Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Preparation of electrospun core–sheath yarn with enhanced bioproperties for biomedical materials

Objectives

To create a multifunctional medical material that combines the advantages of both nanofibers and macroyarns.

Results

A novel electrospinning-based approach was developed for creating polycaprolactone (PCL) nanofiber covered yarns (PCL-NCYs) in which polyglycolic acid multi-strand filaments (PGA-MFs) were used as the core. BALB/3T3 (mouse embryonic fibroblast cell line) cells were cultured on the PCL-NCYs substrate and cell morphology and proliferation were determined by methylthiazol tetrazolium (MTT) assay. Compared with PGA-MFs, PCL-NCYs had a higher porosity and tensile strength of 88 ± 8% and 348 ± 16 MPa and in particular, the porosity was four times higher. BALB/3T3 cells attached more easily onto the nanofiber structure and proliferated along the direction of nanofibers, indicating that PCL-NCYs can achieve better cell differentiation and proliferation.

Conclusions

PCL-NCYs can be created by combining electrospinning covering and textile twisting, and have better mechanical property and higher porosity, and can be used as a novel scaffold in tissue engineering.

     

Maximizing power generation from dark fermentation effluents in microbial fuel cell by selective enrichment of exoelectrogens and optimization of anodic operational parameters

Objective

To selectively enrich an electrogenic mixed consortium capable of utilizing dark fermentative effluents as substrates in microbial fuel cells and to further enhance the power outputs by optimization of influential anodic operational parameters.

Results

A maximum power density of 1.4 W/m³ was obtained by an enriched mixed electrogenic consortium in microbial fuel cells using acetate as substrate. This was further increased to 5.43 W/m³ by optimization of influential anodic parameters. By utilizing dark fermentative effluents as substrates, the maximum power densities ranged from 5.2 to 6.2 W/m³ with an average COD removal efficiency of 75% and a columbic efficiency of 10.6%.

Conclusion

A simple strategy is provided for selective enrichment of electrogenic bacteria that can be used in microbial fuel cells for generating power from various dark fermentative effluents.

     

Performance and diversity of polyvinyl alcohol-degrading bacteria under aerobic and anaerobic conditions

Objectives

To compare the degradation performance and biodiversity of a polyvinyl alcohol-degrading microbial community under aerobic and anaerobic conditions.

Results

An anaerobic–aerobic bioreactor was operated to degrade polyvinyl alcohol (PVA) in simulated wastewater. The degradation performance of the bioreactor during sludge cultivation and the microbial communities in each reactor were compared. Both anaerobic and aerobic bioreactors demonstrated high chemical oxygen demand removal efficiencies of 87.5 and 83.6 %, respectively. Results of 16S rDNA sequencing indicated that Proteobacteria dominated in both reactors and that the microbial community structures varied significantly under different operating conditions. Both reactors obviously differed in bacterial diversity from the phyla Planctomycetes, Chlamydiae, Bacteroidetes, and Chloroflexi. Betaproteobacteria and Alphaproteobacteria dominated, respectively, in the anaerobic and aerobic reactors.

Conclusions

The anaerobic–aerobic system is suitable for PVA wastewater treatment, and the microbial genetic analysis may serve as a reference for PVA biodegradation.

     

Effects of α-zearalenol on the metabolome of two breast cancer cell lines by 1H-NMR approach

Introduction

Zearalenone (ZEN) is one of the most widely distributed toxins that contaminates many crops and foods. Its major metabolites are α-Zearalenol (α-zol) and β-Zearalenol. Previous studies showed that ZEN and α-zol have estrogenic properties and are able to induce growth promoting effect in breast tissues.

Objectivies

Considering that tumorigenesis is dependent on the reprogramming of cellular metabolism and that the evaluation of the cellular metabolome is useful to understand the metabolic changes that can occur during the cancer development and progression or after treatments, aim of our work is to study, for the first time, the effects of α-zol on the metabolomic profile of an estrogen positive breast cancer cell line, MCF-7, and of an estrogen negative breast cancer cell lines MDA-MB231.

Methods

Firstly, we tested the effects of α-zol on the cell viability after 24, 48 and 72 h of treatments with 10^?10, 10^?8 and 10^?6 M concentrations on breast cancer MCF-7 and MDA-MB231 cell lines in comparison to human non-cancerous breast MCF10A cell line. Then, we evaluated cell cycle progression, levels of reactive oxygen species (ROS) and the metabolomic profiling by ¹H-NMR approach on MCF-7 and MDA-MB231 before and after 72 h treatments. Principal component analysis was used to compare the obtained spectra.

