Study in a Region of Central Italy.

The aim of this work is investigate relationship between health-related quality of life and work-related stress and the impact of gender, education level, and age on this relationship. A cross-sectional study was conducted among workers of various setting in Rome and Frosinone. Work-related stress was measured with a demand-control questionnaire and health-related functioning by SF (short form)-12 health survey. There were 611 participants. Men reported high mental composite summary (MCS) and physical composite summary (PCS). In multivariate analysis age, gender (p < 0.001) and job demand (0.045) predicted low PCS. Low MCS predicted poor PCS. Job demand and educational level resulted negatively associated with MCS. In an analysis stratified for age, gender, and educational level, gender and age resulted effect modifier for MCS, gender and education level for PCS. In women increase of decision latitude predict (p = 0.001) an increase in MCS; a low job demand predict high MCS in male (p ≤ 0.001). In younger workers, a lower level of job demand predicted high MCS (<0.001). For PCS, gender and education level resulted effect modifier. In women, high decision latitude predicted higher PCS (p = 0.001) and lower level of job demand results in higher PCS (p ≤ 0.001). Higher educational level resulted predictor of low PCS. Management of risk about work-related stress should consider socio-demographic factors.

The laparoscopic approach in the median arcuate ligament syndrome: report of a case.

A malposition of the median arcuate ligament (MAL) is a rare entity causing the celiac axis compression syndrome (CACS), first described by Harjola in 1963. The presence of anomalous fibrous diaphragmatic bands of the diaphragm compresses the celiac axis, especially during the expiration. In this report we present the fourth case in literature that was ever successfully treated by laparoscopy. A 38-year-old male presented with a history of intermittent epigastric pain, 15 kg weight loss caused by inappetence and frequent diarrhoea, over a 5-year period. The clinical examination revealed only a loud systolic bruit in the epigastrium, with loss of intensity during deep inspiration. Suspecting CACS, a spiral CT angiography was requested. The CT demonstrated the MAL crossing anteriorly to the celiac artery (CA) and the sagittal and the tridimensional reconstructions demonstrated the CA narrowing due to compression, while the superior mesenteric artery (SMA) was normal. The MAL was laparoscopically divided, releasing the celiac axis. The surgical time was 130 minutes, without significant blood losses. At 3-months follow-up, the CT-scan demonstrated no evidence of CACS with complete recovery.

The role of laparoscopy in the diagnosis and the treatment of missed diaphragmatic hernia after penetrating trauma.

BACKGROUND: The traumatic diaphragmatic hernia (TDH) may appear acutely after a blunt or penetrating trauma, or it can remain missed also for many years. The discussion about the utility of a laparoscopic repair in acute and chronic TDH is controversial. METHODS: In this paper, we present two cases of chronic TDH that were successfully treated with laparoscopy. The first patient was treated 1 year after a stab wound and the second one 10 years after a firearm injury. RESULTS: In both cases, the diaphragmatic defects were easily laparoscopically detected and treated. The defects were repaired with a direct running suture owing to the acceptable dimensions of the tears. The mean operative time was 135 minutes (range, 75-195). The blood loss during the operations was unremarkable. No intraoperative complications occurred, and the conversion rate was null. The postoperative course was uneventful in the first patient, whereas the second patient, owing to the intraoperative respiratory problems, needed an accurate respiratory monitoring in the Intensive Care Unit. The mean length of stay after the operation was 7 days (range, 6-8). At follow-up, the clinical examination and the chest X-rays documented no recurrence. CONCLUSIONS: We recommend the use of laparoscopy in left chronic TDH repair, performing a direct suture of the diaphragm when possible.

Carotid endarterectomy with intermediate cervical plexus block.

During carotid endarterectomy, the use of locoregional anesthesia to achieve a combined superficial and deep cervical plexus block can cause cardiovascular, respiratory, and neurologic complications. Seeking to reduce risk and find an easier procedure, we applied locoregional anesthesia and an intermediate cervical plexus block in a series of patients who underwent carotid endarterectomy. From 2006 through 2007, 183 patients underwent primary carotid endarterectomy at our hospital. Mean age was 75.9 +/- 9.9 yr; mean body mass index, 27.3 +/- 6.7 kg/m(2); and median American Society of Anesthesiologists physical status classification, P3 (range, P2-P4). All procedures combined an intermediate cervical plexus block with subcutaneous infiltration of the incision line. We inserted a 15-mm, 25G needle to its full length, perpendicular to the skin along the posterior border of the sternocleidomastoid muscle, midway between the mastoid process and the clavicle. We injected 10 mL of 0.75% ropivacaine solution for 3 to 5 minutes. This block was systematically combined with subcutaneous infiltration of the incision line with the ropivacaine (0.75%, 10 mL), and sometimes also with 2% topical lidocaine intraoperatively. If necessary, intraoperative sedation, analgesia, or both were given to patients to improve their compliance. Intraoperative topical lidocaine was required in 59 patients (32.2%), and intravenous midazolam, fentanyl, or both were required in 29 patients (15.8%). Two procedures were converted to general anesthesia (1.1%). No perioperative deaths or complications occurred. Postoperatively, 2 patients experienced strokes and 1 sustained a myocardial infarction (total rate, 1.6%). We found the intermediate cervical plexus block to be feasible, effective, and safe, with low perioperative and postoperative complication rates. Herein, we report our findings.