Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context

Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).     

International technology transfer of a GCLP-compliant HIV-1 neutralizing antibody assay for human clinical trials

The Collaboration for AIDS Vaccine Discovery/Comprehensive Antibody-Vaccine Immune Monitoring Consortium (CAVD/CA-VIMC) assisted an international network of laboratories in transferring a validated assay used to judge HIV-1 vaccine immunogenicity in compliance with Good Clinical Laboratory Practice (GCLP) with the goal of adding quality to the conduct of endpoint assays for Human Immunodeficiency Virus I (HIV-1) vaccine human clinical trials. Eight Regional Laboratories in the international setting (Regional Laboratories), many located in regions where the HIV-1 epidemic is most prominent, were selected to implement the standardized, GCLP-compliant Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells (TZM-bl NAb Assay). Each laboratory was required to undergo initial training and implementation of the immunologic assay on-site and then perform partial assay re-validation, competency testing, and undergo formal external audits for GCLP compliance. Furthermore, using a newly established external proficiency testing program for the TZM-bl NAb Assay has allowed the Regional Laboratories to assess the comparability of assay results at their site with the results of neutralizing antibody assays performed around the world. As a result, several of the CAVD/CA-VIMC Regional Laboratories are now in the process of conducting or planning to conduct the GCLP-compliant TZM-bl NAb Assay as an indicator of vaccine immunogenicity for ongoing human clinical trials.     

Tigers need cover: multi-scale occupancy study of the big cat in Sumatran forest and plantation landscapes

The critically endangered Sumatran tiger (Panthera tigris sumatrae Pocock, 1929) is generally known as a forest-dependent animal. With large-scale conversion of forests into plantations, however, it is crucial for restoration efforts to understand to what extent tigers use modified habitats. We investigated tiger-habitat relationships at 2 spatial scales: occupancy across the landscape and habitat use within the home range. Across major landcover types in central Sumatra, we conducted systematic detection, non-detection sign surveys in 47, 17×17 km grid cells. Within each cell, we surveyed 40, 1-km transects and recorded tiger detections and habitat variables in 100 m segments totaling 1,857 km surveyed. We found that tigers strongly preferred forest and used plantations of acacia and oilpalm, far less than their availability. Tiger probability of occupancy covaried positively and strongly with altitude, positively with forest area, and negatively with distance-to-forest centroids. At the fine scale, probability of habitat use by tigers across landcover types covaried positively and strongly with understory cover and altitude, and negatively and strongly with human settlement. Within forest areas, tigers strongly preferred sites that are farther from water bodies, higher in altitude, farther from edge, and closer to centroid of large forest block; and strongly preferred sites with thicker understory cover, lower level of disturbance, higher altitude, and steeper slope. These results indicate that to thrive, tigers depend on the existence of large contiguous forest blocks, and that with adjustments in plantation management, tigers could use mosaics of plantations (as additional roaming zones), riparian forests (as corridors) and smaller forest patches (as stepping stones), potentially maintaining a metapopulation structure in fragmented landscapes. This study highlights the importance of a multi-spatial scale analysis and provides crucial information relevant to restoring tigers and other wildlife in forest and plantation landscapes through improvement in habitat extent, quality, and connectivity.     

Production,characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.     

The Submerged Dyslexia Iceberg: How Many School Children Are Not Diagnosed? Results from an Italian Study

Background

Although dyslexia is one of the most common neurobehavioral disorders affecting children, prevalence is uncertain and available data are scanty and dated. The objective of this study is to evaluate the prevalence of dyslexia in an unselected school population using clearly defined and rigorous diagnostic criteria and methods.

Methods

Cross sectional study. We selected a random cluster sample of 94 fourth grade elementary school classes of Friuli Venezia Giulia, a Region of North Eastern Italy. We carried out three consecutive levels of screening: the first two at school and the last at the Neuropsychiatry Unit of a third level Mother and Child Hospital. The main outcome measure was the prevalence of dyslexia, defined as the number of children positive to the third level of screening divided by the total number of children enrolled.

Results

We recruited 1774 children aged 8–10 years, of which 1528 received parents’ consent to participate. After applying exclusion criteria, 1357 pupils constituted the final working sample. The prevalence of dyslexia in the enrolled population ranged from 3.1% (95% CI 2.2–4.1%) to 3.2% (95% CI 2.4–4.3%) depending on different criteria adopted. In two out of three children with dyslexia the disorder had not been previously diagnosed.

Conclusions

This study shows that dyslexia is largely underestimated in Italy and underlines the need for reliable information on prevalence, in order to better allocate resources both to Health Services and Schools.     

The CDK Subunit CKS2 Counteracts CKS1 to Control Cyclin A/CDK2 Activity in Maintaining Replicative Fidelity and Neurodevelopment

CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCF(SKP2) to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2(-/-) knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2(-/-) cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In?vivo, Cks2(-/-) cortical progenitor cells are limited in their capacity to differentiate into mature neurons,?a phenotype akin to animals lacking p27. We propose that?the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation.     

Synthesis and biological evaluation of 2-substituted-4-(3′,4′,5′-trimethoxyphenyl)-5-aryl thiazoles as anticancer agents

Antitumor agents that bind to tubulin and disrupt microtubule dynamics have attracted considerable attention in the last few years. To extend our knowledge of the thiazole ring as a suitable mimic for the cis-olefin present in combretastatin A-4, we fixed the 3,4,5-trimethoxyphenyl at the C4-position of the thiazole core. We found that the substituents at the C2- and C5-positions had a profound effect on antiproliferative activity. Comparing compounds with the same substituents at the C5-position of the thiazole ring, the moiety at the C2-position influenced antiproliferative activities, with the order of potency being NHCH₃ > Me ? N(CH₃)₂. The N-methylamino substituent significantly improved antiproliferative activity on MCF-7 cells with respect to C2-amino counterparts. Increasing steric bulk at the C2-position from N-methylamino to N,N-dimethylamino caused a 1–2 log decrease in activity. The 2-N-methylamino thiazole derivatives 3b, 3d and 3e were the most active compounds as antiproliferative agents, with IC₅₀ values from low micromolar to single digit nanomolar, and, in addition, they are also active on multidrug-resistant cell lines over-expressing P-glycoprotein. Antiproliferative activity was probably caused by the compounds binding to the colchicines site of tubulin polymerization and disrupting microtubule dynamics. Moreover, the most active compound 3e induced apoptosis through the activation of caspase-2, -3 and -8, but 3e did not cause mitochondrial depolarization.     

Signal-to-noise ratio of diffusion weighted magnetic resonance imaging: Estimation methods and in vivo application to spinal cord

Diffusion tensor imaging (DTI) and tractographic reconstruction may be applied for in vivo clinical spinal cord studies. However, this structure represents a challenge to current acquisition and reconstruction strategies, due to its small size, motion artifacts, partial volume effects and low signal-to-noise-ratio (SNR). Aims of this work were to select the best approach for the estimate of SNR and to use it for spinal cord diffusion weighted (DW) sequence optimization.Seven methods for the estimate of SNR were compared on uniform phantom DW images, and the best performing approach (single ROI for signal and noise, difference of images—SNR_diff) was applied for the following in vivo sequence evaluations.Fifteen sequences with different parameters (voxel size, repetition (TR) and echo (TE) times) were compared according to SNR, resolution, fractional anisotropy (FA) and tractography performances on three healthy volunteers. In vivo optimization of DW sequences resulted in: axial sequence, with voxel size = 1.5 mm × 1.5 mm × 3.5 mm, TR = 3200 ms and TE = 89 ms, sagittal sequence with voxel size = 2.2 mm × 2.2 mm × 2 mm, TR = 3000 ms and TE = 84 ms.An objective method tested on phantom and a practical index for in vivo spinal cord DTI SNR estimation allowed to obtain axial and sagittal optimized sequences, providing excellent tractographic results, with acceptable acquisition times for in vivo clinical applications.     

N,N-dimethyl-thioamphetamine and methyl-thioamphetamine, two non-neurotoxic substrates of 5-HT transporters, have scant in vitro efficacy for the induction of transporter-mediated 5-HT release and currents

We studied two non-neurotoxic amphetamine derivatives (methyl-thioamphetamine, MTA and N,N- dimethylMTA, DMMTA) interacting with serotonin (5-HT) transporters (SERTs) with affinities comparable to that of p- Cl-amphetamine (pCA). The rank order for their maximal effects in inducing both [³H]5-HT release from rat brain synaptosomes or hSERT-expressing HEK-293 cells, and currents in hSERT-expressing oocytes, was pCA >> MTA ≥ DMMTA. A correlation between drug-induced release and currents is also strengthened by the similar bell shape of the dose–response curves. Release experiments indicated that MTA and DMMTA are SERT substrates although MTA is taken up by HEK-293 cells with a V _max 40% lower than pCA. The weak effects of MTA and DMMTA in vitro might therefore be due to their properties as 'partial substrates' on the mechanisms, other than translocation, responsible for currents and/or release. After either local or systemic in vivo administration, MTA and DMMTA release 5-HT in a manner comparable to pCA. These findings confirm that the neurotoxic properties of some amphetamine derivatives are independent of their 5-HT-releasing activity in vivo . It is worth noting that only those amphetamine derivatives with high efficiency in inducing 5-HT release and currents in vitro have neurotoxic properties.     

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.