Distinct T cell signatures are associated with Staphylococcus aureus skin infection in pediatric atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin condition with a childhood prevalence of up to 25%. Microbial dysbiosis is characteristic of AD, with Staphylococcus aureus the most frequent pathogen associated with disease flares and increasingly implicated in disease pathogenesis. Therapeutics to mitigate the effects of S. aureus have had limited efficacy and S. aureus-associated temporal disease flares are synonymous with AD. An alternative approach is an anti-S. aureus vaccine, tailored to AD. Experimental vaccines have highlighted the importance of T cells in conferring protective anti-S. aureus responses; however, correlates of T cell immunity against S. aureus in AD have not been identified. We identify a systemic and cutaneous immunological signature associated with S. aureus skin infection (ADS.aureus) in a pediatric AD cohort, using a combined Bayesian multinomial analysis. ADS.aureus was most highly associated with elevated cutaneous chemokines IP10 and TARC, which preferentially direct Th1 and Th2 cells to skin. Systemic CD4+ and CD8+ T cells, except for Th2 cells, were suppressed in ADS.aureus, particularly circulating Th1, memory IL-10+ T cells, and skin-homing memory Th17 cells. Systemic γδ T cell expansion in ADS.aureus was also observed. This study suggests that augmentation of protective T cell subsets is a potential therapeutic strategy in the management of S. aureus in AD.

Chemical and structural approaches to investigate PTEN function and regulation.

Phosphatase and tensin homolog is a lipid phosphatase that serves as the major negative regulator of the PI3K/AKT pathway. It catalyzes the 3'-specific dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to generate PIP2. PTEN's lipid phosphatase function depends on several domains, including an N-terminal segment spanning the first 24 amino acids, which results in a catalytically impaired enzyme when mutated. Furthermore, PTEN is regulated by a cluster of phosphorylation sites located on its C-terminal tail at Ser380, Thr382, Thr383, and Ser385, which drives its conformation from an open to a closed autoinhibited but stable state. Herein, we discuss the protein chemical strategies we used to reveal the structure and mechanism of how PTEN's terminal regions govern its function.

Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.

Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN's biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.

An age-structured SEIR model for COVID-19 incidence in Dublin, Ireland with framework for evaluating health intervention cost.

Strategies adopted globally to mitigate the threat of COVID-19 have primarily involved lockdown measures with substantial economic and social costs with varying degrees of success. Morbidity patterns of COVID-19 variants have a strong association with age, while restrictive lockdown measures have association with negative mental health outcomes in some age groups. Reduced economic prospects may also afflict some age cohorts more than others. Motivated by this, we propose a model to describe COVID-19 community spread incorporating the role of age-specific social interactions. Through a flexible parameterisation of an age-structured deterministic Susceptible Exposed Infectious Removed (SEIR) model, we provide a means for characterising different forms of lockdown which may impact specific age groups differently. Social interactions are represented through age group to age group contact matrices, which can be trained using available data and are thus locally adapted. This framework is easy to interpret and suitable for describing counterfactual scenarios, which could assist policy makers with regard to minimising morbidity balanced with the costs of prospective suppression strategies. Our work originates from an Irish context and we use disease monitoring data from February 29th 2020 to January 31st 2021 gathered by Irish governmental agencies. We demonstrate how Irish lockdown scenarios can be constructed using the proposed model formulation and show results of retrospective fitting to incidence rates and forward planning with relevant "what if / instead of" lockdown counterfactuals. Uncertainty quantification for the predictive approaches is described. Our formulation is agnostic to a specific locale, in that lockdown strategies in other regions can be straightforwardly encoded using this model.

The structural basis of PTEN regulation by multi-site phosphorylation.

Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN's cellular functions.

What is the patient experience of surgical care during the coronavirus disease 2019 (COVID-19) pandemic? A mixed-methods study at a single institution.

BACKGROUND: The coronavirus disease 2019 outbreak has spread worldwide and has resulted in hospital restrictions. The perceived impact of these practices on patients undergoing essential surgeries is less understood. METHODS: Adult (≥18 years) patients who underwent medically necessary surgical procedures spanning multiple surgical specialties from March 23, 2020, to April 24, 2020, during the coronavirus disease 2019 pandemic were identified as eligible for a phone survey. Survey responses were analyzed using a mixed-methods approach involving descriptive statistics and thematic analysis of coded and annotated survey results. RESULTS: Of the 212 patients who underwent medically necessary surgical procedures during the coronavirus disease 2019 pandemic, the majority of these patients were male (61.3%), White (83.5%), married or with a domestic partner (68.9%), and underwent oncologic procedures (69.3%). Of the 46 patients (21.7%) who completed the survey, the majority of these patients indicated that coronavirus disease 2019 pandemic restrictions had no impact on their inpatient hospital stay and were satisfied with their decision to proceed with surgery. Severity of patient condition (44.4%), the risk/benefit discussion with the surgeon (24.4%), and coronavirus disease 2019 education and testing (19.5%) were the most important factors in proceeding with surgery during the pandemic; 34.4% of patients said their inpatient postoperative course was negatively affected by the lack of visitors. CONCLUSION: Medically necessary, time-sensitive surgical procedures, as determined by the surgeon, can be performed during a pandemic with good patient satisfaction provided there is an appropriate discussion between the surgeon and patient about the risks and benefits.

Ubiquitin Ligase Activities of WWP1 Germline Variants K740N and N745S.

WWP1 is an E3 ubiquitin ligase that has been reported to target the tumor suppressor lipid phosphatase PTEN. K740N and N745S are recently identified germline variants of WWP1 that have been linked to PTEN-associated cancers [Lee, Y. R., et al. (2020) N. Engl. J. Med.]. These WWP1 variants have been suggested to release WWP1 from its native autoinhibited state, thereby promoting enhanced PTEN ubiquitination as a mechanism for driving cancer. Using purified proteins and in vitro enzymatic assays, we investigate the possibility that K740N and N745S WWP1 possess enhanced ubiquitin ligase activity and demonstrate that these variants are similar to the wild type (WT) in both autoubiquitination and PTEN ubiquitination. Furthermore, K740N and N745S WWP1 show dependencies similar to those of WT in terms of allosteric activation by an engineered ubiquitin variant, upstream E2 concentration, and substrate ubiquitin concentration. Transfected WWP1 WT and mutants demonstrate comparable effects on cellular PTEN levels. These findings challenge the idea that K740N and N745S WWP1 variants promote cancer by enhanced PTEN ubiquitination.

Impact of COVID-19 Restrictions on Demographics and Outcomes of Patients Undergoing Medically Necessary Non-Emergent Surgeries During the Pandemic.

BACKGROUND: The COVID-19 pandemic has resulted in large-scale healthcare restrictions to control viral spread, reducing operating room censuses to include only medically necessary surgeries. The impact of restrictions on which patients undergo surgical procedures and their perioperative outcomes is less understood. METHODS: Adult patients who underwent medically necessary surgical procedures at our institution during a restricted operative period due to the COVID-19 pandemic (March 23-April 24, 2020) were compared to patients undergoing procedures during a similar time period in the pre-COVID-19 era (March 25-April 26, 2019). Cardinal matching and differences in means were utilized to analyze perioperative outcomes. RESULTS: 857 patients had surgery in 2019 (pre-COVID-19) and 212 patients had surgery in 2020 (COVID-19). The COVID-19 era cohort had a higher proportion of patients who were male (61.3% vs. 44.5%, P < 0.0001), were White (83.5% vs. 68.7%, P < 0.001), had private insurance (62.7% vs. 54.3%, p 0.05), were ASA classification 4 (10.9% vs. 3%, P < 0.0001), and underwent oncologic procedures (69.3% vs. 42.7%, P < 0.0001). Following 1:1 cardinal matching, COVID-19 era patients (N = 157) had a decreased likelihood of discharge to a nursing facility (risk difference-8.3, P < 0.0001) and shorter median length of stay (risk difference-0.6, p 0.04) compared to pre-COVID-19 era patients. There was no difference between the two patient cohorts in overall morbidity and 30-day readmission. CONCLUSIONS: COVID-19 restrictions on surgical operations were associated with a change in the racial and insurance demographics in patients undergoing medically necessary surgical procedures but were not associated with worse postoperative morbidity. Further study is necessary to better identify the causes for patient demographic differences.

Characterization of Stereolithography Printed Soft Tooling for Micro Injection Molding.

The use of microfeature-enabled devices, such as microfluidic platforms and anti-fouling surfaces, has grown in both potential and application in recent years. Injection molding is an attractive method of manufacturing these devices due to its excellent process throughput and commodity-priced raw materials. Still, the manufacture of micro-structured tooling remains a slow and expensive endeavor. This work investigated the feasibility of utilizing additive manufacturing, specifically a Digital Light Processing (DLP)-based inverted stereolithography process, to produce thermoset polymer-based tooling for micro injection molding. Inserts were created with an array of 100-µm wide micro-features, having different heights and thus aspect ratios. These inserts were molded with high flow polypropylene to investigate print process resolution capabilities, channel replication abilities, and insert wear and longevity. Samples were characterized using contact profilometry as well as optical and scanning electron microscopies. Overall, the inserts exhibited a maximum lifetime of 78 molding cycles and failed by cracking of the entire insert. Damage was observed for the higher aspect ratio features but not the lower aspect ratio features. The effect of the tool material on mold temperature distribution was modeled to analyze the impact of processing and mold design.

Selective protein N-terminal labeling with N-hydroxysuccinimide esters.

In order to gain detailed insight into the biochemical behavior of proteins, researchers have developed chemical tools to incorporate new functionality into proteins beyond the canonical 20 amino acids. Important considerations regarding effective chemical modification of proteins include chemoselectivity, near stoichiometric labeling, and reaction conditions that maintain protein stability. Taking these factors into account, we discuss an N-terminal labeling strategy that employs a simple two-step "one-pot" method using N-hydroxysuccinimide (NHS) esters. The first step converts a R-NHS ester into a more chemoselective R-thioester. The second step reacts the in situ generated R-thioester with a protein that harbors an N-terminal cysteine to generate a new amide bond. This labeling reaction is selective for the N-terminus with high stoichiometry. Herein, we provide a detailed description of this method and further highlight its utility with a large protein (>100kDa) and labeling with a commonly used cyanine dye.

Hiatal Hernia after Esophagectomy: An Underappreciated Complication?

BACKGROUND: The natural history of hiatal herniation of small and/or large bowel post-esophagectomy (HHBPE) in the current era of improving long-term survival and evolving surgical technique is unknown. The aim of this study was to describe the rate and risk factors of HHBPE at our hospital. METHODS: Patients undergoing esophagectomy between January 2011 and June 2017 were included if both follow-up information and axial imaging were available beyond 3 months post-esophagectomy. Patient characteristics, disease information, and treatment factors were all included in univariate analysis comparing patients with and without HHBPE, and multivariate regression was used to identify significant independent risk factors associated with HHBPE. RESULTS: Of 310 esophagectomy patients analyzed, 258 patients were included in the study, with 79 patients (31%) showing evidence of an HHBPE and an overall median follow-up of 24 months; 44 of 79 patients (56%) had symptoms possibly referable to HHBPE and 17 of 79 patients (22%) underwent surgical repair. On univariate analysis, neoadjuvant therapy (n = 176), higher clinical stage, minimally invasive approach (n = 154), and transhiatal esophagectomy (n = 189) were significant predictors of HHBPE (p < 0.05). On multivariate analysis, neoadjuvant therapy and transhiatal approach remained significant independent predictors (p < 0.05). The rate of HHBPE was 44% in the 131 patients (51%) that had both factors. CONCLUSIONS: HHBPE in the current era of neoadjuvant therapy and minimally invasive esophagectomy is common. HHBPE can cause gastrointestinal symptoms, but operation to repair HHBPE is uncommon on intermediate follow-up. Additional study and long-term follow-up are required to fully assess the impact of HHBPE and to potentially modify surgical practice to prevent or minimize HHBPE.

Analysis of Site-Specific Phosphorylation of PTEN by Using Enzyme-Catalyzed Expressed Protein Ligation.

The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C terminus. Prior enzymatic characterization of the four monophosphorylated (1p) PTENs by using classical expressed protein ligation (EPL) was complicated by the inclusion of a non-native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here, we apply subtiligase-mediated EPL to create wt 1p-PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with the role of Tyr379 in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p-PTENs, which are poorly recognized by an anti-phospho-PTEN antibody, three of the four wt 1p-PTENs are recognized by a commonly used anti-phospho-PTEN antibody.

Nationwide Analysis of 30-Day Readmissions After Esophagectomy: Causes, Costs, and Risk Factors.

BACKGROUND: Postsurgical readmissions are an increasingly scrutinized marker of health care quality. We sought to estimate the rate, risk factors, causes, and costs associated with readmissions after esophagectomy in a large, nationally representative cohort. METHODS: We studied patients from the Nationwide Readmissions Database undergoing esophagectomy from 2010 to 2014. Data were collected on the prevalence and indications for readmission within 30 days as well as the hospital-, procedure-, and patient-level risk factors as determined by multivariable logistic regression. RESULTS: Among 13,282 cases, the rate of 30-day readmission was 19.4%, with the most common indications for readmission being pulmonary (20.6%) and gastrointestinal complications (20%). Median cost of readmission was $9660 (interquartile range, $5392 to $20,447), and pulmonary complications accounted for the greatest total cost burden at 25.8% of all readmission-related costs. Independent risk factors for readmission on multivariable analysis included perioperative blood transfusion (adjusted odds ratio [AOR] 1.33; 95% confidence interval [CI], 1.08 to 1.65; P = .008), discharge to a nursing facility (AOR 1.83; 95% CI, 1.41 to 2.39; P < .001), high illness severity based on All Patients Refined Diagnosis-Related Groups scoring (AOR 1.49; 95% CI, 1.21 to 1.84; P < .001), chronic renal failure (AOR 1.61; 95% CI, 1.13 to 2.29; P = .009), and comorbid drug abuse (AOR 2.19; 95% CI, 1.08 to 4.41; P = .029). CONCLUSIONS: Nearly 1 in 5 patients undergoing esophagectomy are readmitted within 30 days of discharge, at a median cost of $9660 per readmission. Pulmonary complications account for the greatest number of readmissions and the greatest total cost burden. Targeting the causes of readmission, especially pulmonary causes, may help significantly reduce the total morbidity and health care costs associated with esophagectomy.

Correction to: The Association of Body Mass Index with Postoperative Outcomes After Elective Paraesophageal Hernia Repair.

In this paper, the first author's name was incorrectly tagged.

The Malnourished Patient With Obesity: A Unique Paradox in Bariatric Surgery.

BACKGROUND: Hypoalbuminemia is a known risk factor for poor outcomes following surgery. Obesity can be associated with modest to severe malnutrition. We evaluated the impact of hypoalbuminemia on surgical outcomes in patients with obesity undergoing elective bariatric surgical procedures. MATERIALS AND METHODS: The 2015 metabolic and bariatric surgery accreditation and quality improvement program database was queried. Patients ≥ 18 y with body mass index ≥35 undergoing bariatric surgery were included. Revision procedures were excluded. Patients were classified by albumin level (albumin ≥3.5 g/dL [normal], 3.49-3.0 g/dL [mild], 2.99-2.5 g/dL [moderate], and <2.5 g/dL [severe]). Independent logistic regression models were developed to estimate the adjusted odds of (1) death or serious morbidity (DSM); (2) mild to moderate complications; (3) severe complications; and (4) 30-d readmissions by albumin level. In addition, effect modification by >10% weight loss was examined. RESULTS: A total of 106,577 patients were included in the study. Over 6% of patients had hypoalbuminemia. Fifty-five percent of complications were severe as categorized by the Clavien-Dindo classification. Patients with mild hypoalbuminemia had 20% increased odds of DSM (95% confidence interval: 1.1-1.4). There was increasing likelihood of DSM with severe hypoalbuminemia. Patients with mild hypoalbuminemia had 20% increased odds of 30-d readmission (confidence interval: 1.1-1.3). A >10% weight loss modified the effect of moderate to severe hypoalbuminemia on DSM. CONCLUSIONS: More than 6% of patients with obesity undergoing bariatric surgery are malnourished. Hypoalbuminemia is an important and modifiable risk factor for postoperative adverse outcomes following bariatric surgery. Preoperative weight loss >10% combined with moderate to severe hypoalbuminemia is synergistic for high rates of DSM and should be addressed before proceeding with bariatric surgery.

The Association of Body Mass Index with Postoperative Outcomes After Elective Paraesophageal Hernia Repair.

