Comorbidities Drive the Majority of Overall Mortality in Low-Risk Mucinous Pancreatic Cysts Under Surveillance.

BACKGROUND & AIMS: The Charlson Comorbidity Index (CACI) has been suggested as a tool to determine comorbidity burden and guide management for patients with mucinous pancreatic cysts (Intrapapillary Mucinous Neoplasms and Mucinous Cystic Neoplasms), but has not been studied well among "low-risk" mucinous pancreatic cysts i.e. without worrisome features (WF) and high-risk stigmata (HRS). This study sought to determine the comorbidity burden among surveillance population of low-risk pancreatic cysts and provide their follow-up mortality outcomes. METHODS: A single center study retrospectively reviewed a prospective pancreatic cyst database and included individuals with low-risk cysts undergoing serial imaging during 2016. Electronic medical records were reviewed to determine their baseline age-adjusted CACI (age-CACI). After 4 years, their progression to WF, disease specific (pancreatic malignancy-related, DSM), extra-pancreatic (EPM), and overall mortalities (OM) were determined using Kaplan-Meir Survival Analysis. RESULTS: 502 individuals underwent prospective surveillance. The study included 440 individuals with low-risk suspected or presumed mucinous cysts and excluded 50 and 12 individuals with WF and HRS respectively. Over a median follow-up of 56 months, 12 WF progressions, 2 DSMs, 42 EPMs, and 44 OMs were observed. Baseline age-CACI had good predictive capacity for 4-year EPM (Area-Under Curve: 0.87; p< .0001). The median age-CACI of 4 enabled cohort stratification into Low (age-CACI <4) and High CACI (age-CACI ≥4) groups. A significantly higher OM (p< .001) was observed among the High CACI group as compared to the Low CACI group. CONCLUSION: Through real-time application of CACI to patient outcomes, our analysis supports incorporation of this comorbidity assessment tool in making shared surveillance decisions among low-risk pancreatic cyst population.

Peroral endoscopic myotomy for management of cricopharyngeal bars (CP-POEM): a retrospective evaluation.

BACKGROUND: Cricopharyngeal bars (CPBs) are a unique etiology of oropharyngeal dysphagia. Symptomatic patients are managed with endoscopic dilation or surgical myotomy. Cricopharyngeal peroral endoscopic myotomy (CP-POEM) is an emerging technique for the management of dysphagia due to CPBs. This study evaluated technical success, clinical success, adverse events, and long-term recurrence following CP-POEM. METHODS: Consecutive patients who underwent POEM for management of CPBs between May 2015 and December 2020 at four tertiary care centers were included. Primary outcome was clinical success (defined as improvement of dysphagia score to ≤ 1). Secondary outcomes were technical success, rate and severity of adverse events, procedure duration, and symptom recurrence. RESULTS: 27 patients (mean age 69 years; 10 female) underwent CP-POEM during the study period. The most common presenting symptoms at the time of index procedure were dysphagia (26; 96.3 %) and regurgitation (20; 74.1 %). Clinical and technical success were achieved in all patients. Mild/moderate adverse events occurred in two patients (7.4 %). CP-POEM significantly reduced the median dysphagia score. CONCLUSIONS: CP-POEM was a safe and effective treatment for symptomatic CPBs. Although symptom recurrence was low, long-term outcome data are needed. CP-POEM should be considered as a management option for symptomatic CPBs at centers with POEM expertise.

Zenker's peroral endoscopic myotomy, or flexible or rigid septotomy for Zenker's diverticulum: a multicenter retrospective comparison.

