Optimization of Enzyme Essays to Enhance Reliability of Activity Measurements in Leukocyte Lysates for the Diagnosis of Metachromatic Leukodystrophy and Gangliosidoses.

(1) Lysosomal storage diseases are rare inherited disorders with no standardized or commercially available tests for biochemical diagnosis. We present factors influencing the quality of enzyme assays for metachromatic leukodystrophy (MLD) and gangliosidoses (GM1; GM2 variants B and 0) and validate the reliability and stability of testing in a retrospective analysis of 725 samples. (2) Patient leukocytes were isolated from ethylene-diamine-tetra-acetic acid (EDTA) blood and separated for subpopulation experiments using density gradient centrifugation or magnetic cell separation. Enzyme activities in whole leukocyte lysate and leukocyte subpopulations were determined. (3) The enzyme activities in leukocyte subpopulations differed significantly. Compared to lymphocytes, the respective enzyme activities were 2.31-4.57-fold higher in monocytes and 1.64-2.81-fold higher in granulocytes. During sample preparation, a considerable amount of the lysosomal enzymes was released from granulocytes. Nevertheless, with the sample preparation method used here, total leukocyte count proved to be more accurate than total protein amount as a reference unit for enzyme activities. Subsequent analysis of 725 individuals showed clear discrimination of enzyme activities in patient samples (48 MLD; 21 gangliosidoses), with a sensitivity of 100% and specificity of 98-99%.

Characterization of megahertz X-ray laser beams by multishot desorption imprints in PMMA.

Proper diagnostics of intense free-electron laser (FEL) X-ray pulses is indisputably important for experimental data analysis as well as for the protection of beamline optical elements. New challenges for beam diagnostic methods are introduced by modern FEL facilities capable of delivering powerful pulses at megahertz (MHz) repetition rates. In this paper, we report the first characterization of a defocused MHz 13.5-nm beam generated by the free-electron laser in Hamburg (FLASH) using the method of multi-pulse desorption imprints in poly(methyl methacrylate)(PMMA). The beam fluence profile is reconstructed in a novel and highly accurate way that takes into account the nonlinear response of material removal to total dose delivered by multiple pulses. The algorithm is applied to experimental data of single-shot ablation imprints and multi-shot desorption imprints at both low (10 Hz) and high (1 MHz) repetition rates. Reconstructed response functions show a great agreement with the theoretical desorption response function model.

Changing epidemiology of esophageal stent placement for dysphagia: a decade of trends and the impact of benign indications.

BACKGROUND AND AIMS: In addition to managing malignant obstruction, esophageal stents (ESs) have evolved to address various benign etiologies of dysphagia. We sought to evaluate national trends and changes in practice of ES placement for both benign and malignant etiologies in hospitalized patients with dysphagia. METHODS: The National Inpatient Sample (2003-2013) was used to include all adult inpatients (≥18 years of age) with endoscopy-guided ES placement for a symptom of dysphagia. Multivariable analyses for indications that impact temporal trends (3 time periods: 2003-2005, 2006-2009, and 2010-2013) and for hospital outcomes were performed. RESULTS: A total of 7198 ESs were deployed endoscopically in hospitalized patients with dysphagia. Compared with malignant etiologies, there was a significant increase in ES placement for benign conditions (2013 vs 2003: 32.7% vs 14.5%, respectively; P < .001). Multivariable analysis using 2003 to 2005 as a reference showed that patients with benign etiologies for dysphagia predominantly contributed to the increase of ES placement during the most recent time period (2010-2013: odds ratio, 2.09; 95% confidence interval, 1.40-3.13). Multivariable analysis of hospital outcomes revealed no differences in inpatient mortality, duration of hospital stay, and hospital costs between malignant and benign indications. CONCLUSIONS: In the preceding decade, ES placement for hospitalized patients with dysphagia has increased, driven largely by an over 8-fold rise in stent placement for benign indications. These findings warrant continued efforts to improve stent technology to decrease the risk of migration and review practice guidelines involving ES placement for benign etiologies.

EUS-guided confocal laser endomicroscopy: prediction of dysplasia in intraductal papillary mucinous neoplasms (with video).

