Bacteria Associated with Benthic Diatoms from Lake Constance: Phylogeny and Influences on Diatom Growth and Secretion of Extracellular Polymeric Substances

The composition of diatom-associated bacterial communities was studied with 14 different unialgal xenic diatom cultures isolated from freshwater epilithic biofilms of Lake Constance, Germany. A clear dominance of Alphaproteobacteria was observed, followed by Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. Pure cultures of the diatom Cymbella microcephala, which was found to be dominant in epilithic biofilms in Lake Constance, were cocultivated with six associated bacterial strains. All these bacterial strains were able to grow in C. microcephala cultures in the absence of organic cosubstrates. Diatom growth was generally enhanced in the presence of bacteria, and polysaccharide secretion was generally increased in the presence of Proteobacteria. The monomer composition of extracellular polysaccharides of C. microcephala changed in relation to the presence of different bacteria, but the dominant monomers were less affected. Our results indicate that these changes were caused by the diatom itself rather than by specific bacterial degradation. One Bacteroidetes strain strongly influenced carbohydrate secretion by the alga via extracellular soluble compounds. Biofilms were formed only in the presence of bacteria. Phylogenetic analysis and coculture studies indicate an adaptation of Proteobacteria and Bacteroidetes to the microenvironment created by the diatom biofilm.     

Cultivation of methanotrophic bacteria in opposing gradients of methane and oxygen

In sediments, methane-oxidizing bacteria live in opposing gradients of methane and oxygen. In such a gradient system, the fluxes of methane and oxygen are controlled by diffusion and consumption rates, and the rate-limiting substrate is maintained at a minimum concentration at the layer of consumption. Opposing gradients of methane and oxygen were mimicked in a specific cultivation set-up in which growth of methanotrophic bacteria occurred as a sharp band at either c. 5 or 20 mm below the air-exposed end. Two new strains of methanotrophic bacteria were isolated with this system. One isolate, strain LC 1, belonged to the Methylomonas genus (type I methantroph) and contained soluble methane mono-oxygenase. Another isolate, strain LC 2, was related to the Methylobacter group (type I methantroph), as determined by 16S rRNA gene and pmoA sequence similarities. However, the partial pmoA sequence was only 86% related to cultured Methylobacter species. This strain accumulated significant amounts of formaldehyde in conventional cultivation with methane and oxygen, which may explain why it is preferentially enriched in a gradient cultivation system.     

SMAP-29 has two LPS-binding sites and a central hinge.

The CD spectra of SMAP-29, an antimicrobial peptide from sheep, showed disordered structure in aqueous buffers, and significant helicity in membrane-like environments, including SDS micelles, lipopolysaccharide (LPS) dispersions, and trifluoroethanol buffer systems. A structure determined by NMR in 40% perdeuterated trifluoroethanol indicated that residues 8-17 were helical, residues 18-19 formed a hinge, and residues 20-28 formed an ordered, hydrophobic segment. SMAP-29 was flexible in 40% trifluoroethanol, forming two sets of conformers that differed in the relative orientation of the N-terminal domain. We used a chromogenic Limulus assay to determine the EC50 of the peptide (the concentration that bound 50% of the added LPS). Studies with full-length and truncated SMAP-29 molecules revealed that each end of the holopeptide contained an LPS-binding domain. The higher affinity LPS-binding domain was situated in the flexible N-terminal portion. LPS binding to full-length SMAP-29 showed positive cooperativity, so the EC50 of the peptide (2.6 microm) was considerably lower than that of the individual LPS-binding domains. LPS-binding studies with a mixture of truncated peptides revealed that this cooperativity was primarily intramolecular (i.e. involving the N- and C-terminal LPS-binding sites of the same peptide molecule). CAP-18[106 -142], an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N- and C-terminal LPS-binding domains, had an EC50 of 2.5 microm, and bound LPS with positive cooperativity. We conclude that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity.     

Alleviating CYP and hERG liabilities by structure optimization of dihydrofuran-fused tricyclic benzo[d]imidazole series – Potent,selective and orally efficacious microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors: Part-2

In an effort to identify CYP and hERG clean mPGES-1 inhibitors from the dihydrofuran-fused tricyclic benzo[d]imidazole series lead 7, an extensive structure-activity relationship (SAR) studies were performed. Optimization of A, D and E-rings in 7 afforded many potent compounds with human whole blood potency in the range of 160–950?nM. Selected inhibitors 21d, 21j, 21m, 21n, 21p and 22b provided selectivity against COX-enzymes and mPGES-1 isoforms (mPGES-2 and cPGES) along with sufficient selectivity against prostanoid synthases. Most of the tested analogs demonstrated required metabolic stability in liver microsomes, low hERG and CYP liability. Oral pharmacokinetics and bioavailability of lead compounds 21j, 21m and 21p are discussed in multiple species like rat, guinea pig, dog, and cynomolgus monkey. Besides, these compounds revealed low to moderate activity against human pregnane X receptor (hPXR). The selected lead 21j further demonstrated in vivo efficacy in acute hyperalgesia (ED₅₀: 39.6?mg/kg) and MIA-induced osteoarthritic pain models (ED₅₀: 106?mg/kg).     

MNR2 Regulates Intracellular Magnesium Storage in Saccharomyces cerevisiae

Magnesium (Mg) is an essential enzyme cofactor and a key structural component of biological molecules, but relatively little is known about the molecular components required for Mg homeostasis in eukaryotic cells. The yeast genome encodes four characterized members of the CorA Mg transporter superfamily located in the plasma membrane (Alr1 and Alr2) or the mitochondrial inner membrane (Mrs2 and Lpe10). We describe a fifth yeast CorA homolog (Mnr2) required for Mg homeostasis. MNR2 gene inactivation was associated with an increase in both the Mg requirement and the Mg content of yeast cells. In Mg-replete conditions, wild-type cells accumulated an intracellular store of Mg that supported growth under deficient conditions. An mnr2 mutant was unable to access this store, suggesting that Mg was trapped in an intracellular compartment. Mnr2 was localized to the vacuole membrane, implicating this organelle in Mg storage. The mnr2 mutant growth and Mg-content phenotypes were dependent on vacuolar proton-ATPase activity, but were unaffected by the loss of mitochondrial Mg uptake, indicating a specific dependence on vacuole function. Overexpression of Mnr2 suppressed the growth defect of an alr1 alr2 mutant, indicating that Mnr2 could function independently of the ALR genes. Together, our results implicate a novel eukaryotic CorA homolog in the regulation of intracellular Mg storage.MAGNESIUM (Mg) is a critical factor in a wide variety of biological processes (Elin 1994), and there are at least 300 Mg-dependent enzymes (Williams 1993; Cowan 1995). Given its diverse roles in biology, understanding how cells maintain Mg homeostasis is of fundamental importance. Maintaining a consistent Mg concentration in the cytosol and organelles is likely to require tight regulation of passive influx, active efflux, and sequestration mechanisms. Despite the importance of these mechanisms, relatively little is known about the molecular identity, function, and regulation of Mg transporters in eukaryotic cells.The yeast Alr1 and Alr2 proteins were the first eukaryotic Mg transporters identified. ALR1 inactivation conferred Mg-dependent growth and blocked Mg uptake (MacDiarmid and Gardner 1998; Graschopf et al. 2001). The closely related ortholog Alr2 was not essential for growth, but could compensate for the loss of Alr1 when overexpressed and was found to physically associate with Alr1 in vivo (MacDiarmid and Gardner 1998; Wachek et al. 2006). Further studies identified two related proteins in the mitochondrial inner membrane (Mrs2 and Lpe10). Both proteins were required for the entry of Mg into the mitochondrial matrix, and loss-of-function mutations in either gene caused similar reductions in mitochondrial function and Mg content (Bui et al. 1999; Gregan et al. 2001a,b). All four of these proteins are members of the metal ion transporter (MIT) superfamily, the founder member of which is CorA from Salmonella typhimurium (Gardner 2003; Knoop et al. 2005). In general, MIT proteins mediate rapid, membrane-potential-dependent transport, suggesting that they form Mg-selective channels (MacDiarmid and Gardner 1998; Liu et al. 2002; Kolisek et al. 2003; Froschauer et al. 2004; Schindl et al. 2007). Although divergent in primary sequence, typical MIT proteins possess two conserved structural features: a pair of transmembrane domains close to the C terminus and a triad of conserved residues (glycine–methionine–asparagine) that are essential for Mg transport (Knoop et al. 2005). The low number of transmembrane domains predicted to be present in MIT proteins suggested that oligomerization was required for ion transport (Kolisek et al. 2003; Warren et al. 2004), and independent crystallographic studies of a CorA homolog from Thermotoga maritima support this model (Eshaghi et al. 2006; Lunin et al. 2006; Payandeh and Pai 2006). T. maritima CorA formed a homopentamer of subunits in which the C-terminal transmembrane domains clustered together to form a membrane-spanning pore. The N-terminal regions of the subunits formed a cytosolic “funnel” domain that incorporated several apparent Mg-binding sites, suggesting a regulatory role for this domain. Genetic studies provided evidence that the binding of Mg ions to these sites altered the conformation of the complex and decreased channel activity (Payandeh and Pai 2006; Payandeh et al. 2008). The activity of the Mrs2 protein was also shown to be dependent on Mg concentration (Schindl et al. 2007), suggesting that both prokaryotic and eukaryotic MIT proteins can respond directly to the cytosolic or matrix Mg concentration to promote homeostasis.A fifth CorA homolog (YKL064w) is present in the yeast genome, but has not been characterized (MacDiarmid and Gardner 1998). The Ykl064w protein shares the two predicted transmembrane domains and conserved GMN motif characteristic of Mg-transporting members of the MIT family (MacDiarmid and Gardner 1998; Knoop et al. 2005). Phylogenetic analysis revealed that Ykl064w belongs to a subgroup of fungal MIT proteins in which a tryptophan residue replaces the conserved phenylalanine preceding the GMN motif (Knoop et al. 2005). Most sequenced yeast and fungal genomes include at least one Alr1 and one Ykl064w-type ortholog (Knoop et al. 2005). The presence of two discrete groups of fungal CorA proteins suggested that the Ykl064w-related proteins perform a novel function. For this reason, we decided to investigate the role of Ykl064w in ion homeostasis. Here we report that Ykl064w is a vacuolar membrane protein that is required for Mg homeostasis and present evidence implicating this protein in the regulation of intracellular Mg storage.     

MnSOD activity protects mitochondrial morphology of quiescent fibroblasts from age associated abnormalities

Previously, we have shown manganese superoxide dismutase (MnSOD) activity protects quiescent human normal skin fibroblasts (NHFs) from age associated loss in proliferative capacity. The loss in proliferative capacity of aged vs. young quiescent cells is often characterized as the chronological life span, which is clearly distinct from replicative senescence. We investigate the hypothesis that MnSOD activity protects the mitochondrial morphology from age associated damage and preserves the chronological life span of quiescent fibroblasts. Aged quiescent NHFs exhibited abnormalities in mitochondrial morphology including abnormal cristae formation and increased number of vacuoles. These results correlate with the levels of cellular reactive oxygen species (ROS) and mitochondrial morphology in MnSOD homozygous and heterozygous knockout mouse embryonic fibroblasts. The abnormalities in mitochondrial morphology in aged quiescent NHFs cultured in presence of 21% oxygen concentration were more severe than NHFs cultured in 4% oxygen environment. The alteration in mitochondrial morphology was associated with a significant increase in cell population doubling: 54 h in 21% compared to 44 h in 4% oxygen environment. Overexpression of MnSOD decreased ROS levels, and preserved mitochondrial morphology in aged quiescent NHFs. These results demonstrate that MnSOD activity protects mitochondrial morphology and preserves the proliferative capacities of quiescent NHFs from age associated loss.     

The Effectiveness of Teleglaucoma versus In-Patient Examination for Glaucoma Screening: A Systematic Review and Meta-Analysis

BackgroundGlaucoma is the leading cause of irreversible visual impairment in the world affecting 60.5 million people worldwide in 2010, which is expected to increase to approximately 79.6 million by 2020. Therefore, glaucoma screening is important to detect, diagnose, and treat patients at the earlier stages to prevent disease progression and vision loss. Teleglaucoma uses stereoscopic digital imaging to take ocular images, which are transmitted electronically to an ocular specialist. The purpose is to synthesize literature to evaluate teleglaucoma, its diagnostic accuracy, healthcare system benefits, and cost-effectiveness.MethodsA systematic search was conducted to help locate published and unpublished studies. Studies which evaluate teleglaucoma as a screening device for glaucoma were included. A meta-analysis was conducted to provide estimates of diagnostic accuracy, diagnostic odds ratio, and the relative percentage of glaucoma cases detected. The improvements to healthcare service quality and cost data were assessed.ResultsOf 11237 studies reviewed, 45 were included. Our results indicated that, teleglaucoma is more specific and less sensitive than in-person examination. The pooled estimates of sensitivity was 0.832 [95% CI 0.770, 0.881] and specificity was 0.790 [95% CI 0.668, 0.876]. The relative odds of a positive screen test in glaucoma cases are 18.7 times more likely than a negative screen test in a non-glaucoma cases. Additionally, the mean cost for every case of glaucoma detected was $1098.67 US and of teleglaucoma per patient screened was $922.77 US.ConclusionTeleglaucoma can accurately discriminate between screen test results with greater odds for positive cases. It detects more cases of glaucoma than in-person examination. Both patients and the healthcare systems benefit from early detection, reduction in wait and travel times, increased specialist referral rates, and cost savings. Teleglaucoma is an effective screening tool for glaucoma specifically for remote and under-services communities.     

Regulation of Alr1 Mg transporter activity by intracellular magnesium

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni(2+), Mn(2+), Zn(2+) and Co(2+). Using Ni(2+) uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms.