Retinal ganglion cell degeneration correlates with hippocampal spine loss in experimental Alzheimer's disease.

Neuronal dendritic and synaptic pruning are early features of neurodegenerative diseases, including Alzheimer's disease. In addition to brain pathology, amyloid plaque deposition, microglial activation, and cell loss occur in the retinas of human patients and animal models of Alzheimer's disease. Retinal ganglion cells, the output neurons of the retina, are vulnerable to damage in neurodegenerative diseases and are a potential opportunity for non-invasive clinical diagnosis and monitoring of Alzheimer's progression. However, the extent of retinal involvement in Alzheimer's models and how well this reflects brain pathology is unclear. Here we have quantified changes in retinal ganglion cells dendritic structure and hippocampal dendritic spines in three well-studied Alzheimer's mouse models, Tg2576, 3xTg-AD and APPNL-G-F. Dendritic complexity of DiOlistically labelled retinal ganglion cells from retinal explants was reduced in all three models in an age-, gender-, and receptive field-dependent manner. DiOlistically labelled hippocampal slices showed spine loss in CA1 apical dendrites in all three Alzheimer's models, mirroring the early stages of neurodegeneration as seen in the retina. Morphological classification showed that loss of thin spines predominated in all. The demonstration that retinal ganglion cells dendritic field reduction occurs in parallel with hippocampal dendritic spine loss in all three Alzheimer's models provide compelling support for the use of retinal neurodegeneration. As retinal dendritic changes are within the optical range of current clinical imaging systems (for example optical coherence tomography), our study makes a case for imaging the retina as a non-invasive way to diagnose disease and monitor progression in Alzheimer's disease.

The Feasibility of Electrochemical Ammonia Synthesis in Molten LiCl-KCl Eutectics.

Molten LiCl and related eutectic electrolytes are known to permit direct electrochemical reduction of N2 to N3- with high efficiency. It had been proposed that this could be coupled with H2 oxidation in an electrolytic cell to produce NH3 at ambient pressure. Here, this proposal is tested in a LiCl-KCl-Li3 N cell and is found not to be the case, as the previous assumption of the direct electrochemical oxidation of N3- to NH3 is grossly over-simplified. We find that Li3 N added to the molten electrolyte promotes the spontaneous and simultaneous chemical disproportionation of H2 (H oxidation state 0) into H- (H oxidation state -1) and H+ in the form of NH2- /NH2 - /NH3 (H oxidation state +1) in the absence of applied current, resulting in non-Faradaic release of NH3 . It is further observed that NH2- and NH2 - possess their own redox chemistry. However, these spontaneous reactions allow us to propose an alternative, truly catalytic cycle. By adding LiH, rather than Li3 N, N2 can be reduced to N3- while stoichiometric amounts of H- are oxidised to H2 . The H2 can then react spontaneously with N3- to form NH3 , regenerating H- and closing the catalytic cycle. Initial tests show a peak NH3 synthesis rate of 2.4×10-8  mol cm-2 s-1 at a maximum current efficiency of 4.2 %. Isotopic labelling with 15 N2 confirms the resulting NH3 is from catalytic N2 reduction.

An incoherent feedforward loop facilitates adaptive tuning of gene expression.

We studied adaptive evolution of gene expression using long-term experimental evolution of Saccharomyces cerevisiae in ammonium-limited chemostats. We found repeated selection for non-synonymous variation in the DNA binding domain of the transcriptional activator, GAT1, which functions with the repressor, DAL80 in an incoherent type-1 feedforward loop (I1-FFL) to control expression of the high affinity ammonium transporter gene, MEP2. Missense mutations in the DNA binding domain of GAT1 reduce its binding to the GATAA consensus sequence. However, we show experimentally, and using mathematical modeling, that decreases in GAT1 binding result in increased expression of MEP2 as a consequence of properties of I1-FFLs. Our results show that I1-FFLs, one of the most commonly occurring network motifs in transcriptional networks, can facilitate adaptive tuning of gene expression through modulation of transcription factor binding affinities. Our findings highlight the importance of gene regulatory architectures in the evolution of gene expression.

Probing atomic positions of adsorbed ammonia molecules in zeolite.

Atomic positions and interactions between adsorbed guest molecules, such as ammonia in H-ZSM-5 microporous solids, are for the first time revealed by making use of the change in the periodical scattering parameter using in situ synchrotron powder X-ray diffraction combined with refinement within experimental errors.

Individualization of treatments with drugs metabolized by CES1: combining genetics and metabolomics.

CES1 is involved in the hydrolysis of ester group-containing xenobiotic and endobiotic compounds including several essential and commonly used drugs. The individual variation in the efficacy and tolerability of many drugs metabolized by CES1 is considerable. Hence, there is a large interest in individualizing the treatment with these drugs. The present review addresses the issue of individualized treatment with drugs metabolized by CES1. It describes the composition of the gene encoding CES1, reports variants of this gene with focus upon those with a potential effect on drug metabolism and provides an overview of the protein structure of this enzyme bringing notice to mechanisms involved in the regulation of enzyme activity. Subsequently, the review highlights drugs metabolized by CES1 and argues that individual differences in the pharmacokinetics of these drugs play an important role in determining drug response and tolerability suggesting prospects for individualized drug therapies. Our review also discusses endogenous substrates of CES1 and assesses the potential of using metabolomic profiling of blood to identify proxies for the hepatic activity of CES1 that predict the rate of drug metabolism. Finally, the combination of genetics and metabolomics to obtain an accurate prediction of the individual response to CES1-dependent drugs is discussed.

Timing of transcriptional quiescence during gametogenesis is controlled by global histone H3K4 demethylation.

Gametes are among the most highly specialized cells produced during development. Although gametogenesis culminates in transcriptional quiescence in plants and animals, regulatory mechanisms controlling this are unknown. Here, we confirm that gamete differentiation in the single-celled yeast Saccharomyces cerevisiae is accompanied by global transcriptional shutoff following the completion of meiosis. We show that Jhd2, a highly conserved JARID1-family histone H3K4 demethylase, activates protein-coding gene transcription in opposition to this programmed transcriptional shutoff, sustaining the period of productive transcription during spore differentiation. Moreover, using genome-wide nucleosome, H3K4me, and transcript mapping experiments, we demonstrate that JHD2 globally represses intergenic noncoding transcription during this period. The widespread transcriptional defects of JHD2 mutants are associated with precocious differentiation and the production of stress-sensitive spores, demonstrating that Jhd2 regulation of the global postmeiotic transcriptional program is critical for the production of healthy meiotic progeny.

ELM: the status of the 2010 eukaryotic linear motif resource.

Linear motifs are short segments of multidomain proteins that provide regulatory functions independently of protein tertiary structure. Much of intracellular signalling passes through protein modifications at linear motifs. Many thousands of linear motif instances, most notably phosphorylation sites, have now been reported. Although clearly very abundant, linear motifs are difficult to predict de novo in protein sequences due to the difficulty of obtaining robust statistical assessments. The ELM resource at http://elm.eu.org/ provides an expanding knowledge base, currently covering 146 known motifs, with annotation that includes >1300 experimentally reported instances. ELM is also an exploratory tool for suggesting new candidates of known linear motifs in proteins of interest. Information about protein domains, protein structure and native disorder, cellular and taxonomic contexts is used to reduce or deprecate false positive matches. Results are graphically displayed in a 'Bar Code' format, which also displays known instances from homologous proteins through a novel 'Instance Mapper' protocol based on PHI-BLAST. ELM server output provides links to the ELM annotation as well as to a number of remote resources. Using the links, researchers can explore the motifs, proteins, complex structures and associated literature to evaluate whether candidate motifs might be worth experimental investigation.

