Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide,a Histone Deacetylase Inhibitor

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming.     

Habitat structure and juvenile fish ontogeny shape zooplankton spring dynamics

Macrophytes in shallow lakes have the potential to alter fish–zooplankton interactions considerably. How far predation effects by newly hatched fish (0+ fish) on zooplankton are influenced by different types of aquatic vegetation, and how effects change during the first weeks of fish ontogeny remains, however, less clear. In order to address these issues, we examined the predation effects of 0+ fish on zooplankton in three different habitats during spring and summer in a shallow, eutrophic lake in Sweden. Zooplankton and fish samples were taken along the reed vegetation, in a shallow, unvegetated part of the lake and above dense, submersed vegetation to relate 0+ fish predation effects to vegetation complexity. All the size classes of zooplankton decreased when 0+ fish started to feed on them in all the different habitats. The magnitude of predation effects depended, however, on both the size of zooplankton and the complexity of the vegetation. While small cladocerans could maintain stable populations in the dense Chara vegetation after 0+ fish had started to feed on them, medium and large-sized zooplankton disappeared from all the habitats. Our results suggest that only small cladocerans can use dense vegetation as a refuge against 0+ fish predation, while medium and large zooplankton are not safe from 0+ fish predation in any habitat.     

A study of the distribution of aluminium in human placental tissues based on alkaline solubilization with determination by electrothermal atomic absorption spectrometry

Aluminium (Al) is a nonessential element known to induce neurotoxic effects, such as dialysis dementia, in patients on hemodialysis, with compromised kidney function. The role of Al in the progression of some neurodegenerative diseases, such as Alzheimer's disease (AD), is controversial, and remains unclear. The effects of Al on other vulnerable populations, such as fetuses and infants, have been infrequently studied. In the present study, Al has been measured in human placenta samples, comprising ～160 each of placenta bodies, placenta membranes, and umbilical cords, using electrothermal atomic absorption spectrometry (ETAAS) after atmospheric pressure digestion with tetramethylammonium hydroxide (TMAH) and ethylenediaminetetraacidic acid (EDTA). The sensitivity, or characteristic mass (m(0)), for Al at the 309.3-nm line was found to be 30 ± 4 pg. The instrumental detection limit (IDL) (3s) for Al in solution was calculated as 0.72 μg L(-1) while the method detection limit (MDL) (3s) was 0.25 μg g(-1). Accuracy was assessed through analysis of quality control (QC) materials, including certified reference materials (CRMs), in-house reference materials (RMs), and spike recovery experiments, of varying matrices. Placental tissue analyses revealed geometric mean concentrations of approximately 0.5 μg g(-1) Al in placenta bodies (n = 165) and membranes (n = 155), while Al concentrations in umbilical cords (n = 154) were about 0.3 μg g(-1). Al was detected in 95% of placenta bodies, and 81% of placenta membranes, but only in 46% of umbilical cords.     

Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS)

Background

Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3–10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis.

Methodology/Principal Findings

Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B.

Conclusions/Significance

We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS.We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype.     

Lazarus1, a DUF300 Protein,Contributes to Programmed Cell Death Associated with Arabidopsis acd11 and the Hypersensitive Response

Background

Programmed cell death (PCD) is a necessary part of the life of multi-cellular organisms. A type of plant PCD is the defensive hypersensitive response (HR) elicited via recognition of a pathogen by host resistance (R) proteins. The lethal, recessive accelerated cell death 11 (acd11) mutant exhibits HR-like accelerated cell death, and cell death execution in acd11 shares genetic requirements for HR execution triggered by one subclass of R proteins.

Methodology/Principal Findings

To identify genes required for this PCD pathway, we conducted a genetic screen for suppressors of acd11, here called lazarus (laz) mutants. In addition to known suppressors of R protein-mediated HR, we isolated 13 novel complementation groups of dominant and recessive laz mutants. Here we describe laz1, which encodes a protein with a domain of unknown function (DUF300), and demonstrate that LAZ1 contributes to HR PCD conditioned by the Toll/interleukin-1 (TIR)-type R protein RPS4 and by the coiled-coil (CC)-type R protein RPM1. Using a yeast-based topology assay, we also provide evidence that LAZ1 is a six transmembrane protein with structural similarities to the human tumor suppressor TMEM34. Finally, we demonstrate by transient expression of reporter fusions in protoplasts that localization of LAZ1 is distributed between the cytosol, the plasma membrane and FM4–64 stained vesicles.

Conclusions/Significance

Our findings indicate that LAZ1 functions as a regulator or effector of plant PCD associated with the HR, in addition to its role in acd11-related death. Furthermore, the similar topology of a plant and human DUF300 proteins suggests similar functions in PCD across the eukaryotic kingdoms, although a direct role for TMEM34 in cell death control remains to be established. Finally, the subcellular localization pattern of LAZ1 suggests that it may have transport functions for yet unknown, death-related signaling molecules at the plasma membrane and/or endosomal compartments. In summary, our results validate the utility of the large-scale suppressor screen to identify novel components with functions in plant PCD, which may also have implications for deciphering cell death mechanisms in other organisms.     

The Octarepeat Region of the Prion Protein Is Conformationally Altered in PrPSc

Background

Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP^C. Little is known about the details of the structural rearrangement of physiological PrP^C into a still-elusive disease-associated conformation termed PrP^Sc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.

Methodology/Principal Findings

Here, we report that trypsin digestion of PrP^Sc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP^Sc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP^Sc are exposed in PrP^C.

Conclusions/Significance

We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP^Sc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrP^C participates in PrP^Sc conversion, and may also be exploited for diagnostic purposes.     

Geographical variation in the response of visceral leishmaniasis to paromomycin in East Africa: a multicentre, open-label, randomized trial

Background

Visceral leishmaniasis (VL) is a major health problem in developing countries. The untreated disease is fatal, available treatment is expensive and often toxic, and drug resistance is increasing. Improved treatment options are needed. Paromomycin was shown to be an efficacious first-line treatment with low toxicity in India.

Methods

This was a 3-arm multicentre, open-label, randomized, controlled clinical trial to compare three treatment regimens for VL in East Africa: paromomycin sulphate (PM) at 15 mg/kg/day for 21 days versus sodium stibogluconate (SSG) at 20 mg/kg/day for 30 days; and the combination of both dose regimens for 17 days. The primary efficacy endpoint was cure based on parasite-free tissue aspirates taken 6 months after treatment.

Findings

Overall, 135 patients per arm were enrolled at five centres in Sudan (2 sites), Kenya (1) and Ethiopia (2), when the PM arm had to be discontinued due to poor efficacy. The trial has continued with the higher dose of PM as well as the combination of PM and SSG arms. These results will be reported later. Baseline patient characteristics were similar among treatment arms. The overall cure with PM was significantly inferior to that with SSG (63.8% versus 92.2%; difference 28.5%, 95%CI 18.8% to 38.8%, p<0.001). The efficacy of PM varied among centres and was significantly lower in Sudan (14.3% and 46.7%) than in Kenya (80.0%) and Ethiopia (75.0% and 96.6%). No major safety issues with PM were identified.

Conclusion

The efficacy of PM at 15 mg/kg/day for 21 days was inadequate, particularly in Sudan. The efficacy of higher doses and the combination treatment warrant further studies.     

Meeting report: the terabase metagenomics workshop and the vision of an Earth microbiome project

Between July 18(th) and 24(th) 2010, 26 leading microbial ecology, computation, bioinformatics and statistics researchers came together in Snowbird, Utah (USA) to discuss the challenge of how to best characterize the microbial world using next-generation sequencing technologies. The meeting was entitled "Terabase Metagenomics" and was sponsored by the Institute for Computing in Science (ICiS) summer 2010 workshop program. The aim of the workshop was to explore the fundamental questions relating to microbial ecology that could be addressed using advances in sequencing potential. Technological advances in next-generation sequencing platforms such as the Illumina HiSeq 2000 can generate in excess of 250 billion base pairs of genetic information in 8 days. Thus, the generation of a trillion base pairs of genetic information is becoming a routine matter. The main outcome from this meeting was the birth of a concept and practical approach to exploring microbial life on earth, the Earth Microbiome Project (EMP). Here we briefly describe the highlights of this meeting and provide an overview of the EMP concept and how it can be applied to exploration of the microbiome of each ecosystem on this planet.     

The Parkinson's Disease Associated LRRK2 Exhibits Weaker In Vitro Phosphorylation of 4E-BP Compared to Autophosphorylation

Mutations in the gene encoding Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson''s disease (PD). LRRK2 is a multi-domain protein kinase containing a central catalytic core and a number of protein-protein interaction domains. An important step forward in the understanding of both the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological substrates. In the present study we examined phosphorylation of 4E-BP (eukaryotic initiation factor 4E (eIF4E)-binding protein), a recently proposed substrate for LRRKs. We found that LRRK2 is capable of phosphorylating 4E-BP in vitro. The PD related LRRK2-G2019S mutant was ∼2 fold more active than wild type protein. However, LRRK2 autophosphorylation was stronger than 4E-BP phosphorylation under conditions of molar excess of 4E-BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38α and DAPK2) and found that MAPK14/p38α could efficiently phosphorylate 4E-BP at the same site as LRRK2 in vitro. Finally, we did not see changes in 4E-BP phosphorylation levels using inducible expression of LRRK2 in HEK cell lines. We also found that MAPK14/p38α phosphorylates 4E-BP in transient overexpression experiments whereas LRRK2 did not. We suggest that increased 4E-BP phosphorylation reported in some systems may be related to p38-mediated cell stress rather than direct LRRK2 activity. Overall, our results suggest that 4E-BP is a relatively poor direct substrate for LRRK2.