Abstract
Bacillus circulans Jordan 32352 was isolated from decaying organic matter in the New Jersey soil in the early 1930s. This soil-dwelling bacterium produced an enzyme capable of degrading the ...type 3 capsular polysaccharide (Pn3P) of Streptococcus pneumoniae (Spn). Early reports of this enzyme, Pn3Pase, demonstrated its inducibility by, and specificity for Pn3P. We set out to identify and clone this enzyme for its recombinant expression and characterization. We first sequenced the genome of this bacterial species, and reclassified the Pn3Pase producing bacterium as Paenibacillus species 32352. We identified the putative protein of Pn3Pase through mass spectrometry-based proteomics and cloned the gene for recombinant expression. We then characterized the oligosaccharide products generated upon the enzymatic depolymerization of Pn3P. Sequence analysis suggests that this glycoside hydrolase belongs to a new carbohydrate-active enzyme GH family. To our knowledge, this is the only enzyme to demonstrate Pn3P depolymerization activity.
The recovery and repurposing of biomass are critically important and make a significant contribution to environmental preservation. Lignosulfonate, a by-product of the paper manufacturing industry, ...is an abundant low cost material with unique potential as a sulfur precursor for high performance cathode materials. In this work, we develop a practical green method of using lignosulfonate as both the donor (decomposition of sulfonic groups (–SO 3 H)) of sulfur and the sulfur acceptor in lignosulfonate derived activated carbon. Through a circulatory pyrolysis process with carbon activation and sulfur capture, a high surface area carbon/sulfur composite was obtained. This material was successfully developed into a cathode for a lithium–sulfur battery, which demonstrates outstanding cycling stability with a capacity decay rate as low as 0.1% per cycle over 200 cycles. When the sulfur loading was further increased to 68 wt%, the capacity still reaches as high as 1100 mA h g −1 , suggesting its promising potential for applications in the field of high energy storage devices.
Eggshell matrix can be dissociated into three matrix fractions: acid-insoluble matrix (M1), water-insoluble matrix (M2) and acid-water facultative-soluble matrix (M3). Matrix fractions from pimpled ...and normal eggshells were compared using label-free proteomic method to understand the differences among three matrix fractions and the proteins involved with eggshell quality. A total of 738 and 600 proteins were identified in the pimpled and normal calcified eggshells, respectively. Both eggshells showed a combined proteomic inventory of 769 proteins. In the same type of eggshell, a high similarity was present in the proteomes of three matrix fractions. These triply overlapped common proteins formed the predominant contributor to proteomic abundance in the matrix fractions. In each matrix fraction and between both eggshell models, normal and pimpled eggshells, a majority of the proteomes of the fractions were commonly observed. Forty-two common major proteins (iBAQ-derived abundance ≥0.095% of proteomic abundance) were identified throughout the three matrix fractions and these proteins might act as backbone constituents in chicken eggshell matrix. Finally, using 1.75-fold as up-regulated and using 0.57-fold as down-regulated cutoff values, twenty-five differential major proteins were screened and they all negatively influence and none showed any effect on eggshell quality. Overall, we uncovered the characteristics of proteomics of three eggshell matrix fractions and identified candidate proteins influencing eggshell quality. The next research on differential proteins will uncover the potential mechanisms underlying how proteins affect eggshell quality.
It was reported that the proteins in an eggshell can be divided into insoluble and soluble proteins. The insoluble proteins are thought to be an inter-mineral matrix and acts as a structural framework, while the soluble proteins are thought as intra-mineral matrix that are embedded within the crystal during calcification. However, the difference between matrix fractions is unknown. Cross-analysis of proteomic data of three matrix fractions from the same type of eggshell, uncovered triply overlapped common proteins formed the predominant contributor to proteomic abundance of any matrix fraction, and we suggested that abundance variance of some common proteins between the three matrix fractions might be an important cause of their solubility differences. Moreover, eggshell is formed in hen's uterus, and uterus tend to be considered as unique organ determining eggshell quality. By cross-analysis on proteomic data of three matrix fractions between two eggshell models, normal and pimpled eggshells, the differential proteins were screened as candidates influencing eggshell quality. And we suggested that the liver and spleen or lymphocytes might be the major organs influencing eggshell quality, because the most promising candidates are almost blood and non-collagenous proteins, and originated from above organs.
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•A high similarity was present throughout the proteomes of three matrix fractions from the same eggshell.•In each matrix fraction and between normal and pimpled eggshells, a majority of both proteomes were common.•The differential proteins all showed negative influence on eggshell quality, and almost originated from liver and spleen which might be the major organs influencing eggshell quality.
The emerging, multidrug-resistant yeast pathogen Candida auris is responsible for healthcare-associated outbreaks across the globe with high mortality. The rapid spread of C. auris is linked to its ...successful colonization of human skin, followed by bloodstream infections. We compared glycomics and proteomics of C. auris to closely and distantly related human pathogenic yeasts, C. haemulonii and C. albicans, with the aim to understand the role of cell surface molecules in skin colonization and immune system interactions. Candida auris mannan is distinct from other pathogenic Candida species, as it is highly enriched in β-1,2-linkages. The experimental data showed that C. auris surface mannan β-1,2-linkages were important for the interactions with the immune protein IgG, found in blood and in sweat glands, and with the mannose binding lectin, found in the blood. Candida auris mannan binding to IgG was from 12- to 20-fold stronger than mannan from the more common pathogen C. albicans. The findings suggest unique C. auris mannan could be crucial for the biology and pathogenesis of this emerging pathogen.
Pichia pastoris
is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the
P. pastoris
expression system has become ...one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of
SUT2
could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism.
