Delayed Graft Function After Kidney Transplantation: The Role of Residual Diuresis and Waste Products, as Oxalic Acid and Its Precursors.

Delayed graft function (DGF) after kidney transplantation heralds a worse prognosis. In patients with hyperoxaluria, the incidence of DGF is high. Oxalic acid is a waste product that accumulates when kidney function decreases. We hypothesize that residual diuresis and accumulated waste products influence the DGF incidence. Patients transplanted between 2018-2022 participated in the prospective cohort study. Pre-transplant concentrations of oxalic acid and its precursors were determined. Data on residual diuresis and other recipient, donor or transplant related variables were collected. 496 patients were included, 154 were not on dialysis. Oxalic acid, and glyoxylic acid, were above upper normal concentrations in 98.8%, and 100% of patients. Residual diuresis was ≤150 mL/min in 24% of patients. DGF occurred in 157 patients. Multivariable binary logistic regression analysis demonstrated a significant influence of dialysis type, recipient BMI, donor type, age, and serum creatinine on the DGF risk. Residual diuresis and glycolic acid concentration were inversely proportionally related to this risk, glyoxylic acid directly proportionally. Results in the dialysis population showed the same results, but glyoxylic acid lacked significance. In conclusion, low residual diuresis is associated with increased DGF incidence. Possibly accumulated waste products also play a role. Pre-emptive transplantation may decrease the incidence of DGF.

Metabolomic analysis in Amycolatopsis keratiniphila disrupted the competing ECO0501 pathway for enhancing the accumulation of vancomycin.

Vancomycin is a clinically important glycopeptide antibiotic against Gram-positive pathogenic bacteria, especially methicillin-resistant Staphylococcus aureus. In the mutant strain of Amycolatopsis keratiniphila HCCB10007 Δeco-cds4-27, the production of ECO-0501 was disrupted, but enhanced vancomycin yield by 55% was observed compared with the original strain of A. keratiniphila HCCB10007. To gain insights into the mechanism of the enhanced production of vancomycin in the mutant strain, comparative metabolomics analyses were performed between the mutant strain and the original strain, A. keratiniphila HCCB10007 via GC-TOF-MS and UPLC-HRMS. The results of PCA and OPLS-DA revealed a significant distinction of the intracellular metabolites between the two strains during the fermentation process. 64 intracellular metabolites, which involved in amino acids, fatty acids and central carbon metabolism, were identified as differential metabolites. The high-yield mutant strain maintained high levels of glucose-1-phosphate and glucose-6-phosphate and they declined with the increases of vancomycin production. Particularly, a strong association of fatty acids accumulation as well as 3,5-dihydroxyphenylacetic acid and non-proteinogenic amino acid 3,5-dihydroxyphenylglycine (Dpg) with enhancement of vancomycin production was observed in the high-yield mutant strain, indicating that the consumption of fatty acid pools might be beneficial for giving rise to 3,5-dihydroxyphenylacetic acid and Dpg which further lead to improve vancomycin production. In addition, the lower levels of glyoxylic acid and lactic acid and the higher levels of sulfur amino acids might be beneficial for improving vancomycin production. These findings proposed more advanced elucidation of metabolomic characteristics in the high-yield strain for vancomycin production and could provide potential strategies to enhance the vancomycin production.

Global regulation via modulation of ribosome pausing by the ABC-F protein EttA.

Having multiple rounds of translation of the same mRNA creates dynamic complexities along with opportunities for regulation related to ribosome pausing and stalling at specific sequences. Yet, mechanisms controlling these critical processes and the principles guiding their evolution remain poorly understood. Through genetic, genomic, physiological, and biochemical approaches, we demonstrate that regulating ribosome pausing at specific amino acid sequences can produce ~2-fold changes in protein expression levels which strongly influence cell growth and therefore evolutionary fitness. We demonstrate, both in vivo and in vitro, that the ABC-F protein EttA directly controls the translation of mRNAs coding for a subset of enzymes in the tricarboxylic acid (TCA) cycle and its glyoxylate shunt, which modulates growth in some chemical environments. EttA also modulates expression of specific proteins involved in metabolically related physiological and stress-response pathways. These regulatory activities are mediated by EttA rescuing ribosomes paused at specific patterns of negatively charged residues within the first 30 amino acids of nascent proteins. We thus establish a unique global regulatory paradigm based on sequence-specific modulation of translational pausing.

Multiple levels of transcriptional regulation control glycolate metabolism in Paracoccus denitrificans.

The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described ß-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. Although the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, and the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans. Together, these regulators control glycolate metabolism in P. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.IMPORTANCEAlgae perform ca. 50% of the photosynthetic carbon dioxide fixation on our planet. In the process, they release the two-carbon molecule glycolate. Due to the abundance of algae, massive amounts of glycolate are released. Therefore, this molecule is available as a source of carbon for bacteria in the environment. Here, we describe the regulation of glycolate metabolism in the model organism Paracoccus denitrificans. This bacterium uses the recently characterized ß-hydroxyaspartate cycle to assimilate glycolate in a carbon- and energy-efficient manner. We found that glycolate assimilation is dynamically controlled by three different transcriptional regulators: GlcR, BhcR, and CceR. This allows P. denitrificans to assimilate glycolate together with other carbon substrates in a simultaneous fashion. Overall, this flexible and multi-layered regulation of glycolate metabolism in P. denitrificans represents a resource-efficient strategy to make optimal use of this globally abundant molecule under fluctuating environmental conditions.

Spectrophotometric Assays for Measuring Photorespiratory Glutamate:Glyoxylate and Serine:Glyoxylate Aminotransferase Reactions.

Glutamate:glyoxylate aminotransferase (GGAT; EC 2.6.1.4) and serine:glyoxylate aminotransferase activities (SGAT; EC 2.6.1.45) are central photorespiratory reactions within plant peroxisomes. Both enzymatic reactions convert glyoxylate, a product of glycolate oxidase, to glycine, a substrate of the mitochondrial glycine decarboxylase complex. The GGAT reaction uses glutamate as an amino group donor and also produces α-ketoglutarate, which is recycled to glutamate in plastids by ferredoxin-dependent glutamate synthase. Using serine, a product of mitochondrial serine hydroxymethyltransferase, as an amino group donor, the SGAT reaction also produces hydroxypyruvate, a substrate of hydroxypyruvate reductase. The activities of these photorespiratory aminotransferases can be measured using indirect, coupled, spectrophotometric assays, detailed herein.

Unlocking the formate utilization of wild-type Yarrowia lipolytica through adaptive laboratory evolution.

Synthetic biology is contributing to the advancement of the global net-negative carbon economy, with emphasis on formate as a member of the one-carbon substrate garnering substantial attention. In this study, we employed base editing tools to facilitate adaptive evolution, achieving a formate tolerance of Yarrowia lipolytica to 1 M within 2 months. This effort resulted in two mutant strains, designated as M25-70 and M25-14, both exhibiting significantly enhanced formate utilization capabilities. Transcriptomic analysis revealed the upregulation of nine endogenous genes encoding formate dehydrogenases when cultivated utilizing formate as the sole carbon source. Furthermore, we uncovered the pivotal role of the glyoxylate and threonine-based serine pathway in enhancing glycine supply to promote formate assimilation. The full potential of Y. lipolytica to tolerate and utilize formate establishing the foundation for pyruvate carboxylase-based carbon sequestration pathways. Importantly, this study highlights the existence of a natural formate metabolic pathway in Y. lipolytica.

Metabolic reprogramming during Candida albicans planktonic-biofilm transition is modulated by the transcription factors Zcf15 and Zcf26.

Candida albicans is a commensal of the human microbiota that can form biofilms on implanted medical devices. These biofilms are tolerant to antifungals and to the host immune system. To identify novel genes modulating C. albicans biofilm formation, we performed a large-scale screen with 2,454 C. albicans doxycycline-dependent overexpression strains and identified 16 genes whose overexpression significantly hampered biofilm formation. Among those, overexpression of the ZCF15 and ZCF26 paralogs that encode transcription factors and have orthologs only in biofilm-forming species of the Candida clade, caused impaired biofilm formation both in vitro and in vivo. Interestingly, overexpression of ZCF15 impeded biofilm formation without any defect in hyphal growth. Transcript profiling, transcription factor binding, and phenotypic microarray analyses conducted upon overexpression of ZCF15 and ZCF26 demonstrated their role in reprogramming cellular metabolism by regulating central metabolism including glyoxylate and tricarboxylic acid cycle genes. Taken together, this study has identified a new set of biofilm regulators, including ZCF15 and ZCF26, that appear to control biofilm development through their specific role in metabolic remodeling.

The catabolism of ethylene glycol by Rhodococcus jostii RHA1 and its dependence on mycofactocin.

Ethylene glycol (EG) is a widely used industrial chemical with manifold applications and also generated in the degradation of plastics such as polyethylene terephthalate. Rhodococcus jostii RHA1 (RHA1), a potential biocatalytic chassis, grows on EG. Transcriptomic analyses revealed four clusters of genes potentially involved in EG catabolism: the mad locus, predicted to encode mycofactocin-dependent alcohol degradation, including the catabolism of EG to glycolate; two GCL clusters, predicted to encode glycolate and glyoxylate catabolism; and the mft genes, predicted to specify mycofactocin biosynthesis. Bioinformatic analyses further revealed that the mad and mft genes are widely distributed in mycolic acid-producing bacteria such as RHA1. Neither ΔmadA nor ΔmftC RHA1 mutant strains grew on EG but grew on acetate. In resting cell assays, the ΔmadA mutant depleted glycolaldehyde but not EG from culture media. These results indicate that madA encodes a mycofactocin-dependent alcohol dehydrogenase that initiates EG catabolism. In contrast to some mycobacterial strains, the mad genes did not appear to enable RHA1 to grow on methanol as sole substrate. Finally, a strain of RHA1 adapted to grow ~3× faster on EG contained an overexpressed gene, aldA2, predicted to encode an aldehyde dehydrogenase. When incubated with EG, this strain accumulated lower concentrations of glycolaldehyde than RHA1. Moreover, ecotopically expressed aldA2 increased RHA1's tolerance for EG further suggesting that glycolaldehyde accumulation limits growth of RHA1 on EG. Overall, this study provides insights into the bacterial catabolism of small alcohols and aldehydes and facilitates the engineering of Rhodococcus for the upgrading of plastic waste streams.IMPORTANCEEthylene glycol (EG), a two-carbon (C2) alcohol, is produced in high volumes for use in a wide variety of applications. There is burgeoning interest in understanding and engineering the bacterial catabolism of EG, in part to establish circular economic routes for its use. This study identifies an EG catabolic pathway in Rhodococcus, a genus of bacteria well suited for biocatalysis. This pathway is responsible for the catabolism of methanol, a C1 feedstock, in related bacteria. Finally, we describe strategies to increase the rate of degradation of EG by increasing the transformation of glycolaldehyde, a toxic metabolic intermediate. This work advances the development of biocatalytic strategies to transform C2 feedstocks.

Endogenous ureides are employed as a carbon source in Arabidopsis plants exposed to carbon starvation conditions.

Ureides, the degraded products of purine catabolism in Arabidopsis, have been shown to act as antioxidant and nitrogen sources. Herein we elucidate purine degraded metabolites as a carbon source using the Arabidopsis Atxdh1, Ataln, and Ataah knockout (KO) mutants vis-à-vis wild-type (WT) plants. Plants were grown under short-day conditions on agar plates containing half-strength MS medium with or without 1% sucrose. Notably, the absence of sucrose led to diminished biomass accumulation in both shoot and root tissues of the Atxdh1, Ataln, and Ataah mutants, while no such effect was observed in WT plants. Moreover, the application of sucrose resulted in a reduction of purine degradation metabolite levels, specifically xanthine and allantoin, predominantly within the roots of WT plants. Remarkably, an increase in proteins associated with the purine degradation pathway was observed in WT plants in the presence of sucrose. Lower glyoxylate levels in the roots but not in the shoot of the Atxdh1 mutant in comparison to WT, were observed under sucrose limitation, and improved by sucrose application in root, indicating that purine degradation provided glyoxylate in the root. Furthermore, the deficit of purine-degraded metabolites in the roots of mutants subjected to carbon starvation was partially mitigated through allantoin application. Collectively, these findings signify that under conditions of sucrose limitation and short-day growth, purines are primarily remobilized within the root system to augment the availability of ureides, serving as an additional carbon (as well as nitrogen) source to support plant growth.

Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose.

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.

From genome to evolution: investigating type II methylotrophs using a pangenomic analysis.

A comprehensive pangenomic approach was employed to analyze the genomes of 75 type II methylotrophs spanning various genera. Our investigation revealed 256 exact core gene families shared by all 75 organisms, emphasizing their crucial role in the survival and adaptability of these organisms. Additionally, we predicted the functionality of 12 hypothetical proteins. The analysis unveiled a diverse array of genes associated with key metabolic pathways, including methane, serine, glyoxylate, and ethylmalonyl-CoA (EMC) metabolic pathways. While all selected organisms possessed essential genes for the serine pathway, Methylooceanibacter marginalis lacked serine hydroxymethyltransferase (SHMT), and Methylobacterium variabile exhibited both isozymes of SHMT, suggesting its potential to utilize a broader range of carbon sources. Notably, Methylobrevis sp. displayed a unique serine-glyoxylate transaminase isozyme not found in other organisms. Only nine organisms featured anaplerotic enzymes (isocitrate lyase and malate synthase) for the glyoxylate pathway, with the rest following the EMC pathway. Methylovirgula sp. 4MZ18 stood out by acquiring genes from both glyoxylate and EMC pathways, and Methylocapsa sp. S129 featured an A-form malate synthase, unlike the G-form found in the remaining organisms. Our findings also revealed distinct phylogenetic relationships and clustering patterns among type II methylotrophs, leading to the proposal of a separate genus for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129. This pangenomic study unveils remarkable metabolic diversity, unique gene characteristics, and distinct clustering patterns of type II methylotrophs, providing valuable insights for future carbon sequestration and biotechnological applications. IMPORTANCE: Methylotrophs have played a significant role in methane-based product production for many years. However, a comprehensive investigation into the diverse genetic architectures across different genera of methylotrophs has been lacking. This study fills this knowledge gap by enhancing our understanding of core hypothetical proteins and unique enzymes involved in methane oxidation, serine, glyoxylate, and ethylmalonyl-CoA pathways. These findings provide a valuable reference for researchers working with other methylotrophic species. Furthermore, this study not only unveils distinctive gene characteristics and phylogenetic relationships but also suggests a reclassification for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129 into separate genera due to their unique attributes within their respective genus. Leveraging the synergies among various methylotrophic organisms, the scientific community can potentially optimize metabolite production, increasing the yield of desired end products and overall productivity.

Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Quantitative proteomics analysis reveals an important role of the transcriptional regulator UidR in the bacterial biofilm formation of Aeromonas hydrophila.

Introduction: Bacterial biofilm is a well-known characteristic that plays important roles in diverse physiological functions, whereas the current intrinsic regulatory mechanism of its formation is still largely unknown. Methods: In the present study, a label-free based quantitative proteomics technology was conducted to compare the differentially expressed proteins (DEPs) between ΔuidR and the wild-type strain in the biofilm state. Results: The results showed that the deletion of gene uidR encoding a TetR transcriptional regulator significantly increased the biofilm formation in Aeromonas hydrophila. And there was a total of 220 DEPs, including 120 up-regulated proteins and 100 down-regulated proteins between ΔuidR and the wild-type strain based on the quantitative proteomics. Bioinformatics analysis suggested that uidR may affect bacterial biofilm formation by regulating some related proteins in glyoxylic acid and dicarboxylic acid pathway. The expressions of selected proteins involved in this pathway were further confirmed by q-PCR assay, and the results was in accordance with the quantitative proteomics data. Moreover, the deletion of four genes (AHA_3063, AHA_3062, AHA_4140 and aceB) related to the glyoxylic acid and dicarboxylic acid pathway lead to a significant decrease in the biofilm formation. Discussion: Thus, the results indicated that uidR involved in the regulatory of bacterial biofilm formation, and it may provide a potential target for the drug development and a new clue for the prevention of pathogenic A. hydrophila in the future.

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata.

BACKGROUND: Biofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach. RESULTS: Functional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements. CONCLUSIONS: The study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies.

A molecular journey on the pathogenesis of primary hyperoxaluria.

