Molecular analysis using targeted next generation DNA sequencing and clinical spectrum of Mexican patients with isovaleric acidemia.

Isovaleric acidemia (IVA) is an inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase. IVA clinical picture includes gastroenterological and progressive neurological symptoms which can lead to permanent disability and death. Early detection by newborn screening (NBS) and treatment promotes normal development. In this study, clinical summaries, biochemical measurements and targeted next generation sequencing (tNGS) data from the IVD gene were compared in 13 Mexican patients. The main symptoms were vomiting, feeding refusal, abdominal pain, impaired alertness, lethargy, stupor, coma; hypotonia, ataxia, hallucinations, seizures; anemia, neutropenia and pancytopenia. Mean blood concentration of isovalerylcarnintine was above the reference value (0.5 µM) in symptomatic patients (8.78 µM), as well as in the screen positive newborns (2.23 µM). The molecular spectrum of this cohort was heterogeneous, with 14 different variants identified, seven were previously-described, and seven were novel. The most frequent variant was c.158G > C (p.R53P). In this study, we found a long diagnostic delay (average of 44 months). Thus, it is essential to increase physician awareness of this treatable condition. Biochemical IVA NBS accompanied by molecular studies (e.g. tNGS) will permit identification of potentially asymptomatic forms of the disease, and improve genotype-phenotype relationship, management decisions and follow-up.

Methods for enhancing cyanobacterial stress tolerance to enable improved production of biofuels and industrially relevant chemicals.

Cyanobacteria are photosynthetic prokaryotes that can fix atmospheric CO2 and can be engineered to produce industrially important compounds such as alcohols, free fatty acids, alkanes used in next-generation biofuels, and commodity chemicals such as ethylene or farnesene. They can be easily genetically manipulated, have minimal nutrient requirements, and are quite tolerant to abiotic stress making them an appealing alternative to other biofuel-producing microbes which require additional carbon sources and plants which compete with food crops for arable land. Many of the compounds produced in cyanobacteria are toxic as titers increase which can slow growth, reduce production, and decrease overall biomass. Additionally, many factors associated with outdoor culturing of cyanobacteria such as UV exposure and fluctuations in temperature can also limit the production potential of cyanobacteria. For cyanobacteria to be utilized successfully as biofactories, tolerance to these stressors must be increased and ameliorating stress responses must be enhanced. Genetic manipulation, directed evolution, and supplementation of culture media with antioxidants are all viable strategies for designing more robust cyanobacterial strains that have the potential to meet industrial production goals.

A High-Temperature, High-Throughput Method for Monitoring Residual Formaldehyde in Vaccine Formulations.

Formaldehyde has long been used in the chemical inactivation of viral material during vaccine production. Viral inactivation is required so that the vaccine does not infect the patient. Formaldehyde is diluted during the vaccine manufacturing process, but residual quantities of formaldehyde are still present in some current vaccines. Although formaldehyde is considered safe for use in vaccines by the Food and Drug Administration, excessive exposure to this chemical may lead to cancer or other health-related issues. An assay was developed that is capable of detecting levels of residual formaldehyde in influenza vaccine samples. The assay employs incubation of dosage formulation suspensions with hydralazine hydrochloride under mildly acidic conditions and elevated temperatures, where formaldehyde is derivatized to yield fluorescent s-triazolo-[3,4-a]-phthalazine. The assay has been traditionally run by high-performance liquid chromatography, where runtimes of 15 minutes per sample can be expected. Our laboratory has developed a plate-based version that drastically improved the throughput to a runtime of 96 samples per minute. The assay was characterized and validated with respect to reaction temperature, evaporation, stability, and selectivity to monitor residual formaldehyde in various influenza vaccine samples, including in-process samples. Heat transfer and evaporation will be especially considered in this work. Since the assay is plate based, it is automation friendly. The new assay format has attained detection limits of 0.01 µg/mL residual formaldehyde, which is easily able to detect and quantify formaldehyde at levels used in many current vaccine formulations (<5 µg/0.5-mL dose).