Influences of electrolytes and glucose on formulation of carbonate apatite nanocrystals for efficient gene delivery to mammalian cells

Genetic manipulation of human cells through delivery of a functional gene or a gene-silencing element is an attractive approach to treat critical diseases very precisely and effectively. Extensive research on the genetic basis of human diseases with complete sequencing of human genome has revealed many vital genes as possible targets in gene therapy programs. On the other hand, to facilitate cell- or tissue-directed delivery of genes and gene-silencing nucleic acid sequences, both genetic and chemical engineering approaches have led to the generation of various viral and nonviral carriers. However, considering the issues of both safety and efficacy, none of the existing vectors is an ideal candidate for clinical use. We recently established pH-sensitive inorganic nanocrystals of carbonate apatite with capability of efficient intracellular delivery and release of associated DNA molecules for subsequent protein expression. Here we show a new synthetic approach for carbonate apatite crystals with stronger affinity toward DNA, leading to significant increment in both transgene delivery and expression. Moreover, CaCl₂ and NaCl, existing as the major electrolytes in the bicarbonate-buffered solution, dose-dependently govern particle size and eventually internalization and expression of particle-associated DNA.     

Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

Background

For clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSC's biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed.

Results

Human MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs.

Conclusions

In our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.     

Epidemiology and Genetic Diversity of Rotavirus Strains in Children with Acute Gastroenteritis in Lahore,Pakistan

Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008–2009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n = 447 out of 1306). No significant difference was found between different age groups positive for rotavirus (p>0.05). A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P [4] = 3(6.81%); G1P [6] = 9(20.45%); G1P [8] = 1(2.27%); G2P [4] = 21(47.72%); G2P [8] = 1(2.27%); G9P [4] = 1(2.27%); G9P [6] = 1(2.27%) and G9P [8] = 7(15.90%). Phylogenetic analysis revealed that the VP7 and VP4 sequences clustered closely with the previously detected strains in the country as well as Belgian rotaviruses. Antigenic characterization was performed by analyzing major epitopes in the immunodominant VP7 and VP4 gene segments. Although the neutralization conferring motifs were found variable between the Pakistani strains and the two recommended vaccines strains (Rotarix™ and RotaTeq™), we validate the use of rotavirus vaccine in Pakistan based on the proven and recognized vaccine efficacy across the globe. Our findings constitute the first report on rotavirus’ genotype diversity, their phylogenetic relatedness and epidemiology during the pre-vaccination era in Lahore, Pakistan and support the immediate introduction of rotavirus vaccine in the routine immunization program of the country.     

Neurovascular coupling and oximetry during epileptic events

Epilepsy is an abnormal brain state in which a large population of neurons is synchronously active, causing an enormous increase in metabolic demand. Recent investigations using high-resolution imaging techniques, such as optical recording of intrinsic signals and voltagesensitive dyes, as well as measurements with oxygen-sensitive electrodes have elucidated the spatiotemporal relationship between neuronal activity, cerebral blood volume, and oximetry in vivo. A focal decrease in tissue oxygenation and a focal increase in deoxygenated hemoglobin occurs following both interictal and ictal events. This “epileptic dip” in oxygenation can persist for the duration of an ictal event, suggesting that cerebral blood flow is inadequate to meet metabolic demand. A rapid focal increase in cerebral blood flow and cerebral blood volume also accompanies epileptic events; however, this increase in perfusion soon (>2 s) spreads to a larger area of the cortex than the excitatory change in membrane potential. Investigations in humans during neurosurgical operations have confirmed the laboratory data derived from animal studies. These data not only have clinical implications for the interpretation of noninvasive imaging studies such as positron emission tomography, single-photon emission tomography, and functional magnetic resonance imaging but also provide a mechanism for the cognitive decline in patients with chronic epilepsy.     

Representational geometry of perceptual decisions in the monkey parietal cortex

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Identification of Small Molecule and Genetic Modulators of AON-Induced Dystrophin Exon Skipping by High-Throughput Screening

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened ∼10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.     

Effectiveness of revascularization of the ulcerated foot in diabetic patients with peripheral artery disease for one year follow-up

Background

Diabetes is an important risk factor for atherosclerosis. The diabetic foot is characterized by the presence of arteriopathy and neuropathy. When ischemia is diagnosed, restoration of pulsatile blood flow by revascularization may be considered for salvaging the limb. The treatment options are angioplasty with or without stenting and surgical bypass or hybrid procedures combining the two.

Aims

To evaluate the outcomes of severe ischemic diabetic foot ulcers for which percutaneous transluminal angioplasty (PTA) was considered as the first-line vascular procedure. Factors associated with successful PTA were also evaluated.

Methods

In 80 consecutive diabetic patients with foot ulcers and severe limb ischemia, PTAwas performed if feasible. All patients were followed until healing or for one year. Clinical and angiographic factors in fluencing outcomes after PTA were sought by univariate and multivariate analysis.

Results

PTAwas done in 73 of the 80 (91.2%) patients, and considered clinically succe ssful in 58(79.9%). Successful PTA was significantly higher in patients with Superficial femoral artery, posterior Tibialis and dorsalis pedis arteries involvement in the univariate analysis. Seven patients were expired during the study follow up due to MI, pulmonary thromboembolism and GI bleeding.

Conclusion

PTA in diabetic patients with severe ischemic foot ulcers provided favorable. Some parameters could be used for predicting PTA successfulness.