Preparation of hydrogels via ultrasonic polymerization.

Several acrylic hydrogels were prepared via ultrasonic polymerization of water soluble monomers and macromonomers. Ultrasound was used to create initiating radicals in viscous aqueous monomer solutions using the additives glycerol, sorbitol or glucose in an open system at 37 degrees C. The water soluble additives were essential for the hydrogel production, glycerol being the most effective. Hydrogels were prepared from the monomers 2-hydroxyethyl methacrylate, poly(ethylene glycol) dimethacrylate, dextran methacrylate, acrylic acid/ethylene glycol dimethacrylate and acrylamide/bis-acrylamide. For example a 5% w/w solution of dextran methacrylate formed a hydrogel in 6.5min in a 70% w/w solution of glycerol in water at 37 degrees C with 20kHz ultrasound, 56Wcm(-2). The ultrasonic polymerization method described here has a wide range of applications such a biomaterial synthesis where initiators are not desired.

Feasibility of T2* mapping for the evaluation of hip joint cartilage at 1.5T using a three-dimensional (3D), gradient-echo (GRE) sequence: a prospective study.

This study defines the feasibility of utilizing three-dimensional (3D) gradient-echo (GRE) MRI at 1.5T for T(2)* mapping to assess hip joint cartilage degenerative changes using standard morphological MR grading while comparing it to delayed gadolinium-enhanced MRI of cartilage (dGEMRIC). MRI was obtained from 10 asymptomatic young adult volunteers and 33 patients with symptomatic femoroacetabular impingement (FAI). The protocol included T(2)* mapping without gadolinium-enhancement utilizing a 3D-GRE sequence with six echoes, and after gadolinium injection, routine hip sequences, and a dual-flip-angle 3D-GRE sequence for dGEMRIC T(1) mapping. Cartilage was classified as normal, with mild changes, or with severe degenerative changes based on morphological MRI. T(1) and T(2)* findings were subsequently correlated. There were significant differences between volunteers and patients in normally-rated cartilage only for T(1) values. Both T(1) and T(2)* values decreased significantly with the various grades of cartilage damage. There was a statistically significant correlation between standard MRI and T(2)* (T(1)) (P < 0.05). High intraclass correlation was noted for both T(1) and T(2)*. Correlation factor was 0.860 to 0.954 (T(2)*-T(1) intraobserver) and 0.826 to 0.867 (T(2)*-T(1) interobserver). It is feasible to gather further information about cartilage status within the hip joint using GRE T(2)* mapping at 1.5T.

Myeloid leukaemia treatment and survival--the South Australian experience, 1977 to 2002.

OBJECTIVE: To evaluate trends in survival and treatment for myeloid leukaemia in South Australia during 1977-2002, using population-based survival data plus data on survival and treatment of patients at three teaching hospitals. METHODS: Population data were analysed using relative survival methods and hospital registry data using disease-specific survival. Univariate and multivariable analyses were undertaken. Multiple logistic regression analysis was used to investigate factors associated with first-line chemotherapy. RESULTS: South Australia recorded 1,572 new cases of acute myeloid leukaemia (AML) in 1977-2002, together with 536 cases of chronic myeloid leukaemia (CML). Of these cases, 42.6% were recorded in teaching hospital registries. The five-year survival for AML at the teaching hospitals of 14.5% was similar to the corresponding 12.0% for South Australia as a whole. The five-year survival for CML at these hospitals was higher, however, at 48.1% compared with 37.5% for all South Australian cases. Younger patients had higher survivals, both for AML and CML. An increase in survival was evident for more recently diagnosed cases for both leukaemia types, after adjusting for age. This increase in survival was accompanied by an increase over time in the proportion of patients at teaching hospitals having a primary course of chemotherapy. Cytarabine in combination with other agents was the most common induction therapy for AML. While hydroxyurea was the most common first-line treatment of CML, there were changes in clinical policies towards higher-dose treatments, plus trials of new agents and combination therapies. CONCLUSIONS: Secular gains in survival have occurred from AML and CML in association with an increased use of chemotherapy.

