Proteomic profiling of extracellular vesicles derived from human serum for the discovery of biomarkers in Avascular necrosis.

BACKGROUND: Avascular necrosis (AVN) is a medical condition characterized by the destruction of bone tissue due to a diminished blood supply. When the rate of tissue destruction surpasses the rate of regeneration, effective treatment becomes challenging, leading to escalating pain, arthritis, and bone fragility as the disease advances. A timely diagnosis is imperative to prevent and initiate proactive treatment for osteonecrosis. We explored the potential of differentially expressed proteins in serum-derived extracellular vesicles (EVs) as biomarkers for AVN of the femoral head in humans. We analyzed the genetic material contained in serum-derived exosomes from patients for early diagnosis, treatment, and prognosis of avascular necrosis. METHODS: EVs were isolated from the serum of both patients with AVN and a control group of healthy individuals. Proteomic analyses were conducted to compare the expression patterns of these proteins by proteomic analysis using LC-MS/MS. RESULTS: Our results show that the levels of IGHV3-23, FN1, VWF, FGB, PRG4, FCGBP, and ZSWIM9 were upregulated in the EVs of patients with AVN compared with those of healthy controls. ELISA results showed that VWF and PRG4 were significantly upregulated in the patients with AVN. CONCLUSIONS: These findings suggest that these EV proteins could serve as promising biomarkers for the early detection and diagnosis of AVN. Early diagnosis is paramount for effective treatment, and the identification of new osteonecrosis biomarkers is essential to facilitate swift diagnosis and proactive intervention. Our study provides novel insights into the identification of AVN-related biomarkers that can enhance clinical management and treatment outcomes.

Hepatitis B virus e antigen induces atypical metabolism and differentially regulates programmed cell deaths of macrophages.

Macrophages can undergo M1-like proinflammatory polarization with low oxidative phosphorylation (OXPHOS) and high glycolytic activities or M2-like anti-inflammatory polarization with the opposite metabolic activities. Here we show that M1-like macrophages induced by hepatitis B virus (HBV) display high OXPHOS and low glycolytic activities. This atypical metabolism induced by HBV attenuates the antiviral response of M1-like macrophages and is mediated by HBV e antigen (HBeAg), which induces death receptor 5 (DR5) via toll-like receptor 4 (TLR4) to induce death-associated protein 3 (DAP3). DAP3 then induces the expression of mitochondrial genes to promote OXPHOS. HBeAg also enhances the expression of glutaminases and increases the level of glutamate, which is converted to α-ketoglutarate, an important metabolic intermediate of the tricarboxylic acid cycle, to promote OXPHOS. The induction of DR5 by HBeAg leads to apoptosis of M1-like and M2-like macrophages, although HBeAg also induces pyroptosis of the former. These findings reveal novel activities of HBeAg, which can reprogram mitochondrial metabolism and trigger different programmed cell death responses of macrophages depending on their phenotypes to promote HBV persistence.

Targeting Mycobacterium tuberculosis Persistence through Inhibition of the Trehalose Catalytic Shift.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb's ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.

Central carbon metabolism remodeling as a mechanism to develop drug tolerance and drug resistance in Mycobacterium tuberculosis.

Suboptimal efficacy of the current antibiotic regimens and frequent emergence of antibiotic-resistant Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), render TB the world's deadliest infectious disease before the COVID-19 outbreak. Our outdated TB treatment method is designed to eradicate actively replicating populations of Mtb. Unfortunately, accumulating evidence suggests that a small population of Mtb can survive antimycobacterial pressure of antibiotics by entering a "persister" state (slowly replicating or non-replicating and lacking a stably heritable antibiotic resistance, termed drug tolerance). The formation of drug-tolerant Mtb persisters is associated with TB treatment failure and is thought to be an adaptive strategy for eventual development of permanent genetic mutation-mediated drug resistance. Thus, the molecular mechanisms behind persister formation and drug tolerance acquisition are a source of new antibiotic targets to eradicate both Mtb persisters and drug-resistant Mtb. As Mtb persisters are genetically identical to antibiotic susceptible populations, metabolomics has emerged as a vital biochemical tool to differentiate these populations by determining phenotypic shifts and metabolic reprogramming. Metabolomics, which provides detailed insights into the molecular basis of drug tolerance and resistance in Mtb, has unique advantages over other techniques by its ability to identify specific metabolic differences between the two genetically identical populations. This review summarizes the recent advances in our understanding of the metabolic adaptations used by Mtb persisters to achieve intrinsic drug tolerance and facilitate the emergence of drug resistance. These findings present metabolomics as a powerful tool to identify previously unexplored antibiotic targets and improved combinations of drug regimens against drug-resistant TB infection.

