Evaluation of "Toolkit" consisting of handheld and portable analytical devices for detecting active pharmaceutical ingredients in drug products collected during a simultaneous nation-wide mail blitz.

A "toolkit" consisting of a handheld Raman spectrometer equipped with a 1064 nm laser, a portable Fourier transform infrared (FT-IR) spectrometer and a portable direct analysis in real-time mass spectrometer (DART-MS) was employed in a laboratory setting to examine 82 representative products collected during a nationwide mail blitz for the presence of APIs. These results were compared to those obtained using laboratory-based methods; 8 of the products were not found to contain APIs and 74 of the products were found to contain a total of 88 APIs (65 of the 88 APIs were unique). The individual performance of each device and combined performance of the three-device toolkit were evaluated with regard to true positives, true negatives, false positives and false negatives. Using this toolkit, 81 (92.0 %) of the APIs were detected by at least one technique and 47 (64.8 %) of the APIs were detected by at least two techniques. Seven false negatives (8.0 %) were encountered and while the toolkit yielded 12 false positives, no false positives were detected by more than one technique. Overall, this study demonstrated that when the toolkit detects an API using two or more devices, the results are as reliable as those generated by a full-service laboratory.

Evaluation of four field portable devices for the rapid detection of mitragynine in suspected kratom products.

The development of a method for the rapid screening of food and drug products for constituents such as mitragynine, the most abundant alkaloid found in Mitragyna speciosa (kratom) plant leaves, has become increasingly important. The use of kratom is said to produce stimulant or narcotic effects and poses risks of addiction, abuse, and dependence, much like other opioids. Direct Analysis in Real Time with thermal desorption mass spectrometry (DART-TD-MS), hand-held mass spectrometry, portable ion mobility spectrometry (IMS), and portable Fourier-transform infrared spectroscopy (FT-IR) were each evaluated as field-deployable screening techniques for the detection of mitragynine in food and drug products. These devices offer the potential for rapid, early detection of mitragynine in suspect products entering the United States through international mail facilities and other ports of entry. Ninety-six kratom products, including capsules, bulk powder, and bulk plant material, were analyzed by either direct sampling of the solid material or by solvent extraction. True and false positive and negative results are reported, based on comparison to results from qualitative screening using gas chromatography with mass spectral detection (GC-MS), liquid chromatography with mass spectral detection (LC-MS), and/or quantitative screening using high-performance liquid chromatography with ultraviolet detection (HPLC-UV), with a discussion of the assessment of each technique for use in the field. Each device demonstrated attributes that would be favorable for use in screening of suspected mitragynine-containing products at places like ports of entry, and simultaneous deployment of two or more of these devices as part of a workflow would be the most effective for rapid screening of these products. This combination of rapid screening orthogonal techniques suited to a non-laboratory environment will allow onsite destruction of products found to contain mitragynine.

Capturing Multicellular System Designs Using Synthetic Biology Open Language (SBOL).

Synthetic biology aims to develop novel biological systems and increase their reproducibility using engineering principles such as standardization and modularization. It is important that these systems can be represented and shared in a standard way to ensure they can be easily understood, reproduced, and utilized by other researchers. The Synthetic Biology Open Language (SBOL) is a data standard for sharing biological designs and information about their implementation and characterization. Previously, this standard has only been used to represent designs in systems where the same design is implemented in every cell; however, there is also much interest in multicellular systems, in which designs involve a mixture of different types of cells with differing genotype and phenotype. Here, we show how the SBOL standard can be used to represent multicellular systems, and, hence, how researchers can better share designs with the community and reliably document intended system functionality.

Metabolite profiles of repeatedly sampled urine from male fathead minnows (Pimephales promelas) contain unique lipid signatures following exposure to anti-androgens.

The purpose of this study was twofold. First, we sought to identify candidate markers of exposure to anti-androgens by analyzing endogenous metabolite profiles in the urine of male fathead minnows (mFHM, Pimephales promelas). Based on earlier work, we hypothesized that unidentified lipids in the urine of mFHM were selectively responsive to exposure to androgen receptor antagonists, which is otherwise difficult to confirm using established fish toxicity assays. A second goal was to evaluate the feasibility of non-lethally and repeatedly sampling urine from individual mFHMs over the time course of response to a chemical exposure. Accordingly, we exposed mFHM to the model anti-androgens vinclozolin or flutamide. Urine was collected from each fish at 48hour intervals over the course of a 14day exposure. Parallel experiments were conducted with mFHM exposed to bisphenol A or control water. The frequent handling/sampling regime did not cause apparent adverse effects on the fish. Endogenous metabolite profiling was conducted with gas chromatography-mass spectrometry (GC-MS), which exhibited lower variation for the urinary metabolome than was found in earlier work with nuclear magnetic resonance (NMR) spectroscopy. Specifically, for inter- and intra-individual variations, the median spectrum-wide relative standard deviation (RSD) was 32.6% and 33.3%, respectively, for GC-MS analysis of urine from unexposed mFHM. These results compared favorably with similar measurements of urine from other model species, including the Sprague Dawley rat. In addition, GC-MS allowed us to identify several lipids (e.g., certain saturated fatty acids) in mFHM urine as candidate markers of exposure to androgen receptor antagonists.

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells.

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-¹³C-glucose and ¹5N-glutamate as labeled precursors. This study suggests that uniformly ¹5N,¹³C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

Pathogenesis of metaphyseal radiolucent changes following ischemic necrosis of the capital femoral epiphysis in immature pigs. A preliminary report.

BACKGROUND: Although metaphyseal radiolucent changes are often seen in patients with Legg-Calvé-Perthes disease, the pathogenesis of these changes remains controversial. The purpose of the present study was to determine the prevalence and histopathological characteristics of these metaphyseal radiolucent changes in a piglet model of ischemic necrosis of the capital femoral epiphysis. METHODS: Ischemic necrosis of the right femoral head was produced in fifty piglets by surgically placing a ligature tightly around the femoral neck. The contralateral, left hip of each animal was used as a control. Radiographs and histological sections of the femoral heads were examined at two, four, and eight weeks. The radiographs were used to measure the femoral neck length in order to assess growth disturbance. RESULTS: Thirteen of the fifty animals were found to have radiolucent changes in the proximal femoral metaphysis on the side of the infarcted femoral head. These changes were observed in none of the twelve animals that were evaluated at two weeks, in one of the fourteen animals that were evaluated at four weeks, and in twelve of the twenty-four animals that were evaluated at eight weeks. The radiolucent changes ranged from a focal cystic lesion to a diffuse area of radiolucency around the proximal femoral physis. Three distinct types of histological changes were observed in the metaphysis. Type-I changes were characterized by focal thickening of the physeal cartilage extending down into the metaphysis. Some of these lesions demonstrated cystic degeneration of the thickened cartilage. Type-II changes were characterized by central disruption of the physis and resorption and replacement of the metaphyseal bone in the region by fibrovascular tissue. Type-III changes were characterized by diffuse resorption of the physeal cartilage and resorption of the adjacent metaphyseal and epiphyseal bone. The mean femoral neck length on the infarcted side in animals with metaphyseal radiolucent changes was significantly shorter than that in animals without metaphyseal radiolucent changes (p = 0.02). CONCLUSIONS: Metaphyseal radiolucent changes frequently were observed in this piglet model at eight weeks after the induction of ischemia. The metaphyseal radiolucent changes were associated with histopathological lesions of the physis. The present study suggests that the presence of metaphyseal lesions can result in a greater growth disturbance of the proximal femoral physis than is seen in the absence of metaphyseal lesions. CLINICAL RELEVANCE: The present study supports the clinical observation that the presence of diffuse metaphyseal radiolucent changes may be associated with substantial growth disturbance of the proximal part of the femur in patients with Legg-Calvé-Perthes disease. The study provides a histopathological basis for proximal femoral physeal growth disturbance that has not been clearly demonstrated in the past. These findings also may provide a histopathological basis for the fluidfilled metaphyseal cysts that sometimes are observed on the magnetic resonance imaging scans of these patients.

Anxiety and depression in hospitalized patients in resistant organism isolation.

BACKGROUND: Previous studies have reported an increase in psychiatric symptoms in seriously ill patients who were placed in resistant organism isolation. We conducted this study to assess whether there is an increase in symptoms of anxiety and depression in patients who are not critically ill and are placed in isolation. METHODS: Patients hospitalized with methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus species infections were evaluated with the Hamilton Anxiety Rating Scale and the Hamilton Depression Rating Scale at baseline and again during hospitalization. The results were then compared with the results of patients who were hospitalized for infectious diseases that did not require isolation. RESULTS: Patients in isolation had significantly higher scores on both the anxiety and depression scales at the time of follow-up than did patients who were not isolated. There was no significant difference between the scores of the two groups before isolation. CONCLUSION: The results of this preliminary study suggest that placement in resistant organism isolation may increase hospitalized patients' levels of anxiety and depression.