Epigenetic Reprogramming of Autophagy Drives Mutant IDH1 Glioma Progression and Response to Radiation.

Mutant isocitrate dehydrogenase 1 (mIDH1; IDH1 R132H ) exhibits a gain of function mutation enabling 2-hydroxyglutarate (2HG) production. 2HG inhibits DNA and histone demethylases, inducing epigenetic reprogramming and corresponding changes to the transcriptome. We previously demonstrated 2HG-mediated epigenetic reprogramming enhances DNA-damage response and confers radioresistance in mIDH1 gliomas harboring p53 and ATRX loss of function mutations. In this study, RNA-seq and ChIP-seq data revealed human and mouse mIDH1 glioma neurospheres have downregulated gene ontologies related to mitochondrial metabolism and upregulated autophagy. Further analysis revealed that the decreased mitochondrial metabolism was paralleled by a decrease in glycolysis, rendering autophagy as a source of energy in mIDH1 glioma cells. Analysis of autophagy pathways showed that mIDH1 glioma cells exhibited increased expression of pULK1-S555 and enhanced LC3 I/II conversion, indicating augmented autophagy activity. This dependence is reflected by increased sensitivity of mIDH1 glioma cells to autophagy inhibition. Blocking autophagy selectively impairs the growth of cultured mIDH1 glioma cells but not wild-type IDH1 (wtIDH1) glioma cells. Targeting autophagy by systemic administration of synthetic protein nanoparticles packaged with siRNA targeting Atg7 (SPNP-siRNA-Atg7) sensitized mIDH1 glioma cells to radiation-induced cell death, resulting in tumor regression, long-term survival, and immunological memory, when used in combination with IR. Our results indicate autophagy as a critical pathway for survival and maintenance of mIDH1 glioma cells, a strategy that has significant potential for future clinical translation. One Sentence Summary: The inhibition of autophagy sensitizes mIDH1 glioma cells to radiation, thus creating a promising therapeutic strategy for mIDH1 glioma patients. Graphical abstract: Our genetically engineered mIDH1 mouse glioma model harbors IDH1 R132H in the context of ATRX and TP53 knockdown. The production of 2-HG elicited an epigenetic reprogramming associated with a disruption in mitochondrial activity and an enhancement of autophagy in mIDH1 glioma cells. Autophagy is a mechanism involved in cell homeostasis related with cell survival under energetic stress and DNA damage protection. Autophagy has been associated with radio resistance. The inhibition of autophagy thus radio sensitizes mIDH1 glioma cells and enhances survival of mIDH1 glioma-bearing mice, representing a novel therapeutic target for this glioma subtype with potential applicability in combined clinical strategies.

Current Status and Future Perspectives on Distribution of Fungal Endophytes and Their Utilization for Plant Growth Promotion and Management of Grapevine Diseases.

Grapevine is one of the economically most important fruit crops cultivated worldwide. Grape production is significantly affected by biotic constraints leading to heavy crop losses. Changing climatic conditions leading to widespread occurrence of different foliar diseases in grapevine. Chemical products are used for managing these diseases through preventive and curative application in the vineyard. High disease pressure and indiscriminate use of chemicals leading to residue in the final harvest and resistance development in phytopathogens. To mitigate these challenges, the adoption of potential biocontrol control agents is necessary. Moreover, multifaceted benefits of endophytes made them eco-friendly, and environmentally safe approach. The genetic composition, physiological conditions, and ecology of their host plant have an impact on their dispersion patterns and population diversity. Worldwide, a total of more than 164 fungal endophytes (FEs) have been characterized originating from different tissues, varieties, crop growth stages, and geographical regions of grapevine. These diverse FEs have been used extensively for management of different phytopathogens globally. The FEs produce secondary metabolites, lytic enzymes, and organic compounds which are known to possess antimicrobial and antifungal properties. The aim of this review was to understand diversity, distribution, host-pathogen-endophyte interaction, role of endophytes in disease management and for enhanced, and quality production.