PURPOSE OF REVIEW: Primary hyperoxalurias (PHs) are rare disorders caused by the deficit of liver enzymes involved in glyoxylate metabolism. Their main hallmark is the increased excretion of oxalate leading to the deposition of calcium oxalate stones in the urinary tract. This review describes the molecular aspects of PHs and their relevance for the clinical management of patients. RECENT FINDINGS: Recently, the study of PHs pathogenesis has received great attention. The development of novel in vitro and in vivo models has allowed to elucidate how inherited mutations lead to enzyme deficit, as well as to confirm the pathogenicity of newly-identified mutations. In addition, a better knowledge of the metabolic consequences in disorders of liver glyoxylate detoxification has been crucial to identify the key players in liver oxalate production, thus leading to the identification and validation of new drug targets. SUMMARY: The research on PHs at basic, translational and clinical level has improved our knowledge on the critical factors that modulate disease severity and the response to the available treatments, leading to the development of new drugs, either in preclinical stage or, very recently, approved for patient treatment.

Co-exposure of petrochemical workers to noise and mixture of benzene, toluene, ethylbenzene, xylene, and styrene: Impact on mild renal impairment and interaction.

Epidemiological evidence concerning effects of simultaneous exposure to noise and benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS) on renal function remains uncertain. In 2020, a cross-sectional study was conducted among 1160 petrochemical workers in southern China to investigate effects of their co-exposure on estimated glomerular filtration rate (eGFR) and mild renal impairment (MRI). Noise levels were assessed using cumulative noise exposure (CNE). Urinary biomarkers for BTEXS were quantified. We found the majority of workers had exposure levels to noise and BTEXS below China's occupational exposure limits. CNE, trans, trans-muconic acid (tt-MA), and the sum of mandelic acid and phenylglyoxylic acid (PGMA) were linearly associated with decreased eGFR and increased MRI risk. We observed U-shaped associations for both N-acetyl-S-phenyl-L-cysteine (SPMA) and o-methylhippuric acid (2-MHA) with MRI. In further assessing the joint effect of BTEXS (ß, -0.164 [95% CI, -0.296 to -0.033]) per quartile increase in all BTEXS metabolites on eGFR using quantile g-computation models, we found SPMA, tt-MA, 2-MHA, and PGMA played pivotal roles. Additionally, the risk of MRI associated with tt-MA was more pronounced in workers with lower CNE levels (P = 0.004). Multiplicative interaction analysis revealed antagonisms of CNE and PGMA on MRI risk (P = 0.034). Thus, our findings reveal negative dose-effect associations between noise and BTEXS mixture exposure and renal function in petrochemical workers. With the exception of toluene, benzene, xylene, ethylbenzene, and styrene are all concerning pollutants for renal dysfunction. Effects of benzene, ethylbenzene, and styrene exposure on renal dysfunction were more pronounced in workers with lower CNE.

Mrc1+ macrophage-derived IGF1 mitigates crystal nephropathy by promoting renal tubule cell proliferation via the AKT/Rb signaling pathway.

Rationale: The present understanding of the cellular characteristics and communications in crystal nephropathy is limited. Here, molecular and cellular studies combined with single-cell RNA sequencing (scRNA-seq) were performed to investigate the changes in cell components and their interactions in glyoxylate-induced crystallized kidneys to provide promising treatments for crystal nephropathy. Methods: The transcriptomes of single cells from mouse kidneys treated with glyoxylate for 0, 1, 4, or 7 days were analyzed via 10× Genomics, and the single cells were clustered and characterized by the Seurat pipeline. The potential cellular interactions between specific cell types were explored by CellChat. Molecular and cellular findings related to macrophage-to-epithelium crosstalk were validated in sodium oxalate (NaOx)-induced renal tubular epithelial cell injury in vitro and in glyoxylate-induced crystal nephropathy in vivo. Results: Our established scRNA atlas of glyoxylate-induced crystalline nephropathy contained 15 cell populations with more than 40000 single cells, including relatively stable tubular cells of different segments, proliferating and injured proximal tubular cells, T cells, B cells, and myeloid and mesenchymal cells. In this study, we found that Mrc1+ macrophages, as a subtype of myeloid cells, increased in both the number and percentage within the myeloid population as crystal-induced injury progresses, and distinctly express IGF1, which induces the activation of a signal pathway to dominate a significant information flow towards injured and proliferating tubule cells. IGF1 promoted the repair of damaged tubular epithelial cells induced by NaOx in vitro, as well as the repair of damaged tubular epithelial cells and the recovery of disease outcomes in glyoxylate-induced nephrolithic mice in vivo. Conclusion: After constructing a cellular atlas of glyoxylate-induced crystal nephropathy, we found that IGF1 derived from Mrc1+ macrophages attenuated crystal nephropathy through promoting renal tubule cell proliferation via the AKT/Rb signaling pathway. These findings could lead to the identification of potential therapeutic targets for the treatment of crystal nephropathy.

Model-guided metabolic rewiring to bypass pyruvate oxidation for pyruvate derivative synthesis by minimizing carbon loss.

Engineering microbial hosts to synthesize pyruvate derivatives depends on blocking pyruvate oxidation, thereby causing severe growth defects in aerobic glucose-based bioprocesses. To decouple pyruvate metabolism from cell growth to improve pyruvate availability, a genome-scale metabolic model combined with constraint-based flux balance analysis, geometric flux balance analysis, and flux variable analysis was used to identify genetic targets for strain design. Using translation elements from a ~3,000 cistronic library to modulate fxpK expression in a bicistronic cassette, a bifido shunt pathway was introduced to generate three molecules of non-pyruvate-derived acetyl-CoA from one molecule of glucose, bypassing pyruvate oxidation and carbon dioxide generation. The dynamic control of flux distribution by T7 RNAP-mediated synthetic small RNA decoupled pyruvate catabolism from cell growth. Adaptive laboratory evolution and multi-omics analysis revealed that a mutated isocitrate dehydrogenase functioned as a metabolic switch to activate the glyoxylate shunt as the only C4 anaplerotic pathway to generate malate from two molecules of acetyl-CoA input and bypass two decarboxylation reactions in the tricarboxylic acid cycle. A chassis strain for pyruvate derivative synthesis was constructed to reduce carbon loss by using the glyoxylate shunt as the only C4 anaplerotic pathway and the bifido shunt as a non-pyruvate-derived acetyl-CoA synthetic pathway and produced 22.46, 27.62, and 6.28 g/L of l-leucine, l-alanine, and l-valine by a controlled small RNA switch, respectively. Our study establishes a novel metabolic pattern of glucose-grown bacteria to minimize carbon loss under aerobic conditions and provides valuable insights into cell design for manufacturing pyruvate-derived products.IMPORTANCEBio-manufacturing from biomass-derived carbon sources using microbes as a cell factory provides an eco-friendly alternative to petrochemical-based processes. Pyruvate serves as a crucial building block for the biosynthesis of industrial chemicals; however, it is different to improve pyruvate availability in vivo due to the coupling of pyruvate-derived acetyl-CoA with microbial growth and energy metabolism via the oxidative tricarboxylic acid cycle. A genome-scale metabolic model combined with three algorithm analyses was used for strain design. Carbon metabolism was reprogrammed using two genetic control tools to fine-tune gene expression. Adaptive laboratory evolution and multi-omics analysis screened the growth-related regulatory targets beyond rational design. A novel metabolic pattern of glucose-grown bacteria is established to maintain growth fitness and minimize carbon loss under aerobic conditions for the synthesis of pyruvate-derived products. This study provides valuable insights into the design of a microbial cell factory for synthetic biology to produce industrial bio-products of interest.

Glyoxylate reductase: Definitive identification in human liver mitochondria, its importance for the compartment-specific detoxification of glyoxylate.

Glyoxylate is a key metabolite generated from various precursor substrates in different subcellular compartments including mitochondria, peroxisomes, and the cytosol. The fact that glyoxylate is a good substrate for the ubiquitously expressed enzyme lactate dehydrogenase (LDH) requires the presence of efficient glyoxylate detoxification systems to avoid the formation of oxalate. Furthermore, this detoxification needs to be compartment-specific since LDH is actively present in multiple subcellular compartments including peroxisomes, mitochondria, and the cytosol. Whereas the identity of these protection systems has been established for both peroxisomes and the cytosol as concluded from the deficiency of alanine glyoxylate aminotransferase (AGT) in primary hyperoxaluria type 1 (PH1) and glyoxylate reductase (GR) in PH2, the glyoxylate protection system in mitochondria has remained less well defined. In this manuscript, we show that the enzyme glyoxylate reductase has a bimodal distribution in human embryonic kidney (HEK293), hepatocellular carcinoma (HepG2), and cervical carcinoma (HeLa) cells and more importantly, in human liver, and is actively present in both the mitochondrial and cytosolic compartments. We conclude that the metabolism of glyoxylate in humans requires the complicated interaction between different subcellular compartments within the cell and discuss the implications for the different primary hyperoxalurias.