Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.     

Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings

In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.     

3 Beta-hydroxysteroid dehydrogenase-isomerase activity in bovine adrenocortical cells in culture: lack of response to ACTH treatment

Primary cultures of bovine adrenocortical cells (BAC) were used to determine whether the adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase complex (3 beta-HSD), like the 17 alpha-hydroxylase (17-OHase), responded to ACTH treatment with an increase in activity. Both enzymes influence the steroidogenic path leading to 17 alpha-hydroxyprogesterone formation and thus could affect adrenal androgen biosynthesis. 3 beta-HSD Activity in postmitochondrial supernatant fluid, homogenates or cell monolayers remained unchanged after cells had been maintained in 1 microM ACTH up to 48 h. Since ACTH exposure led to a marked increase in 17-OHase activity over the same time period, it is concluded that, under the conditions used, the 3 beta-HSD-isomerase complex in BAC is nonresponsive to tropic hormone treatment.     

Microbes make the meal: oligolectic bees require microbes within their host pollen to thrive

1. For solitary bees that specialise on select pollen types (oligoleges), larval development depends on the availability of forage pollen from appropriate host plants and the naturally occurring microbiota present therein. While access to host pollen may be critical for the development of oligolectic bees, the extent to which pollen microbiota contribute to their brood success is unknown. 2. To investigate, we used a diet manipulation experiment to rear larvae of the oligolege, Osmia ribifloris, under in-vitro conditions. Larvae were reared either on host pollen provisioned by their mother or on non-host pollen collected by honey bees, in the presence or absence of the respective pollen-associated microbiota. We assessed impacts on components of larval fitness: developmental time, biomass, and survivorship. 3. Our results revealed a significant interaction between pollen type and pollen-associated microbes. The relative effect of microbes on larval performance was substantially greater than that of pollen type. Host pollen substrate produced the fittest larvae but only when combined with its full complement of naturally occurring microbiota. In contrast, host pollen without microbes resulted in a marked decline in fitness components. Larvae consuming non-host pollen showed intermediate fitness, regardless of whether microbes were present or not. 4. These findings imply that the microbiota associated with maternally provisioned host pollen perform critical functions in larval nutrition and survival. For oligoleges in particular, the ability to develop on poorer quality host pollen likely derives from this sustained symbiosis with their microbial exosymbionts, rather than the biochemical characteristics of pollen type alone.     

An Integrated Approach for Analyzing Clinical Genomic Variant Data from Next-Generation Sequencing

Next-generation sequencing (NGS) technologies provide the potential for developing high-throughput and low-cost platforms for clinical diagnostics. A limiting factor to clinical applications of genomic NGS is downstream bioinformatics analysis for data interpretation. We have developed an integrated approach for end-to-end clinical NGS data analysis from variant detection to functional profiling. Robust bioinformatics pipelines were implemented for genome alignment, single nucleotide polymorphism (SNP), small insertion/deletion (InDel), and copy number variation (CNV) detection of whole exome sequencing (WES) data from the Illumina platform. Quality-control metrics were analyzed at each step of the pipeline by use of a validated training dataset to ensure data integrity for clinical applications. We annotate the variants with data regarding the disease population and variant impact. Custom algorithms were developed to filter variants based on criteria, such as quality of variant, inheritance pattern, and impact of variant on protein function. The developed clinical variant pipeline links the identified rare variants to Integrated Genome Viewer for visualization in a genomic context and to the Protein Information Resource’s iProXpress for rich protein and disease information. With the application of our system of annotations, prioritizations, inheritance filters, and functional profiling and analysis, we have created a unique methodology for downstream variant filtering that empowers clinicians and researchers to interpret more effectively the relevance of genomic alterations within a rare genetic disease.     

Effects of individual Bacillus thuringiensis insecticidal crystal proteins on adult Heliothis virescens (F.) and Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Bacillus thuringiensis (Bt) is a grampositive, spore forming bacterium, which is principally distinguishedfrom other bacilli by the production of large, insecticidal,protein crystals (Insecticidal Crystal Proteins, or ICPs). Theseproteins are usually thought to act only on the actively feedinglarvae of susceptible species by a mechanism which involvesconsumption and proteolytic processing of the protein followed bybinding to, and lysis of, midgut epithelial cells. However, few authorshave reported Bt toxicity to adult insects. In the followingpaper, we expand on previous reports of toxicity to adult insects andpresent data which demonstrate that: (1) proteolytically activated ICPssignificantly reduce the lifespans of adult Heliothis virescensand Spodoptera exigua at concentrations of 500 g/ml, butnot 167 or 25 g/ml, (2) individual activated ICPs are differentiallytoxic to adult H. virescens and S. exigua, and (3)adult S. exigua are sensitive to Cry1C protoxin at aconcentration of 1 mg/ml.Deceased     

Characterization of Viral Load,Viability and Persistence of Influenza A Virus in Air and on Surfaces of Swine Production Facilities

Indirect transmission of influenza A virus (IAV) in swine is poorly understood and information is lacking on levels of environmental exposure encountered by swine and people during outbreaks of IAV in swine barns. We characterized viral load, viability and persistence of IAV in air and on surfaces during outbreaks in swine barns. IAV was detected in pigs, air and surfaces from five confirmed outbreaks with 48% (47/98) of oral fluid, 38% (32/84) of pen railing and 43% (35/82) of indoor air samples testing positive by IAV RT-PCR. IAV was isolated from air and oral fluids yielding a mixture of subtypes (H1N1, H1N2 and H3N2). Detection of IAV RNA from air was sustained during the outbreaks with maximum levels estimated between 7 and 11 days from reported onset. Our results indicate that during outbreaks of IAV in swine, aerosols and surfaces in barns contain significant levels of IAV potentially representing an exposure hazard to both swine and people.