Steven R. Gullans

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. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg-1 LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg-1 LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.

Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice

Minocycline mediates neuroprotection in experimental models of neurodegeneration. It inhibits the activity of caspase-1, caspase-3, inducible form of nitric oxide synthetase (iNOS) and p38 mitogen-activated protein kinase (MAPK). Although minocycline does not directly inhibit these enzymes, the effects may result from interference with upstream mechanisms resulting in their secondary activation. Because the above-mentioned factors are important in amyotrophic lateral sclerosis (ALS), we tested minocycline in mice with ALS. Here we report that minocycline delays disease onset and extends survival in ALS mice. Given the broad efficacy of minocycline, understanding its mechanisms of action is of great importance. We find that minocycline inhibits mitochondrial permeability-transition-mediated cytochrome c release. Minocycline-mediated inhibition of cytochrome c release is demonstrated in vivo, in cells, and in isolated mitochondria. Understanding the mechanism of action of minocycline will assist in the development and testing of more powerful and effective analogues. Because of the safety record of minocycline, and its ability to penetrate the blood–brain barrier, this drug may be a novel therapy for ALS.

Ascorbic Acid Enhances Differentiation of Embryonic Stem Cells Into Cardiac Myocytes

Background—Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germ layers. In appropriate culture conditions, ES cells can differentiate into specialized cells, including cardiac myocytes, but the efficiency is typically low and the process is incompletely understood. Methods and Results—We evaluated a chemical library for its potential to induce cardiac differentiation of ES cells in the absence of embryoid body formation. Using ES cells stably transfected with cardiac-specific &agr;-cardiac myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP), 880 compounds approved for human use were screened for their ability to induce cardiac differentiation. Treatment with ascorbic acid, also known as vitamin C, markedly increased the number of EGFP-positive cells, which displayed spontaneous and rhythmic contractile activity and stained positively for sarcomeric myosin and &agr;-actinin. Furthermore, ascorbic acid induced the expression of cardiac genes, including GATA4, &agr;-MHC, and &bgr;-MHC in untransfected ES cells in a developmentally controlled manner. This effect of ascorbic acid on cardiac differentiation was not mimicked by the other antioxidants such as N-acetylcysteine, Tiron, or vitamin E. Conclusions—Ascorbic acid induces cardiac differentiation in ES cells. This study demonstrates the potential for chemically modifying the cardiac differentiation program of ES cells.

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

Parkinsons 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. The molecular multigene marker here identified is associated with risk of PD in 66 samples of the training set comprising healthy and disease controls [third tertile cross-validated odds ratio of 5.7 (P for trend 0.005)]. It is further validated in 39 independent test samples [third tertile odds ratio of 5.1 (P for trend 0.04)]. Insights into disease-linked processes detectable in peripheral blood are offered by 22 unique genes differentially expressed in patients with PD versus healthy individuals. These include the cochaperone ST13, which stabilizes heat-shock protein 70, a modifier of α-synuclein misfolding and toxicity. ST13 messenger RNA copies are lower in patients with PD (mean ± SE 0.59 ± 0.05) than in controls (0.96 ± 0.09) (P = 0.002) in two independent populations. Thus, gene expression signals measured in blood can facilitate the development of biomarkers for PD.

Hyponatremia causes large sustained reductions in brain content of multiple organic osmolytes in rats

Brain adaptation to hypoosmolality is known to involve volume regulatory losses of both extracellular and intracellular electrolytes. We studied the effects of acute and chronic hypoosmolality on brain content of organic osmolytes as well as electrolytes in rats to ascertain the relative contributions of different brain solutes to the brain volume regulation that occurs under these conditions. Brains were dissected from rats after 2, 7 and 14 d of sustained hyponatremia induced by continuous infusion of 1-deamino-[8-D-arginine]-vasopressin (DDVAP) in combination with a liquid formula, along with control rats fed the same formula in the absence of DDAVP infusions. One half of each brain was analyzed for organic osmolyte contents and the other half for water and electrolyte contents. Brain Na+, K+ and Cl- and multiple organic osmolytes (glutamate, creatine, taurine, myo-inositol, glutamine and glycerophosphoryl-choline) decreased markedly by 2 d of hyponatremia, and brain electrolyte and most organic osmolyte contents then remained at these reduced levels throughout the duration of the hyponatremia. Although the absolute magnitude of the brain electrolyte losses was greater than the magnitude of the brain organic osmolyte losses, the organic osmolyte losses accounted for approximately 35% of the total measured brain solute losses during sustained hyponatremia. These results demonstrate that organic osmolytes constitute a significant proportion of the brain solute losses that take place during hyponatremia, and indicate that reductions in both organic osmolyte and electrolyte contents are necessary to accomplish brain volume regulation during adaptation to sustained hypoosmolality.