Difluoromethylornithine (DFMO) and AMXT 1501 inhibit capsule biosynthesis in pneumococci.

Polyamines are small cationic molecules that have been linked to various cellular processes including replication, translation, stress response and recently, capsule regulation in Streptococcus pneumoniae (Spn, pneumococcus). Pneumococcal-associated diseases such as pneumonia, meningitis, and sepsis are some of the leading causes of death worldwide and capsule remains the principal virulence factor of this versatile pathogen. α-Difluoromethyl-ornithine (DFMO) is an irreversible inhibitor of the polyamine biosynthesis pathway catalyzed by ornithine decarboxylase and has a long history in modulating cell growth, polyamine levels, and disease outcomes in eukaryotic systems. Recent evidence shows that DFMO can also target arginine decarboxylation. Interestingly, DFMO-treated cells often escape polyamine depletion via increased polyamine uptake from extracellular sources. Here, we examined the potential capsule-crippling ability of DFMO and the possible synergistic effects of the polyamine transport inhibitor, AMXT 1501, on pneumococci. We characterized the changes in pneumococcal metabolites in response to DFMO and AMXT 1501, and also measured the impact of DFMO on amino acid decarboxylase activities. Our findings show that DFMO inhibited pneumococcal polyamine and capsule biosynthesis as well as decarboxylase activities, albeit, at a high concentration. AMXT 1501 at physiologically relevant concentration could inhibit both polyamine and capsule biosynthesis, however, in a serotype-dependent manner. In summary, this study demonstrates the utility of targeting polyamine biosynthesis and transport for pneumococcal capsule inhibition. Since targeting capsule biosynthesis is a promising way for the eradication of the diverse and pathogenic pneumococcal strains, future work will identify small molecules similar to DFMO/AMXT 1501, which act in a serotype-independent manner.

Fatty acid metabolism of Mycobacterium tuberculosis: A double-edged sword.

Unlike other heterotrophic bacteria, Mycobacterium tuberculosis (Mtb) can co-catabolize a range of carbon sources simultaneously. Evolution of Mtb within host nutrient environment allows Mtb to consume the host's fatty acids as a main carbon source during infection. The fatty acid-induced metabolic advantage greatly contributes to Mtb's pathogenicity and virulence. Thus, the identification of key enzymes involved in Mtb's fatty acid metabolism is urgently needed to aid new drug development. Two fatty acid metabolism enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and isocitrate lyase (ICL) have been intensively studied as promising drug targets, but recently, Quinonez et al. (mBio, doi: 10.1128/mbio.03559-21) highlighted a link between the fatty acid-induced dormancy-like state and drug tolerance. Using metabolomics profiling of a PEPCK-deficient mutant, Quinonez et al. identified that over-accumulation of methylcitrate cycle (MCC) intermediates are phenotypically associated with enhanced drug tolerance against first- and second- line TB antibiotics. This finding was further corroborated by metabolomics and phenotypic characterization of Mtb mutants lacking either ICL or 2-methylcitrate dehydratase. Fatty acid metabolism induced drug-tolerance was also recapitulated in wildtype Mtb after treatment with authentic 2-methylisocitrate, an MCC intermediate. Together, the fatty acid-induced dormancy-like state and drug tolerance are attributed to dysregulated MCC activity.

Human Epidural AD-MSC Exosomes Improve Function Recovery after Spinal Cord Injury in Rats.

