Large-gap peripheral nerve repair using xenogeneic transplants in rhesus macaques.

Surgical intervention is required to successfully treat severe, large-gap (≥4 cm) peripheral nerve injuries. However, all existing treatments have shortcomings and an alternative to the use of autologous nerves is needed. Human and porcine nerves are physiologically similar, with comparable dimensions and architecture, presence and distribution of Schwann cells, and conserved features of the extracellular matrix (ECM).

Novel function of the C-terminal region of the Hsp110 family member Osp94 in unfolded protein refolding.

Osp94 (also known as HSPA4L or HSPH3), a member of the Hsp110/Sse1 family of heat-shock proteins, has a longer C-terminus than found in Hsc70/Hsp70 family proteins, composed of the loop region with a partial substrate-binding domain (SBD) β (L), and the SBDα and the C-terminal extension (H), but the functions of these domains are poorly understood. Here, we found that Osp94 suppressed heat-induced aggregation of luciferase (Luc). Osp94-bound heat-inactivated Luc was reactivated in the presence of rabbit reticulocyte lysate (RRL) and/or a combination of Hsc70 and Hsp40 (also known as HSPA8 and DNAJB1, respectively).

Second generation sequencing of the mesothelioma tumor genome.

The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.

LNA-mediated microRNA silencing in non-human primates.

microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease and therefore represent a potential new class of targets for therapeutic intervention. Despite recent progress in silencing of miRNAs in rodents, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates.

Transcriptome sequencing of malignant pleural mesothelioma tumors.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue.

ATF3 protects against renal ischemia-reperfusion injury.

Oxidative stress-induced cell death plays a major role in the progression of ischemic acute renal failure. Using microarrays, we sought to identify a stress-induced gene that may be a therapeutic candidate. Human proximal tubule (HK2) cells were treated with hydrogen peroxide (H2O2) and RNA was applied to an Affymetrix gene chip.

Identification, coassembly, and activity of gamma-aminobutyric acid receptor subunits in renal proximal tubular cells.

Although the properties and functions of GABA(A) receptors in the mammalian central nervous system have been well studied, the presence and significance of GABA(A) receptors in non-neural tissues are less clear. The goal of this study was to examine the expression of GABA(A) receptor alpha(1), alpha(2), alpha(4), alpha(5), beta(1), gamma(1), gamma(2), and delta subunits in the kidney and to determine whether these subunits coassemble to form an active renal epithelial cell GABA(A) receptor. Using reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and brain or cerebellum through polymerase chain reaction (PCR) and sequencing of the PCR products, we revealed that rat kidney cortex contained the alpha(1), alpha(5), beta(1), gamma(1), and gamma(2) subunits and that they were similar to the neuronal subunits.

Molecular markers of early Parkinson's disease based on gene expression in blood.

Parkinson's disease (PD) progresses relentlessly and affects five million people worldwide. Laboratory tests for PD are critically needed for developing treatments designed to slow or prevent progression of the disease. We performed a transcriptome-wide scan in 105 individuals to interrogate the molecular processes perturbed in cellular blood of patients with early-stage PD.

Single-molecule detection of transcription factor binding to DNA in real time: specificity, equilibrium, and kinetic parameters.

Specificity and temporal control of transcriptional machinery are encoded within sequence-specific transcription factors, of which there are thousands in mammalian genomes. Efforts to completely decipher this code will require an understanding of the DNA binding thermodynamic and kinetic properties displayed by each transcription factor, a daunting task given the current methodologies for measuring these interactions. Here, we present a novel methodology to quantify the binding of proteins to target DNA molecules based on single-molecule detection and real-time counting of individual free and bound fluorescently tagged molecules flowing past a detection device.

Single DNA molecule stretching in sudden mixed shear and elongational microflows.

High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels.

Acute injury directs the migration, proliferation, and differentiation of solid organ stem cells: evidence from the effect of hypoxia-ischemia in the CNS on clonal "reporter" neural stem cells.

