Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system

High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.

The amino acid transporter SLC6A14 in cancer and its potential use in chemotherapy

Tumor cells have an increased demand for glucose and amino acids to support their rapid growth,and also exhibit alterations in biochemical pathways that metabolize these nutrients.Transport across the plasma membrane is essential to feed glucose and amino acids into these tumor cell-selective metabolic pathways.Transfer of amino acids across biological membranes occurs via a multitude of transporters;tumor cells must upregulate one or more of these transporters to satisfy their increased demand for amino acids.Among the amino acid transporters,SLC6A14 stands out with specific functional features uniquely suited for the biological needs of the tumor cells.This transporter is indeed upregulated in tumors of epithelial origin,including colon cancer,cervical cancer,breast cancer,and pancreatic cancer.Since normal cells express this transporter only at low levels,blockade of this transporter should lead to amino acid starvation selectively in tumor cells,thus having little effect on normal cells.This offers a novel,yet logical,strategy for the treatment of cancers that are associated with upregulation of SLC6A14.In addition,a variety of amino acid-based prodrugs are recognized as substrates by SLC6A14,thus raising the possibility that anticancer drugs can be delivered into tumor cells selectively via this transporter in the form of amino acid prodrugs.This strategy allows exposure of SLC6A14-positive tumor cells to chemotherapy with minimal off-target effects.In conclusion,the amino acid transporter SLC6A14 holds great potential not only as a direct drug target for cancer therapy but also for tumor cell-selective delivery of anticancer drugs.

Function, transport, and regulation of amino acids: What is missing in rice?

Amino acids are essential plant compounds serving as the building blocks of proteins,the predominant forms of nitrogen(N)distribution,and signaling molecules.Plant amino acids derive from root acquisition,nitrate reduction,and ammonium assimilation.Many amino acid transporters(AATs)mediating transfer processes of amino acids have been functionally characterized in Arabidopsis,whereas the function and regulation of the vast majority of AATs in rice(Oryza sativa L.)and other crops remain unknown.In this review,we summarize the current understanding of amino acids in the rhizosphere and in metabolism.We describe their function as signal molecules and in regulating plant architecture,flowering time,and defense against abiotic stress and pathogen attack.AATs not only function in root acquisition and translocation of amino acids from source to sink organs,regulating N uptake and use efficiency,but also as transporters of non-amino acid substrates or as amino acid sensors.Several AAT genes show natural variations in their promoter and coding regions that are associated with altered uptake rate of amino acids,grain N content,and tiller number.Development of an amino acid transfer model in plants will advance the manipulation of AATs for improving rice architecture,grain yield and quality,and N-use efficiency.

Glucose supplementation improves intestinal amino acid transport and muscle amino acid pool in pigs during chronic cold exposure

Mammals in northern regions chronically suffer from low temperatures during autumn-winter seasons.The aim of this study was to investigate the response of intestinal amino acid transport and the amino acid pool in muscle to chronic cold exposure via Min pig models(cold adaptation)and Yorkshire pig models(non-cold adaptation).Furthermore,this study explored the beneficial effects of glucose supplementation on small intestinal amino acid transport and amino acid pool in muscle of cold-exposed Yorkshire pigs.Min pigs(Exp.1)and Yorkshire pigs(Exp.2)were divided into a control group(17℃,n=6)and chronic cold exposure group(7℃,n=6),respectively.Twelve Yorkshire pigs(Exp.3)were divided into a cold control group and cold glucose supplementation group(8℃).The results showed that chronic cold exposure inhibited peptide transporter protein 1(PepT1)and excitatory amino acid transporter 3(EAAT3)expression in ileal mucosa and cationic amino acid transporter-1(CAT-1)in the jejunal mucosa of Yorkshire pigs(P<0.05).In contrast,CAT-1,PepT1 and EAAT3 expression was enhanced in the duodenal mucosa of Min pigs(P<0.05).Branched amino acids(BCAA)in the muscle of Yorkshire pigs were consumed by chronic cold exposure,accompanied by increased muscle RING-finger protein-1(MuRF1)and muscle atrophy F-box(atrogin-1)expression(P<0.05).More importantly,reduced concentrations of dystrophin were detected in the muscle of Yorkshire pigs(P<0.05).However,glycine concentration in the muscle of Min pigs was raised(P<0.05).In the absence of interaction between chronic cold exposure and glucose supplementation,glucose supplementation improved CAT-1 expression in the jejunal mucosa and PepT1 expression in the ileal mucosa of cold-exposed Yorkshire pigs(P<0.05).It also improved BCAA and inhibited MuRF1 and atrogin-1 expression in muscle(P<0.05).Moreover,dystrophin concentration was improved by glucose supplementation(P<0.05).In summary,chronic cold exposure inhibits amino acid absorption in the small intestine,depletes BCAA and promotes protein degradation in muscle.Glucose supplementation ameliorates the negative effects of chronic cold exposure on amino acid transport and the amino acid pool in muscle.