BACKGROUND AND AIMS: Previous studies have validated EUS-guided needle-based confocal laser endomicroscopy (nCLE) diagnosis of intraductal papillary mucinous neoplasms (IPMNs). We sought to derive EUS-guided nCLE criteria for differentiating IPMNs with high-grade dysplasia/adenocarcinoma (HGD-Ca) from those with low/intermediate-grade dysplasia (LGD). METHODS: We performed a post hoc analysis of consecutive IPMNs with a definitive diagnosis from a prospective study evaluating EUS-guided nCLE in the diagnosis of pancreatic cysts. Three internal endosonographers reviewed all nCLE videos for the patients and identified potential discriminatory EUS-guided nCLE variables to differentiate HGD-Ca from LGD IPMNs (phase 1). Next, an interobserver agreement (IOA) analysis of variables from phase 1 was performed among 6 blinded external nCLE experts (phase 2). Last, 7 blinded nCLE-naïve observers underwent training and quantified variables with the highest IOA from phase 2 using dedicated software (phase 3). RESULTS: Among 26 IPMNs (HGD-Ca in 16), the reference standard was surgical histopathology in 24 and cytology confirmation of metastatic liver lesions in 2 patients. EUS-guided nCLE characteristics of increased papillary epithelial "width" and "darkness" were the most sensitive variables (90%; 95% confidence interval [CI], 84%-94% and 91%; 95% CI, 85%-95%, respectively) and accurate (85%; 95% CI, 78%-90% and 84%; 95% CI, 77%-89%, respectively) with substantial (κ = 0.61; 95% CI, 0.51-0.71) and moderate (κ = 0.55; 95% CI, 0.45-0.65) IOAs for detecting HGD-Ca, respectively (phase 2). Logistic regression models were fit for the outcome of HGD-Ca as predictor variables (phase 3). For papillary width (cut-off ≥50 µm), the sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) for detection of HGD-Ca were 87.5% (95% CI, 62%-99%), 100% (95% CI, 69%-100%), and 0.95, respectively. For papillary darkness (cut-off ≤90 pixel intensity), the sensitivity, specificity, and AUC for detection of HGD-Ca were 87.5% (95% CI, 62%-99%), 100% (95% CI, 69%-100%), and 0.90, respectively. CONCLUSIONS: In this derivation study, quantification of papillary epithelial width and darkness identified HGD-Ca in IPMNs with high accuracy. These quantifiable variables can be used in multicenter studies for risk stratification of IPMNs. (Clinical trial registration number: NCT02516488.).

Acute Biliary Pancreatitis is Associated With Adverse Outcomes in the Elderly: A Propensity Score-Matched Analysis.

GOALS AND BACKGROUND: In the elderly (age, 65 y or older), acute pancreatitis is most frequently because of gallstones; however, there is a paucity of national estimates evaluating outcomes of acute biliary pancreatitis (ABP). Hence, we utilized a representative population database to evaluate the outcomes of ABP among the elderly. STUDY: The National Readmission Database provides longitudinal follow-up of inpatients for 1 calendar-year. All adult inpatients (18 y or older) with an index primary admission for ABP between 2011 and 2014 were evaluated for clinical outcomes of mortality, severe acute pancreatitis (SAP), and 30-day readmission. Outcomes between age groups (≥65 vs. <65 y) were compared using multivariate and one-to-one propensity score-matched analyses. RESULTS: Among 184,763 ABP admissions, 41% were elderly. Index mortality and SAP rates in the elderly were 1.96% and 21.5%, respectively. Elderly patients underwent more ERCPs (27.5% vs. 23.6%; P<0.001) and less frequent cholecystectomies (44.4% vs. 58.7%; P<0.001). Elderly patients had increased odds of mortality and SAP along with an age-dependent increase in the odds of adverse outcomes; patients aged 85 years or older demonstrated the highest odds of SAP [odds ratio (OR), 1.3; 95% confidence interval (CI): 1.2, 1.4] and mortality (OR, 2.2; 95% CI: 1.7, 2.9) within in the elderly cohort. Propensity score-matched analysis substantiated that mortality (OR, 2.8; 95% CI: 2.2, 3.5) and SAP (OR, 1.2; 95% CI: 1.1, 1.3) were increased in the elderly. CONCLUSIONS: Current national survey reveals adverse clinical outcomes among elderly patients hospitalized with ABP. Consequently, there is a need for effective management strategies for this demographic as the aging population is increasing nationally.

Endoscopic intervention and cholecystectomy in pregnant women with acute biliary pancreatitis decrease early readmissions.

BACKGROUND AND AIMS: Acute biliary pancreatitis (ABP) is associated with increased rates of morbidity in pregnancy. Because there is a paucity of population-based studies evaluating ABP in pregnancy, we sought to investigate clinical outcomes in hospitalized pregnant women on a national level. METHODS: By using the Nationwide Readmission Database (2011-2014), we identified all women (age ≥18 years) with an index admission for ABP in the United States. Multivariate and propensity-score matched analyses were performed to evaluate the impact of pregnancy on the clinical outcomes of early readmission and severe acute pancreatitis (SAP) in ABP. RESULTS: There were 7787 hospitalizations for ABP in pregnant women during the study period. The rate of 30-day readmission was 16.26%; 57% of these early readmissions were due to adverse events of ABP. Compared with nonpregnant women with ABP, ERCP (21.1% vs 25.2%; P < .001) and cholecystectomy (52.8% vs 55.2%; P = .02) were performed less frequently during pregnancy. Propensity-score matched analysis revealed an increased risk of 30-day readmissions in pregnancy (odds ratio [OR], 1.96; 95% confidence interval [CI], 1.67-2.30), whereas there was no difference in the risk of SAP (OR, 1.09; 95% CI, 0.76-1.57). Multivariate analysis demonstrated that weekend admission (OR, 1.40; 95% CI, 1.10-1.79) and >1 week of hospitalization (OR, 1.75; 95% CI, 1.24-2.48) increased the risk of 30-day readmission, whereas ERCP (OR, 0.40; 95% CI, 0.27-0.57) and cholecystectomy (OR, 0.13; 95% CI, 0.10-0.18) reduced the odds of early readmission in pregnancy. CONCLUSIONS: Pregnant women with ABP less frequently undergo timely endoscopic biliary decompression and cholecystectomy. These modifiable factors can potentially lower early readmissions in pregnant women.

Mechanism of single-shot damage of Ru thin films irradiated by femtosecond extreme UV free-electron laser.

Ruthenium is a perspective material to be used for XUV mirrors at free-electron laser facilities. Yet, it is still poorly studied in the context of ultrafast laser-matter interaction. In this work, we present single-shot damage studies of thin Ru films irradiated by femtosecond XUV free-electron laser pulses at FLASH. Ex-situ analysis of the damaged spots, performed by different types of microscopy, shows that the weakest detected damage is surface roughening. For higher fluences we observe ablation of Ru. Combined simulations using Monte-Carlo code XCASCADE(3D) and the two-temperature model reveal that the damage mechanism is photomechanical spallation, similar to the case of irradiating the target with optical lasers. The analogy with the optical damage studies enables us to explain the observed damage morphologies.

Experimental study of EUV mirror radiation damage resistance under long-term free-electron laser exposures below the single-shot damage threshold.

The durability of grazing- and normal-incidence optical coatings has been experimentally assessed under free-electron laser irradiation at various numbers of pulses up to 16 million shots and various fluence levels below 10% of the single-shot damage threshold. The experiment was performed at FLASH, the Free-electron LASer in Hamburg, using 13.5 nm extreme UV (EUV) radiation with 100 fs pulse duration. Polycrystalline ruthenium and amorphous carbon 50 nm thin films on silicon substrates were tested at total external reflection angles of 20° and 10° grazing incidence, respectively. Mo/Si periodical multilayer structures were tested in the Bragg reflection condition at 16° off-normal angle of incidence. The exposed areas were analysed post-mortem using differential contrast visible light microscopy, EUV reflectivity mapping and scanning X-ray photoelectron spectroscopy. The analysis revealed that Ru and Mo/Si coatings exposed to the highest dose and fluence level show a few per cent drop in their EUV reflectivity, which is explained by EUV-induced oxidation of the surface.

Role of heat accumulation in the multi-shot damage of silicon irradiated with femtosecond XUV pulses at a 1 MHz repetition rate.

