30-Minute Highly Multiplexed VaxArray Immunoassay for Pneumococcal Vaccine Antigen Characterization.

Pneumonia accounts for over 20% of deaths worldwide in children aged 1 to 5 years, disproportionately affecting lower- and middle-income countries. Effective, highly multivalent pneumococcal vaccines are available to decrease disease burden, with numerous new vaccines currently under development to serve a variety of worldwide markets. However, pneumococcal conjugate vaccines are among the hardest biologics to manufacture and characterize due to their complexity and heterogeneity.

Multiplexed VaxArray immunoassay for rapid antigen quantification in measles and rubella vaccine manufacturing.

Measles-containing vaccines (MCV), specifically vaccines against measles and rubella (MR), are extremely effective and critical for the eradication of measles and rubella diseases. In developed countries, vaccination rates are high and vaccines are readily available, but continued high prevalence of both diseases in developing countries and surges in measles deaths in recent years have highlighted the need to expand vaccination efforts. To meet demand for additional vaccines at a globally affordable price, it is highly desirable to streamline vaccine production thereby reducing cost and speeding up time to delivery.

A neuraminidase potency assay for quantitative assessment of neuraminidase in influenza vaccines.

Neuraminidase (NA) immunity leads to decreased viral shedding and reduced severity of influenza disease; however, NA content in influenza vaccines is currently not regulated, resulting in inconsistent quality and quantity of NA that can vary from manufacturer to manufacturer, from year to year, and from lot to lot. To address this problem, we have developed an assay for NA quantification that could be used by the industry to move toward developing influenza vaccines that induce a predictable immune response to NA. The VaxArray Influenza Seasonal NA Potency Assay (VXI-sNA) is a multiplexed sandwich immunoassay that relies on six subtype-specific monoclonal antibodies printed in microarray format and a suite of fluor-conjugated "label" antibodies.

VaxArray potency assay for rapid assessment of "pandemic" influenza vaccines.

The VaxArray Influenza Pandemic HA (VXI-pHA) potency assay is a multiplexed sandwich immunoassay that consists of nine broadly reactive yet subtype-specific monoclonal capture antibodies printed in microarray format and a suite of fluor-labeled secondary antibodies that were selected to probe conserved HA epitopes. VXI-pHA was designed to optimize the probability that the ready-to-use assay would work for the most concerning, emergent influenza A strains, eliminating the need for the time-consuming process of reference reagents production. The performance of this new potency test was evaluated using a panel of 48 potentially pandemic strains of influenza viruses and vaccines spanning 16 years of antigenic drift, including the most recent pre-pandemic vaccine being developed against the "5 wave" A/H7N9 virus.

MicroRNA-335-5p and -3p synergize to inhibit estrogen receptor alpha expression and promote tamoxifen resistance.

microRNAs (miRNAs) are small noncoding RNA molecules involved in the regulation of gene expression and play critical roles in human malignancies. Next-generation sequencing analysis of the MCF-7 breast cancer cell line overexpressing miR-335-5p and miR-335-3p demonstrated that the miRNA duplex repressed genes involved in the ERα signaling pathway, and enhanced resistance of MCF-7 cells to the growth inhibitory effects of tamoxifen. These data suggest that despite its conventional role in tumor suppression, the miR-335 transcript can also play an oncogenic role in promoting agonistic estrogen signaling in a cancerous setting.

Complete Transcriptome RNA-Seq.

RNA-Seq is the leading technology for analyzing gene expression on a global scale across a broad spectrum of sample types. However, due to chemical modifications by fixation or degradation due to collection methods, samples often contain an abundance of RNA that is no longer intact, and the capability of current RNA-Seq protocols to accurately quantify such samples is often limited. We have developed an RNA-Seq protocol to address these key issues as well as quantify gene expression from the whole transcriptome.

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer.

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer.

The novel, small-molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer.

Purpose: To investigate SGI-110 as a "chemosensitizer" in ovarian cancer and to assess its effects on tumor suppressor genes (TSG) and chemoresponsiveness-associated genes silenced by DNA methylation in ovarian cancer.

Experimental Design: Several ovarian cancer cell lines were used for in vitro and in vivo platinum resensitization studies. Changes in DNA methylation and expression levels of TSG and other cancer-related genes in response to SGI-110 were measured by pyrosequencing and RT-PCR.

Effects of the endocrine-disrupting chemical DDT on self-renewal and differentiation of human mesenchymal stem cells.

Background: Although the global use of the endocrine-disrupting chemical DDT has decreased, its persistence in the environment has resulted in continued human exposure. Accumulating evidence suggests that DDT exposure has long-term adverse effects on development, yet the impact on growth and differentiation of adult stem cells remains unclear.

Objectives: Human mesenchymal stem cells (MSCs) exposed to DDT were used to evaluate the impact on stem cell biology.

A new method for stranded whole transcriptome RNA-seq.

This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis.

Adenoviral-delivered HE4-HSV-tk sensitizes ovarian cancer cells to ganciclovir.

Ovarian cancer (OC) is most often contained within the peritoneal cavity, making it an ideal disease for adenoviral-delivered gene therapies. In effort to develop a safe and effective gene therapy for OC, we created a replication deficient adenovirus bearing the herpes simplex thymidine kinase (HSV-tk) gene under direction of the tumor specific promoter human epididymis protein 4 (HE4). The purpose of this study was to investigate the ability of our adenoviral construct to transduce OC cells and mediate transgene expression of HSV-tk, thereby sensitizing OC to the pro-drug ganciclovir.

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity.

Proteome profiling for assessing diversity: analysis of individual heads of Drosophila melanogaster using LC-ion mobility-MS.

The proteomes of three heads of individual Drosophila melanogaster organisms have been analyzed and compared by a combination of liquid chromatography, ion mobility spectrometry, and mass spectrometry approaches. In total, 197 proteins are identified among all three individuals (an average of 120 +/- 20 proteins per individual), of which at least 101 proteins are present in all three individuals. Within all three datasets, more than 25 000 molecular ions (an average of 9000 +/- 2000 per individual) corresponding to protonated precursor ions of individual peptides have been observed.

Mapping the proteome of Drosophila melanogaster: analysis of embryos and adult heads by LC-IMS-MS methods.

Multidimensional separations combined with mass spectrometry are used to study the proteins that are present in two states of Drosophila melanogaster: the whole embryo and the adult head. The approach includes the incorporation of a gas-phase separation dimension in which ions are dispersed according to differences in their mobilities and is described as a means of providing a detailed analytical map of the proteins that are present. Overall, we find evidence for 1133 unique proteins.

Hox gene cross-regulatory interactions in the embryonic brain of Drosophila.

During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere. In order to gain further insight into the mechanisms of Hox gene action in the CNS, we have studied the molecular and genetic basis of cross-regulatory interactions between labial and other more posterior Hox genes using the GAL4/UAS system for targeted misexpression. Misexpression of posterior Hox genes in the embryonic neuroectoderm results in a labial loss-of function phenotype and a corresponding lack of Labial protein expression in the tritocerebrum.

Development of high-sensitivity ion trap ion mobility spectrometry time-of-flight techniques: a high-throughput nano-LC-IMS-TOF separation of peptides arising from a Drosophila protein extract.

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.

Customized microinjection glass capillary needles for P-element transformations in Drosophila melanogaster.

Here we describe how to generate customized microinjection needles from glass capillary tubes. Controls demonstrate the range of variables and effects on needle tip shape using a standard Flaming/Brown micropipet needle puller. Needles generated with two-cycle pulls provide a wider range of needle shapes in a predictable fashion.