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Purpose: To evaluate VEGF-C-induced lymphoproliferation in conjunction with 5-fluorouracil (5-FU) antimetabolite treatment in a rabbit glaucoma filtration surgery (GFS) model., Methods: Thirty-two rabbits underwent GFS and were assigned to four groups (n = 8 each) defined by subconjunctival drug treatment: (a) VEGF-C combined with 5-FU, (b) 5-FU, (c) VEGF-C, (d) and control. Bleb survival, bleb measurements, and IOP were evaluated over 30 days. At the end, histology and anterior segment OCT were performed on some eyes. mRNA was isolated from the remaining eyes for RT-PCR evaluation of vessel-specific markers (lymphatics, podoplanin and LYVE-1; and blood vessels, CD31)., Results: Qualitatively and quantitatively, VEGF-C combined with 5-FU resulted in blebs which were posteriorly longer and wider than the other conditions: vs. 5-FU (P = 0.043 for longer, P = 0.046 for wider), vs. VEGF-C (P < 0.001, P < 0.001) and vs. control (P < 0.001, P < 0.001). After 30 days, the VEGF-C combined with 5-FU condition resulted in longer bleb survival compared with 5-FU (P = 0.025), VEGF-C (P < 0.001), and control (P < 0.001). Only the VEGF-C combined with 5-FU condition showed a negative correlation between IOP and time that was statistically significant (r = -0.533; P = 0.034). Anterior segment OCT and histology demonstrated larger blebs for the VEGF-C combined with 5-FU condition. Only conditions including VEGF-C led to increased expression of lymphatic markers (LYVE-1, P < 0.001-0.008 and podoplanin, P = 0.002-0.011). Expression of CD31 was not different between the groups (P = 0.978)., Conclusions: Adding VEGF-C lymphoproliferation to standard antimetabolite treatment improved rabbit GFS success and may suggest a future strategy to improve human GFSs.

Phenylpyrazolo[3,4- d ]pyrimidine is considered a milestone scaffold known to possess various biological activities such as antiparasitic, antifungal, antimicrobial, and antiproliferative activities. In addition, the urgent need for selective and potent novel anticancer agents represents a major route in the drug discovery process. Herein, new aryl analogs were synthesized and evaluated for their anticancer effects on a panel of cancer cell lines: MCF-7, HCT116, and HePG-2. Some of these compounds showed potent cytotoxicity, with variable degrees of potency and cell line selectivity in antiproliferative assays with low resistance. As the analogs carry the pyrazolopyrimidine scaffold, which looks structurally very similar to tyrosine and receptor kinase inhibitors, the potent compounds were evaluated for their inhibitory effects on three essential cancer targets: EGFR WT , EGFR T790M , VGFR2, and Top-II. The data obtained revealed that most of these compounds were potent, with variable degrees of target selectivity and dual EGFR/VGFR2 inhibitors at the IC 50 value range, i.e., 0.3-24 µM. Among these, compound 5i was the most potent non-selective dual EGFR/VGFR2 inhibitor, with inhibitory concentrations of 0.3 and 7.60 µM, respectively. When 5i was tested in an MCF-7 model, it effectively inhibited tumor growth, strongly induced cancer cell apoptosis, inhibited cell migration, and suppressed cell cycle progression leading to DNA fragmentation. Molecular docking studies were performed to explore the binding mode and mechanism of such compounds on protein targets and mapped with reference ligands. The results of our studies indicate that the newly discovered phenylpyrazolo[3,4- d ]pyrimidine-based multitarget inhibitors have significant potential for anticancer treatment.

Nucleoside-based drugs, recognized as purine or pyrimidine analogs, have been potent therapeutic agents since their introduction in 1950, deployed widely in the treatment of diverse diseases such as cancers, myelodysplastic syndromes, multiple sclerosis, and viral infections. These antimetabolites establish complex interactions with cellular molecular constituents, primarily via activation of phosphorylation cascades leading to consequential interactions with nucleic acids. However, the therapeutic efficacy of these agents is frequently compromised by the development of drug resistance, a continually emerging challenge in their clinical application. This comprehensive review explores the mechanisms of resistance to nucleoside-based drugs, encompassing a wide spectrum of phenomena from alterations in membrane transporters and activating kinases to changes in drug elimination strategies and DNA damage repair mechanisms. The critical analysis in this review underlines complex interactions of drug and cell and also guides towards novel therapeutic strategies to counteract resistance. The development of targeted therapies, novel nucleoside analogs, and synergistic drug combinations are promising approaches to restore tumor sensitivity and improve patient outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Antimetabolites, especially 5-fluorouracil, are commonly used clinically to treat breast, colon, and other cancers. However, their side effects and inefficiency in monotherapy have prompted further searches for new combinations. Thus, the anticancer effect of 5-fluorouracil (5-FU) and the sulforaphane analogue, 4-isoselenocyanato-1-butyl 4'-fluorobenzyl sulfoxide (ISC), were tested in in vitro and in vivo models of triple-negative breast cancer (TNBC) as a new option for this treatment-resistant and aggressive type of breast cancer. A synergic interaction between 5-FU and ISC was observed in the TNBC in vitro model MDA-MB-231 cell line, which led to enhanced antiproliferative effects. The results of in vitro studies were confirmed by in vivo tests, which demonstrated stronger tumor growth inhibition and additive interactions between 5-FU and ISC in the murine TNBC model. Moreover, the results of the body mass and blood analysis showed the safety of the tested combination. The mechanistic study revealed that the combined treatment triggered apoptosis and necrosis, as well as inhibited cell migration.

Methotrexate (MTX) is a commonly used antimetabolite in cancer treatment. Its intensive use is linked with skeletal adverse effects such as reduced bone formation and bone loss, and yet little information is available on molecular mechanisms underlying MTX-induced impaired bone formation. This study investigated the effects of MTX treatment at a clinical chemotherapy relevant dose on osteogenic differentiation in MC3T3E1 osteoblastic cells. To investigate the potential mechanisms, the expression of 87 genes regulating osteoblast differentiation and bone homeostasis was screened in MTX-treated versus untreated cells by polymerase chain reaction (PCR) arrays and results illustrated significant upregulation of Notch2 and Notch target genes at both early and late stages of MC3T3E1 differentiation following MTX treatment. To confirm the roles of Notch2 pathway and its potential action mechanisms, MC3T3E1 cells were treated with MTX with an anti-Notch2 neutralizing antibody or control IgG and effects were examined on osteogenesis and activation of the Wnt/β-catenin pathway. Our results demonstrated that induction of Notch2 activity is associated with MTX adverse effects on osteogenic differentiation and blocking Notch2 rescues osteoblast differentiation by preserving activation of the Wnt/β-catenin pathway., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Purpose: To characterize and pharmacologically influence subconjunctival lymphatics in rabbit and mouse eyes., Methods: Rabbits received subconjunctival injections of trypan blue or fixable fluorescent dextrans. Bleb-related outflow pathways were quantified. Immunofluorescence for vessel-specific markers (lymphatics [podoplanin and LYVE-1] and blood vessels [CD31]) were performed in native rabbit conjunctiva and after fixable fluorescent dextran injection. Vascular endothelial cell growth factor-C (VEGFC) was injected subconjunctivally in rabbits. mRNA and protein were assessed for the above markers using RT-PCR and Western blot. Alternatively, mouse studies used Prox1-tdTomato transgenic reporter mice. Subconjunctival injection conditions included: no injection, balanced salt solution (BSS), VEGFC, 5-fluorouracil (5FU) and two concentrations of mitomycin-C (MMC). Two mouse injection protocols (short and long) with different follow-up times and number of injections were performed. Mouse eyes were enucleated, flat mounts created, and subconjunctival branching and length assessed., Results: Rabbit eyes demonstrated clear bleb-related subconjunctival outflow pathways that were distinct from blood vessels and were without nasal/temporal predilection. Immunofluorescence against vessel-specific markers showed lymphatics and blood vessels in rabbit conjunctiva, and these lymphatics overlapped with bleb-related subconjunctival outflow pathways. Subconjunctival VEGFC increased lymphatic (P = 0.004-0.04) but not blood vessel (P = 0.77-0.84) mRNA or protein in rabbits. Prox1-tdTomato transgenic reporter mice demonstrated natively fluorescent lymphatics. Subconjunctival VEGFC increased murine lymphatic branching and length (P ≤ 0.001-0.004) while antimetabolites (P ≤ 0.001-0.043) did the opposite for the long protocol., Discussion: Subconjunctival lymphatics are pharmacologically responsive to both VEGFC and antimetabolites in two animal models studied using different methodologies. These results may be important for bleb-forming glaucoma surgeries or ocular drug delivery.

