Pellet Fraction Research Articles

Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research

BackgroundAerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbiome profiling. Recent studies have adopted shotgun metagenomic sequencing (SMS) to address this issue. However, SMS requires relatively large DNA inputs, which are challenging when studying low biomass air environments, and puts high requirements on air sampling, sample processing and DNA isolation protocols. Previous SMS studies have consequently adopted various mitigation strategies, including long-duration sampling, sample pooling, and whole genome amplification, each associated with some inherent drawbacks/limitations.ResultsHere, we demonstrate a new custom, multi-component DNA isolation method optimized for SMS-based aerosol microbiome research. The method achieves improved DNA yields from filter-collected air samples by isolating DNA from the entire filter extract, and ensures a more comprehensive microbiome representation by combining chemical, enzymatic and mechanical lysis. Benchmarking against two state-of-the-art DNA isolation methods was performed with a mock microbial community and real-world air samples. All methods demonstrated similar performance regarding DNA yield and community representation with the mock community. However, with subway samples, the new method obtained drastically improved DNA yields, while SMS revealed that the new method reported higher diversity. The new method involves intermediate filter extract separation into a pellet and supernatant fraction. Using subway samples, we demonstrate that supernatant inclusion results in improved DNA yields. Furthermore, SMS of pellet and supernatant fractions revealed overall similar taxonomic composition but also identified differences that could bias the microbiome profile, emphasizing the importance of processing the entire filter extract.ConclusionsBy demonstrating and benchmarking a new DNA isolation method optimized for SMS-based aerosol microbiome research with both a mock microbial community and real-world air samples, this study contributes to improved selection, harmonization, and standardization of DNA isolation methods. Our findings highlight the importance of ensuring end-to-end sample integrity and using methods with well-defined performance characteristics. Taken together, the demonstrated performance characteristics suggest the new method could be used to improve the quality of SMS-based aerosol microbiome research in low biomass air environments.

Proteomic Analysis of Whole Saliva in Relation to Dental Caries Resistance

Saliva contains possible biomarkers that are associated with dental caries. The present study aimed to analyse differences in the abundance of proteins in the saliva between caries-positive (CP; N = 15) and caries-free (CF; N = 12) males and to compare differences in the abundance of proteins between two saliva sample fractions (supernatant and pellet). We found 14 differently significantly expressed proteins in the CF group when comparing the supernatant fractions of the CP and CF groups, and three proteins in the pellet fractions had significantly higher expression in the CP group. Our results indicate very specific protein compositions of the saliva in relation to dental caries resistance (the saliva of the CP group mainly contained pellet proteins and the saliva of the CF group mainly contained supernatant proteins). This was the first time that the saliva pellet fraction was analysed in relation to the dental caries status. We detected specific calcium-binding proteins that could have decalcified enamel in the saliva pellet of the CP group. We also observed significantly up-regulated immune proteins in the saliva supernatant of the CF group that could play an important role in the caries prevention. The particular protein compositions of the saliva pellet and supernatant in the groups with different susceptibilities to tooth decay is a promising finding for future research.

Separation of brewer pellets in a vibratory-centrifugal centrifuge

The article reflects the process of dehydration of brewer pellets, as one of the ways to solve the problem of their utilization in order to obtain a highly concentrated feed for farm animals, which is an urgent task. The purpose of the study is to substantiate the interrelation between the technological and physical properties of the brewer pellets during the separation of brewer pellets into dense and liquid fractions in a continuous vibratory-centrifugal centrifuge and its main design and operating parameters. As a result of the research with the analytical method, process and physical properties of brewer pellets are determined when they are interacting with the perforated rotor blades of a vibratory-centrifugal centrifuge. The equations for the yield of the liquid fraction of brewer pellets through the rectilinear and curvilinear rotor blades are obtained, indicating the interrelation between process and physical properties of brewer pellets, which are necessary for determining the rational basic parameters of the proposed vibratory-centrifugal centrifuge.

Application of zinc and calcium acetate to precipitate milk fat globule membrane components from a dairy by-product

There has been a great deal of interest in developing isolated dairy lipid fractions that are rich in phospholipids (PL), due to their health benefits and functional properties. Dairy by-products that contain elevated levels of PL and milk fat globule membrane (MFGM) proteins can be an excellent source for these isolates. The β stream, a by-product of anhydrous milk fat production, is an excellent candidate because it contains a higher concentration of PL than many other dairy by-products. In this study, we investigated an economically feasible processing method to obtain these valuable components from the β stream. The use of zinc acetate and calcium acetate, along with mild heat treatment and pH adjustment, was effective in precipitating PL and proteins into a pellet fraction. With an additional extraction from the pellet using ethanol (90% at 70°C), a PL-enriched lipid fraction was obtained. The effective precipitation conditions were zinc acetate of 25 mM concentration at pH greater than 6.5 at 30°C, and calcium acetate of greater than 75 mM concentration at pH greater than 6.5 at 60°C. With ethanol extraction, PL recovery of 97.7 ± 1.7% from the zinc acetate precipitate and 94.9 ± 3.7% from calcium acetate precipitate were achieved.

Calumenin-1 Interacts with Climp63 to Cooperatively Determine the Luminal Width and Distribution of Endoplasmic Reticulum Sheets.

SummaryThe ER is composed of distinct structures like tubules, matrices, and sheets, all of which are important for its various functions. However, how these distinct ER structures, especially the perinuclear ER sheets, are formed remains unclear. We report here that the ER membrane protein Climp63 and the ER luminal protein calumenin-1 (Calu1) collaboratively maintain ER sheet morphology. We show that the luminal length of Climp63 is positively correlated with the luminal width of ER sheets. Moreover, the lumen-only mutant of Climp63 dominant-negatively narrows the lumen of ER sheets, demonstrating that Climp63 acts as an ER luminal bridge. We also reveal that Calu1 specifically interacts with Climp63 and antagonizes Climp63 in terms of both ER sheet distribution and luminal width. Together, our data provide insight into how the structure of ER sheets is maintained and regulated.

Antioxidant Enzyme-Mimetic Activity and Neuroprotective Effects of Cerium Oxide Nanoparticles Stabilized with Various Ratios of Citric Acid and EDTA.

Cerium oxide (CeO2) nanoparticles (CeNPs) are potent antioxidants that are being explored as potential therapies for diseases in which oxidative stress plays an important pathological role. However, both beneficial and toxic effects of CeNPs have been reported, and the method of synthesis as well as physico-chemical, biological, and environmental factors can impact the ultimate biological effects of CeNPs. In the present study, we explored the effect of different ratios of citric acid (CA) and EDTA (CA/EDTA), which are used as stabilizers during synthesis of CeNPs, on the antioxidant enzyme-mimetic and biological activity of the CeNPs. We separated the CeNPs into supernatant and pellet fractions and used commercially available enzymatic assays to measure the catalase-, superoxide dismutase (SOD)-, and oxidase-mimetic activity of each fraction. We tested the effects of these CeNPs in a mouse hippocampal brain slice model of ischemia to induce oxidative stress where the fluorescence indicator SYTOX green was used to assess cell death. Our results demonstrate that CeNPs stabilized with various ratios of CA/EDTA display different enzyme-mimetic activities. CeNPs with intermediate CA/EDTA stabilization ratios demonstrated greater neuroprotection in ischemic mouse brain slices, and the neuroprotective activity resides in the pellet fraction of the CeNPs. The neuroprotective effects of CeNPs stabilized with equal proportions of CA/EDTA (50/50) were also demonstrated in two other models of ischemia/reperfusion in mice and rats. Thus, CeNPs merit further development as a neuroprotective therapy for use in diseases associated with oxidative stress in the nervous system.

Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-β/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.

Postsynaptic densities fragment into subcomplexes upon sonication

Postsynaptic density (PSD) fractions were isolated from rat forebrain and sonicated. Pellets from sonicated samples examined by electron microscopy revealed particles with an electron density similar to PSDs that appeared to be fragments of PSDs. Immuno-gold labeling confirmed that some of these contained PSD-95 and/or SynGAP. Biochemical analysis of supernatant and pellet fractions from sonicated samples showed almost complete recovery of several major PSD components (SynGAP, PSD-95, Shank3, Homer and Glutamate receptors) in the pellet, while the supernatant contained known contaminants of PSD fractions, such as glial acidic fibrillary protein and neurofilament protein, as well as actin and α-actinin, indicating susceptibility of these cytoskeletal elements to mechanical disruption. Size distributions of particulate material in control and sonicated samples were clearly different, with particles in the 40–90 nm range observed only in sonicated samples. Fragmentation of the PSD into subcomplexes containing major constituents suggests a patchwork structure consisting of weakly bound modules, that can be readily dissociated from each other through mechanical disruption. Modular organization and weak association between modules would endow the PSD with lateral structural flexibility.

A novel population of extracellular vesicles smaller than exosomes promotes cell proliferation

BackgroundExtracellular vesicles (EVs) play important roles in intercellular communication by delivering RNA, lipid, and proteins to neighboring or distant cells. Identification and classification of EVs secreted from diverse cell types are essential for understanding their signaling properties.MethodsIn this study, EVs from the culture media were isolated by ultracentrifugation and analyzed by electron microscopy (EM) and nanoparticle tracking analyses. Conditioned media (CM) from HEK293 cells culture grown either in serum-free (SF) or 10% fetal bovine serum (FBS) containing media were centrifuged at 100,000×g to separate the SNΔ supernatant and the P100 pellet in which exosomes are enriched. Then, the SNΔ fraction was centrifuged at 200,000×g to yield the P200 pellet fraction containing novel EVs smaller than exosomes. The exosomal markers in the EV subgroups were examined by western blotting and immune-EM, and the functional analyses of EVs were conducted on HEK293 and THP-1 cell culture.ResultsWe identified a new group of EVs in the P200 fraction that was smaller than exosomes in size. Typical exosome markers such as Hsp70, TSG101, and CD63 were found in both P100 exosomes and the P200 vesicles, but CD81 was highly enriched in exosomes but not in the P200 vesicles. Furthermore, chemicals that inhibit the major exosome production pathway did not decrease the level of P200 vesicles. Therefore, these small EVs indeed belong to a distinguished group of EVs. Exosomes and the P200 vesicles were found in CM of human cell lines as well as FBS. Addition of the exosomes and the P200 vesicles to human cell cultures enhanced exosome production and cell proliferation, respectively.ConclusionsOur study identifies a novel population of EVs present in the P200 fraction. This EV population is distinguished from exosomes in size, protein contents, and biogenesis pathway. Furthermore, exosomes promote their own production whereas the P200 vesicles support cell proliferation. In sum, we report a new group of EVs that are distinct physically, biologically and functionally from exosomes.

Effects of 1% Mouse Serum on the Catalase‐ and Superoxide Dismutase‐Mimetic Activity of Custom‐Synthesized Cerium Oxide Nanoparticles

Due to their potent antioxidant activity, cerium oxide nanoparticles (CeNPs) are being investigated as a potential therapeutic for neurodegenerative diseases. CeNPs are small, regenerative and display both catalase‐ and superoxide dismutase‐mimetic activity. However, toxic effects of CeNPs in biological systems have also been reported. Our working hypothesis is that the synthetic and/or biological identity of CeNPs could account for these disparate reports. In particular, the biological identity of CeNPs can be influenced by serum proteins that adsorb onto the nanoparticle surface. This can then impact the catalytic activity as well as pharmacokinetics properties of CeNPs, potentially contributing to toxic effects in an organism. The aim of the present study was to investigate the effects of serum on the catalytic activity of CeNPs. Custom‐synthesized CeNPs were incubated with or without 1% mouse serum for 30 minutes. CeNPs were then spun in an ultracentrifuge at 434, 513 x g for one hour in order to separate the supernatant (SUP) and pellet (PEL) fractions. We used commercially available enzymatic assay kits to assess the catalase‐ and superoxide dismutase (SOD)‐mimetic activity of each fraction. We also assayed oxidase activity, which could be indicative of promiscuous toxic activity. We observed that pre‐incubation with 1% serum did not significantly impact the catalase‐ or SOD‐mimetic activity of the CeNPs. On the other hand, serum pre‐incubation resulted in significantly enhanced oxidase activity that, upon further analysis, was revealed to be an artifact of the serum rather than attributable to promiscuous CeNP activity. In summary, we demonstrate that pre‐incubation with serum does not negatively affect the antioxidant activity of CeNPs, an important step in continuing to develop CeNPs as neuroprotective agents in oxidative stress diseases.Support or Funding InformationSupported by St. Lawrence University and Cerion, LLC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modeling the Impact of Zooplankton Diel Vertical Migration on the Carbon Export Flux of the Biological Pump

One pathway of the biological pump that remains largely unquantified in many export models is the active transport of carbon from the surface ocean to the mesopelagic by zooplankton diel vertical migration (DVM). Here, we develop a simple representation of zooplankton DVM and implement it in a global export model as a thought experiment to illustrate the effects of DVM on carbon export and mesopelagic biogeochemistry. The model is driven by diagnostic satellite measurements of net primary production, algal biomass, and phytoplankton size structure. Due to constraints on available satellite data, the results are restricted to the latitude range from 60°N to 60°S. The modeled global export flux from the base of the euphotic zone was 6.5 PgC/year, which represents a 14% increase over the export flux in model runs without DVM. The mean (± standard deviation, SD) proportional contribution of the DVM‐mediated export flux to total carbon export, averaged over the global domain and the climatological seasonal cycle, was 0.16 ± 0.04 and the proportional contribution of DVM activity to total respiration within the twilight zone was 0.16 ± 0.06. Adding DVM activity to the model also resulted in a deep local maximum in the oxygen utilization profile. The model results were most sensitive to the assumptions for the fraction of individuals participating in DVM, the fraction of fecal pellets produced in the euphotic zone, and the fraction of grazed carbon that is metabolized.

Irreversibility of arsenic trioxide induced PML/RARα fusion protein solubility changes.

Arsenic trioxide (As2O3) is one of the most effective drugs for the treatment of acute promyelocytic leukemia (APL), and induces the degradation of chimeric oncoprotein PML/RARα (P/R) and APL cell differentiation. Recent evidence has suggested that P/R fusion protein degradation by arsenic occurs through two steps, namely, rapid solubility change/shift of the P/R fusion protein following arsenic treatment (i.e., transfer of P/R protein from the soluble fraction to the insoluble pellet fraction), and subsequent degradation of these insoluble proteins. However, there is little information regarding the reversibility of arsenic induced P/R fusion protein solubility change as well as protein degradation in the insoluble fraction after removing arsenic. In this study, we used APL cell line NB4 or P/R and PML over-expressed 293T cells as well as HeLa cells to reveal the solubility change of P/R and PML by arsenic exposure, and further determined the fate of these insoluble proteins after the removal of arsenic. Here, for the first time, we found that arsenic induced P/R or PML protein solubility change is an irreversible process. Once arsenic induces a P/R or PML protein solubility change, these insoluble proteins could be degraded by the proteasomal pathway even without continuous arsenic treatment. However, PML and P/R proteins can be newly synthesized after the removal of arsenic, suggesting that great caution should be taken in the clinical therapy of APL patients before ending arsenic treatment.

ANTISICKLING ACTIVITY EVALUATION OF FRACTIONS OBTAINED FROM WHOLE EXTRACTS OF NEWBOULDIA LAEVIS P. BEAUV (BIGNONIACEAE)

Objective: This study aims to evaluate the fractions from whole extracts of roots and stem barks of Newbouldia laevis and their effect on sickling.
 Methods: Hydroethanolic extracts of stem barks and roots of Newbouldia laevis were fractionated by the technics of cold precipitation in ethanol. The fractions obtained after phytochemical screening were subjected to Emmel test to evaluate their anti-sickling activity. Active fractions were tested for DPPH and AAPH assay (AAPH induced membrane lipoperoxidation and evaluation of reduction of hemolysis).
 Results: Two fractions were obtained from each whole extract: supernatant and pellet fractions. Supernatants fractions obtained from whole roots barks extract and stem barks extract at a concentration of 30 mg/ml reduced sickling up respectively to 7% and 10% against 86% for the control. Pellets fractions obtained from the both extracts induced coagulation of SS blood at 30 mg/ml against 86% for the control.
 Conclusion: Supernatants fractions of hydroethanolic whole extract of roots and stem backs of Newbouldia laevis promise as the potential source of active molecules against sickle cell disease.

Mass spectrometric discrimination of phospholipid patterns in cisplatin-resistant and -sensitive cancer cells.

Development of therapy-resistant cancer is a major problem in clinical oncology, and there is an urgent need for novel markers identifying development of the resistant phenotype. Lipidomics represents a promising approach to discriminate lipid profiles of malignant phenotype cells. Alterations in phospholipid distribution or chemical composition have been reported in various pathologies including cancer. Here we were curious whether quantitative differences in phospholipid composition between cisplatin-resistant and -sensitive model cancer cell lines could be revealed by mass spectrometric means. The phospholipid contents of cell membranes of the cancer cell lines CCRF-CEM and A2780, both responsive and resistant to cisplatin, were analyzed by solid-phase extraction (SPE) and electrospray ionization mass spectrometry (ESI-MS and tandem mass spectrometry (MS/MS)). Extracts were obtained by disruption of cells with a dounce tissue grinder set followed by centrifugation. To minimize the enzymatic activity, phospholipids were extracted from cell extracts by SPE immediately after the cell lysis and analyzed by MS. Both supernatant and pellet fractions of cell extracts were analyzed. A phospholipid profile specific for cell lines and their phenotypes was revealed. We have documented by quantitative analysis that phosphocholines PC P-34:0, PC 34:1, PC 20:2_16:0, LPC 18:1 and LPC 16:0 PLs were present in the 200-400 μM concentration range in CCRF-CEM cisplatin-responsive cells, but absent in their cisplatin-resistant cells. Similarly, PC 34:1, LPC 18:1 and LPC 16:0 were increased in cisplatin-responsive A2780 cells, and PC 20:2_16:0 was downregulated in cisplatin-resistant A2780 cells. In this work we showed that the ESI-MS analysis of the lipid content of the therapy-resistant and -sensitive cells can clearly distinguish the phenotypic pattern and determine the potential tumor response to cytotoxic therapy. Lipid entities revealed by mass spectrometry and associated with development of therapy resistance can thus support molecular diagnosis and provide a potential complementary cancer biomarker.

Novel DRAQ5™/SYTOX® Blue Based Flow Cytometric Strategy to Identify and Characterize Stem Cells in Human Breast Milk.

Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples. Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5™ and SYTOX® blue dye as well as antibodies to characterize cells within the pellet fraction. Flow cytometry analysis identified viable DRAQ5™+ /SYTOX® Blue- cells and determined the content of CD11b+ monocytes and TRA-1-81+ putative stem cells in human breast milk samples. Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content. © 2018 International Clinical Cytometry Society.

Real-time measurement of particle volume fraction in centrifugal fields by wireless electrical resistance detector

A wireless electrical resistance detector (WERD) has been developed to measure the real-time particle volume fraction in centrifugal fields. The developed WERD is composed of an AC (alternating current) generation device, a measurement circuit and a wireless transmission module. The effective sensing area of measurement electrodes of WERD was clarified by the three dimensional electrostatic field simulations. In order to evaluate the measurement accuracy, the developed WERD is attached around a lab-scale centrifuge, and the real-time volume fraction of polymer pellets in the centrifugal field is measured at various positions zm under various rotational velocity ω and initial particle loading height hp. The measured particle volume fraction αp at various measurement positions indicates a parabolic separation interface between the particle phase and the sodium chloride solution phase, which are in good agreement with the numerical simulation by Euler-Euler method for validation with an average deviation of 2.47%. As the rotational velocity ω is increased, particles are pushed outward to be raised along the cylindrical vessel because of the increased centrifugal force, which causes sharper paraboloid interface. Moreover, as the initial particle loading height hp is increased, the parabolic phase interface shifts upward while the parabolic surface profiles of the phase boundary almost keep constant for the same rotational velocity ω.

Neuronal serine racemase associates with Disrupted-In-Schizophrenia-1 and DISC1 agglomerates: Implications for schizophrenia

D-Serine, an endogenous coagonist of N-methyl-d-aspartate receptors (NMDARs) at the glycine binding site, is synthesized by serine racemase (SR) through conversion of l-Serine. Dysregulation of SR/D-Serine and Disrupted-In-Schizophrenia-1 (DISC1) contributes to the pathogenesis of schizophrenia at converging pathways, as perturbation of SR-DISC1 binding in astrocytes elicits schizophrenia-like behaviors in mice. However, an association of neuronal SR with DISC1 remains elusive. Here we report that SR associates with DISC1 and its agglomerates in cortical neurons, which can be modulated by NMDAR activity. Endogenous SR colocalizes with DISC1 large agglomerates in the soma and with smaller puncta in the nucleus and dendrites of cortical neurons. Co-immunoprecipitation assays demonstrate SR interaction with DISC1 in cortical neuronal lysates, suggesting the physiological presence of functional SR-DISC1 complexes in neurons. Moreover, exogenous d-Serine application significantly increases the interaction of SR with DISC1, the number of DISC1-SR large agglomerates and the levels of DISC1 agglomerated form along with SR in the triton-insoluble pellet fraction, whereas application of glycine with a glycine transporter inhibitor fails to increase their interactions, abundance of DISC1-SR large agglomerates and levels of DISC1 agglomerated form. This increase by d-Serine application is blocked by 7-chlorokynurenic acid, a specific antagonist at the glycine site of NMDARs, suggesting mediation through NMDARs. Our findings thus demonstrate neuronal SR association with DISC1 and its agglomerates, which can be modulated by d-Serine, thereby validating a novel neuronal SR-DISC1 complex responsive to NMDAR activation and providing a molecular mechanism by which pathways implicated in schizophrenia converge.

Autophagosome immunoisolation from GFP-LC3B mouse tissue

ABSTRACTWe describe a protocol for rapid and efficient enrichment of autophagosomes from various tissues of the GFP-LC3 mouse. In order to increase the number of autophagosomes, we block autophagy flux in the GFP-LC3 mouse tissue with a single intraperitoneal injection of leupeptin 4–5 h before tissue harvesting. We homogenize dissected tissue samples using a Dounce homogenizer followed by passing the slurry through needles of different sizes to dissociate the cells and disrupt their outer membranes. The post-nuclear supernatant fraction of the cell lysate is further centrifuged and the supernatant fraction is discarded to remove residual cytosolic GFP-LC3 that is not associated with autophagosomes. The pellet fraction is resuspended and incubated with magnetic microbeads coated with anti-GFP antibodies for 1 h on ice. The lysate-bead mixture is then applied to a column that is placed in a magnetic separator. After washes, the autophagosome fraction is eluted from the column for morphological and protein analysis.Abbreviations: EDTA: ethylenediaminetetraacetic acid; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LC3: microtubule-associated protein 1 light chain 3 beta; MES: 4-morpholineethanesulfonic acid (MES); SQSTM1: sequestosome 1; TEM: transmission electron microscopy

Identification of an apiosyltransferase in the plant pathogen Xanthomonas pisi.

The rare branched-chain sugar apiose, once thought to only be present in the plant kingdom, was found in two bacterial species: Geminicoccus roseus and Xanthomonas pisi. Glycans with apiose residues were detected in aqueous methanol-soluble fractions as well as in the insoluble pellet fraction of X. pisi. Genes encoding bacterial uridine diphosphate apiose (UDP-apiose) synthases (bUASs) were characterized in these bacterial species, but the enzyme(s) involved in the incorporation of the apiose into glycans remained unknown. In the X. pisi genome two genes flanking the XpUAS were annotated as hypothetical glycosyltransferase (GT) proteins. The first GT (here on named XpApiT) belongs to GT family 90 and has a Leloir type B fold and a putative lipopolysaccharide-modifying (LPS) domain. The second GT (here on XpXylT) belongs to GT family 2 and has a type A fold. The XpXylT and XpApiT genes were cloned and heterologously expressed in E. coli. Analysis of nucleotide sugar extracts from E. coli expressing XpXylT or XpApiT with UAS showed that recombinant XpApiT utilized UDP-apiose and XpXylT utilized UDP-xylose as substrate. Indirect activity assay (UDP-Glo) revealed that XpApiT is an apiosyltransferase (ApiT) able to specifically use UDP-apiose. Further support for the apiosyltransferase activity was demonstrated by in microbe co-expression of UAS and XpApiT in E. coli showing the utilization of UDP-apiose to generate an apioside detectable in the pellet fraction. This work provides evidence that X. pisi developed the ability to synthesize an apioside of indeterminate function; however, the evolution of the bacterial ApiT remains to be determined. From genetic and evolutionary perspectives, the apiose operon may provide a unique opportunity to examine how genomic changes reflect ecological adaptation during the divergence of a bacterial group.

A gravitational approach to modeling the representative volume geometry of particle-reinforced metal matrix composites

Computational models of representative volumes of metal matrix composites are crucial for investigating material behavior on the microscale. Numerical simulations often employ finite element models of representative volumes. Such models are based on observations of material microstructural properties, for example, by means of electron microscopy. Constructing a geometrical model of a representative volume for further computations can be a tedious task. This paper presents a new approach to creating geometrical models of this kind. The approach is based on the simulation of rigid body motion in a gravitational field and allows one to automatically generate geometrical models of representative volumes. The approach capabilities are exemplified by two geometrical models of representative volume. One model describes a metal matrix composite with high volume fraction of prismatic reinforcement particles, produced by liquid metal infiltration. The other model describes a metal matrix composite with high volume fraction of metal pellets, produced by powder metallurgy.