Enzymatic hydrolysis of esterified diarrhetic shellfish poisoning toxins and pectenotoxins

Okadaic acid (OA) and dinophysistoxins-1 and -2 (DTX1, DTX2), the toxins responsible for incidents of diarrhetic shellfish poisoning (DSP), can occur as complex mixtures of ester derivatives in both plankton and shellfish. Alkaline hydrolysis is usually employed to release parent OA/DTX toxins, and analyses are conducted before and after hydrolysis to determine the concentrations of nonesterified and esterified toxins. Recent research has shown that other toxins, including pectenotoxins and spirolides, can also exist as esters in shellfish, but these toxins cannot survive alkaline hydrolysis. A promising alternative approach is enzymatic hydrolysis. In this study, two enzymatic methods were developed for the hydrolysis of 7-O-acyl esters, “DTX3,” and the carboxylate esters of OA, “diol-esters.” Porcine pancreatic lipase induced complete conversion of DTX3 to OA and DTXs within one hour for reference solutions. The presence of mussel tissue matrix reduced the rate of hydrolysis, but an optimized lipase concentration resulted in greater than 95% conversion within four hours. OA-diol-ester was hydrolyzed by porcine liver esterase and was completely converted to OA in less than 30 min, even in the presence of mussel tissue matrix. Esters and OA/DTX toxins were all monitored by LC–MS. Further experiments with pectenotoxin esters indicated that enzymatic hydrolysis could also be applied to esters of other toxins. Enzymatic hydrolysis has excellent potential as an alternative to the conventional alkaline hydrolysis procedure used in the preparation of shellfish samples for the analysis of toxins.     

In Situ Visualization of Block Copolymer Self‐Assembly in Organic Media by Super‐Resolution Fluorescence Microscopy

Analytical methods that enable visualization of nanomaterials derived from solution self‐assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media at the sub‐diffraction scale. Four different dyes were successfully used for single‐colour super‐resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual‐colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well‐established for aqueous systems, the results highlight the potential of super‐resolution microscopy techniques for the interrogation of self‐assembly processes in organic media.     

Vitamin D and Skin Cancer

Vitamin D signaling plays a key role in many important processes, including cellular proliferation, differentiation and apoptosis, immune regulation, hormone secretion and skeletal health. Furthermore, vitamin D production and supplementation have been shown to exert protective effects via an unknown signaling mechanism involving the vitamin D receptor (VDR) in several diseases and cancer types, including skin cancer. With over 3.5 million new diagnoses in 2 million patients annually, skin cancer is the most common cancer type in the United States. While ultraviolet B (UVB) radiation is the main etiologic factor for nonmelanoma skin cancer (NMSC), UVB also induces cutaneous vitamin D production. This paradox has been the subject of contradictory findings in the literature in regards to amount of sun exposure necessary for appropriate vitamin D production, as well as any beneficial or detrimental effects of vitamin D supplementation for disease prevention. Further clinical and epidemiological studies are necessary to elucidate the role of vitamin D in skin carcinogenesis.     

The interaction of a screw dislocation with point defects in bcc iron

In this study, we calculate the interaction energy of intrinsic point defects vacancies and interstitials) with screw dislocations in body-centered cubic iron. First (we calculate the dipole tensor of a defect in the bulk crystal using molecular statics. Using a formulation based on linear elasticity theory, we calculate the interaction energy of the defect and the dislocation using both isotropic and anisotropic strain fields. Second, we perform atomistic calculations using molecular statics methods to directly calculate the interaction energy. Results from these two methods are compared. We verify that continuum methods alone are unable to correctly predict the interactions of defects and dislocations near the core. Although anisotropic theory agrees qualitatively with atomistics far from the core, it cannot predict which dumbbell orientations are stable and any continuum calculations must be used with caution. Spontaneous absorption by the core of both vacancies and dumbbells is seen. This paper demonstrates and discusses the differences between continuum and atomistic calculations of interaction energy between a dislocation core and a point defect.     

Isotope hydrology and baseflow geochemistry in natural and human-altered watersheds in the Inland Pacific Northwest,USA

This study presents a stable isotope hydrology and geochemical analysis in the inland Pacific Northwest (PNW) of the USA. Isotope ratios were used to estimate mean transit times (MTTs) in natural and human-altered watersheds using the FLOWPC program. Isotope ratios in precipitation resulted in a regional meteoric water line of δ²H?=?7.42·δ¹⁸O?+?0.88 (n?=?316; r²?=?0.97). Isotope compositions exhibited a strong temperature-dependent seasonality. Despite this seasonal variation, the stream δ¹⁸O variation was small. A significant regression (τ?=?0.11D^?1.09; r²?=?0.83) between baseflow MTTs and the damping ratio was found. Baseflow MTTs ranged from 0.4 to 0.6 years (human-altered), 0.7 to 1.7 years (mining-altered), and 0.7 to 3.2 years (forested). Greater MTTs were represented by more homogenous aqueous chemistry whereas smaller MTTs resulted in more dynamic compositions. The isotope and geochemical data presented provide a baseline for future hydrological modelling in the inland PNW.     

Modular synthesis of functionalized bis-bispidine tetraazamacrocycles

An effective synthesis is reported for the construction of highly rigid and preorganized bis-bispidine tetraazamacrocycles bearing either identical or different functionalities. Using essential building blocks derived from N-Boc-N'-allylbispidinone, the modular approach facilitates independent incorporation of the functional groups to the macrocyclic framework as well as selective derivatization of one functionality in the presence of another.     

Siloxyl ether functionalized resins for chemoselective enrichment of carboxylic acids

Although the carboxylic acid moiety is prevalent in many biologically produced molecules, including natural products and proteins, methods to chemoselectively target this functional group have remained elusive. Generally, strategies that utilize carboxylate nucleophilicity also promote reactions with other nucleophiles such as amines and hydroxyls. A reagent was sought to facilitate the selective isolation of carboxylic acid containing compounds from complex mixtures. Here, the development of siloxyl ether functionalized solid supports is described.     

Evaluation of VIDAS Salmonella (SLM) easy Salmonella method for the detection of Salmonella in a variety of foods: collaborative study

The VIDAS Salmonella (SLM) Easy Salmonella method is a specific enzyme-linked fluorescent immunoassay performed in the automated VIDAS instrument. The VIDAS Easy Salmonella method is a simple 2-step enrichment procedure, using pre-enrichment followed by selective enrichment in a newly formulated broth, SX2 broth. This new method was compared in a multilaboratory collaborative study to the U.S. Food and Drug Administration's Bacteriological Analytical Manual, Chapter 5 method for five food matrixes (liquid egg, vanilla ice cream, spinach, raw shrimp, and peanut butter) and the U.S. Department of Agriculture's Microbiology Laboratory Guidebook 4.04 method for deli turkey. Each food type was artificially contaminated with Salmonella at three inoculation levels. A total of 15 laboratories representing government, academia, and industry, throughout the United States, participated. In this study, 1583 samples were analyzed, of which 792 were paired replicates and 791 were unpaired replicates. Of the 792 paired replicates, 285 were positive by both the VIDAS and reference methods. Of the 791 unpaired replicates, 341 were positive by the VIDAS method and 325 were positive by the cultural reference method. A Chi-square analysis of each of the six food types was performed at the three inoculation levels tested. For all foods evaluated, the VIDAS Easy SLM method demonstrated results comparable to those of the reference methods for the detection of Salmonella.     

Contactless dielectrophoretic spectroscopy: examination of the dielectric properties of cells found in blood

The use of non-invasive methods to detect and enrich circulating tumor cells (CTCs) independent of their genotype is critical for early diagnostic and treatment purposes. The key to using CTCs as predictive clinical biomarkers is their separation and enrichment. This work presents the use of a contactless dielectrophoresis (cDEP) device to investigate the frequency response of cells and calculate their area-specific membrane capacitance. This is the first demonstration of a cDEP device which is capable of operating between 10 and 100 kHz. Positive and negative dielectrophoretic responses were observed in red blood cells, macrophages, breast cancer, and leukemia cells. The area-specific membrane capacitances of MDA-MB231, THP-1 and PC1 cells were determined to be 0.01518 ± 0.0013, 0.01719 ± 0.0020, 0.01275 ± 0.0018 (F/m(2)), respectively. By first establishing the dielectrophoretic responses of cancerous cells within this cDEP device, conditions to detect and enrich tumor cells from mixtures with non-transformed cells can be determined providing further information to develop methods to isolate these rare cells.