Trypanosoma brucei , the causative agent for human African trypanosomiasis, is an emerging ergosterol-dependent parasite that produces chokepoint enzymes, sterol methyltransferases (SMT), not synthesized in their animal hosts that can regulate cell viability. Here, we report the lethal effects of two recently described natural product antimetabolites that disrupt Acanthamoeba sterol methylation and growth, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT) that can equally target T. brucei . We found that CHT/ERGT inhibited cell growth in vitro, yielding EC 50 values in the low nanomolar range with washout experiments showing cidal activity against the bloodstream form, consistent with their predicted mode of suicide inhibition on SMT activity and ergosterol production. Antimetabolite treatment generated altered T. brucei cell morphology and death rapidly within hours. Notably, in vivo ERGT/CHT protected mice infected with T. brucei , doubling their survival time following daily treatment for 8-10 days at 50 mg/kg or 100 mg/kg. The current study demonstrates a new class of lead antibiotics, in the form of common fungal sterols, for antitrypanosomal drug development.

Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 patients with relapsing-remitting multiple sclerosis (RRMS) and 14 healthy age-matched controls. Of 632 known metabolites detected, 60 were significantly altered in RRMS. Bioinformatics analysis identified an altered metabotype in patients with RRMS, represented by four changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these four pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient-derived peripheral blood mononuclear cells showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the antiinflammatory phenotype of monocytes/macrophage in the central nervous system. Our study provided a proof of principle for how a blood-based metabolomic approach using patient samples could lead to the identification of a therapeutic target for developing potential therapy.

Purpose: The purpose of this study was to investigate the effect of antimetabolite drugs on T-cell responses and intestinal microbial composition in autoimmune uveitis., Methods: Experimental autoimmune uveitis (EAU) was induced in C57BL/6J mice treated with 0.00625 mg/mL methotrexate (MTX) or 0.625 mg/mL mycophenolate mofetil (MMF) in drinking water for 4 weeks prior to immunization and 2 weeks thereafter. The effector T cell (Teff) and regulatory T cell (Treg) populations were identified using flow cytometry. The 16S rRNA gene sequencing was applied for gut microbiome characterization. DESeq2 analysis was used to discriminate relative abundances of taxa and PLS-DA to integrate cytometric and microbiome data between groups., Results: Both MTX and MMF abrogated uveitis in EAU without clinical signs of toxicity as compared to water-fed controls. MTX reduced Teff and Treg expansion in peripheral tissues and eyes. MTX decreased alpha diversity, increased Akkermansia, and reduced Lachnoclostridium abundances. Conversely, MMF enhanced Tregs in the mesenteric lymph node and the eyes. In parallel, MMF increased the gut alpha diversity, including an increased abundance of Lachnospiraceae NK4A136 group and a decreased abundance of Lachnospiraceae UCG-001. A significant congruent correlation among intestinal microbial changes, T-cell responses, and clinical scores was observed for both antimetabolites., Conclusions: Although MTX and MMF both abrogated uveitis in EAU, they showed different effects on T-cell subsets and the intestinal bacterial composition. This work indicates unique immunomodulation by each drug and is the first to demonstrate potential microbiota-related mechanisms.

Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5'-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5'-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.

Recent studies have shown the significance of metabolic reprogramming in immune and stromal cell function. Yet, the metabolic reconfiguration of RA macrophages (MΦs) is incompletely understood during active disease and in crosstalk with other cell types in experimental arthritis. This study elucidates a distinct regulation of glycolysis and oxidative phosphorylation in RA MΦs compared to fibroblast (FLS), although PPP (Pentose Phosphate pathway) is similarly reconfigured in both cell types. 2-DG treatment showed a more robust impact on impairing the RA M1 MΦ-mediated inflammatory phenotype than IACS-010759 (IACS, complexli), by reversing ERK, AKT and STAT1 signaling, IRF8/3 transcription and CCL2 or CCL5 secretion. This broader inhibitory effect of 2-DG therapy on RA M1 MΦs was linked to dysregulation of glycolysis (GLUT1, PFKFB3, LDHA, lactate) and oxidative PPP (NADP conversion to NADPH), while both compounds were ineffective on oxidative phosphorylation. Distinctly, in RA FLS, 2-DG and IACS therapies constrained LPS/IFNγ-induced AKT and JNK signaling, IRF5/7 and fibrokine expression. Disruption of RA FLS metabolic rewiring by 2-DG or IACS therapy was accompanied by a reduction of glycolysis (HIF1α, PFKFB3) and suppression of citrate or succinate buildup. We found that 2-DG therapy mitigated CIA pathology by intercepting joint F480 + iNOS + MΦ, Vimentin + fibroblast and CD3 + T cell trafficking along with downregulation of IRFs and glycolytic intermediates. Surprisingly, IACS treatment was inconsequential on CIA swelling, cell infiltration, M1 and Th1/Th17 cytokines (IFN-γ/IL-17) and joint glycolytic mediators. Collectively, our results indicate that blockade of glycolysis is more effective than inhibition of complex 1 in CIA, in part due to its effectiveness on the MΦ inflammatory phenotype., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Activation of ALK leads to a high level of aerobic glycolysis related to crizotinib insensitivity in anaplastic lymphoma kinase-positive non-small cell lung cancer (ALK + NSCLC). The strategy and mechanism of glycolysis inhibition in sensitizing ALK + NSCLC cells to crizotinib requires further investigation., Methods: The levels of glycolysis in H3122 and H2228 cells were evaluated through detection of glucose consumption and lactate production. MTT assay was used to explore the effects of glycolytic inhibitors on crizotinib sensitivity, and the potential mechanism of action were detected by colony formation, Ki67 incorporation assay, transwell assay, small interfering RNA technology and western blot analysis., Results: ALK + NSCLC cells exhibited significantly higher levels of glycolysis compared to ALK - NSCLC cells. Long-term exposure to crizotinib could decrease the sensitivity of ALK + NSCLC cells to crizotinib via increasing the levels of glycolysis related to hexokinases II (HK2). Crizotinib in combination with glycolysis inhibitor 2-deoxy-D-glucose (2DG) synergistically inhibited proliferation, glycolysis, colony formation and invasion ability of ALK + NSCLC cells. 2DG sensitization crizotinib might be associated with the inhibition of HK2-mediated glycolysis and P-ALK/AKT/mTOR signaling pathway in H3122 and H2228 cells., Conclusions: These results indicate that HK2-mediated glycolysis plays a crucial role in the increased tolerance of ALK + NSCLC cells to crizotinib. 2DG may sensitize ALK + NSCLC to crizotinib via suppression of HK2-mediated glycolysis and the AKT/mTOR signaling pathway., (© 2021 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Programmed cell death protein 1 (PD-1) and PD-ligand 1 (PD-L1) expression is upregulated in cluster of differentiation 34 (CD34) + bone marrow cells from patients with myelodysplastic syndromes (MDS). Hypomethylating agent (HMA) treatment results in further increased expression of these immune checkpoints. We hypothesised that combining an anti-PD-1 antibody with HMAs may have efficacy in patients with MDS. To test this concept, we designed a phase II trial of the combination of azacitidine and pembrolizumab with two cohorts. In the 17 previously untreated patients, the overall response rate (ORR) was 76%, with a complete response (CR) rate of 18% and median overall survival (mOS) not reached after a median follow-up of 12·8 months. For the HMA-failure cohort (n = 20), the ORR was 25% and CR rate was 5%; with a median follow-up of 6·0 months, the mOS was 5·8 months. The most observed toxicities were pneumonia (32%), arthralgias (24%) and constipation (24%). Immune-related adverse events requiring corticosteroids were required in 43%. Overall, this phase II trial suggests that azacitidine and pembrolizumab is safe with manageable toxicities in patients with higher-risk MDS. This combined therapy may have anti-tumour activity in a subset of patients and merits further studies in the front-line setting., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Autoimmune disease has presented an insurmountable barrier to restoration of durable immune tolerance. Previous studies indicate that chronic therapy with metabolic inhibitors can reduce autoimmune inflammation, but it remains unknown whether acute metabolic modulation enables permanent immune tolerance to be established. In an animal model of lupus, we determined that targeting glucose metabolism with 2-deoxyglucose (2DG) and mitochondrial metabolism with metformin enables endogenous immune tolerance mechanisms to respond to tolerance induction. A 2-week course of 2DG and metformin, when combined with tolerance-inducing therapy anti-CD45RB, prevented renal deposition of autoantibodies for 6 months after initial treatment and restored tolerance induction to allografts in lupus-prone mice. The restoration of durable immune tolerance was linked to changes in T cell surface glycosylation patterns, illustrating a role for glycoregulation in immune tolerance. These findings indicate that metabolic therapy may be applied as a powerful preconditioning to reinvigorate tolerance mechanisms in autoimmune and transplant settings that resist current immune therapies.

Thiopurines, immunomodulating drugs used in the management of different chronic autoimmune conditions and as anti-leukemic agents, may exert in some cases gastrointestinal toxicity. Moreover, since these agents are administered orally, they are absorbed across the gastrointestinal tract epithelium. On these premises, cellular and molecular events occurring in intestinal cells may be important to understand thiopurine effects. However, quantitative information on the biotransformation of thiopurines in intestinal tissues is still limited. To shed light on biotransformation processes specific of the intestinal tissue, in this study thiopurine metabolites concentrations were analyzed by an in vitro model of human healthy colon, the HCEC cell line, upon exposure to cytotoxic concentrations of azathioprine or mercaptopurine; the investigation was carried out using an innovative mass spectrometry method, that allowed the simultaneous quantification of 11 mono-, di-, and triphosphate thionucleotides. Among the 11 metabolites evaluated, TIMP, TGMP, TGDP, TGTP, MeTIMP, MeTIDP and MeTITP were detectable in HCEC cells treated with azathioprine or mercaptopurine, considering two different incubation times before the addition of the drugs (4 and 48 h). Different associations between metabolites concentrations and cytotoxicity were detected. In particular, the cytotoxicity was dependent on the TGMP, TGDP, TGTP and MeTITP concentrations after the 4 h incubation before the addition of thiopurines. This may be an indication that, to study the association between thiopurine metabolite concentrations and the cytotoxicity activity in vitro, short growth times before treatment should be used. Moreover, for the first time our findings highlight the strong correlation between cytotoxicity and thiopurine pharmacokinetics in HCEC intestinal cells in vitro suggesting that these cells could be a suitable in vitro model for studying thiopurine intestinal cytotoxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cancer cells have an increased need for glucose and, despite aerobic conditions, obtain their energy through aerobic oxidation and lactate fermentation, instead of aerobic oxidation alone. Glutamine is an essential amino acid in the human body. Glutaminolysis and glycolysis are crucial for cancer cell survival. In the therapy of estrogen receptor α (ERα)-positive breast cancer (BC), the focus lies on hormone sensitivity targeting therapy with selective estrogen receptor modulators (SERMs) such as 4-hydroxytamoxifen (4-OHT), although this therapy is partially limited by the development of resistance. Therefore, further targets for therapy improvement of ERα-positive BC with secondary 4-OHT resistance are needed. Hence, increased glucose requirement and upregulated glutaminolysis in BC cells could be used. We have established sublines of ERα-positive MCF7 and T47D BC cells, which were developed to be resistant to 4-OHT. Further, glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) and glutaminase inhibitor CB-839 were analyzed. Co-treatments using 4-OHT and CB-839, 2-DG and CB-839, or 4-OHT, 2-DG and CB-839, respectively, showed significantly stronger inhibitory effects on viability compared to single treatments. It could be shown that tamoxifen-resistant BC cell lines, compared to the non-resistant cell lines, exhibited a stronger reducing effect on cell viability under co-treatments. In addition, the tamoxifen-resistant BC cell lines showed increased expression of proto-oncogene c-Myc compared to the parental cell lines. This could be reduced depending on the treatment. Suppression of c-Myc expression using specific siRNA completely abolished resistance to 4OH-tamoxifen. In summary, our data suggest that combined treatments affecting the metabolism of BC are suitable depending on the cellularity and resistance status. In addition, the anti-metabolic treatments affected the expression of the proto-oncogene c-Myc, a key player in the regulation of cancer cell metabolism.

Macrophages deploy numerous strategies to combat invasion by microbes. One tactic is to restrict acquisition of diverse nutrients, including trace metals, a process termed nutritional immunity. Intracellular pathogens adapt to a resource-poor environment by marshaling mechanisms to harvest nutrients. Carbon acquisition is crucial for pathogen survival; compounds that reduce availability are a potential strategy to control intracellular replication. Treatment of macrophages with the glucose analog 2-deoxy-D-glucose (2-DG) armed phagocytes to eliminate the intracellular fungal pathogen Histoplasma capsulatum in vitro and in vivo. Killing did not rely on altering access to carbon-containing molecules or changes in ATP, ER stress, or autophagy. Unexpectedly, 2-DG undermined import of exogenous zinc into macrophages, decreasing the quantity of cytosolic and phagosomal zinc. The fungus perished as a result of zinc starvation. This change in metal ingress was not ascribed to a defect in a single importer; rather, there was a collective impairment in transporter activity. This effect promoted the antifungal machinery of macrophages and expanded the complexity of 2-DG activities far beyond manipulating glycolysis. Mechanistic metabolic studies employing 2-DG will have to consider its effect on zinc transport. Our preclinical data support consideration of this agent as a possible adjunctive therapy for histoplasmosis.

It has been proposed that carbon monoxide (CO) is a chemical light carrier that is transferred by the humoral pathway from the retina to the brain. Here, we aimed to study how deeply CO is involved in regulating the expression of Period2 gene ( PER2 ), one of the genes maintaining the intrinsic biological clock. In our in vivo experiment, we studied whether CO may be a chemical signal and is also equivalent to natural light in three groups of pigs: Normal: housed in natural conditions without any procedures, Control: adapted and kept in constant darkness, infused with blank plasma, and CO treated: adapted and kept in constant darkness infused with CO-enriched plasma. After the experiment, the animals were slaughtered at two times of day: 12 p.m. and 12 a.m. Next, hypothalamus samples were collected. Quantitative PCR, the DNA methylation of the promoter sequence containing enhancers (E-box) and a functional analysis of the PER2 promoter was performed. qPCR showed a differential pattern of PER2 mRNA expression at daytime oscillation in the examined groups. Pyrosequencing revealed daytime changes in the methylation level of regulatory sites of the examined sequence. Luciferase reporter assay confirmed that E-boxes (CANNTG) drive the expression of the porcine PER2 in vitro. In conclusion, changes in methylation over 24 h may regulate the oscillatory manner of PER2 expression.

Background: Although Amyloid beta (Aβ) and N - methyl d- aspartate receptors (NMDARs are involved in Ca 2+ neurotoxicity, the function of mitochondrial calcium uniporter in cognition deficit remain uncertain. Here, we examined the effect of mitochondrial calcium uniporter (MCU) blocker, together with NMDA receptor agonist d-cycloserine (DCS) on memory impairment in a rat model of AD., Methods: Forty adult male Wistar rats underwent stereotaxic cannulation for inducing AD by intracerebroventricular (ICV) injection of Aβ 1-42 (5 μg /8 μl/rat). Then animals were divided into 5 groups of: Saline + Saline, Aβ + Saline, Aβ + RU360, Aβ + DCS, Aβ + RU360 + DCS. Two weeks after the treatments, Morris Water Maze (MWM) and step through passive avoidance learning (SPL) were undertaken for evaluating of spatial and associative memories, respectively. Hippocampal level of cyclic-AMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) were measured by western blot and ELISA., Results: Co - administration of RU360 and DCS significantly improved both acquisition and retrieval of spatial memory as evident by decreased escape latency and increased time spent in the target quadrant (TTS) in MWM, together with increase in step-through latency, but reduced time spent in the dark compartment in SPL. Furthermore, there was a significant rise in the hippocampal level of CREB and BDNF in comparison with Aβ + Saline., Conclusion: The present study supports the idea that co- administration of RU360 and DCS ameliorate memory impairment induced by Aβ 1-42 probably via CREB / BDNF signaling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bone marrow adipocytes (BMAs) have recently been recognized as a niche component with a suppressive function. Obese individuals with abundant BMAs exhibit impaired hematopoietic regeneration after hematopoietic stem cell transplantation (HSCT). We hypothesized that plasminogen activator inhibitor type-1 (PAI-1), an adipokine that regulates the fibrinolytic system, contributes to impaired hematopoiesis in bone marrow (BM) microenvironment with abundant BMAs. We demonstrated that BMAs differentiated in vitro could secrete PAI-1 and were positive for PAI-1 in vivo. In addition, the abundance of BMAs was associated with high levels of PAI-1 expression. The BMA-rich microenvironment exhibited impaired hematopoietic regeneration after HSCT when compared with a BMA-less microenvironment. The impaired hematopoietic regeneration in BMA-rich microenvironment was significantly alleviated by PAI-1 knockout or PAI-1 inhibitor treatment. Obese mice with abundant BMAs, compared with normal-weight mice, exhibited higher bone marrow PAI-1 concentrations, increased fibrinolytic system suppression, and lower stem cell factor (SCF) concentrations after HSCT. PAI-1 inhibitor administration significantly activated the fibrinolytic system in obese mice, contributing to the higher SCF concentration. Moreover, PAI-1 inhibitor treatment significantly alleviated the impaired hematopoietic regeneration in obese mice both after 5-fluorouracil injection and HSCT. These results indicate that PAI-1 hinders hematopoietic regeneration in BMA-rich microenvironments. The blockade of PAI-1 activity could be a novel therapeutic means of facilitating hematopoietic reconstitution in BMA-rich patients., Competing Interests: Declaration of competing interest The PAI-1 inhibitor used in this study is protected as intellectual property. T.M, K.A., and T.Y are listed as inventors in the patent application., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: Senescence, characterized by permanent cycle arrest, plays an important role in diabetic nephropathy (DN). However, the mechanism of renal senescence is still unclear, and the treatment targeting it remains to be further explored., Materials and Methods: The DN mice were induced by HFD and STZ, and 3 types of renal cells were treated with high glucose (HG) to establish in vitro model. Senescence-related and autophagy-related markers were detected by qRT-PCR and Western blot. Further, autophagy inhibitors and co-immunoprecipitation were used to clarify the mechanism of CO. Additionally, the specific relationship between autophagy and senescence was explored by immunofluorescence triple co-localization and ELISA., Results: We unravelled that senescence occurred in vivo and in vitro, which could be reversed by CO. Mechanistically, we demonstrated that CO inhibited the dysfunction of autophagy in DN mice partly through dissociating Beclin-1-Bcl-2 complex. Further results showed that autophagy inhibitors blocked the improvement of CO on senescence. In addition, the data revealed that autophagy regulated the degradation of senescence-related secretory phenotype (SASP) including Il-1β, Il-6, Tgf-β and Vegf., Conclusions: These results suggested that CO protects DN mice from renal senescence and function loss via improving autophagy partly mediated by dissociating Beclin-1-Bcl-2 complex, which is possibly ascribed to the degradation of SASP. These findings bring new ideas for the prevention and treatment of DN and the regulation of senescence., (© 2021 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

This study was the first to investigate the synthesis of near-infrared light-sensitive NO prodrug [Mn(PaPy2Q)(NO)]ClO 4 , and detection the amount of NO released by the drug in different time and near infrared light (10 mW, 20 mW). It showed that with the increase of light power, the time required for the drug to release NO was shortened, and we selected 20 mW, 10 min as a follow-up study of light power and irradiation time while ensuring the near-infrared light did not affect tumor cells. The cells were irradiated with 20 mW of near-infrared light for 10 min at 6 h after treatment with the drug on PC-3, LNCaP and 22RV1 cells, and NO concentration and cell survival rate were tested at 12 h, 24 h and 48 h. Experiments showed that NO concentration remained stable within 48 h and [Mn(PaPy2Q)(NO)]ClO 4 inhibited the proliferation of cells in a concentration and time-dependent manner. Then we also found that [Mn(PaPy2Q)(NO)]ClO 4 increased the expression of apoptosis-related proteins (PARP, Bax, Caspase 3/9), inhibited the expression of BCl-2 and increased the activity level of Caspase 3/7, which showed [Mn(PaPy2Q)(NO)]ClO 4 promoted prostate cancer cells apoptosis. Next, the results in xenograft mouse model showed that [Mn(PaPy2Q)(NO)]ClO 4 also had anti-prostate cancer effects in vivo, and the NO concentration increased in the tumor after near-infrared light irradiation. After [Mn(PaPy2Q)(NO)]ClO 4 treatment 6 weeks, tumor volume was significantly reduced, Ki67 and BrdU protein expression was significantly reduced. TUNEL assay results showed that [Mn(PaPy2Q)(NO)]ClO 4 could promote the apoptosis of solid tumors in vivo and in a concentration-dependent manner., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Mutations in lysyl oxidase (LOX) genes cause severe vascular anomalies in mice and humans. LOX activity can be irreversibly inhibited by the administration of β-aminoproprionitrile (BAPN). We investigated the mechanisms underlying the damage to the ascending thoracic aorta induced by LOX deficiency and evaluated whether 6-propylthiouracil (PTU) can afford protection in rats. BAPN administration caused disruption of the ascending aortic wall, increased the number of apoptotic cells, stimulated TGF-β signaling (increase of nuclear p-SMAD2 staining), and up-regulated the expression of metalloproteinases-2 and -9. In BAPN-treated animals, PTU reduced apoptosis, p-SMAD2 staining, MMP-2, and -9 expression, and markedly decreased the damage to the aortic wall. Our results suggest that, as in some heritable vascular diseases, enhanced TGF-β signaling and upregulation of MMP-2 and -9 can contribute to the pathogenesis of ascending aorta damage caused by LOX deficiency. We have also shown that PTU, a drug already in clinical use, protects against the effects of LOX inhibition. MMP-2 and -9 might be potential targets of new therapeutic strategies for the treatment of vascular diseases caused by LOX deficiency.

The enzyme-free nucleic acid amplification circuit, for example, hybridization chain reaction (HCR), has paved a broad avenue for evaluating various enzyme-involved biotransformations, including DNA methyltransferases (MTases). The nonenzymatic MTase-sensing platform has supplemented a versatile toolbox for monitoring aberrant methylation in intricate biological samples, yet their amplification efficiency is always constrained by the initiator-depletion paradigm. Herein, the autonomously initiator-replicated HCR (IR-HCR) was developed as a versatile amplification system for detecting MTase with ∼100-fold sensitivity of the conventional HCR system. The initiator I -triggered HCR leads the assembly of a tandem DNAzyme concatemer that cleaves its substrate. This leads to the cyclic replication of a new initiator I for reversely motivating the initial HCR circuit, resulting in a dramatic Förster resonance energy transfer (FRET) readout. Without M.SssI MTase, hairpin H M can be recognized and digested by restriction endonuclease HpaII to release initiator I for stimulating a high FRET signal. While the M.SssI-methylated H M prohibits the HpaII-mediated cleavage of H M , the caged initiator I fails to trigger the IR-HCR circuit. Based on a systematic investigation, the IR-HCR circuit readily achieves selective and sensitive analysis of M.SssI MTase and its inhibitors. As a general MTase-sensing platform, the IR-HCR principle was further applied to analyze another MTase (Dam) by redesigning H M with the Dam recognition sequence. Overall, the versatile homogeneous MTase sensing platform was achieved via an efficient and robust initiator replication amplification circuit and may have enormous potential for early disease diagnosis.

By late 2020, the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused tens of millions of infections and over 1 million deaths worldwide. A protective vaccine and more effective therapeutics are urgently needed. We evaluated a new poly(ADP-ribose) polymerase (PARP) inhibitor, stenoparib, that recently advanced to phase II clinical trials for treatment of ovarian cancer, for activity against human respiratory coronaviruses, including SARS-CoV-2, in vitro Stenoparib exhibits dose-dependent suppression of SARS-CoV-2 multiplication and spread in Vero E6 monkey kidney and Calu-3 human lung adenocarcinoma cells. Stenoparib was also strongly inhibitory to the human seasonal respiratory coronavirus HCoV-NL63. Compared to remdesivir, which inhibits viral replication downstream of cell entry, stenoparib impedes entry and postentry processes, as determined by time-of-addition (TOA) experiments. Moreover, a 10 μM dosage of stenoparib-below the approximated 25.5 μM half-maximally effective concentration (EC 50 )-combined with 0.5 μM remdesivir suppressed coronavirus growth by more than 90%, indicating a potentially synergistic effect for this drug combination. Stenoparib as a stand-alone or as part of combinatorial therapy with remdesivir should be a valuable addition to the arsenal against COVID-19. IMPORTANCE New therapeutics are urgently needed in the fight against COVID-19. Repurposing drugs that are either already approved for human use or are in advanced stages of the approval process can facilitate more rapid advances toward this goal. The PARP inhibitor stenoparib may be such a drug, as it is currently in phase II clinical trials for the treatment of ovarian cancer and its safety and dosage in humans have already been established. Our results indicate that stenoparib possesses strong antiviral activity against SARS-CoV-2 and other coronaviruses in vitro. This activity appears to be based on multiple modes of action, where both pre-entry and postentry viral replication processes are impeded. This may provide a therapeutic advantage over many current options that have a narrower target range. Moreover, our results suggest that stenoparib and remdesivir in combination may be especially potent against coronavirus infection., (Copyright © 2021 Stone et al.)

Objectives: Mesenchymal Stem/Stromal Cells (MSC) are promising therapeutic tools for inflammatory diseases due to their potent immunoregulatory capacities. Their suppressive activity mainly depends on inflammatory cues that have been recently associated with changes in MSC bioenergetic status towards a glycolytic metabolism. However, the molecular mechanisms behind this metabolic reprogramming and its impact on MSC therapeutic properties have not been investigated. Methods: Human and murine-derived MSC were metabolically reprogramed using pro-inflammatory cytokines, an inhibitor of ATP synthase (oligomycin), or 2-deoxy-D-glucose (2DG). The immunosuppressive activity of these cells was tested in vitro using co-culture experiments with pro-inflammatory T cells and in vivo with the Delayed-Type Hypersensitivity (DTH) and the Graph versus Host Disease (GVHD) murine models. Results: We found that the oligomycin-mediated pro-glycolytic switch of MSC significantly enhanced their immunosuppressive properties in vitro . Conversely, glycolysis inhibition using 2DG significantly reduced MSC immunoregulatory effects. Moreover, in vivo , MSC glycolytic reprogramming significantly increased their therapeutic benefit in the DTH and GVHD mouse models. Finally, we demonstrated that the MSC glycolytic switch effect partly depends on the activation of the AMPK signaling pathway. Conclusion: Altogether, our findings show that AMPK-dependent glycolytic reprogramming of MSC using an ATP synthase inhibitor contributes to their immunosuppressive and therapeutic functions, and suggest that pro-glycolytic drugs might be used to improve MSC-based therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

We fabricated polysaccharide-based hydrogels, which are biocompatible, self-recoverable and pH-sensitive. Succinoglycan dialdehyde (SGDA) was first synthesized from bacterial succinoglycan directly isolated from Sinorhizobium meliloti and then hydrazine-functionalized alginate (HZ-Alg) was prepared to form SGDA-crosslinked alginate hydrogels (SGDA/HZ-Alg) without any catalyst. Due to structural characteristics of SGDA, SGDA/HZ-Alg were effectively obtained in a short time even at low concentrations (0.94-1.57 wt%) where they exhibited self-recoverable and tunable rheological properties corresponding to efficiency of recovery from 93.2%-97.9%. Moreover, SGDA/HZ-Alg showed the pH-responsive degradation as well as pH-controlled release behavior for 5-fluorouracil. 5-Fluorouracil was released approximately 98 % at pH 2.0 within 12 h, but not completely released even after 24 h at pH 7.4. The WST-8 assay results also demonstrated that SGDA/HZ-Alg did not show any cytotoxicity against HEK-293 cells. Since the suggested hydrogels are biocompatible, rheologically self-recoverable and tunable, and pH-controllable, they would be potential biomaterials for the hydrogel-based drug delivery systems., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Osteoclasts are specialised bone resorbing cells that control both physiological and pathological bone turnover. Functional changes in the differentiation and activity of osteoclasts are accompanied by active metabolic reprogramming. However, the biological significance and the in vivo relevance of these events has remained unclear. Here we show that bone resorption of differentiated osteoclasts heavily relies on increased aerobic glycolysis and glycolysis-derived lactate production. While pharmacological inhibition of glycolysis did not affect osteoclast differentiation or viability, it efficiently blocked bone resorption in vitro and in vivo and consequently ameliorated ovariectomy-induced bone loss. Our experiments thus highlight the therapeutic potential of interfering with osteoclast-intrinsic metabolic pathways as possible strategy for the treatment of diseases characterized by accelerated bone loss.

The recently delineated structure- and reactivity-based concept of antivitamins B 12 has begun to bear fruit by the generation, and study, of a range of such B 12 -dummies, either vitamin B 12 -derived, or transition metal analogues that also represent potential antivitamins B 12 or specific B 12 -antimetabolites. As reviewed here, this has opened up new research avenues in organometallic B 12 -chemistry and bioinorganic coordination chemistry. Exploratory studies with antivitamins B 12 have, furthermore, revealed some of their potential, as pharmacologically interesting compounds, for inducing B 12 -deficiency in a range of organisms, from hospital resistant bacteria to laboratory mice. The derived capacity of antivitamins B 12 to induce functional B 12 -deficiency in mammalian cells and organs also suggest their valuable potential as growth inhibitors of cancerous human and animal cells., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Background: The specific and accurate pathogenesis of diarrhea-type irritable bowel syndrome is still unclear., Aims: We explored the mechanism of heat shock protein 27 (HSP27) in diarrhea-type irritable bowel syndrome to identify the key targets for the disease., Methods: The human colonic epithelial cell lines Caco-2 and NCM460 were pretreated with KRIBB3 (a phosphorylation inhibitor of HSP27) and then stimulated with lipopolysaccharide for different times. The apoptosis ratios of Caco-2 and NCM460 cells were examined with Annexin V/PI assays. Cell growth was determined using the cell counting kit-8 assay, and the expression levels of IL-1β and IL-6 in the cell supernatant were analyzed by ELISA. In addition, the expression levels of HSP27 and the nuclear factor-κB (NF-κB) signaling pathway were examined by Western blot assay., Results: Stimulation with lipopolysaccharide promoted the expression of HSP27 in colonic epithelial cells. HSP27 was phosphorylated at serine 78 and 82 after exposure to LPS. Apoptosis, growth inhibition, and inflammatory factor expression of lipopolysaccharide-induced colonic epithelial cells were greatly exacerbated by KRIBB3 treatment. In addition, KRIBB3 inhibited the phosphorylation of IκB-α and the activation of NF-κB. Gene silencing by small interfering RNA indicated that phosphorylation of HSP27 may regulate the NF-κB pathway., Conclusions: HSP27 plays an important role in the inflammatory response of intestinal human colonic epithelial cells. HSP27 may protect intestinal epithelial cells against damage by regulating the NF-κB pathway.

