Shunt Pathway Research Articles

Deciphering the metabolic capabilities of Bifidobacteria using genome-scale metabolic models

Bifidobacteria, the initial colonisers of breastfed infant guts, are considered as the key commensals that promote a healthy gastrointestinal tract. However, little is known about the key metabolic differences between different strains of these bifidobacteria, and consequently, their suitability for their varied commercial applications. In this context, the present study applies a constraint-based modelling approach to differentiate between 36 important bifidobacterial strains, enhancing their genome-scale metabolic models obtained from the AGORA (Assembly of Gut Organisms through Reconstruction and Analysis) resource. By studying various growth and metabolic capabilities in these enhanced genome-scale models across 30 different nutrient environments, we classified the bifidobacteria into three specific groups. We also studied the ability of the different strains to produce short-chain fatty acids, finding that acetate production is niche- and strain-specific, unlike lactate. Further, we captured the role of critical enzymes from the bifid shunt pathway, which was found to be essential for a subset of bifidobacterial strains. Our findings underline the significance of analysing metabolic capabilities as a powerful approach to explore distinct properties of the gut microbiome. Overall, our study presents several insights into the nutritional lifestyles of bifidobacteria and could potentially be leveraged to design species/strain-specific probiotics or prebiotics.

A new adaptation strategy to glucose starvation: modulation of the gluconate shunt and pentose phosphate pathway by the transcriptional repressor Rsv1.

Survival upon glucose starvation requires a delicate balance between different metabolic pathways. A recent work by the Roe laboratory provides a mechanistic link between glucose deprivation and the regulation of the pentose phosphate pathway, with the transcriptional repressor Rsv1 playing a key role in the process. Rsv1 regulates the flow of glucose into its possible metabolic fates and promotes long-term survival under low glucose.

TiO2/WO3 Bilayer as Electron Transport Layer for Efficient Planar Perovskite Solar Cell with Efficiency Exceeding 20%

AbstractIt is crucial to retard the carrier recombination and minimize the energy loss at the transparent electrode/electron transport layer (ETL)/perovskite absorber interfaces to improve the performance of the perovskite solar cells (PSCs). Here, a bilayered TiO2/WO3 film is designed as ETL by combining atomic layer deposition (ALD) technology and spin‐coating process. The ALD‐TiO2 underlayer fills the fluorine‐doped tin oxide (FTO) valleys and makes the surface smoother, which effectively avoids the shunt pathways between perovskite layer and FTO substrate and thereby suppresses electron–hole recombination at the interface. Moreover, the presence of hydrophilic TiO2 underlayer is helpful in forming a uniform and compact WO3 layer which is beneficial for extracting electron from perovskite to ETL. Meanwhile, the lower valance band minimum level of TiO2 relative to WO3 can efficiently enhance the hole‐blocking ability. By employing the optimized TiO2 (7 nm)/WO3 bilayer as ETL, the resulting cell exhibits an obviously enhanced power conversion efficiency of up to 20.14%, which is much better than the single WO3 or TiO2 ETL based device. This work is expected to provide a viable path to design ultrathin and compact ETL for efficient PSCs.

Long non-coding RNA GAS5 acts as proliferation \u201cbrakes\u201d in CD133+ cells responsible for tumor recurrence

Presence of quiescent, therapy evasive population often described as cancer stem cells (CSC) or tumor initiating cells (TIC) is often attributed to extreme metastasis and tumor recurrence. This population is typically enriched in a tumor as a result of microenvironment or chemotherapy induced stress. The TIC population adapts to this stress by turning on cell cycle arrest programs that is a “fail-safe” mechanism to prevent expansion of malignant cells to prevent further injury. Upon removal of the “stress” conditions, these cells restart their cell cycle and regain their proliferative nature thereby resulting in tumor relapse. Growth Arrest Specific 5 (GAS5) is a long-non-coding RNA that plays a vital role in this process. In pancreatic cancer, CD133+ population is a typical representation of the TIC population that is responsible for tumor relapse. In this study, we show for the first time that emergence of CD133+ population coincides with upregulation of GAS5, that reprograms the cell cycle to slow proliferation by inhibiting GR mediated cell cycle control. The CD133+ population further routed metabolites like glucose to shunt pathways like pentose phosphate pathway, that were predominantly biosynthetic in spite of being quiescent in nature but did not use it immediately for nucleic acid synthesis. Upon inhibiting GAS5, these cells were released from their growth arrest and restarted the nucleic acid synthesis and proliferation. Our study thus showed that GAS5 acts as a molecular switch for regulating quiescence and growth arrest in CD133+ population, that is responsible for aggressive biology of pancreatic tumors.