PURPOSE: To evaluate the association between body mass index (BMI) and postoperative outcomes in elective paraesophageal hernia (PEH) repairs. METHODS: A retrospective review of patients who underwent elective PEH repair in the ACS NSQIP database (2005-2015) was performed. Patients were stratified into BMI groups (< 18.5, 18.5-24.9, 25.0-29.9, 30.0-34.9, 35-39.9, and ≥ 40.0 kg/m2) according to the World Health Organization classification criteria. A multivariable logistic regression model was developed to characterize the association between BMI class and outcomes, including readmission, reoperation, postoperative complications, and mortality. RESULTS: The median (IQR) age of the 9641 patients who met inclusion criteria was 64 (55-72) and 72.7% were women. Across each BMI class, age, race, gender, type of procedure, frailty index, smoking, and ASA class varied (p < 0.05). Underweight patients (BMI < 18.5 kg/m2) had an increased risk of mortality (OR = 6.35, p < 0.05). Patients with a BMI 35-39.9 kg/m2 (OR = 0.65, p < 0.05) and ≥ 40 kg/m2 (OR = 0.36, p < 0.001) were associated with a decreased risk for readmissions. CONCLUSION: Underweight patients have an increased risk for postoperative mortality after elective PEH repair. Higher BMI was associated with a diminished risk for readmission, but not for mortality, reoperations, or overall complications.

Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis.

Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.

Protein Chemical Approaches to Understanding PTEN Lipid Phosphatase Regulation.

Since the discovery of C-tail phosphorylation of PTEN almost 20 years ago, much progress has been made in understanding its regulatory influences on the cellular function of PTEN. Phosphorylation of Ser380, Thr382, Thr383, and Ser385 drives a PTEN conformational change from an open to closed state where catalytic function is impaired, plasma membrane binding is reduced, and cellular stability is enhanced. Despite these advances, a detailed structural and mechanistic model of how these phosphorylations impact PTEN function is lacking. We discuss here several recent approaches to analyzing PTEN phosphorylation and highlight several insights that have come from this work. We also discuss remaining challenges for the PTEN regulation field and potential directions for future research.

Site-Specific Protein Labeling with N-Hydroxysuccinimide-Esters and the Analysis of Ubiquitin Ligase Mechanisms.

N-Hydroxysuccinimide (NHS)-esters are widely used to label proteins nonselectively on free amino groups. Such broad labeling can be disadvantageous because it can interfere with protein structure or function and because stoichiometry is poorly controlled. Here we describe a simple method to transform NHS-esters into site-specific protein labeling on N-terminal Cys residues. MESNA addition converts NHS-esters to chemoselective thioesters for N-Cys modification. This labeling strategy was applied to clarify mechanistic features of the ubiquitin E3 ligase WWP2 including its interaction with one of its substrates, the tumor suppressor PTEN, as well as its autoubiquitination molecularity. We propose that this convenient protein labeling strategy will allow for an expanded application of NHS-esters in biochemical investigation.

Resection of gallbladder remnants after subtotal cholecystectomy: presentation and management.

BACKGROUND: Subtotal cholecystectomy (SC) involves removal of a portion of the gallbladder typically due to hazardous inflammation. While this technique reliably prevents common bile duct (CBD) injury, future procedures can be required if the gallbladder remnant becomes symptomatic. The morbidity associated with resection of gallbladder remnants in patients that previously underwent SC is reviewed. METHODS: Records for patients having undergone redo cholecystectomy for symptomatic gallbladder remnants in a tertiary care system from 2013 to 2017 were retrospectively reviewed. RESULTS: Fourteen patients underwent repeat cholecystectomy. Five surgeons dictated the initial procedure as a subtotal cholecystectomy. All patients returned with symptomatic cholelithiasis between zero months and seven years after the index cholecystectomy. Redo cholecystectomy was attempted laparoscopically in two patients but ultimately required an open approach in all. One patient had a recognized CBD injury requiring a hepaticojejunostomy, and a second patient had a minor wound infection. Symptoms resolved in 13/14 patients. CONCLUSIONS: Redocholecystectomy (RC) for gallbladder remnants has been detailed in case reports, but no sizable North American series have been presented. These results illustrate a drawback to the reconstituting technique of SC. RC effectively resolves symptoms but requires adherence to safe principles of cholecystectomy and is one indication for an open approach.