Approaches to improving the biocompatibility of porous perfluoropolyethers for ophthalmic applications.

Porous perfluoropolyether (PFPE) membranes for ophthalmic applications were prepared with a zwitterion monomer, 3-[[2-(methacryloxy) ethyl](N,N-dimethyl)ammonio]-propane-1-sulphonate, copolymerized in weight ratios of 0-10%. The polymer samples were assessed for a range of physical properties, including equilibrium water content, bovine serum albumin permeability, transparency, refractive index and the ability to support corneal epithelial cell and tissue attachment, growth and migration. In vitro assessment of the polymers using bovine corneal epithelial cells and tissue showed that a zwitterion incorporation level of between 0% and 6% in the PFPE membranes supported the migration of an intact sheet of epithelial tissue without compromising epithelial cell attachment and growth, with 4-6% being the optimal level for these properties. Binding patterns of the cell adhesion glycoprotein fibronectin were also found to reflect the cell and tissue response. Effective nutrient permeability, refractive index and optical transparency were also maintained by the porous PFPE polymers containing this concentration of zwitterionic monomer. The presence of amounts of zwitterion greater than 6% was inhibitory to both tissue migration and cell growth and was associated with increased optical haze. These results demonstrated that it is possible to achieve the potential for increased biocompatibility in zwitterion-containing PFPE polymers without compromising existing beneficial characteristics.

Real-time quantitative PCR analysis can be used as a primary screen to identify patients with CML treated with imatinib who have BCR-ABL kinase domain mutations.

Mutations within the BCR-ABL kinase domain in imatinib-treated chronic myeloid leukemia (CML) are the main mechanism of acquired resistance. The early detection of mutations should provide clinical benefit by allowing early intervention. Quantitative polymerase chain reaction (RQ-PCR) results of BCR-ABL mRNA were correlated with mutation analysis in 214 patients treated with imatinib. We determined whether there was a difference in the incidence of mutations between the patients with a more than 2-fold rise in BCR-ABL and patients with stable or decreasing levels. Of the 56 patients with a more than 2-fold rise, 34 (61%) had detectable mutations (median rise, 3.0-fold; 25th-75th percentiles, 2.3-5.2). In 31 (91%) of these 34 patients, the mutation was present at the time of the rise and became detectable within 3 months in the remaining patients. Only 1 (0.6%) of 158 patients with stable or decreasing BCR-ABL levels had a detectable mutation, P less than .0001. Thus, a more than 2-fold rise identified 34 (97%) of 35 patients with a mutation. We conclude that a rise in BCR-ABL of more than 2-fold can be used as a primary indicator to test patients for BCR-ABL kinase domain mutations.

Old drugs, new tricks: using genetically sensitized yeast to reveal drug targets.

A study in this issue of Cell illustrates the power of applying genomic approaches with model systems to characterize the biological activity of small molecules and to identify their cellular targets, which can clarify the mode of action of human therapeutics.

Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia.

