Amino Acid Transport Research Articles

Cancer-associated fibroblasts-derived exosomal METTL3 promotes the proliferation, invasion, stemness and glutaminolysis in non-small cell lung cancer cells by eliciting SLC7A5 m6A modification.

Cancer-associated fibroblasts (CAFs) can promote the crosstalk between cancer cells and tumor microenvironment by exosomes. METTL3-mediated N6-methyladenine (m6A) modification has been proved to promote the progression of non-small cell lung cancer (NSCLC). Here, we focused on the impacts of CAFs-derived exosomes and METTL3-mediated m6A modification on NSCLC progression. Functional analyses were conducted using Cell Counting Kit-8, EdU, colony formation, sphere formation and transwell assays, respectively. Glutamine metabolism was evaluated by detecting glutamate consumption, and the production of intercellular glutamate and α-ketoglutarate (α-KG). qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. Exosomes were isolated by kits. The methylated RNA immunoprecipitation assay detected the m6A modification profile of Amino acid transporter LAT1 (SLC7A5) mRNA. The NSCLC mouse model was established to conduct in vivo experiments. We found that CAFs promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells. METTL3 was enriched in CAFs and was packaged into exosomes. After knockdown of METTL3 in CAF exosomes, it was found the oncogenic effects of CAFs on NSCLC cells were suppressed. CAFs elevated m6A levels in NSCLC cells. Mechanistically, exosomal METTL3-induced m6A modification in SLC7A5 mRNA and stabilized its expression in NSCLC cells. Moreover, SLC7A5 overexpression abolished the inhibitory effects of exosomal METTL3-decreased CAFs on NSCLC cells. In addition, METTL3 inhibition in CAF exosomes impeded NSCLC growth in vivo. In all, CAFs-derived exosomal METTL3 promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells by inducing SLC7A5 m6A modification.

A CRISPRi/a screening platform to study cellular nutrient transport in diverse microenvironments.

Blocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogate the contribution of nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumours. We applied this platform to identify the transporters of amino acids in leukaemia cells and found that amino acid transport involves high bidirectional flux dependent on the microenvironment composition. While investigating the role of transporters in cystine starved cells, we uncovered a role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumours and found that levels of glucose and amino acids can restrain proliferation in that environment. This study establishes a framework for systematically identifying critical cellular nutrient transporters, characterizing their function and exploring how the tumour microenvironment impacts cancer metabolism.

Comprehensive Characterization of LAT1 Cholesterol-Binding Sites.

The human L-type amino acid transporter 1 (LAT1; SLC7A5), is an amino acid exchanger protein, primarily found in the blood-brain barrier, placenta, and testis, where it plays a key role in amino acid homeostasis. Cholesterol is an essential lipid that has been highlighted to play a role in regulating the activity of membrane transporters, such as LAT1, yet little is known about the molecular mechanisms driving this phenomenon. Here we perform a comprehensive computational analysis to investigate cholesterol's role in LAT1 structure and function, focusing on four cholesterol-binding sites (CHOL1-4) identified in a recent LAT1-apo inward-open conformation cryo-EM structure. Through a series of independent molecular dynamics (MD) simulations, molecular docking, MM/GBSA free energy calculations, and other analysis tools, we explored the interactions between LAT1 and cholesterol. Our findings suggest that CHOL3 forms the most stable and favorable interactions with LAT1. Principal component analysis (PCA) and center of mass (COM) distance assessments show that CHOL3 binding stabilizes the inward-open state of LAT1 by preserving the spatial arrangement of the hash and bundle domains. Additionally, we propose an alternative cholesterol-binding site for originally assigned CHOL1. Overall, this study improves the understanding of cholesterol's modulatory effect on LAT1 and proposes candidate sites for the discovery of future allosteric ligands with rational design.

Cryo-EM structure of the human Asc-1 transporter complex

The Alanine-Serine-Cysteine transporter 1 (Asc-1 or SLC7A10) forms a crucial heterodimeric transporter complex with 4F2hc (SLC3A2) through a covalent disulfide bridge. This complex enables the sodium-independent transport of small neutral amino acids, including L-Alanine (L-Ala), Glycine (Gly), and D-Serine (D-Ser), within the central nervous system (CNS). D-Ser and Gly are two key endogenous glutamate co-agonists that activate N-methyl-d-aspartate (NMDA) receptors by binding to the allosteric site. Mice deficient in Asc-1 display severe symptoms such as tremors, ataxia, and seizures, leading to early postnatal death. Despite its physiological importance, the functional mechanism of the Asc-1-4F2hc complex has remained elusive. Here, we present cryo-electron microscopy (cryo-EM) structures of the human Asc-1-4F2hc complex in its apo state, D-Ser bound state, and L-Ala bound state, resolved at 3.6 Å, 3.5 Å, and 3.4 Å, respectively. Through detailed structural analysis and transport assays, we uncover a comprehensive alternating access mechanism that underlies conformational changes in the complex. In summary, our findings reveal the architecture of the Asc-1 and 4F2hc complex and provide valuable insights into substrate recognition and the functional cycle of this essential transporter complex.

Structure and mechanisms of transport of human Asc1/CD98hc amino acid transporter

Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4–3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.

Abstract LB425: A tumor microenvironment-targeting CD98-directed ADC confers robust anti-tumor activity in multiple cancers with favorable pharmacokinetics and safety profiles in preclinical models

Abstract CD98, a subunit of the LAT1/CD98 amino acid transporter complex, is intimately involved in cell adhesion, migration, proliferation, and signal transduction by enhancing amino acid transport and amplifying integrin signaling. CD98 is expressed at aberrantly high levels in many solid tumors and hematological malignancies, and this is frequently associated with poor prognosis, highlighting its potential as a cancer therapy target. However, CD98 is also expressed in various normal tissues hence complicating the development of antibody-based therapies owing to lack of tumor selectivity, poor pharmacokinetics (PK), and safety issues. We previously reported a pH-dependent anti-CD98 monoclonal antibody (mAb) that selectively targets the acidic tumor microenvironment (Tian, X., et al. Nat Biomed Eng 7(1): 8-23.). In the present report, we further developed that mAb into an antibody-drug conjugate (ADC; HH018-sesutecan), in which the mAb is conjugated to a topoisomerase I inhibitor-based linker-drug, sesutecan, at a drug:antibody ratio (DAR) of approximately 8. Sesutecan previously demonstrated promising characteristics in preclinical studies with multiple targets and conferred an encouraging benefit:risk profile in the clinic with a FRα-directed ADC (rinatabart sesutecan). HH018-sesutecan exhibited similar pH-dependent binding as its parental mAb in vitro, and markedly improved PK compared to the non-pH-dependent ADC control in CD98-humanized C57BL/6 mice. HH018-sesutecan demonstrated potent cytotoxicity against CD98-expressing cancer cell lines, strong bystander effect, as well as dose-dependent anti-tumor activity in mouse models representing multiple tumor types (colorectal cancer, ovarian cancer, renal cell carcinoma, lymphoma, hypopharyngeal carcinoma, and oral adeno-squamous carcinoma). Notably, sustained tumor regression was achieved with a single dose of 3-5 mg/kg in ~8 tumor models. A pilot toxicity study was conducted for HH018-sesutecan at two repeat-dose of 30 mg/kg in cynomolgus monkeys. HH018-sesutecan was well-tolerated with the principal toxicity (myelosuppression) being payload driven and reversible, accompanied with a favorable PK profile (half-life of 7-9 days). In summary, our studies demonstrate that the pH-dependent CD98-directed HH018- sesutecan exerts wide-ranging and potent anti-tumor efficacy, as well as favorable PK and safety profiles in preclinical models. HH018-sesutecan is a promising agent for further development in a broad range of CD98-expressing tumors. Citation Format: Fang Yang, Lei Wang, Xinxin Tian, Xuan Qiu, Jianhe Chen, Wenke Qi, Guangyu Li, Haidong Liu, Juan Liu, Suping Huang, Bin Ye, Qilin Wu, Baiteng Zhao, Wenhui Li, Jianhua Sui, Zhu Chen. A tumor microenvironment-targeting CD98-directed ADC confers robust anti-tumor activity in multiple cancers with favorable pharmacokinetics and safety profiles in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB425.

3,5-Dimethyladamantan-1-amine Restores Short-term Synaptic Plasticity by Changing Function of Excitatory Amino Acid Transporters in Mouse Model of Spinocerebellar Ataxia Type 1

Introduction. Memantine is an agent that used for treatment of Alzheimer's type dementia. Memantine considerably reduces the effects of neurodegeneration, may potentially slow down the neurodegenerative changes in the cerebellum and may act as treatment of choice for spinocerebellar ataxia type 1 (SCA 1).
 Our objective was to study molecular mechanisms of the short-term synaptic plasticity improvement associated with long-term memantine use in SCA 1 transgenic mice.
 Materials and methods. The experiments were performed on 12-week-old CD1 mice. We created a mouse model of cerebellar astrogliosis after expression of mutant ataxin-1 (ATXN1[Q85]) in the Bergmann glia (BG). To model the astrocyte-mediated neurodegeneration in the cerebellum, the mice were injected with LVV GFAP-Flag-ATXN1[Q85] lentiviral vector (LVV) constructs intracortically. Some of the mice received 0.35 mg/kg memantine dissolved in drink water once daily for 9 weeks. The control animals were administered LVV GFAP-ATXN1[Q2]-Flag. Changes of the excitatory postsynaptic currents amplitudes from Purkinje cells (PC) were recorded by patch clamp. Expression of anti-EAAT1 in the cerebellar cortex was assessed using immunohistochemistry.
 Results. The reactive glia of the cerebellar cortex in SCA1 mice is characterized by a decrease in the immunoreactivity of anti-EAAT1, while chronic memantine use restores this capacity. The decay time of the excitatory postsynaptic current amplitude in the parallel fiber-Purkinje cell (PF-PC) synapses of the SCA1 mice is considerably longer, which indicates the slowing of glutamate reuptake and EAAT1 dysfunction. The prolonged presence of increased neurotransmitter levels in the synaptic cleft facilitates activation of the mGluR1 signaling and restoration of mGluR1-dependent synaptic plasticity in Purkinje cells of the SCA1 mice.
 Conclusions. The slowing of neurotransmitter reuptake associated with long-term memantine treatment improves mGluR1-dependent short-term synaptic plasticity of the Purkinje cells in the SCA1 mice. Restoration of synaptic plasticity in these animals may underlie partial reduction of ataxic syndrome.

Effects of low protein diets on acid-base balance, electrolyte balance, intestinal structure, and amino acid transport in piglets.

Reducing the dietary crude protein (CP) could effectively reduce pressure on protein ingredient supplies. However, few data have been reported about the extent to which CP can be reduced and whether limiting the use of soybean meal leads to electrolyte imbalance. In this experiment, using the low protein (LP) diet [2% lower than NRC (2012)], seventy-two piglets (35 days old) were randomly divided into 2 groups with 6 replicates of 6 piglets each: CON group (CP = 18.5%) and LP group (CP = 16.5%), to investigate the effect of the LP diet on electrolyte balance, acid-base balance, intestinal structure and amino acid transport in piglets. The results revealed that the LP diet decreased the average daily gain and dietary CP digestibility, and damaged the villi structure of the small intestine. Compared with the CON diet, the potassium content decreased and the chlorine content increased in the LP diet, and similar trends were shown in piglet serum. The arterial pH, pCO2, HCO3 -, and base excess of piglets in the LP group were lower than those in the CON group, while pO2 was higher than those in the CON group. Interestingly, the LP diet significantly increased the lysine content in piglet serum and significantly decreased the levels of arginine, leucine, and glutamic acid. Furthermore, the LP diet significantly affected the expression of some amino acid transport vectors (B0AT1, EAAC1, and y+LAT1). In summary, these findings suggested that the LP diet leads to acid-base imbalance, amino acid transport disorder and amino acids imbalance in piglets, and the dietary electrolyte may be a key factor in the impact of the LP diet on piglet growth performance and intestinal health.

TSG-6 mediated extracellular matrix modifications regulate hypoxic-ischemic brain injury.

Proteoglycans containing link-domains modify the extracellular matrix (ECM) to regulate cellular homeostasis and can also sensitize tissues/organs to injury and stress. Hypoxic-Ischemic (H-I) injury disrupts cellular homeostasis by activating inflammation and attenuating regeneration and repair pathways. In the brain, the main component of the ECM is the glycosaminoglycan (GAG), Hyaluronic Acid (HA), but whether HA modifications of the ECM regulate cellular homeostasis and response to H-I injury is not known. In this report, employing both male and female mice, we demonstrate that link-domain containing proteoglycan, TNFα-stimulated gene-6 (TSG-6), is active in the brain from birth onwards and differentially modifies ECM HA during discrete neurodevelopmental windows. ECM HA modification by TSG-6 enables it to serve as a developmental switch to regulate activity of the Hippo pathway effector protein, Yes Associated Protein 1 (YAP1) in the maturing brain and in response to H-I injury. Mice that lack TSG-6 expression display dysregulated expression of YAP1 targets, excitatory amino acid transporter 1 (EAAT1; GLAST) and 2 (EAAT2; GLT-1). Dysregulation of YAP1 activation in TSG-6-/- mice coincides with age- and sex-dependent sensitization of the brain to H-I injury such that 1-week-old neonates display an anti-inflammatory response in contrast to an enhanced pro-inflammatory injury reaction in 3-month-old adult males but not females. Our findings thus support that a key regulator of age- and sex-dependent H-I injury response in the mouse brain is modulation of the Hippo-YAP1 pathway by TSG-6 dependent ECM modifications.Significance Statement Hypoxic-Ischemic (H-I) injury is a common cause of morbidity and mortality worldwide. Numerous genomic and proteomic screens have identified changes in extracellular matrix (ECM) as the most common finding, yet the downstream mechanistic insights remain obscure. We found that proteoglycan, TNF stimulated gene-6 (TSG-6) dependent ECM modifications regulate the activity of the Hippo pathway effector, the RNA transcription co-activator, Yes Associated Protein 1 (YAP1). Dysregulation of YAP1 activation in TSG-6-/- mice coincided with age- and sex-dependent sensitization of the brain to H-I injury such that neonates displayed an anti-inflammatory response in contrast to an enhanced pro-inflammatory injury reaction in adults. Thus, our findings establish TSG-6-dependent ECM modifications as a key regulator of age- and sex-dependent H-I injury responses.

Functional evaluation of Bacillus licheniformis PF9 for its potential in controlling enterotoxigenic Escherichia coli in weaned piglets

Abstract During the bacterial selection, isolate PF9 demonstrated tolerance to low pH and high bile salt and an ability to extend the lifespan of C. elegans infected with enterotoxigenic Escherichia coli (ETEC) (P < 0.05). Thirty-two weaned piglets susceptible to ETEC F4 were randomly allocated to 4 treatments as follows: 1) non-challenged negative control group (NNC; basal diet and piglets gavaged with phosphate-buffered saline (PBS)); 2) negative control group (NC; basal diet and piglets challenged with ETEC F4, 3 × 107 CFU per pig); 3) positive control (PC; basal diet + 80 mg·kg-1 of avilamycin and piglets challenged with ETEC F4); and 4) probiotic candidate (PF9; control basal diet + 2.5 × 109 CFU·kg-1 diet of B. licheniformis PF9 and piglets challenged with ETEC F4). The infection of ETEC F4 decreased average daily gain (ADG) and gain:feed in the NC group when compared to the NNC group (P < 0.05). The inoculation of ETEC F4 induced severe diarrhea at 3 h post-inoculum (hpi), 36 hpi, 40 hpi in the NC group when compared to the NNC group (P < 0.05). The supplementation of B. licheniformis PF9 significantly relieved diarrhea severity at 3 hpi when compared to the NC group (P < 0.05). The inoculation of ETEC F4 reduced duodenal, jejunal and ileal villus height (VH) in the NC group when compared to the NNC group. A significant (P < 0.05) decrease was detected in the duodenal VH in the PC and NNC groups. Moreover, the NNC group had a reduced relative mRNA level of Na+-glucose cotransporter 1 (SGLT1) when compared to the NC group (P < 0.05). Compared to the NC and NNC groups, the supplementation of B. licheniformis PF9 increased the relative mRNA levels of aminopeptidase N (APN), occludin (OCLN), zonula occludens-1 (ZO-1), and SGLT1 (P < 0.05). The supplementation of B. licheniformis PF9 also significantly increased the relative mRNA level of excitatory amino acid transporter 1 (EAAC1) when compared to the NC group (P < 0.05). Piglets supplemented with B. licheniformis PF9 showed lower relative abundance of Bacteroidetes in the colon than piglets from the NNC group (P < 0.05). The NNC group had a higher relative abundance of Firmicutes in the ileum than all the challenged piglets (P < 0.05); however, a lower relative abundance of Proteobacteria in the ileum and colon was observed in the NC group (P < 0.05). This study provides evidence that B. licheniformis PF9 has the potential to improve the gut health of piglets under challenging conditions.

Advancing apoptosis induction in triple negative breast cancer: Empowering treatment with tyrosine-stapled mixed micelles of lapatinib

The available treatment modalities for triple-negative breast cancer (TNBC) face several challenges, including discouraging overall survival outcomes, unavoidable toxicities, and the emergence of drug resistance. Further, there is an amplified requirement for various amino acids to support the uncontrolled proliferation of TNBC. Hence, targeted therapy for enhancing the treatment of TNBC was explored which encompassed hydrophobized (using α-linolenic acid) Pluronic-based mixed micelles having inherent L-type/Large neutral amino acid transporter 1 targeting (tyrosine-stapled) ability (LPT T-MM). LPT T-MM had optimal size (142.34 ± 30.23 nm), polydispersity index (0.224 ± 0.01), and enhanced drug loading capacity (∼10 %). Transmission electron microscopy confirmed their spherical shape. In vitro release studies showed sustained drug release without hemolysis risk. LPT T-MM had higher cellular uptake and cytotoxicity than free LPT. IC50 values for LPT T-MM were significantly reduced in MDA-MB-231 (∼1.64-fold) and 4 T1 (∼1.88-fold) cell lines; apoptosis index was superior in MDA-MB-231 (∼2.04-fold) and 4 T1 cell line (∼2.09-fold) compared to free LPT. LPT T-MM generated more reactive oxygen species and caused mitochondrial membrane depolarization, indicating increased cell death by apoptosis. Thus, LPT T-MM had the ability to promote apoptosis, showed promise in enhancing the payload capabilities and targeting in TNBC.

Dietary crude protein and protein solubility manipulation enhances intestinal nitrogen absorption and mitigates reactive nitrogen emissions through gut microbiota and metabolome reprogramming in sheep

Dietary nutrient manipulation (e.g. protein fractions) could lower the environmental footprints of ruminants, especially reactive nitrogen (N). This study investigated the impacts of dietary soluble protein (SP) levels with decreased crude protein (CP) on intestinal N absorption, hindgut N metabolism, fecal microbiota and metabolites, and their linkage with N metabolism phenotype. Thirty-two male Hu sheep, with an age of six months and an initial BW of 40.37 ± 1.18 kg, were randomly assigned to four dietary groups. The control diet (CON), aligning with NRC standards, maintained a CP content of 16.7% on a dry matter basis. Conversely, the experimental diets (LPA, LPB, and LPC) featured a 10% reduction in CP compared with CON, accompanied by SP adjustments to 21.2%, 25.9%, and 29.4% of CP, respectively. Our results showed that low-protein diets led to significant reductions in the concentrations of plasma creatinine, ammonia, urea N, and fecal total short-chain fatty acids (SCFA) (P < 0.05). Notably, LPB and LPC exhibited increased total SCFA and propionate concentrations compared with LPA (P < 0.05). The enrichment of the Prevotella genus in fecal microbiota associated with energy metabolism and amino acid (AA) biosynthesis pathways was evident with SP levels in low-protein diets of approximately 25% to 30%. Moreover, LPB and LPC diets demonstrated a decrease in fecal NH4+-N and NO2--N contents as well as urease activity, compared with CON (P < 0.05). Concomitantly, reductions in fecal glutamic acid dehydrogenase gene (gdh), nitrite reductase gene (nirS), and nitric oxide reductase gene (norB) abundances were observed (P < 0.05), pointing towards a potential reduction in reactive N production at the source. Of significance, the up-regulation of mRNA abundance of AA and peptide transporters in the small intestine (duodenum, jejunum, and ileum) and the elevated concentration of plasma AA (e.g. arginine, methionine, aspartate, glutamate, etc.) underscored the enhancement of N absorption and N efficiency. In summary, a 10% reduction in CP, coupled with an SP level of approximately 25% to 30%, demonstrated the potential to curtail reactive N emissions through fecal Prevotella enrichment and improve intestinal energy and N utilization efficiency.

