Design, Synthesis, Antibacterial Activity, and Mechanisms of Novel Benzofuran Derivatives Containing Disulfide Moieties.

The unsatisfactory effects of conventional bactericides and antimicrobial resistance have increased the challenges in managing plant diseases caused by bacterial pests. Here, we report the successful design and synthesis of benzofuran derivatives using benzofuran as the core skeleton and splicing the disulfide moieties commonly seen in natural substances with antibacterial properties. Most of our developed benzofurans displayed remarkable antibacterial activities to frequently encountered pathogens, including Xanthomonas oryzae pv oryzae (Xoo), Xanthomonas oryzae pv oryzicola (Xoc), and Xanthomonas axonopodis pv citri (Xac). With the assistance of the three-dimensional quantitative constitutive relationship (3D-QSAR) model, the optimal compound V40 was obtained, which has better in vitro antibacterial activity with EC50 values of 0.28, 0.56, and 10.43 µg/mL against Xoo, Xoc, and Xac, respectively, than those of positive control, TC (66.41, 78.49, and 120.36 µg/mL) and allicin (8.40, 28.22, and 88.04 µg/mL). Combining the results of proteomic analysis and enzyme activity assay allows the antibacterial mechanism of V40 to be preliminarily revealed, suggesting its potential as a versatile bactericide in combating bacterial pests in the future.

LINC01806 Promotes Breast Cancer Growth and Metastasis via Sponging miR-1286 to Disinhibit ZEB1 Expression.

Breast cancer (BC) is the most abundant and aggressive cancer that impacts millions of women with poorly understood mechanisms. Here, we aimed to investigate the function of LINC01806 in BC development. Human BC tissues and nearby normal specimens were taken from diagnosed BC patients. The expression levels of LINC01806, miR-1286, ZEB1, and EMT-related markers were evaluated by qRT-PCR and western blotting. FISH was used to visualize the subcellular localization of LINC01806. The viability, proliferation, migration and invasion capacities of BC cells were assessed by MTT, colony formation, and transwell assays. Interactions among LINC01806, miR-1286 and ZEB1 were validated by dual luciferase assay. The unpaired Student t-test (for two groups) or one-way ANOVA following with Tukey post-hoc test (for more than three groups) was employed for statistical analysis. LINC01806 level was elevated in BC tissues. Knockdown of LINC01806 suppressed EMT process and BC cell proliferation, migration, and invasion. LINC01806 co-localized and directly bound with miR-1286 in the cytoplasm. MiR-1286 inhibitor blocked the effects of LINC01806 knockdown on BC cell EMT, proliferation and migration. MiR-1286 targeted ZEB1 and overexpression of ZEB1 blocked the regulatory functions of miR-1286 mimics in BC. LINC01806 facilitates EMT and accelerates BC cell proliferation, migration, and invasion via acting as miR-1286 sponge to disinhibit ZEB1 expression.

Pulse-shaped high-energy and high-average-power fiber laser in the nanosecond regime.

A high-energy and high-average-power pulsed fiber laser has been investigated in a master oscillator power amplifier (MOPA) configuration seeding with a diode laser at a programmed pulse duration of ∼250ns. The fiber amplifier successfully demonstrates the pulse with 21.4 mJ at the repetition rate of 50 kHz and a maximum average output power of 1535 W with a slope efficiency of 81.6% at 250 kHz. To overcome fiber nonlinearities such as stimulated Raman scattering (SRS) and self-phase modulation (SPM), extra-large mode area ytterbium (Yb)-doped step-index dual cladding fiber has been utilized as gain fiber in the MOPA laser system. The gain saturation effect in the power amplifier was greatly mitigated by the programmed seed signal. This pulse-shaped MOPA system can provide practical applications in many fields such as laser cleaning, paint stripping, and other applications requiring special pulse shapes.

Ethylicin Inhibition of Xanthomonas oryzae pv. oryzicola In Vitro and In Vivo.

