Gram-staining characterisation of activated sludge filamentous bacteria by automated colour analysis

An automated image analysis method has been developed for the monitoring of the Gram-staining characteristics of filamentous bacteria in activated sludge. The binary method of pixel classification agreed with manual estimation (level of correlation of 0.9 for Gram-positive bacteria). Its robustness has been assessed by repeatability tests. Population shifts in terms of Gram-staining characteristics have been monitored in laboratory-scale experiments with two feeding schedules using this technique.Revisions requested 22 September 2004; Revisions received 11 October 2004     

New Short Tandem Repeat-Based Molecular Typing Method for Pneumocystis jirovecii Reveals Intrahospital Transmission between Patients from Different Wards

Pneumocystis pneumonia is a severe opportunistic infection in immunocompromised patients caused by the unusual fungus Pneumocystis jirovecii. Transmission is airborne, with both immunocompromised and immunocompetent individuals acting as a reservoir for the fungus. Numerous reports of outbreaks in renal transplant units demonstrate the need for valid genotyping methods to detect transmission of a given genotype. Here, we developed a short tandem repeat (STR)-based molecular typing method for P. jirovecii. We analyzed the P. jirovecii genome and selected six genomic STR markers located on different contigs of the genome. We then tested these markers in 106 P. jirovecii PCR-positive respiratory samples collected between October 2010 and November 2013 from 91 patients with various underlying medical conditions. Unique (one allele per marker) and multiple (more than one allele per marker) genotypes were observed in 34 (32%) and 72 (68%) samples, respectively. A genotype could be assigned to 55 samples (54 patients) and 61 different genotypes were identified in total with a discriminatory power of 0.992. Analysis of the allelic distribution of the six markers and minimum spanning tree analysis of the 61 genotypes identified a specific genotype (Gt21) in our hospital, which may have been transmitted between 10 patients including six renal transplant recipients. Our STR-based molecular typing method is a quick, cheap and reliable approach to genotype Pneumocystis jirovecii in hospital settings and is sensitive enough to detect minor genotypes, thus enabling the study of the transmission and pathophysiology of Pneumocystis pneumonia.     

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.     

Inspiratory resistances facilitate the diaphragm response to transcranial stimulation in humans

Background  

Breathing in humans is dually controlled for metabolic (brainstem commands) and behavioral purposes (suprapontine commands) with reciprocal modulation through spinal integration. Whereas the ventilatory response to chemical stimuli arises from the brainstem, the compensation of mechanical loads in awake humans is thought to involve suprapontine mechanisms. The aim of this study was to test this hypothesis by examining the effects of inspiratory resistive loading on the response of the diaphragm to transcranial magnetic stimulation.     

cAMP-positive regulation of angiotensinogen gene expression and protein secretion in rat adipose tissue

The adipose renin-angiotensin system (RAS) has been assigned to participate in the control of adipose tissue development and in the pathogenesis of obesity-related hypertension. In adipose cells, the biological responses to beta-adrenergic stimulation are mediated by an increase in intracellular cAMP. Because cAMP is known to promote adipogenesis and because an association exists between body fat mass, hypertension, and increased sympathetic stimulation, we examined the influence of cAMP on angiotensinogen (ATG) expression and secretion in rat adipose tissue. Exposure of primary cultured differentiated preadipocytes to the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or cAMP-stimulating agents (forskolin and IBMX) results in a significant increase in ATG mRNA levels. In adipose tissue fragments, 8-BrcAMP also increases ATG mRNA levels and protein secretion, but not in the presence of the protein kinase A inhibitor H89. The addition of isoproterenol, known to stimulate the synthesis of intracellular cAMP via beta-adrenoreceptors, had the same stimulatory effect on ATG expression and secretion. These results indicate that cAMP in vitro upregulates ATG expression and secretion in rat adipose tissue via the protein kinase A-dependent pathway. Further studies are required to determine whether this regulatory pathway is activated in human obesity, where increased sympathetic tone is frequently observed, and to elucidate the importance of adipose ATG to the elevated blood pressure observed in this pathological state.     

The Spatiotemporal Program of DNA Replication Is Associated with Specific Combinations of Chromatin Marks in Human Cells

The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS). The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those marks as potential key regulators of replication origins.     

Suppression of Dwarf and irregular xylem Phenotypes Generates Low-Acetylated Biomass Lines in Arabidopsis

eskimo1-5 (esk1-5) is a dwarf Arabidopsis (Arabidopsis thaliana) mutant that has a constitutive drought syndrome and collapsed xylem vessels, along with low acetylation levels in xylan and mannan. ESK1 has xylan O-acetyltransferase activity in vitro. We used a suppressor strategy on esk1-5 to screen for variants with wild-type growth and low acetylation levels, a favorable combination for ethanol production. We found a recessive mutation in the KAKTUS (KAK) gene that suppressed dwarfism and the collapsed xylem character, the cause of decreased hydraulic conductivity in the esk1-5 mutant. Backcrosses between esk1-5 and two independent knockout kak mutants confirmed suppression of the esk1-5 effect. kak single mutants showed larger stem diameters than the wild type. The KAK promoter fused with a reporter gene showed activity in the vascular cambium, phloem, and primary xylem in the stem and hypocotyl. However, suppression of the collapsed xylem phenotype in esk1 kak double mutants was not associated with the recovery of cell wall O-acetylation or any major cell wall modifications. Therefore, our results indicate that, in addition to its described activity as a repressor of endoreduplication, KAK may play a role in vascular development. Furthermore, orthologous esk1 kak double mutants may hold promise for ethanol production in crop plants.Today, the fields of agriculture and forestry must address challenging issues, particularly in the context of fluctuating environmental conditions, such as ensuring that food and feed production remain efficient, meeting societal demands to reduce inputs, including water, and creating new products, such as biofuel. The demand for biofuel, a renewable alternative to fossil fuel, further increases the need to develop biomass amenable to alcohol fermentation. Second-generation biofuels are based on the fermentation of sugars extracted from lignocellulosic biomass, which is produced from the residues of food crops or from nonfood crops; therefore, its production does not compete with food crops (Sims et al., 2010). This lignocellulosic biomass is made up of secondary cell walls, mostly found in vascular tissues.Vascular tissues consist of a network of conduits, spanning an entire plant and connecting biosynthetically active leaves to the soil via the root and the shoot. There are two main vascular tissues, the xylem and the phloem, which arise from a lateral meristem called the procambium during primary growth. When dicot plants undergo secondary growth and cell walls are thickening, a secondary meristem, called the cambium or vascular cambium, emerges, giving rise to secondary vascular tissues (Esau, 1965; Buvat, 1989). The xylem is responsible for the upward transport of water and nutrients from the soil to the whole plant. The phloem, positioned parallel to the xylem, supplies sink organs (roots, etc.) with leaf photoassimilates. In angiosperms, such as Arabidopsis (Arabidopsis thaliana), the xylem is composed of two main cellular types, tracheary elements, involved in the transport of water, and fibers, playing a major role in plant support (Turner and Sieburth, 2003). The function of the xylem depends on the plant’s capacity to form thick secondary cell walls that confer mechanical strength to resist gravity and withstand negative pressure, allowing sap to travel upward through vessels. Xylem tissue is a major carbon sink that incorporates sugars into biopolymers (Ragni et al., 2011). Xylem secondary cell walls are mainly composed of cellulose embedded in a matrix of lignin and hemicelluloses. Xylan is a major hemicellulose in monocot and dicot secondary cell walls (Faik, 2010). Xylan has a linear backbone of β-(1,4)-linked d-Xyl residues that can be mono- or di-O-acetylated at positions O-2 and O-3 of the xylosyl residues (Ebringerova and Heinze, 2000).O-Acetylation of polysaccharides reportedly has a negative effect on the utilization of lignocellulose, such as in the production of paper and bioethanol (Biely, 1985; Grohmann et al., 1989). A major xylan acetyltransferase was recently identified in Arabidopsis: TRICHOME BIREFRINGENCE-LIKE29/ESKIMO1 (TBL29/ESK1; Urbanowicz et al., 2014). esk1 knockout mutants show a 60% reduction in xylan acetylation and a lesser reduction in mannan acetylation (Xiong et al., 2013; Yuan et al., 2013). esk1 has been described previously as a genotype with drought stress symptoms (Bouchabke-Coussa et al., 2008; Lugan et al., 2009); its collapsed xylem vessels (irregular xylem [irx] phenotype) are assumed to be the cause of the drastic hydraulic conductivity drop, and thus the drought stress syndrome, including dwarfism (Lefebvre et al., 2011).To identify new mutations that restore plant stature but maintain a low xylan O-acetylation level, we explored the possibility of producing this combination by screening an esk1-5 ethyl methanesulfonate (EMS)-mutagenized population for nondwarf phenotypes. The suppressors of esk1 were called beem, for biomass enhancement in esk1-5 mutation background. Here, we describe the identification of one BEEM gene as KAKTUS/UBIQUITIN PROTEIN LIGASE3 (KAK/UPL3), which encodes a protein belonging to the E3-ubiquitin protein ligase family (Downes et al., 2003).