Quantifying Methane Influx from Sewer into Wastewater Treatment Processes.

Wastewater treatment contributes substantially to methane (CH4) emissions, yet monitoring and tracing face challenges because the treatment processes are often treated as a "black box". Particularly, despite growing interest, the amount of CH4 carryover and influx from the sewer and its impacts on overall emissions remain unclear. This study quantified CH4 emissions from six wastewater treatment plants (WWTPs) across China, utilizing existing multizonal odor control systems, with a focus on Beijing and Guiyang WWTPs. In the Beijing WWTP, almost 90% of CH4 emissions from the wastewater treatment process were conveyed through sewer pipes, affecting emissions even in the aerobic zone of biological treatment. In the Guiyang WWTP, where most CH4 from the sewer was released at the inlet well, a 24 h online monitoring revealed CH4 fluctuations linked to neighborhood water consumption and a strong correlation to influent COD inputs. CH4 emission factors monitored in six WWTPs range from 1.5 to 13.4 gCH4/kgCODrem, higher than those observed in previous studies using A2O technology. This underscores the importance of considering CH4 influx from sewer systems to avoid underestimation. The odor control system in WWTPs demonstrates its potential as a cost-effective approach for tracing, monitoring, and mitigating CH4.

Identifying Potential Lyme Disease Cases Using Self-Reported Worldwide Tweets: Deep Learning Modeling Approach Enhanced With Sentimental Words Through Emojis.

BACKGROUND: Lyme disease is among the most reported tick-borne diseases worldwide, making it a major ongoing public health concern. An effective Lyme disease case reporting system depends on timely diagnosis and reporting by health care professionals, and accurate laboratory testing and interpretation for clinical diagnosis validation. A lack of these can lead to delayed diagnosis and treatment, which can exacerbate the severity of Lyme disease symptoms. Therefore, there is a need to improve the monitoring of Lyme disease by using other data sources, such as web-based data. OBJECTIVE: We analyzed global Twitter data to understand its potential and limitations as a tool for Lyme disease surveillance. We propose a transformer-based classification system to identify potential Lyme disease cases using self-reported tweets. METHODS: Our initial sample included 20,000 tweets collected worldwide from a database of over 1.3 million Lyme disease tweets. After preprocessing and geolocating tweets, tweets in a subset of the initial sample were manually labeled as potential Lyme disease cases or non-Lyme disease cases using carefully selected keywords. Emojis were converted to sentiment words, which were then replaced in the tweets. This labeled tweet set was used for the training, validation, and performance testing of DistilBERT (distilled version of BERT [Bidirectional Encoder Representations from Transformers]), ALBERT (A Lite BERT), and BERTweet (BERT for English Tweets) classifiers. RESULTS: The empirical results showed that BERTweet was the best classifier among all evaluated models (average F1-score of 89.3%, classification accuracy of 90.0%, and precision of 97.1%). However, for recall, term frequency-inverse document frequency and k-nearest neighbors performed better (93.2% and 82.6%, respectively). On using emojis to enrich the tweet embeddings, BERTweet had an increased recall (8% increase), DistilBERT had an increased F1-score of 93.8% (4% increase) and classification accuracy of 94.1% (4% increase), and ALBERT had an increased F1-score of 93.1% (5% increase) and classification accuracy of 93.9% (5% increase). The general awareness of Lyme disease was high in the United States, the United Kingdom, Australia, and Canada, with self-reported potential cases of Lyme disease from these countries accounting for around 50% (9939/20,000) of the collected English-language tweets, whereas Lyme disease-related tweets were rare in countries from Africa and Asia. The most reported Lyme disease-related symptoms in the data were rash, fatigue, fever, and arthritis, while symptoms, such as lymphadenopathy, palpitations, swollen lymph nodes, neck stiffness, and arrythmia, were uncommon, in accordance with Lyme disease symptom frequency. CONCLUSIONS: The study highlights the robustness of BERTweet and DistilBERT as classifiers for potential cases of Lyme disease from self-reported data. The results demonstrated that emojis are effective for enrichment, thereby improving the accuracy of tweet embeddings and the performance of classifiers. Specifically, emojis reflecting sadness, empathy, and encouragement can reduce false negatives.

CoT-UNet++: A medical image segmentation method based on contextual transformer and dense connection.