Clonal neural cells with stem-like features integrate appropriately into the developing and degenerating central and peripheral nervous system throughout the neuraxis. In response to hypoxic-ischemic (HI) injury, previously engrafted, integrated, and quiescent clonal neural stem cells (NSCs) transiently re-enter the cell cycle, migrate preferentially to the site of ischemia, and differentiate into neurons and oligodendrocytes, the neural cell types typically lost following HI brain injury. They also replenish the supply of immature uncommitted resident stem/progenitor cells.

A single-molecule method for the quantitation of microRNA gene expression.

MicroRNAs (miRNA) are short endogenous noncoding RNA molecules that regulate fundamental cellular processes such as cell differentiation, cell proliferation and apoptosis through modulation of gene expression. Critical to understanding the role of miRNAs in this regulation is a method to rapidly and accurately quantitate miRNA gene expression. Existing methods lack sensitivity, specificity and typically require upfront enrichment, ligation and/or amplification steps.

Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA.

Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs.

Differences in apolipoprotein E3/3 and E4/4 allele-specific gene expression in hippocampus in Alzheimer disease.

Apolipoprotein E4 (APOE4) allele is a major risk factor for late-onset familial and sporadic Alzheimer disease (AD). The mechanism of action of APOE in the etiology of AD remains unclear. Using gene expression (microarray) analysis of human hippocampus from APOE3/3 AD and APOE4/4 AD cases, we found different gene transcription patterns between APOE4/4 and APOE3/3 AD cases.

Endogenous ouabain: upregulation of steroidogenic genes in hypertensive hypothalamus but not adrenal.

Background: Mammalian tissues contain a presumed endogenous Na+, K(+)-ATPase inhibitor that binds reversibly to the Na+ pump with high affinity and specificity. The inhibitor has been linked to the pathogenesis of experimental volume-expanded and human essential hypertension. This compound has been isolated from mammalian hypothalamus and appears to be an isomer of the plant-derived cardiac glycoside ouabain, if not ouabain itself.

Expression profiling of substantia nigra in Parkinson disease, progressive supranuclear palsy, and frontotemporal dementia with parkinsonism.

Background: Parkinson disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra. Genes contributing to rare mendelian forms of PD have been identified, but the genes involved in the more common idiopathic PD are not well understood.

Objectives: To identify genes important to PD pathogenesis using microarrays and to investigate their potential to aid in diagnosing parkinsonism.

Osmoadaptation-related genes in inner medulla of mouse kidney using microarray.

To distinguish biological molecular processes of osmotic stress occurring in inner medulla, we utilized microarrays to monitor expression profiles. RNAs from three segments (cortex, outer medulla, and inner medulla) of mouse kidney were isolated and applied to microarrays. We found 35 genes expressed highly in inner medulla.

Limited agreement among three global gene expression methods highlights the requirement for non-global validation.

Motivation: DNA microarrays have revolutionized biological research, but their reliability and accuracy have not been extensively evaluated. Thorough testing of microarrays through comparison to dissimilar gene expression methods is necessary in order to determine their accuracy.

Results: We have systematically compared three global gene expression methods on all available histologically normal samples from five human organ types.

DNA mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags.

We have developed a rapid molecular mapping technology--Direct Linear Analysis (DLA)--on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. Double-stranded DNA molecules were tagged at sequence-specific motif sites with fluorescent bisPNA (Peptide Nucleic Acid) tags.

Regulation of expression of the stress response gene, Osp94: identification of the tonicity response element and intracellular signalling pathways.

Osp94 (osmotic stress protein of 94 kDa) is known to be up-regulated by hypertonic and heat-shock stresses in mouse renal inner medullary collecting duct (mIMCD3) cells. To investigate the molecular mechanism of transcriptional regulation of the Osp94 gene under these stresses, we cloned and characterized the 5'-flanking region of the gene. Sequence analysis of the proximal 4 kb 5'-flanking region revealed a TATA-less G/C-rich promoter region containing a cluster of Sp1 sites.

Spatiotemporal patterns of gene expression during fetal monkey brain development.

Human DNA microarrays are used to study the spatiotemporal patterns of gene expression during the course of fetal monkey brain development. The 444 most dynamically expressed genes in four major brain areas are reported at five different fetal ages. The spatiotemporal profiles of gene expression show both regional specificity as well as waves of gene expression across the developing brain.