Characterizing the protein-protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization.

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.

Circadian regulation of liver metabolism: experimental approaches in human, rodent, and cellular models.

Circadian rhythms are endogenous oscillations with approximately a 24-h period that allow organisms to anticipate the change between day and night. Disruptions that desynchronize or misalign circadian rhythms are associated with an increased risk of cardiometabolic disease. This review focuses on the liver circadian clock as relevant to the risk of developing metabolic diseases including nonalcoholic fatty liver disease (NAFLD), insulin resistance, and type 2 diabetes (T2D). Many liver functions exhibit rhythmicity. Approximately 40% of the hepatic transcriptome exhibits 24-h rhythms, along with rhythms in protein levels, posttranslational modification, and various metabolites. The liver circadian clock is critical for maintaining glucose and lipid homeostasis. Most of the attention in the metabolic field has been directed toward diet, exercise, and rather little to modifiable risks due to circadian misalignment or disruption. Therefore, the aim of this review is to systematically analyze the various approaches that study liver circadian pathways, targeting metabolic liver diseases, such as diabetes, nonalcoholic fatty liver disease, using human, rodent, and cell biology models.NEW & NOTEWORTHY Over the past decade, there has been an increased interest in understanding the intricate relationship between circadian rhythm and liver metabolism. In this review, we have systematically searched the literature to analyze the various experimental approaches utilizing human, rodent, and in vitro cellular approaches to dissect the link between liver circadian rhythms and metabolic disease.

Tracking the mechanism of covalent molecular glue stabilization using native mass spectrometry.

Molecular glues are powerful tools for the control of protein-protein interactions. Yet, the mechanisms underlying multi-component protein complex formation remain poorly understood. Native mass spectrometry (MS) detects multiple protein species simultaneously, providing an entry to elucidate these mechanisms. Here, for the first time, covalent molecular glue stabilization was kinetically investigated by combining native MS with biophysical and structural techniques. This approach elucidated the stoichiometry of a multi-component protein-ligand complex, the assembly order, and the contributions of covalent versus non-covalent binding events that govern molecular glue activity. Aldehyde-based molecular glue activity is initially regulated by cooperative non-covalent binding, followed by slow covalent ligation, further enhancing stabilization. This study provides a framework to investigate the mechanisms of covalent small molecule ligation and informs (covalent) molecular glue development.

The association of social vulnerability with colorectal enhanced recovery program failure.

Background: Enhanced recovery programs (ERPs) improve outcomes, but over 20 % of patients fail ERP and the contribution of social vulnerability is unknown. This study aimed to characterize the association between social vulnerability and ERP adherence and failure. Methods: This was a retrospective cohort study of colorectal surgery patients between 2015 and 2020 utilizing ACS-NSQIP data. Patients who failed ERP (LOS > 6 days) were compared to patients not failing ERP. The CDC's social vulnerability index (SVI) was used to assess social vulnerability. Result: 273 of 1191 patients (22.9 %) failed ERP. SVI was a significant predictor of ERP failure (OR 4.6, 95 % CI 1.3-16.8) among those with >70 % ERP component adherence. SVI scores were significantly higher among patients non-adherent with 3 key ERP components: preoperative block (0.58 vs. 0.51, p < 0.01), early diet (0.57 vs. 0.52, p = 0.04) and early foley removal (0.55 vs. 0.50, p < 0.01). Conclusions: Higher social vulnerability was associated with non-adherence to 3 key ERP components as well as ERP failure among those who were adherent with >70 % of ERP components. Social vulnerability needs to be recognized, addressed, and included in efforts to further improve ERPs. Key message: Social vulnerability is associated with non-adherence to enhanced recovery components and ERP failure among those with high ERP adherence. Social vulnerability needs to be addressed in efforts to improve ERPs.

Robotic Anastomotic Technique.

The first robotic colectomy was performed 20 years ago. Since that time, the robotic surgery platform has made significant advancements and become increasingly prevalent in colorectal surgery. The da Vinci Xi system (Intuitive Surgical, Sunnyvale, CA) and technology such as integrated table motion has facilitated multiquadrant procedures. Intracorporeal anastomoses (ICAs) have proven benefit in the literature, including decreased length of stay, decreased narcotic requirements, and lower rate of postoperative wound infections and hernias. Additional studies have shown a lower conversion to open rate in robotic surgery compared with laparoscopy. In this article, we will describe techniques for creation of robotic ICAs.

Circadian clock function does not require the histone methyltransferase MLL3.

The circadian clock controls the physiological function of tissues through the regulation of thousands of genes in a cell-type-specific manner. The core cellular circadian clock is a transcription-translation negative feedback loop, which can recruit epigenetic regulators to facilitate temporal control of gene expression. Histone methyltransferase, mixed lineage leukemia gene 3 (MLL3) was reported to be required for the maintenance of circadian oscillations in cultured cells. Here, we test the role of MLL3 in circadian organization in whole animals. Using mice expressing catalytically inactive MLL3, we show that MLL3 methyltransferase activity is in fact not required for circadian oscillations in vitro in a range of tissues, nor for the maintenance of circadian behavioral rhythms in vivo. In contrast to a previous report, loss of MLL3-dependent methylation did not affect the global levels of H3K4 methylation in liver, indicating substantial compensation from other methyltransferases. Furthermore, we found little evidence of genomic repositioning of H3K4me3 marks. We did, however, observe repositioning of H3K4me1 from intronic regions to intergenic regions and gene promoters; however, there were no changes in H3K4me1 mark abundance around core circadian clock genes. Output functions of the circadian clock, such as control of inflammation, were largely intact in MLL3-methyltransferase-deficient mice, although some gene-specific changes were observed, with sexually dimorphic loss of circadian regulation of specific cytokines. Taken together, these observations indicate that MLL3-directed histone methylation is not essential for core circadian clock function; however, it may influence the inflammatory response.

Abdominal Wall Thickness is a Predictor for Surgical Site Infections in Patients Undergoing Colorectal Operations.

BACKGROUND: Surgical site infections (SSIs) are an established complication following colorectal operations, with rates up to 30% reported in the literature. Obesity is a known risk factor for SSI; however, body mass index (BMI), body fat percentage, waist-hip ratio, or abdominal circumference are imperfect measures. The purpose of our study was to determine whether abdominal wall thickness (AWT) is predictive of SSI. METHODS: We queried our American College of Surgeons National Surgical Quality Improvement Project (ACS-NSQIP) database for patients (age ≥18 years) undergoing a colectomy at the University of Kentucky (UK) from January 1, 2013 to December 31, 2018. The exclusion criteria included patients with open abdomens or the lack of preoperative computed tomography (CT) within 3 months of their operation. AWT was measured at the level of the anterior superior iliac spine (ASIS) on abdominal CT. SSI was defined by superficial SSI, deep SSI, and wound dehiscence. RESULTS: Of 1261 patients enrolled, 52.2% were female, with an average age of 57.4 years. More patients had laparoscopic operations (51%), and the median length of stay was 7 days. Our study demonstrated an SSI rate of 9.4% and a 30-day readmission rate of 11%. The overall mean AWT was 2.6 cm (range .1-13.1), and patients with the highest AWT quintile were more likely to develop an SSI than the lowest quintile (12% vs. 5%). After controlling for risk factors and confounders, the odds of an SSI were 3.6 times higher for patients with the highest AWT than patients with the lowest AWT. CONCLUSIONS: Among colorectal surgery patients, AWT is an independent risk factor predictive for SSI.

Novel Technique to Reduce the Incidence of SSI after Colorectal Surgery.

SSI is a leading cause of morbidity and increases health-care cost after colorectal operations. It is a key hospital-level patient safety indicator. Previous literature has identified perioperative risk factors associated with SSI and interventions to decrease rate of infection. The purpose of this study was to evaluate the impact of blowhole closure on the rate of superficial and deep SSI. The ACS-NSQIP database was queried for patients undergoing colectomy at the University of Kentucky from 2013 to 2016. Retrospective chart review was performed to gather demographic data and perioperative variables. Wounds left open and packed were excluded. Rates of postoperative SSI were measured between the groups. One thousand eighty-three patients undergoing elective and emergent colectomy were reviewed. Nine hundred and forty-five had closed incision and 138 had blowhole closure. Patient characteristics between the groups were well matched. Patients with a blowhole closure were more likely to have an open procedure (P = 0.037) and a higher wound class (P < 0.001). The rate of superficial and deep SSI was 9.1 per cent in patients with a closed incision and 5.1 per cent in patients with blowhole closure (P = 0.142). With adjustment for approach and wound class, blowhole closure decreased the incidence of SSI (P = 0.04). There was no significant difference in morbidity or mortality. Patients undergoing elective and emergent colectomy had decreased incidence of SSI when blowhole closure was used. Given that it does not increase resource usage and its technical ease, blowhole closure should become the standard method of surgical wound closure.

Decision Tool for Predicting Outcomes in Geriatric Acute Mesenteric Ischemia.

Acute mesenteric ischemia is a morbid disease process that is most common in elderly patients who often have multiple medical comorbidities. Intervention can progress to costly and futile care. The goal of this study was to develop a tool for practitioners to assess the risk of mortality. Patients treated at our institution over the past decade diagnosed with acute mesenteric ischemia were identified. Patients aged less than 65 years were excluded. Data were collected by retrospective chart review. Univariate analysis was used to identify significant risk factors for death. Decision tree analysis yielded a prognostic tool to assess death risk. Univariate analysis demonstrated that lactate (P ≤ 0.001) and pressor requirement (P ≤ 0.001) were predictive of death. Decision tree analysis showed that 79 per cent of patients with day of surgery (DOS) lactate ≥5.4 died postoperatively. Seventy per cent of patients with DOS lactate <5.4 progressed to death if they required pressors and had a creatinine >1.18. Only 6.1 per cent patients with a DOS lactate <5.4, creatinine <1.54, and no pressor requirement progressed to death. Several variables can be used to set expectations for families and help guide decision-making. Our tool was predictive of outcomes in 82 per cent of our study population.

Rumination Syndrome and Dental Erosions in Children.

Rumination syndrome is the effortless regurgitation of recently ingested food with subsequent reswallowing or spitting out. Dental erosion (DE) affects 2% to 5% of the population. DE is defined as loss of tooth structure by a chemical process that does not involve bacteria. Our objective was to compare the frequency of DE among children with rumination syndrome with healthy controls. We enrolled 30 patients 4 to 21 years of age diagnosed with rumination syndrome, and 30 age- and sex-matched healthy control subjects. Patients were evaluated by pediatric dentists for presence of DE with Taji et al a validated grading system. Patients with rumination were more likely to have DE (P < 0.001). Of patients with rumination syndrome, 23 (77%) had DE, compared with 4 (13%) control subjects. DEs are more frequent in patients with rumination syndrome.

Myogenic Akt signaling attenuates muscular degeneration, promotes myofiber regeneration and improves muscle function in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy, the most common form of childhood muscular dystrophy, is caused by X-linked inherited mutations in the dystrophin gene. Dystrophin deficiencies result in the loss of the dystrophin-glycoprotein complex at the plasma membrane, which leads to structural instability and muscle degeneration. Previously, we induced muscle-specific overexpression of Akt, a regulator of cellular metabolism and survival, in mdx mice at pre-necrotic (<3.5 weeks) ages and demonstrated upregulation of the utrophin-glycoprotein complex and protection against contractile-induced stress. Here, we found that delaying exogenous Akt treatment of mdx mice after the onset of peak pathology (>6 weeks) similarly increased the abundance of compensatory adhesion complexes at the extrasynaptic sarcolemma. Akt introduction after onset of pathology reverses the mdx histopathological measures, including decreases in blood serum albumin infiltration. Akt also improves muscle function in mdx mice as demonstrated through in vivo grip strength tests and in vitro contraction measurements of the extensor digitorum longus muscle. To further explore the significance of Akt in myofiber regeneration, we injured wild-type muscle with cardiotoxin and found that Akt induced a faster regenerative response relative to controls at equivalent time points. We demonstrate that Akt signaling pathways counteract mdx pathogenesis by enhancing endogenous compensatory mechanisms. These findings provide a rationale for investigating the therapeutic activation of the Akt pathway to counteract muscle wasting.

Dinuclear alkynyllanthanoid(II) dications with pentaphenylcyclopentadienyl or tri-tert-butyldiphosphacyclopentadienyl counter ions.

Reaction of [Yb(CpPh5)(C[triple bond]CPh)(thf)]2 (CpPh5 = pentaphenylcyclopentadienyl), prepared from Yb(C triple bond CPh)2 and HCpPh5 or Yb metal, HgPh(C[triple bond]CPh) and HCpPh5, with a controlled amount of diglyme (dig), and of Eu(C triple bond CPh)2, P triple bond CBut and dig, yield the unusual organolanthanoid(II) dicationic complexes [Yb(C[triple bond]CPh)(dig)(thf)2]2[CpPh5]2.4thf and [Eu(C triple bond CPh)(dig)2]2[P2C3But3]2 respectively.