Dietary rumen-protected L-arginine or N-carbamylglutamate enhances placental amino acid transport and suppresses angiogenesis and steroid anabolism in underfed pregnant ewes

This study aimed to investigate the effects of dietary supplementation of underfed Hu ewes from d 35 to110 of gestation with either rumen-protected L-arginine(RP-Arg)or N-carbamylglutamate(NCG)on placental amino acid(AA)transport,angiogenic gene expression,and steroid anabolism.On d 35 of gestation,32 Hu ewes carrying twin fetuses were randomly divided into four treatment groups,each consisting of eight ewes,and were fed the following diets:A diet providing 100%of NRC’s nutrient requirements for pregnant ewes(CON);A diet providing 50%of NRC’s nutrient requirements for pregnant ewes(RES);RES diet plus 5 g/d NCG(RES+NCG);or RES diet plus 20 g/d RP-Arg(RES+ARG).On the d 110 of pregnancy,blood samples were taken from the mother,and samples were collected from type A cotyledons(COT;the fetal portions of the placenta).The levels of 17β-estradiol and progesterone in the maternal serum and both the capillary area density(CAD)and capillary surface density(CSD)in type A COT were decreased in response to Arg or NCG supplementation when compared to the RES group.The concentrations of arginine,leucine,putrescine and spermidine in type A COT were higher(P<0.05)in the RES+ARG or RES+NCG group than in the RES group.The mRNA expression levels of inducible nitric oxide synthase(iNOS)and solute carrier family 15,member 1(SLC15A1)were increased(P<0.05)while those of progesterone receptor(PGR)and fibroblast growth factor 2(FGF2)were decreased in type A COT by supplementation with either NCG or RP-Arg compared to the RES group.The results suggest that providing underfed pregnant ewes from d 35 to 110 of gestation with a diet supplemented with NCG or RP-Arg improves placental AA transport,and reduces the expression of angiogenic growth factor genes and steroid anabolism,leading to better fetal development.

Amino acid sensing in the gut and its mediation in gut-brain signal transduction

Animal gastrointestinal tract is not only a digestive organ, but also a nutrient sensing organ which detects luminal nutrient and thus can regulate food intake. There are many amino acid sensing receptors and transporters in the gut. Amino acids sensing by these receptors and transporters can stimulate the intestinal endocrine cells to release a variety of gut hormones. These hormones trigger a series of physiological effects via the nerve system. This review summarized the recent advance on the amino acid sensing receptors and transporters in the gastrointestinal tract, the gut hormones released from the intestinal endocrine cells and the hormones-induced signal transduction between the gut and brain. A better understanding of these processes may help to gain further insight into the specific role of amino acids in digestion and provide guidelines in developing strategy for the better use of amino acids in the diet.

Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma

Objective: Solute carrier family 38(SLC38 s) transporters play important roles in amino acid transportation and signaling transduction. However, their genetic alterations and biological roles in tumors are still largely unclear.This study aimed to elucidate the genetic signatures of SLC38 s transporters and their implications in esophageal squamous cell carcinoma(ESCC).Methods: Analyses on somatic mutation and copy number alterations(CNAs) of SLC38 A3 were performed as described. Immunohistochemistry(IHC) assay and Western blot assay were used to detect the protein expression level. MTS assay, colony formation assay, transwell assay and wound healing assay were used to explore the malignant phenotypes of ESCC cells. Immunofluorescence assay was used to verify the colocalization of two indicated proteins and immunopreciptation assay was performed to confirm the interaction of proteins.Results: Our findings revealed that SLC38 s family was significantly disrupted in ESCC, with high frequent CNAs and few somatic mutations. SLC38 A3 was the most frequent loss gene among them and was linked to poor survival and lymph node metastasis. The expression of SLC38 A3 was lower in tumor tissues compared to that in normal tissues, which was also significantly associated with worse clinical outcome. Further experiments revealed that depletion of SLC38 A3 could promote EMT in ESCC cell lines, and the interaction of SLC38 A3 and SETDB1 might lead to the reduced transcription of Snail. Pharmacogenomic analyses demonstrated that fifteen inhibitors were showed significantly correlated with SLC38 A3 expression.Conclusions: Our investigations have provided insights that SLC38 A3 could act as a suppressor in EMT pathway and serve as a prognostic factor and predictor of differential drug sensitivities in ESCC.