All-vacuum deposited and thermally stable perovskite solar cells with F4-TCNQ/CuPc hole transport layer

Hole transporting layers (HTLs) play a crucial role in the realization of efficient and stable perovskite solar cells (PSCs). Copper phthalocyanine (CuPc) is a promising HTL owing to its thermal stability and favorable band alignment with the perovskite absorber. However, the power conversion efficiency (PCE) of PSCs with a CuPc HTL is still lagging behind highly efficient solar cells. Herein, a p-type tetrafluoro-tetracyanoquinodimethane (F4-TCNQ) is employed as an interlayer between the perovskite and CuPc HTL in all-vacuum deposited PSCs. The F4-TCNQ interlayer improves the conductivity of both MAPbI3 and CuPc, reduces the shunt pathway and facilitates an efficient photoexcited holes transfer from the valance band of the MAPbI3 to the LUMO of the F4-TCNQ. Consequently, the best solar cell device with an F4-TCNQ interlayer achieved a PCE of 13.03% with a remarkable improvement in fill factor. Moreover, the device showed superior stability against thermal stress at 85 °C over 250 h and retained ∼95% of its initial efficiency. This work demonstrates a significant step towards all-vacuum deposited perovskite solar cells with high thermal stability.

GATA-type transcriptional factor Gat1 regulates nitrogen uptake and polymalic acid biosynthesis in polyextremotolerant fungus Aureobasidium pullulans.

Polymalic acid (PMA) is a novel biopolymer produced by the polyextremotolerant fungus Aureobasidium pullulans. In this study, a GATA-family transcriptional factor, Gat1, which regulates nitrogen uptake and PMA biosynthesis, was investigated. PMA production increased to 11.2% in the mutant overexpressing gat1 but decreased to 49.1% of the PMA titre when gat1 was knocked out from the genome of A. pullulans. Comparative transcriptome analysis of wild-type and mutant strains (∆gat1 and OE::gat1) revealed that 23 common differentially expressed genes were related to oxidative phosphorylation, ribosome biogenesis, and nitrogen metabolism. Under nitrogen-limited stress, regardless of the preferred nitrogen (glutamine, Gln) or non-preferred nitrogen (proline, Pro), 70% of Gat1 in the cells was located in the nucleus-cytoplasm, which resulted in an increase in nitrogen uptake and PMA biosynthesis regulation. Quantitative RT-PCR revealed that glucosekinase (GLK) in the glycolytic pathway and malate synthase (MLS) in the glyoxylate shunt pathway may be cross-regulated by Gat1 and nitrogen concentration (Gln or Pro), Therefore, glk was overexpressed in mutant strain (OE::gat1), which resulted in an increased PMA titre and yield of 12.6% and 13.0% respectively. These findings indicate that Gat1 may play an important role in the dual regulation of the nitrogen and carbon metabolisms in PMA biosynthesis.

Rewiring of Metabolic Network in Mycobacterium tuberculosis During Adaptation to Different Stresses.

Metabolic adaptation of Mycobacterium tuberculosis (M. tuberculosis) to microbicidal intracellular environment of host macrophages is fundamental to its pathogenicity. However, an in-depth understanding of metabolic adjustments through key reaction pathways and networks is limited. To understand how such changes occur, we measured the cellular metabolome of M. tuberculosis subjected to four microbicidal stresses using liquid chromatography-mass spectrometric multiple reactions monitoring (LC-MRM/MS). Overall, 87 metabolites were identified. The metabolites best describing the separation between stresses were identified through multivariate analysis. The coupling of the metabolite measurements with existing genome-scale metabolic model, and using constraint-based simulation led to several new concepts and unreported observations in M. tuberculosis; such as (i) the high levels of released ammonia as an adaptive response to acidic stress was due to increased flux through L-asparaginase rather than urease activity; (ii) nutrient starvation-induced anaplerotic pathway for generation of TCA intermediates from phosphoenolpyruvate using phosphoenolpyruvate kinase; (iii) quenching of protons through GABA shunt pathway or sugar alcohols as possible mechanisms of early adaptation to acidic and oxidative stresses; and (iv) usage of alternate cofactors by the same enzyme as a possible mechanism of rewiring metabolic pathways to overcome stresses. Besides providing new leads and important nodes that can be used for designing intervention strategies, the study advocates the strength of applying flux balance analyses coupled with metabolomics to get a global picture of complex metabolic adjustments.

The role of Rsv1 in the transcriptional regulation of genes involved in sugar metabolism for long-term survival.

Glucose limitation is a major stress condition that cells must respond to by altering their metabolism to ensure survival. Rsv1 is a zinc finger protein previously shown to be required for survival during stationary phase. In this study, we present a novel mechanism regulated by Rsv1 in the fission yeast Schizosaccharomycespombe that is involved in altering glucose metabolic flux. We found that rsv1 gene expression is induced by Rst2 and Atf1, two transcription factors regulated by the cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) cascade, respectively. The downstream target genes of Rsv1 were identified by genome-wide ChIP sequencing of Rsv1-bound DNA sites and RNA sequencing analysis of Rsv1-dependent transcripts that were differentially expressed under glucose starvation. Rsv1 directly regulated the expression of at least 21 genes that mostly encode transporters and proteins related to sugar metabolism. Among these, gcd1, which encodes glucose dehydrogenase in the gluconate shunt for the pentose phosphate pathway, was most remarkably repressed by Rsv1. The defect in survival of Δrsv1 mutant under glucose starvation condition was mitigated by additional deletion of a gcd1, idn1, or a gene for a putative lactonase (SPCC16c4.10), suggesting the critical importance of downregulating the gluconate shunt and pentose phosphate pathway for long-term survival. These results show an intricate response to glucose starvation: increasing the synthesis of a transcription factor via two signal transduction pathways, which sheds light on the importance of remodeling a metabolic circuit to secure glucose for cell survival.

Developing an l-threonine-producing strain from wild-type Escherichia coli by modifying the glucose uptake, glyoxylate shunt, and l-threonine biosynthetic pathway.

Wild-type Escherichia coli MG1655 usually does not accumulate l-threonine. In this study, the effects of 13 genes related to the glucose uptake, glycolysis, TCA cycle, l-threonine biosynthesis, or their regulation on l-threonine accumulation in E. coli MG1655 were investigated. Sixteen E. coli mutant strains were constructed by chromosomal deletion or overexpression of one or more genes of rsd, ptsG, ptsH, ptsI, crr, galP, glk, iclR, and gltA; the plasmid pFW01-thrA*BC-rhtC harboring the key genes for l-threonine biosynthesis and secretion was introduced into these mutants. The analyses on cell growth, glucose consumption, and l-threonine production of these recombinant strains showed that most of these strains could accumulate l-threonine, and the highest yield was obtained in WMZ016/pFW01-thrA*BC-rhtC. WMZ016 was derived from MG1655 by deleting crr and iclR and enhancing the expression of gltA. WMZ016/pFW01-thrA*BC-rhtC could produce 17.98 g/L l-threonine with a yield of 0.346 g/g glucose, whereas the control strain MG1655/pFW01-thrA*BC-rhtC could only produce 0.68 g/L l-threonine. In addition, WMZ016/pFW01-thrA*BC-rhtC could tolerate the high concentration of glucose and produced no detectable by-products; therefore, it should be an ideal platform strain for further development. The results indicate that manipulating the glucose uptake and TCA cycle could efficiently increase l-threonine production in E. coli.

Engineering Pseudomonas putida for isoprenoid production by manipulating endogenous and shunt pathways supplying precursors

BackgroundThe soil bacterium Pseudomonas putida is a promising platform for the production of industrially valuable natural compounds. In the case of isoprenoids, the availability of biosynthetic precursors is a major limiting factor. In P. putida and most other bacteria, these precursors are produced from pyruvate and glyceraldehyde 3-phosphate by the methylerythritol 4-phosphate (MEP) pathway, whereas other bacteria synthesize the same precursors from acetyl-CoA using the unrelated mevalonate (MVA) pathway.ResultsHere we explored different strategies to increase the supply of isoprenoid precursors in P. putida cells using lycopene as a read-out. Because we were not aiming at producing high isoprenoid titers but were primarily interested in finding ways to enhance the metabolic flux to isoprenoids, we engineered the well-characterized P. putida strain KT2440 to produce low but detectable levels of lycopene under conditions in which MEP pathway steps were not saturated. Then, we compared lycopene production in cells expressing the Myxococcus xanthus MVA pathway genes or endogenous MEP pathway genes (dxs, dxr, idi) under the control of IPTG-induced and stress-regulated promoters. We also tested a shunt pathway producing isoprenoid precursors from ribulose 5-phosphate using a mutant version of the Escherichia coli ribB gene.ConclusionsThe most successful combination led to a 50-fold increase in lycopene levels, indicating that P. putida can be successfully engineered to substantially increase the supply of metabolic substrates for the production of industrially valuable isoprenoids.

Role of GABA transaminase in the regulation of development and senescence in Arabidopsis thaliana

GABA (gamma amino butyric acid) transaminase (GABA-T) shows significant regulatory function during plant developmental process. It is involved in the reduction of GABA to succinic semialdehyde and participates in GABA shunt pathway, which critically contributes to nitrogen metabolism during senescence stage of plant’s life cycle. Thus, to study the function of GABA-T gene in the development and senescence processes, Arabidopsis thaliana GABA-T knock out mutants (pop2-1 and pop2-3) were characterized by the assays of leaf senescence parameters (survival efficacy, total chlorophyll content, electrolyte leakage of membrane, and lipid peroxidation) and GABA shunt components in different developmental stages. GABA-T gene mutants (pop2-1 and pop2-3) impact the overall plant morphology, leaf development, flowering time and display early onset of senescence. It was observed that pop2 mutants declined the leaf survival efficacy, total chlorophyll, GABA, GABA-T as well as glutamate decarboxylase (GAD) enzymes activities. Knockout mutation in GABA-T gene adversely elevated the electrolyte leakage of membrane, lipid peroxidation at S3 stage (30 days old plant) rather than S5 stage (50 days old plant). ROS generation and cell death was also increased in pop2 mutant leaf earlier. Taken together, our results suggest a prominent role of GABA-T gene in leaf senescence and development processes in Arabidopsis thaliana.

Amelioration of biomass and lipid in marine alga by an endophytic fungus Piriformospora indica

BackgroundMany studies have been carried out on the growth-modulating efficiency of plants by the colonization of an endophytic fungus Piriformospora indica. However, studies involving the co-culture of alga with endophytic fungal strains for enhanced biodiesel production are rare. In this study, the interaction between P. indica and Parachlorella kessleri-I, a marine algal strain, was assessed at metabolic level.ResultsIn association with an endophytic fungus, the algal biomass enhanced from 471.6 to 704 mg/L, and the fatty acid methyl ester (FAME) profile of P. kessleri-I increased substantially. In case of FAME profile of co-cultured P. kessleri-I, two essential components of biodiesel, i.e. elaidic acid and oleic acid, increased by 1.4- and 1.8-fold, respectively. To ascertain changes in the metabolic profile of P. kessleri-I by P. indica co-culture, gas chromatography–mass spectrometry (GC–MS)-based untargeted metabolomics study was performed to identify the metabolites involved; and differential nature of the essential metabolites was also confirmed using HPLC and LC–MS. Significant modulation of the bioactive metabolites such as succinate, oxo-propanoate, l-alanine, glutamate, acetate and 1,2 propanediol, hydroxy butane was observed.ConclusionThe metabolites like glutamate and succinate that usually belong to the GABA shunt pathway were observed to be upregulated. The pathway links nitrogen metabolism and carbon metabolism, thus influencing the growth and lipid profile of the algae. These differential metabolites thus indicated the important commensal association between the endophytic fungus and autotrophic marine alga, and established that endophytic fungus can be handy for the sustainability of algal biofuel industries.

Disruption of cyanobacterial \u03b3-aminobutyric acid shunt pathway reduces metabolites levels in tricarboxylic acid cycle, but enhances pyruvate and poly(3-hydroxybutyrate) accumulation

The photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 assimilates carbon dioxide as the sole carbon source, and a major portion of the assimilated carbon is metabolically consumed by the tricarboxylic acid (TCA) cycle. Effects of partial interference of TCA cycle metabolic activity on other carbon metabolism have yet to be examined. Here, the γ-aminobutyric acid (GABA) shunt, one of the metabolic pathways for completing TCA cycle in Synechocystis, was disrupted via inactivating the glutamate decarboxylase gene (gdc). Under normal photoautotrophic condition, cell growth and the level of the TCA cycle metabolites succinate, malate and citrate were decreased by 25%, 35%, 19% and 28%, respectively, in Δgdc mutant relative to those in the wild type (WT). The cellular levels of glycogen and total lipids of the Δgdc mutant were comparable to those of the WT, but the intracellular levels of pyruvate and bioplastic poly(3-hydroxybutyrate) (PHB) were 1.23- and 2.50-fold higher, respectively, in Δgdc mutant. Thus, disruption of the GABA shunt pathway reduced the TCA cycle metabolites levels, but positively enhanced the bioaccumulation of pyruvate and PHB. The PHB production rate in Δgdc mutant was 2.0-fold higher than in the WT under normal photoautotrophy.

Correlation Analysis of Lignin Accumulation and Expression of Key Genes Involved in Lignin Biosynthesis of Ramie (Boehmeria nivea).

The phloem of the stem of ramie (Boehmeria nivea) is an important source of natural fiber for the textile industry. However, the lignin content in the phloem affects the quality of ramie phloem fiber. In this study, the lignin content and related key gene expression levels were analyzed in the phloem and xylem at different developmental periods. The results showed that the relative expression levels of lignin synthesis-related key genes in the xylem and phloem of the stem gradually decreased from the fast-growing period to the late maturation period, but the corresponding lignin content increased significantly. However, the relative expression levels of a few genes were the highest during the maturation period. During all three periods, the lignin content in ramie stems was positively correlated with the expression of genes, including PAL, C4H and 4CL1 in the phenylpropanoid pathway, F5H and CCoAOMT in the lignin-specific synthetic pathway, and CAD in the downstream pathway of lignin synthesis, but the lignin content was negatively correlated with the expression of genes including 4CL3 in the phenylpropanoid pathway and UDP-GT in the shunt pathway of lignin monomer synthesis. The ramie 4CL3 recombinant protein prefers cinnamic acid as a substrate during catalysis, and it negatively regulates lignin synthesis. It is speculated that ramie 4CL3 is mainly involved in the synthesis of ramie flavonoid compounds, and that 4CL1 is mainly involved in lignin synthesis.

Melatonin treatment promotes endogenous melatonin accumulation and triggers GABA shunt pathway activity in tomato fruits during cold storage

Higher H2O2 accumulation in tomato fruits during storage at 4 °C for 7 days by melatonin treatment at 100 μM may serve as signaling molecule for promoting endogenous melatonin accumulation by trigerring melatonin biosynthesis TDC, T5H, SNAT, and ASMT genes expression during storage at 4 °C for 28 days. Also, higher GABA shunt pathway activity demonstrating by higher GAD, GABA-T and SSADH enzymes activity in tomato fruits treated with melatonin may be responsible for promoting phenylpropanoid pathway activity demonstrating by higher PAL gene expression and enzyme activity giving rise to higher phenols accumulation and higher DPPH scavenging capacity during storage at 4 °C for 28 days. Hence, exogenous melatonin treatment may serve as beneficial procedure for response to chilling stress in tomato fruits by signaling H2O2 accumulation responsible for promoting endogenous melatonin accumulation accompanying by stimulating GABA shunt pathway activity.