Spinal cord injury (SCI) interferes with the normal function of the autonomic nervous system by blocking circuits between the sensory and motor nerves. Although many studies focus on functional recovery after neurological injury, effective neuroregeneration is still being explored. Recently, extracellular vesicles such as exosomes have emerged as cell-free therapeutic agents owing to their ability of cell-to-cell communication. In particular, exosomes released from mesenchymal stem cells (MSCs) have the potential for tissue regeneration and exhibit therapeutic effectiveness in neurological disorders. In this study, we isolated exosomes from human epidural adipose tissue-derived MSCs (hEpi AD-MSCs) using the tangential flow filtration method. The isolated exosomes were analyzed for size, concentration, shape, and major surface markers using nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. To evaluate their effect on SCI recovery, hEpi AD-MSC exosomes were injected intravenously in SCI-induced rats. hEpi AD-MSC exosomes improved the locomotor function of SCI-induced rats. The results of histopathological and cytokine assays showed that hEpi AD-MSC exosomes regulated inflammatory response. Genetic profiling of the rat spinal cord tissues revealed changes in the expression of inflammation-related genes after exosome administration. Collectively, hEpi AD-MSC exosomes are effective in restoring spinal functions by reducing the inflammatory response.

The Role of Fatty Acid Metabolism in Drug Tolerance of Mycobacterium tuberculosis.

Mycobacterium tuberculosis can cocatabolize a range of carbon sources. Fatty acids are among the carbons available inside the host's macrophages. Here, we investigated the metabolic changes of the fatty acid-induced dormancy-like state of M. tuberculosis and its involvement in the acquisition of drug tolerance. We conducted metabolomics profiling using a phosphoenolpyruvate carboxykinase (PEPCK)-deficient M. tuberculosis strain in an acetate-induced dormancy-like state, highlighting an overaccumulation of methylcitrate cycle (MCC) intermediates that correlates with enhanced drug tolerance against isoniazid and bedaquiline. Further metabolomics analyses of two M. tuberculosis mutants, an ICL knockdown (KD) strain and PrpD knockout (KO) strain, each lacking an MCC enzyme-isocitrate lyase (ICL) and 2-methylcitrate dehydratase (PrpD), respectively-were conducted after treatment with antibiotics. The ICL KD strain, which lacks the last enzyme of the MCC, showed an overaccumulation of MCC intermediates and a high level of drug tolerance. The PrpD KO strain, however, failed to accumulate MCC intermediates as it lacks the second step of the MCC and showed only a minor level of drug tolerance compared to the ICL KD mutant and its parental strain (CDC1551). Notably, addition of authentic 2-methylisocitrate, an MCC intermediate, improved the M. tuberculosis drug tolerance against antibiotics even in glycerol medium. Furthermore, wild-type M. tuberculosis displayed levels of drug tolerance when cultured in acetate medium significantly greater than those in glycerol medium. Taken together, the fatty acid-induced dormancy-like state remodels the central carbon metabolism of M. tuberculosis that is functionally relevant to acquisition of M. tuberculosis drug tolerance. IMPORTANCE Understanding the mechanisms underlying M. tuberculosis adaptive strategies to achieve drug tolerance is crucial for the identification of new targets and the development of new drugs. Here, we show that acetate medium triggers a drug-tolerant state in M. tuberculosis when challenged with antituberculosis (anti-TB) drugs. This carbon-induced drug-tolerant state is linked to an accumulation of the methylcitrate cycle (MCC) intermediates, whose role was previously known as a detox pathway for propionate metabolism. Three mutant strains with mutations in gluconeogenesis and MCC were used to investigate the correlation between drug tolerance and the accumulation of MCC metabolites. We herein report a new role of the MCC used to provide a survival advantage to M. tuberculosis as a species against both anti-TB drugs upon specific carbon sources.

Isolation and Characterization of Extracellular Vesicle from Mesenchymal Stem Cells of the Epidural Fat of the Spine.

STUDY DESIGN: An experimental study with extracellular vesicles (EVs) from mesenchymal stem cell (MSC) of the epidural fat (EF) of the spine. PURPOSE: This study aims to isolate the exosomes from epidural fat-derived mesenchymal stem cells (EF-MSCs) and fully characterize the EF-MSC-EVs. OVERVIEW OF LITERATURE: EF-MSCs were reported in 2019, and a few studies have shown the positive outcomes of using EF-MSCs to treat specific spine pathologies. However, MSCs have significant limitations for conducting basic studies or developing therapeutic agents. Although EVs are an emerging research topic, no studies have focused on EVs, especially exosomes, from EF and EF-MSCs. METHODS: In this study, we isolated the exosomes using the tangential flow filtration (TFF) system with exosome-depleted fetal bovine serum and performed the characterization tests via western blotting, reverse transcription-polymerase chain reaction, nanoparticle tracking analysis (NTA), and transmission electron microscopy. RESULTS: In transmission electron microscopy, the exosome had a diameter of approximately 100-200 nm and had a spherical shape, whereas in the NTA, the exosome had an average diameter of 142.8 nm with a concentration of 1.27×1010 particles/mL. The flow cytometry analysis showed the expression of CD63 and CD81. The western blotting analysis showed the positive markers. CONCLUSIONS: These findings showed that isolating the exosomes via TFF resulted in high-quality EF-MSC exosome yield. Further studies with exosomes from EF-MSC are needed to evaluate the function and role of the EF tissue.

Therapeutic Potential of Mesenchymal Stem Cells (MSCs) and MSC-Derived Extracellular Vesicles for the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is a life-threatening condition that leads to permanent disability with partial or complete loss of motor, sensory, and autonomic functions. SCI is usually caused by initial mechanical insult, followed by a cascade of several neuroinflammation and structural changes. For ameliorating the neuroinflammatory cascades, MSC has been regarded as a therapeutic agent. The animal SCI research has demonstrated that MSC can be a valuable therapeutic agent with several growth factors and cytokines that may induce anti-inflammatory and regenerative effects. However, the therapeutic efficacy of MSCs in animal SCI models is inconsistent, and the optimal method of MSCs remains debatable. Moreover, there are several limitations to developing these therapeutic agents for humans. Therefore, identifying novel agents for regenerative medicine is necessary. Extracellular vesicles are a novel source for regenerative medicine; they possess nucleic acids, functional proteins, and bioactive lipids and perform various functions, including damaged tissue repair, immune response regulation, and reduction of inflammation. MSC-derived exosomes have advantages over MSCs, including small dimensions, low immunogenicity, and no need for additional procedures for culture expansion or delivery. Certain studies have demonstrated that MSC-derived extracellular vesicles (EVs), including exosomes, exhibit outstanding chondroprotective and anti-inflammatory effects. Therefore, we reviewed the principles and patho-mechanisms and summarized the research outcomes of MSCs and MSC-derived EVs for SCI, reported to date.

Serum-Derived Neuronal Exosomal microRNAs as Stress-Related Biomarkers in an Atopic Dermatitis Model.

Chronic allergic inflammatory skin disease-atopic dermatitis (AD)-is characterized by eczema, pruritus, xeroderma, and lichenification. Psychological stress is one cause of this disease; however, psychological stress will also result from the presence of AD symptoms. Previous studies have shown that psychological stress triggers neuroinflammation in the brain, where microRNAs (miRNAs) in the neuronal exosomes (nEVs) were analyzed to identify the composition of the miRNAs in the nEVs and how they were altered by AD. In this study, the AD model was induced by treatment with 2,4-dinitrochlorobenzene (DNCB). The expression patterns of neuroinflammation markers, such as brain-derived neurotrophic factor, cyclooxygenase-2, and glial fibrillary acidic protein, were subsequently evaluated over time. Among these groups, there was a significant difference in DNCB 14 days expression compared with the control; therefore, nEVs were isolated from serum and next-generation sequencing was performed. The results demonstrate that 9 miRNAs were upregulated and 16 were downregulated in the DNCB 14 days compared with the control. Previous studies have shown that some of these miRNAs are associated with stress and stress-induced depression, which suggests that the miRNAs in nEVs may also be stress-related biomarkers.