BACKGROUND: In a randomized trial, 1106 patients with chronic myeloid leukemia (CML) in chronic phase were assigned to imatinib or interferon alfa plus cytarabine as initial therapy. We measured levels of BCR-ABL transcripts in the blood of all patients in this trial who had a complete cytogenetic remission. METHODS: Levels of BCR-ABL transcripts were measured by a quantitative real-time polymerase-chain-reaction assay. Results were expressed relative to the median level of BCR-ABL transcripts in the blood of 30 patients with untreated CML in chronic phase. RESULTS: In patients who had a complete cytogenetic remission, levels of BCR-ABL transcripts after 12 months of treatment had fallen by at least 3 log in 57 percent of those in the imatinib group and 24 percent of those in the group given interferon plus cytarabine (P=0.003). On the basis of the rates of complete cytogenetic remission of 68 percent in the imatinib group and 7 percent in the group given interferon plus cytarabine at 12 months, an estimated 39 percent of all patients treated with imatinib but only 2 percent of all those given interferon plus cytarabine had a reduction in BCR-ABL transcript levels of at least 3 log (P<0.001). For patients who had a complete cytogenetic remission and a reduction in transcript levels of at least 3 log at 12 months, the probability of remaining progression-free was 100 percent at 24 months, as compared with 95 percent for such patients with a reduction of less than 3 log and 85 percent for patients who were not in complete cytogenetic remission at 12 months (P<0.001). CONCLUSIONS: The proportion of patients with CML who had a reduction in BCR-ABL transcript levels of at least 3 log by 12 months of therapy was far greater with imatinib treatment than with treatment with interferon plus cytarabine. Patients in the imatinib group with this degree of molecular response had a negligible risk of disease progression during the subsequent 12 months.

Molecular monitoring of chronic myeloid leukemia.

The tyrosine kinase inhibitor imatinib mesylate (Gleevec) (formerly STI571) has proven to be an effective and safe new therapy for patients with chronic myeloid leukemia (CML). It has induced short-term hematologic control in many patients with advanced-phase CML, with some patients achieving durable responses. In chronic-phase patients it induces significantly better cytogenetic responses and lower progression rates than interferon-alpha. However, relapse is a significant problem, especially for advanced-phase patients, and imatinib alone appears unlikely to be curative in any patient group. Real-time quantitative polymerase chain reaction (Q-PCR) provides an accurate, sensitive, and noninvasive measure of residual leukemia in patients on imatinib. Levels of BCR-ABL in the blood correlate strongly with the bone marrow cytogenetic results and early measurement can predict subsequent cytogenetic response. Complete molecular responses (no BCR-ABL detected by real-time Q-PCR) are rarely achieved. Sequential real-time Q-PCR studies should facilitate rational patient management and allow comparison of different imatinib-based treatment strategies. It may be possible to define levels of molecular response that predict long-term disease control. In addition, by defining patterns of response, an early indication of imatinib resistance may be detected.

Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis.

Imatinib-treated chronic myeloid leukemia (CML) patients with acquired resistance commonly have detectable BCR-ABL kinase domain mutations. It is unclear whether patients who remain sensitive to imatinib also have a significant incidence of mutations. We evaluated 144 patients treated with imatinib for BCR-ABL kinase domain mutations by direct sequencing of 40 accelerated phase (AP), 64 late chronic phase (> or = 12 months from diagnosis, late-CP), and 40 early-CP patients. Mutations were detected in 27 patients at 17 different residues, 13 (33%) of 40 in AP, 14 (22%) of 64 in late-CP, and 0 of 40 in early-CP. Acquired resistance was evident in 24 (89%) of 27 patients with mutations. Twelve (92%) of 13 patients with mutations in the adenosine triphosphate (ATP) binding loop (P-loop) died (median survival of 4.5 months after the mutation was detected). In contrast, only 3 (21%) of 14 patients with mutations outside the P-loop died (median follow-up of 11 months). As the detection of mutations was strongly associated with imatinib resistance, we analyzed features that predicted for their detection. Patients who commenced imatinib more than 4 years from diagnosis had a significantly higher incidence of mutations (18 [41%] of 44) compared with those treated within 4 years (9 [9%] of 100), P <.0001. Lack of a major cytogenetic response (MCR) was also associated with a higher likelihood of detecting a mutation; 19 (38%) of 50 patients without a MCR had mutations compared with 8 (8.5%) of 94 with an MCR, P <.0001. In conclusion, the detection of kinase domain mutations using a direct sequencing technique was almost always associated with imatinib resistance, and patients with mutations in the P-loop had a particularly poor prognosis.