MSC-Derived Small Extracellular Vesicles Exert Cardioprotective Effect Through Reducing VLCFAs and Apoptosis in Human Cardiac Organoid IRI Model.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs' ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

Cottonseed Meal Protein Isolate as a New Source of Alternative Proteins: A Proteomics Perspective.

Cottonseed meal (CSM) is a good source of dietary proteins but is unsuitable for human consumption due to its gossypol content. To unlock its potential, we developed a protein extraction process with a gossypol removal treatment to generate CSM protein isolate (CSMPI) with ultra-low gossypol content. This process successfully reduced the free and total gossypol content to 4.8 ppm and 147.2 ppm, respectively, far below the US FDA limit. In addition, the functional characterisation of CSMPI revealed a better oil absorption capacity and water solubility than pea protein isolate. Proteome profiling showed that the treatment improved protein identification, while SDS-PAGE analysis indicated that the treatment did not induce protein degradation. Amino acid analysis revealed that post-treated CSMPI was rich in branched-chain amino acids (BCAAs). Mass spectrometry analysis of various protein fractions obtained from an in vitro digestibility assay helped to establish the digestibility profile of CSM proteins. Several potential allergens in CSMPI were also found using allergenic prediction software, but further evaluation based on their digestibility profiles and literature reviews suggests that the likelihood of CSMPI allergenicity remains low. Overall, our results help to navigate and direct the application of CSMPIs as alternative proteins toward nutritive human food application.

Global Profiling of Metabolite and Lipid Soluble Microbial Products in Anaerobic Wastewater Reactor Supernatant Using UPLC-MSE.

Identification of soluble microbial products (SMPs) released during bacterial metabolism in mixed cultures in bioreactors is essential to understanding fundamental mechanisms of their biological production. SMPs constitute one of the main foulants (together with colloids and bacterial flocs) in membrane bioreactors widely used to treat and ultimately recycle wastewater. More importantly, the composition and origin of potentially toxic, carcinogenic, or mutagenic SMPs in renewable/reused water supplies must be determined and controlled. Certain classes of SMPs have previously been studied by GC-MS, LC-MS, and MALDI-ToF MS; however, a more comprehensive LC-MS-based method for SMP identification is currently lacking. Here we develop a UPLC-MS approach to profile and identify metabolite SMPs in the supernatant of an anaerobic batch bioreactor. The small biomolecules were extracted into two fractions based on their polarity, and separate methods were then used for the polar and nonpolar metabolites in the aqueous and lipid fractions, respectively. SMPs that increased in the supernatant after feed addition were identified primarily as phospholipids, ceramides, with cardiolipins in the highest relative abundance, and these lipids have not been previously reported in wastewater effluent.

Chemical Characterization of Low Molecular Weight Soluble Microbial Products in an Anaerobic Membrane Bioreactor.

Effluents from wastewater treatment systems contain a variety of organic compounds, including end products from the degradation of influent substrates, nonbiodegradable feed compounds, and soluble microbial products (SMPs) produced by microbial metabolism. It is important to identify the major components of these SMPs to understand what is in wastewater effluents. In this study, physical pretreatments to extract and concentrate low molecular weight SMPs (MW< 580 Da) from effluents were optimized. Liquid-liquid extraction (LLE) of a 200 mL effluent sample showed the best performance using a mixture of n-hexane, chloroform, and dichloromethane (70 mL) for extraction. For solid phase extraction (SPE), two OasisHLB cartridges were connected in-line to optimize recovery, and the eluted samples from each cartridge were analyzed separately to avoid overlapping peaks. Four solvents varying from polar to nonpolar (methanol, acetone, dichloromethane, and n-hexane) were selected to maximize the number of compound peaks eluted. A combination of SPE (OasisHLB) followed by LLE was shown to maximize compound identification and quantification. However, the compounds identified accounted for only 2.1 mg of chemical oxygen demand (COD)/L (16% of total SMP as COD) because many SMPs have considerably higher MWs. Finally, the method was validated by analyzing a variety of different reactor effluents and feeds.

Lipopolysaccharide neutralizing peptide-porphyrin conjugates for effective photoinactivation and intracellular imaging of gram-negative bacteria strains.

A simple and specific strategy based on the bioconjugation of a photosensitizer protophophyrin IX (PpIX) with a lipopolysaccharide (LPS) binding antimicrobial peptide YI13WF (YVLWKRKRKFCFI-Amide) has been developed for the effective fluorescent imaging and photodynamic inactivation of Gram-negative bacterial strains. The intracellular fluorescent imaging and photodynamic antimicrobial chemotherapy (PACT) studies supported our hypothesis that the PpIX-YI13WF conjugates could serve as efficient probes to image the bacterial strains and meanwhile indicated the potent activities against Gram-negative bacterial pathogens especially for those with antibiotics resistance when exposed to the white light irradiation. Compared to the monomeric PpIX-YI13WF conjugate, the dimeric conjugate indicated the stronger fluorescent imaging signals and higher photoinactivation toward the Gram-negative bacterial pathogens throughout the whole concentration range. In addition, the photodynamic bacterial inactivation also demonstrated more potent activity than the minimum inhibitory concentration (MIC) values of dimeric PpIX-YI13WF conjugate itself observed for E. coli DH5a (~4 times), S. enterica (~8 times), and other Gram-negative strains including antibiotic-resistant E. coli BL21 (~8 times) and K. pneumoniae (~16 times). Moreover, both fluorescent imaging and photoinactivation measurements also demonstrated that the dimeric PpIX-YI13WF conjugate could selectively recognize bacterial strains over mammalian cells and generate less photo damage to mammalian cells. We believed that the enhanced fluorescence and bacterial inactivation were probably attributed to the higher binding affinity between dimeric photosensitizer peptide conjugate and LPS components on the surface of bacterial strains, which were the results of efficient multivalent interactions.

Comparison of soluble microbial product (SMP) production in full-scale anaerobic/aerobic industrial wastewater treatment and a laboratory based synthetic feed anaerobic membrane system.

This study focused on the characterisation of soluble microbial products (SMPs) produced from a full-scale multi-stage (anaerobic/aerobic) industrial wastewater treatment plant, and contrasted them to the SMPs detected in the effluent of a lab-scale AnMBR treating synthetic wastewater to determine if there were any common solutes detected irrespective of the feed organics. Recently developed analytical methods using gas chromatography coupled mass spectrometry (GC-MS) and liquid chromatography coupled quadrupole-time-of-flight (LC-Q-ToF) for SMP characterisation in a wide molecular weight (MW) range of 30-2000 Da (Da) were applied. Samples collected from the Industrial Wastewater plant were the upflow anaerobic sludge blanket (UASB) influent and effluent, and aerobic membrane bioreactor (MBR) effluent before discharge. The GC-MS detected a spike in cyclooctasulphur in the UASB effluent, an indicator of shock-loading, which disappeared after the MBR process. Alkanes, acids and nitrogenous compounds were found to be the end-products from the GC-MS results, while LC-Q-ToF analysis revealed that eicosanoids, a group of cell-signalling molecules, were produced in the aerobic MBR, and made up 71% of its effluent. A comparison of the submerged anaerobic membrane bioreactor (SAMBR) and aerobic MBR effluents using GC-MS showed that there was only a small degree of similarity between the SMPs, comprising mainly long chain alkanes and phthalate. On the other hand, LC-Q-ToF showed a large contrast in compound composition, mostly having cell-signalling functions, which deepened our understanding of the different metabolic processes occurring in aerobic and anaerobic systems. These data could be useful for future work in various areas such as controlling quorum-sensing and biofilm formation, process optimisation and control, and microbial ecology.

Micro-level evaluation of organic compounds transformation in anaerobic digestion under feast and famine conditions assisted by iron-based materials - Revealing the true mechanism of AD enhancement.

Conductive materials have been applied to assist syntrophic metabolism in anaerobic digestion. However, their role in the transformation of organic compounds, particularly recalcitrant compounds, has not been revealed. In this study, iron-based materials - magnetite nanoparticles and Fe2+- were employed to explore their effects on the transformation of different organic matters in anaerobic system. Prompted methane production rates and quantity in iron-based materials groups were found due to the improved solubilization of organic particles, enhanced degradation of recalcitrant compounds, and maintained microbial activity under substrate-limited conditions. Specifically, the proportion of the reducing functional groups (C-C/H or CC) and O/C ratio were always significantly lower in iron-based materials supplemented groups (Fe groups) compared to Control group, despite hydrolysis was greatly enhanced in Fe groups. The greater dehydrogenation oxidation was confirmed in the presence of iron-based materials. The remaining humic-like substances (HS), a typical type of recalcitrant compound, was about 2.5 times higher in Control group (221.2 ± 5.3 mg/L-C) compared to Fe groups after 30 days degradation. By tracking the aromaticity of HS and individual compounds at molecular level, this study reveals that iron-based materials were more effective in stimulating the degradation of aliphatic moieties than the aromatic moieties of recalcitrant compounds. When readily biodegradable substrates were limited, Fe groups continued methane generation by using recalcitrant compounds (e.g. thiethylperazine and fluvoxamino acid) as carbon source, and the microbial activity was maintained according to higher relative abundance of protonated nitrogen and continuous methanogenesis activity at starvation phase.

Biotransformation of phosphorus in enhanced biological phosphorus removal sludge biochar.

Biochar derived from enhanced biological phosphorus removal (EBPR) sludge could be a potential phosphorus (P) fertilizer. Soil microorganisms play a regulating role on the turnover of P in soil. When the EBPR sludge biochar is added to soil, it would inevitably interact with soil microorganisms. Thus, for the wise use of the EBPR sludge biochar, it is imperative to understand the interaction between the biochar and soil microorganisms. In this study, Pseudomonas putida (P. putida), a common soil microorganism, was applied to investigate the biotransformation of P in two EBPR sludge biochars. The results reveal that P released from biochar produced at 700 °C (E700) was more easily absorbed by P. putida than that released from biochar produced at 400 °C (E400). This is attributed to the higher polyphosphates (poly-P) content in E700 and poly-P has higher affinity to P. putida surface compared to orthophosphates. Furthermore, E400 has a negative effect on intracellular poly-P formation in P. putida, which is probably caused by the oxidative stress induced by the free radicals from E400. As intracellular poly-P plays a critical role on bacteria survival and their interaction with surrounding environment, high-temperature biochar (E700) in this case would be more suitable for soil remediation.

Composition and biotransformational changes in soluble microbial products (SMPs) along an anaerobic baffled reactor (ABR).

This work examined the production and catabolism/biotransformation dynamics of SMPs down the length of an eight-compartment-anaerobic baffled reactor (ABR) which physically separates the biological processes, in contrast to completely mixed reactors which do not enable these dynamics to measured, and this is totally novel. SMPs were extracted and characterised by gas and liquid chromatography coupled mass spectrometry to determine their composition and production/catabolism. 60%-70% of the feed compounds decreased from the first to fourth compartment; the increase in SMPs after the fourth compartment suggested a mixture of degraded and biotransformed compounds, and microbial products. High concentrations of low MW alkanes and alkenes, and higher MW (up to 2000 Da) lipids and amino acid derivatives accumulate in the last compartment at pseudo-steady state, and past work identifying polysaccharides/peptides as major membrane biofoulants have excluded these lipids. In addition, lipids and changes detected during feed transients have not been noted before in previous work. Finally, feed step-increases also increased some amino acid derivatives used in cell-signalling. Interestingly, some natural products from plant and fungal extracts were also found in the fourth compartment, where methanogenesis was the dominant process.

Identification of the production and biotransformational changes of soluble microbial products (SMP) in wastewater treatment processes: A short review.

While the definition of soluble microbial products (SMP) remains somewhat contentious, they have been widely accepted to be the pool of organic compounds which are released by cells into their surroundings (liquid or otherwise) due to substrate metabolism and biomass decay. SMPs are also potential precursors of disinfection by-products, and are known to be important in membrane fouling. With recent developments in analytical methodologies, many of the low molecular weight (MW) compounds can now be identified, although they are often incorrectly identified as recalcitrant compounds present in the influent. The old hypothesis of "microbial infallibility" suggested that all organic compounds produced by bacteria will eventually be degraded by microorganisms. However, there are some limitations to this hypothesis due to; the time available for degradation, the rate of activity of the microorganisms themselves, synergistic effects, as well as the degree of complexity of the chemical substance. Therefore, it is important to identify and characterise the SMPs involved in these processes, which can then in turn support the research and development of improving wastewater treatment efficiency and effectiveness, and eventually reduce environmental damage. In addition, it is still unclear what the evolutionary purpose of these compounds are. This paper reviews the work that has been done on the production and biotransformation of chemical compounds up to now and which were reported to be found in wastewater treatment systems.

Transformation of dissolved organic matters produced from alkaline-ultrasonic sludge pretreatment in anaerobic digestion: From macro to micro.

Soluble organic compounds released by alkaline (ALK), ultrasonic (ULS) and combined alkaline-ultrasonic (ALK-ULS) pretreatment as well as their transformation in the anaerobic digestion systems were investigated. The maximum methane production of 197.1 ±â€¯3.0 mL CH4/g tCODfeed was observed with ALK-ULS pretreated sludge (pH 12 and specific energy input of 24 kJ/g TS). The combined treatment likely enhanced the sludge solubilization and produced more low molecular weight (LMW) substances, which were beneficial to improve the biogas generation rate. However, such pretreatment released not only easily biodegradable substances but also more recalcitrants, such as humic substances (HS) and complex high molecular weight (HMW) proteins. Thus, more residual dissolved organic matters (DOMs) were detected after digestion, which may pose adverse effects on the downstream water treatment. Refractory HS and hydrophobic dissolved organic carbon (HO DOC) were the main components of the residual DOMs, which accounted up to 35.0% and 22.3% respectively. At the molecular level, a large amount of residual polycyclic steroid-like matters, alkanes and aromatics were identified. Specific higher MW residual compounds, e.g. polar metabolites (like dipeptide, benzene and substituted derivatives), and non-polar lipids (like diacylglycerols, long chain fatty acids, alkenes, flavonoids, sphingolipids, glycerolipids, glycerophospholipids and their derivatives) were also identified. The results indicate that further polishing steps should be considered to remove the remaining soluble recalcitrant compounds. This study helps to understand the insight of sludge treatment from macro to micro level.

An investigation of the stability of emerging new psychoactive substances.

The evolving nature of new psychoactive substances (NPS) - often referred to as 'legal highs', 'designer drugs' or 'bath salts' - presents an evolving challenge for toxicologists. Apart from the detection and identification of these compounds, further analytical challenges may arise from the presence of possible metabolites or degradation products which may have to be considered when devising an analytical strategy. Whilst there has been some stability research for some more established drugs of abuse and medicinal products, data on emerging NPS are less abundant. In order to address this need, 13 NPS (4-MEC, MDAI, methoxetamine, 5-MeO-DALT, 6-APB, MPA, 5-IAI, MDAT, 2-AI, AMT, 25C-NBOMe, AH-7921, 5-MAPB) were spiked in blood and plasma and kept at room temperature (20-23 °C). Detection and identification of the suspected breakdown products were carried out by high performance liquid chromatography with diode-array detection (HPLC-DAD), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and ultra high performance liquid chromatography with high mass accuracy quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). 4-MEC became undetectable in blood within 14 days with a corresponding loss of 54% in plasma. A breakdown product was identified as dihydro-4-MEC which was also found in vivo in a case work sample. Storage of AMT led to a range of potential breakdown products which were also found in vivo. The remaining substances were found to be stable for at least 21 days in blood and plasma. This is the first time stability data have been published for these emerging substances and showed that additional compounds found during forensic casework were potential metabolites rather than instability products. In particular, presumptive metabolites of 25C-NBOMe and AH-7921 are presented.