Negative regulation of thyroid adenoma-associated protein (THADA) in the cardiac glycoside-induced anti-cancer effect

Cardiac glycosides, known as inhibitors of Na+,K+-ATPase, have anti-cancer effects such as suppression of cancer cell proliferation and induction of cancer cell death. Here, we examined the signaling pathway elicited by cardiac glycosides in the human hepatocellular carcinoma HepG2 cells and human epidermoid carcinoma KB cells. Three kinds of cardiac glycosides (ouabain, oleandrin, and digoxin) inhibited the cancer cell proliferation and decreased the expression level of thyroid adenoma-associated protein (THADA). Interestingly, the knockdown of THADA inhibited cancer cell proliferation, and the proliferation was significantly rescued by re-expression of THADA in the THADA-knockdown cells. In addition, the THADA-knockdown markedly decreased the expression level of L-type amino acid transporter LAT1. Cardiac glycosides also reduced the LAT1 expression. The LAT1 inhibitor, JPH203, significantly weakened the cancer cell proliferation. These results suggest that the binding of cardiac glycosides to Na+,K+-ATPase negatively regulates the THADA-LAT1 pathway, exerting the anti-proliferative effect in cancer cells.

TFE3-SLC36A1 axis promotes resistance to glucose starvation in kidney cancer cells

Higher demand for nutrients including glucose is characteristic of cancer. "Starving cancer" has been pursued to curb tumor progression. An ,intriguing regime is to inhibit glucose transporter GLUT1 in cancer cells. In addition, during cancer progression, cancer cells may suffer from insufficient glucose supply. Yet cancer cells can somehow tolerate glucose starvation. Uncovering the underlying mechanisms shall not only shed insight into cancer progression but also benefit cancer therapy. TFE3 is a transcription factor known to activate autophagic genes. Physiological TFE3 activity is regulated by phosphorylation-triggered translocation responsive to nutrient status. We recently reported TFE3 constitutively localizes to the cell nucleus and promotes cell proliferation in kidney cancer even under nutrient replete condition. Whether and how TFE3 responds to glucose starvation remain unclear. In this study, we show TFE3 promotes kidney cancer cell resistance to glucose starvation by exposing cells to physiologically relevant glucose concentration. We find glucose starvation triggers TFE3 protein stabilization through increasing its O-GlcNAcylation. Furthermore, through an unbiased functional genomic study, we identify SLC36A1, a lysosomal amino acid transporter, as a TFE3 target gene sensitive to TFE3 protein level. We find SLC36A1 is overexpressed in kidney cancer, which promotes mTOR activity and kidney cancer cell proliferation. Importantly, SLC36A1 level is induced by glucose starvation through TFE3, which enhances cellular resistance to glucose starvation. Suppressing TFE3 or SLC36A1 significantly increases cellular sensitivity to GLUT1 inhibitor in kidney cancer cells. Collectively, we uncover a functional TFE3-SLC36A1 axis that responds to glucose starvation and enhances starvation tolerance in kidney cancer.