SUT2
encodes a putative transcription factor-like protein harboring a Gal4-like Zn
2
Cys
6
DNA-binding domain in
Pichia pastoris
, and its homolog in
Saccharomyces cerevisiae
regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative
SUT2
promoter at diverse loci, the − 973 base pair (bp) to − 547 bp region to the ATG was shown to be the core element of the inducible promoter
P
SUT2
, which strongly responds to the methanol signal. The transcriptional start site of
SUT2
, “A” at the 22nd bp upstream of ATG, was determined with 5′-rapid amplification of cDNA ends. A forward-loop cassette was constructed with
MXR1
(Methanol Expression Regulator 1, a positive transcription factor of
P
AOX1
) promoted by
P
SUT2
, enabling moderate elevation in the expression level of Mxr1 and high activity of
P
AOX1
without damaging cellular robustness further boosting the production of heterologous proteins. The
P
AOX1
-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis.
P
SUT2
was characterized as a novel inducible promoter in
P. pastoris
and a
P
SUT2
-driven MXR1 forward-loop cassette was constructed to enhance the
P
AOX1
activity, laying a foundation for further development and application of
P. pastoris
expression system.
The sulfo groups of glycosaminoglycans contribute to their high charge densities, and are critical for the role they play in various physiological and pathophysiological processes. Unfortunately, the ...sulfo groups can be hydrolyzed to inorganic sulfate. Thus, it is important to monitor the presence of these sulfo groups. In addition, free anions, including chloride, sulfate and acetate, are often present in glycosaminoglycans as a result of multiple purification steps, and their presence also needs to be monitored. In this report, ion chromatography with conductivity detection is used to analyze the anions present in glycosaminoglycans, including heparin, heparan sulfate, chondroitin sulfate and dermatan sulfate. This method allows quantitation over a wide range of concentrations, affording a limit of quantitation of 0.1 ppm and a limit of detection of 0.05 ppm for most anions of interest. The stability of heparin was also studied, providing data on the formation of both sulfate and acetate anions.
A heparin oligosaccharide having a completely natural structure was successfully synthesized through a chemoenzymatic approach using an unnatural glycosyl acceptor, p-nitrophenyl glucuronide ...(GlcA-pNP). The use of an inexpensive and commercially available GlcA-pNP acceptor facilitates oligosaccharide recovery and purification on C-18 resin during chemoenzymatic synthesis. Oligosaccharide chain extension and modification afforded a heptasaccharide with gluconic acid residues at its reducing and non-reducing ends. Treatment with periodate oxidation followed by Smith degradation or alkaline elimination resulted in the selective cleavage of vicinal diol-containing glucuronic acid residues affording highly sulfated heparin pentasaccharides having a completely natural structure. This methodology should facilitate the chemoenzymatic synthesis of a family of highly sulfated heparin oligosaccharides with unmodified structures for biological evaluation.
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A series of 5,7-dihydroxyflavanone derivatives were efficiently synthesized. Their antimicrobial efficacy on Gram-negative, Gram-positive bacteria and yeast were evaluated. Among ...these compounds, most of the halogenated derivatives exhibited the best antimicrobial activity against Gram-positive bacteria, the yeast Saccharomyces cerevisiae, and the Gram-negative bacterium Vibrio cholerae. The cytotoxicities of these compounds were low as evaluated on HepG2 cells using a cell viability assay. This study suggests that halogenated flavanones might represent promising pharmacological candidates for further drug development.
A high-resolution method for the separation and analysis of disaccharides prepared from heparin and heparan sulfate (HS) using heparin lyases is described. Ultra-performance liquid chromatography in ...a reverse-phase ion-pairing mode efficiently separates eight heparin/HS disaccharides. The disaccharides can then be detected and quantified using electrospray ionization mass spectrometry. This method is particularly useful in the analysis of small amounts of biological samples, including cells, tissues, and biological fluids, because it provides high sensitivity without being subject to interference from proteins, peptides, and other sample impurities.
We developed a three-dimensional (3D) cellular microarray platform for the high-throughput (HT) analysis of human neural stem cell (hNSC) growth and differentiation. The growth of an immortalized ...hNSC line, ReNcell VM, was evaluated on a miniaturized cell culture chip consisting of 60nl spots of cells encapsulated in alginate, and compared to standard 2D well plate culture conditions. Using a live/dead cell viability assay, we demonstrated that the hNSCs are able to expand on-chip, albeit with lower proliferation rates and viabilities than in conventional 2D culture platforms. Using an in-cell, on-chip immunofluorescence assay, which provides quantitative information on cellular levels of proteins involved in neural fate, we demonstrated that ReNcell VM can preserve its multipotent state during on-chip expansion. Moreover, differentiation of the hNSCs into glial progeny was achieved both off- and on-chip six days after growth factor removal, accompanied by a decrease in the neural progenitor markers. The versatility of the platform was further demonstrated by complementing the cell culture chip with a chamber system that allowed us to screen for differential toxicity of small molecules to hNSCs. Using this approach, we showed differential toxicity when evaluating three neurotoxic compounds and one antiproliferative compound, and the null effect of a non-toxic compound at relevant concentrations. Thus, our 3D high-throughput microarray platform may help predict, in vitro, which compounds pose an increased threat to neural development and should therefore be prioritized for further screening and evaluation.
•We employed a new 3D platform for neural stem cell growth and differentiation.•We showed that this microplatform can discern differential small molecule toxicity.•We followed stem cell differentiation using an on-chip immunofluorescence assay.•We studied differential responses of stem cells vs. differentiated cells.
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