Better therapeutics through microarrays

DNA microarrays are an integral part of the process for therapeutic discovery, optimization and clinical validation. At an early stage, investigators use arrays to prioritize a few genes as potential therapeutic targets on the basis of various criteria. Subsequently, gene expression analysis assists in drug discovery and toxicology by eliminating poor compounds and optimizing the selection of promising leads. Integral to this process is the use of sophisticated statistics, mathematics and bioinformatics to define statistically valid observations and to deduce complex patterns of phenotypes and biological pathways. In short, microarrays are redefining the drug discovery process by providing greater knowledge at each step and by illuminating the complex workings of biological systems.

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. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.

A combination of NaCl and urea enhances survival of IMCD cells to hyperosmolality

Physiological adaptation to the hyperosmolar milieu of the renal medulla involves a complex series of signaling and gene expression events in which NaCl and urea activate different cellular processes. In the present study, we evaluated the effects of NaCl and urea, individually and in combination, on the viability of murine inner medullary collecting duct (mIMCD3) cells. Exposure to hyperosmolar NaCl or urea caused comparable dose- and time-dependent decreases in cell viability, such that 700 mosmol/kgH2O killed >90% of the cells within 24 h. In both cases, cell death was an apoptotic event. For NaCl, loss of viability at 24 h paralleled decreases in RNA and protein synthesis at 4 h, whereas lethal doses of urea had little or no effect on these biosynthetic processes. Cell cycle analysis showed both solutes caused a slowing of the G2/M phase. Surprisingly, cells exposed to a combination of NaCl + urea were significantly more osmotolerant such that 40% survived 900 mosmol/kgH2O. Madin-Darby canine kidney cells but not human umbilical vein endothelial cells also exhibited a similar osmotolerance response. Enhanced survival was not associated with a restoration of normal biosynthetic rates or cell cycle progression. However, the combination of NaCl + urea resulted in a shift in Hsp70 expression that appeared to correlate with survival. In conclusion, hyperosmolar NaCl and urea activate independent and complementary cellular programs that confer enhanced osmotolerance to renal medullary epithelial cells.Physiological adaptation to the hyperosmolar milieu of the renal medulla involves a complex series of signaling and gene expression events in which NaCl and urea activate different cellular processes. In the present study, we evaluated the effects of NaCl and urea, individually and in combination, on the viability of murine inner medullary collecting duct (mIMCD3) cells. Exposure to hyperosmolar NaCl or urea caused comparable dose- and time-dependent decreases in cell viability, such that 700 mosmol/kgH2O killed >90% of the cells within 24 h. In both cases, cell death was an apoptotic event. For NaCl, loss of viability at 24 h paralleled decreases in RNA and protein synthesis at 4h, whereas lethal doses of urea had little or no effect on these biosynthetic processes. Cell cycle analysis showed both solutes caused a slowing of the G2/M phase. Surprisingly, cells exposed to a combination of NaCl + urea were significantly more osmotolerant such that 40% survived 900 mosmol/kgH2O. Madin-Darby canine kidney cells but not human umbilical vein endothelial cells also exhibited a similar osmotolerance response. Enhanced survival was not associated with a restoration of normal biosynthetic rates or cell cycle progression. However, the combination of NaCl + urea resulted in a shift in Hsp70 expression that appeared to correlate with survival. In conclusion, hyperosmolar NaCl and urea activate independent and complementary cellular programs that confer enhanced osmotolerance to renal medullary epithelial cells.

Osmotic Stress Protein 94 (Osp94) A NEW MEMBER OF THE Hsp110/SSE GENE SUBFAMILY

Preservation of cell viability and function in the hyperosmolar environment of the renal medulla is a complex process that requires selective gene expression. We have identified a new member of the heat shock protein (hsp) 70 superfamily that is up-regulated in renal inner medullary collecting duct cells (mIMCD3 cells) during exposure to hyperosmotic NaCl stress. Known as osmotic stress protein 94, or Osp94, this 2935-base pair cDNA encodes an 838-amino acid protein that shows greatest homology to the recently discovered hsp110/SSE gene subfamily. Like the hsps, Osp94 has a putative amino-terminal ATP-binding domain and a putative carboxyl-terminal peptide-binding domain. The in vitro translated Osp94 product migrated as a 105-110-kDa protein on SDS-polyacrylamide gel electrophoresis. In mIMCD3 cells, Osp94 mRNA expression was greatly up-regulated by hyperosmotic NaCl or heat stress. In mouse kidney, Osp94 mRNA expression paralleled the known corticomedullary osmolality gradient showing highest expression in the inner medulla. Moreover, inner medullary Osp94 expression was increased during water restriction when osmolality is known to increase. Thus, Osp94 is a new member of the hsp110/SSE stress protein subfamily and likely acts as a molecular chaperone.

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. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.