Accurate depiction of individual teeth from CBCT images is a critical step in the diagnosis of oral diseases, and the traditional methods are very tedious and laborious, so automatic segmentation of individual teeth in CBCT images is important to assist physicians in diagnosis and treatment. TransUNet has achieved success in medical image segmentation tasks, which combines the advantages of Transformer and CNN. However, the skip connection taken by TransUNet leads to unnecessary restrictive fusion and also ignores the rich context between adjacent keys. To solve these problems, this paper proposes a context-transformed TransUNet++ (CoT-UNet++) architecture, which consists of a hybrid encoder, a dense connection, and a decoder. To be specific, a hybrid encoder is first used to obtain the contextual information between adjacent keys by CoTNet and the global context encoded by Transformer. Then the decoder upsamples the encoded features by cascading upsamplers to recover the original resolution. Finally, the multi-scale fusion between the encoded and decoded features at different levels is performed by dense concatenation to obtain more accurate location information. In addition, we employ a weighted loss function consisting of focal, dice, and cross-entropy to reduce the training error and achieve pixel-level optimization. Experimental results demonstrate that the proposed CoT-UNet++ method outperforms the baseline models and can obtain better performance in tooth segmentation.

Environmental behaviors of emerging contaminants in freshwater ecosystem dominated by submerged plants: A review.

Persistent exposure of emerging contaminants (ECs) in freshwater ecosystem has initiated intense global concerns. Freshwater ecosystem dominated by submerged plants (SP-FES) has been widely constructed to control eutrophic water. However, the environmental behaviors (e.g. migration, transformation, and degradation) of ECs in SP-FES have rarely been concerned and summarized. This review briefly introduced the sources of ECs, the pathways of ECs entering into SP-FES, and the constituent elements of SP-FES. And then the environmental behaviors of dissolved ECs and refractory solid ECs in SP-FES were comprehensively summarized, and the feasibility of removing ECs from SP-FES was critically evaluated. Finally, the challenges and perspectives on the future development for ECs removal from SP-FES were prospected, giving possible research gaps and key directions. This review will provide theoretical and technical support for the effective removal of ECs in freshwater ecosystem, especially in SP-FES.

Effect of allelochemicals sustained-release microspheres on the ingestion, incorporation, and digestion abilities of Daphnia magna Straus.

Allelochemicals sustained-release microspheres (ACs-SMs) exhibited great inhibition effect on algae, however, few studies have focused on ACs-SMs toxicity on invertebrate. In this study, the effects of single high-concentration ACs (15 mg/L, SH-ACs), repeated low-concentration ACs (3 × 5 mg/L, RL-ACs) and ACs-SMs containing 15 mg/L ACs exposure on the ingestion, incorporation, and digestion of Daphnia magna Straus (DS) were investigated by stable isotope 15N labeling method. Meanwhile, the diversity and abundance of microflora in DS guts were determined by 16S rRNA genes and cloning methods. The results showed that SH-ACs exposure caused 50% and 33.3% death rates for newborn and adult DS, while RL-ACs exposure caused 10% death rate for newborn DS and no obvious effect on the activity of adult DS. And ACs-SMs exposure did not diminish the motility of both newborn and adult DS, indicating the lower acute toxicity of ACs-SMs. Furthermore, SH-ACs inhibited the ingestion (-6.45%), incorporation (-47.1%) and digestion (-53.8%) abilities of DS and reduced the microbial abundance (-27.7%) in DS guts. Compared with SH-ACs, RL-ACs showed relatively low impact on the ingestion (-3.23%), incorporation (-5.89%) and digestion (-23.9%) abilities of DS. Interestingly, ACs-SMs enhanced the ingestion (+9.68%), incorporation (+52.9%) and digestion (+51.3%) abilities of DS and increased the microbial abundance (+10.7%) in DS guts. Overall ACs and ACs-SMs reduced the diversity of microflora in DS guts. In conclusion, ACs-SMs can release ACs sustainably and prolong the sustained release time, which not only effectively reduce the toxicity of ACs, but also had positive effects on DS.

Physiological analysis and transcriptome sequencing of a delayed-green leaf mutant 'Duojiao' of ornamental crabapple (Malus sp.).

