Matrin 3 and HIV Rev Regulation of mRNA

The nuclear matrix protein, MATR3, is a newly-described Rev cofactor whose mechanism of action is only starting to be revealed

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids

T-DNA insertion mutants reveal complex expression patterns of the aldehyde dehydrogenase 3H1 locus in Arabidopsis thaliana

The Arabidopsis thaliana aldehyde dehydrogenase 3H1 gene (ALDH3H1; AT1G44170) belongs to family 3 of the plant aldehyde dehydrogenase superfamily. The full-length transcript of the corresponding gene comprises an open reading frame of 1583 bp and encodes a protein of 484 amino acid residues. Gene expression studies have shown that this transcript accumulates mainly in the roots of 4-week-old plants following abscisic acid, dehydration, and NaCl treatments. The current study provided experimental data that the ALDH3H1 locus generates at least five alternative transcript variants in addition to the previously described ALDH3H1 mRNA. The alternative transcripts accumulated in wild-type plants at a low level but were upregulated in a mutant that carried a T-DNA insertion in the first exon of the gene. Expression of the transcript isoforms involved alternative gene splicing combined with an alternative promoter. The transcript isoforms were differentially expressed in the roots and shoots and showed developmental stage- and tissue-specific expression patterns. These data support the hypothesis that alternative isoforms produced by gene splicing or alternative promoters regulate the abundance of the constitutively spliced and functional variants

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform

Matrin3: connecting gene expression with the nuclear matrix.

As indicated by its name, Matrin3 was discovered as a component of the nuclear matrix, an insoluble fibrogranular network that structurally organizes the nucleus. Matrin3 possesses both DNA- and RNA-binding domains and, consistent with this, has been shown to function at a number of stages in the life cycle of messenger RNAs. These numerous activities indicate that Matrin3, and indeed the nuclear matrix, do not just provide a structural framework for nuclear activities but also play direct functional roles in these activities. Here, we review the structure, functions, and molecular interactions of Matrin3 and of Matrin3-related proteins, and the pathologies that can arise upon mutation of Matrin3. WIREs RNA 2016, 7:303-315. doi: 10.1002/wrna.1336 For further resources related to this article, please visit the WIREs website.We thank Clare Gooding and Dipen Rajgor for critical comments on the manuscript. Work in the CWJS lab on Matrin3 is funded by a grant from the Wellcome Trust (092900). JA was funded by a Boehringer Ingelheim Fonds studentship.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/wrna.133

Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization

FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts. A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin

Underwater Application of Quantitative PCR on an Ocean Mooring

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions