Methanol Oxidation Genes in the Marine Methanotroph Methylomonas sp. Strain A4

Methanol dehydrogenase has been purified from the type I marine methanotroph Methylomonas sp. strain A4 and found to be similar to other methanol dehydrogenase enzymes in subunit composition, molecular mass, and N-terminal sequence of the two subunits. A heterologous gene probe and a homologous oligonucleotide have been used to identify a DNA fragment from Methylomonas sp. strain A4 which contains moxF, the gene encoding the large subunit of methanol dehydrogenase. Protein expression experiments with Escherichia coli, immunoblotting of expression extracts, and partial DNA sequence determination have confirmed the presence of moxF on this DNA fragment. In addition, expression and immunoblot experiments have shown the presence of the genes for the small subunit of methanol dehydrogenase (moxI) and for the methanol dehydrogenase-specific cytochrome c (moxG). The moxG gene product has been shown to be cytochrome c552. The expression experiments have also shown that two other genes are present on this DNA fragment, and our evidence suggests that these are the homologs of moxJ and moxR, whose functions are unknown. Our data suggest that the order of these genes in Methylomonas sp. strain A4 is moxFJGIR, the same as in the facultative methylotrophs. The transcriptional start site for moxF was mapped. The sequence 5' to the transcriptional start does not resemble other promoter sequences, including the putative moxF promoter sequence of facultative methylotrophs. These results suggest that although the order of these genes and the N-terminal amino acid sequence of MoxF and MoxI are conserved between distantly related methylotrophs, the promoters for this gene cluster differ substantially

The chaperone protein clusterin may serve as a cerebrospinal fluid biomarker for chronic spinal cord disorders in the dog

Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent condition such as cIVDD

Practical, Microfabrication-Free Device for Single-Cell Isolation

Microfabricated devices have great potential in cell-level studies, but are not easily accessible for the broad biology community. This paper introduces the Microscale Oil-Covered Cell Array (MOCCA) as a low-cost device for high throughput single-cell analysis that can be easily produced by researchers without microengineering knowledge. Instead of using microfabricated structures to capture cells, MOCCA isolates cells in discrete aqueous droplets that are separated by oil on patterned hydrophilic areas across a relatively more hydrophobic substrate. The number of randomly seeded Escherichia coli bacteria in each discrete droplet approaches single-cell levels. The cell distribution on MOCCA is well-fit with Poisson distribution. In this pioneer study, we created an array of 900-picoliter droplets. The total time needed to seed cells in ∼3000 droplets was less than 10 minutes. Compared to traditional microfabrication techniques, MOCCA dramatically lowers the cost of microscale cell arrays, yet enhances the fabrication and operational efficiency for single-cell analysis

Modern microwave methods in solid state inorganic materials chemistry: from fundamentals to manufacturing

No abstract available

HIV-1 Drug Resistance Emergence among Breastfeeding Infants Born to HIV-Infected Mothers during a Single-Arm Trial of Triple-Antiretroviral Prophylaxis for Prevention of Mother-To-Child Transmission: A Secondary Analysis

Analysis of a substudy of the Kisumu breastfeeding trial by Clement Zeh and colleagues reveals the emergence of HIV drug resistance in HIV-positive infants born to HIV-infected mothers treated with antiretroviral drugs

Force spectroscopy in studying infection

Biophysical force spectroscopy tools - for example optical tweezers, magnetic tweezers, atomic force microscopy, - have been used to study elastic, mechanical, conformational and dynamic properties of single biological specimens from single proteins to whole cells to reveal information not accessible by ensemble average methods such as X-ray crystallography, mass spectroscopy, gel electrophoresis and so on. Here we review the application of these tools on a range of infection-related questions from antibody-inhibited protein processivity to virus-cell adhesion. In each case we focus on how the instrumental design tailored to the biological system in question translates into the functionality suitable for that particular study. The unique insights that force spectroscopy has gained to complement knowledge learned through population averaging techniques in interrogating biomolecular details prove to be instrumental in therapeutic innovations such as those in structure-based drug design

Facultative methanotrophy: false leads, true results, and suggestions for future research

Methanotrophs are a group of phylogenetically diverse microorganisms characterized by their ability to utilize methane as their sole source of carbon and energy. Early studies suggested that growth on methane could be stimulated with the addition of some small organic acids, but initial efforts to find facultative methanotrophs, i.e., methanotrophs able to utilize compounds with carbon–carbon bonds as sole growth substrates were inconclusive. Recently, however, facultative methanotrophs in the genera Methylocella , Methylocapsa , and Methylocystis have been reported that can grow on acetate, as well as on larger organic acids or ethanol for some species. All identified facultative methanotrophs group within the Alphaproteobacteria and utilize the serine cycle for carbon assimilation from formaldehyde. It is possible that facultative methanotrophs are able to convert acetate into intermediates of the serine cycle (e.g. malate and glyoxylate), because a variety of acetate assimilation pathways convert acetate into these compounds (e.g. the glyoxylate shunt of the tricarboxylic acid cycle, the ethylmalonyl‐CoA pathway, the citramalate cycle, and the methylaspartate cycle). In this review, we summarize the history of facultative methanotrophy, describe scenarios for the basis of facultative methanotrophy, and pose several topics for future research in this area.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86841/1/fml2315.pd

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. Methodology/Principal Findings The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).Conclusion/Significance These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.Organismic and Evolutionary Biolog