Loss of LAT1 sex-dependently delays recovery after caerulein-induced acute pancreatitis

BACKGROUND The expression of amino acid transporters is known to vary during acute pancreatitis(AP)except for LAT1(slc7a5),the expression of which remains stable.LAT1 supports cell growth by importing leucine and thereby stimulates mammalian target of rapamycin(mTOR)activity,a phenomenon often observed in cancer cells.The mechanisms by which LAT1 influences physiological and pathophysiological processes and affects disease progression in the pancreas are not yet known.AIM To evaluate the role of LAT1 in the development of and recovery from AP.METHODS AP was induced with caerulein(cae)injections in female and male mice expressing LAT1 or after its knockout(LAT1 Cre/LoxP).The development of the initial AP injury and its recovery were followed for seven days after cae injections by daily measuring body weight,assessing microscopical tissue architecture,mRNA and protein expression,protein synthesis,and enzyme activity levels,as well as by testing the recruitment of immune cells by FACS and ELISA.RESULTS The initial injury,evaluated by measurements of plasma amylase,lipase,and trypsin activity,as well as the gene expression of dedifferentiation markers,did not differ between the groups.However,early metabolic adaptations that support regeneration at later stages were blunted in LAT1 knockout mice.Especially in females,we observed less mTOR reactivation and dysfunctional autophagy.The later regeneration phase was clearly delayed in female LAT1 knockout mice,which did not regain normal expression of the pancreas-specific differentiation markers recombining binding protein suppressor of hairless-like protein(rbpjl)and basic helixloop-helix family member A15(mist1).Amylase mRNA and protein levels remained lower,and,strikingly,female LAT1 knockout mice presented signs of fibrosis lasting until day seven.In contrast,pancreas morphology had returned to normal in wild-type littermates.CONCLUSION LAT1 supports the regeneration of acinar cells after AP.Female mice lacking LAT1 exhibited more pronounced alterations than male mice,indicating a sexual dimorphism of amino acid metabolism.

An emerging role of vitamin D_(3) in amino acid absorption in different intestinal segments of on-growing grass carp(Ctenopharyngodon idella)

Vitamin D_(3)(VD_(3)),an essential nutrient for animals,has been demonstrated to stimulate the uptake of certain amino acids.However,the role of VD_(3) in the intestine,the primary site for digestion and absorption of nutrients,remains poorly characterized.Here,the grass carp(Ctenopharyngodon idella)was studied to assess the influence of different doses of VD_(3)(15.2,364.3,782.5,1,167.9,1,573.8,and 1,980.1 IU/kg)on growth performance,intestinal morphology,digestive absorption,amino acid transport,and potential signaling molecule levels in a feeding experiment.As a result,dietary VD_(3) improved growth performance,intestinal structure,and digestive and brush border enzyme activities.Additionally,most intestinal free amino acids and their transporters were upregulated after VD_(3) intake,except for Ala,Lys,Asp,Leu,solute carrier(SLC)7A7,SLC1A5,and SLC1A3 mRNA in different segments,Leu and SLC6A14 mRNA in the proximal intestine,and SLC7A5 mRNA in the mid and distal intestine.In the crucial target of rapamycin(TOR)signal pathway of amino acid transport,the gene and protein expression of TOR,S6 kinase 1,and activating transcription factor 4 were elevated,whereas 4E-binding protein 1 was decreased,further suggesting an advanced amino acid absorption capacity in the fish due to VD_(3) supplementation.Based on percentage weight gain,feed efficiency,and trypsin activity,the VD_(3) requirements of on-growing grass carp were estimated to be 968.33,1,005.00,and 1,166.67 IU/kg,respectively.Our findings provide novel recommendations for VD_(3) supplementation to promote digestion and absorption capacities of fish,contributing to the overall productivity of aquaculture.

Probiotic Lactobacillus brevis CLB3 prevents azoxymethane/dextran sulfate sodium-induced colon carcinogenesis in mice by reducing amino acid transport and IL-17A levels and repressing the IL-6/AKT/p-STAT3 signaling pathway

Amino acid intake plays a crucial role in the Warburg effect of cancer.Gut microbes can regulate intestinal amino acid metabolism.However,it is still unknown whether probiotic therapy can protect the host from intestinal tumor invasion by reducing amino acid intake.With in vitro methods,three acid-tolerant strains from fermented pickles were screened out.Using azoxymethane/dextran sulfate sodium-induced colon cancer models,we evaluated the therapeutic effects of Lactobacillus brevis CLB3,Lactobacillus plantarum XLP,and Lactobacillus johnsonii CM on model mice.Their functional mechanisms were further explained through anatomy section,quantitative reverse transcription polymerase chain reaction(qRT-PCR),Western blot,and immunohistochemical staining analyses as well as database mining and gut culturomics.The Lactobacillus brevis(L.brevis)CLB3 treatment significantly improved the clinical signs and symptoms of colon cancer,alleviated colon damage,and inhibited colon carcinogenesis in mice.In addition,this treatment significantly increased gut cultivable Lactobacillus abundance,inhibited the expression and translation levels of the tumor metabolism-related solute carrier(SLC)amino acid transporter including SLC7A5 and SLC7A11,lowered circulating interleukin-6(IL-6)and interleukin-17A(IL-17A)levels,and improved the accumulation of tumor-infiltrating lymphocytes and cancer proliferation factors.These findings suggest that L.brevis CLB3 can reduce amino acid transport,inhibit mammalian target of rapamycin(mTOR)signaling and enhance intestinal anti-tumor immune responses,which provides a potential targeting amino acid transporter strategy for preventing colorectal cancer.

Engineering blood-brain barrier-permeable and tumor cell-ingestible pro-proteins for glioblastoma treatment

Intracellular protein therapeutics holds great potentials for the treatment of glioblastoma, which however, is greatly challenged by the unmet demands to concomitantly penetrate the blood-brain barrier(BBB) and glioblastoma cell membrane barrier with high efficiency and selectivity. Herein, a unique pro-protein platform was developed via facile green synthesis, which allowed efficient and selective delivery into glioblastoma cells in a carrier-free manner. Pro-proteins were engineered via reversible modification of native proteins in the aqueous buffer with 3,4-dihydroxy-phenylalanine, the substrate of L-type amino acid transporter(LAT1), bridged with a phenylboronic acid-containing linker. By harnessing the LAT1-mediated direct transport mechanism, the optimized pro-protein, named protein-M2-D, can efficiently penetrate BBB after i.v. injection, and subsequently enable selective and endocytosis-free delivery of various proteins including enzymes, toxins, and antibodies into glioblastoma cells, wherein intracellular H_(2)O_(2) triggered traceless restoration of the native protein structure. Systemic administration of saporin-M2-D provoked potent anti-tumor efficacy against orthotopic U87 glioblastoma in mice, without inducing systemic toxicity. Such a facile, versatile, and robust platform renders a promising paradigm for cytosolic protein delivery and glioblastoma treatment.

LAT1 targeted brain delivery of temozolomide and sorafenib for effective glioma therapy

Glioma is the most common primary craniocerebral tumor caused by the cancerous growth of glial cells in the brain and spinal cord.Currently,standard treatment is the surgical resection followed by concurrent radiation and chemotherapy.However,the blood-brain barrier(BBB)prevents most antitumor drugs from entering the brain and reduces their efficacy,especially in lowgrade glioma.Since L-type amino acid transporter 1(LAT1)is highly expressed in glioma cells and mediates drug transport across the BBB,it is a promising target for drug delivery and treatment of glioma.Temozolomide(TMZ)is the first-line treatment for glioma,however,patients often exhibit drug resistance at advanced stage.A multikinase inhibitor and inducer of ferroptosis,sorafenib can improve the therapeutic effects of TMZ.Therefore,to optimize the glioma treatment and cross the BBB,we designed LAT1-targeting nanoparticles co-loaded with TMZ and sorafenib.Our results from both in vitro and in vivo studies confirmed that LAT1-targeting nanoparticles significantly increased the cellular uptake,cytotoxicity,accumulation at tumor site,and the anti-tumor efficacy compared to the non-target nanoparticles.Therefore,LAT1 can be used as a potential target for braintargeted drug delivery,and sorafenib-induced ferroptosis can aid the anti-glioma efficacy of TMZ.