The role played by heat accumulation in multi-shot damage of silicon was studied. Bulk silicon samples were exposed to intense XUV monochromatic radiation of a 13.5 nm wavelength in a series of 400 femtosecond pulses, repeated with a 1 MHz rate (pulse trains) at the FLASH facility in Hamburg. The observed surface morphological and structural modifications are formed as a result of sample surface melting. Modifications are threshold dependent on the mean fluence of the incident pulse train, with all threshold values in the range of approximately 36-40 mJ/cm2. Experimental data is supported by a theoretical model described by the heat diffusion equation. The threshold for reaching the melting temperature (45 mJ/cm2) and liquid state (54 mJ/cm2), estimated from this model, is in accordance with experimental values within measurement error. The model indicates a significant role of heat accumulation in surface modification processes.

Whole-Cell Biosensors as Tools for the Detection of Quorum-Sensing Molecules: Uses in Diagnostics and the Investigation of the Quorum-Sensing Mechanism.

Genetically engineered bacterial whole-cell biosensors are powerful tools that take advantage of bacterial proteins and pathways to allow for detection of a specific analyte. These biosensors have been employed for a broad range of applications, including the detection of bacterial quorum-sensing molecules (QSMs). Bacterial QSMs are the small molecules bacteria use for population density-dependent communication, a process referred to as quorum sensing (QS). Various research groups have investigated the presence of QSMs, including N-acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2), in physiological samples in attempts to enhance our knowledge of the role of bacteria and QS in disease states. Continued studies in these fields may allow for improved patient care and therapeutics based upon QSMs. Furthermore, bacterial whole-cell biosensors have elucidated the roles of some antibiotics as QS agonists and antagonists. Graphical Abstract.

Update on idiopathic colitides.

PURPOSE OF REVIEW: This article summarizes the latest developments for three types of idiopathic colitis: ulcerative colitis, Crohn's disease, and microscopic colitis. RECENT FINDINGS: Crohn's disease and ulcerative colitis are highly related genetically. Colonoscopy remains the gold standard for making a diagnosis. The addition of chromoendoscopy can aid in identification and removal of colonic dysplasia in both disorders. The therapy for ulcerative colitis and Crohn's disease has been transformed with the introduction of anti-TNF treatment. For ulcerative colitis, recent data show that prolonged use of infliximab is effective, well tolerated and that early mucosal healing is associated with decreased risk of colectomy. There is no evidence of a significant increased risk for anti-TNF-induced malignancies. Combination therapy with azathioprine and infliximab for ulcerative colitis has now been shown to be superior to monotherapy. Natalizumab is effective in anti-TNF failures and patients can be risk stratified for progressive multifocal leukoencephalopathy by testing for John Cunningham virus antibodies. Microscopic colitis can often be related to medications, and symptoms may be worsened by coexisting celiac disease. SUMMARY: Combination therapy with an anti-TNF and a thiopurine is currently the most effective treatment for moderate-to-severe Crohn's disease or ulcerative colitis. Stem cell therapy for perianal fistulas in patients with Crohn's disease is a promising new therapy approach.

IBS patients' willingness to take risks with medications.

OBJECTIVES: We explored irritable bowel syndrome (IBS) patients' impulsivity and risk-taking behavior and their willingness to take medication risks. METHODS: A validated questionnaire assessed the illness experience of IBS patients. A standard gamble evaluated respondents' willingness to take medication risks. RESULTS: IBS patients with severe symptoms were more willing to take significant medication risks than those with mild or moderate symptoms. Impulsivity scores were not associated with an increased likelihood of taking medication risks. Age, gender, and years of IBS symptoms were not associated with medication risk-taking behavior. IBS patients reported they would accept a median 1% risk of sudden death for a 99% chance of cure for their symptoms using a hypothetical medication. CONCLUSIONS: IBS patients are willing to take significant medication risks to cure their symptoms. To counsel patients effectively, physicians must determine and understand IBS patients' risk aversion.

Cancer in inflammatory bowel disease: lessons from animal models.