Infestation of rice with the bacterium Xanthomonas oryzae pv. oryzicola (Xoc) causes the serious disease bacterial leaf streak (BLS). We studied the effect of ethylicin, a broad-spectrum bactericide, on Xoc both in vivo and in vitro. Ethylicin increases the defensive enzyme activities and defensive genes expression of rice. Ethylicin also significantly inhibited Xoc activity in vitro compared with other commercial bactericides. The half-maximal effective concentration (EC50) of ethylicin was 2.12 µg/mL. It has been shown that ethylicin can inhibit Xoc quorum sensing through the production of extracellular polysaccharides and enzymes, which disrupt the Xoc cell membrane. We used proteomic analysis to identify two oxidative phosphorylation pathway proteins (ACU12_RS13405 and ACU12_RS13355) which affected the virulence of Xoc and validated them using quantitative real-time polymerase chain reaction (qRT-PCR). The results indicate that ethylicin can increase the defense responses of rice and control Xoc proliferation.

YAP-VGLL4 antagonism defines the major physiological function of the Hippo signaling effector YAP.

The Hippo-YAP signaling pathway plays a critical role in development, homeostasis, regeneration, and tumorigenesis by converging on YAP, a coactivator for the TEAD family DNA-binding transcription factors, to regulate downstream transcription programs. Given its pivotal role as the nuclear effector of the Hippo pathway, YAP is indispensable in multiple developmental and tissue contexts. Here we report that the essentiality of YAP in liver and lung development can be genetically bypassed by simultaneous inactivation of the TEAD corepressor VGLL4. This striking antagonistic epistasis suggests that the major physiological function of YAP is to antagonize VGLL4. We further show that the YAP-VGLL4 antagonism plays a widespread role in regulating Hippo pathway output beyond normal development, as inactivation of Vgll4 dramatically enhanced intrahepatic cholangiocarcinoma formation in Nf2-deficient livers and ameliorated CCl4-induced damage in normal livers. Interestingly, Vgll4 expression is temporally regulated in development and regeneration and, in certain contexts, provides a better indication of overall Hippo pathway output than YAP phosphorylation. Together, these findings highlight the central importance of VGLL4-mediated transcriptional repression in Hippo pathway regulation and inform potential strategies to modulate Hippo signaling in cancer and regenerative medicine.

Multiphase coalescence mediates Hippo pathway activation.

The function of biomolecular condensates is often restricted by condensate dissolution. Whether condensates can be suppressed without condensate dissolution is unclear. Here, we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and their coalescence into a common phase provides a mode of counteracting the function of biomolecular condensates without condensate dissolution. Specifically, the negative regulator SLMAP forms Hippo-inactivating condensates to facilitate pathway inhibition by the STRIPAK complex. In response to cell-cell contact or osmotic stress, the positive regulators AMOT and KIBRA form Hippo-activating condensates to facilitate pathway activation. The functionally antagonizing SLMAP and AMOT/KIBRA condensates further coalesce into a common phase to inhibit STRIPAK function. These findings provide a paradigm for restricting the activity of biomolecular condensates without condensate dissolution, shed light on the molecular principles of multiphase organization, and offer a conceptual framework for understanding upstream regulation of the Hippo signaling pathway.

Synthesis, Antibacterial Activity, and Mechanisms of Novel Indole Derivatives Containing Pyridinium Moieties.

The development of effective antibacterial agents equipped with novel action modes and unique skeletons starting from natural compounds serves as an important strategy in the modern pesticide industry. Disclosed here are a series of novel indole derivatives containing pyridinium moieties and their antibacterial activity evaluation against two prevalent phytopathogenic bacteria, Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo). A three-dimensional (3D)-QSAR model was adopted to discover higher activity like title compounds based on the Xoc antibacterial activity of the tested compounds. Compound 43 was consequently designed, and it displayed higher antibacterial activity as expected with the half-maximal effective concentration EC50 values of 1.0 and 1.9 µg/mL for Xoo and Xoc, respectively, which were better than those of the commercial drug thiodiazole copper (TC) (72.9 and 87.5 µg/mL). Under greenhouse conditions, the results of a rice in vivo pot experiment indicated that the protective and curative activities of compound 43 against rice bacterial leaf streak (BLS) and rice bacterial blight (BLB) were 45.0 and 44.0% and 42.0 and 39.3%, respectively, which were better than those of the commercial agent thiodiazole copper (38.0 and 37.9%, 38.6 and 37.0%) as well. Scanning electron microscopy images, defense enzyme activity tests, and proteomic techniques were utilized in a preliminary mechanism study, suggesting that compound 43 shall modulate and interfere with the physiological processes and functions of pathogenic bacteria.

Synthesis, Antibacterial Activity, and Action Mechanism of Novel Sulfonamides Containing Oxyacetal and Pyrimidine.

Bacterial leaf blight (BLB) and bacterial leaf streak (BLS) are two serious bacterial diseases caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively. However, the control of these diseases by conventional pesticides remains challenging due to development of resistances. We aimed to address this pending problem and developed a series of novel pyrimidine sulfonamide derivatives. Structurally, title compounds bear a unique oxyacetal group, which has a proven immune-activating effect. Compound E35 designed based on the 3D-QSAR model was demonstrated as the optimal in vitro activity against Xoo and Xoc, with EC50 values of 26.7 and 30.8 mg/L, respectively, which were higher than the positive controls bismerthiazol (29.9 and 32.7 mg/L) and thiodiazole copper (30.5 and 36.4 mg/L). On the prevention level, the biological activity test showed compound E35 had superior protective activity (43.7%) on BLS to thiodiazole copper (32.1%). The defense enzymes and proteomics results suggested that compound E35 could be a versatile candidate as it improved plant's resistance to disease.

Synthesis and Antibacterial Activities of 2-Oxo-N-phenylacetamide Derivatives Containing a Dissulfone Moiety Target on Clp.

Rice bacterial blight and rice bacterial streak are two serious rice diseases and have caused great harm to the production of rice all over the world. To develop an efficient antibacterial agent with a novel target, a series of novel 2-oxo-N-phenylacetamide derivatives containing a dissulfone moiety were synthesized, and their antibacterial activities were evaluated. Among them, compound D14 exhibited the best antibacterial activities, especially against Xoo and Xoc with EC50 values of 0.63 and 0.79 mg/L, respectively, which were much better than the commercial control of bismerthiazol (BT) (76.59 and 83.35 mg/L, respectively) and thiodiazole copper (TC) (91.72 and 114.00 mg/L, respectively). Meanwhile, compound D14 can interact with a CRP-like protein (Clp) of Pxo99A and show strong binding activity with Xoo-Clp with a Kd value of 0.52 µM, which was far superior to the corresponding Kd values of BT (183.94 µM) and TC (222.58 µM). Treatment of D14 and deletion of the clp gene could significantly reduce the expression of the clp gene and attenuate the virulence of pathogenic bacteria. These results indicated that compound D14 could be used as a potential novel agricultural bactericide and Clp can be used as a target protein for the control of plant bacterial diseases. This work provided reliable support for developing novel antibacterial agents based on Clp as a target protein.

Erratum: Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy: Erratum.

[This corrects the article DOI: 10.7150/thno.46006.].

YAP/TAZ drives cell proliferation and tumour growth via a polyamine-eIF5A hypusination-LSD1 axis.

Metabolic reprogramming is central to oncogene-induced tumorigenesis by providing the necessary building blocks and energy sources, but how oncogenic signalling controls metabolites that play regulatory roles in driving cell proliferation and tumour growth is less understood. Here we show that oncogene YAP/TAZ promotes polyamine biosynthesis by activating the transcription of the rate-limiting enzyme ornithine decarboxylase 1. The increased polyamine levels, in turn, promote the hypusination of eukaryotic translation factor 5A (eIF5A) to support efficient translation of histone demethylase LSD1, a transcriptional repressor that mediates a bulk of YAP/TAZ-downregulated genes including tumour suppressors in YAP/TAZ-activated cells. Accentuating the importance of the YAP/TAZ-polyamine-eIF5A hypusination-LSD1 axis, inhibiting polyamine biosynthesis or LSD1 suppressed YAP/TAZ-induced cell proliferation and tumour growth. Given the frequent upregulation of YAP/TAZ activity and polyamine levels in diverse cancers, our identification of YAP/TAZ as an upstream regulator and LSD1 as a downstream effector of the oncometabolite polyamine offers a molecular framework in which oncogene-induced metabolic and epigenetic reprogramming coordinately drives tumorigenesis, and suggests potential therapeutic strategies in YAP/TAZ- or polyamine-dependent human malignancies.

()-Epigallocatechin-3-Gallate Inhibits EBV Lytic Replication via Targeting LMP1-Mediated MAPK Signal Axes.

EpsteinBarr virus (EBV)-encoded latent membrane protein 1 (LMP1) plays an important oncogenic role in the viral latent infection. Recently, increasing evidence indicates that the high expression of LMP1 during EBV lytic cycle is related to the viral lytic replication. However, the mechanism by which LMP1 regulates EBV lytic replication remains unclear. ()-Epigallocatechin-3-gallate (EGCG) prevents carcinogenesis by directly targeting numerous membrane proteins and effectively inhibits EBV lytic cascade. Here, we demonstrated that LMP1 promotes EBV lytic replication through the downstream signal molecules MAPKs, including ERKs, p38, and JNKs. LMP1 induces the phosphorylation of p53 through MAPKs to enhance the ability of wild-type p53 (wt-p53) to activate expression of BZLF1 gene, while the JNKs/c-Jun signal axis appears to be involved in EBV lytic replication induced by LMP1 in p53 mutant manner. We provided the first evidence that EGCG directly targets the viral membrane LMP1 (K d=0.36 M, n=1) using fluorescence quenching, isothermal titration calorimetry (ITC) assay, and CNBR-activated Sepharose 4B pull-down affinity chromatography. Furthermore, we revealed that EGCG inhibits EBV lytic replication via suppressing LMP1 and thus blocking the downstream MAPKs/wt-p53 signal axis in AGS-EBV cells and JNKs/c-Jun signal axis in p53 mutant B95.8 cells. Our study, for the first time, reports the binding and inhibitory efficacy of EGCG to the LMP1, which is a key oncoprotein encoded by EBV. These findings suggest the novel function of LMP1 in the regulation of EBV lytic cycle and reveal the new role of EGCG in EBV-associated malignancies through suppressing viral reactivation.

Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy.

Generating oxidative stress is a critical mechanism by which host cells defend against infection by pathogenic microorganisms. Radiation resistance is a critical problem in radiotherapy against cancer. Epstein-Barr virus (EBV) is a cancer-causing virus and its reactivation plays an important role in the development of EBV-related tumors. This study aimed to explore the inner relationship and regulatory mechanism among oxidative stress, EBV reactivation, and radioresistance and to identify new molecular subtyping models and treatment strategies to improve the therapeutic effects of radiotherapy. Methods: ROS, NADP+/NADPH, and GSSG/GSH were detected to evaluate the oxidative stress of cells. 8-OHdG is a reliable oxidative stress marker to evaluate the oxidative stress in patients. Its concentration in serum was detected using an ELISA method and in biopsies was detected using IHC. qPCR array was performed to evaluate the expression of essential oxidative stress genes. qPCR, Western blot, and IHC were used to measure the level of EBV reactivation in vitro and in vivo. A Rta-IgG ELISA kit and EBV DNA detection kit were used to analyze the reactivation of EBV in serum from NPC patients. NPC tumor tissue microarrays was used to investigate the prognostic role of oxidative stress and EBV reactivation. Radiation resistance was evaluated by a colony formation assay. Xenografts were treated with NAC, radiation, or a combination of NAC and radiation. EBV DNA load of tumor tissue was evaluated using an EBV DNA detection kit. Oxidative stress, EBV reactivation, and the apoptosis rate in tumor tissues were detected by using 8-OHdG, EAD, and TUNEL assays, respectively. Results: We found that EBV can induce high oxidative stress, which promotes its reactivation and thus leads to radioresistance. Basically, EBV caused NPC cells to undergo a process of 'Redox Resetting' to acquire a new redox status with higher levels of ROS accumulation and stronger antioxidant systems by increasing the expression of the ROS-producing enzyme, NOX2, and the cellular master antioxidant regulator, Nrf2. Also, EBV encoded driving protein LMP1 promotes EBV reactivation through production of ROS. Furthermore, high oxidative stress and EBV reactivation were positively associated with poor overall survival of patients following radiation therapy and were significant related to NPC patients' recurrence and clinical stage. By decreasing oxidative stress using an FDA approved antioxidant drug, NAC, sensitivity of tumors to radiation was increased. Additionally, 8-OHdG and EBV DNA could be dual prognostic markers for NPC patients. Conclusions: Oxidative stress mediates EBV reactivation and leads to radioresistance. Targeting oxidative stress can provide therapeutic benefits to cancer patients with radiation resistance. Clinically, we, for the first time, generated a molecular subtyping model for NPC relying on 8-OHdG and EBV DNA level. These dual markers could identify patients who are at a high risk of poor outcomes but who might benefit from the sequential therapy of reactive oxygen blockade followed by radiation therapy, which provides novel perspectives for the precise treatment of NPC.

The oncometabolite L-2-hydroxyglutarate is a common product of dipteran larval development.

The oncometabolite L-2-hydroxyglutarate (L-2HG) is considered an abnormal product of central carbon metabolism that is capable of disrupting chromatin architecture, mitochondrial metabolism, and cellular differentiation. Under most circumstances, mammalian tissues readily dispose of this compound, as aberrant L-2HG accumulation induces neurometabolic disorders and promotes renal cell carcinomas. Intriguingly, Drosophila melanogaster larvae were recently found to accumulate high L-2HG levels under normal growth conditions, raising the possibility that L-2HG plays a unique role in insect metabolism. Here we explore this hypothesis by analyzing L-2HG levels in 18 insect species. While L-2HG was present at low-to-moderate levels in most of these species (<100 pmol/mg; comparable to mouse liver), dipteran larvae exhibited a tendency to accumulate high L-2HG concentrations (>100 pmol/mg), with the mosquito Aedes aegypti, the blow fly Phormia regina, and three representative Drosophila species harboring concentrations that exceed 1 nmol/mg - levels comparable to those measured in mutant mice that are unable to degrade L-2HG. Overall, our findings suggest that one of the largest groups of animals on earth commonly generate high concentrations of an oncometabolite during juvenile growth, hint at a role for L-2HG in the evolution of dipteran development, and raise the possibility that L-2HG metabolism could be targeted to restrict the growth of key disease vectors and agricultural pests.

Mouse Umbilical Cord Mesenchymal Stem Cell Paracrine Alleviates Renal Fibrosis in Diabetic Nephropathy by Reducing Myofibroblast Transdifferentiation and Cell Proliferation and Upregulating MMPs in Mesangial Cells.

Transplantation of umbilical cord mesenchymal stem cells (UC-MSCs) is currently considered a novel therapeutic strategy for diabetic nephropathy (DN). However, the mechanisms by which UC-MSCs ameliorate renal fibrosis in DN are not well understood. Herein, we firstly investigated the therapeutic effects of mouse UC-MSC infusion on kidney structural and functional impairment in streptozotocin- (STZ-) induced diabetic mice. We found that the repeated injection with mUC-MSCs alleviates albuminuria, glomerulus injury, and fibrosis in DN mouse models. Next, mesangial cells were exposed to 5.6 mM glucose, 30 mM glucose, or mUC-MSC-conditioned medium, and then we performed western blotting, immunofluorescence, wound healing assay, and cell proliferation assay to measure extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs), myofibroblast transdifferentiation (MFT), and cell proliferation. We demonstrated that mUC-MSC paracrine decreased the deposition of fibronectin and collagen I by inhibiting TGF-ß1-triggered MFT and cell proliferation mediated by PI3K/Akt and MAPK signaling pathways, and elevating the levels of MMP2 and MMP9. Importantly, we provided evidence that the antifibrosis role of mUC-MSC paracrine in DN might be determined by exosomes shed by MSCs. Together, these findings reveal the mechanisms underlying the therapeutic effects of UC-MSCs on renal fibrosis in DN and provide the evidence for DN cell-free therapy based on UC-MSCs in the future.

Human mesenchymal stem cells-derived conditioned medium inhibits hypoxia-induced death of neonatal porcine islets by inducing autophagy.

BACKGROUND: The dysfunction of islet grafts is generally attributed to hypoxia-induced damage. Mesenchymal stem cells (MSCs) are currently thought to effectively protect cells from various risk factors via regulating autophagy. In our study, we investigated if human umbilical cord-derived MSCs could ameliorate hypoxia-induced apoptosis in porcine islets by modulating autophagy, and we explored the underlying mechanisms. METHODS: Neonatal porcine islet cell clusters (NICCs) were cultured with human umbilical cord-derived MSC conditioned medium (huc-MSC-CM) and RPMI-1640 medium (control) under hypoxic conditions (1% O2 ) in vitro. NICCs were treated with 3-methyladenine (3-MA) and chloroquine (CQ) to examine the role of huc-MSC-CM in regulating autophagy. Finally, the levels of several cytokines secreted by huc-MSCs were detected by ELISAs, and the corresponding inhibitors were applied to investigate which cytokine mediates the protective effects of huc-MSC-CM. The effects of huc-MSC-CM on NICCs viability and autophagy were examined using AO/PI staining, flow cytometry analysis, transmission electron microscopy (TEM) and confocal fluorescence microscopy analysis. The insulin secretion of NICCs was tested with an insulin immunoradiometric assay kit. RESULTS: Compared to the control, the huc-MSC-CM treatment improved the viability of NICCs, inhibited apoptosis, increased autophagic activity and the levels of PI3K class III and phosphorylated Akt, while the ratio of phosphorylated mTOR/mTOR was reduced. These changes were reversed by CQ and 3-MA treatments. High concentrations of IL-6 were detected in hu-MSC-CM. Furthermore, recombinant IL-6 pre-treatment exerted similar effects as huc-MSC-CM, and these effects were reversed by a specific inhibitor of IL-6 (Sarilumab). CONCLUSIONS: Our results demonstrated that huc-MSC-CM improved islet viability and function by increasing autophagy through the PI3K/Akt/mTOR pathway under hypoxic conditions. Additionally, IL-6 plays an important role in the function of huc-MSC-CM.

FADS3 is a Δ14Z sphingoid base desaturase that contributes to gender differences in the human plasma sphingolipidome.

Sphingolipids (SLs) are structurally diverse lipids that are defined by the presence of a long-chain base (LCB) backbone. Typically, LCBs contain a single Δ4E double bond (DB) (mostly d18:1), whereas the dienic LCB sphingadienine (d18:2) contains a second DB at the Δ14Z position. The enzyme introducing the Δ14Z DB is unknown. We analyzed the LCB plasma profile in a gender-, age-, and BMI-matched subgroup of the CoLaus cohort (n = 658). Sphingadienine levels showed a significant association with gender, being on average ∼30% higher in females. A genome-wide association study (GWAS) revealed variants in the fatty acid desaturase 3 (FADS3) gene to be significantly associated with the plasma d18:2/d18:1 ratio (p = -log 7.9). Metabolic labeling assays, FADS3 overexpression and knockdown approaches, and plasma LCB profiling in FADS3-deficient mice confirmed that FADS3 is a bona fide LCB desaturase and required for the introduction of the Δ14Z double bond. Moreover, we showed that FADS3 is required for the conversion of the atypical cytotoxic 1-deoxysphinganine (1-deoxySA, m18:0) to 1-deoxysphingosine (1-deoxySO, m18:1). HEK293 cells overexpressing FADS3 were more resistant to m18:0 toxicity than WT cells. In summary, using a combination of metabolic profiling and GWAS, we identified FADS3 to be essential for forming Δ14Z DB containing LCBs, such as d18:2 and m18:1. Our results unravel FADS3 as a Δ14Z LCB desaturase, thereby disclosing the last missing enzyme of the SL de novo synthesis pathway.

Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase cooperatively regulate growth and carbohydrate metabolism during Drosophila melanogaster larval development.

The dramatic growth that occurs during Drosophila larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands by increasing carbohydrate metabolism and lactate dehydrogenase (LDH) activity. The resulting metabolic program is ideally suited for synthesis of macromolecules and mimics the manner by which cancer cells rely on aerobic glycolysis. To explore the potential role of Drosophila LDH in promoting biosynthesis, we examined how Ldh mutations influence larval development. Our studies unexpectedly found that Ldh mutants grow at a normal rate, indicating that LDH is dispensable for larval biomass production. However, subsequent metabolomic analyses suggested that Ldh mutants compensate for the inability to produce lactate by generating excess glycerol-3-phosphate (G3P), the production of which also influences larval redox balance. Consistent with this possibility, larvae lacking both LDH and G3P dehydrogenase (GPDH1) exhibit growth defects, synthetic lethality and decreased glycolytic flux. Considering that human cells also generate G3P upon inhibition of lactate dehydrogenase A (LDHA), our findings hint at a conserved mechanism in which the coordinate regulation of lactate and G3P synthesis imparts metabolic robustness to growing animal tissues.

Quantification of D- and L-2-Hydroxyglutarate in Drosophila melanogaster Tissue Samples Using Gas Chromatography-Mass Spectrometry.

The fruit fly Drosophila melanogaster has emerged as an ideal system in which to study 2-hydroxyglutarate (2HG) metabolism. Unlike many mammalian tissues and cell lines, which primarily accumulate D- or L-2HG as the result of genetic mutations or metabolic stress, Drosophila larvae accumulate high concentrations of L-2HG during normal larval growth. As a result, flies represent one of the few model systems that allows for studies of endogenous L-2HG metabolism. Moreover, the Drosophila genome not only encodes key enzymes involved in the synthesis and degradation of D-2HG, but the fly has also been used as to investigate the in vivo effects of oncogenic isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations. All of these studies, however, rely on mass spectrometry-based methods to distinguish between the D- and L-2HG enantiomers. While such approaches are common among labs studying mammalian cell culture, few Drosophila studies have attempted to resolve and measure the individual 2HG enantiomers. Here we describe a highly reproducible gas chromatography-mass spectrometry (GC-MS)-based protocol that allows for quantitative measurements of both 2HG enantiomers in Drosophila homogenates.

Paracrine effect of mesenchymal stem cell as a novel therapeutic strategy for diabetic nephropathy.

Diabetic nephropathy (DN) is a microvascular complication of diabetes mellitus (DM) and the main reason for end-stage renal diseases (ESRD). Based on the role of mesenchymal stem cells (MSCs) in regenerative medicine, the MSC therapy has been considered a promising strategy to ameliorate the progression of DN. In this article, we review the therapeutic potential of MSCs in DN, mainly involving MSC paracrine mechanism based on trophic factors and extracellular vesicles. Knowledge of mechanism underlying the therapeutic action of MSCs on DN can provide much needed new drug targets for this disease.