Unpicking neurodegeneration in a dish with human pluripotent stem cells

Eukaryotic cell division is an orderly and timely process involving the error-free segregation of chromosomes and cytoplasmic components to give rise to two separate daughter cells. Defects in genome maintenance mechanisms such as cell cycle checkpoints and DNA repair can impact the segregation of the genome during mitosis leading to multiple chromosomal imbalances. In mammals, the DNA damage checkpoint effector Checkpoint Kinase 1 (Chk1) is essential for responses to DNA replication errors, external DNA damage, and chromatin breaks. We reported recently that Chk1 also was essential for chromosome segregation and completion of cytokinesis to prevent genomic instability. Our studies demonstrated that Chk1 deficiency in mitotic cells causes chromosome mis-alignment, lagging chromosomes, chromosome mis- segregation, cytokinetic regression, and binucleation. In addition, abrogation of Chk1 resulted in aberrant localization of mitotic Aurora B kinase at the metaphase plate, anaphase spindle midzone, and cytokinetic midbody as studied both in various cell lines and in a mouse model. Therefore, inappropriate regulation of Chk1 levels during cell cycle progression will result in failed cell division and enhanced genomic instability.     

Vitreous from idiopathic epiretinal membrane patients induces glial-to-mesenchymal transition in Müller cells

Idiopathic epiretinal membranes (ERMs) are fibrocellular membranes containing extracellular matrix proteins and epiretinal cells of retinal and extraretinal origin. iERMs lead to decreased visual acuity and their pathogenesis has not been completely defined. Macroglial Müller cells appear to play a pivotal role in the pathogenesis of iERM where they may undergo glial-to-mesenchymal transition (GMT), a transdifferentiation process characterized by the downregulation of Müller cell markers, paralleled by the upregulation of pro-fibrotic myofibroblast markers. Previous observations from our laboratory allowed the molecular identification of two major clusters of iERM patients (named iERM-A and iERM-B), iERM-A patients being characterized by less severe clinical features and a more “quiescent” iERM gene expression profile when compared to iERM-B patients. In the present work, Müller MIO-M1 cells were exposed to vitreous samples obtained before membrane peeling from the same cohort of iERM-A and iERM-B patients. The results demonstrate that iERM vitreous induces proliferation, migration, and GMT in MIO-M1 cells, a phenotype consistent with Müller cell behavior during iERM progression. However, even though the vitreous samples obtained from iERM-A patients were able to induce a complete GMT in MIO-M1 cells, iERM-B samples caused only a partial GMT, characterized by the downregulation of Müller cell markers in the absence of upregulation of pro-fibrotic myofibroblast markers. Together, the results indicate that a relationship may exist among the ability of iERM vitreous to modulate GMT in Müller cells, the molecular profile of the corresponding iERMs, and the clinical features of iERM patients.     

Decreased glutamine synthetase,increased citrulline–nitric oxide cycle activities,and oxidative stress in different regions of brain in epilepsy rat model

To understand their role in epilepsy, the nitric oxide synthetase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite (NOx), thiobarbituric acid reactive substances (TBARS), and total antioxidant status (TAS), were estimated in different regions of brain in rats subjected to experimental epilepsy induced by subcutaneous administration of kainic acid (KA). The short-term (acute) group animals were killed after 2 h and the long term (chronic) group animals were killed after 5 days of single injection of KA (15 mg/kg body weight). After decapitation of rats, the brain regions were separated and in their homogenates, the concentration of NOx, TBARS and TAS and the activities of NOS, AS, AL, arginase and glutamine synthetase were assayed by colorimetric methods. The results of the study demonstrated the increased activity of NOS and formation of NO in acute and chronic groups epilepsy. The activities of AS and AL were increased and indicate the effective recycling of citrulline to arginine. The activity of glutamine synthetase was decreased in acute and chronic groups of epilepsy compared to control group and indicate the modulation of its activity by NO in epilepsy. The activity of arginase was not changed in acute group; however it was decreased in chronic group and may favor increased production of NO in this condition. The concentration TBARS were increased and TAS decreased in acute and chronic groups of epilepsy and supports the oxidative stress in epilepsy.     

Immunohistochemical staining of slit2 in primary and metastatic prostatic adenocarcinoma

BACKGROUND: Conflicting roles for Slit2, a protein involved in mediating the processes of cell migration and chemotactic response, have been previously described in prostate cancer. Here we use immunohistochemistry to evaluate the expression of Slit2 in normal donor prostate (NDP), benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets). METHODS: Tissue microarrays were immunostained for Slit2. The staining intensities were quantified using automated image analysis software. The data was statistically analyzed using one-way analysis of variance with subsequent Tukey tests for multiple comparisons or a nonparametric equivalent. Eleven cases of NDP, 35 cases of NAC, 15 cases of BPH, 35 cases of HGPIN, 106 cases of PCa, and 37 cases of Mets were analyzed. RESULTS: Specimens of PCa and HGPIN had the highest absolute staining for Slit2. Significant differences were seen between PCa and NDP (P < .05), PCa and NAC (P < .05), HGPIN and NDP (P < .05), and HGPIN and NAC (P < .05). Whereas the average Mets staining was not significantly different from NDP or NAC, several individual Mets cases featured intense staining. CONCLUSIONS: To our knowledge, this represents the first study comparing the immunohistochemical profiles of Slit2 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC. These findings suggest that Slit2 expression can be increased in HGPIN, PCa, and Mets, making it a potentially important biomarker for prostate cancer.     

