Synthesis Pathway Research Articles

One-pot chemobiological cascade strategy for synthesizing furyl hydroxymethyl ketone from corncob

Furyl hydroxymethyl ketone (FHK) is essential in synthesizing pharmaceuticals and biologically active molecules. However, green and sustainable methods for synthesizing FHK from renewable feedstocks remain challenging. This research proposed a simple one-pot chemobiocatalytic cascade strategy for effectually transforming renewable biomass into FHK via solid acid catalysis and whole-cells hydroxymethylation. Using whole-cells (SMPDC cells) containing pyruvate decarboxylase and small ubiquitin-like modifier (SUMO) fusion tag as biocatalysts, non-natural substrates furfural and formaldehyde were introduced into a C-C ligation bioreaction, establishing a biocatalytic pathway for FHK synthesis. SMPDC cells exhibited excellent biocatalytic activity and high tolerance to high furfural concentrations (up to 200mM), a well-known effective microbial inhibitor. Optimal reaction conditions were identified, enabling the manufacturing of FHK from corncob- and D-xylose-derived furfural with productivities of 0.12g FHK/(1g corncob + 0.09g HCHO) and 0.39g FHK/(1g D-xylose + 0.28g HCHO), respectively. This strategy demonstrated the potential for synthesizing valuable chemicals from low-cost biomass, providing a sustainable alternative to traditional chemical synthesis.

GhLCYε-3 characterized as a lycopene cyclase gene responding to drought stress in cotton

Drought stress significantly affects crop productivity. Carotenoids are essential photosynthetic pigment for plants, bacteria, and algae, with signaling and antioxidant functions. Lutein is a crucial branch product in the carotenoid synthesis pathway, which effectively improves the stress tolerance of higher plants. lycopene cyclase, a central enzyme for lutein synthesis, holds great significance in regulating lutein production. This research establishes a correlation between lutein content and stress resistance by measuring the drought resistance and lutein content of various cotton materials. To identify which crucial genes are associated with lutein, the lycopene cyclase family (LCYs) was analyzed. The research found that LCYs form a highly conserved family divided into two subfamilies, LCY-ε (lycopene ε-cyclase) and LCY-β (lycopene β-cyclase). Most members of the LCY family contain photoresponsive elements and abscisic acid elements. qRT-PCR demonstrates showed that most genes responded positively to drought stress, and GhLCYε-3 was expressed significantly differently under drought stress. Virus-induced gene silencing (VIGS) assay showed that the content of GhLCYε-3 was significantly increased with MDA and PRO, and the contents of chlorophyll and lutein were significantly decreased in pYL156 plants. The decrease in GhLCYε-3 expression is speculated to lead to reduced lutein content in vivo, resulting in the accumulation of reactive oxygen species (ROS) and decreased drought tolerance. This research enriched the understanding of LCY gene family and lutein function, and provided a new reference for cotton planting in arid areas. SynopsisLycopene cyclase plays an important role in enhancing the ability of scavenging ROS and drought resistance of plants.

Enduring effects of acute prenatal ischemia in rat soleus muscle, and protective role of erythropoietin.

Motor disorders are considered to originate mainly from brain lesions. Placental dysfunction or maternal exposure to a persistently hypoxic environment is a major cause of further motor disorders such as cerebral palsy. Our main goal was to determine the long-term effects of mild intrauterine acute ischemic stress on rat soleus myofibres and whether erythropoietin treatment could prevent these changes. Rat embryos were subjected to ischemic stress at embryonic day E17. They then received an intraperitoneal erythropoietin injection at postnatal days 1-5. Soleus muscles were collected at postnatal day 28. Prenatal ischemic stress durably affected muscle structure, as indicated by the greater fiber cross-sectional area (+ 18%) and the greater number of mature vessels (i.e. vessels with mature endothelial cells) per myofibres (+ 43%), and muscle biochemistry, as shown by changes in signaling pathways involved in protein synthesis/degradation balance (-81% for 4EBP1; -58% for AKT) and Hif1α expression levels (+ 95%). Erythropoietin injection in ischemic pups had a weak protective effect: it increased muscle mass (+ 25% with respect to ischemic pups) and partially prevented the increase in muscle degradation pathways and mature vascularization, whereas it exacerbated the decrease in synthesis pathways. Hence, erythropoietin treatment after acute ischemic stress contributes to muscle adaptation to ischemic conditions.

Enhancement of the Photoluminescent Intensity of YVO4: Er, Yb UCNPs Using a Simple Coprecipitation Synthesis Method.

Yttrium vanadate (YVO4) is a non-toxic ceramic matrix that, when doped with lanthanides, can be used as a photoluminescent biosensor. In this study, we meticulously synthesized upconversion nanoparticles (UCNPs) of YVO4 via chemical coprecipitation, using Er3+ and Yb3+ ions for codoping. The light emission achieved through upconversion mechanisms enables the excitation of nanoparticles with infrared light rather than ultraviolet light, enhancing the potential of current bioimaging techniques. The light emission intensity of our YVO4: Er, Yb UCNPs, a key factor in their effectiveness, depended on various easily adjustable factors during the synthesis, such as the dopant concentration, the heat treatment, and the cleaning process. The UCNPs were characterized using a range of advanced techniques, including X-ray diffraction (XRD) and Rietveld refinements, as well as Raman, photoluminescence (PL), and ultraviolet-visible (UV-vis) spectroscopies, and high-resolution transmission electron microscopy (HRTEM). We found the most convenient stoichiometry to obtain the YVO4: Er, Yb UCNPs and showed that a rigorous thermal treatment was necessary to achieve light emission through upconversion mechanisms. We also discovered that some porosity characteristics can be promoted in the YVO4: Er, Yb UCNPs during the cleaning process, depending on the solvent employed. The porosity and morphology of the nanoparticles could be predicted using the microstrain values obtained from the refinement of the crystalline structures. All these meticulous steps in our research have enabled us to develop an efficient synthesis pathway to produce YVO4: Er, Yb UCNPs with high photoluminescent intensity.

Cytomegalovirus infection of the fetal brain: intake of aspirin during pregnancy blunts neurodevelopmental pathogenesis in the offspring.

Congenital cytomegalovirus (CMV) infections represent one leading cause of human neurodevelopmental disorders. Despite their high prevalence and severity, no satisfactory therapy is available and pathophysiology remains elusive. The pathogenic involvement of immune processes occurring in infected developing brains has been increasingly documented. Here, we have used our previously validated rat model of CMV infection of the fetal brain in utero to test whether the maternal administration of four different drugs with immunomodulatory properties would have an impact on the detrimental postnatal outcome of CMV infection. CMV infection of the rat fetal brain was done intracerebroventricularly. Each of the drugs, including acetylsalicylic acid (aspirin, ASA), a classical inhibitor of cyclooxygenases Cox-1 and Cox-2, the two key rate-limiting enzymes of the arachidonic acid-to-prostaglandins (PG) synthesis pathway, was administered to pregnant dams until delivery. ASA was selected for subsequent analyses based on the improvement in postnatal survival. A combination of qRT-PCR, mass spectrometry-based targeted lipidomics, immunohistochemistry experiments, monitoring of neurologic phenotypes and electrophysiological recordings was used to assess the impact of ASA in CMV-infected samples and pups. The postnatal consequences of CMV infection were also analyzed in rats knocked-out (KO) for Cox-1. Increased PGE2 levels and increased proportions of Cox-1+ and Cox-2+ microglia were detected in CMV-infected developing brains. Maternal intake of ASA led to decreased proportion of Cox-1+ fetal, but not neonatal, microglia, while leaving the proportions of Cox-2+ microglia unchanged. Maternal intake of ASA also improved the key postnatal in vivo phenotypes caused by CMV infection and dramatically prevented against the spontaneous epileptiform activity recorded in neocortical slices from CMV-infected pups. In contrast with maternal intake of ASA, Cox-1 KO pups displayed no improvement in the in vivo phenotypes after CMV infection. However, as with ASA administration, the spontaneous epileptiform activity was dramatically inhibited in neocortical slices from CMV-infected, Cox-1 KO pups. Overall, our data indicate that, in the context of CMV infection of the fetal brain, maternal intake of ASA during pregnancy improved CMV-related neurodevelopmental alterations in the offspring, likely via both Cox-1 dependent and Cox-1 independent mechanisms, and provide proof-of-principle for the use of ASA against the detrimental outcomes of congenital CMV infections.

Mutation of rice EARLY LEAF LESION AND SENESCENCE 1(ELS1), which encodes an anthranilate synthase α-subunit, induces ROS accumulation and cell death through activating the tryptophan synthesis pathway in rice.

Lesion-mimic mutants (LMMs) serve as valuable resources for uncovering the molecular mechanisms that govern programmed cell death (PCD) in plants. Despite extensive research, the regulatory mechanisms of PCD and lesion formation in various LMMs remain to be fully elucidated. In this study, we identified a rice LMM named early leaf lesion and senescence 1 (els1), cloned the causal gene through map-based cloning, and confirmed its function through complementation. ELS1 encodes an anthranilate synthase α-subunit involved in anthranilate biosynthesis. It is predominantly localized in chloroplasts and is primarily expressed in light-exposed tissues. Mutation of ELS1 triggers upregulation of its homologous gene, ASA1, via a genetic compensation response, leading to the activation of the tryptophan (Trp) synthesis pathway and amino acid metabolism. The accumulation of abnormal Trp-derived intermediate metabolites results in reactive oxygen species (ROS) production and abnormal PCD in the els1 mutant, ultimately causing the leaf lesion phenotype. The els1 mutant also exhibits reduced chlorophyll content, upregulation of genes related to chloroplast degradation and leaf senescence, and decreased activity of photosynthetic proteins, indicating that ELS1 plays a role in chloroplast development. These factors collectively contribute to the premature leaf senescence observed in the els1 mutant. Our findings shed light on the role of ELS1 in regulating ROS accumulation and PCD in rice, providing further genetic insights into the molecular mechanisms governing leaf lesions and senescence.

The metabolic engineering of Escherichia coli for the high-yield production of hypoxanthine.

Hypoxanthine, prevalent in animals and plants, is used in the production of food additives, nucleoside antiviral drugs, and disease diagnosis. Current biological fermentation methods synthesize quantities insufficient to meet industrial demands. Therefore, this study aimed to develop a strain capable of industrial-scale production of hypoxanthine. De novo synthesis of hypoxanthine was achieved by blocking the hypoxanthine decomposition pathway, thus alleviating transcriptional repression and multiple feedback inhibition, and introducing a purine operon from Bacillus subtilis to construct a chassis strain. The effects of knocking out the IMP(Inosine 5'-monophosphate) branch on the growth status and titer of the strain were then investigated, and the effectiveness of adenosine deaminase and adenine deaminase was verified. Overexpressing these enzymes created a dual pathway for hypoxanthine synthesis, enhancing the metabolic flow of hypoxanthine synthesis and preventing auxotrophic strain formation. Introducing IMP-specific 5' -nucleotidase addressed the issue of adenylate accumulation. In addition, the metabolic flow of the guanine branch was dynamically regulated by the guaB gene. The supply of glutamine and aspartic acid precursors was enhanced by introducing an exogenous glnA mutant gene, overexpressing aspC, and replacing the weaker promoter to regulate the aspartic acid branching pathway. Ultimately, fermentation in a 5L bioreactor for 48h produced 30.6g/L hypoxanthine, with a maximum real-time productivity of 1.4g/L/h, the highest value of hypoxanthine production by microbial fermentation reported so far. The intracellular purine biosynthesis pathway is extensive and regulated at multiple levels in cells. The IMP branch in the hypoxanthine synthesis pathway has a higher metabolic flux. The current challenge lies in systematically allocating the metabolic flux within the branch pathway to achieve substantial product accumulation. In this study, E. coli was used as the chassis strain to construct a dual pathway for IMP and AMP(Adenosine 5'-monophosphate) synergistic hypoxanthine synthesis and dynamically regulate the guanine branch pathway. Overall, our experimental efforts culminated in a high-yield, plasmid- and defect-free engineered hypoxanthine strain.

Betaine lipids overproduced in seed plants are excluded from plastid membranes and promote endomembrane expansion

Abstract Plants and algae have to adapt to environmental changes and face various stresses that negatively affect their growth and development. One common stress is phosphate (Pi) deficiency, which is often present in the environment at limiting levels. In response to Pi deficiency, these organisms increase Pi uptake and remobilize intracellular Pi. Phospholipids are degraded to provide Pi and replaced by non-phosphorus lipids, such as glycolipids or betaine lipids. During evolution, seed plants lost the ability to synthesize betaine lipids. By expressing BTA1 genes, which are involved in the synthesis of diacylglyceryl-N,N,N-trimethyl-homoserine (DGTS), from different species, our work shows that DGTS can be produced in seed plants. In Arabidopsis, expression of BTA1 under a phosphate starvation-inducible promoter resulted in limited DGTS production without having any impact on plant growth or lipid remodeling. In transient expression systems in Nicotiana benthamiana, leaves were able to accumulate DGTS up to 30 % of their glycerolipid content at a slight expense of galactolipid and phospholipid production. At the subcellular level, we showed that DGTS is absent from the plastids and seems to be enriched in the endomembranes, driving an ER membrane proliferation. Finally, the DGTS synthesis pathway seems to compete with PC synthesis via the Kennedy pathway but does not seem to be derived from the PC diacylglycerol backbone and therefore does not interfere with the eukaryotic pathway involved in galactolipid synthesis.

Catalytic Strategies for the Selective Synthesis of α‐ and β‐Functionalized Amines via Hydrofunctionalization of Enamides

Abstractα‐ and β‐Functionalized amines are two classes of important compounds, whose structural units are widely present in approved drugs. The hydrofunctionalization of enamides is the most direct and efficient method for constructing these two types of compounds. Regioselectivity is the focus of this conversion, and is largely influenced by the catalytic mode. Therefore, to selectively synthesize α‐ or β‐functionalized amines, the progress in the catalytic hydrofunctionalization of enamides over the past decade is reviewed, and the catalytic pathways for this process discussed in detail. This account aims to objectively evaluate the effects of various catalytic pathways on the regioselectivity of hydrofunctionalization, to provide a new perspective on the selection of catalytic methods and the design of catalytic pathways for the selective synthesis of α‐ or β‐functionalized amines.

Sertraline/chloroquine combination therapy to target hypoxic and immunosuppressive serine/glycine synthesis-dependent glioblastomas.

The serine/glycine (ser/gly) synthesis pathway branches from glycolysis and is hyperactivated in approximately 30% of cancers. In ~13% of glioblastoma cases, we observed frequent amplifications and rare mutations in the gene encoding the enzyme PSPH, which catalyzes the last step in the synthesis of serine. This urged us to unveil the relevance of PSPH genetic alterations and subsequent ser/gly metabolism deregulation in the pathogenesis of glioblastoma. Primary glioblastoma cells overexpressing PSPH and PSPHV116I showed an increased clonogenic capacity, cell proliferation, and migration, supported by elevated nucleotide synthesis and utilization of reductive NAD(P). We previously identified sertraline as an inhibitor of ser/gly synthesis and explored its efficacy at suboptimal dosages in combination with the clinically pretested chloroquine to target ser/glyhigh glioblastoma models. Interestingly, ser/glyhigh glioblastomas, including PSPHamp and PSPHV116I, displayed selective synergistic inhibition of proliferation in response to combination therapy. PSPH knockdown severely affected ser/glyhigh glioblastoma clonogenicity and proliferation, while simultaneously increasing its sensitivity to chloroquine treatment. Metabolite landscaping revealed that sertraline/chloroquine combination treatment blocks NADH and ATP generation and restricts nucleotide synthesis, thereby inhibiting glioblastoma proliferation. Our previous studies highlight ser/glyhigh cancer cell modulation of its microenvironment at the level of immune suppression. To this end, high PSPH expression predicts poor immune checkpoint therapy responses in glioblastoma patients. Interestingly, we show that PSPH amplifications in glioblastoma facilitate the expression of immune suppressor galectin-1, which can be inhibited by sertraline treatment. Collectively, we revealed that ser/glyhigh glioblastomas are characterized by enhanced clonogenicity, migration, and suppression of the immune system, which could be tackled using combined sertraline/chloroquine treatment, revealing novel therapeutic opportunities for this subgroup of GBM patients.

Regulation of Milk Fat Synthesis: Key Genes and Microbial Functions

Milk is rich in a variety of essential nutrients, including fats, proteins, and trace elements that are important for human health. In particular, milk fat has an alleviating effect on diseases such as heart disease and diabetes. Fatty acids, the basic units of milk fat, play an important role in many biological reactions in the body, including the involvement of glycerophospholipids and sphingolipids in the formation of cell membranes. However, milk fat synthesis is a complex biological process involving multiple organs and tissues, and how to improve milk fat of dairy cows has been a hot research issue in the industry. There exists a close relationship between milk fat synthesis, genes, and microbial functions, as a result of the organic integration between the different tissues of the cow’s organism and the external environment. This review paper aims (1) to highlight the synthesis and regulation of milk fat by the first and second genomes (gastrointestinal microbial genome) and (2) to discuss the effects of ruminal microorganisms and host metabolites on milk fat synthesis. Through exploring the interactions between the first and second genomes, and discovering the relationship between microbial and host metabolite in the milk fat synthesis pathway, it may become a new direction for future research on the mechanism of milk fat synthesis in dairy cows.

Biological Characteristics of a Novel Bibenzyl Synthase (DoBS1) Gene from Dendrobium officinale Catalyzing Dihydroresveratrol Synthesis

Bibenzyl compounds are one of the most important bioactive components of natural medicine. However, Dendrobium officinale as a traditional herbal medicine is rich in bibenzyl compounds and performs functions such as acting as an antioxidant, inhibiting cancer cell growth, and assisting in neuro-protection. The biosynthesis of bibenzyl products is regulated by bibenzyl synthase (BBS). In this study, we have cloned the cDNA gene of the bibenzyl synthase (DoBS1) from D. officinale using PCR with degenerate primers, and we have identified a novel type III polyketide synthase (PKS) gene by phylogenetic analyses. In a series of perfect experiments, DoBS1 was expressed in Escherichia coli, purified and some catalytic properties of the recombinant protein were investigated. The molecular weight of the recombinant protein was verified to be approximately 42.7 kDa. An enzyme activity analysis indicated that the recombinant DoBS1-HisTag protein was capable of using 4-coumaryol-CoA and 3 malonyl-CoA as substrates for dihydroresveratrol (DHR) in vitro. The Vmax and Km of the recombinant protein for DHR were 3.57 ± 0.23 nmol·min−1·mg−1 and 0.30 ± 0.08 mmol, respectively. The present study provides further insights into the catalytic mechanism of the active site in the biosynthetic pathway for the catalytic production of dihydroresveratrol by bibenzylase in D. officinale. The results can be used to optimize a novel biosynthetic pathway for the industrial synthesis of DHR.

Multi-Omics Analysis Uncovers the Mechanism for Enhanced Organic Acid Accumulation in Peach (Prunus persica L.) Fruit from High-Altitude Areas

The early-ripening peach industry has undergone rapid development in the Panxi region of the Sichuan Basin in recent years. However, after the introduction of some new peach varieties to the high-altitude peach-producing areas in Panxi, the titratable acid content in peach fruit has significantly increased. This study compared the fruit quality indicators of early-ripening peach varieties cultivated in Xide County (a high-altitude peach-producing area) and Longquanyi District (a low-altitude peach-producing area) in Sichuan Province and analyzed the differences in organic acid metabolism by combining primary metabolomic and transcriptomic approaches. The results showed that the ‘Zhongtaohongyu’ fruit from the high-altitude peach-producing area had a much higher accumulation of malic acid and, accordingly, a significantly higher organic acid content than the other samples. The lower annual average temperature and stronger ultraviolet radiation in high-altitude peach-producing areas may lead to the increased expression of genes (PpNAD-ME1, PpNADP-ME3, and PpPEPC1) in the organic acid synthesis pathway and the decreased expression of genes (PpACO2, PpNAD-MDH2/3/4/5, and PpPEPCK2) in the organic acid degradation pathway in peach fruit, ultimately resulting in the accumulation of more organic acids. Among them, the downregulation of the key genes PpNAD-MDH3/4/5 involved in malic acid metabolism may be the main reason for the higher malic acid accumulation in peach fruit from high-altitude peach-producing areas. Overall, this study elucidates the mechanism by which environmental factors enhance the accumulation of organic acids in peach fruit from high-altitude peach-producing areas from a multi-omics perspective, as well as providing a theoretical basis for screening key genes involved in organic acid metabolism in peach fruit.

The Development, Essence and Perspective of Nitrogen Reduction to Ammonia.

Ammonia plays a pivotal role in agriculture and meanwhile holds promising potential as an energy vector for the hydrogen economy, where the nitrogen reduction to ammonia is a critical pathway for achieving sustainable development. Over the past hundred years, ammonia synthesis has undergone several breakthrough developments from Haber-Bosch process to photo/electro-catalysis and Li-mediated strategy, but still faces the challenges of low yield rate, selectivity and efficiency. Therefore, there is a pressing demand to develop efficient and green ammonia synthesis from nitrogen. This review summarizes the development of the nitrogen reduction to ammonia, highlighting six milestones during the whole journey. From the development direction, this work finds and extracts the essence of ammonia synthesis, that is the reaction pathways are affected by the energy barrier of reaction intermediates, which can be altered by proton sources, auxiliaries and catalysts. Then this work discusses the detailed overview of the significant development of proton source, auxiliaries and catalysts. Finally, based on the essence, the possible opportunities of ammonia synthesis from nitrogen reduction are presented, including the design of new ammonia synthesis pathways and efficient catalysts. The deep insight of nitrogen reduction to ammonia will provide a design guidance for efficient ammonia synthesis.

STEM-08. LIPID SATURATION HOMEOSTASIS PRESENTS A TARGETABLE VULNERABILITY IN MYC-AMPLIFIED GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain cancer, originating from the cerebellum. Despite advancements in standard of care (SoC), MB remains fatal for 30% of patients. Tumor relapse, spinal metastasis and treatment resistance are the most prevalent in MYC-driven Group 3 MB (G3-MB). Patients surviving SoC are faced with life-long neurocognitive and neurodevelopmental deficits. These issues highlight the urgent need for improved treatment modalities. Reprogramming of cellular lipid metabolism is an emerging hallmark of cancer and may yield novel cancer-specific therapeutic options. Here we explore the lipidome of both G3-MB and its proposed cell of origin, human neural stem cells (hNSCs) by comparing untargeted lipidomics using Liquid-Chromatography-Mass Spectrometry (LC-MS). Comparative analyses revealed a differential abundance of distinct lipid species in G3-MB, with an overall reduced saturation level of fatty acids (FAs). These findings implicate the de novo lipid synthesis (DNL) pathway. We identified the enzymes involved in DNL to be essential for MB survival in our genome-wide CRISPR KO screen. Additionally, mRNA expression of the DNL enzymes increases at relapse in our SoC-adapted murine patient-derived xenograft (PDX) model. Pharmacological and genetic targeting of the DNL enzyme Stearoyl-CoA Desaturase 1 (SCD1) selectively targets G3-MB. Furthermore, small molecule treatment of SCD1 demonstrates efficacy against G3-MB PDX models in vivo. We identified SCD expression as a prognostic marker in Group 3 and 4 MB patients and identified possible pathways for MB treatment. Overall, these findings indicate that SCD1 is a potent target for the treatment of G3-MB.

Identification of the new allele ptc1-2 and analysis of the regulatory role of PTC1 gene in rice anther development.

Anther development involves a series of important biological events that are precisely regulated by many genes. Although several important genes involved in rice anther development have been identified, the regulatory network involved in tapetal development and pollen wall formation is still largely unclear. PERSISTENT TAPETAL CELL 1 (PTC1) encodes a PHD-Finger protein, which plays a critical role in the regulation of tapetal cell death and pollen development in rice. Here, we report the isolation and characterization of a new allele ptc1-2 with 2-base deletion in the third exon, causing the absent of the PHD domain due to the sequence change. Cytological analysis revealed delayed tapetal PCD, defective pollen exine formation and abnormal ubisch bodies development. Transcriptome analysis revealed that genes related to pollen wall formation (secondary metabolism, phenylalanine synthesis, and cutin and wax biosynthesis pathways), cell death (cysteine and methionine metabolism and DNA repair pathways), and carbohydrate synthesis (starch and sucrose metabolism pathways) were significantly altered in ptc1-2 mutant. A total of 13 reported anther development genes exhibited significant expression changes in the ptc1-2 mutant. Yeast two-hybrid and BiFC analyses showed that PTC1 could interact with API5, an inhibitor of apoptosis, and the citrin-binding enzyme EDT1. This work is helpful in deepening the understanding of the regulatory network of male reproductive development in rice.

TMET-10. TARGETING PGM3 TO INHIBIT HEXOSAMINE PRODUCTION IS AN EFFECTIVE APPROACH TO TREAT GLIOBLASTOMA

Abstract Hexosamine biosynthesis pathway (HBP) mediated by a series of enzymes is highly upregulated in many cancers to promote the glycosylation of proteins and lipids, while which enzyme in this pathway is better to serve as anti-tumor target is unclear. Here, we found targeting GFAT1, the initial and rate-limiting enzyme in HBP, fails to inhibit the growth of glioblastoma (GBM), the most lethal brain tumor, due to this cancer dramatically elevating NAGK-mediated hexosamine salvage pathway. In contrast, targeting PGM3, the latter enzyme in HBP, blocks both de novo hexosamine synthesis and salvage pathways, effectively inhibiting GBM growth. We further identified HBP enzymes are upregulated by SREBP-1, a key lipogenic transcriptional factor. In turn, this upregulation promotes SREBP-1 activation to stimulate lipogenesis via stabilization of its key transporter SCAP by N-glycosylation modification. Together, our study uncovered a previously unrecognized HBP-SCAP-SREBP-1 feedforward loop and demonstrated targeting PGM3 is an effective approach to treat GBM.

TMET-08. RADIATION-INDUCED SLC25A22 CONTRIBUTES TO GLIOBLASTOMA RADIORESISTANCE ACQUISITION THROUGH REMODELING GLUTAMATE METABOLISM

Abstract Glioblastoma Multiforme (GBM), a malignant primary brain tumor, is treated by surgical resection followed by chemo- and radiotherapy. Nevertheless, most GBM patients show dismal prognosis due to the radioresistance acquisition of GBM cells via metabolic rewiring. Therefore, it is urgently required to discover how metabolic rewiring attributes to GBM radioresistance, and its targeting strategies. To identify GBM radioresistance driver genes, we previously established radioresistance GBM cells (RGCs) via repetitive in vivo selection, followed by mRNA sequencing, and selected candidate genes that are altered in RGCs (fold change>2 or <0.5, p-value<0.05) and also related with GBM patients’ prognosis. Here, we focused on amino acid transmembrane transporter (Gene Ontology: 0003333) and found that SLC25A22 was the most increased in RGCs. SLC25A22 exhibits uni-directional properties, exporting glutamate to cytosol, resulting in cytosolic glutamate accumulation in RGCs. Using 13C glutamine isotope tracing analysis following mitochondrial fractionation, we quantified the mitochondrial metabolic flux of glutamine. Despite decreased 13C glutamate efflux from RGCs mitochondria after genetic inhibition of SLC25A22, glutamine metabolites (a-ketoglutarate, succinate, malate, fumarate) remained constant. Cytosolic glutamate, as a substrate, is incorporated into various biosynthetic pathways, especially glutathione (GSH) and proline synthesis pathways in radioresistant GBM cells. GSH protects GBM cells by scavenging radiation-induced reactive oxygen species. Whereas, proline, a rate-limiting substrate for collagen biosynthesis, leads GBM cells to show an invasiveness phenotype via extracellular matrix remodeling. Genetic inhibition of SLC25A22 using short hairpin RNA (shRNA) or microRNA-184 mimic suppresses RGCs radioresistance and aggressiveness. Additionally, intranasal administration of microRNA-184 mimic prolonged the survival (median survival: 26 days after irradiation) of GBM-bearing mice, compared with non-target control (median survival: 16 days after irradiation). Together, our study demonstrates that SLC25A22 upregulation confers GBM radioresistance by inducing cytosolic glutamate-driven biosynthesis and suggests SLC25A22 as a therapeutic target to overcome GBM radioresistance.

CapD deletion in the Elizabethkingia miricola capsular locus leads to capsule production deficiency and reduced virulence.

Elizabethkingia miricola is a multidrug-resistant pathogen that can cause life-threatening infections in immunocompromised humans and outbreaks in amphibians. However, the specific virulence factors of this microorganism have not been described. In this study, we identified the polysaccharide biosynthesis protein-encoding gene capD, which is located in the conserved region of the Wzy-dependent capsule synthesis gene cluster in the E. miricola strain FL160902, and investigated its role in the pathogenesis of E. miricola. Our results revealed that the capD deletion strain (ΔcapD) lost its typical encapsulated structure, with a 45% reduction in cell wall thickness. CapD affects wza expression in the capsule polysaccharide synthesis pathway. Furthermore, the survival rates were significantly reduced in ΔcapD in response to complement-mediated killing, desiccation stress, and macrophage phagocytosis, whereas biofilm formation, surface hydrophobicity, and adherence to both endothelial and epithelial cells were increased. Additionally, the deletion of capD sharply attenuated the virulence of E. miricola in a frog infection model. Complementation of the capD gene restored the biological properties and virulence to wild-type levels. Overall, these findings suggest that CapD contributes to polysaccharide synthesis and plays a crucial role in the pathogenesis of E. miricola.

Chromosome-level assembly of the Clinopodium gracile genome

Clinopodium gracile is an important medicinal herb in the Lamiaceae family. This species lacks corresponding genomic resources, which significantly limits the study of its active compound synthesis pathways, breeding practices, and assessment of natural genetic variations. We assembled the chromosomal-level genome of C. gracile using Oxford Nanopore (ONT) technology and Hi-C sequence. The assembled genome is 307.3 Mb in size and consists of 9 chromosomes. The scaffold N50 was 36.3 Mb. The BUSCO completeness (Embryophyta_db10) of the genome was 97.2%. The genome annotates 40,083 protein coding genes. C. gracile and S. miltiorrhiza diverged approximately 30.615 million years ago. C. gracile has not undergone recent species-specific WGD events. A high proportion of young LTRs indicates a recent transposable element (TE) transposition burst in C. gracile.