Deciphering the Roles of Interfacial Amino Acids in Inter-Protein Charge Transport.

Elucidating charge transport (CT) through proteins is critical for gaining insights into ubiquitous CT chain reactions in biological systems and developing high-performance bioelectronic devices. While intra-protein CT has been extensively studied, crucial knowledge about inter-protein CT via interfacial amino acids is still absent due to the structural complexity. Herein, by loading cytochrome c (Cyt c) on well-defined peptide self-assembled monolayers to mimic the protein-protein interface, we provide a precisely controlled platform for identifying the roles of interfacial amino acids in solid-state CT via peptide-Cyt c junctions. The terminal amino acid of peptides serves as a fine-tuning factor for both the interfacial interaction between peptides and Cyt c and the immobilized Cyt c orientation, resulting in a nearly 10-fold difference in current through peptide-Cyt c junctions with varied asymmetry. This work provides a valuable platform for studying CT across proteins and contributes to the understanding of fundamental principles governing inter-protein CT.

The Conventional and Breakthrough Tool for the Study of L-Glutamate Transporters

In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific field, but they are still attractive experimental systems for neuropharmacological studies. We will therefore summarize the pharmacological significance, advantages (especially in the study of EAAT2), and experimental techniques that can be applied to Xenopus oocytes; our new findings concerning L-glutamate (L-Glu) transporters and PUFAs; and the significant outcomes of our data. The data obtained from electrophysiological and molecular biological studies of Xenopus oocytes have provided us with further important questions, such as whether or not some PUFAs can modulate EAATs as allosteric modulators and to what extent docosahexaenoic acid (DHA) affects neurotransmission and thereby affects brain functions. Xenopus oocytes have great advantages in the studies about the interactions between molecules and functional proteins, especially in the case when the expression levels of the proteins are small in cell culture systems without transfections. These are also proper to study the mechanisms underlying the interactions. Based on the data collected in Xenopus oocyte experiments, we can proceed to the next step, i.e., the physiological roles of the compounds and their significances. In the case of EAAT2, the effects on the neurotransmission should be examined by electrophysiological approach using acute brain slices. For new drug development, pharmacokinetics pharmacodynamics (PKPD) data and blood brain barrier (BBB) penetration data are also necessary. In order not to miss the promising candidate compounds at the primary stages of drug development, we should reconsider using Xenopus oocytes in the early phase of drug development.

OCTN2- and ATB0,+-targeted nanoemulsions for improving ocular drug delivery

Traditional eye drops are administered via topical instillation. However, frequent dosing is needed due to their relatively rapid precorneal removal and low ocular bioavailability. To address these issues, stearoyl L-carnitine-modified nanoemulsions (SC-NEs) were fabricated. The physicochemical properties of SC-NEs in terms of size, morphology, zeta potential, encapsulation efficiency, and in vitro drug release behavior were characterized. The cellular uptake and mechanisms of SC-NEs were comprehensively studied in human corneal epithelial cells and the stearoyl L-carnitine ratio in SC-NEs was optimized. The optimized SC-NEs could target the novel organic cation/carnitine transporter 2 (OCTN2) and amino acid transporter B (0 +) (ATB0,+) on the corneal epithelium, which led to superior corneal permeation, ocular surface retention ability, ocular bioavailability. Furthermore, SC-NEs showed excellent in vivo anti-inflammatory efficacy in a rabbit model of endotoxin-induced uveitis. The ocular safety test indicated that the SC-NEs were biocompatible. In general, the current study demonstrated that OCTN2 and ATB0,+-targeted nanoemulsions were promising ophthalmologic drug delivery systems that can improve ocular drug bioavailability and boost the therapeutic effects of drugs for eye diseases.Graphical

Metabolic and Bioprocess Engineering of Clostridium tyrobutyricum for Butyl Butyrate Production on Xylose and Shrimp Shell Waste.

Microbial conversion of agri-food waste to valuable compounds offers a sustainable route to develop the bioeconomy and contribute to sustainable biorefinery. Clostridium tyrobutyricum displays a series of native traits suitable for high productivity conversion of agri-food waste, which make it a promising host for the production of various compounds, such as the short-chain fatty acids and their derivative esters products. In this study, a butanol synthetic pathway was constructed in C. tyrobutyricum, and then efficient butyl butyrate production through in situ esterification was achieved by the supplementation of lipase into the fermentation. The butyryl-CoA/acyl-CoA transferase (cat1) was overexpressed to balance the ratio between precursors butyrate and butanol. Then, a suitable fermentation medium for butyl butyrate production was obtained with xylose as the sole carbon source and shrimp shell waste as the sole nitrogen source. Ultimately, 5.9 g/L of butyl butyrate with a selectivity of 100%, and a productivity of 0.03 g/L·h was achieved under xylose and shrimp shell waste with batch fermentation in a 5 L bioreactor. Transcriptome analyses exhibited an increase in the expression of genes related to the xylose metabolism, nitrogen metabolism, and amino acid metabolism and transport, which reveal the mechanism for the synergistic utilization of xylose and shrimp shell waste. This study presents a novel approach for utilizing xylose and shrimp shell waste to produce butyl butyrate by using an anaerobic fermentative platform based on C. tyrobutyricum. This innovative fermentation medium could save the cost of nitrogen sources (~97%) and open up possibilities for converting agri-food waste into other high-value products.

The Probiotic Lactobacillus reuteri Preferentially Synthesizes Kynurenic Acid from Kynurenine.

The gut-brain axis is increasingly understood to play a role in neuropsychiatric disorders. The probiotic bacterium Lactobacillus (L.) reuteri and products of tryptophan degradation, specifically the neuroactive kynurenine pathway (KP) metabolite kynurenic acid (KYNA), have received special attention in this context. We, therefore, assessed relevant features of KP metabolism, namely, the cellular uptake of the pivotal metabolite kynurenine and its conversion to its primary products KYNA, 3-hydroxykynurenine and anthranilic acid in L. reuteri by incubating the bacteria in Hank's Balanced Salt solution in vitro. Kynurenine readily entered the bacterial cells and was preferentially converted to KYNA, which was promptly released into the extracellular milieu. De novo production of KYNA increased linearly with increasing concentrations of kynurenine (up to 1 mM) and bacteria (107 to 109 CFU/mL) and with incubation time (1-3 h). KYNA neosynthesis was blocked by two selective inhibitors of mammalian kynurenine aminotransferase II (PF-048559989 and BFF-122). In contrast to mammals, however, kynurenine uptake was not influenced by other substrates of the mammalian large neutral amino acid transporter, and KYNA production was not affected by the presumed competitive enzyme substrates (glutamine and α-aminoadipate). Taken together, these results reveal substantive qualitative differences between bacterial and mammalian KP metabolism.

Isolation, identification and transcriptome analysis of triadimefon-degrading strain Enterobacter hormaechei TY18.

Triadimefon, a type of triazole systemic fungicide, has been extensively used to control various fungal diseases. However, triadimefon could lead to severe environmental pollution, and even threatens human health. To eliminate triadimefon residues, a triadimefon-degrading bacterial strain TY18 was isolated from a long-term polluted site and was identified as Enterobacter hormaechei. Strain TY18 could grow well in a carbon salt medium with triadimefon as the sole nitrogen source, and could efficiently degrade triadimefon. Under triadimefon stress, a total of 430 differentially expressed genes (DEGs), including 197 up-regulated and 233 down-regulated DEGs, were identified in strain TY18 using transcriptome sequencing (RNA-Seq). Functional classification and enrichment analysis revealed that these DEGs were mainly related to amino acid transport and metabolism, carbohydrate transport and metabolism, small molecule and pyrimidine metabolism. Interestingly, the DEGs encoding monooxygenase and hydrolase activity acting on carbon-nitrogen were highly up-regulated, might be mainly responsible for the metabolism in triadimefon. Our findings in this work suggest that strain E. hormaechei TY18 could efficiently degrade triadimefon for the first time. They provide a great potential to manage triadimefon biodegradation in the environment successfully.