Malus spectabilis 'Duojiao' is a spontaneous delayed-green leaf color mutant of M. spectabilis 'Riversii' and has chloroplasts with irregularly arranged vesicles and indistinct stromal lamellae. The yellow leaves of mutant have less chlorophyll (Chl), carotenoids, and flavonoids. Measurement of photosynthetic gas exchange indicated that the mutant has lower photosynthetic activity than 'Riversii' plants. Transcriptome sequencing with the Illumina platform was used to characterize differences in gene expression between the leaves of plants with yellow and green colors and elucidate the molecular mechanisms responsible for variation in leaf color in ornamental crabapple. In the comparison group of mutant yellow leaves and the maternal green leaves, 1848 differentially significant expressed genes (DEGs) were annotated by transcriptomic analysis. Many DEGs and transcription factors were identified related to chloroplast development, Chl synthesis and degradation, photosynthesis, carotenoid biosynthesis, flavonoid biosynthesis and other pathways related to plant leaf color formation. Among these, the Chl biosynthesis-related coproporphyrinogen gene, oxidative decarboxylase gene, and Chl a oxygenase gene were down-regulated, indicating that Chl biosynthesis was blocked. GLK1, which regulates chloroplast development, was down-regulated in yellow leaves. Parallel experiments showed that the content of the Chl synthesis precursors, protoporphyrinogen IX, chlorophyllide a, and chlorophyllide b and the activity of chlorophyllogen III oxidase and chlorophyllide a oxygenase in the yellow leaves of 'Duojiao' were lower than those in the green leaves of 'Riversii'. Thus, leaf color formation is greatly affected by Chl metabolism and chloroplast development. The reliability of the RNA-sequencing data was confirmed by quantitative real-time PCR analysis with 12 selected DEGs. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01248-7.

Effects of Organic Acid Root Exudates of Malus hupehensis Rehd. Derived from Soil and Root Leaching Liquor from Orchards with Apple Replant Disease.

Organic acids secreted by plants, such as p-hydroxybenzoic acid, ferulic acid, cinnamic acid, and benzoic acid, can inhibit seed germination and root growth. The effects of root and soil leaching liquor from orchards on the growth of M. hupehensis Rehd. seedlings under sand culture are studied; the seedlings are sampled at 15, 30, 45, and 60 d. Changes in the amount of root exudates are determined using HPLC. Low concentrations of root leaching liquor (A1) and soil leaching liquor (B1) significantly promoted plant growth and chlorophyll synthesis; high concentrations of root leaching liquor (A6) and soil leaching liquor (B4-6) inhibited growth. Low concentrations of soil leaching liquor had no significant effect on the POD, SOD, and CAT activities. A5-6 and B5-6 significantly decreased Fv/Fm and qP values, respectively, and increased NPQ values. All root and soil leaching liquor treatments inhibited the secretion of gallic acid, hydroxybenzoic acid, benzoic acid, and phloridzin, and promoted the secretion of caffeic acid. The root leaching liquor treatments inhibited the secretion of catechin and promoted the secretion of phloretin. The soil leaching liquor treatments promoted the secretion of cinnamic acid. The secretion of other phenolic acids is likely associated with the different concentrations of leaching liquor.

De novo assembly provides new insights into the evolution of Elaeagnus angustifolia L.

BACKGROUND: Elaeagnus angustifolia L. is a deciduous tree in the family Elaeagnaceae. It is widely used to study abiotic stress tolerance in plants and to improve desertification-affected land because of its ability to withstand diverse types of environmental stress, such as drought, salt, cold, and wind. However, no studies have examined the mechanisms underlying the resistance of E. angustifolia to environmental stress and its adaptive evolution. METHODS: Here, we used PacBio, Hi-C, resequencing, and RNA-seq to construct the genome and transcriptome of E. angustifolia and explore its adaptive evolution. RESULTS: The reconstructed genome of E. angustifolia was 526.80 Mb, with a contig N50 of 12.60 Mb and estimated divergence time of 84.24 Mya. Gene family expansion and resequencing analyses showed that the evolution of E. angustifolia was closely related to environmental conditions. After exposure to salt stress, GO pathway analysis showed that new genes identified from the transcriptome were related to ATP-binding, metal ion binding, and nucleic acid binding. CONCLUSION: The genome sequence of E. angustifolia could be used for comparative genomic analyses of Elaeagnaceae family members and could help elucidate the mechanisms underlying the response of E. angustifolia to drought, salt, cold, and wind stress. Generally, these results provide new insights that could be used to improve desertification-affected land.

EpsRAc is a copper-sensing MarR family transcriptional repressor from Acidithiobacillus caldus.