Metabolism of host-targeted drugs by the microbiome can substantially impact host treatment success. However, since many host-targeted drugs inadvertently hamper microbiome growth, repeated drug administration can lead to microbiome evolutionary adaptation. We tested if evolved bacterial resistance against host-targeted drugs alters their drug metabolism and impacts host treatment success. We used a model system of Caenorhabditis elegans , its bacterial diet, and two fluoropyrimidine chemotherapies. Genetic screens revealed that most of loss-of-function resistance mutations in Escherichia coli also reduced drug toxicity in the host. We found that resistance rapidly emerged in E. coli under natural selection and converged to a handful of resistance mechanisms. Surprisingly, we discovered that nutrient availability during bacterial evolution dictated the dietary effect on the host - only bacteria evolving in nutrient-poor media reduced host drug toxicity. Our work suggests that bacteria can rapidly adapt to host-targeted drugs and by doing so may also impact the host., Competing Interests: BR, SS, PO, AG, HM, AW, AM No competing interests declared, (© 2020, Rosener et al.)

Glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) is a rate-limiting enzyme catalyzing triglyceride synthesis. Recently, we demonstrated that the anti-viral drug ribavirin (RBV) reduces GPAM expression by downregulating CCAAT/enhancer-binding protein α (C/EBPα). However, the precise mechanisms of GPAM suppression have remained unclear. Here, we found that RBV suppressed GPAM expression by downregulating not only C/EBPα, but also sterol regulatory element-binding protein-1c (SREBP-1c). We also found that cis-elements regulated by C/EBPα and SREBP-1c functioned as distal and proximal enhancers, respectively, to express hepatocyte- and adipocytes-specific GPAM variants. These results imply that RBV disrupts formation of the enhancer machineries on the GPAM genome by downregulating both transcription factors. Our findings may contribute to the development of treatments for fatty liver diseases caused by aberrant triglyceride synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coronaviruses have been reported previously due to their association with the severe acute respiratory syndrome (SARS). After SARS, these viruses were known to be causing Middle East respiratory syndrome (MERS) and caused 35% evanescence amid victims pursuing remedial care. Nowadays, beta coronaviruses, members of Coronaviridae , family order Nidovirales , have become subjects of great importance due to their latest pandemic originating from Wuhan, China. The virus named as human-SARS-like coronavirus-2 contains four structural as well as sixteen nonstructural proteins encoded by single-stranded ribonucleic acid of positive polarity. As there is no vaccine available to treat the infection caused by these viruses, there is a dire need for taking necessary steps against this virus. Herein, we have targeted two nonstructural proteins of SARS-CoV-2, namely, methyltransferase (nsp16) and helicase (nsp13), respectively, due to their substantial activity in viral pathogenesis. A total of 2035 compounds were analyzed for their pharmacokinetics and pharmacological properties. The screened 108 compounds were docked against both targeted proteins and were compared with previously reported known compounds. Compounds with high binding affinity were analyzed for their reactivity through DFT analysis, and binding was analyzed using molecular dynamics simulations. Through the analyses performed in this study, it is concluded that EryvarinM, Silydianin, Osajin, and Raddeanine can be considered potential inhibitors for MTase, while TomentodiplaconeB, Osajin, Sesquiterpene Glycoside, Rhamnetin, and Silydianin for helicase after these compounds are validated thoroughly using in vitro and in vivo protocols., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Kafila Kousar et al.)

Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and are also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty of reaching sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defense peptide of the African clawed frog and the first AMP developed into an antibacterial drug, we studied whether sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of Staphylococcus aureus at sub-lethal concentrations using quantitative proteomics. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that Staphylococcus aureus is highly susceptible to antimetabolite nucleoside analogs when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides., (Copyright © 2020 Rodríguez-Rojas, Nath, El Shazely, Santi, Kim, Weise, Kuropka and Rolff.)

High temperature is a major threat to Pleurotus ostreatus cultivation. In this study, a potential mechanism by which P. ostreatus mycelia growth is inhibited under heat stress was explored. Lactate, as a microbial fermentation product, was found unexpectedly in the mycelia of P. ostreatus under heat stress, and the time-dependent accumulation and corresponding inhibitory effect of lactate on mycelial growth was further confirmed. The addition of a glycolysis inhibitor, 2-deoxy-D-glucose (2DG), reduced the lactate content in mycelia and slightly restored mycelial growth under high-temperature conditions, which indicated the accumulation of lactate can be inhibited by glycolysis inhibition. Further data revealed mitochondrial dysfunction under high-temperature conditions, with evidence of decreased oxygen consumption and adenosine triphosphate (ATP) synthesis and increased reactive oxygen species (ROS). The removal of ROS with ascorbic acid decreased the lactate content, and mycelial growth recovered to a certain extent, indicating lactate accumulation could be affected by the mitochondrial ROS. Moreover, metabolic data showed that glycolysis and the tricarboxylic acid cycle were enhanced. This study reported the accumulation of lactate in P. ostreatus mycelia under heat stress and the inhibitory effect of lactate on the growth of mycelia, which might provide further insights into the stress response mechanism of edible fungi. Key Points • Lactate can accumulate in Pleurotus ostreatus mycelia under heat stress and inhibit its growth. • The accumulation of lactate may be due to the acceleration of glycolysis and the dysfunction of mitochondria of P. ostreatus mycelia under high-temperature stress. • The glycolysis and tricarboxylic acid cycle of P. ostreatus mycelia were accelerated under high-temperature stress.

Previously, we have shown that Staphylococcus (S) aureus induces a glycolytic response in retinal residential (microglia) and infiltrated cells (neutrophils and macrophages) during endophthalmitis. In this study, we sought to investigate the physiological role of glycolysis in bacterial endophthalmitis using a glycolytic inhibitor, 2-deoxyglucose (2DG). Our data showed that 2DG treatment attenuated the inflammatory responses of mouse bone marrow-derived macrophages (BMDM) and neutrophils (BMDN) when challenged with either live or heat-killed S. aureus (HKSA). Among the inflammatory mediators, 2DG caused a significant reduction in levels of cytokines (TNF-α, IL-1β, IL-6) and chemokines (CXCL1 and CXCL2). Western blot analysis of 2DG treated cells showed downregulation of bacterial-induced MEK/ERK pathways. In vivo, intravitreal administration of 2DG both pre- and post-bacterial infection resulted in a significant reduction in intraocular inflammation in C57BL/6 mouse eyes and downregulation of ERK phosphorylation in retinal tissue. Collectively, our study demonstrates that 2DG attenuates inflammatory response in bacterial endophthalmitis and cultured innate immune cells via inhibition of ERK signaling. Thus glycolytic inhibitors in combination with antibiotics could mitigate inflammation-mediated tissue damage in ocular infections., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cancer cells alter their metabolism by switching from glycolysis to oxidative phosphorylation (OXPHOS), regardless of oxygen availability. Metabolism may be a molecular target in acute myeloid leukemia (AML), where mutations in metabolic genes have been described. This study evaluated glycolysis and OXPHOS as therapeutic targets. The sensitivity to 2-deoxy-D-glucose (2-DG; glycolysis inhibitor) and oligomycin (OXPHOS inhibitor) was tested in six AML cell lines (HEL, HL-60, K-562, KG-1, NB-4, THP-1). These cells were characterized for IDH1/2 exon 4 mutations, reactive oxygen species, and mitochondrial membrane potential. Metabolic activity was assessed by resazurin assay, whereas cell death and cell cycle were assessed by flow cytometry. Glucose uptake and metabolism-related gene expression were analyzed by 18 F-FDG and RT-PCR/qPCR, respectively. No IDH1/2 exon 4 mutations were detected. HEL cells had the highest 18 F-FDG uptake and peroxides/superoxide anion levels, whereas THP-1 showed the lowest. 2-DG reduced metabolic activity in all cell lines with HEL, KG-1, and NB-4 being the most sensitive cells. Oligomycin decreased metabolic activity in a cell line-dependent manner, the THP-1 resistant and HL-60 being the most sensitive. Both inhibitors induced apoptosis and cell cycle arrest in a cell line- and compound-dependent manner. 2-DG decreased 18 F-FDG uptake in HEL, HL-60, KG-1, and NB-4, while oligomycin increased the uptake in K-562. Metabolism gene expression had different responses to treatments. In conclusion, HEL and KG-1 show to be more glycolytic, whereas HL-60 was more OXPHOS dependent. Results suggest that AML cells reprogram their metabolism to overcome OXPHOS inhibition suggesting that glycolysis may be a better therapeutic target.

Glucocorticoids (GCS) are known to modulate cardiovascular response during stress conditions. The present study was aimed to test the hypothesis that permissive and/or stimulating effect of GCs is essential for the maintenance of peripheral vascular resistance and for the adequate response of cardiovascular system to stressor exposure. The effects of acute pharmacological adrenalectomy (PhADX) on humoral and cardiovascular parameters were studied in adult Wistar rats under the basal conditions and during the acute restraint stress. Acute PhADX was performed by the administration of metyrapone and aminoglutethimide (100 mg/kg s.c. of each drug) resulting in a suppression of endogenous glucocorticoid synthesis. Blood pressure (BP), heart rate (HR) and core body temperature were measured using radiotelemetry. BP responses to administration of vasoactive agents were determined in pentobarbital-anesthetized animals. PhADX considerably attenuated stress-induced increase of BP, HR and core body temperature. PhADX did not abolish BP and HR lowering effects of ganglionic blocker pentolinium indicating preserved sympathetic function in PhADX rats. BP response to exogenous norepinephrine administration was attenuated in PhADX rats, suggesting reduced sensitivity of cardiovascular system. Suppression of corticosterone synthesis by PhADX increased basal plasma levels of ACTH, aldosterone and plasma renin activity in unstressed animals but there was no further increase of these hormones following stressor exposure. In conclusion, PhADX attenuated stress-induced rise of blood pressure, heart rate and core body temperature indicating an important permissive and/or stimulating role of glucocorticoids in the maintenance of the adequate response of cardiovascular system and thermoregulation to several stimuli including acute exposure to stressor.

Objective: Neonatal status epilepticus (SE) is a life-threatening medical emergency. Unfortunately, up to 50% of neonates with SE are resistant to current antiseizure drugs, highlighting the need for better treatments. This study aims to explore a novel metabolic approach as a potential alternative treatment to control neonatal SE, using the glycolytic inhibitor 2-deoxyglucose (2-DG)., Methods: SE was induced by pilocarpine (300 mg/kg, intraperitoneally [ip]) in neonatal Sprague Dawley rats (postnatal day 10 [P10]-P17) and was monitored by video-electroencephalography (V-EEG). After 30 minutes of SE, 2-DG or one of two conventional antiseizure drugs with different mechanisms of action, phenobarbital or levetiracetam, was administrated ip, and V-EEG recording was continued for ~60 additional minutes. The time to seizure cessation after drug injection, EEG scores, and power spectra before and after drug or saline treatment were used to assess drug effects., Results: Once SE became sustained, administration of 2-DG (50, 100, or 500 mg/kg, ip) consistently stopped behavioral and electrographic seizures within 10-15 minutes; lower doses took longer (25-30 minutes) to stop SE, demonstrating a dose-dependent effect. Administration of phenobarbital (30 mg/kg, ip) or levetiracetam (100 mg/kg, ip) also stopped SE within 10-15 minutes in neonatal rats., Significance: Our results suggest that the glycolysis inhibitor 2-DG acts quickly to reduce neuronal hyperexcitability and effectively suppress ongoing seizure activity, which may provide translational value in the treatment of neonatal SE., (© 2020 International League Against Epilepsy.)

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.

The rapid global emergence of SARS-CoV-2 has been the cause of significant health concern, highlighting the immediate need for antivirals. Viral RNA-dependent RNA polymerases (RdRp) play essential roles in viral RNA synthesis, and thus remains the target of choice for the prophylactic or curative treatment of several viral diseases, due to high sequence and structural conservation. To date, the most promising broad-spectrum class of viral RdRp inhibitors are nucleoside analogues (NAs), with over 25 approved for the treatment of several medically important viral diseases. However, Coronaviruses stand out as a particularly challenging case for NA drug design due to the presence of an exonuclease (ExoN) domain capable of excising incorporated NAs and thus providing resistance to many of these available antivirals. Here we use the available structures of the SARS-CoV RdRp and ExoN proteins, as well as Lassa virus N exonuclease to derive models of catalytically competent SARS-CoV-2 enzymes. We then map a promising NA candidate, GS-441524 (the active metabolite of Remdesivir) to the nucleoside active site of both proteins, identifying the residues important for nucleotide recognition, discrimination, and excision. Interestingly, GS-441524 addresses both enzyme active sites in a manner consistent with significant incorporation, delayed chain termination, and altered excision due to the ribose 1'-CN group, which may account for the increased antiviral effect compared to other available analogues. Additionally, we propose structural and function implications of two previously identified RdRp resistance mutations in relation to resistance against Remdesivir. This study highlights the importance of considering the balance between incorporation and excision properties of NAs between the RdRp and ExoN., (Copyright © 2020 Elsevier B.V. All rights reserved.)

An escalating pandemic by the novel SARS-CoV-2 virus is impacting global health and effective therapeutic options are urgently needed. We evaluated the in vitro antiviral effect of compounds that were previously reported to inhibit coronavirus replication and compounds that are currently under evaluation in clinical trials for SARS-CoV-2 patients. We report the antiviral effect of remdesivir, lopinavir, homorringtonine, and emetine against SARS-CoV-2 virus in Vero E6 cells with the estimated 50% effective concentration at 23.15 μM, 26.63 μM, 2.55 μM and 0.46 μM, respectively. Ribavirin or favipiravir that are currently evaluated under clinical trials showed no inhibition at 100 μM. Synergy between remdesivir and emetine was observed, and remdesivir at 6.25 μM in combination with emetine at 0.195 μM may achieve 64.9% inhibition in viral yield. Combinational therapy may help to reduce the effective concentration of compounds below the therapeutic plasma concentrations and provide better clinical benefits., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cellular starvation is typically a consequence of tissue injury that disrupts the local blood supply but can also occur where cell populations outgrow the local vasculature, as observed in solid tumors. Cells react to nutrient deprivation by adapting their metabolism, or, if starvation is prolonged, it can result in cell death. Cell starvation also triggers adaptive responses, like angiogenesis, that promote tissue reorganization and repair, but other adaptive responses and their mediators are still poorly characterized. To explore this issue, we analyzed secretomes from glucose-deprived cells, which revealed up-regulation of multiple cytokines and chemokines, including IL-6 and IL-8, in response to starvation stress. Starvation-induced cytokines were cell type-dependent, and they were also released from primary epithelial cells. Most cytokines were up-regulated in a manner dependent on NF-κB and the transcription factor of the integrated stress response ATF4, which bound directly to the IL-8 promoter. Furthermore, glutamine deprivation, as well as the antimetabolic drugs 2-deoxyglucose and metformin, also promoted the release of IL-6 and IL-8. Finally, some of the factors released from starved cells induced chemotaxis of B cells, macrophages, and neutrophils, suggesting that nutrient deprivation in the tumor environment can serve as an initiator of tumor inflammation., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here, we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes tumorigenicity of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and that KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Glioblastoma (GBM) remains one of the most lethal primary brain tumors in children and adults. Targeting tumor metabolism has emerged as a promising-targeted therapeutic strategy for GBM and characteristically resistant GBM stem-like cells (GSCs)., Methods: Gene expression data was obtained from the online patient-histology database, GlioVis. GSC mitochondria morphology was examined by TEM. Cell viability and effect on GSC self-renewal was determined via MTS assay and neurosphere assay, respectively. Proteins were evaluated by Western Blot., Results: Enzymes necessary for ketone catabolism (BDH1, OXCT1 and ACAT1) are significantly downregulated in adult and pediatric GBM. GSC mitochondrial ultrastructure suggested defects in oxidative phosphorylation. Treatment of both GBM and GSC cell lines resulted in dose-dependent decreases in viability in response to glycolytic inhibitor 2-deoxy-D-glucose (2-DG), and ketone body Acetoacetate (AA), but not β-hydroxybutyrate (βHB). AA induced apoptosis was confirmed by western blot analysis, indicating robust caspase activation and PARP cleavage. AA reduced neurosphere formation at concentrations as low as 1 mM. Combined treatment of low dose 2-DG (50 μM) with AA resulted in more cell death than either treatment alone. The effect was greater than additive at low concentrations of AA, reducing viability approximately 50% at 1 mM AA. AA was found to directly upregulate mitochondrial uncoupling protein 2 (UCP2), which may explain this potential drug synergism via multi-faceted inhibition of the glycolytic pathway., Conclusion: Targeting the metabolic pathway of GBM via glycolytic inhibition in conjunction with ketogenic diet or exogenous ketone body supplementation warrants further investigation as a promising adjunctive treatment to conventional therapy.

Increasing rates of drug-resistant Gram-negative (GN) infections, combined with a lack of new GN-effective antibiotic classes, are driving the need for the discovery of new agents. Bacterial metabolism represents an underutilized mechanism of action in current antimicrobial therapies. Therefore, we sought to identify novel antimetabolites that disrupt key metabolic pathways and explore the specific impacts of these agents on bacterial metabolism. This study describes the successful application of this approach to discover a new series of chemical probes, N -(phenyl)thioacetamide-linked 1,2,3-triazoles (TAT), that target cysteine synthase A (CysK), an enzyme unique to bacteria that is positioned at a key juncture between several fundamental pathways. The TAT class was identified using a high-throughput screen against Escherichia coli designed to identify modulators of pathways related to folate biosynthesis. TAT analog synthesis demonstrated a clear structure-activity relationship, and activity was confirmed against GN antifolate-resistant clinical isolates. Spontaneous TAT resistance mutations were tracked to CysK, and mode of action studies led to the identification of a false product formation mechanism between the CysK substrate O -acetyl-l-serine and the TATs. Global transcriptional responses to TAT treatment revealed that these antimetabolites impose substantial disruption of key metabolic networks beyond cysteine biosynthesis. This study highlights the potential of antimetabolite drug discovery as a promising approach to the discovery of novel GN antibiotics and the pharmacological promise of TAT CysK probes.

Prolonging the clinical effectiveness of β-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between β-lactams and DCS. DCS resensitized MRSA to β-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to β-lactam antibiotics., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

D-cycloserine (DCS), a partial NMDA-receptor agonist, seems to be a promising enhancer for exposure therapy in anxiety disorders. It has been tested successfully in animal models of fear extinction, where DCS enhanced extinction learning. Applied in clinical studies, results of DCS-augmented exposure therapy remain ambiguous, calling for a deeper understanding of the underlying mechanisms of DCS and its exact effect on extinction learning and return of fear (ROF) in humans. In the present study, we investigated the effect of DCS-augmented extinction learning on behavioral, psychophysiological, and neural indices of ROF during a 24-h delayed recall test. Thirty-seven participants entered a randomized, placebo-controlled, double-blind, 3-day fear conditioning and delayed extinction fMRI design. One hour before extinction training, participants received an oral dose of 50 mg of DCS or a placebo. Behavioral arousal ratings revealed a generalized ROF during extinction recall in the placebo but not DCS group. Furthermore, participants receiving DCS compared to placebo showed attenuated differential BOLD responses in left posterior hippocampus and amygdala from extinction learning to extinction recall, due to increased hippocampal recruitment in placebo and trendwise decreased amygdala responding in DCS subjects. Our finding that DCS reduces ROF in arousal ratings and neural structures subserving defensive reactions support a role for NMDA receptors in extinction memory consolidation and encourage further translational research.

Orexins are neuropeptides that are implicated in a number of functions. With regard to the gastrointestinal functions, orexin acts centrally to regulate gastric secretion, gastrointestinal motility and visceral sensation. Little is however known about a role of central orexin in the control of intestinal barrier function. The present study was performed to clarify whether brain orexin plays a role in the control of intestinal permeability. Colonic permeability was estimated in vivo by quantifying the absorbed Evans blue in colonic tissue in rats. Intracisternally administered orexin-A but not orexin-B dose-dependently blocked the increased intestinal permeability by lipopolysaccharide (LPS) or corticotropin-releasing factor while intraperitoneally injected orexin-A failed to block it. Atropine or vagotomy abolished the action by central orexin-A. Intravenous injection of 2-deoxy-D-glucose (2-DG), a central vagal stimulant, significantly blocked the LPS-induced increase in intestinal permeability and atropine prevented the action of 2-DG. Intracisternal injection of SB-334687, a selective orexin 1 receptor antagonist, significantly blocked the action of 2-DG-induced improvement of intestinal hyperpermeability. These results suggest that exogenously administered or endogenously released orexin acts centrally to improve the intestinal hyperpermeability by LPS via the vagal cholinergic pathway. The findings also suggest for the first time that the brain could control intestinal permeability. The neuronal rapid protective advantage to the host by improving the intestinal barrier function by the neuropeptide may help us understand the brain-gut interaction in stress sensitive gastrointestinal disorders like irritable bowel syndrome associated with the altered intestinal permeability., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pre-gestational diabetes is a risk factor for preeclampsia, a condition associated with inflammatory markers, a dysregulated angiogenic profile, and impaired placentation. Using an in vitro model, we previously reported that hyperglycemic levels of glucose induced a pro-inflammatory (IL-1β, IL-8, RANTES, GRO-α), anti-angiogenic (sFlt-1) and anti-migratory profile in a human trophoblast cell line. The IL-1β response to excess glucose was mediated by uric acid-induced activation of the NLRP3 inflammasome. Allopurinol is a xanthine oxidase inhibitor that inhibits uric acid and reactive oxygen species (ROS) production. Thus, we sought to test the effects of allopurinol on the IL-1β and other inflammatory, angiogenic and migratory responses that are triggered in the trophoblast by excess glucose. Under excess glucose conditions, allopurinol significantly inhibited trophoblast secretion of inflammatory IL-1β; caspase-1 activity; IL-8; RANTES; and GRO-α. Allopurinol also significantly inhibited excess glucose-induced trophoblast secretion of anti-angiogenic sFlt-1. The presence of IL1Ra significantly inhibited excess glucose-induced trophoblast IL-8 and GRO-α secretion but had no effect on RANTES or sFlt-1. Conversely, DPI, a ROS inhibitor, significantly inhibited excess glucose-induced trophoblast GRO-α and sFlt-1 secretion, but had no effect on IL-8 or RANTES. Together, our findings indicate that the xanthine oxidase inhibitor allopurinol inhibited excess glucose-induced trophoblast IL-1β secretion. Additionally, through its inhibition of both IL-1β and ROS production by the trophoblast, allopurinol reduced the additional pro-inflammatory and anti-angiogenic responses to excess glucose. Thus, allopurinol may be a candidate medication to prevent placental dysfunction and adverse pregnancy outcomes, such as preeclampsia, in pregnant women with diabetes.