BACKGROUND: Treatment of Zenker's diverticulum has evolved from open surgery to endoscopic techniques, including flexible and rigid endoscopic septotomy, and more recently, peroral endoscopic myotomy (Z-POEM). This study compared the effectiveness of flexible and rigid endoscopic septotomy with that of Z-POEM. METHODS: Consecutive patients who underwent endoscopic septotomy (flexible/rigid) or Z-POEM for Zenker's diverticulum between 1/2016 and 9/2019 were included. Primary outcomes were clinical success (decrease in Dakkak and Bennett dysphagia score to ≤ 1), clinical failure, and clinical recurrence. Secondary outcomes included technical success and rate/severity of adverse events. RESULTS: 245 patients (110 females, mean age 72.63 years, standard deviation [SD] 12.37 years) from 12 centers were included. Z-POEM was the most common management modality (n = 119), followed by flexible (n = 86) and rigid (n = 40) endoscopic septotomy. Clinical success was 92.7 % for Z-POEM, 89.2 % for rigid septotomy, and 86.7 % for flexible septotomy (P = 0.26). Symptoms recurred in 24 patients (15 Z-POEM during a mean follow-up of 282.04 [SD 300.48] days, 6 flexible, 3 rigid [P = 0.47]). Adverse events occurred in 30.0 % rigid septotomy patients, 16.8 % Z-POEM patients, and 2.3 % flexible septotomy patients (P < 0.05). CONCLUSIONS: There was no difference in outcomes between the three treatment approaches for symptomatic Zenker's diverticulum. Rigid endoscopic septotomy was associated with the highest rate of complications, while flexible endoscopic septotomy appeared to be the safest. Recurrence following Z-POEM was similar to flexible and rigid endoscopic septotomy. Prospective studies with long-term follow-up are required.

General Dialdehyde Click Chemistry for Amine Bioconjugation.

The development of methods for conjugating a range of molecules to primary amine functional groups has revolutionized the fields of chemistry, biology, and material science. The primary amine is a key functional group and one of the most important nucleophiles and bases used in all of synthetic chemistry. Therefore, tremendous interest in the synthesis of molecules containing primary amines and strategies to devise chemical reactions to react with primary amines has been at the core of chemical research. In particular, primary amines are a ubiquitous functional group found in biological systems as free amino acids, as key side chain lysines in proteins, and in signaling molecules and metabolites and are also present in many natural product classes. Due to its abundance, the primary amine is the most convenient functional group handle in molecules for ligation to other molecules for a broad range of applications that impact all scientific fields. Because of the primary amine's central importance in synthetic chemistry, acid-base chemistry, redox chemistry, and biology, many methods have been developed to efficiently react with primary amines, including activated carboxylic acids, isothiocyanates, Michael addition type systems, and reaction with ketones or aldehydes followed by in situ reductive amination. Herein, we introduce a new traceless, high-yield, fast click-chemistry method based on the rapid and efficient trapping of amine groups via a functionalized dialdehyde group. The click reaction occurs in mild conditions in organic solvents or aqueous media and proceeds in high yield, and the starting dialdehyde reagent and resulting dialdehyde click conjugates are stable. Moreover, no catalyst or dialdehyde-activating group is required, and the only byproduct is water. The initial dialdehyde and the resulting conjugate are both straightforward to characterize, and the reaction proceeds with high atom economy. To demonstrate the broad scope of this new click-conjugation strategy, we designed a straightforward scheme to synthesize a suite of dialdehyde reagents. The dialdehyde molecules were used for applications in cell-surface engineering and for tailoring surfaces for material science applications. We anticipate the broad utility of the general dialdehyde click chemistry to primary amines in all areas of chemical research, ranging from polymers and bioconjugation to material science and nanoscience.

Generation of a Scaffold-Free Three-Dimensional Liver Tissue via a Rapid Cell-to-Cell Click Assembly Process.

There has been tremendous interest in constructing in vitro liver organ models for a range of fundamental studies of cell signaling, metabolism, and infectious diseases, and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress toward studying two-dimensional hepatic function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free three-dimensional multiple cell line coculture tissue models of liver. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts among multiple hepatic cell lines to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a three cell line coculture 3D liver tissue model by assembling hepatocytes, hepatic endothelial cells, and hepatic stellate cells via a rapid intercell click ligation process. We compare and analyze the function of the superior 3D liver tissue chips with 2D coculture monolayer by assessing mitochondrial metabolic activity and evaluating drug toxicity.

Rewiring Gram-Negative Bacteria Cell Surfaces with Bio-Orthogonal Chemistry via Liposome Fusion.

The ability to tailor bacteria cell surfaces with non-native molecules is critical to advance the study of bacteria communication, cell behavior, and for next-generation therapeutics to improve livestock and human health. Such modifications would allow for novel control over cell behavior, cell-cell interactions, biofilm formation, adjuvant conjugation, and imaging. Current methods to engineer bacteria surfaces have made major advances but rely on complicated, slow, and often expensive molecular biology and metabolic manipulation methods with limited scope on the type of molecules installed onto the surface. In this report, we introduce a new straightforward method based on liposome fusion to engineer Gram-negative bacteria cells with bio-orthogonal groups that can subsequently be conjugated to a range of molecules (biomolecules, small molecules, probes, proteins, nucleic acids, ligands, and radiolabels) for further studies and programmed behavior of bacteria. This method is fast, efficient, inexpensive, and useful for installing a broad scope of ligands and biomolecules to Gram-negative bacteria surfaces.

Spheroid and Tissue Assembly via Click Chemistry in Microfluidic Flow.

Proper cell-cell contact and communication are essential for the correct development and survival of higher order organisms. In order to study complex cell interactions that occur in vivo, model systems that are able to recapitulate 3D cell-cell interactions in vitro are key to advancing new biotechnologies, therapeutics, and tissue engineering applications. Herein, we show a new strategy to rapidly and efficiently generate complex multiple cell line containing spheroids and tissues in microfluidic flow without the use of scaffolds, molecular biology, or metabolic biosynthesis. The method relies on the integration of microfluidics, liposome fusion, bio-orthogonal chemistry, and cell surface engineering to rapidly click coculture cell assemblies in flow. We demonstrate this strategy by assembling various combinations of cell types with an interfacial cell to cell click chemistry in microfluidic flow to generate a range of spheroid types and oriented tissue multilayers.

General chemoselective and redox-responsive ligation and release strategy.

We report a switchable redox click and cleave reaction strategy for conjugating and releasing a range of molecules on demand. This chemoselective redox-responsive ligation (CRRL) and release strategy is based on a redox switchable oxime linkage that is controlled by mild chemical or electrochemical redox signals and can be performed at physiological conditions without the use of a catalyst. Both conjugation and release reactions are kinetically well behaved and quantitative. The CRRL strategy is synthetically modular and easily monitored and characterized by routine analytical techniques. We demonstrate how the CRRL strategy can be used for the dynamic generation of cyclic peptides and the ligation of two different peptides that are stable but can be selectively cleaved upon changes in the redox environment. We also demonstrate a new redox based delivery of cargoes to live cells strategy via the CRRL methodology by synthesizing a FRET redox-responsive probe that is selectively activated within a cellular environment. We believe the ease of the CRRL strategy should find wide use in a range of applications in biology, tissue engineering, nanoscience, synthetic chemistry, and material science and will expand the suite of current conjugation and release strategies.

In situ modulation of cell behavior via smart dual-ligand surfaces.

Due to the highly complex nature of the extracellular matrix (ECM), the design and implementation of dynamic, stimuli-responsive surfaces that present well-defined ligands and serve as model ECM substrates have been of tremendous interest to biomaterials, biosensor, and cell biology communities. Such tools provide strategies for identifying specific ligand-receptor interactions that induce vital biological consequences. Herein, we report a novel dual-ligand-presenting surface methodology that modulates dynamic ECM properties to investigate various cell behaviors. Peptides PHSRN, cRGD, and KKKTTK, which mimic the cell- and heparan sulfate-binding domains of fibronectin, and carbohydrates Gal and Man were combined with cell adhesive RGD to survey possible synergistic or antagonist ligand effects on cell adhesion, spreading, growth, and migration. Soluble molecule and enzymatic inhibition assays were also performed, and the levels of focal adhesion kinase in cells subjected to different ligand combinations were quantified. A redox-responsive trigger was incorporated into this surface strategy to spontaneously release ligands in the presence of adhered cells, and cell spreading, growth, and migration responses were measured and compared. The identity and nature of the dual-ligand combination directly influenced cell behavior.

PI3 kinase enzymology on fluid lipid bilayers.