Results

α-zol is resulted able to induce: (i) an increase of the cell viability on MCF-7 cells mainly after 72 h treatment, (ii) a slight decrease of the cell viability on MDA-MB231 cells, and (iii) an increase of cells in S phase of the cell cycle and of ROS only in MCF-7 cells. Moreover, the evaluation of metabolomics profile evidenced that after treatment with α-zol the levels of some metabolites increased in MCF-7 cells whereas decreased slightly in MDA-MB231 cells.

Conclusions

Our results showed that α-zol was able to increase the protein biosynthesis as well as the lipid metabolism in MCF-7 cells, and, hence, to induce an estrogen positive breast cancer progression.

     

Biodegradation of saline phenolic wastewater in a biological contact oxidation reactor with immobilized cells of <Emphasis Type="Italic">Oceanimonas</Emphasis> sp.

Objective

To develop a method to treat saline phenolic wastewater in a biological contact oxidation reactor (BCOR) with immobilized cells of a marine microorganism, Oceanimonas sp., isolated from seawater.

Results

Cells were immobilized on fibre carriers in the BCOR. Saline wastewater with phenol at 1.5 g/l and NaCl at 6 % (w/v) was treated. In continuous assays, 99 % removal of phenol was achieved and a kinetic model for the phenol degradation is presented based on Monod’s equation.

Conclusion

The BOCR system using immobilized cells of Oceanimonas efficiently treats saline phenolic wastewaters without having decrease the salinity of the wastewater.

     

Comparison of different methods for erythroid differentiation in the K562 cell line

Objective

To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis.

Results

Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001).

Conclusions

Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

     

Comparison of adult stem cells derived from multiple stem cell niches

Objectives

Adult stem cells (ASCs) have great potential for tissue regeneration; however, comparative studies of ASCs from different niches are required to understand the characteristics of each population for their potential therapeutic uses.

Results

We compared the proliferation, stem cell marker expression, and differentiation potential of ASCs from bone marrow, skin dermis, and adipose tissue. ASCs from bone marrow and skin dermis showed 50–100 % increased proliferation in comparison to the ASCs from adipose tissues. Furthermore, ASCs from each stem cell niche showed differential expression of stem cell marker genes, and preferentially differentiated into cell types of their tissue of origin.

Conclusion

Different characters of each ASC might be major factors for their effective use for therapeutics and tissue regeneration.

     

Impact of transduction towards the proliferation and migration as well as the transduction efficiency of human umbilical cord-derived late endothelial progenitor cells with nine recombinant adeno-associated virus serotypes

Objectives

To evaluate the transduction efficiency of human umbilical cord-derived, late endothelial progenitor cells late (HUCB-late EPCs) with nine recombinant adeno-associated virus (rAAV) serotypes and the ability of proliferation and migration of the cells after transduction.

Results

rAAV2 and rAAV6 showed a greater ability than other serotypes to transduce late EPCs (P < 0.05). After transduction, cell proliferation ability weakened (P < 0.05), but the ability of migration to stromal cell-derived factor (SDF-1) unchanged.

Conclusion

There is an advantage of choosing the optimal rAAV serotype as a gene vector to alter the biologic characteristics of late EPCs.

     

PEGylation of cytochrome <Emphasis Type="Italic">c</Emphasis> at the level of lysine residues mediated by a microbial <Emphasis Type="Italic">trans</Emphasis>glutaminase

Objectives

To establish a method for microbial transglutaminase (mTG)-mediated PEGylation of proteins at the level of lysine (Lys) residues.

Results

Carboxybenzyl-glutaminyl–glycinyl-methoxypolyethylene glycol (CBZ-QG-mPEG) was prepared by introducing carboxybenzyl-glutaminyl-glycine (CBZ-QG) to mPEG amine. The analysis by Fourier transform infrared spectroscopy and SDS-PAGE showed that CBZ-QG-mPEG was successfully synthesized and can be recognized by mTG as an acyl donor to modify therapeutic protein, cytochrome c (cyt c). Finally, under an optimized condition (cyt c 0.5 mg/ml, CBZ-QG-mPEG 11.25 mg/ml, mTG 0.5 mg/ml, 37 °C, 2 h), the PEGylation yield reached 76.5 %.

Conclusions

This is the first study regarding the PEGylation of protein at the level of Lys residues catalyzed by mTG. The novel method could be employed to immobilize active proteins and modify therapeutic proteins.

     

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> expressing human UDP-glucose 6-dehydrogenase

Objectives

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Results

In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD⁺. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.

Conclusions

Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

     

Differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in fibrin scaffold by a histone deacetylase inhibitor

Background

Human adipose-derived stem cells (hADSCs) are capable of differentiating into many cells such as cardiac cells. Different types of inducers are used for cardiac cell differentiation, but this question still remains to be investigated, which one is the best. The aim of this paper was to investigate the effect of combination of fibrin scaffold and trichostatin A (TSA), for differentiation of hADSCs into cardiomyocyte-like cells.

Methods

After approval of characteristics of hADSCs and fibrin scaffold, hADSCs were cultured in fibrin scaffold with 10 µM TSA for 72 h and kept in standard conditions for 4 weeks. QRT-PCR and immunostaining assay were performed for evaluating the expression pattern of special cardiac genes and proteins.

Results

In particular, our study showed that fibrin scaffold alongside TSA enhanced expression of the selected genes and proteins.

Conclusions

We concluded that the TSA alone or with fibrin scaffold can lead to the generation of cardiac like cells in a short period of time.

     

Contribution of direct electron transfer mechanisms to overall electron transfer in microbial fuel cells utilising <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> as biocatalyst

Objectives

To investigate the contribution of direct electron transfer mechanisms to electricity production in microbial fuel cells by physically retaining Shewanella oneidensis cells close to or away from the anode electrode.

Results

A maximum power output of 114 ± 6 mWm^?2 was obtained when cells were retained close to the anode using a dialysis membrane. This was 3.5 times more than when the cells were separated away from the anode. Without the membrane the maximum power output was 129 ± 6 mWm^?2. The direct mechanisms of electron transfer contributed significantly to overall electron transfer from S. oneidensis to electrodes, a result that was corroborated by another experiment where S. oneidensis cells were entrapped in alginate gels.

Conclusion

S. oneidensis transfers electrons primarily by direct electron transfer as opposed to mediated electron transfer.

     

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

     

Altering bioelectricity on inhibition of human breast cancer cells

Background

Membrane depolarization is associated with breast cancer. Depolarization-activated voltage-gated ion channels are directly implicated in the initiation, proliferation, and metastasis of breast cancer.

Methods

In this study, the role of voltage-gated potassium and calcium ion channel modulation was explored in two different invasive ductal human carcinoma cell lines, MDA-MB-231 (triple-negative) and MCF7 (estrogen-receptor-positive).

Results

Resting membrane potential is more depolarized in MCF7 and MDA-MB-231 cells compared to normal human mammary epithelial cells. Increasing extracellular potassium concentration up to 50 mM depolarized membrane potential and greatly increased cell growth. Tetraethylammonium (TEA), a non-specific blocker of voltage-gated potassium channels, stimulated growth of MCF7 cells (control group grew by 201 %, 1 mM TEA group grew 376 %). Depolarization-induced calcium influx was hypothesized as a requirement for growth of human breast cancer. Removing calcium from culture medium stopped growth of MDA and MCF7 cells, leading to cell death after 1 week. Verapamil, a blocker of voltage-gated calcium channels clinically used in treating hypertension and coronary disease, inhibited growth of MDA cells at low concentration (10–20 μM) by 73 and 92 % after 1 and 2 days, respectively. At high concentration (100 μM), verapamil killed >90 % of MDA and MCF7 cells after 1 day. Immunoblotting experiments demonstrated that an increased expression of caspase-3, critical in apoptosis signaling, positively correlated with verapamil concentration in MDA cells. In MCF7, caspase-9 expression is increased in response to verapamil.

Conclusions

Our results support our hypotheses that membrane depolarization and depolarization-induced calcium influx stimulate proliferation of human breast cancer cells, independently of cancer subtypes. The underlying mechanism of verapamil-induced cell death involves different caspases in MCF7 and MDA-MB-231. These data suggest that voltage-gated potassium and calcium channels may be putative targets for pharmaceutical remediation in human invasive ductal carcinomas.

     

Dynamics of plant nutrient uptake as affected by biopore-associated root growth in arable subsoil

Background and aims

Soil microbial communities influence nutrient cycling, chemistry and structure of soil, and plant productivity. In turn, agronomic practices such as fertilization and crop rotation alter soil physical and chemical properties and consequently soil microbiomes. Understanding the long-term effects of agronomic practices on soil microbiomes is essential for improving agronomic practices to optimize these microbial communities for agricultural sustainability. We examine the composition and substrate-utilization profiles of microbial communities at the Morrow Plots in Illinois.

Methods

Microbial community composition is assessed with 16S rRNA gene sequencing and subsequent bioinformatic analyses. Community- level substrate utilization is characterized with the BIOLOG EcoPlate.

Results

Fertilizer and rotation treatments significantly affected microbial community structure, while substrate utilization was affected by fertilizer, but not crop-rotation treatments. Differences in relative abundance and occurrence of bacterial taxa found in fertilizer treatments can explain the observed differences in community level substrate utilization.

Conclusion

Long-term fertilization and crop-rotation treatments affect soil microbial community composition and physiology, specifically through chronic nutrient limitation, long-term influx of microbes and organic matter via manure application, as well as through changes in soil chemistry. Relatively greater abundance of Koribacteraceae and Solibacterales taxa in soils might prove useful as indicators of soil degradation.

     

Transient expression of recombinant tissue plasminogen activator (rt-PA) gene in cucurbit plants using viral vector

Objective

To use a transient expression system to express a truncated human tissue plasminogen activator (K2S) gene in cucurbit plants.

Results

The recombinant tissue plasminogen activator protein (K2S form) was expressed in active form in cucurbit plants. Its molecular weight was 43 kDa. The plant-derived rt-PA was determined using goat anti-rabbit antibody by western blotting. Among the infected lines, the highest expression of rt-PA was 62 ng/100 mg per leaf tissue as measured by ELISA. The enzymatic activity of the plant-derived rt-PA was 0.8 IU/ml.

Conclusions

The K25 form of rt-PA was expressed for the first time using the viral expression system. Plant-derived rt-PA showed similar potency to commercially-available PA.

     

Daily variation and effect of dietary folate on urinary pteridines

Introduction

Urinary pteridines are putative molecular biomarkers for noninvasive cancer screening and prognostication. Central to their translational biomarker development is the need to understand the sources and extent of their non-epidemiological variation.

Objectives

This study was designed to characterize the two primary sources of urinary pteridine variance: daily variation and the effect of dietary folate.

Methods

Daily variation was studied by collecting urine specimens (n = 81) three times daily for 3 days. The effect of dietary folate was investigated in a treatment study in which urine specimens (n = 168) were collected daily during a control week and a treatment week during which participants received dietary folate supplements. Measurements of six urinary pteridines were made using high-performance liquid chromatography–tandem mass spectrometry. Coefficients of variation were calculated to characterize daily variance between and within subjects, while nearest neighbor non-parametric analyses were used to identify diurnal patterns and measure dietary folate effects.

Results

Daily variance was approximately 35 % RSD for both within-day and between-day periods for most pteridines. Diurnal patterns in response to circadian rhythms were similarly observed for urinary pteridines. Folate supplementation was shown to alter urinary pteridine profiles in a pathway dependent manner, suggesting that dietary folate may regulate endogenous neopterin and biopterin biosynthesis.

Conclusions

Urinary pteridine levels were found to be responsive to both daily variation and folate supplementation. These findings provide new insights into pteridine biosynthesis and regulation as well as useful information for the design of future clinical translational research.

     

Prediction of intravenous busulfan clearance by endogenous plasma biomarkers using global pharmacometabolomics

Introduction

High-dose busulfan (busulfan) is an integral part of the majority of hematopoietic cell transplantation conditioning regimens. Intravenous (IV) busulfan doses are personalized using pharmacokinetics (PK)-guided dosing where the patient’s IV busulfan clearance is calculated after the first dose and is used to personalize subsequent doses to a target plasma exposure. PK-guided dosing has improved patient outcomes and is clinically accepted but highly resource-intensive.

Objective

We sought to discover endogenous plasma biomarkers predictive of IV busulfan clearance using a global pharmacometabolomics-based approach

Methods

Using LC-QTOF, we analyzed 59 (discovery) and 88 (validation) plasma samples obtained before IV busulfan administration.

Results

In the discovery dataset, we evaluated the association of the relative abundance of 1885 ions with IV busulfan clearance and found 21 ions that were associated with IV busulfan clearance tertiles (r² ≥ 0.3). Identified compounds were deoxycholic acid and/or chenodeoxycholic acid, and linoleic acid. We used these 21 ions to develop a parsimonious seven-ion linear predictive model that accurately predicted IV busulfan clearance in 93 % (discovery) and 78 % (validation) of samples.

Conclusion

IV busulfan clearance was significantly correlated with the relative abundance of 21 ions, seven of which were included in a predictive model that accurately predicted IV busulfan clearance in the majority of the validation samples. These results reinforce the potential of pharmacometabolomics as a critical tool in personalized medicine, with the potential to improve the personalized dosing of drugs with a narrow therapeutic index such as busulfan.

     

Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Background

Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.

Aim

Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.

Methods

Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.

Results

Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.

Conclusion

Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.