Authentication of the species identity of squid rings using UHPLC-Q-Orbitrap MS/MS-based lipidome fingerprinting and chemoinformatics.

Species mislabeling of commercial loliginidae squid can undermine important conservation efforts and prevent consumers from making informed decisions. A comprehensive lipidomic fingerprint of Uroteuthis singhalensis, Uroteuthis edulis, and Uroteuthis duvauceli rings was established using high-resolution mass spectrometry-based lipidomics and chemoinformatics analysis. The principal component analysis showed a clear separation of sample groups, with R2X and Q2 values of 0.97 and 0.85 for ESI+ and 0.96 and 0.86 for ESI-, indicating a good model fit. The optimized OPLS-DA and PLS-DA models could discriminate the species identity of validation samples with 100 % accuracy. A total of 67 and 90 lipid molecules were putatively identified as biomarkers in ESI+ and ESI-, respectively. Identified lipids, including PC(40:6), C14 sphingomyelin, PS(O-36:0), and PE(41:4), played an important role in species discrimination. For the first time, this study provides a detailed lipidomics profile of commercially important loliginidae squid and establishes a faster workflow for species authentication.

Automated, cryogen-free headspace-trap with gas chromatography-mass spectrometry analysis of ethylene oxide and 2-chloroethanol as residual fumigants in foods.

Ethylene oxide (EtO), although banned for use, is still being detected in foodstuffs that have been fumigated to eradicate pests during storage and transport. Residual levels over the European Union's (EU) maximum residue limit (MRL) pose severe health concerns. Recent detection of EtO and its by-product 2-chloroethanol (2-CE) at alarming levels have led to product recalls throughout the EU. Here, a simple, automated headspace (HS)-trap method for the simultaneous determination of EtO and its derivative 2-CE by gas chromatography-mass spectrometry (GC-MS) at the required MRL of ≤ 0.05 mg/kg has been implemented. Syringe-based HS combined with backflushed trapping technology provided enrichment of multiple extractions from the same sample vial (known as multi-step enrichment or MSE®) to increase sensitivity for EtO and 2-CE analysis by GC-MS using single-ion-monitoring (SIM) mode. Method detection limits (MDLs) of 0.00059 mg/kg and 0.00219 mg/kg for EtO and 2-CE, respectively, were obtained without the need for manual handling, solvent extraction or derivatization methods. Recoveries were shown to average (n = 5) at 98% and 107% for EtO and 2-CE, respectively, and the reproducibility was <10% for both compounds.

Untargeted metabolomics reveals potential health risks associated with chronic exposure to environmentally relevant concentrations of 2-Phenylphenol.

Chronic exposure to endocrine-disrupting chemicals through foods of aquatic origin, at levels that are commonly found in the environment, can affect metabolic health and lead to metabolic diseases. One such chemical is 2-phenylphenol (2-PP), a suspected endocrine disruptor that is used extensively in agriculture and industry, and has become a widespread pollutant in aquatic environments. This study evaluated the risk of exposure to 2-PP through foods of aquatic origin from Vembanad Lake, using a Target Hazard Quotient (THQ) and an untargeted metabolomics approach. The study found that 2-PP content was higher in samples from areas with intense industrial, tourism, and agricultural activities. The average concentration of 2-PP in fish, crustaceans, and mollusks from the Vembanad estuary ranged from 0.012 to 0.017 mg/kg. The mean concentration of 2-PP was used to assess the THQ of exposure to the coastal population. The results showed that the THQ value was <1, indicating a low to moderate health risk for both adults and children. Furthermore, an untargeted metabolomics approach using HPLC-Q-Orbitrap MS was used to study the metabolome changes associated with chronic exposure to 2-PP (at the environmentally relevant concentration) over 60 days in the Wistar albino rat model. The findings indicated significant alterations in the phospholipid, fatty acid, sterol lipid, and amino acid profiles, suggesting that chronic exposure to 2-PP at environmentally relevant concentrations could affect purine, phenylalanine, tyrosine, and cholesterol metabolism.

Dynamic headspace GC-MS/MS analysis of ethylene oxide and 2-chloroethanol in dry food commodities: a novel approach.

With frequent RASFF notifications from the EU countries, the residue testing of ethylene oxide (EtO) and its metabolite 2-chloroethanol (2-CE) in food commodities has become essential to check their compliance with MRLs. This study, for the first time, aimed at establishing a dynamic headspace-GC-MS/MS method for the simultaneous determination of these two analytes in acetonitrile extracts of cumin, ashwagandha, chilli powder, turmeric powder, guar gum, locust bean gum, and ginger powder. The samples (4 g) were extracted using acetonitrile (10 mL). A dispersive-solid phase extraction cleanup step with primary secondary amine sorbent (50 mg/mL) reduced the interfering signal of (matrix-derived) acetaldehyde by >40% in chilli powder, ginger, turmeric, and guar gum. This cleanup was not required for sesame seeds. With high selectivity and sensitivity, the GC-MS/MS approach identified and quantified both compounds simultaneously. At the spiking levels of 0.01, 0.02, and 0.05 mg/kg, the recoveries and precision were satisfactory (70-120%, RSDs, ≤15%). The headspace method-performance was similar to liquid injections. The method provided reproducible results when evaluated by two different laboratories. The method provided high-precision results for incurred residue analysis. Given its efficiency, the validated method is anticipated to improve the effectiveness of monitoring of EtO residues in food commodities.

H3.3-G34R Mutation-Mediated Epigenetic Reprogramming Leads to Enhanced Efficacy of Immune Stimulatory Gene Therapy in Pediatric High-Grade Gliomas.

Pediatric high-grade gliomas (pHGGs) are diffuse and highly aggressive CNS tumors which remain incurable, with a 5-year overall survival of less than 20%. Within glioma, mutations in the genes encoding the histones H3.1 and H3.3 have been discovered to be age-restricted and specific of pHGGs. This work focuses on the study of pHGGs harboring the H3.3-G34R mutation. H3.3-G34R tumors represent the 9-15% of pHGGs, are restricted to the cerebral hemispheres, and are found predominantly in the adolescent population (median 15.0 years). We have utilized a genetically engineered immunocompetent mouse model for this subtype of pHGG generated via the Sleeping Beauty-transposon system. The analysis of H3.3-G34R genetically engineered brain tumors by RNA-Sequencing and ChIP-Sequencing revealed alterations in the molecular landscape associated to H3.3-G34R expression. In particular, the expression of H3.3-G34R modifies the histone marks deposited at the regulatory elements of genes belonging to the JAK/STAT pathway, leading to an increased activation of this pathway. This histone G34R-mediated epigenetic modifications lead to changes in the tumor immune microenvironment of these tumors, towards an immune-permissive phenotype, making these gliomas susceptible to TK/Flt3L immune-stimulatory gene therapy. The application of this therapeutic approach increased median survival of H3.3-G34R tumor bearing animals, while stimulating the development of anti-tumor immune response and immunological memory. Our data suggests that the proposed immune-mediated gene therapy has potential for clinical translation for the treatment of patients harboring H3.3-G34R high grade gliomas.

Method development and validation in the curry leaf matrix employing advanced mass spectrometry: quantitative screening of 490 multiclass pesticides by buffered ethyl acetate technique.

Curry leaf is an evergreen herb with culinary, pharmaceutical, and nutraceutical applications. As pesticide residue in curry leaf has garnered significant regulatory attention in recent years, here we report a reliable method, which was validated for the determination of 265 and 225 pesticides using LC-MS/MS and GC-MS/MS, respectively. At first, the sample was comminuted after adding water (1:2). The sample preparation workflow included extraction of 10 g homogenized sample with 10 mL ethyl acetate (+1% acetic acid), cleanup by dispersive solid phase extraction (d-SPE, 50 mg PSA + 50 mg C18 + 10 mg GCB + 150 mg Na2SO4) and the final analysis by tandem mass spectrometry. The cleanup step adeptly removed co-extractives. The method effectively reduced matrix effects and offered an LOQ of 0.01 mg kg-1 for most compounds. The method's accuracy and precision results fulfilled the requirements of SANTE/11312/2021 guidelines at 0.01 mg kg-1 and higher levels of fortification. The accuracy and precision results were comparable for all pesticides. The successful screening of market samples indicates its high extraction efficiency and precision for incurred residue analysis. Due to its robustness and conformity with regulatory criteria, food testing laboratories worldwide can use the method to monitor pesticide levels in curry leaves.

Regulatory landscape of risk assessment of pesticide residues in processed foods in India: a perspective.

In India, the levels of pesticide residues in Raw Agricultural Commodities (RAC) are being subjected to adequate legal regulations, and the health-risks associated with them are determined from time to time adhering to global standards. Since RACs are generally consumed by humans as the processed foods (PF), it is imperative to monitor the levels of pesticide residues in them in order to approach a realistic analysis of dietary exposure and concomitant health risk assessment. In India, production and consumption of PFs have a rising trend and hence it is indispensable to monitor the residue levels of pesticides in largely consumed PFs. Depending on the processing methods and physicochemical properties of pesticides, the residue levels may decrease or increase in a PF when compared to the corresponding RAC. While obtaining data on processing factors (Pf), it is pragmatic to focus on those situations in which the residues get concentrated following the processing step. Currently, regulatory agencies of several countries and the CODEX have determined the levels of pesticide residues in processed agriculture commodities, arrived at the Pfs, and fixed the maximum residue levels. Since consumption of PFs in India has tremendously increased in recent times and there is paucity of data about their health risks/benefits, it is imminent to deliberate on the complexities associated with the issues of adopting the Pfs generated by other regulatory agencies and subsequently examine the possibilities of generating the required data on Pfs on a priority basis to enable a comprehensive risk assessment.

Manjari Medika Grape Seed Extract Protects Methotrexate-Induced Hepatic Inflammation: Involvement of NF-κB/NLRP3 and Nrf2/HO-1 Signaling System.

Objective: Grape Seed Extract is a natural source of various polyphenols, which have been shown to possess potent antioxidant and free radical-scavenging activities. The earlier studies have reported that grape seed extract exhibits broad-spectrum pharmacological activities. Therefore, studying the hepatoprotective effects and elucidation of mechanisms of action of the Indian Variety, Manjari Medika grape seed extract (GSE), may give an insight into therapeutic benefits. Methotrexate (MTX) is the first-line pharmacological therapy for different rheumatic diseases. The major adverse events such as hepatotoxicity are evident even in the low doses used for the treatment. The present study investigated the role of MTX on hepatic damage in murine liver and the plausible protective effects of the Indian grape variety, Manjari Medika grape seed extract, in ameliorating it. Methods and Results: To assess the hepatological modulation, mice were divided into eight groups to investigate the ameliorative potential of this GSE (75 and 125 mg/kg) and correlate the experimental findings. The active components of the extract were assessed through UPLC-(ESI)-QToF-MS analysis. On the other hand, various biochemical and immunological indices were carried out to correlate the experimental data. The result demonstrated that the prophylactic administration of GSE reduced MTX-induced hepatic toxicity indices, which subsequently restored the hepatic morphological architecture. Moreover, the application of GSE in a dual dosage (75 and 125 mg/kg) suppressed MTX-induced reactive oxygen species generation, followed by lipid peroxidation and cellular nitrite formation. MTX-induced inflammasome activation through the redox-assisted cascade of TLR4/NF-κB signaling was further reduced by applying the GSE. The results showed that the activation of cytoprotective transcription factor Nrf2 enhanced the level of endogenous antioxidants. Furthermore, through the regulation of TLR4/NF-κB and Nrf2/HO-1 axis, this extract could reduce the MTX-mediated hepatic damage. Conclusion: Our findings suggest that Manjari Medika seed extract could be used as a therapeutic agent to relieve the side effects of MTX and other hepatic disorders.

Mycotoxin Monitoring, Regulation and Analysis in India: A Success Story.

Mycotoxins are deleterious fungal secondary metabolites that contaminate food and feed, thereby creating concerns regarding food safety. Common fungal genera can easily proliferate in Indian tropical and sub-tropical conditions, and scientific attention is warranted to curb their growth. To address this, two nodal governmental agencies, namely the Agricultural and Processed Food Products Export Development Authority (APEDA) and the Food Safety and Standards Authority of India (FSSAI), have developed and implemented analytical methods and quality control procedures to monitor mycotoxin levels in a range of food matrices and assess risks to human health over the last two decades. However, comprehensive information on such advancements in mycotoxin testing and issues in implementing these regulations has been inadequately covered in the recent literature. The aim of this review is thus to uphold a systematic picture of the role played by the FSSAI and APEDA for mycotoxin control at the domestic level and for the promotion of international trade, along with certain challenges in dealing with mycotoxin monitoring. Additionally, it unfolds various regulatory concerns regarding mycotoxin mitigation in India. Overall, it provides valuable insights for the Indian farming community, food supply chain stakeholders and researchers about India's success story in arresting mycotoxins throughout the food supply chain.

Multiresidue analysis of pesticides in three Indian soils: method development and validation using gas chromatography tandem mass spectrometry.

The paper reports a multiresidue method that was validated on 220 multi-class pesticides in three major Indian soils, namely, (i) new alluvial soil (NAS); (ii) red lateritic soil (RS) and (iii) black soil (BS) from three different regions. An ethyl acetate-based extraction method with a freezing-out cleanup step was employed for sample preparation, followed by gas chromatography-tandem mass spectrometric analysis. The method that was initially optimized on BS worked satisfactorily for the other two soil matrices. At the spiking level of 10 µg/kg (LOQ), the recoveries were satisfactory (within 70-120%) with precision-RSDs, ≤20% (n = 6) for 85, 88.6, and 89% of compounds in BS, RS, and NAS respectively. At 20 µg/kg, the method performance was satisfactory in each soil for all pesticides. When this validated method was applied to analyse 25 field samples, 6 pesticides were detected in them. In each case, precision (RSD) was <20%. The method sensitivity, accuracy and precision complied with the SANTE/2020/12830 guidelines. The method can be applied for environmental monitoring and risk assessment purposes, thus aiding in regulating pesticide usage in agricultural fields. The limitations and future scope of the study are also discussed.HighlightsA multiresidue method is reported for simultaneous analysis of multi-class pesticides in diverse soilsThe method was validated on 220 pesticides in new alluvial, red lateritic and black soilsSample preparation involved extraction with ethyl acetate and cleanup by a freezing stepThe residues were estimated by gas chromatography tandem mass spectrometry (GC-MS/MS)The method accuracy and precision complied with the EU's SANTE guidelines.

Zinc Finger MYND-Type Containing 8 (ZMYND8) Is Epigenetically Regulated in Mutant Isocitrate Dehydrogenase 1 (IDH1) Glioma to Promote Radioresistance.

PURPOSE: Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR). EXPERIMENTAL DESIGN: We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP. RESULTS: Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. CONCLUSIONS: These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648.

Multiresidue analysis of pesticides in four different pomegranate cultivars: Investigating matrix effect variability by GC-MS/MS and LC-MS/MS.

Matrix effect (ME) is unavoidable in multiresidue pesticide analysis, even when using highly advanced instruments, and differences in MEs can affect residue analytical accuracy due to pomegranate cultivar composition variations. However, literature to support this claim is limited.The study used GC-MS/MS and LC-MS/MS to investigate four different Indian pomegranate cultivar extracts and their MEs on multi-class pesticides.The whole fruit and arils of all cultivarswere tested for 22 GC-amenable and 21 LC-amenable pesticides. Principal component analysis of the data confirmed that each cultivar had unique MEs for each pesticide.The majority of pesticides showed acute variations in recovery rates with 95% confidence, while GC-MS/MS-amenablepesticides showed more variation. Although extrapolative dilution reduced the influence of MEs on analytical accuracy, a generalized matrix-matching for all cultivars was not possible to achieve.To reduce the variability in MEs, it is recommended that a cultivar-specific matrix-matched standard should be used.

Catalytic dye degradation by novel phytofabricated silver/zinc oxide composites.

Phytofabrication of the nanoparticles with exotic shape and size is an attractive area where nanostructures with noteworthy physicochemical and optoelectronic properties that can be significantly employed for photocatalytic dye degradation. In this study a medicinal plant, Plumbago auriculata leaf extract (PALE) was used to synthesize zinc oxide particles (ZnOPs) and silver mixed zinc oxide particles (ZnOAg1Ps, ZnOAg10Ps, ZnO10Ag1Ps) by varying the concentration of the metal precursor salts, i.e. zinc acetate and silver nitrate. The PALE showed significantly high concentrations of polyphenols, flavonoids, reducing sugar, starch, citric acid and plumbagin up to 314.3 ± 0.33, 960.0 ± 2.88, 121.3 ± 4.60, 150.3 ± 3.17, 109.4 ± 2.36, and 260.4 ± 8.90 µg/ml, respectively which might play an important role for green synthesis and capping of the phytogenic nanoparticles. The resulting particles were polydispersed which were mostly irregular, spherical, hexagonal and rod like in shape. The pristine ZnOPs exhibited a UV absorption band at 352 nm which shifted around 370 in the Ag mixed ZnOPs with concomitant appearance of peaks at 560 and 635 nm in ZnO10Ag1Ps and ZnOAg1Ps, respectively. The majority of the ZnOPs, ZnOAg1Ps, ZnOAg10Ps, and ZnO10Ag1Ps were 407, 98, 231, and 90 nm in size, respectively. Energy dispersive spectra confirmed the elemental composition of the particles while Fourier transform infrared spectra showed the involvement of the peptide and methyl functional groups in the synthesis and capping of the particles. The composites exhibited superior photocatalytic degradation of methylene blue dye, maximum being 95.7% by the ZnOAg10Ps with a rate constant of 0.0463 s-1 following a first order kinetic model. The present result clearly highlights that Ag mixed ZnOPs synthesized using Plumbago auriculata leaf extract (PALE) can play a critical role in removal of hazardous dyes from effluents of textile and dye industries. Further expanding the application of these phytofabricated composites will promote a significant complementary and alternative strategy for treating refractory pollutants from wastewater.

Development of immunotherapy for high-grade gliomas: Overcoming the immunosuppressive tumor microenvironment.

The preclinical and clinical development of novel immunotherapies for the treatment of central nervous system (CNS) tumors is advancing at a rapid pace. High-grade gliomas (HGG) are aggressive tumors with poor prognoses in both adult and pediatric patients, and innovative and effective therapies are greatly needed. The use of cytotoxic chemotherapies has marginally improved survival in some HGG patient populations. Although several challenges exist for the successful development of immunotherapies for CNS tumors, recent insights into the genetic alterations that define the pathogenesis of HGG and their direct effects on the tumor microenvironment (TME) may allow for a more refined and targeted therapeutic approach. This review will focus on the TME in HGG, the genetic drivers frequently found in these tumors and their effect on the TME, the development of immunotherapy for HGG, and the practical challenges in clinical trials employing immunotherapy for HGG. Herein, we will discuss broadly the TME and immunotherapy development in HGG, with a specific focus on glioblastoma multiforme (GBM) as well as additional discussion in the context of the pediatric HGG diagnoses of diffuse midline glioma (DMG) and diffuse hemispheric glioma (DHG).

H3.3-G34 mutations impair DNA repair and promote cGAS/STING-mediated immune responses in pediatric high-grade glioma models.

Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation led to the downregulation of DNA repair pathways. This resulted in enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG led to the accumulation of extrachromosomal DNA, which activated the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier-permeable PARP inhibitor pamiparib and the cell-cycle checkpoint CHK1/2 inhibitor AZD7762), and these combinations resulted in long-term survival for approximately 50% of the mice. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induced immune-mediated therapeutic efficacy in G34-mutant pHGG.

Genetic Evidence in Favor of a Polyketide Origin of Acremeremophilanes, the Fungal "Sesquiterpene" Metabolites.

Eremophilanes are a large group of "sesquiterpenes" produced by plants and fungi, with more than 180 compounds being known in fungi alone. Many of these compounds are phytotoxic, antimicrobial, anticancer and immunomodulators, and hence are of great economic values. Acremeremophilanes A to O have earlier been reported in a marine isolate of Acremonium sp. We report here the presence of Acremeremophilane I, G, K, N, and O, in a plant beneficial fungus Trichoderma virens, in a strain-specific manner. We also describe a novel, P strain-specific polyketide synthase (PKS) gene cluster in T. virens. This gene cluster, designated amm cluster, is absent in the genome of a Q strain of T. virens, and in other Trichoderma spp.; instead, a near identical cluster is present in the genome of the toxic mold Stachybotrys chartarum. Using gene knockout, we provide evidence that acremeremophilanes are biosynthesized via a polyketide route, and not via the mevalonate/terpene synthesis route as believed. We propose here that the 10-carbon skeleton is a product of polyketide synthase, to which a five-carbon isoprene unit is added by a prenyl transferase (PT), a gene for which is present next to the PKS gene in the genome. Based on this evidence, we propose that at least some of the eremophilanes classified in literature as sesquiterpenes (catalyzed by terpene cyclase) are actually meroterpenes (catalyzed by PKSs and PTs), and that the core moiety is not a sesquiterpene, but a hybrid polyketide/isoprene unit. IMPORTANCE The article contradicts the established fact that acremeremophilane metabolites produced by fungi are sesquiterpenes; instead, our findings suggest that at least some of these well-studied metabolites are of polyketide origin. Acremeremophilane metabolites are of medicinal significance, and the present findings have implications for the metabolic engineering of these metabolites and also their overproduction in microbial cell factories.

Analysis of aflatoxins and ochratoxin a in chilli powder using ultrahigh performance liquid chromatography with fluorescence detection and tandem mass spectrometry.

Chilli powder, a popular spice, is predominantly contaminated with aflatoxins (AFs) and ochratoxin A (OTA), posing a menace to public health. As no validated method exists for the simultaneous and direct analysis of AFs and OTA in chilli powder, it was imperative to develop one to ensure their effective monitoring and promote trade. In this research, we developed and validated a multi-mycotoxin analysis method that allows the simultaneous determination of AFs (AFB1, AFB2, AFG1 and AFG2) and OTA in chilli powder with high sensitivity, accuracy and precision. The optimised sample preparation workflow started with the extraction of chilli powder (25 g) with methanol-water (100 mL, 80:20). An aliquot (3 mL) was cleaned on a multi-mycotoxin, immunoaffinity column (AFLAOCHRA PREP®) and analysed using ultrahigh performance liquid chromatography with fluorescence (UHPLC-FLD) and tandem mass spectrometric (LC-MS/MS) detection in a single chromatographic run. The method performance was evaluated through intra- and inter-laboratory validation (ILV) studies, and also by analysing a certified reference material. A direct analysis using UHPLC-FLD (without derivatisation) provided the limits of quantification (LOQ) of 0.25 and 1 ng/g for AFs and OTA, respectively, while the LOQ for all these mycotoxins in LC-MS/MS was 0.5 ng/g. These LOQs are much lower than the maximum levels (MLs) specified by the European Commission. The recoveries of these analytes at LOQ and higher levels were above 75% (RSDr < 12%). The ILV study demonstrated satisfactory method-reproducibility (RSDR < 25%). The analysis of the certified reference material provided accuracies of AFs and OTA in the range of 83-101%. The analysis by UHPLC-FLD and LC-MS/MS provided very similar results. The incurred levels of B1 in market samples were estimated with a precision-RSD of < 6%. Considering its efficiency and alignment with the regulatory requirements, this method can be implemented for the routine analysis of AFs and OTA in chilli powder.