We report the use of fluid lipid bilayer membrane as a model platform to study the influence of the bilayer microenvironment and composition on the enzymology in membrane. As a model system we determined the enzyme kinetics on membranes for the transformation of bilayers containing phosphoinositol(4,5)-bisphosphate (PI(4,5)P2) to phosphoinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) by the enzyme phosphoinositol-3-kinase (PI3K) using radiolabeled ATP. The activity of the enzyme was monitored as a function of the radioactivity incorporated within the bilayer. The transformation of PI(4,5)P2 to PI(3,4,5)P3 was determined using a mass strip assay. The fluidity of the bilayer was confirmed by Fluorescence Recovery After Photobleaching (FRAP) experiments. Kinetic simulations were performed based on Langmuir adsorption and Michaelis-Menton kinetics equations to generate the rate constants for the enzymatic reaction. The effect of cholesterol on the enzyme kinetics was studied by doping the bilayer with 1% cholesterol. This leads to significant reduction in reaction rate due to change in membrane microenvironment. This strategy provides a method to study the enzymology of various kinases and phosphatases occurring at the membrane and also how these reactions are affected by the membrane composition and surface microenvironment.

Cell-surface engineering by a conjugation-and-release approach based on the formation and cleavage of oxime linkages upon mild electrochemical oxidation and reduction.

We report a strategy to rewire cell surfaces for the dynamic control of ligand composition on cell membranes and the modulation of cell-cell interactions to generate three-dimensional (3D) tissue structures applied to stem-cell differentiation, cell-surface tailoring, and tissue engineering. We tailored cell surfaces with bioorthogonal chemical groups on the basis of a liposome-fusion and -delivery method to create dynamic, electroactive, and switchable cell-tissue assemblies through chemistry involving chemoselective conjugation and release. Each step to modify the cell surface: activation, conjugation, release, and regeneration, can be monitored and modulated by noninvasive, label-free analytical techniques. We demonstrate the utility of this methodology by the conjugation and release of small molecules to and from cell surfaces and by the generation of 3D coculture spheroids and multilayered cell tissues that can be programmed to undergo assembly and disassembly on demand.

Overlapping Hepatotoxicity and Colitis Associated with Immune Checkpoint Inhibitors.

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that are widely used for the management of many solid-organ and hematologic cancers. These agents work by inhibition of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), programmed cell death-1 (PD-1), and PD ligand 1 (PD-L1). Hyperactivation of immune system results in ICI-associated adverse events. Simultaneous hepatotoxicity and colitis associated with ICIs is rare and potentially overlooked, as clinical symptoms are often nonspecific. A 73-year-old man with metastatic squamous cell carcinoma presented six weeks after starting pembrolizumab with abdominal discomfort and diarrhea. Pembrolizumab therapy was held, and supportive therapy with antidiarrheals provided partial relief. After initial workup, ICI-associated hepatitis (ICIH) and ICI-related colitis (ICIC) were diagnosed. Colitis resolution required corticosteroids. This case illustrates the importance of high index of clinical suspensions for gastrointestinal and hepatic adverse events associated with ICIs, which may be overlooked and result in severe complications. While isolated ICIH and ICIC are well known adverse events, overlapping ICIH and ICIC is rare. Prompt recognition, cessation of the inciting agent, and initiation of early supportive therapy are essential. Treatment may require corticosteroids or mycophenolate mofetil.

EUS-guided versus percutaneous liver biopsy: A prospective randomized clinical trial.

Background and Objectives: Prospective studies comparing EUS-guided liver biopsy (EUS-LB) to percutaneous LB (PC-LB) are scarce. We compared the efficacy and safety of EUS-LB with those of PC-LB in a prospective randomized clinical trial. Methods: Between 2020 and 2021, patients were enrolled and randomized (1:1 ratio). The primary outcome was defined as the proportion of patients with ≥11 complete portal tracts (CPTs). The sample size (n = 80) was calculated based on the assumption that 60% of those in the EUS-LB and 90% of those in the PC-LB group will have LB with ≥11 CPTs. The secondary outcomes included proportion of patients in whom a diagnosis was established, number of CPTs, pain severity (Numeric Rating Scale-Pain Intensity), duration of hospital stay, and adverse events. Results: Eighty patients were enrolled (median age, 53 years); 67.5% were female. Sixty percent of those in the EUS-LB and 75.0% of those in the PC-LB group met the primary outcome (P = 0.232). The median number of CPTs was higher in the PC-LB (17 vs 13; P = 0.031). The proportion of patients in whom a diagnosis was established was similar between the groups (92.5% [EUS-LB] vs 95.0% [PC-LB]; P = 1.0). Patients in the EUS-LB group had less pain severity (median Numeric Rating Scale-Pain Intensity, 2.0 vs 3.0; P = 0.003) and shorter hospital stay (2.0 vs 4.0 hours; P < 0.0001) compared with the PC-LB group. No patient experienced a serious adverse event. Conclusions: EUS-guided liver biopsy was safe, effective, better tolerated, and associated with a shorter hospital stay.

Respiratory Syncytial Virus Associated Hepatitis in Pregnancy.

Respiratory syncytial virus (RSV) predominantly affects children and typically manifests as an upper respiratory tract infection. Primary RSV infection in immunosuppressed adults may increase risks of disseminated infection manifesting as RSV hepatitis. A 29-year-old pregnant woman of 10 weeks gestation presented with mild right upper quadrant abdominal pain, intractable nausea, and vomiting, requiring hospitalization. Due to initial lab work showing significantly elevated liver transaminases, she underwent a thorough workup to evaluate for causes of hepatitis. Common viral and autoimmune etiologies of hepatitis were excluded with appropriate serologies. A respiratory viral molecular panel (RVP) was obtained to evaluate for SARS-CoV-2/coronavirus disease 2019 (COVID-19) infection, despite lack of typical respiratory symptoms. No structural pathologies were detected on abdominal imaging with ultrasound and magnetic resonance imaging. No other etiologies for the patient's hepatitis were detected other than RSV infection detected on RVP. The patient's care required close coordination between multiple different subspecialties. Her condition improved due to the early detection of RSV infection and prompt initiation of supportive care. This case highlights the need for providers to consider obtaining an RVP early in workup of hepatitis to evaluate for RSV infection, even when patients have minimal respiratory symptoms. A high index of suspicion is required for early identification of RSV hepatitis as timely supportive care may prevent progression to acute liver failure.

International external validation of a stratification tool to identify branch-duct intraductal papillary mucinous neoplasms at lowest risk of progression.

BACKGROUND: Identifying branch-duct intraductal papillary mucinous neoplasms (BD-IPMNs) at lowest risk of progression may allow for a reduced intensity of surveillance. OBJECTIVE: We aimed to externally validate the previously developed Dutch-American Risk stratification Tool (DART-1; https://rtools.mayo.edu/DART/), which identifies cysts at low risk of developing worrisome features (WFs) or high-risk stigmata (HRS). METHODS: Three prospective cohorts of individuals under surveillance for BD-IPMNs were combined, independent from the original development cohort. We assessed the performance (discrimination and calibration) of DART-1, a multivariable Cox-proportional logistic regression model with five predictors for the development of WFs or HRS. RESULTS: Of 832 individuals (mean age 77 years, SD 11.5) under surveillance for a median of 40 months (IQR 44), 163 (20%) developed WFs or HRS. DART-1's discriminative ability (C-statistic 0.68) was similar to that in the development cohort (0.64-0.72) and showed moderate calibration. DART-1 adequately estimated the risk for patients in the middle risk quintile, and slightly underestimated it in the lowest quintiles. Their range of predicted versus observed 3-year risk was 0%-0% versus 0%-3.7% for Q1; 0.3%-0.4% versus 3%-11% for Q2; and 2.6%-3% versus 2.4%-9.8% for Q3. The development of WFs or HRS was associated with pancreatic cancer (p < 0.001). Vice versa, in absence of WFs or HRS, the risk of malignancy was low (0.3%). CONCLUSIONS: The performance of DART-1 to predict the development of WFs or HRS in BD-IPMN was validated in an external international cohort, with a discriminative ability equal as in the development cohort. Risk estimations were most accurate for patients with BD-IPMNs in the middle risk quintile and slightly underestimated in the lowest quintiles.