Serum-Derived Neuronal Exosomal miRNAs as Biomarkers of Acute Severe Stress.

Stress is the physical and psychological tension felt by an individual while adapting to difficult situations. Stress is known to alter the expression of stress hormones and cause neuroinflammation in the brain. In this study, miRNAs in serum-derived neuronal exosomes (nEVs) were analyzed to determine whether differentially expressed miRNAs could be used as biomarkers of acute stress. Specifically, acute severe stress was induced in Sprague-Dawley rats via electric foot-shock treatment. In this acute severe-stress model, time-dependent changes in the expression levels of stress hormones and neuroinflammation-related markers were analyzed. In addition, nEVs were isolated from the serum of control mice and stressed mice at various time points to determine when brain damage was most prominent; this was found to be 7 days after foot shock. Next-generation sequencing was performed to compare neuronal exosomal miRNA at day 7 with the neuronal exosomal miRNA of the control group. From this analysis, 13 upregulated and 11 downregulated miRNAs were detected. These results show that specific miRNAs are differentially expressed in nEVs from an acute severe-stress animal model. Thus, this study provides novel insights into potential stress-related biomarkers.

Mesenchymal Stem Cell Exosomes Derived from Feline Adipose Tissue Enhance the Effects of Anti-Inflammation Compared to Fibroblasts-Derived Exosomes.

Adipose tissue-derived mesenchymal stem cells (AD-MSCs) release extracellular vesicles such as exosomes, apoptotic bodies, and microparticles. In particular, exosomes are formed inside cells via multivesicular bodies (MVBs), thus their protein, DNA, and RNA content are similar to those of the parent cells. Exosome research is rapidly expanding, with an increase in the number of related publications observed in recent years; therefore, the function and application of MSC-derived exosomes could emerge as cell-free therapeutics. Exosomes have been isolated from feline AD-MSCs and feline fibroblast cell culture media using ultracentrifugation. Feline exosomes have been characterized by FACS, nanoparticle tracking analysis, and transmission electron microscopy imaging. Moreover, cytokine levels were detected by sandwich enzyme-linked immunosorbent assay in exosomes and LPS-induced THP-1 macrophages. The size of the isolated exosomes was that of a typical exosome, i.e., approximately 150 nm, and they expressed tetraspanins CD9 and CD81. The anti-inflammatory factor IL-10 was increased in feline AD-MSC-derived exosomes. However, pro-inflammatory factors such as IL-1ß, IL-8, IL-2, RANTES, and IFN-gamma were significantly decreased in feline AD-MSC-derived exosomes. This was the first demonstration that feline AD-MSC-derived exosomes enhance the inflammatory suppressive effects and have potential for the treatment of immune diseases or as an inflammation-inhibition therapy.

Phosphoenolpyruvate depletion mediates both growth arrest and drug tolerance of Mycobacterium tuberculosis in hypoxia.

Mycobacterium tuberculosis (Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic effects and can mutate to become drug resistant (DR), our conventional tuberculosis (TB) treatment is not effective. Thus, a novel strategy to kill NR Mtb is required. Accumulating evidence has shown that repetitive exposure to sublethal doses of antibiotics enhances the level of drug tolerance, implying that NR Mtb is formed by adaptive metabolic remodeling. As such, metabolic modulation strategies to block the metabolic remodeling needed to form NR Mtb have emerged as new therapeutic options. Here, we modeled in vitro NR Mtb using hypoxia, applied isotope metabolomics, and revealed that phosphoenolpyruvate (PEP) is nearly completely depleted in NR Mtb. This near loss of PEP reduces PEP-carbon flux toward multiple pathways essential for replication and drug sensitivity. Inversely, supplementing with PEP restored the carbon flux and the activities of the foregoing pathways, resulting in growth and heightened drug susceptibility of NR Mtb, which ultimately prevented the development of DR. Taken together, PEP depletion in NR Mtb is associated with the acquisition of drug tolerance and subsequent emergence of DR, demonstrating that PEP treatment is a possible metabolic modulation strategy to resensitize NR Mtb to conventional TB treatment and prevent the emergence of DR.

Transcriptomic and Metabolomic Analysis Revealed Roles of Yck2 in Carbon Metabolism and Morphogenesis of Candida albicans.

Candida albicans is a part of the normal microbiome of human mucosa and is able to thrive in a wide range of host environments. As an opportunistic pathogen, the virulence of C. albicans is tied to its ability to switch between yeast and hyphal morphologies in response to various environmental cues, one of which includes nutrient availability. Thus, metabolic flexibility plays an important role in the virulence of the pathogen. Our previous study has shown that C. albicans Yeast Casein Kinase 2 (CaYck2) regulates the yeast-to-hyphal switch, but its regulatory mechanisms remain unknown. This study further elucidated the role of Yck2 in governing morphology and carbon metabolism by analyzing the transcriptome and metabolome of the C. albicans YCK2 deletion mutant strain (yck2Δ strain) in comparison to the wild type strain. Our study revealed that loss of CaYck2 perturbs carbon metabolism, leading to a transcriptional response that resembles a transcriptional response to glucose starvation with coinciding intracellular accumulation of glucose and depletion of TCA cycle metabolites. This shift in the metabolome is likely mediated by derepression of glucose-repressed genes in the Mig1/2-mediated glucose sensing pathway and by downregulation of glycolytic genes, possibly through the Rgt1-mediated SRR pathway. In addition, genes involved in beta-oxidation, glyoxylate cycle, oxidative stress response, and arginine biosynthesis were upregulated in the yck2Δ strain, which is highly reminiscent of C. albicans engulfment by macrophages. This coincides with an increase in arginine degradation intermediates in the yck2Δ strain, suggesting arginine catabolism as a potential mechanism of CaYck2-mediated filamentation as seen during C. albicans escape from macrophages. Transcriptome analysis also shows differential expression of hyphal transcriptional regulators Nrg1 and Ume6. This suggests dysregulation of hyphal initiation and elongation in the yck2Δ strain which may lead to the constitutive pseudohyphal phenotype of this strain. Metabolome analysis also detected a high abundance of methyl citrate cycle intermediates in the yck2Δ strain, suggesting the importance of CaYck2 in this pathway. Taken together, we discovered that CaYck2 is an integral piece of carbon metabolism and morphogenesis of C. albicans.

Comparisons of Extracellular Vesicles from Human Epidural Fat-Derived Mesenchymal Stem Cells and Fibroblast Cells.

Extracellular vesicles (EVs) are generated and secreted by cells into the circulatory system. Stem cell-derived EVs have a therapeutic effect similar to that of stem cells and are considered an alternative method for cell therapy. Accordingly, research on the characteristics of EVs is emerging. EVs were isolated from human epidural fat-derived mesenchymal stem cells (MSCs) and human fibroblast culture media by ultracentrifugation. The characterization of EVs involved the typical evaluation of cluster of differentiation (CD antigens) marker expression by fluorescence-activated cell sorting, size analysis with dynamic laser scattering, and morphology analysis with transmission electron microscopy. Lastly, the secreted levels of cytokines and chemokines in EVs were determined by a cytokine assay. The isolated EVs had a typical size of approximately 30-200 nm, and the surface proteins CD9 and CD81 were expressed on human epidural fat MSCs and human fibroblast cells. The secreted levels of cytokines and chemokines were compared between human epidural fat MSC-derived EVs and human fibroblast-derived EVs. Human epidural fat MSC-derived EVs showed anti-inflammatory effects and promoted macrophage polarization. In this study, we demonstrated for the first time that human epidural fat MSC-derived EVs exhibit inflammatory suppressive potency relative to human fibroblast-derived EVs, which may be useful for the treatment of inflammation-related diseases.

SP_0916 Is an Arginine Decarboxylase That Catalyzes the Synthesis of Agmatine, Which Is Critical for Capsule Biosynthesis in Streptococcus pneumoniae.

