Overexpression of torsinA in PC12 cells protects against toxicity

Childhood-onset dystonia is an autosomal dominant movement disorder associated with a three base pair (GAG) deletion mutation in the DYT1 gene. This gene encodes a novel ATP-binding protein called torsinA, which in the central nervous system is expressed exclusively in neurons. Neither the function of torsinA nor its role in the pathophysiology of DYT1 dystonia is known. In order to better understand the cellular functions of torsinA, we established PC12 cell lines overexpressing wild-type or mutant torsinA and subjected them to various conditions deleterious to cell survival. Treatment of control PC12 cells with an inhibitor of proteasomal activity, an oxidizing agent, or trophic withdrawal, resulted in cell death, whereas PC12 cells that overexpressed torsinA were significantly protected against each of these treatments. Overexpression of mutant torsinA failed to protect cells against trophic withdrawal. These results suggest that torsinA may play a protective role in neurons against a variety of cellular insults.     

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Here we show that many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. We show that following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a "PGC niche".     

Finger Millet processing in East Africa

Although archaeobotanical sampling and recovery programmes are a relatively recent implementation in East African archaeology, results from sites where they have been carried out follow a similar trend. This is one of abundant recovery of wood charcoal, but very little in the way of other macroscopic plant remains. Restricted archaeological evidence and ethnographic interviews show the importance of grains, in particular finger millet (Eleusine coracana), for the Bunyoro people of Uganda in pre-colonial times. It has been suggested that one of the possible reasons why finger millet is not being recovered in quantity from archaeological contexts is because the processing of this crop does not involve heating and hence there is not the chance of being deposited in charred form in the archaeological record. Recent ethnographic work on finger millet processing in Uganda shows that it is exposed to heat and potential charring during cleaning and preparation of the grain for either storage or cooking, and this regime is discussed in terms of its activities and products. These findings reinforce the need for archaeobotanists and archaeologists working in this region to look for other possible causes of the scarcity of macroscopic plant remains, and also the importance of considering integrated evidence for agricultural activity on prehistoric sites.     

Hierarchical Phosphorylation of Recombinant Tau by the Paired-Helical Filament-Associated Protein Kinase Is Dependent on Cyclic AMP-Dependent Protein Kinase

Abstract : Immunoaffinity-purified paired helical filaments (PHFs) from Alzheimer's disease (AD) brain homogenates contain an associated protein kinase activity that is able to induce the phosphorylation of PHF proteins on addition of exogenous MgCl₂ and ATP. PHF kinase activity is shown to be present in immunoaffinity-purified PHFs from both sporadic and familial AD, Down's syndrome, and Pick's disease but not from normal brain homogenates. Although initial studies failed to show that the kinase was able to induce the phosphorylation of tau, additional studies presented in this article show that only cyclic AMP-dependent protein kinase-pretreated recombinant tau is a substrate for the PHF kinase activity. Deletional mutagenesis, phosphopeptide mapping, and site-directed mutagenesis have identified the PHF kinase phosphorylation sites as amino acids Thr³⁶¹ and Ser⁴¹² in htau40. In addition, the cyclic AMP-dependent protein kinase phosphorylation sites that direct the PHF kinase have been mapped to amino acids Ser³⁵⁶ and Ser⁴⁰⁹ in htau40. Additional data demonstrate that these hierarchical phosphorylations in the extreme C terminus of tau allow for the incorporation of recombinant tau into exogenously added AD-derived PHFs, providing evidence that certain unique phosphorylations of tau may play a role in the pathogenesis of neurofibrillary pathology in AD.     

DNA damage, p14ARF, Nucleophosmin (NPM/B23), and cancer

The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF’s nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.     

Elucidating Emergence and Transmission of Multidrug-Resistant Tuberculosis in Treatment Experienced Patients by Whole Genome Sequencing

Background

Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.

Methods and Findings

We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.

Conclusions

Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.     

Amplified Fragment Length Polymorphism Fingerprinting of 16 Banana Cultivars (Musa cvs.)

Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits.     

How do we overcome abrupt degradation of marine ecosystems and meet the challenge of heat waves and climate extremes?

Extreme heat wave events are now causing ecosystem degradation across marine ecosystems. The consequences of this heat‐induced damage range from the rapid loss of habitat‐forming organisms, through to a reduction in the services that ecosystems support, and ultimately to impacts on human health and society. How we tackle the sudden emergence of ecosystem‐wide degradation has not yet been addressed in the context of marine heat waves. An examination of recent marine heat waves from around Australia points to the potential important role that respite or refuge from environmental extremes can play in enabling organismal survival. However, most ecological interventions are being devised with a target of mid to late‐century implementation, at which time many of the ecosystems, that the interventions are targeted towards, will have already undergone repeated and widespread heat wave induced degradation. Here, our assessment of the merits of proposed ecological interventions, across a spectrum of approaches, to counter marine environmental extremes, reveals a lack preparedness to counter the effects of extreme conditions on marine ecosystems. The ecological influence of these extremes are projected to continue to impact marine ecosystems in the coming years, long before these interventions can be developed. Our assessment reveals that approaches which are technologically ready and likely to be socially acceptable are locally deployable only, whereas those which are scalable—for example to features as large as major reef systems—are not close to being testable, and are unlikely to obtain social licence for deployment. Knowledge of the environmental timescales for survival of extremes, via respite or refuge, inferred from field observations will help test such intervention tools. The growing frequency of extreme events such as marine heat waves increases the urgency to consider mitigation and intervention tools that support organismal and ecosystem survival in the immediate future, while global climate mitigation and/or intervention are formulated.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.