The MarR family, as multiple antibiotic resistance regulators, is associated with the resistance of organisms to unfavorable conditions. MarR family extracellular polymeric substances (EPS)-associated transcriptional regulator (EpsRAc) was closely associated with copper resistance in Acidithiobacillus caldus (A. caldus). Transcriptional analysis showed high activity of the epsR promoter (PI) in Escherichia coli and differential response to metal ions. The copper content and UV absorption spectrum of the co-purified protein did not increase, but a stoichiometry of 0.667 mol Cu(I) per EpsRAc monomer was observed in vitro in copper titration experiments, suggesting that Cu(II) acts with low affinity in binding to the EpsRAc protein. Electrophoretic mobility shift assays (EMSA) demonstrated that EpsRAc could bind to its own promoter in vitro, and the binding region was the palindrome sequence TGTTCATCGTGTGTGAGCACACA. EpsRAc negatively regulated its own gene expression, whereas Cu(II) mitigates this negative effect. EpsRAc did not bind to other neighboring gene promoters. Finally, we developed a working model to illustrate the regulatory mechanism of A. caldus in response to extreme copper stress. KEY POINTS: • Identification of a MarR family EPS-associated transcriptional regulator, named EpsRAc. • Cu(I) can bind to the EpsRAc protein with low affinity. • EpsRAc negatively regulates the expression of epsR, and Cu(II) can alleviate this negative regulation.

Homologous Cloning of Potassium Channel Genes From the Superior Apple Rootstock Line 12-2, Which is Tolerant to Apple Replant Disease.

Potassium channels are important ion channels that are responsible for the absorption of potassium in the plant nutrient uptake system. In this study, we used homologous molecular cloning to obtain 8 K+ channel genes from the superior apple rootstock line 12-2 (self-named): MsAKT1-1, MsKAT3-2, MsKAT1-3, MsK2P3-4, MsK2P3-5, MsK2P5-6, MsK2P3-7, and MsK2P3-8. Their lengths varied from 942 bp (MsK2P5-6) to 2625 bp (MsAKT1-1), and the number of encoded amino acids varied from 314 (MsK2P5-6) to 874 (MsAKT1-1). Subcellular localization predictions showed that MsAKT1-1, MsKAT3-2, and MsKAT1-3 were localized on the plasma membrane, and MsK2P3-4, MsK2P3-5, MsK2P5-6, MsK2P3-7, and MsK2P3-8 were localized on the vacuole and plasma membrane. The 8 K+ channel proteins contained α helices, extended strands, ß turns, and random coils. MsKAT1-3 had four transmembrane structures, MsKAT3-2 had six, and the other six K+ channel genes had five. Protein structure domain analysis showed that MsAKT1-1 contained nine protein domains, followed by MsKAT3-2 with four, MsKAT1-3 with three, and the other five two-pore domain K+ channel proteins with two. Semi-quantitative RT-PCR detection of the K+ channel genes showed that their expression levels were high in roots. qRT-PCR analysis showed that the relative expression levels of the 8 genes changed after exposure to ARD stress. The above results provide a theoretical basis for further research on the functions of potassium channel genes in 12-2 and a scientific basis for the breeding of ARD-resistant rootstock.

The adaptation mechanisms of Acidithiobacillus caldus CCTCC M 2018054 to extreme acid stress: Bioleaching performance, physiology, and transcriptomics.

To understand the acid-resistant mechanism of bioleaching microorganism Acidithiobacillus caldus CCTCC M 2018054, its physiology and metabolic changes at the transcriptional level under extreme acid stress were systemically studied. Scanning electron microscopy (SEM), Fourier transform infrared reflection (FTIR) and X-ray diffraction (XRD) showed that with an increase in acidity, the absorption peak of sulfur oxidation-related functional groups such as S-O decreased significantly, and a dense sulfur passivation film appeared on the surface of the ore. Confocal laser scanning microscopy (CLSM) revealed that coverage scale of extracellular polymeric substance (EPS) and biofilm fluctuated accordingly along with the increasing acid stress (pH-stat 1.5, 1.2 0.9 and 0.6) during the bioleaching process. In response to acid stress, the increased levels of intracellular glutamic acid, alanine, cysteine, and proline contributed to the maintenance of intracellular pH homeostasis via decarboxylation and alkaline neutralization. Higher unsaturated fatty acid content was closely related to membrane fluidity. Up to 490 and 447 differentially expressed genes (DEGs) were identified at pH 1.5 vs pH 1.2 and pH 1.2 vs pH 0.9, respectively, and 177 common DEGs were associated with two-component system (TCS) regulation, transporter regulation, energy metabolism, and stress response. The upregulation of kdpB helped cells defend against proton invasion, whereas the downregulation of cysB and cbl implied stronger oxidation of sulfur compounds. The transcriptional level of sqr, sor, and soxA was significantly increased and consolidated the energy supply needed for resisting acid stress. Furthermore, eight of the identified DEGs (sor, cbl, ompA, atpF, nuoH, nuoC, sqr, grxB) were verified as being related to the acid stress response process. This study contributes toward expanding the application of these acidophiles in industrial bioleaching.

Simultaneously enhance iron/sulfur metabolism in column bioleaching of chalcocite by pyrite and sulfur oxidizers based on joint utilization of waste resource.

In chalcocite (Cu2S) bioleaching, the lack of iron metabolism is a key restricting factor. As the most common sulfide mineral, pyrite (FeS2) can release Fe(Ⅱ) and compensate for the iron metabolism deficiency in chalcocite bioleaching. The bioleaching of chalcocite in an imitated industrial system was improved by enhancing the iron-sulfur metabolism simultaneously using pyrite and sulfur oxidizers based on the joint utilization of waste resources, while the bioleaching performance and community structure in the leachate were systematically investigated. Due to the active sulfur/iron metabolism, the pH reached 1.2, and Fe3+ was increased by 77.78%, while the biomass of planktonic cells was improved to 2.19 × 107 cells/mL. Fourier transform infrared reflection (FTIR) and X-ray diffraction (XRD) analysis results showed that more iron-sulfur crystals were produced due to more active iron-sulfur metabolism. Scanning electron microscopy (SEM) revealed that many derivative particles and corrosion marks appeared on the surface of the ore, implying that the mineral-microbe interaction was strengthened. Confocal laser scanning microscopy (CLSM) showed the accumulation of cells and extracellular polymeric substances (EPS) on the ore surface, indicating a stronger contact leaching mechanism. Furthermore, the community structure and canonical correspondence analysis (CCA) demonstrated that the introduction of sulfur-oxidizing bacteria and pyrite could maintain the diversity of dominant leaching microorganisms at a high level. Sulfobacillus (27.75%) and Leptospirllillum (20.26%) were the dominant sulfur-oxidizing and iron-oxidizing bacteria during the bioleaching process. With the accumulation of multiple positive effects, the copper ion leaching rate was improved by 44.8%. In general, this new type of multiple intervention strategy can provide an important guide for the bioleaching of low-grade ores.

Detection of Root Physiological Parameters and Potassium and Calcium Currents in the Rhizoplane of the Apple Rootstock Superior Line 12-2 With Improved Apple Replant Disease Resistance.

The cultivation of resistant rootstocks is one of the more effective ways to mitigate apple replant disease (ARD). We performed an ion current test, a pot experiment, and a pathogen infection test on the apple rootstocks 12-2 (self-named), T337, and M26. The ion current test showed that exposure to ARD soil extract for 30 min had a significant effect on K+ ion currents at the meristem, elongation, and mature zones of the M26 rhizoplane and on Ca2+ currents in the meristem and elongation zones. ARD also had a significant effect on Ca2+ currents in the meristem, elongation, and mature zones of the T337 rhizoplane. Exposure to ARD soil extract for 5 min had a significant effect on K+ currents in the meristem, elongation, and mature zones of 12-2 and on the Ca2+ currents in the elongation and mature zones. Compared to a 5-min exposure, a 30-min exposure to ARD extract had a less pronounced effect on K+ and Ca2+ currents in the 12-2 rhizoplane. The pot experiment showed that ARD soil had no significant effect on any root architectural or physiological parameters of 12-2. By contrast, ARD soil significantly reduced some root growth indices and the dry and fresh weights of T337 and M26 compared with controls on sterilized soil. ARD also had a significant effect on root metabolic activity, root antioxidant enzyme activity (except superoxide dismutase for T337), and malondialdehyde content of T337 and M26. Pathogen infection tests showed that Fusarium proliferatum MR5 significantly affected the root structure and reduced the root metabolic activity of T337 and M26. It also reduced their root antioxidant enzyme activities (except catalase for T337) and significantly increased the root malondialdehyde content, reactive oxygen levels, and proline and soluble sugar contents. By contrast, MR5 had no such effects on 12-2. Based on these results, 12-2 has the potential to serve as an important ARD-resistant rootstock.