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process ¹. Over the years many improvements and tools for genetic manipulation have become available in Drosophila². Soon after the initial discovery by Frie and Mello ³ that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development ^{4, 5}.Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases ^{6, 7}. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs ⁸. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion ^{9, 10}. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.Download video file.^{(52M, mov)}     

Factors influencing the density of aerobic granular sludge

In the present study, the factors influencing density of granular sludge particles were evaluated. Granules consist of microbes, precipitates and of extracellular polymeric substance. The volume fractions of the bacterial layers were experimentally estimated by fluorescent in situ hybridisation staining. The volume fraction occupied by precipitates was determined by computed tomography scanning. PHREEQC was used to estimate potential formation of precipitates to determine a density of the inorganic fraction. Densities of bacteria were investigated by Percoll density centrifugation. The volume fractions were then coupled with the corresponding densities and the total density of a granule was calculated. The sensitivity of the density of the entire granule on the corresponding settling velocity was evaluated by changing the volume fractions of precipitates or bacteria in a settling model. Results from granules originating from a Nereda reactor for simultaneous phosphate COD and nitrogen removal revealed that phosphate-accumulating organisms (PAOs) had a higher density than glycogen-accumulating organisms leading to significantly higher settling velocities for PAO-dominated granules explaining earlier observations of the segregation of the granular sludge bed inside reactors. The model showed that a small increase in the volume fraction of precipitates (1–5 %) strongly increased the granular density and thereby the settling velocity. For nitritation–anammox granular sludge, mainly granular diameter and not density differences are causing a segregation of the biomass in the bed.     

Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems

Chemolithoautotrophic AOB (ammonia-oxidizing bacteria) form a crucial component in microbial nitrogen cycling in both natural and engineered systems. Under specific conditions, including transitions from anoxic to oxic conditions and/or excessive ammonia loading, and the presence of high nitrite (NO??) concentrations, these bacteria are also documented to produce nitric oxide (NO) and nitrous oxide (N?O) gases. Essentially, ammonia oxidation in the presence of non-limiting substrate concentrations (ammonia and O?) is associated with N?O production. An exceptional scenario that leads to such conditions is the periodical switch between anoxic and oxic conditions, which is rather common in engineered nitrogen-removal systems. In particular, the recovery from, rather than imposition of, anoxic conditions has been demonstrated to result in N?O production. However, applied engineering perspectives, so far, have largely ignored the contribution of nitrification to N?O emissions in greenhouse gas inventories from wastewater-treatment plants. Recent field-scale measurements have revealed that nitrification-related N?O emissions are generally far higher than emissions assigned to heterotrophic denitrification. In the present paper, the metabolic pathways, which could potentially contribute to NO and N?O production by AOB have been conceptually reconstructed under conditions especially relevant to engineered nitrogen-removal systems. Taken together, the reconstructed pathways, field- and laboratory-scale results suggest that engineering designs that achieve low effluent aqueous nitrogen concentrations also minimize gaseous nitrogen emissions.     

An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect

Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(-)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.     

A specific miRNA signature in the peripheral blood of glioblastoma patients

The prognosis of patients afflicted by glioblastoma remains poor. Biomarkers for the disease would be desirable in order to allow for an early detection of tumor progression or to indicate rapidly growing tumor subtypes requiring more intensive therapy. In this study, we investigated whether a blood-derived specific miRNA fingerprint can be defined in patients with glioblastoma. To this end, miRNA profiles from the blood of 20 patients with glioblastoma and 20 age- and sex-matched healthy controls were compared. Of 1158 tested miRNAs, 52 were significantly deregulated, as assessed by unadjusted Student's t-test at an alpha level of 0.05. Of these, two candidates, miR-128 (up-regulated) and miR-342-3p (down-regulated), remained significant after correcting for multiple testing by Benjamini-Hochberg adjustment with a p-value of 0.025. The altered expression of these two biomarkers was confirmed in a second cohort of glioblastoma patients and healthy controls by real-time PCR and validated for patients who had received neither radio- nor chemotherapy and for patients who had their glioblastomas resected more than 6 months ago. Moreover, using machine learning, a comprehensive miRNA signature was obtained that allowed for the discrimination between blood samples of glioblastoma patients and healthy controls with an accuracy of 81% [95% confidence interval (CI) 78-84%], specificity of 79% (95% CI 75-83%) and sensitivity of 83% (95% CI 71-85%). In summary, our proof-of-concept study demonstrates that blood-derived glioblastoma-associated characteristic miRNA fingerprints may be suitable biomarkers and warrant further exploration.