The global burden of invasive pneumococcal diseases, including pneumonia and sepsis, caused by Streptococcus pneumoniae, a Gram-positive bacterial pathogen, remains a major global health risk. The success of pneumococcus as a pathogen can be attributed to its ability to regulate the synthesis of capsular polysaccharide (CPS) during invasive disease. We previously reported that deletion of a putative lysine decarboxylase (LDC; ΔSP_0916) in pneumococcal serotype 4 (TIGR4) results in reduced CPS. SP_0916 locus is annotated as either an arginine or a LDC in pneumococcal genomes. In this study, by biochemical characterization of the recombinant SP_0916, we determined the substrate specificity of SP_0916 and show that it is an arginine decarboxylase (speA/ADC). We also show that deletion of the polyamine transporter (potABCD) predicted to import putrescine and spermidine results in reduced CPS, while deletion of spermidine synthase (speE) for the conversion of putrescine to spermidine had no impact on the capsule. Targeted metabolomics identified a correlation between reduced levels of agmatine and loss of capsule in ΔspeA and ΔpotABCD, while agmatine levels were comparable between the encapsulated TIGR4 and ΔspeE. Exogenous supplementation of agmatine restored CPS in both ΔpotABCD and ΔspeA. These results demonstrate that agmatine is critical for regulating the CPS, a predominant virulence factor in pneumococci.

Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries.

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.

A nutrient-limited screen unmasks rifabutin hyperactivity for extensively drug-resistant Acinetobacter baumannii.

Industry screens of large chemical libraries have traditionally relied on rich media to ensure rapid bacterial growth in high-throughput testing. We used eukaryotic, nutrient-limited growth media in a compound screen that unmasked a previously unknown hyperactivity of the old antibiotic, rifabutin (RBT), against highly resistant Acinetobacter baumannii. In nutrient-limited, but not rich, media, RBT was 200-fold more potent than rifampin. RBT was also substantially more effective in vivo. The mechanism of enhanced efficacy was a Trojan horse-like import of RBT, but not rifampin, through fhuE, only in nutrient-limited conditions. These results are of fundamental importance to efforts to discover antibacterial agents.

Selection and Characterization of Monoclonal Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through a Human Synthetic Fab Phage Display Library Panning.

Since its first report in the Middle East in 2012, the Middle East respiratory syndrome-coronavirus (MERS-CoV) has become a global concern due to the high morbidity and mortality of individuals infected with the virus. Although the majority of MERS-CoV cases have been reported in Saudi Arabia, the overall risk in areas outside the Middle East remains significant as inside Saudi Arabia. Additional pandemics of MERS-CoV are expected, and thus novel tools and reagents for therapy and diagnosis are urgently needed. Here, we used phage display to develop novel monoclonal antibodies (mAbs) that target MERS-CoV. A human Fab phage display library was panned against the S2 subunit of the MERS-CoV spike protein (MERS-S2P), yielding three unique Fabs (S2A3, S2A6, and S2D5). The Fabs had moderate apparent affinities (Half maximal effective concentration (EC50 = 123-421 nM) for MERS-S2P, showed no cross-reactivity to spike proteins from other CoVs, and were non-aggregating and thermostable (Tm = 61.5-80.4 °C). Reformatting the Fabs into IgGs (Immunoglobulin Gs) greatly increased their apparent affinities (KD = 0.17-1.2 nM), presumably due to the effects of avidity. These apparent affinities were notably higher than that of a previously reported anti-MERS-CoV S2 reference mAb (KD = 8.7 nM). Furthermore, two of the three mAbs (S2A3 and S2D5) bound only MERS-CoV (Erasmus Medical Center (EMC)) and not other CoVs, reflecting their high binding specificity. However, the mAbs lacked MERS-CoV neutralizing activity. Given their high affinity, specificity, and desirable stabilities, we anticipate that these anti-MERS-CoV mAbs would be suitable reagents for developing antibody-based diagnostics in laboratory or hospital settings for point-of-care testing.