A review: Application of allelochemicals in water ecological restoration--algal inhibition.

Problems caused by harmful algal blooms have attracted worldwide attention due to their severe harm to aquatic ecosystems, prompting researchers to study applicable measures to inhibit the growth of algae. Allelochemicals, as secondary metabolites secreted by plants, have excellent biocompatibility, biodegradability, obvious algal inhibiting effect and little ecological harm, and have promising application prospect in the field of water ecological restoration. This review summarized the research progress of allelochemicals, including (i) definition, development, and classification, (ii) influencing factors and mechanism of algal inhibition, (iii) the preparation methods of algal inhibitors based on allelochemicals. The future research directions of allelochemicals sustained-released microspheres (SRMs) were also prospected. In the future, it is urgent to explore more efficient allelochemicals, to study the regulation mechanism of allelochemicals in natural water bodies, and to improve the preparation method of allelopathic algal suppressant. This paper proposed a feasible direction for the development of allelochemicals SRMs which exhibited certain guiding significance for their application in water ecological restoration.

Quality Measures for Managing Prescription of Antipsychotic Medication Among Youths: Factors Associated With Health Plan Performance.

OBJECTIVE: This study examined the performance of health plans on two HEDIS measures: metabolic monitoring of children and adolescents prescribed an antipsychotic and use of first-line psychosocial care for children and adolescents prescribed an antipsychotic for a nonindicated use. Plan characteristics and other contextual factors that may be associated with health plan performance were examined to identify potential strategies for improving care. METHODS: The study population included 279 commercial and 169 Medicaid health plans that voluntarily submitted data for care provided in 2016. Bivariate associations between performance on the two measures and each plan characteristic (eligible population size, region, profit status, model type, and operating in a state with legislation on prior authorization for antipsychotics) were examined. Main-effects multivariable linear regression models were used to examine the combined association of plan characteristics with each measure. RESULTS: Performance rates on both measures were comparable for commercial and Medicaid plans. Among commercial plans, not-for-profit plans outperformed for-profit plans on both measures. Commercial and Medicaid plans in the North performed significantly better on the metabolic monitoring measure. Commercial plans in the South and Medicaid plans in the West performed significantly worse on the first-line psychosocial care measure. Plans operating in states requiring prior authorization performed significantly better on the metabolic monitoring measure. CONCLUSIONS: This study identified key plan characteristics and other contextual factors associated with health plan performance on quality measures related to pediatric antipsychotic prescribing. Findings suggest that quality measures, in conjunction with policies such as prior authorization, can encourage better care delivery to vulnerable populations.

Immunotherapeutic potential of extracellular vesicles.

Extracellular vesicle or EV is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes, the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized endosome-derived vesicles secreted by many cell types and their immunomodulatory potential is independent of their cell source. Besides immune cells such as dendritic cells, macrophages, and T cells, cancer and stem cells also secrete immunologically active exosomes that could influence both physiological and pathological processes. The immunological activities of exosomes affect both innate and adaptive immunity and include antigen presentation, T cell activation, T cell polarization to regulatory T cells, immune suppression, and anti-inflammation. As such, exosomes carry much immunotherapeutic potential as a therapeutic agent and a therapeutic target.

Mesenchymal stem cells secrete immunologically active exosomes.

Mesenchymal stem cells (MSCs) have been shown to secrete exosomes that are cardioprotective. Here, we demonstrated that MSC exosome, a secreted membrane vesicle, is immunologically active. MSC exosomes induced polymyxin-resistant, MYD88-dependent secreted embryonic alkaline phosphatase (SEAP) expression in a THP1-Xblue, a THP-1 reporter cell line with an NFκB-SEAP reporter gene. In contrast to lipopolysaccharide, they induced high levels of anti-inflammatory IL10 and TGFß1 transcript at 3 and 72 h, and much attenuated levels of pro-inflammatory IL1B, IL6, TNFA and IL12P40 transcript at 3-h. The 3-h but not 72-h induction of cytokine transcript was abrogated by MyD88 deficiency. Primary human and mouse monocytes exhibited a similar exosome-induced cytokine transcript profile. Exosome-treated THP-1 but not MyD88-deficient THP-1 cells polarized activated CD4(+) T cells to CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) at a ratio of one exosome-treated THP-1 cell to 1,000 CD4(+) T cells. Infusion of MSC exosomes enhanced the survival of allogenic skin graft in mice and increased Tregs.

