Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1+ gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1¿ strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression

Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves hepatic production of acute-phase proteins, including serum amyloid A (SAA). Extrahepatically, SAA immunoreactivity is found in axonal myelin sheaths of cortex in Alzheimer's disease and multiple sclerosis (MS), although its cellular origin is unclear. We examined the responses of cultured rat cortical astrocytes, microglia and oligodendrocyte precursor cells (OPCs) to master pro-inflammatory cytokine tumour necrosis factor (TNF)-\u3b1 and lipopolysaccaride (LPS). TNF-\u3b1 time-dependently increased Saa1 (but not Saa3) mRNA expression in purified microglia, enriched astrocytes, and OPCs (as did LPS for microglia and astrocytes). Astrocytes depleted of microglia were markedly less responsive to TNF-\u3b1 and LPS, even after re-addition of microglia. Microglia and enriched astrocytes showed complementary Saa1 expression profiles following TNF-\u3b1 or LPS challenge, being higher in microglia with TNF-\u3b1 and higher in astrocytes with LPS. Recombinant human apo-SAA stimulated production of both inflammatory mediators and its own mRNA in microglia and enriched, but not microglia-depleted astrocytes. Co-ultramicronized palmitoylethanolamide/luteolin, an established anti-inflammatory/neuroprotective agent, reduced Saa1 expression in OPCs subjected to TNF-\u3b1 treatment. These last data, together with past findings suggest that co-ultramicronized palmitoylethanolamide/luteolin may be a novel approach in the treatment of inflammatory demyelinating disorders like MS

Reduced Health-Related Quality of Life in Elders with Frailty: A Cross-Sectional Study of Community-Dwelling Elders in Taiwan

PURPOSE: Exploring the domains and degrees of health-related quality of life (HRQOL) that are affected by the frailty of elders will help clinicians understand the impact of frailty. This association has not been investigated in community-dwelling elders. Therefore, we examined the domains and degree of HRQOL of elders with frailty in the community in Taiwan. METHODS: A total of 933 subjects aged 65 years and over were recruited in 2009 from a metropolitan city in Taiwan. Using an adoption of the Fried criteria, frailty was defined by five components: shrinking, weakness, poor endurance and energy, slowness, and low physical activity level. HRQOL was assessed by the short form 36 (SF-36). The multiple linear regression model was used to test the independent effects of frailty on HRQOL. RESULTS: After multivariate adjustment, elders without frailty reported significantly better health than did the pre-frail and frail elders on all scales, and the pre-frail elders reported better health than did the frail elders for all scales except the scales of role limitation due to physical and emotional problems and the Mental Component Summary (MCS). The significantly negative differences between frail and robust elders ranged from 3.58 points for the MCS to 22.92 points for the physical functioning scale. The magnitude of the effects of frail components was largest for poor endurance and energy, and next was for slowness. The percentages of the variations of these 10 scales explained by all factors in the models ranged from 11.1% (scale of role limitation due to emotional problems) to 49.1% (scale of bodily pain). CONCLUSIONS: Our study demonstrates that the disabilities in physical health inherent in frailty are linked to a reduction in HRQOL. Such an association between clinical measures and a generic measure of the HRQOL may offer clinicians new information to understand frailty and to conceptualize it within the broader context of disability

CCAAT/enhancer binding proteins in normal mammary development and breast cancer

CCAAT/enhancer binding proteins (C/EBPs) are a family of leucine zipper, transcription factors that bind to DNA as homodimers and heterodimers. They regulate cellular proliferation, differentiation and apoptosis in the mammary gland. Multiple protein isoforms, including truncated, dominant negatives, are generated by translation of the C/EBPβ transcript or via proteolytic cleavage of the full-length C/EBPβ protein. Gene deletion of individual C/EBP family members has demonstrated an essential role for C/EBPβ in normal mammary development, while transgenic and overexpression studies provide evidence that the dominant-negative C/EBPβ-liver-enriched inhibitory protein isoform induces proliferation in mammary epithelial cells. Mounting evidence suggests that alterations in the ratio of the C/EBPβ-liver-enriched inhibitory protein isoform and the C/EBPβ-liver-enriched activating protein isoform may play a role in the development of breast cancer. This review will consequently focus on C/EBP actions in normal mammary development and on the emerging data that supports a role in breast cancer

GLUT 5 Is Not Over-Expressed in Breast Cancer Cells and Patient Breast Cancer Tissues

F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues

Molecular classification improves risk assessment in adult BCR-ABL1–negative B-ALL

Genomic classification has improved risk assignment of pediatric but not adult B-lineage acute lymphoblastic leukemia (B-ALL). The international UKALLXII/ECOG-ACRIN E2993 (NCT00002514) trial accrued 1229 BCR-ABL1-negative adolescent/adult B-ALL patients (aged 14-65 years). While 93% of patients achieved remission, 41% relapsed at a median of 13 months (range 28 days to 12 years). Five-year overall survival (5yr-OS) was 42% (95% CI, 39, 44). Transcriptome sequencing (n=238), gene expression profiling (n=210), cytogenetics (n=197) and fusion PCR (n=274) enabled genomic subtyping of 282 patient samples, of which 264 were eligible for trial, accounting for 64.5% of E2993 patients. Among patients in the outcome analysis, 29.5% of cases had favorable outcomes with 5yr-OS of 65-80% and were deemed standard-risk (DUX4-rearranged [9.2%], ETV6-RUNX1/-like [2.3%], TCF3-PBX1 [6.9%], PAX5 P80R [4.1%], high-hyperdiploid [6.9%]); 50.2% had high-risk genotypes with 5yr-OS of 0-27% (Ph-like [21.2%], KMT2A-AFF1 [12%], low-hypodiploid/near-haploid [14.3%], BCL2/MYC-rearranged [2.8%]); and 20.3% had intermediate-risk genotypes with 5yr-OS of 33-45% (PAX5alt [12.4%], ZNF384/-like [5.1%], MEF2D-rearranged [2.8%]). IKZF1 alterations occurred in 86% of Ph-like and TP53 mutations occurred in low-hypodiploid (54%) and BCL2/MYC-rearranged patients (33%), but were not independently associated with outcome. Of patients considered high-risk for relapse based on presenting age and WBC count, 40% harbored subtype-defining genetic alterations associated with standard- or intermediate-risk outcomes. We identified distinct immunophenotypic features for DUX4-rearranged, PAX5 P80R, ZNF384-R/-like and Ph-like genotypes. These data in a large adult B-ALL cohort treated with a non-risk-adapted approach on a single trial show the prognostic importance of genomic analyses which may translate into future therapeutic benefits

A Nitrile Hydratase in the Eukaryote Monosiga brevicollis

Bacterial nitrile hydratase (NHases) are important industrial catalysts and waste water remediation tools. In a global computational screening of conventional and metagenomic sequence data for NHases, we detected the two usually separated NHase subunits fused in one protein of the choanoflagellate Monosiga brevicollis, a recently sequenced unicellular model organism from the closest sister group of Metazoa. This is the first time that an NHase is found in eukaryotes and the first time it is observed as a fusion protein. The presence of an intron, subunit fusion and expressed sequence tags covering parts of the gene exclude contamination and suggest a functional gene. Phylogenetic analyses and genomic context imply a probable ancient horizontal gene transfer (HGT) from proteobacteria. The newly discovered NHase might open biotechnological routes due to its unconventional structure, its new type of host and its apparent integration into eukaryotic protein networks

Role of C/EBPβ Transcription Factor in Adult Hippocampal Neurogenesis

[Background]: The dentate gyrus of the hippocampus is one of the regions in which neurogenesis takes place in the adult brain. We have previously demonstrated that CCAAT/enhancer binding protein β (C/EBPβ) is expressed in the granular layer of the dentate gyrus of the adult mouse hippocampus. Taking into account the important role of C/EBPβ in the consolidation of long term memory, the fact that newborn neurons in the hippocampus contribute to learning and memory processes, and the role of this transcription factor, previously demonstrated by our group, in regulating neuronal differentiation, we speculated that this transcription factor could regulate stem/progenitor cells in this region of the brain. [Methodologu/Principal Findings]: Here, we show, using C/EBPβ knockout mice, that C/EBPβ expression is observed in the subset of newborn cells that proliferate in the hippocampus of the adult brain. Mice lacking C/EBPβ present reduced survival of newborn cells in the hippocampus, a decrease in the number of these cells that differentiate into neurons and a diminished number of cells that are proliferating in the subgranular zone of the dentate gyrus. These results were further confirmed in vitro. Neurosphere cultures from adult mice deficient in C/EBPβ present less proliferation and neuronal differentiation than neurospheres derived from wild type mice. [Conclusions/Significance]: In summary, using in vivo and in vitro strategies, we have identified C/EBPβ as a key player in the proliferation and survival of the new neurons produced in the adult mouse hippocampus. Our results support a novel role of C/EBPβ in the processes of adult hippocampal neurogenesis, providing new insights into the mechanisms that control neurogenesis in this region of the brain.This work was supported by a postdoctoral fellowship of the Consejo Superior de Investigaciones Cientificas (M.C.-C.) Grant Sponsor: Ministerio de Investigación y Ciencia; Grant numbers: SAF2007-62811 and SAF2010-16365. CIBERNED is funded by the Instituto de Salud Carlos III.Peer reviewe

Clinical and radiological evaluation of Trabecular Metal and the Smith–Robinson technique in anterior cervical fusion for degenerative disease: a prospective, randomized, controlled study with 2-year follow-up

A prospective, randomized, controlled study was carried out to compare the radiological and clinical outcomes after anterior cervical decompression and fusion (ACDF) with Trabecular Metal™ (TM) to the traditional Smith–Robinson (SR) procedure with autograft. The clinical results of cervical fusion with autograft from the iliac crest are typically satisfactory, but implications from the donor site are frequently reported. Alternative materials for cervical body interfusion have shown lower fusion rates. Trabecular Metal is a porous tantalum biomaterial with structure and mechanical properties similar to that of trabecular bone and with proven osteoconductivity. As much as 80 consecutive patients planned for ACDF were randomized for fusion with either TM or tricortical autograft from the iliac crest (SR) after discectomy and decompression. Digitized plain radiographic images of 78 (98%) patients were obtained preoperatively and at 2-year follow-up and were subsequently evaluated by two senior radiologists. Fusion/non-fusion was classified by visual evaluation of the A–P and lateral views in forced flexion/extension of the cervical spine and by measuring the mobility between the fused vertebrae. MRI of 20 TM cases at 2 years was successfully used to assess the decompression of the neural structures, but was not helpful in determining fusion/non-fusion. Pain intensity in the neck, arms and pelvis/hip were rated by patients on a visual analog scale (VAS) and neck function was rated using the Neck Disability Index (NDI) the day before surgery and 4, 12 and 24 months postoperatively. Follow-ups at 12 and 24 months were performed by an unbiased observer, when patients also assessed their global outcome. Fusion rate in the SR group was 92%, and in the TM group 69% (P < 0.05). The accuracy of the measurements was calculated to be 2.4°. Operating time was shorter for fusion with TM compared with autograft; mean times were 100 min (SD 18) and 123 min (SD 23), respectively (P = 0.001). The patients’ global assessments of their neck and arm symptoms 2 years postoperatively for the TM group were rated as 79% much better or better after fusion with TM and 75% using autograft. Pain scores and NDI scores were significantly improved in both groups when compared with baseline at all follow-ups, except for neck pain at 1 year for the TM group. There was no statistically significant difference in clinical outcomes between fusion techniques or between patients who appeared radiologically fused or non-fused. There was no difference in pelvic/hip pain between patients operated on with or without autograft. In our study, Trabecular Metal showed a lower fusion rate than the Smith–Robinson technique with autograft after single-level anterior cervical fusion without plating. There was no difference in clinical outcomes between the groups. The operative time was shorter with Trabecular Metal implants

The benefits of participatory methodologies to develop effective community dialogue in the context of a microbicide trial feasibility study in Mwanza, Tanzania

BACKGROUND: As part of a microbicide trial feasibility study among women at high-risk of HIV and sexually transmitted infections in Mwanza City, northern Tanzania we used participatory research tools to facilitate open dialogue and partnership between researchers and study participants. METHODS: A mobile community-based sexual & reproductive health service was established in ten city wards. Wards were divided into seventy-eight geographical clusters and representatives at cluster and ward level elected in a process facilitated by the projects Community Liaison Officer. A city-level Community Advisory Committee (CAC) with representatives from each ward was established. Workshops and community meetings at ward and city-level were conducted to explore project-related concerns using tools adapted from participatory learning and action techniques such as listing, scoring, ranking, chapatti diagrams and pair-wise matrices. RESULTS: Key issues identified included beliefs that blood specimens were being sold for witchcraft purposes; worries about specula not being clean; inadequacy of transport allowances; and delays in reporting laboratory test results to participants. To date, the project has responded by inviting members of the CAC to visit the laboratory to observe how blood and genital specimens are prepared; demonstrated the use of the autoclave to community representatives; raised reimbursement levels; introduced HIV rapid testing in the clinic; and streamlined laboratory reporting procedures. CONCLUSIONS: Participatory techniques were instrumental in promoting meaningful dialogue between the research team, study participants and community representatives in Mwanza, allowing researchers and community representatives to gain a shared understanding of project-related priority areas for intervention