Application of Differential Pulse Voltammetry to Determine the Efficiency of Stripping Tocopherols from Commercial Fish Oil

There has been an increase in the use of electrochemical methods for monitoring antioxidant levels in a variety of disciplines due to the sensitivity, low detection limits, ease of use, low cost and rapid analysis time offered by these techniques. One technique that has received specific attention is differential pulse voltammetry. We describe a novel application of differential pulse voltammetry to quantitatively and qualitatively determine the efficiency of removing tocopherols from commercial fish oil via column chromatographic separation. The relative limits of detection and quantitation of differential pulse voltammetry are compared to HPLC for determining the removal of tocopherols from commercial fish oil. It was determined that differential pulse voltammetry can monitor the separation of commercially added antioxidants from the bulk sample via a decrease in antioxidant oxidation currents. Furthermore, the limits of detection and quantitation were found to be comparable with values obtained using HPLC for tocopherol identification and quantitation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141339/1/aocs0527.pd

Determination of the Cyanide Metabolite 2-Aminothiazoline-4-Carboxylic Acid in Urine and Plasma by Gas Chromatography–mass Spectrometry

The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography–mass spectrometry (GC–MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d2), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra- and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females

Constructing a man-made c-type cytochrome maquette in vivo:electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette

The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo

The L&E of Intellectual Property – Do we get maximum innovation with the current regime?

Innovation is crucial to economic growth – the essential path for lifting much of the world population out of dire poverty and for maintaining the living standard of those who already have. To stimulate innovation, the legal system has to support the means through which innovators seek to get rewarded for their efforts. Amongst these means, some, such as the first mover advantage or 'lead time,' are not directly legal; but secrets and intellectual property rights are legal institutions supported for the specific purpose of stimulating innovation. Whilst the protection of secrets has not changed very much over recent years, intellectual property (or IP) has. IP borrows some features from ordinary property rights, but is also distinct, in that, unlike physical goods, information, the object of IP, is not inherently scarce; indeed as information and communication technologies expand, the creation and distribution of information is becoming ever cheaper and in many circumstances abundant, so that selection is of the essence ('on the internet, point of view is everything'). Where rights on information extend too far, their monopolising effect may hamper innovation. The paper investigates the underlying structure of IP rights and surveys what we know empirically about the incentive effects of IP as about industries that flourish without formal IP

Isolation of Human Islets for Autologous Islet Transplantation in Children and Adolescents with Chronic Pancreatitis

Chronic pancreatitis is an inflammatory disease of the pancreas that causes permanent changes in the function and structure of the pancreas. It is most commonly a complication of cystic fibrosis or due to a genetic predisposition. Chronic pancreatitis generally presents symptomatically as recurrent abdominal pain, which becomes persistent over time. The pain eventually becomes disabling. Once specific medical treatments and endoscopic interventions are no longer efficacious, total pancreatectomy is the alternative of choice for helping the patient achieve pain control. While daily administrations of digestive enzymes cannot be avoided, insulin-dependent diabetes can be prevented by transplanting the isolated pancreatic islets back to the patient. The greater the number of islets infused, the greater the chance to prevent or at least control the effects of surgical diabetes. We present here a technical approach for the isolation and preservation of the islets proven to be efficient to obtain high numbers of islets, favoring the successful treatment of young patients

Outcomes and risk score for distal pancreatectomy with celiac axis resection (DP-CAR) : an international multicenter analysis

Background: Distal pancreatectomy with celiac axis resection (DP-CAR) is a treatment option for selected patients with pancreatic cancer involving the celiac axis. A recent multicenter European study reported a 90-day mortality rate of 16%, highlighting the importance of patient selection. The authors constructed a risk score to predict 90-day mortality and assessed oncologic outcomes. Methods: This multicenter retrospective cohort study investigated patients undergoing DP-CAR at 20 European centers from 12 countries (model design 2000-2016) and three very-high-volume international centers in the United States and Japan (model validation 2004-2017). The area under receiver operator curve (AUC) and calibration plots were used for validation of the 90-day mortality risk model. Secondary outcomes included resection margin status, adjuvant therapy, and survival. Results: For 191 DP-CAR patients, the 90-day mortality rate was 5.5% (95 confidence interval [CI], 2.2-11%) at 5 high-volume (1 DP-CAR/year) and 18% (95 CI, 9-30%) at 18 low-volume DP-CAR centers (P=0.015). A risk score with age, sex, body mass index (BMI), American Society of Anesthesiologists (ASA) score, multivisceral resection, open versus minimally invasive surgery, and low- versus high-volume center performed well in both the design and validation cohorts (AUC, 0.79 vs 0.74; P=0.642). For 174 patients with pancreatic ductal adenocarcinoma, the R0 resection rate was 60%, neoadjuvant and adjuvant therapies were applied for respectively 69% and 67% of the patients, and the median overall survival period was 19months (95 CI, 15-25months). Conclusions: When performed for selected patients at high-volume centers, DP-CAR is associated with acceptable 90-day mortality and overall survival. The authors propose a 90-day mortality risk score to improve patient selection and outcomes, with DP-CAR volume as the dominant predictor

Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep

siRNAs comprise a class of drugs that can be programmed to silence any target gene. Chemical engineering efforts resulted in development of divalent siRNAs (di-siRNAs), which support robust and long-term efficacy in rodent and nonhuman primate brains upon direct cerebrospinal fluid (CSF) administration. Oligonucleotide distribution in the CNS is nonuniform, limiting clinical applications. The contribution of CSF infusion placement and dosing regimen on relative accumulation, specifically in the context of large animals, is not well characterized. To our knowledge, we report the first systemic, comparative study investigating the effects of 3 routes of administration - intrastriatal (i.s.), i.c.v., and intrathecal catheter to the cisterna magna (ITC) - and 2 dosing regimens - single and repetitive via an implanted reservoir device - on di-siRNA distribution and accumulation in the CNS of Dorset sheep. CSF injections (i.c.v. and ITC) resulted in similar distribution and accumulation across brain regions. Repeated dosing increased homogeneity, with greater relative deep brain accumulation. Conversely, i.s. administration supported region-specific delivery. These results suggest that dosing regimen, not CSF infusion placement, may equalize siRNA accumulation and efficacy throughout the brain. These findings inform the planning and execution of preclinical and clinical studies using siRNA therapeutics in the CNS

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories