Search Results (18)

Soybean (Glycine max) nitrogen fixation is inhibited by nitrate, which has been linked to a reduction in carbon allocation and metabolism within nodules. However, the underlying mechanisms remain unclear. In this study, we tested the hypothesis that the nitrate-induced suppression of nitrogen fixation is mediated through altered sucrose allocation and catabolism in nodules. Using unilaterally nodulated dual-root soybean plants in sand-based systems, we applied 200 mg·L⁻¹ nitrate exclusively to the non-nodulated roots for 14 days. Nitrate supply enhanced the proportion of dry weight in leaves but reduced it in nodules at 3, 7, and 14 days. Similarly, nodule dry weight, nodule number, acetylene reduction activity (ARA), and specific nodule activity (SNA) all declined significantly during the same intervals. Notably, sucrose content in the nodules decreased significantly by 20.4% after 3 days but recovered at 7 and 14 days. In contrast, sucrose synthase (SuSy) cleavage activity and malate content in nodules decreased significantly following nitrate treatment, with reductions of 27.8% and 30.7% observed at 7 days, and further decreased to 38.5% and 39.2% at 14 days, respectively. These results suggest that transient sucrose scarcity may drive the initial decline in nitrogen fixation capacity, while restricted sucrose catabolism and decreased malate levels may be a consequence rather than a cause. Full article

CRISPR/Cas9, as a well-established gene editing technology, has been applied in numerous model organisms, but its application in wild-type E. coli remains limited. Pathogenic wild-type E. coli, a major cause of foodborne illnesses and intestinal inflammation in humans and animals, poses a significant global public health threat. The valine-glycine repeat protein G (VgrG) is a key virulence factor that enhances E. coli pathogenicity. In this study, PCR was used to identify 50 strains carrying the virulence gene VgrG2 out of 83 wild pathogenic E. coli strains, with only one strain sensitive to kanamycin and spectinomycin. A homologous repair template for VgrG2 was constructed using overlap PCR. A dual-plasmid CRISPR/Cas9 system, combining pTarget (spectinomycin resistance) and pCas (kanamycin resistance) with Red homologous recombination, was then used to induce genomic cleavage and knock out VgrG2. PCR and sequencing confirmed the deletion of a 1708 bp fragment of the VgrG2 gene in wild-type E. coli. IPEC-J2 cells were infected with E. coli-WT and E. coli ∆VgrG2, and treated with the mTOR inhibitor rapamycin to study the effects of VgrG2 on the mTOR signaling pathway. The qPCR results showed that VgrG2 activated the mTOR pathway, suppressed mTOR and p62 mRNA levels, and upregulated the autophagy-related genes and LC3-II protein expression. In conclusion, we utilized CRISPR/Cas9 technology to achieve large-fragment deletions in wild-type E. coli, revealing that VgrG2 activates the mTOR signaling pathway and upregulates autophagy markers. These findings offer new insights into E. coli genome editing and clarifies the pathogenic mechanisms through which VgrG2 induces cellular damage. Full article

Alzheimer’s disease (AD) pathogenesis is correlated with the membrane content of various lipid species, including cholesterol, whose interactions with amyloid precursor protein (APP) have been extensively explored. Amyloid-β peptides triggering AD are products of APP cleavage by secretases, which differ depending on the APP and secretase location relative to ordered or disordered membrane microdomains. We used high-resolution NMR to probe the interactions of the cholesterol analog with APP transmembrane domain in two membrane-mimicking systems resembling ordered or perturbed lipid environments (bicelles/micelles). In bicelles, spin-labeled sterol interacted with the peptide near the amphiphilic juxtamembrane region and N-terminal part of APP transmembrane helix, as described earlier for cholesterol. Upon transition into micellar environment, another interaction site appeared where sterol polar head was buried in the hydrophobic core near the hinge region. In MD simulations, sterol moved between three interaction sites, sliding along the polar groove formed by glycine residues composing the dimerization interfaces and flexible hinge of the APP transmembrane domain. Because the lipid environment modulates interactions, the role of lipids in the AD pathogenesis is defined by the state of the entire lipid subsystem rather than the effects of individual lipid species. Cholesterol can interplay with other lipids (polyunsaturated, gangliosides, etc.), determining the outcome of amyloid-β production cascades. Full article