Systematic analysis of the effects of different nitrogen source and ICDH knockout on glycolate synthesis in Escherichia coli

BackgroundGlycolate is an important α-hydroxy carboxylic acid widely used in industrial and consumer applications. The production of glycolate from glucose in Escherichia coli is generally carried out by glycolysis and glyoxylate shunt pathways, followed by reduction to glycolate. Glycolate accumulation was significantly affected by nitrogen sources and isocitrate dehydrogenase (ICDH), which influenced carbon flux distribution between the tricarboxylic acid (TCA) cycle and the glyoxylate shunt, however, the mechanism was unclear.ResultsHerein, we used RNA-Seq to explore the effects of nitrogen sources and ICDH knockout on glycolate production. The Mgly534 strain and the Mgly624 strain (with the ICDH deletion in Mgly534), displaying different phenotypes on organic nitrogen sources, were also adopted for the exploration. Though the growth of Mgly534 was improved on organic nitrogen sources, glycolate production decreased and acetate accumulated, while Mgly624 achieved a balance between cell growth and glycolate production, reaching 0.81 g glycolate/OD (2.6-fold higher than Mgly534). To further study Mgly624, the significant changed genes related to N-regulation, oxidative stress response and iron transport were analyzed. Glutamate and serine were found to increase the biomass and productivity respectively. Meanwhile, overexpressing the arginine transport gene argT accelerated the cell growth rate and increased the biomass. Further, the presence of Fe2+ also speeded up the cells growth and compensated for the lack of reducing equivalents.ConclusionOur studies identified that ICDH knockout strain was more suitable for glycolate production. RNA-Seq provided a better understanding of the ICDH knockout on cellular physiology and glycolate production.

Mitochondrial Topoisomerase Dysfunction Contributes to Myocardial Mitochondrial Dysfunction and Mitochondrial DNA (mtDNA) Degradation

Diabetes promotes mtDNA degradation as an antecedent event to cardiomyocyte cell death and we have reported that mitochondrial topoisomerase dysfunction participates in this pathway. In post mitotic cells, the mitochondrial genome continues to replicate about once a month and resolving the topology of mtDNA replication would be almost insurmountable in the absence of the mitochondrial topoisomerases. It remains unclear how chronic hyperglycemia induces this shift in mitochondrial topoisomerase function from physiologic to pathologic. To that end, we hypothesize that low levels of ROS alter mitochondrial topoisomerase function to accelerate mtDNA damage. DNA relaxation of supercoiled DNA is a hallmark measurement of topoisomerase function. Supercoiled DNA incubated with isolated myocardial mitochondria or purified topoisomerase 1 generated the same isopane pattern of relaxation. In the absence of mitochondrial extracts, H2O2 (125–1000 μM) did not alter DNA relaxation of supercoiled DNA. In contrast, in the presence of myocardial mitochondrial extracts, H2O2 (125–1000 μM) significantly increased DNA relaxation in a dose dependent manner. Similar results were obtained following the DNA cleavage protocol. Using the iron chelator EDTA, we determined that DNA relaxation was not due to a Fenton effect. Following immunoprecipitation using mtTop1 or Topo‐2beta antibodies, mitochondrial extracts retained DNA relaxation function indicating the overlapping presence of functional mitochondrial topoisomerases. Using isolated intact mitochondria, incubation with H2O2 significantly increased mitochondrial superoxide levels; an effect that could be blocked with MitoTempo (mitochondrial specific antioxidant). NOX4 is a member of the NADPH oxidase family, but is unique in its preferential generation of H2O2 over superoxide (O2¾). Others have reported that NOX4 is localized to the outer mitochondrial membrane. Overexpression of NOX4 in H9c2 cells exacerbated intracellular and mitochondrial superoxide levels when incubated in the present of high glucose. Diabetes increases pentose shunt pathway activity, which in turn may fuel mitochondrial localized NOX4 to promote the generation of H2O2 a diffusible ROS species. Collectively our results suggest that increased mitochondrial ROS presentation alters mitochondrial topoisomerase function. Separate from a direct impact of oxidative stress, our studies find that alteration of mitochondrial topoisomerase function accelerates and propagates an increase in mtDNA damage. These findings indicate that the functional state of the mitochondrial topoisomerases participates in the pathophysiology leading to the development of diabetic cardiomyopathy.Support or Funding InformationSupported in part by NIH RO3HD065555101 and TCUS Biomedical Education FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic Reprogramming in Astrocytes Distinguishes Region-Specific Neuronal Susceptibility in Huntington Mice

The basis for region-specific neuronal toxicity in Huntington disease is unknown. Here, we show that region-specific neuronal vulnerability is a substrate-driven response in astrocytes. Glucose is low in HdhQ(150/150) animals, and astrocytes in each brain region adapt by metabolically reprogramming their mitochondria to use endogenous, non-glycolytic metabolites as an alternative fuel. Each region is characterized by distinct metabolic pools, and astrocytes adapt accordingly. The vulnerable striatum is enriched in fatty acids, and mitochondria reprogram by oxidizing them as an energy source but at the cost of escalating reactive oxygen species (ROS)-induced damage. The cerebellum is replete with amino acids, which are precursors for glucose regeneration through the pentose phosphate shunt or gluconeogenesis pathways. ROS is not elevated, and this region sustains little damage. While mhtt expression imposes disease stress throughout the brain, sensitivity or resistance arises from an adaptive stress response, which is inherently region specific. Metabolic reprogramming may have relevance to other diseases.

Effects of exogenous calcium chloride (CaCl2) and ascorbic acid (AsA) on the γ-aminobutyric acid (GABA) metabolism in shredded carrots

CaCl2 or AsA treatment was applied to shredded carrots to explore their influences on GABA content and its metabolic pathway. The metabolites, enzyme activity and gene expression associated with GABA shunt pathway and polyamine degradation were measured. Results demonstrated that treatment with exogenous CaCl2 or AsA alone not only significantly promoted the activities of glutamate decarboxylase (GAD), GABA transaminase (GABA-T), diamine oxidase (DAO), polyamine oxidase (PAO) and aminoaldehyde dehydrogenase (AMADH), but up-regulated the expressions of DcGAD1, DcGAD2, DcGABA-T1 and DcPAO. Meanwhile, glutamate (Glu) content, a substrate for GABA shunt, was significantly reduced in both two exogenous treatments. In addition, lower putrescine (Put) and spermine (Spm) contents were observed in AsA treatment only. Taken together, our results suggested that both CaCl2 and AsA treatments could activate GABA shunt pathway and polyamine degradation. CaCl2 seemed to be more effective to GABA shunt regulation, while AsA to polyamine degradation.

'Candidatus Liberibacter asiaticus' and Its Vector, Diaphorina citri, Augment the Tricarboxylic Acid Cycle of Their Host via the γ-Aminobutyric Acid Shunt and Polyamines Pathway.

Huanglongbing (HLB), a destructive citrus disease, is associated with 'Candidatus Liberibacter asiaticus', which is transmitted by the Asian citrus psyllid Diaphorina citri. Both 'Ca. L. asiaticus' and its vector manipulate the host metabolism for their benefit, to meet their nutritional needs and neutralize the host defense responses. We used a targeted gas chromatography-mass spectrometry-based method to explore the connection between the tricarboxylic acid (TCA) cycle, γ-aminobutyric acid (GABA) shunt, and polyamines (PAs) pathways in citrus. 'Ca. L. asiaticus' and D. citri accelerated the conversion of α-ketoglutarate to glutamate, then to GABA, causing an accumulation of GABA in the cytosol. In silico analysis showed that the citrus genome possesses a putative GABA permease that connects the GABA shunt with the TCA cycle and supports the accumulation of succinate, fumarate, and citrate. Additionally, the PAs biosynthetic pathway might be connected directly to the TCA cycle, through the production of fumarate, or indirectly, via enhancement of GABA shunt. Taken together, we suggest that GABA shunt and PAs pathways are alternative pathways that contribute to the flux toward succinate rather than an intact TCA cycle in citrus. Both 'Ca. L. asiaticus' and its vector enhance these pathways. This study provides more insights into citrus responses to the HLB pathosystem and could be a further step toward clues for understanding the nutritional needs of 'Ca. L. asiaticus', which could help in culturing 'Ca. L. asiaticus'.