A Microfluidics Workflow for Sample Preparation for Next-Generation DNA Sequencing.

Next-generation sequencing technology requires amplified, short DNA fragments with known end sequences. Samples must undergo processing steps, including extraction and purification of genomic DNA (gDNA), fragmentation, end repair, adapter ligation, and amplification, to prepare a sequencing library. The process of sample preparation requires careful control of temperature and buffer conditions, as well as the stringent removal of contaminants.

HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo.

Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of -acting RNA binding proteins (RBPs) with myriad -regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein-coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on noncoding regions (introns).

IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC.

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) expression correlates with malignancy, but its role(s) in pathogenesis remains enigmatic. We interrogated the IGF2BP3-RNA interaction network in pancreatic ductal adenocarcinoma (PDAC) cells. Using a combination of genome-wide approaches, we have identified 164 direct mRNA targets of IGF2BP3.

RNA-Seq methods for transcriptome analysis.

Deep sequencing has been revolutionizing biology and medicine in recent years, providing single base-level precision for our understanding of nucleic acid sequences in high throughput fashion. Sequencing of RNA, or RNA-Seq, is now a common method to analyze gene expression and to uncover novel RNA species. Aspects of RNA biogenesis and metabolism can be interrogated with specialized methods for cDNA library preparation.

RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation.

Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia-rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse.

Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA.

Eukaryotic cells carry two genomes, nuclear (nDNA) and mitochondrial (mtDNA), which are ostensibly decoupled in their replication, segregation and inheritance. It is increasingly appreciated that heteroplasmy, the occurrence of multiple mtDNA haplotypes in a cell, plays an important biological role, but its features are not well understood. Accurately determining the diversity of mtDNA has been difficult, due to the relatively small amount of mtDNA in each cell (<1% of the total DNA), the intercellular variability of mtDNA content and mtDNA pseudogenes (Numts) in nDNA.

Evaluation of the in vivo anti-inflammatory and analgesic and in vitro anti-cancer activities of curcumin and its derivatives.

Curcumin, obtained from turmeric, has several biological properties to make it a desirable template for drug development. A lipophilic derivative of curcumin, diacetyl curcumin (DAC) and a hydrophilic derivative, diglutaryl curcumin (DGC) were synthesized and their in vivo analgesic and anti-inflammatory activities were compared with those of curcumin and aspirin. The in vitro anti-cancer activities of curcumin and the two derivatives against three cell cancer lines were compared with those against a non-cancerous cell line.

Delivery of RNAi mediators.

Delivering polynucleotides into animals has been a major challenge facing their success as therapeutic agents. Given the matured understanding of antibody-mediated delivery techniques, it is possible to rationally design delivery vehicles that circulate in the blood stream and are specifically delivered into target organs. If the targeting moiety is designed to contain the cargo of an RNAi mediator without impacting its paratope, directed delivery can be achieved.

Antibody targeted siRNA delivery.

It is very clear that RNA interference (RNAi) is a potent and versatile tool for gene silencing. One of the hurdles to making siRNA/miRNA a human therapeutic includes effective in vivo delivery and being able to deliver drugs to target cells only. The commercial success of in vivo applications of RNAi hinges on the development of new delivery methods.

Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.

Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexagonal symmetry in a connexin hemichannel, particularly in the cytoplasmic domain regions at the ends of the transmembrane helices.

Connexin mutation that causes dominant congenital cataracts inhibits gap junctions, but not hemichannels, in a dominant negative manner.

The connexin (Cx) 50, E48K, mutation is associated with a human dominant congenital cataract; however, the underlying molecular mechanism has not been characterized. The glutamate (E) residue at position 48 is highly conserved across animal species and types of connexins. When expressed in paired Xenopus oocytes, human (h) and chicken (ch) Cx50 E48K mutants showed no electrical coupling.

Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel.

Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix.