Molecular approach to the nucleo-melanosomal interaction in human melanoma cells

This paper presents evidence that L-tyrosine oxidation products and 5,6-dihydroxyindole, an intermediate of melanin synthesis bind to and modify DNA structure, as tested by extracting cell DNA, using topoisomerase I and denaturation assays. When supercoiled plasmid pCU18 or pBR322 DNAs are treated with 5,6-dihydroxyindole the supercoiled species disappear and are converted to species less mobile in a gel retardation test with respect to relaxed DNA. 5,6-Dihydroxyindole causes an easier acid denaturation of the double helix. The results, that are dose dependent,would point to both intercalation and cross-linking of DNA by 5,6-dihydroxyindole and its oxidation product(s). ³H-L-tyrosine deriving radioactivity, bound to nuclear DNA, is higher at low pH, (5.6) if compared to pH 6.8. The highest radioactivity bound to cell DNA is found during the transition from the amelanotic to the melanotic phenotype in human melanoma cell lines. As a control, the binding of ³H-L-tyrosine radioactivity to human prostate fibroblast DNA was investigated.     

Adaptive response of human melanoma cells to methylglyoxal injury

The effects of methylglyoxal on the growth of a line of human melanoma cells are investigated. Methylglyoxal inhibits cell growth in a dose- dependent manner and causes an increase in glyceraldehyde 3-phosphate dehydrogenase, and glyoxalase 1 and glyoxalase 2 specific activities. The cellular response to increasing concentrations of methylglyoxal in the culture medium is also studied by measuring L-lactate production, reduced-oxidized glutathione levels and apoptotic cell death. Methylglyoxal seems to promote a change of cell population phenotypic repertoire toward a more monomorphic phenotype. In conclusion, methylglyoxal seems to induce an enzymatic cellular response that lowers methylglyoxal levels and selects the most resistant cells.      

Exploiting test structure: case series, case-control comparison, and dissociation

Most neuropsychological tests consist of multiple items, and a subject's test score is the sum of the item scores. The test results for each subject thus comprise multiple data-points, and any data-set with test results from more than one subject has at least a two-level structure, with the test item as the first level and the subject as the second level. This structure may be exploited to yield more nuanced statistical analyses than those that treat each subject's test score as a single data-point. Exploiting this structure allows us to take into account the effect of test length and dispersion on score variance and may enhance statistical power. Focusing on tests for which the score can be regarded as a binomial random variable, and using the binomial general linear model, we describe appropriate statistical methods for exploiting test structure in analysing a case series, comparing a case with a control sample, and testing for dissociation. These methods also allow multiple predictors, both categorical and continuous, to be taken into account, thereby enhancing the capacity of researchers to test hypotheses in a case series and to investigate other explanatory factors, in addition to case-control status.     

Unbiased optical mapping of telomere-integrated endogenous human herpesvirus 6

Next-generation sequencing technologies allowed sequencing of thousands of genomes. However, there are genomic regions that remain difficult to characterize, including telomeres, centromeres, and other low-complexity regions, as well as transposable elements and endogenous viruses. Human herpesvirus 6A and 6B (HHV-6A and HHV-6B) are closely related viruses that infect most humans and can integrate their genomes into the telomeres of infected cells. Integration also occurs in germ cells, meaning that the virus can be inherited and result in individuals harboring the virus in every cell of their body. The integrated virus can reactivate and cause disease in humans. While it is well established that the virus resides in the telomere region, the integration locus is poorly defined due to the low sequence complexity (TTAGGG)n of telomeres that cannot be easily resolved through sequencing. We therefore employed genome imaging of the integrated HHV-6A and HHV-6B genomes using whole-genome optical site mapping technology. Using this technology, we identified which chromosome arm harbors the virus genome and obtained a high-resolution map of the integration loci of multiple patients. Surprisingly, this revealed long telomere sequences at the virus−subtelomere junction that were previously missed using PCR-based approaches. Contrary to what was previously thought, our technique revealed that the telomere lengths of chromosomes harboring the integrated virus genome were comparable to the other chromosomes. Taken together, our data shed light on the genetic structure of the HHV-6A and HHV-6B integration locus, demonstrating the utility of optical mapping for the analysis of genomic regions that are difficult to sequence.

Human herpesvirus 6A and 6B (HHV-6A and HHV-6B) are closely related virus species that infect humans (1). HHV-6B infects almost all humans within the first years of life and causes the febrile illness exanthema subitum (2). Infection with HHV-6A is thought to occur later in life, but the epidemiology of the virus is poorly characterized. Like all herpesviruses, HHV-6A and HHV-6B establish latency upon primary infection, allowing the virus to persist in the host for life. In contrast to other human herpesviruses, HHV-6A and HHV-6B integrate their genomes into the telomere region of host chromosomes of latently infected cells (3–6). The viruses can also heritably integrate into germ cells, resulting in individuals and their offspring that harbor the virus in every nucleated cell (3, 7–11). This phenomenon is referred to as inherited chromosomally integrated HHV-6 (iciHHV-6). Importantly, HHV-6 reactivation from the integrated state is associated with various diseases, including graft-versus-host disease, encephalitis, and heart disease (12–18). More recently, a study indicated that telomeres carrying integrated HHV-6 are shorter and more unstable (19), which, in turn, could influence aging and/or diseases of individuals with iciHHV-6.It remains unknown whether certain integration events or iciHHV-6 genomes at specific loci are responsible for, or contribute directly to, disease in humans. Although we can identify individuals that harbor iciHHV-6A and iciHHV-6B through qPCR (20), a major challenge in studying iciHHV-6 is that we cannot readily examine the integration site with high resolution. Integration loci of HHV-6A and HHV-6B were first detected by fluorescent in situ hybridization (FISH) (3), and the junction has since been sequenced using a PCR-based approach for three different integrations. Sanger sequencing of these PCR fragments indicated that the right direct repeat (DR-R) is fused to the subtelomeres with only a very short stretch of telomere sequences between the virus and host chromosome (4, 5, 19, 21).However, FISH is a challenging and laborious technique, offering very low spatial resolution that does not allow us to directly observe the junction. Although PCR amplification and Sanger sequencing does offer sequence information, this approach requires previous knowledge of the chromosomal location of the virus genome (19, 22), is prone to amplification errors, and has not succeeded in determining structures of junctions. Next-generation sequencing (NGS) using various platforms has not succeeded in sequencing the junction, as either the coverage or the read length was not sufficient to resolve the complex and repetitive nature of the telomere region harboring the virus genome.We therefore set out to develop an approach to study the virus−host junction because understanding its composition is key to understanding the effect of iciHHV-6 on human biology. Using a whole-genome optical mapping technology (23, 24), we were able to generate unbiased, high-resolution maps of HHV-6A and HHV-6B integration sites, which allowed the identification of the chromosomes harboring the integrated HHV-6A and HHV-6B genome in four different iciHHV-6 patient-derived cell lines. We also know, from our recent work on the evolutionary history of the virus, that most known integrations are derived from very few ancestral genome invasion events (25). This means that, for every sample we determine the chromosomal location for, this can be extrapolated to many orthologs without the expense and effort of further experimental work. Our results conclusively determine the orientation of the virus genome, as well as the length of the telomeric virus−chromosome junction and the distal telomeres on the end of the virus genome. Furthermore, the ability to determine the chromosomal location of the virus will be crucial to decipher the role of the integrated virus genome in human diseases.     

Pathways involved in human conscious vision contribute to obstacle-avoidance behaviour

Human patients with visual field defects following damage to their primary visual cortex (V1) will often misperceive the midpoint of a horizontal line. They tend to shift the midpoint away from the real position towards their blind field. In patients with unilateral neglect, where midpoint shifts can also be observed, these perceptual errors do not lead to errors in an obstacle-avoidance task, which also requires the ability to find the midpoint between two obstacles. This dissociation in neglect patients was taken as evidence that obstacle-avoidance performance is guided by visual information from the dorsal visual stream. Recently it was shown that a patient with hemianopia could avoid an obstacle presented in his blind field. This suggests that obstacle-avoidance behaviour can be guided by subconscious vision alone involving a direct route from extrageniculate structures in the brain to dorsal stream areas. To investigate whether obstacle avoidance relies only on this subconscious route or also uses information from pathways involved in conscious vision, we examined the effect of the hemianopic shift on obstacle-avoidance behaviour. This shift is found in tasks where a conscious visual judgement is required and presumably arises in pathways underlying conscious vision (V1 and ventral stream areas). We compared the performance of six patients with left hemianopia with the performance of six patients with right hemianopia. We found a clear bias in both groups, which also affected obstacle-avoidance performance. It is thus concluded that obstacle avoidance does not bypass the system for conscious vision completely.     

Correlation of acetylcholinesterase activity in the brain and blood of wistar rats acutely infected with Trypanosoma congolense

ObjectiveTo investigate the neurotransmitter enzyme Acetylcholinesterase (AChE) activity in the brain and blood of rats infected with Trypanosoma congolense (T. congo).MethodsPresence and degree of parasitemia was determined daily for each rat by the rapid matching method. AChE activity was determined by preparing a reaction mixture of brain homogenate and whole blood with 5, 5-dithiobisnitrobenzioc acid (DTNB or Ellman's reagent) and Acetylthiocholine (ATC). The increase in absorbance was recorded at 436 nm over 10 min at 2 min intervals. Trypanosome species identification (before inoculation and on day 10 post infection) was done by Polymerase chain reaction using specific primers.ResultsThe AChE activity in the brain and blood decreased significantly as compared with the uninfected control. The AChE activity dropped to 0.32 from 2.20 μmol ACTC min^?1mg protein^?1 in the brain and 4.57 to 0.76 μmol ACTC min-1mg protein^?1 in the blood. The animals treated with Diminaveto at 3.5 mg/kg/d were observed to have recovered significantly from parasitemia and were able to regain AChE activity in the blood but not in the brain as compared to the control groups. We also observed, that progressive parasitemia resulted to alterations in PCV, Hb, RBC, WBC, neurophils, total protein, lymphocytes, monocytes and eosinophil in acute infections of T. congo. Polymerase chain reaction (PCR) of infected blood before inoculation and on day 10 post infection revealed 600 bp on agarose gel electrophoresis.ConclusionsThis finding suggest that decrease in AChE activity increases acetylcholine concentration in the synaptic cleft resulting to neurological failures in impulse transfer in T. congo infection rats.     

Attentional blink deficits observed in dyslexia depend on task demands

The attentional blink (AB) refers to a deficit in the ability to identify a second target following a first target when both appear randomly within a rapid sequence of distractor items. The AB of five adults with dyslexia (ADys) was compared with that of a group of normal adult readers. Two tasks were completed which differed in the conceptual category of the target items (a red digit or letter) relative to the distractor items (all black digits). In the digit condition, all ADys cases showed a longer AB compared to the control group. In the letter condition, all participants showed improvement in accuracy compared to the digit condition, but three ADys cases continued to have a longer AB. The results suggest that (a) AB performance depends on task requirements, and (b) the attentional system is compromised in dyslexia. However, examination of individual case performance suggests that prolonged attentional dwell time is not a core deficit in dyslexia. The results also illustrate the limitations of group comparisons in small sample studies.     

Epigenome-derived drugs: recent advances and future perspectives

This review highlights the current knowledge of epigenetic targets of anticancer therapy and outlines the current limitations of epigenetic approaches and the difficulties in defining preventive tools and strategies. Promising strategies towards achieving the goal of developing effective epigenetic treatments are discussed, including restoration or enhancement of sensitivity to other treatment modalities, and combinations with other agents and new therapeutic areas.     

Touch and feel? Using the rubber hand paradigm to investigate self-touch enhancement in right-hemisphere stroke patients

Following stroke, a patient may fail to report touch administered by another person but claim that she feels touch when it is self-administered. We investigated three explanations for self-touch enhancement: (1) proprioceptive information from the administering hand, (2) attentional modulation, and (3) temporal expectation. Tactile sensation was assessed with vision precluded, and with the affected hand positioned in the left and right hemispace. In four of six experiments, the somatic rubber hand paradigm was used: the Examiner administered stimulation to the patient's affected left hand while guiding the patient's right hand to administer synchronous stimulation to a prosthetic hand. Even though the patient's two hands were not in contact, patients detected the same number of stimulations as when they touched their own hand directly (self-administered condition). Moreover, there was no decline in rates of detection when potentially informative movements of the administering hand were restricted. This demonstrates that patients feel rather than infer stimulation under conditions of self-touch. When patients received stimulation to the affected hand in the opposite hemispace to the hand administering touch to the prosthetic hand, all but one showed self-touch enhancement. Thus, neither proprioceptive information nor attentional modulation at the spatial region of the administering hand provided a sufficient explanation for self-touch enhancement. A follow-up experiment indicated an important role for temporal expectation: a delay, between the patient's stimulation of the prosthetic hand and the Examiner's stimulation of the patient's affected hand, eliminated the self-touch enhancement effect.