PURPOSE OF REVIEW: Human colitis-associated cancers (CAC) represent a heterogeneous group of conditions in which multiple oncogenic pathways are involved. In this article, we review the latest studies using genetic, chemical, bacterial and innate immune-mediated experimental models of CAC. RECENT FINDINGS: Using the azoxymethane-dextran sodium sulfate model, wound healing pathways seem to be required in the development of CAC. There is also an emerging understanding that commensal and/or pathogenic bacteria can promote tumorigenesis, through T cell and TLR-mediated inflammation. Using specific transgenic mice (villin-CD98, T cell SMAD7, villin-TLR4) or specific knockout mice, investigators have determined that derangements in epithelial or innate and adaptive immune pathways can result in CAC. Subtle perturbations in epithelial repair - both too little or too exuberant - can render mice susceptible to tumorigenesis. SUMMARY: With the aid of animal models, we have witnessed a rapid expansion of our knowledge of the molecular and immunologic mechanisms underlying inflammatory cancers. Though animal models have contributed a discrete amount of information to our understanding of tumorigenesis in the setting of intestinal inflammation, it is clear that no single animal model will be able to adequately recapitulate the pathogenesis of complex colorectal cancers, but each model gets us one step closer to comprehending the nature of CAC.

Electrochemical development of hydrogen silsesquioxane by applying an electrical potential.

We present a new method for developing hydrogen silsesquioxane (HSQ) by using electrical potentials and deionized water. Nested-L test structures with a pitch as small as 9 nm were developed using this electrochemical technique in saline solution without adding hydroxyl ions. Furthermore, we showed that high-resolution structures can be electrochemically developed in deionized water alone. Electrochemical development is controlled by the applied voltage and may overcome several of the limitations discussed for alkaline developers, such as poor hydroxyl anion diffusion and charge repulsion effects in small trenches.

Dielectrophoretic trapping of DNA-coated gold nanoparticles on silicon based vertical nanogap devices.

We report on the successful dielectrophoretic trapping and electrical characterization of DNA-coated gold nanoparticles on vertical nanogap devices (VNDs). The nanogap devices with an electrode distance of 13 nm were fabricated from Silicon-on-Insulator (SOI) material using a combination of anisotropic reactive ion etching (RIE), selective wet chemical etching and metal thin-film deposition. Au nanoparticles (diameter 40 nm) coated with a monolayer of dithiolated 8 base pairs double stranded DNA were dielectrophoretically trapped into the nanogap from electrolyte buffer solution at MHz frequencies as verified by scanning and transmission electron microscopy (SEM/TEM) analysis. First electrical transport measurements through the formed DNA-Au-DNA junctions partially revealed an approximately linear current-voltage characteristic with resistance in the range of 2-4 GΩ when measured in solution. Our findings point to the importance of strong covalent bonding to the electrodes in order to observe DNA conductance, both in solution and in the dry state. We propose our setup for novel applications in biosensing, addressing the direct interaction of biomolecular species with DNA in aqueous electrolyte media.

Predictors of aggressive inflammatory bowel disease.

Inflammatory bowel disease comprises a group of conditions characterized by idiopathic inflammation of the gastrointestinal tract. The natural course of disease can range from an indolent course with prolonged periods of remission to aggressive, incapacitating disease. Predicting which patients are more susceptible to developing severe disease is important, especially when choosing therapeutic agents and treatment strategies. This paper reviews current evidence on the main demographic, clinical, endoscopic, histologic, serologic, and genetic markers that predict aggressive inflammatory bowel disease. In ulcerative colitis, we considered disease to be aggressive when patients had a high relapse rate, need for admission and/or surgery, development of colon cancer, or extraintestinal manifestations. We defined aggressive Crohn's disease as having a high relapse rate, development of penetrating disease, need for repeat surgery, or multiple admissions for flares. In Crohn's disease, involvement of the upper gastrointestinal tract and ileum, penetrating disease, early age at diagnosis, smoking, extensive ulceration of the mucosa, high titers of serum antibodies, and mutations of the NOD2 gene are markers of aggressive disease. In ulcerative colitis, patients with more extensive involvement of the colon (pancolitis) have more symptomatology and are at higher risk for needing a colectomy and developing colon cancer. Also, plasmocytic infiltration of the colonic mucosa and crypt atrophy predict treatment failure. As with diagnosis, no single method can predict disease aggressiveness. Multiple serologic and genetic tests are being developed to refine the accuracy of prediction. Endoscopic findings can also predict the future course of disease. At present, clinical manifestations are the most useful way to make therapeutic decisions.

Sub-10 nm structures on silicon by thermal dewetting of platinum.

A study of the dewetting behavior of platinum-thin-films on silicon was carried out to determine how variation of dewetting parameters affects the evolution of film morphology and to pinpoint which parameters yielded the smallest, most circular features. Platinum film thickness as well as dewetting time and temperature were varied and the film morphology characterized by means of scanning electron microscopy (SEM) analysis. Two different pathways of dewetting predicted in the literature (Vrij 1966 Discuss. Faraday Soc. 42 23, Becker et al 2003 Nat. Mater. 2 59-63) were observed. Depending on the initial criteria, restructuring of the film occurred via hole or droplet formation. With increased annealing time, a transition from an intermediate network structure to separated islands occurred. In addition, the formation of multilayered films, silicide crystals and nanowires occurred for certain parameters. Nevertheless, the dewetting behavior witnessed could be related to physical processes. Droplets with a mean diameter of 9 nm were formed by using a 1.5 nm thick platinum film annealed at 800 °C for 30 s. To demonstrate the suitability of the annealed films for further processing, we then used the dewetted films as masks for reactive ion etching to transfer the pattern into the silicon substrate, forming tapered nanopillars.

Endoscopic versus surgical therapy for early cancer in Barrett's esophagus: a decision analysis.

BACKGROUND: Esophagectomy for early esophageal adenocarcinoma is associated with increased operative mortality and morbidity, but possibly a decreased recurrence rate compared with endoscopic therapy when using EMR and radiofrequency ablation. OBJECTIVE: To compare the cost-effectiveness of esophagectomy and endoscopic therapy in the treatment of early esophageal adenocarcinoma. DESIGN: Decision analysis model. MAIN OUTCOME MEASUREMENTS: Incremental cost-effectiveness ratio. RESULTS: During the 5-year study period, endoscopic therapy cost $17,000.00 and yielded 4.88 quality-adjusted life years, compared with $28,000.00 and 4.59, respectively, for esophagectomy. Varying the recurrence rates of cancer or Barrett's esophagus metaplasia after endoscopic therapy did not change the overall outcome. The sensitivity analysis demonstrated, however, that the outcome depended on the rate of lymph node involvement and operative mortality. Under the best of circumstances in favor of esophagectomy, such as 2% operative mortality, no reduced quality of life after esophagectomy, and a low 5-year survival rate after recurrence of endoscopic ablation, the risk of positive lymph nodes still needed to exceed 25% before esophagectomy became the preferred treatment option. This threshold is twice as high as the values reported for early submucosal cancer invasion. LIMITATIONS: Limited data are available about the long-term outcome of EMR and radiofrequency ablation. CONCLUSIONS: Endoscopic therapy for early Barrett's esophagus adenocarcinoma is more effective and less expensive than esophagectomy. Even in early esophageal adenocarcinoma with submucosal invasion, endoscopic therapy is a cost-effective alternative to esophagectomy, especially in patients with a high operative risk.

Silicon based nanogap device for studying electrical transport phenomena in molecule-nanoparticle hybrids.

We report the fabrication and characterization of vertical nanogap electrode devices using silicon-on-insulator substrates. Using only standard silicon microelectronic process technology, nanogaps down to 26 nm electrode separation were prepared. Transmission electron microscopy cross-sectional analysis revealed the well defined material architecture of the nanogap, comprising two electrodes of dissimilar geometrical shape. This asymmetry is directly reflected in transport measurements on molecule-nanoparticle hybrid systems formed by self-assembling a monolayer of mercaptohexanol on the electrode surface and the subsequent dielectrophoretic trapping of 30 nm diameter Au nanoparticles. The observed Coulomb staircase I-V characteristic measured at T = 4.2 K is in excellent agreement with theoretical modelling, whereby junction capacitances of the order of a few 10(-18) farad and asymmetric resistances of 30 and 300 MΩ, respectively, are also supported well by our independent estimates for the formed double barrier tunnelling system. We propose our nanoelectrode system for integrating novel functional electronic devices such as molecular junctions or nanoparticle hybrids into existing silicon microelectronic process technology.