Divergence in regulatory mechanisms of GR-RBP genes in different plants under abiotic stress.

The IVa subfamily of glycine-rich proteins (GRPs) comprises a group of glycine-rich RNA binding proteins referred to as GR-RBPa here. Previous studies have demonstrated functions of GR-RBPa proteins in regulating stress response in plants. However, the mechanisms responsible for the differential regulatory functions of GR-RBPa proteins in different plant species have not been fully elucidated. In this study, we identified and comprehensively studied a total of 34 GR-RBPa proteins from five plant species. Our analysis revealed that GR-RBPa proteins were further classified into two branches, with proteins in branch I being relatively more conserved than those in branch II. When subjected to identical stresses, these genes exhibited intensive and differential expression regulation in different plant species, corresponding to the enrichment of cis-acting regulatory elements involving in environmental and internal signaling in these genes. Unexpectedly, all GR-RBPa genes in branch I underwent intensive alternative splicing (AS) regulation, while almost all genes in branch II were only constitutively spliced, despite having more introns. This study highlights the complex and divergent regulations of a group of conserved RNA binding proteins in different plants when exposed to identical stress conditions. These species-specific regulations may have implications for stress responses and adaptations in different plant species.

Structure and function of GluN1-3A NMDA receptor excitatory glycine receptor channel.

N-methyl-d-aspartate receptors (NMDARs) and other ionotropic glutamate receptors (iGluRs) mediate most of the excitatory signaling in the mammalian brains in response to the neurotransmitter glutamate. Uniquely, NMDARs composed of GluN1 and GluN3 are activated exclusively by glycine, the neurotransmitter conventionally mediating inhibitory signaling when it binds to pentameric glycine receptors. The GluN1-3 NMDARs are vital for regulating neuronal excitability, circuit function, and specific behaviors, yet our understanding of their functional mechanism at the molecular level has remained limited. Here, we present cryo-electron microscopy structures of GluN1-3A NMDARs bound to an antagonist, CNQX, and an agonist, glycine. The structures show a 1-3-1-3 subunit heterotetrameric arrangement and an unprecedented pattern of GluN3A subunit orientation shift between the glycine-bound and CNQX-bound structures. Site-directed disruption of the unique subunit interface in the glycine-bound structure mitigated desensitization. Our study provides a foundation for understanding the distinct structural dynamics of GluN3 that are linked to the unique function of GluN1-3 NMDARs.

Transport mechanism and pharmacology of the human GlyT1.

The glycine transporter 1 (GlyT1) plays a crucial role in the regulation of both inhibitory and excitatory neurotransmission by removing glycine from the synaptic cleft. Given its close association with glutamate/glycine co-activated NMDA receptors (NMDARs), GlyT1 has emerged as a central target for the treatment of schizophrenia, which is often linked to hypofunctional NMDARs. Here, we report the cryo-EM structures of GlyT1 bound with substrate glycine and drugs ALX-5407, SSR504734, and PF-03463275. These structures, captured at three fundamental states of the transport cycle-outward-facing, occluded, and inward-facing-enable us to illustrate a comprehensive blueprint of the conformational change associated with glycine reuptake. Additionally, we identified three specific pockets accommodating drugs, providing clear insights into the structural basis of their inhibitory mechanism and selectivity. Collectively, these structures offer significant insights into the transport mechanism and recognition of substrate and anti-schizophrenia drugs, thus providing a platform to design small molecules to treat schizophrenia.

The Relative Contribution of Glycine-GABA Cotransmission in the Core of the Respiratory Network.

The preBötzinger complex (preBötC) and the Bötzinger complex (BötC) are interconnected neural circuits that are involved in the regulation of breathing in mammals. Fast inhibitory neurotransmission is known to play an important role in the interaction of these two regions. Moreover, the corelease of glycine and GABA has been described in the respiratory network, but the contribution of the individual neurotransmitter in different pathways remains elusive. In sagittal brainstem slices of neonatal mice, we employed a laser point illumination system to activate glycinergic neurons expressing channelrhodopsin-2 (ChR2). This approach allowed us to discern the contribution of glycine and GABA to postsynaptic currents of individual whole-cell clamped neurons in the preBötC and BötC through the application of glycine and GABA receptor-specific antagonists. In more than 90% of the recordings, both transmitters contributed to the evoked IPSCs, with the glycinergic component being larger than the GABAergic component. The GABAergic component appeared to be most prominent when stimulation and recording were both performed within the preBötC. Taken together, our data suggest that GABA-glycine cotransmission is the default mode in the respiratory network of neonatal mice with regional differences that may be important in tuning the network activity.

The Glycine soja cytochrome P450 gene GsCYP82C4 confers alkaline tolerance by promoting reactive oxygen species scavenging.

Recent studies have demonstrated the crucial role of Cytochrome P450 enzymes (CYPs) in the production of secondary metabolites, phytohormones and antioxidants in plants. However, their functional characterization specifically under alkaline stress remains elusive. CYP82C4 was the key gene screened from a family of wild soybean CYPs in our previous studies. The aim of this present study was to clone the Glycine soja GsCYP82C4 gene and characterize its functions in Arabidopsis and Glycine max. The results showed that the GsCYP82C4 gene displayed a high expression in different plant tissues at mature stages compared to young stages. Further, higher temporal expression of the GsCYP82C4 gene was noted at 6, 12 and 24 h time points after alkali treatment in leaves compared to roots. In addition, overexpression of GsCYP82C4 improved alkaline stress tolerance in Arabidopsis via increased root lengths and fresh biomass and strengthened the antioxidant defense system via a reduction in superoxide radicals in transgenic lines compared to wild type (WT) and atcyp82c4 mutants. Further, the expression levels of stress-related marker genes were up-regulated in GsCYP82C4 OX lines under alkali stress. The functional analysis of GsCYP82C4 overexpression in soybean displayed better hairy root growth, increased fresh weight, higher antioxidant enzyme activities and reduced lipid peroxidation rates in OX lines compared to the soybean WT (K599) line. In total, our study displayed positive roles of GsCYP82C4 overexpression in both Arabidopsis and Glycine max to alleviate alkaline stress via altering expression abundance of stress responsive genes, stronger roots, higher antioxidant enzyme activities as well as reduced rates of lipid peroxidation and superoxide radicals.

The potential of Rhodobacter sphaeroides extract as an alternative supplement for cell culture systems.

It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.IMPORTANCEThe choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract's abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.

Co-treatment with melatonin and ortho-topolin riboside reduces cell viability by altering metabolic profiles in non-small cell lung cancer cells.

Lung cancer is a highly prevalent and lethal malignancy worldwide, with non-small cell lung cancer (NSCLC) accounting for 85% of cancer-related deaths. In this study, the effects of co-treatment with melatonin and ortho-topolin riboside (oTR) on the cell viability and alteration of metabolites and transcripts were investigated in NSCLC cells using gas chromatography-mass spectrometry (GC-MS) and next-generation sequencing (NGS). The co-treatment of melatonin and oTR exhibited synergistic effects on the reduction of cell viability and alteration of metabolic and transcriptomic profiles in NSCLC cells. We observed that the co-treatment inhibited glycolytic function and mitochondria respiration, and downregulated glycine, serine and threonine metabolism alongside tyrosine metabolism in NSCLC cells. In the glycine, serine and threonine metabolism pathway, the co-treatment resulted in a significant 8.4-fold reduction in the expression level of the SDS gene, which encodes the enzyme responsible for the breakdown of serine to pyruvate. Moreover, co-treatment decreased the gene expression of TH, DDC, and CYP1A1 in tyrosine metabolism. Additionally, we observed that the co-treatment resulted in a significant 146.9-fold reduction in the expression of the DISC1 gene. The alteration in metabolites and transcript expressions might provide information to explain the cytotoxicity of co-treatment of melatonin and oTR in NSCLC cells. Our study presents insights into the synergistic anticancer effect of the co-treatment of melatonin and oTR, which could be a potential future therapeutic strategy for the treatment of NSCLC patients.

Nitrogen-fertilizer addition to an agricultural soil enhances biogenic non-extractable residue formation from 2-13C,15N-glyphosate.

Glyphosate and nitrogen (N) or (P) phosphorus fertilizers are often applied in combination to agricultural fields. The additional P or N supply to microorganisms might drive glyphosate degradation towards sarcosine/glycine or aminomethylphosphonic acid (AMPA), and consequently determine the speciation of non-extractable residues (NERs): harmless biogenic NERs (bioNERs) or potentially hazardous xenobiotic NERs (xenoNERs). We therefore investigated the effect of P or N-fertilizers on microbial degradation of glyphosate and bioNER formation in an agricultural soil. Four different treatments were incubated at 20 °C for 75 days as follows; I: no fertilizer (2-13C,15N-glyphosate only, control), II: P-fertilizer (superphosphate + 2-13C,15N-glyphosate, effect of P-supply), III: N-fertilizer (ammonium nitrate + 2-13C,15N-glyphosate, effect of N-supply) and IV: 15N-fertilizer (15N-ammonium nitrate + 2-13C-glyphosate, differentiation between microbial assimilations of 15N: 15N-fertilizer versus 15N-glyphosate). We quantified 13C or 15N in mineralization, extractable residues, NERs and in amino acids (AAs). At the end, mineralization (36-41 % of the 13C), extractable 2-13C,15N-glyphosate/2-13C-glyphosate (0.42-0.49 %) & 15N-AMPA (1.2 %), and 13C/15N-NERs (40-43 % of the 13C, 40-50 % of the 15N) were comparable among treatments. Contrastingly, the 15N-NERs from 15N-fertlizer amounted to only 6.6 % of the 15N. Notably, N-fertilizer promoted an incorporation of 13C/15N from 2-13C,15N-glyphosate into AAs and thus the formation of 13C/15N-bioNERs. The 13C/15N-AAs were as follows: 16-21 % (N-fertilizer) > 11-13 % (control) > 7.2-7.3 % (P-fertilizer) of the initially added isotope. 2-13C,15N-glyphosate was degraded via the sarcosine/glycine and AMPA simultaneously in all treatments, regardless of the treatment type. The percentage share of bioNERs within the NERs in the N-fertilized soil was highest (13C: 80-82 %, 15N: 100 %) compared to 53 % (13C & 15N, control) and to only 30 % (13C & 15N, P-fertilizer). We thus concluded simultaneous N & glyphosate addition to soils could be beneficial for the environment due to the enhanced bioNER formation, while P & glyphosate application disadvantageous since it promoted xenoNER formation.

Serine synthesis via reversed SHMT2 activity drives glycine depletion and acetaminophen hepatotoxicity in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one-third of the global population. Understanding the metabolic pathways involved can provide insights into disease progression and treatment. Untargeted metabolomics of livers from mice with early-stage steatosis uncovered decreased methylated metabolites, suggesting altered one-carbon metabolism. The levels of glycine, a central component of one-carbon metabolism, were lower in mice with hepatic steatosis, consistent with clinical evidence. Stable-isotope tracing demonstrated that increased serine synthesis from glycine via reverse serine hydroxymethyltransferase (SHMT) is the underlying cause for decreased glycine in steatotic livers. Consequently, limited glycine availability in steatotic livers impaired glutathione synthesis under acetaminophen-induced oxidative stress, enhancing acute hepatotoxicity. Glycine supplementation or hepatocyte-specific ablation of the mitochondrial SHMT2 isoform in mice with hepatic steatosis mitigated acetaminophen-induced hepatotoxicity by supporting de novo glutathione synthesis. Thus, early metabolic changes in MASLD that limit glycine availability sensitize mice to xenobiotics even at the reversible stage of this disease.

Canagliflozin regulates metabolic reprogramming in diabetic kidney disease by inducing fasting-like and aestivation-like metabolic patterns.

AIMS/HYPOTHESIS: Sodium-glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) are antihyperglycaemic drugs that protect the kidneys of individuals with type 2 diabetes mellitus. However, the underlying mechanisms mediating the renal benefits of SGLT2i are not fully understood. Considering the fuel switches that occur during therapeutic SGLT2 inhibition, we hypothesised that SGLT2i induce fasting-like and aestivation-like metabolic patterns, both of which contribute to the regulation of metabolic reprogramming in diabetic kidney disease (DKD). METHODS: Untargeted and targeted metabolomics assays were performed on plasma samples from participants with type 2 diabetes and kidney disease (n=35, 11 women) receiving canagliflozin (CANA) 100 mg/day at baseline and 12 week follow-up. Next, a systematic snapshot of the effect of CANA on key metabolites and pathways in the kidney was obtained using db/db mice. Moreover, the effects of glycine supplementation in db/db mice and human proximal tubular epithelial cells (human kidney-2 [HK-2]) cells were studied. RESULTS: Treatment of DKD patients with CANA for 12 weeks significantly reduced HbA1c from a median (interquartile range 25-75%) of 49.0 (44.0-57.0) mmol/mol (7.9%, [7.10-9.20%]) to 42.2 (39.7-47.7) mmol/mol (6.8%, [6.40-7.70%]), and reduced urinary albumin/creatinine ratio from 67.8 (45.9-159.0) mg/mmol to 47.0 (26.0-93.6) mg/mmol. The untargeted metabolomics assay showed downregulated glycolysis and upregulated fatty acid oxidation. The targeted metabolomics assay revealed significant upregulation of glycine. The kidneys of db/db mice undergo significant metabolic reprogramming, with changes in sugar, lipid and amino acid metabolism; CANA regulated the metabolic reprogramming in the kidneys of db/db mice. In particular, the pathways for glycine, serine and threonine metabolism, as well as the metabolite of glycine, were significantly upregulated in CANA-treated kidneys. Glycine supplementation ameliorated renal lesions in db/db mice by inhibiting food intake, improving insulin sensitivity and reducing blood glucose levels. Glycine supplementation improved apoptosis of human proximal tubule cells via the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. CONCLUSIONS/INTERPRETATION: In conclusion, our study shows that CANA ameliorates DKD by inducing fasting-like and aestivation-like metabolic patterns. Furthermore, DKD was ameliorated by glycine supplementation, and the beneficial effects of glycine were probably due to the activation of the AMPK/mTOR pathway.

Protective effect of tretinoin on cervical cancer growth and proliferation through downregulation of pFAK2 expression.

BACKGROUND: Cervical cancer, a life-threatening disease, is the seventh most commonly detected cancer among women throughout the world. The present study investigated the effect of tretinoin on cervical cancer growth and metastasis in vitro and in vivo in the mice model. MATERIALS AND METHODS: Cell Counting Kit-8, clonogenic survival, and transwell chamber assays were used for determination cells proliferation, colony formation, and invasiveness. Western blotting assay was used for assessment of protein expression whereas AutoDock Vina and Discovery studio software for in silico studies. RESULTS: Tretinoin treatment significantly (p < .05) reduced the proliferation of HT-3 and Caski cells in concentration-based manner. Incubation with tretinoin caused a significant decrease in clonogenic survival of HT-3 and Caski cells compared with the control cultures. The invasive potential of HT-3 cells was decreased to 18%, whereas that of Caski cells to 21% on treatment with 8 µM concentration of tretinoin. In HT-3 cells, tretinoin treatment led to a prominent reduction in p-focal adhesion kinase (FAK), matrix metalloproteinases (MMP)-2, and MMP-9 expression in HT-3 cells. Treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. The metastasis of tumor in model cervical cancer mice group was effectively inhibited in spleen, intestines, and peritoneal cavity. In silico studies showed that tretinoin interacts with alanine, proline, isoleucine, and glycine amino acid residues of FAK protein to block its activation. The 2-dimensional diagram of interaction of tretinoin with FAK protein revealed that tretinoin binds to alanine and glycine amino acids through conventional hydrogen bonding. CONCLUSION: In summary, tretinoin suppressed the proliferation, colony formation, and invasiveness of cervical cancer cells in vitro. It decreased the expression of activated focal adhesion kinase, MMP-2, and MMP-9 in HT-3 cells in dose-dependent manner. In silico studies showed that tretinoin interacts with alanine and glycine amino acids through conventional hydrogen bonding. In vivo data demonstrated that treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. Therefore, tretinoin can be developed as an effective therapeutic agent for cervical cancer treatment.

Response characterization and target site mechanism study in glyphosate-resistant populations of Lolium multiflorum L. from Brazil.

Italian ryegrass (Lolium multiflorum L.) is an invasive species widely spread in croplands worldwide. The intensive use of glyphosate has resulted in the selection of resistance to this herbicide in Italian ryegrass. This work characterized the response to glyphosate of Italian ryegrass populations from the South and Southwest regions of Paraná, Brazil. A total of 44 Italian ryegrass populations were collected in farming areas, and were classified for glyphosate resistance with 75% of populations resistant to gloyphosate. Of these, 3 resistant (VT05AR, MR20AR and RN01AR) and three susceptible (VT07AS, MR05AS and RN01AS) of these populations were selected to determine the resistance level and the involvement of the target site mechanisms for glyphosate resistance. Susceptible populations GR50 ranged from 165.66 to 218.17 g.e.a. ha-1 and resistant populations from 569.37 to 925.94, providing RI ranging from 2.88 and 4.70. No mutation in EPSPS was observed in the populations, however, in two (MR20AR and RN02AR) of the three resistant populations, an increase in the number of copies of the EPSPs gene (11 to 57×) was detected. The number of copies showed a positive correlation with the gene expression (R2 = 0.86) and with the GR50 of the populations (R2 = 0.81). The increase in EPSPS gene copies contributes to glyphosate resistance in Italian ryegrass populations from Brazil.

Transposable elements contribute to the establishment of the glycine shuttle in Brassicaceae species.

C3 -C4 intermediate photosynthesis has evolved at least five times convergently in the Brassicaceae, despite this family lacking bona fide C4 species. The establishment of this carbon concentrating mechanism is known to require a complex suite of ultrastructural modifications, as well as changes in spatial expression patterns, which are both thought to be underpinned by a reconfiguration of existing gene-regulatory networks. However, to date, the mechanisms which underpin the reconfiguration of these gene networks are largely unknown. In this study, we used a pan-genomic association approach to identify genomic features that could confer differential gene expression towards the C3 -C4 intermediate state by analysing eight C3 species and seven C3 -C4 species from five independent origins in the Brassicaceae. We found a strong correlation between transposable element (TE) insertions in cis-regulatory regions and C3 -C4 intermediacy. Specifically, our study revealed 113 gene models in which the presence of a TE within a gene correlates with C3 -C4 intermediate photosynthesis. In this set, genes involved in the photorespiratory glycine shuttle are enriched, including the glycine decarboxylase P-protein whose expression domain undergoes a spatial shift during the transition to C3 -C4 photosynthesis. When further interrogating this gene, we discovered independent TE insertions in its upstream region which we conclude to be responsible for causing the spatial shift in GLDP1 gene expression. Our findings hint at a pivotal role of TEs in the evolution of C3 -C4 intermediacy, especially in mediating differential spatial gene expression.

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.

HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor.

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.

Glycine recalibrates iron homeostasis of lens epithelial cells by blocking lysosome-dependent ferritin degradation.

One of the major pathological processes in cataracts has been identified as ferroptosis. However, studies on the iron metabolism mechanism in lens epithelial cells (LECs) and the methods of effectively alleviating ferroptosis in LECs are scarce. Along these lines, we found that in the ultraviolet radiation b (UVB) induced cataract model in vitro and in vivo, the ferritin of LECs is over-degraded by lysosomes, resulting in the occurrence of iron homeostasis disorder. Glycine can affect the ferritin degradation through the proton-coupled amino acid transporter (PAT1) on the lysosome membrane, further upregulating the content of nuclear factor erythrocyte 2 related factor 2 (Nrf2) to reduce the damage of LECs from two aspects of regulating iron homeostasis and alleviating oxidative stress. By co-staining, we further demonstrate that there is a more sensitive poly-(rC)-binding protein 2 (PCBP2) transportation of iron ions in LECs after UVB irradiation. Additionally, this study illustrated the increased expression of nuclear receptor coactivator 4 (NCOA4) in NRF2-KO mice, indicating that Nrf2 may affect ferritin degradation by decreasing the expression of NCOA4. Collectively, glycine can effectively regulate cellular iron homeostasis by synergistically affecting the lysosome-dependent ferritin degradation and PCBP2-mediated ferrous ion transportation, ultimately delaying the development of cataracts.

Engineering and evolution of the complete Reductive Glycine Pathway in Saccharomyces cerevisiae for formate and CO2 assimilation.

Using captured CO2 and C1-feedstocks like formate and methanol derived from electrochemical activation of CO2 are key solutions for transforming industrial processes towards a circular carbon economy. Engineering formate and CO2-based growth in the biotechnologically relevant yeast Saccharomyces cerevisiae could boost the emergence of a formate-mediated circular bio-economy. This study adopts a growth-coupled selection scheme for modular implementation of the Reductive Glycine Pathway (RGP) and subsequent Adaptive Laboratory Evolution (ALE) to enable formate and CO2 assimilation for biomass formation in yeast. We first constructed a serine biosensor strain and then implemented the serine synthesis module of the RGP into yeast, establishing glycine and serine synthesis from formate and CO2. ALE improved the RGP-dependent growth by 8-fold. 13C-labeling experiments reveal glycine, serine, and pyruvate synthesis via the RGP, demonstrating the complete pathway activity. Further, we re-established formate and CO2-dependent growth in non-evolved biosensor strains via reverse-engineering a mutation in GDH1 identified from ALE. This mutation led to significantly more 13C-formate assimilation than in WT without any selection or overexpression of the RGP. Overall, we demonstrated the activity of the complete RGP, showing evidence for carbon transfer from formate to pyruvate coupled with CO2 assimilation.

Role of the Glycine Receptor ß Subunit in Synaptic Localization and Pathogenicity in Severe Startle Disease.

Startle disease is due to the disruption of recurrent inhibition in the spinal cord. Most common causes are genetic variants in genes (GLRA1, GLRB) encoding inhibitory glycine receptor (GlyR) subunits. The adult GlyR is a heteropentameric complex composed of α1 and ß subunits that localizes at postsynaptic sites and replaces embryonically expressed GlyRα2 homomers. The human GlyR variants of GLRA1 and GLRB, dominant and recessive, have been intensively studied in vitro. However, the role of unaffected GlyRß, essential for synaptic GlyR localization, in the presence of mutated GlyRα1 in vivo is not fully understood. Here, we used knock-in mice expressing endogenous mEos4b-tagged GlyRß that were crossed with mouse Glra1 startle disease mutants. We explored the role of GlyRß under disease conditions in mice carrying a missense mutation (shaky) or resulting from the loss of GlyRα1 (oscillator). Interestingly, synaptic targeting of GlyRß was largely unaffected in both mouse mutants. While synaptic morphology appears unaltered in shaky animals, synapses were notably smaller in homozygous oscillator animals. Hence, GlyRß enables transport of functionally impaired GlyRα1 missense variants to synaptic sites in shaky animals, which has an impact on the efficacy of possible compensatory mechanisms. The observed enhanced GlyRα2 expression in oscillator animals points to a compensation by other GlyRα subunits. However, trafficking of GlyRα2ß complexes to synaptic sites remains functionally insufficient, and homozygous oscillator mice still die at 3 weeks after birth. Thus, both functional and structural deficits can affect glycinergic neurotransmission in severe startle disease, eliciting different compensatory mechanisms in vivo.

Pre-optimization and one-step preparation of cascade enzymes system with broad substrates by model guidance: Application of chiral L-norvaline and L-phenylglycine biosynthesis.

Cascade biocatalyst systems with catalytic promiscuity can be used for synthesis of a class of chiral chemicals but the optimization of these systems by model guidance is poorly explored. In this study, a cascade system with broad substrate spectrum was characterized and simulated by kinetic model with substrates of DL-Norvaline (DL-Nor) and DL-Phenylglycine (DL-Phg) as examples. To evaluate the optimal cascade system, maximum accumulation of intermediate products and conversion rate in the process were investigated by simultaneous solution of the rate equations for varying enzyme quantities. According to the simulation results, the cascade system was optimized by regulating the expression of D-amino acid oxidase and formate dehydrogenase and was prepared by one-step. The conversion efficiency of DL-Nor and DL-Phg have been significantly improved compared with that of before optimization. Moreover, the total of L-Nor and L-Phg were reached 498.2 mM and 79.5 mM through a gradient fed-batch conversion strategy, respectively.

Aging-disturbed FUS phase transition impairs hematopoietic stem cells by altering chromatin structure.

ABSTRACT: Aged hematopoietic stem cells (HSCs) exhibit compromised reconstitution capacity. The molecular mechanisms behind this phenomenon are not fully understood. Here, we observed that the expression of FUS is increased in aged HSCs, and enforced FUS recapitulates the phenotype of aged HSCs through arginine-glycine-glycine-mediated aberrant FUS phase transition. By using Fus-gfp mice, we observed that FUShigh HSCs exhibit compromised FUS mobility and resemble aged HSCs both functionally and transcriptionally. The percentage of FUShigh HSCs is increased upon physiological aging and replication stress, and FUSlow HSCs of aged mice exhibit youthful function. Mechanistically, FUShigh HSCs exhibit a different global chromatin organization compared with FUSlow HSCs, which is observed in aged HSCs. Many topologically associating domains (TADs) are merged in aged HSCs because of the compromised binding of CCCTC-binding factor with chromatin, which is invoked by aberrant FUS condensates. It is notable that the transcriptional alteration between FUShigh and FUSlow HSCs originates from the merged TADs and is enriched in HSC aging-related genes. Collectively, this study reveals for the first time that aberrant FUS mobility promotes HSC aging by altering chromatin structure.