Tissue morphing control on dynamic gradient surfaces.

In this report, we develop smart surfaces for the spatial and temporal control of mammalian cell behavior. We integrate a bioactive surface strategy with a photo-electroactive surface strategy to generate dynamic ligand surface gradients for controlling cell adhesion, tissue shape morphing, and cell tissue migration.

Redox-switchable surface for controlling peptide structure.

A general surface chemistry strategy is described for the development of a new switchable material. The method modulates a surface-immobilized-molecules structure by using two orthogonal "click" reactions based on Huisgen cycloaddition and oxime chemistry, where the oxime linkage is redox active and switchable. We demonstrate this strategy by developing a noninvasive, biocompatible, in situ surface chemistry that is able to modulate the affinity of a cell-adhesive peptide to cell integrin receptors to study dynamic cell adhesion and cell migration in real time and as a new hide-and-reveal strategy for application in new types of smart biofouling biomaterials.

Synthetic chemoselective rewiring of cell surfaces: generation of three-dimensional tissue structures.

Proper cell-cell communication through physical contact is crucial for a range of fundamental biological processes including, cell proliferation, migration, differentiation, and apoptosis and for the correct function of organs and other multicellular tissues. The spatial and temporal arrangements of these cellular interactions in vivo are dynamic and lead to higher-order function that is extremely difficult to recapitulate in vitro. The development of three-dimensional (3D), in vitro model systems to investigate these complex, in vivo interconnectivities would generate novel methods to study the biochemical signaling of these processes, as well as provide platforms for tissue engineering technologies. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. This strategy uses liposome fusion for the delivery of ketone or oxyamine groups to different populations of cells for subsequent cell assembly via oxime ligation. We demonstrate how this method can be used for several applications including, the delivery of reagents to cells for fluorescent labeling and cell-surface engineering, the formation of small, 3D spheroid cell assemblies, and the generation of large and dense, 3D multilayered tissue-like structures for tissue engineering applications.

Engineering cell surfaces via liposome fusion.

In this study, we have rewired cell surfaces with ketone and oxyamine molecules based on liposome fusion for applications in cell-surface engineering. Lipid vesicles, functionalized with ketone and oxyamine molecules, display complementary chemistry and undergo recognition, docking, and subsequent fusion upon covalent oxime bond formation. Liposome fusion was characterized by several techniques including matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), light scattering, fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). When cultured with cells, ketone- and oxyamine-containing liposomes undergo spontaneous membrane fusion to present the respective molecules from cell surfaces. Ketone-functionalized cell surfaces serve as sites for chemoselective ligation with oxyamine-conjugated molecules. We tailored and fluorescently labeled cell surfaces with an oxyamine-conjugated rhodamine dye. As an application of this cell-surface engineering strategy, ketone- and oxyamine-functionalized cells were patterned on oxyamine- and ketone-presenting surfaces, respectively. Cells adhered, spread, and proliferated in the patterned regions via interfacial oxime linkage. The number of ketone molecules on the cell surface was also quantified by flow cytometry.

Biomolecular modification of carbon nanotubes for studies of cell adhesion and migration.

We report a strategy for tailoring and patterning carbon nanotubes (CNTs) for biospecific cell studies. We synthesized a new electroactive hydroquinone terminated pyrene molecule to tailor CNTs. These modified CNTs can be oxidized and chemoselectively reacted with oxyamine tethered ligands to generate various ligand tethered CNTs. A cell adhesive Arg-Gly-Asp peptide (RGD) is immobilized to the CNTs and a new microfluidic patterning method is employed to generate multiplex patterned surfaces for biospecific cell adhesion and migration studies. This work demonstrates the integration of a new functionalization strategy to immobilize a variety of ligands to CNTs for a range of potential drug delivery, tissue imaging and cellular behavior studies and a microfluidic patterning strategy for generating complex high-throughput surfaces for biotechnological and cell based assay applications.