Therapeutic MSC exosomes are derived from lipid raft microdomains in the plasma membrane.

BACKGROUND: Mesenchymal stem cell (MSC) was previously shown to secrete lipid vesicles that when purified by high performance liquid chromatography as a population of homogenously sized particles with a hydrodynamic radius of 55-65 nm reduce infarct size in a mouse model of myocardial ischemia/reperfusion injury. As these vesicles exhibit many biophysical and biochemical properties of exosomes, they were identified as exosomes. Here we investigated if these lipid vesicles were indeed exosomes that have an endosomal biogenesis. METHOD: In most cells, endocytosis is thought to occur at specialized microdomains known as lipid rafts. To demonstrate an endosomal origin for MSC exosomes, MSCs were pulsed with ligands e.g. transferrin (Tfs) and Cholera Toxin B (CTB) that bind receptors in lipid rafts. The endocytosed ligands were then chased to determine if they were incorporated into the exosomes. RESULTS: A fraction of exogenous Tfs was found to recycle into MSC exosomes. When MSCs were pulsed with labelled Tfs in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, Tf incorporation in CD81-immunoprecipitate was reduced during the chase. CTB which binds GM1 gangliosides that are enriched in lipid rafts extracted exosome-associated proteins, CD81, CD9, Alix and Tsg101 from MSC-conditioned medium. Exogenous CTBs were pulse-chased into secreted vesicles. Extraction of Tf- or CTB-binding vesicles in an exosome preparation mutually depleted each other. Inhibition of sphingomyelinases reduced CTB-binding vesicles. CONCLUSION: Together, our data demonstrated that MSC exosomes are derived from endocytosed lipid rafts and that their protein cargo includes exosome-associated proteins CD81, CD9, Alix and Tsg101.

Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery.

Advances in biomedical research have generated an unprecedented number of potential targets for therapeutic intervention to treat disease or delay disease progression. Unfortunately, many of these targets are not druggable as they are intracellular, present in many cell types, poorly soluble or rapidly inactivated. Although synthetic drug vehicles have successfully circumvented many of these problems, natural particulates such as exosomes that intrinsically possess many attributes of a drug delivery vehicle are highly attractive as potentially better alternatives. Of the cell types known to produce exosomes, the readily available proliferative, immunosuppressive and clinically tested human mesenchymal stem cell (MSC) is the most prolific producer. Its exosomes are therapeutic in animal model of disease and exhibit immunosuppressive activity. The quality and quantity of exosome production is not compromised by immortalization to create a permanent MSC cell line. Therefore, MSC is well suited for mass production of exosomes that are ideal for drug delivery.

Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs.

BACKGROUND: Exosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hESC-MSCs) have been shown to reduce myocardial ischemia/reperfusion injury in animal models. However, as hESC-MSCs are not infinitely expansible, large scale production of these exosomes would require replenishment of hESC-MSC through derivation from hESCs and incur recurring costs for testing and validation of each new batch. Our aim was therefore to investigate if MYC immortalization of hESC-MSC would circumvent this constraint without compromising the production of therapeutically efficacious exosomes. METHODS: The hESC-MSCs were transfected by lentivirus carrying a MYC gene. The transformed cells were analyzed for MYC transgene integration, transcript and protein levels, and surface markers, rate of cell cycling, telomerase activity, karyotype, genome-wide gene expression and differentiation potential. The exosomes were isolated by HPLC fractionation and tested in a mouse model of myocardial ischemia/reperfusion injury, and infarct sizes were further assessed by using Evans' blue dye injection and TTC staining. RESULTS: MYC-transformed MSCs largely resembled the parental hESC-MSCs with major differences being reduced plastic adherence, faster growth, failure to senesce, increased MYC protein expression, and loss of in vitro adipogenic potential that technically rendered the transformed cells as non-MSCs. Unexpectedly, exosomes from MYC-transformed MSCs were able to reduce relative infarct size in a mouse model of myocardial ischemia/reperfusion injury indicating that the capacity for producing therapeutic exosomes was preserved. CONCLUSION: Our results demonstrated that MYC transformation is a practical strategy in ensuring an infinite supply of cells for the production of exosomes in the milligram range as either therapeutic agents or delivery vehicles. In addition, the increased proliferative rate by MYC transformation reduces the time for cell production and thereby reduces production costs.