Comparative genomics reveals adaptation by Alteromonas sp. SN2 to marine tidal-flat conditions: cold tolerance and aromatic hydrocarbon metabolism

Alteromonas species are globally distributed copiotrophic bacteria in marine habitats. Among these, sea-tidal flats are distinctive: undergoing seasonal temperature and oxygen-tension changes, plus periodic exposure to petroleum hydrocarbons. Strain SN2 of the genus Alteromonas was isolated from hydrocarbon-contaminated sea-tidal flat sediment and has been shown to metabolize aromatic hydrocarbons there. Strain SN2's genomic features were analyzed bioinformatically and compared to those of Alteromonas macleodii ecotypes: AltDE and ATCC 27126. Strain SN2's genome differs from that of the other two strains in: size, average nucleotide identity value, tRNA genes, noncoding RNAs, dioxygenase gene content, signal transduction genes, and the degree to which genes collected during the Global Ocean Sampling project are represented. Patterns in genetic characteristics (e.g., GC content, GC skew, Karlin signature, CRISPR gene homology) indicate that strain SN2's genome architecture has been altered via horizontal gene transfer (HGT). Experiments proved that strain SN2 was far more cold tolerant, especially at 5°C, than the other two strains. Consistent with the HGT hypothesis, a total of 15 genomic islands in strain SN2 likely confer ecological fitness traits (especially membrane transport, aromatic hydrocarbon metabolism, and fatty acid biosynthesis) specific to the adaptation of strain SN2 to its seasonally cold sea-tidal flat habitat.     

Palmitate promotes the paracrine effects of macrophages on vascular smooth muscle cells: the role of bone morphogenetic proteins

Saturated fatty acids are known to activate macrophages and induce vascular inflammation. Although cytokines from activated macrophage influence other vascular cells, the influence of saturated fatty acids on the paracrine effect of macrophages is not fully understood yet. Here we examined the impact of palmitate on the effect of macrophages on vascular smooth muscle cells (SMCs) and their mediators. SMCs proliferation increased significantly after treatment with conditioned media from palmitate-stimulated RAW264.7 cells. SMC migration was found to be greater after treatment with palmitate-conditioned media. SM α-actin and SM22α were decreased in SMCs treated with palmitate-conditioned media. When stimulated with palmitate, RAW264.7 cells secreted more bone morphogenetic protein (BMP)2 and BMP4 into the cell culture media. SMC proliferation, migration, and phenotypic changes were attenuated after treatment of neutralizing antibodies against BMPs or knockdown of BMPs with siRNA. The influences of these proteins were further confirmed by direct treatment of recombinant BMP2 and BMP4 on SMCs. Particularly, the effects of BMPs on SMC migration on phenotypic change were obvious, whereas their effect on SMC proliferation seemed not significant or modest. In conclusion, palmitate promoted macrophages' paracrine effects on SMC proliferation, migration, and phenotypic change. The effect of stimulated macrophages was mediated, at least in part, by BMP2 and BMP4. These results suggest a novel mechanism linking saturated fatty acids and the progression of vascular diseases that is possibly mediated by BMPs from macrophages.     

Raman spectroscopy provides a powerful diagnostic tool for accurate determination of albumin glycation

We present the first demonstration of glycated albumin detection and quantification using Raman spectroscopy without the addition of reagents. Glycated albumin is an important marker for monitoring the long-term glycemic history of diabetics, especially as its concentrations, in contrast to glycated hemoglobin levels, are unaffected by changes in erythrocyte life times. Clinically, glycated albumin concentrations show a strong correlation with the development of serious diabetes complications including nephropathy and retinopathy. In this article, we propose and evaluate the efficacy of Raman spectroscopy for determination of this important analyte. By utilizing the pre-concentration obtained through drop-coating deposition, we show that glycation of albumin leads to subtle, but consistent, changes in vibrational features, which with the help of multivariate classification techniques can be used to discriminate glycated albumin from the unglycated variant with 100% accuracy. Moreover, we demonstrate that the calibration model developed on the glycated albumin spectral dataset shows high predictive power, even at substantially lower concentrations than those typically encountered in clinical practice. In fact, the limit of detection for glycated albumin measurements is calculated to be approximately four times lower than its minimum physiological concentration. Importantly, in relation to the existing detection methods for glycated albumin, the proposed method is also completely reagent-free, requires barely any sample preparation and has the potential for simultaneous determination of glycated hemoglobin levels as well. Given these key advantages, we believe that the proposed approach can provide a uniquely powerful tool for quantification of glycation status of proteins in biopharmaceutical development as well as for glycemic marker determination in routine clinical diagnostics in the future.     

Portable optical fiber probe-based spectroscopic scanner for rapid cancer diagnosis: a new tool for intraoperative margin assessment

There continues to be a significant clinical need for rapid and reliable intraoperative margin assessment during cancer surgery. Here we describe a portable, quantitative, optical fiber probe-based, spectroscopic tissue scanner designed for intraoperative diagnostic imaging of surgical margins, which we tested in a proof of concept study in human tissue for breast cancer diagnosis. The tissue scanner combines both diffuse reflectance spectroscopy (DRS) and intrinsic fluorescence spectroscopy (IFS), and has hyperspectral imaging capability, acquiring full DRS and IFS spectra for each scanned image pixel. Modeling of the DRS and IFS spectra yields quantitative parameters that reflect the metabolic, biochemical and morphological state of tissue, which are translated into disease diagnosis. The tissue scanner has high spatial resolution (0.25 mm) over a wide field of view (10 cm × 10 cm), and both high spectral resolution (2 nm) and high spectral contrast, readily distinguishing tissues with widely varying optical properties (bone, skeletal muscle, fat and connective tissue). Tissue-simulating phantom experiments confirm that the tissue scanner can quantitatively measure spectral parameters, such as hemoglobin concentration, in a physiologically relevant range with a high degree of accuracy (<5% error). Finally, studies using human breast tissues showed that the tissue scanner can detect small foci of breast cancer in a background of normal breast tissue. This tissue scanner is simpler in design, images a larger field of view at higher resolution and provides a more physically meaningful tissue diagnosis than other spectroscopic imaging systems currently reported in literatures. We believe this spectroscopic tissue scanner can provide real-time, comprehensive diagnostic imaging of surgical margins in excised tissues, overcoming the sampling limitation in current histopathology margin assessment. As such it is a significant step in the development of a platform technology for intraoperative management of cancer, a clinical problem that has been inadequately addressed to date.     

HDAC6 Inhibitor Blocks Amyloid Beta-Induced Impairment of Mitochondrial Transport in Hippocampal Neurons

Even though the disruption of axonal transport is an important pathophysiological factor in neurodegenerative diseases including Alzheimer's disease (AD), the relationship between disruption of axonal transport and pathogenesis of AD is poorly understood. Considering that α-tubulin acetylation is an important factor in axonal transport and that Aβ impairs mitochondrial axonal transport, we manipulated the level of α-tubulin acetylation in hippocampal neurons with Aβ cultured in a microfluidic system and examined its effect on mitochondrial axonal transport. We found that inhibiting histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, significantly restored the velocity and motility of the mitochondria in both anterograde and retrograde axonal transports, which would be otherwise compromised by Aβ. The inhibition of HDAC6 also recovered the length of the mitochondria that had been shortened by Aβ to a normal level. These results suggest that the inhibition of HDAC6 significantly rescues hippocampal neurons from Aβ-induced impairment of mitochondrial axonal transport as well as mitochondrial length. The results presented in this paper identify HDAC6 as an important regulator of mitochondrial transport as well as elongation and, thus, a potential target whose pharmacological inhibition contributes to improving mitochondrial dynamics in Aβ treated neurons.     

Production of functional agarolytic nano-complex for the synergistic hydrolysis of marine biomass and its potential application in carbohydrate-binding module-utilizing one-step purification

Marine algal polysaccharides are promising alternative resources to terrestrial biomass. Agarolytic enzymes degrade agarose to various kinds of oligosaccharides. In this study, we expressed miniCbpA, a recombinant scaffolding protein from Clostridium cellulovorans, in Escherichia coli. To assemble the agarolytic complex via cohesin–dockerin interaction, we constructed a chimeric agarase cAgaB from Zobellia galactanivorans containing the catalytic domain of AgaB fused with a dockerin domain from C. cellulovorans EngB. The assembly of functional agarolytic complexes increased the activity against the agar substrate approximately 1.4-fold compared with that for the corresponding enzymes alone. The carbohydrate-binding module (CBM) of miniCbpA was used as a tag for CBM-utilizing one-step purification using cellulose as a support. This is the first report on the formation of agarolytic complexes using the cohesin–dockerin interaction system. The assembly of agar-degrading complexes will lead to the commercial production of useful products from agar biomass at low costs.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

Identification of the first archaeal arylsulfatase from Pyrococcus furiosus and its application to desulfatation of agar

A gene encoding a putative arylsulfatase from the hyperthermophilic archaeon Pyrococcus furiosus was identified, cloned, and expressed as a fusion protein with a Sce VMA intein and chitin binding domain (CBD) residue. The gene (PF1345) from P. furiosus encoding a 35 kDa protein showed some similarity (17 ～ 19%) with other arylsulfatases from the bacteria. The recombinant fusion arylsulfatase was overexpressed in E. coli and partially purified. Its molecular mass was estimated to be 90 kDa by SDS-PAGE. The optimal temperature and pH for arylsulfatase activity were found to be 45°C and 9.5, respectively. Various divalent cations (Ca²⁺, Mg²⁺, Co²⁺, Cu²⁺, Zn²⁺, and Mn²⁺) slightly activated the arylsulfatase activity in a narrow range of concentrations (below 0.5 mM), whereas Zn²⁺ concentrations above 2.0 mM significantly inhibited the activity. After the reaction of agar with recombinant fusion arylsulfatase for 12 h at 50°C, 75% of the sulfate in the agar was removed, and the DNA migration was greatly enhanced. Therefore, the arylsulfatase in this study could be applicable for the production of electrophoretic grade agarose by removing sulfate groups in agar.     

Characterization, metabolites and gas formation of fumarate reducing bacteria isolated from Korean native goat (Capra hircus coreanae)

Fumarate reducing bacteria, able to convert fumarate to succinate, are possible to use for the methane reduction in rumen because they can compete for H₂ with methanogens. In this, we isolated fumarate reducing bacteria from a rumen of Korean native goat and characterized their molecular properties [fumarate reductase A gene (frdA)], fumarate reductase activities, and productions of volatile fatty acids and gas. Eight fumarate reducing bacteria belonging to Firmicutes were isolated from rumen fluid samples of slaughtered Korean black goats and characterized their phylogenetic positions based on 16S rRNA gene sequences. PCR based analyses showed that only one strain, closely related to Mitsuokella jalaludinii, harbored frdA. The growths of M. jalaludinii and Veillonella parvula strains were tested for different media. Interestingly, M. jalaludinii grew very well in the presence of hydrogen alone, while V. parvula grew well in response of fumarate and fumarate plus hydrogen. M. jalaludinii produced higher levels of lactate (P≤0.05) than did V. parvula. Additionally, M. jalaludinii produced acetate, but not butyrate, whereas V. parvula produced butyrate, not acetate. The fumarate reductase activities of M. jalaludinii and V. parvula were 16.8 ± 0.34 and 16.9 ± 1.21 mmol NADH oxidized/min/mg of cellular N, respectively. In conclusion, this showed that M. jalaludinii can be used as an efficient methane reducing agent in rumen.