Detection of Elevated Signaling Amino Acids in Human Diabetic Vitreous by Rapid Capillary Electrophoresis

Elevated glutamate is implicated in the pathology of PDR. The ability to rapidly assess the glutamate and amino acid content of vitreous provides a more complete picture of the chemical changes occurring at the diabetic retina and may lead to a better understanding of the pathology of PDR. Vitreous humor was collected following vitrectomies of patients with PDR and control conditions of macular hole or epiretinal membrane. A capillary electrophoresis method was developed to quantify glutamate and arginine. The analysis is relatively fast (<6 minutes) and utilizes a poly(ethylene)oxide and sodium dodecylsulfate run buffer. Both amino acid levels show significant increases in PDR patients versus controls and are comparable to other reports. The levels of vitreal glutamate vary inversely with the degree of observed hemorrhage. The results demonstrate a rapid method for assessment of a number of amino acids to characterize the chemical changes at the diabetic retina to better understand tissue changes and potentially identify new treatments

Improved annotation of the insect vector of citrus greening disease: Biocuration by a diverse genomics community

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models.Peer reviewedBiochemistry and Molecular BiologyEntomology and Plant Patholog

Development of Assays for Neuronal Peptide Content and Release

101 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997.Neuropeptide release is also studied with MALDI TOF MS. In this method, a small chamber (10 $\mu$l) is used to hold an isolated bag cell cluster in a small volume of physiological saline. The chamber consists of a standard pipette tip with the narrow end melted around two sections of fused silica capillaries. Physiological saline flows into and from the chamber via capillaries and gravimetric pressure flow. Following electrical stimulation of a bag cell cluster, 50-100 nl samples are collected from the outlet end of the sampling capillary for analysis. MALDI MS is performed on the sample for peptide detection. Even in high salt concentrations peptides are detected by MALDI MS.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Development of a Simplified Microfluidic Injector for Analysis of Droplet Content via Capillary Electrophoresis

Droplet-based microfluidic platforms sequester nanoliter to picoliter samples in an immiscible carrier phase and have gained notoriety for their ability to be used in laboratory procedures on a miniaturized scale. Recently, droplet microfluidics has been used to prevent zone diffusion in time-resolved sample collection methods and in separation techniques. The assay of droplets remains challenging, however, because the carrier phase is often incompatible with separation techniques. In this work, we report the development of a droplet injector for capillary electrophoresis (CE) which delivers 750 pL droplets to a channel for separation while excluding the fluorous carrier phase. This design is simple compared to previous reports, consisting of only two straight channels and no additional working parts such as membranes or valves. To demonstrate a proof-of-concept and characterize performance, riboflavin was used as a biologically relevant model molecule. Droplets containing a step change in riboflavin concentration were injected and mobilized by CE. The current method is capable of riboflavin peak % relative standard deviations (RSDs) down to 4.4% and temporal resolutions down to 15 s. Human urine samples containing riboflavin and its photolysis products were successfully separated and found to be chemically compatible with the injector. Our simplified design could improve robustness and ruggedness and may allow device construction via nontraditional fabrication techniques

Hemolymph amino acid analysis of individual Drosophila larvae

International audienceOne of the most widely used transgenic animal models in biology is Drosophila melanogaster, the fruit fly. Chemical information from this exceedingly small organism is usually accomplished by studying populations to attain sample volumes suitable for standard analysis methods. This paper describes a direct sampling technique capable of obtaining 50-300 nL of hemolymph from individual Drosophila larvae. Hemolymph sampling performed under mineral oil and in air at 30 s intervals up to 120 s after piercing larvae revealed that the effect of evaporation on amino acid concentrations is insignificant when the sample was collected within 60 s. Qualitative and quantitative amino acid analyses of obtained hemolymph were carried out in two optimized buffer conditions by capillary electrophoresis with laser-induced fluorescence detection after derivatizing with fluorescamine. Thirteen amino acids were identified from individual hemolymph samples of both wild-type (WT) control and the genderblind (gb) mutant larvae. The levels of glutamine, glutamate, and taurine in the gb hemolymph were significantly lower at 35%, 38%, and 57% of WT levels, respectively. The developed technique that samples only the hemolymph fluid is efficient and enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations, estimations, and effects of evaporation compared to the traditional hemolymph-sampling techniques