Abnormal cytoplasmic aggregates containing the TDP-43 protein and its fragments are present in the central nervous system of the majority of patients with amyotrophic lateral sclerosis (ALS) and in patients with frontotemporal lobar degeneration (FTLD). Many studies have focused on the C-terminal cleavage products of TDP-43 (CTFs), but few have focused on the N-terminal products (NTFs), yet several works and their protein domain composition support the involvement of NTFs in pathophysiology. In the present study, we expressed six NTFs of TDP-43, normally generated in vivo by proteases or following the presence of pathogenic genetic truncating variants, in HEK-293T cells. The N-terminal domain (NTD) alone was not sufficient to produce aggregates. Fragments containing the NTD and all or part of the RRM1 domain produced nuclear aggregates without affecting cell viability. Only large fragments also containing the RRM2 domain, with or without the glycine-rich domain, produced cytoplasmic aggregates. Of these, only NTFs containing even a very short portion of the glycine-rich domain caused a reduction in cell viability. Our results provide insights into the involvement of different TDP-43 domains in the formation of nuclear or cytoplasmic aggregates and support the idea that work on the development of therapeutic molecules targeting TDP-43 must also take into account NTFs and, in particular, those containing even a small part of the glycine-rich domain. Full article

Abiotic humification, dominated by catalytic oxidation, is one of the critical mechanisms for organic carbon preservation in nature. However, the effects of biochar catalysis on abiotic humification have not yet been elucidated. This study investigated the catalytic power of biochar from walnut shells at different temperatures (300 °C, 600 °C, and 900 °C) for the abiotic transformation of hydroquinone (HQ) as a representative polyphenol. All the biochar samples catalyzed HQ polymerization, resulting in the formation of humic polymers such as fulvic acids (FAs) and humic acids (HAs). Light and oxygen promoted HA formation. HO^• was detected in the BC600–HQ reaction system, and HO^• quenching resulted in a 41.22% decrease in HA production, indicating that HO^• plays a major role in the oxidative polymerization. In the proposed pathway for the abiotic humification, biochar active sites and generated reactive oxygen species accept an electron from HQ, resulting in oxidation to (semi)quinone radicals, which subsequently undergo cleavage or a coupling reaction to form the oligomerized products. Under BC600 catalysis, the weight-average molecular weight (Mw) of the reaction products of HQ, glucose, and glycine reached 14,449 Da. These findings provide new insights into the application potential of biochar for promoting soil carbon sequestration. Full article

Methylotrophic bacteria are widely distributed in nature and can be applied in bioconversion because of their ability to use one-carbon source. The aim of this study was to investigate the mechanism underlying utilization of high methanol content and other carbon sources by Methylorubrum rhodesianum strain MB200 via comparative genomics and analysis of carbon metabolism pathway. The genomic analysis revealed that the strain MB200 had a genome size of 5.7 Mb and two plasmids. Its genome was presented and compared with that of the 25 fully sequenced strains of Methylobacterium genus. Comparative genomics revealed that the Methylorubrum strains had closer collinearity, more shared orthogroups, and more conservative MDH cluster. The transcriptome analysis of the strain MB200 in the presence of various carbon sources revealed that a battery of genes was involved in the methanol metabolism. These genes are involved in the following functions: carbon fixation, electron transfer chain, ATP energy release, and resistance to oxidation. Particularly, the central carbon metabolism pathway of the strain MB200 was reconstructed to reflect the possible reality of the carbon metabolism, including ethanol metabolism. Partial propionate metabolism involved in ethyl malonyl-CoA (EMC) pathway might help to relieve the restriction of the serine cycle. In addition, the glycine cleavage system (GCS) was observed to participate in the central carbon metabolism pathway. The study revealed the coordination of several metabolic pathways, where various carbon sources could induce associated metabolic pathways. To the best of our knowledge, this is the first study providing a more comprehensive understanding of the central carbon metabolism in Methylorubrum. This study provided a reference for potential synthetic and industrial applications of this genus and its use as chassis cells. Full article

The glycine cleavage system (GCS) is a complex located on the mitochondrial membrane that is responsible for regulating glycine levels and contributing one-carbon units to folate metabolism. Congenital mutations in GCS components, such as glycine decarboxylase (gldc), cause an elevation in glycine levels and the rare disease, nonketotic hyperglycinemia (NKH). NKH patients suffer from pleiotropic symptoms including seizures, lethargy, mental retardation, and early death. Therefore, it is imperative to fully elucidate the pathological effects of gldc dysfunction and glycine accumulation during development. Here, we describe a zebrafish model of gldc deficiency that recapitulates phenotypes seen in humans and mice. gldc deficient embryos displayed impaired fluid homeostasis suggesting renal abnormalities, as well as aberrant craniofacial morphology and neural development defects. Whole mount in situ hybridization (WISH) revealed that gldc transcripts were highly expressed in the embryonic kidney, as seen in mouse and human repository data, and that formation of several nephron segments was disrupted in gldc deficient embryos, including proximal and distal tubule populations. These kidney defects were caused by alterations in renal progenitor populations, revealing that the proper function of Gldc is essential for the patterning of this organ. Additionally, further analysis of the urogenital tract revealed altered collecting duct and cloaca morphology in gldc deficient embryos. Finally, to gain insight into the molecular mechanisms underlying these disruptions, we examined the effects of exogenous glycine treatment and observed analogous renal and cloacal defects. Taken together, these studies indicate for the first time that gldc function serves an essential role in regulating renal progenitor development by modulating glycine levels. Full article

Chlortetracycline (CTC), which has been frequently detected in surface water, is generated primarily by the discharge of high-concentration CTC wastewater from pharmaceutical and livestock plants. The development of effective CTC degradation technology is critical. In this study, the extent of CTC degradation at 80 mg/L was investigated by combining hydrodynamic cavitation (HC) and hydrogen peroxide (H₂O₂). The results indicate degradation ratios of 88.7% and 93.8% at 5 and 30 min, respectively. Furthermore, the possible mechanisms of CTC degradation were determined via HPLC-MS. The CTC degradation pathways include ring openings, C–N bond cleavage, demethylation, dehydroxylation, and desaturation in the sole system of HC, and a series of additional reactions, such as glycine conjugation and the cleavage of C–C double bonds, occurs in the binary system of HC + H₂O₂. Nevertheless, the treated water poses ecological risks and cannot be directly discharged into the environment. Therefore, HC + H₂O₂ treatment may be a rapid and effective primary method for the degradation of high-concentration CTC in pharmaceutical factories. Full article

Given the popularity of ketogenic diets, their potential long-term consequences deserve to be more carefully monitored. Mitochondrially derived formate has a critical role in mammalian one-carbon (1C) metabolism and development. The glycine cleavage system (GCS) accounts for another substantial source for mitochondrially derived 1C units. Objective: We investigated how the ketogenic state modulates mitochondrial formate generation and partitioning of 1C metabolic fluxes. Design: HepG2 cells treated with physiological doses (1 mM and 10 mM) of β-hydroxybutyrate (βHB) were utilized as the in vitro ketogenic model. Eight-week male C57BL/6JNarl mice received either a medium-chain fatty-acid-enriched ketogenic diet (MCT-KD) or a control diet AIN 93M for 8 weeks. Stable isotopic labeling experiments were conducted. Results and Conclusions: MCT-KD is effective in weight and fat loss. Deoxythymidine (dTMP) synthesis from the mitochondrial GCS-derived formate was significantly suppressed by βHB and consumption of MCT-KD. Consistently, plasma formate concentrations, as well as the metabolic fluxes from serine and glycine, were suppressed by MCT-KD. MCT-KD also decreased the fractional contribution of mitochondrially derived formate in methionine synthesis from serine. With the worldwide application, people and medical professionals should be more aware of the potential metabolic perturbations when practicing a long-term ketogenic diet. Full article

In this review we discuss the beneficial effects of amino acid transport and metabolism on pre- and peri-implantation embryo development, and we consider how disturbances in these processes lead to undesirable health outcomes in adults. Proline, glutamine, glycine, and methionine transport each foster cleavage-stage development, whereas leucine uptake by blastocysts via transport system B^0,+ promotes the development of trophoblast motility and the penetration of the uterine epithelium in mammalian species exhibiting invasive implantation. (Amino acid transport systems and transporters, such as B^0,+, are often oddly named. The reader is urged to focus on the transporters’ functions, not their names.) B^0,+ also accumulates leucine and other amino acids in oocytes of species with noninvasive implantation, thus helping them to produce proteins to support later development. This difference in the timing of the expression of system B^0,+ is termed heterochrony—a process employed in evolution. Disturbances in leucine uptake via system B^0,+ in blastocysts appear to alter the subsequent development of embryos, fetuses, and placentae, with undesirable consequences for offspring. These consequences may include greater adiposity, cardiovascular dysfunction, hypertension, neural abnormalities, and altered bone growth in adults. Similarly, alterations in amino acid transport and metabolism in pluripotent cells in the blastocyst inner cell mass likely lead to epigenetic DNA and histone modifications that produce unwanted transgenerational health outcomes. Such outcomes might be avoided if we learn more about the mechanisms of these effects. Full article

(1) Background: Antifolate methotrexate (MTX) is the most common disease-modifying antirheumatic drug (DMARD) for treating human rheumatoid arthritis (RA). The mitochondrial-produced formate is essential for folate-mediated one carbon (1C) metabolism. The impacts of MTX on formate homeostasis in unknown, and rigorously controlled kinetic studies can greatly help in this regard. (2) Methods: Combining animal model (8-week old female C57BL/6JNarl mice, n = 18), cell models, stable isotopic tracer studies with gas chromatography/mass spectrometry (GC/MS) platforms, we systematically investigated how MTX interferes with the partitioning of mitochondrial and cytosolic formate metabolism. (3) Results: MTX significantly reduced de novo deoxythymidylate (dTMP) and methionine biosyntheses from mitochondrial-derived formate in cells, mouse liver, and bone marrow, supporting our postulation that MTX depletes mitochondrial 1C supply. Furthermore, MTX inhibited formate generation from mitochondria glycine cleavage system (GCS) both in vitro and in vivo. Folinate selectively rescued 1C metabolic pathways in a tissue-, cellular compartment-, and pathway-specific manner: folinate effectively reversed the inhibition of mitochondrial formate-dependent 1C metabolism in mouse bone marrow (dTMP, methionine, and GCS) and cells (dTMP and GCS) but not methionine synthesis in liver/liver-derived cells. Folinate failed to fully recover hepatic mitochondrial-formate utilization for methionine synthesis, suggesting that the efficacy of clinical folinate rescue in MTX therapy on hepatic methionine metabolism is poor. (4) Conclusion: Conducting studies in mouse and cell models, we demonstrate novel findings that MTX specifically depletes mitochondrial 1C supply that can be ameliorated by folinate supplementation except for hepatic transmethylation. These results imply that clinical use of low-dose MTX may particularly impede 1C metabolism via depletion of mitochondrial formate. The MTX induced systematic and tissue-specific formate depletion needs to be addressed more carefully, and the efficacy of folinate with respect to protecting against such depletion deserves to be evaluated in medical practice. Full article

Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO₂ and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO₂ production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-¹³C]glycine and [2-¹³C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway. Full article

Photoresponsive polymers have attracted increasing interest for a variety of applications. Here, we report a family of photoresponsive polypeptoid-based copolymer poly(ethylene glycol)-b-poly(N-(S-(o-nitrobenzyl)-thioethyl) glycine)-co-poly(N-(2-phenylethyl) glycine) (PEG-b-PNSN-co-PNPE) synthesized by the controlled ring-opening polymerization (ROP) technique. The key feature of the design is to incorporate both o-nitrobenzyl group moiety to offer the photoresponsive property and phenethyl residues to tune the structural and amphiphilic property of the system. We demonstrate that the cleavage degree of the o-nitrobenzyl group can reach to 100% upon UV-irradiation. With delicate design, a photoresponsive vesicle-to-sphere transition has been observed that facilitates the release of the encapsulants. This work provides a facile approach to prepare a type of photoresponsive polymers with tunable properties for drug delivery. Full article

The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression. Adding N⁵, N¹⁰-methylene tetrahydrofolate (N⁵, N¹⁰-mTHF), which is the product of GCS-catalyzed glycine, was able to activate cas3 expression. In addition, a cAMP receptor protein (CRP) encoded by crp activated cas3 expression via binding to the cas3 promoter in response to cAMP concentration. Since N⁵, N¹⁰-mTHF provides one-carbon unit for purine, we assumed GCS regulates cas3 through associating with CRP. It was evident that the mutation of gcvP failed to further reduce the cas3 expression with the crp deletion. These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements, where CRP is indispensable for GCS regulation of cas3 expression. Full article

At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean. Full article