Rapid exchange of mammalian topoisomerase IIα at kinetochores and chromosome arms in mitosis

Astable cell line (GT2-LPk) derived from LLC-Pk was created in which endogenous DNA topoisomerase IIα (topoIIα) protein was downregulated and replaced by the expression of topoIIα fused with enhanced green fluorescent protein (EGFP–topoIIα). The EGFP–topoIIα faithfully mimicked the distribution of the endogenous protein in both interphase and mitosis. In early stages of mitosis, EGFP–topoIIα accumulated at kinetochores and in axial lines extending along the chromosome arms. During anaphase, EGFP–topoIIα diminished at kinetochores and increased in the cytoplasm with a portion accumulating into large circular foci that were mobile and appeared to fuse with the reforming nuclei. These cytoplasmic foci appearing at anaphase were coincident with precursor organelles of the reforming nucleolus called nucleolus-derived foci (NDF). Photobleaching of EGFP–topoIIα associated with kinetochores and chromosome arms showed that the majority of the protein rapidly exchanges (t1/2 of 16 s). Catalytic activity of topoIIα was essential for rapid dynamics, as ICRF-187, an inhibitor of topoIIα, blocked recovery after photobleaching. Although some topoIIα may be stably associated with chromosomes, these studies indicate that the majority undergoes rapid dynamic exchange. Rapid mobility of topoIIα in chromosomes may be essential to resolve strain imparted during chromosome condensation and segregation

Starvation and recovery in the deep-sea methanotroph Methyloprofundus sedimenti

In the deep ocean, the conversion of methane into derived carbon and energy drives the establishment of diverse faunal communities. Yet specific biological mechanisms underlying the introduction of methane-derived carbon into the food web remain poorly described, due to a lack of cultured representative deep-sea methanotrophic prokaryotes. Here, the response of the deep-sea aerobic methanotroph Methyloprofundus sedimenti to methane starvation and recovery was characterized. By combining lipid analysis, RNA analysis, and electron cryotomography, it was shown that M. sedimenti undergoes discrete cellular shifts in response to methane starvation, including changes in headgroup-specific fatty acid saturation levels, and reductions in cytoplasmic storage granules. Methane starvation is associated with a significant increase in the abundance of gene transcripts pertinent to methane oxidation. Methane reintroduction to starved cells stimulates a rapid, transient extracellular accumulation of methanol, revealing a way in which methane-derived carbon may be routed to community members. This study provides new understanding of methanotrophic responses to methane starvation and recovery, and lays the initial groundwork to develop Methyloprofundus as a model chemosynthesizing bacterium from the deep sea

The rise and fall of methanotrophy following a deepwater oil-well blowout

The blowout of the Macondo oil well in the Gulf of Mexico in April 2010 injected up to 500,000 tonnes of natural gas, mainly methane, into the deep sea1. Most of the methane released was thought to have been consumed by marine microbes between July and August 20102, 3. Here, we report spatially extensive measurements of methane concentrations and oxidation rates in the nine months following the spill. We show that although gas-rich deepwater plumes were a short-lived feature, water column concentrations of methane remained above background levels throughout the rest of the year. Rates of microbial methane oxidation peaked in the deepwater plumes in May and early June, coincident with a rapid rise in the abundance of known and new methane-oxidizing microbes. At this time, rates of methane oxidation reached up to 5,900 nmol l−1 d−1—the highest rates documented in the global pelagic ocean before the blowout4. Rates of methane oxidation fell to less than 50 nmol l−1 d−1 in late June, and continued to decline throughout the remainder of the year. We suggest the precipitous drop in methane consumption in late June, despite the persistence of methane in the water column, underscores the important role that physiological and environmental factors play in constraining the activity of methane-oxidizing bacteria in the Gulf of Mexico

In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for L-methionine, followed by fluorescent labeling of AHA containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater, and anoxic sediment. We also developed combined assays that couple BONCAT with rRNA-targeted FISH, enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labeling by nanoSIMS (^(15)NH_4^+ assimilation) for individual cells within a sediment sourced enrichment culture showed concordance between AHA positive cells and ^(15)N enrichment. BONCAT-FISH offers a fast, inexpensive, and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single cell level

Community singing, wellbeing and older people: implementing and evaluating an English singing tool for health intervention in Rome

Aim: The aim of this research was to explore the transferability and effectiveness of the English Silver Song Clubs model for older people in a different social and cultural context, i.e. in the capital city of Italy, Rome. Methods: A single condition, pre-test, post-test design was implemented. Participants completed two questionnaires: EQ-5D and York SF-12. Results: After the singing experience, participants showed a decrease in their levels of anxiety and depression. An improvement was also found from baseline to follow up in reported performance of usual activities. The English study showed a difference between the singing and non-singing groups at three and six months on mental health, and after three months on specific anxiety and depression measures. The current (Rome) study shows similar findings with an improvement on specific anxiety and depression items. Conclusions: Policy makers in different national contexts should consider social singing activities to promote the health and wellbeing of older adults as they are inexpensive to run and have been shown to be enjoyable and effective

Pseudocisti pancreatiche infette: Note di tecnica

Introduction. Starting from the observation of 9 cases of giant infected pancreatic cysts, which occurred from 1994 to 2004 at the Department of Oncological and Surgical Studies, the Authors' aim has been to evaluate whether a more thorough necrosectomy, carried-out under videoendoscopic control, associated with a nose-gastro-cavity tube, which ensures a continuous cleansing of the newly-formed cavity, and an appropriate positioning of the drainages, could reduce the morbility and allow a shorter recovery of the infected psuodocysts. Patients and methods. Of 73 cases of acute pancreatitis, observed from 1994 to 2004, 9 showed severe and acute pancreatitis, which included giant pseudocysts, as revealed by the abdomen angio-TC. Our nine septic patients underwent cysto-gastro-anastomosis, necrosectomy, intraoperative cleansing of the cavity with an antibiotic solution and positioning of multiple drainages. Three of these patients also underwent a thorough and targeted nectrosectomy, assisted by a trans-anastomotic videoendoscopy. A nose-gastro-cavity tube has been placed in all the patients. Results. The disappearance of the septic state in our three patients who underwent a targeted video-assisted necrosectomy occurred after three days of treatment; moreover, the abdomen angio-TC on the 5th postoperative day showed the disappearance of the necrotic areas. The recovery of these three patients was significantly shorter, compared to those undergoing traditional treatment (cysto-gastro-anastomosis, standard necrosectomy and positioning of abdominal drainages). Conclusions. Our surgical video-assisted technique demonstrated that, with a slight increase in the operative time, a better control over sepsis may be accomplished, as well as a reduction of he post-operative morbidity, which leads to shorter hospitalisation of patients with infected pancreatic pseudocysts

Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα

© 2012 Samejima et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication dateMitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic "X" shape. Here we report results of a detailed morphological analysis, which revealed that chromokinesin KIF4 cooperated in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Depletion of either caused sister chromatids to expand and compromised the "intrinsic structure" of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. In these experiments, topoisomerase IIα contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology

Allosteric control of the RNA polymerase by the elongation factor RfaH

Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptional pausing at certain positions rather than by accelerating RNAP at all sites. We show that ‘fast’ RNAP variants are characterized by pause-free RNA chain elongation and are resistant to RfaH action. Similarly, the wild-type RNAP is insensitive to RfaH in the absence of pauses. In contrast, those enzymes that may be prone to falling into a paused state are hypersensitive to RfaH. RfaH inhibits pyrophosphorolysis of the nascent RNA and reduces the apparent Michaelis–Menten constant for nucleotides, suggesting that it stabilizes the post-translocated, active RNAP state. Given that the RfaH-binding site is located 75 Å away from the RNAP catalytic center, these results strongly indicate that RfaH acts allosterically. We argue that despite the apparent differences in the nucleic acid targets, the time of recruitment and the binding sites on RNAP, unrelated antiterminators (such as RfaH and λQ) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex

Metagenomic and Metatranscriptomic Analysis of Microbial Community Structure and Gene Expression of Activated Sludge

The present study applied both metagenomic and metatranscriptomic approaches to characterize microbial structure and gene expression of an activated sludge community from a municipal wastewater treatment plant in Hong Kong. DNA and cDNA were sequenced by Illumina Hi-seq2000 at a depth of 2.4 Gbp. Taxonomic analysis by MG-RAST showed bacteria were dominant in both DNA and cDNA datasets. The taxonomic profile obtained by BLAST against SILVA SSUref database and annotation by MEGAN showed that activated sludge was dominated by Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia phyla in both DNA and cDNA datasets. Global gene expression annotation based on KEGG metabolism pathway displayed slight disagreement between the DNA and cDNA datasets. Further gene expression annotation focusing on nitrogen removal revealed that denitrification-related genes sequences dominated in both DNA and cDNA datasets, while nitrifying genes were also expressed in relative high levels. Specially, ammonia monooxygenase and hydroxylamine oxidase demonstrated the high cDNA/DNA ratios in the present study, indicating strong nitrification activity. Enzyme subunits gene sequences annotation discovered that subunits of ammonia monooxygenase (amoA, amoB, amoC) and hydroxylamine oxygenase had higher expression levels compared with subunits of the other enzymes genes. Taxonomic profiles of selected enzymes (ammonia monooxygenase and hydroxylamine oxygenase) showed that ammonia-oxidizing bacteria present mainly belonged to Nitrosomonas and Nitrosospira species and no ammonia-oxidizing Archaea sequences were detected in both DNA and cDNA datasets