Effects of autoimmune abnormalities on fertility and placental morphology in mice.

Autoimmune diseases (AIDs) alter the placental immune environment leading to fetal loss. This study investigated the effects of AIDs on pregnancy and the placenta in AID-prone MRL/MpJ-Faslpr/lpr mice and wild-type MRL/MpJ, which were mated with male MRL/MpJ and MRL/MpJ-Faslpr/lpr at five months and defined as moLpr and moMpJ, respectively. AID indices (spleen weight and serum autoantibody levels) and fertility status (number and size of fetuses, morphology, and comprehensive gene expression of placentas) were evaluated on gestational day 15.5. Both strains showed equivalent fertility, but moLpr showed lighter placentas and fetuses than moMpJ, and decreased fertility with AID severity. moLpr placentas had a higher number of T cells, higher expression of genes associated with T helper 2 and T follicular helper functions, and altered expression of genes (Krt15, Slc7a3, Sprr2a3) that significantly regulate pregnancy or immunity. The gene expression of T cell migration-associated chemokines (Ccl5, Cxcl9) was significantly increased in moLpr placentas, and CCL5 and CXCL9 were detected in moLpr placentas, particularly in T cells and placenta-component cells, respectively. Thus, AID altered placental morphofunction and fertility in mice; however, fertility was maintained at the examined time points. This study enhances our understanding of placental alterations and gestational risk due to AIDs.

Elevated SLC7A2 expression is associated with an abnormal neuroinflammatory response and nitrosative stress in Huntington's disease.

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for L-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington's disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR-Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients.

Baicalein improves the chemoresistance of ovarian cancer through regulation of CirSLC7A6.

PURPOSE: The present study aimed to investigate whether baicalein improves the sensitivity of resistant ovarian cancer cells to cisplatin. METHODS: Transcriptomic sequencing and bioinformatics analysis were used to screen differentially expressed CirSLC7A6 in A2780 and A2780/CDDP cells. RT-qPCR was performed to examine the expression levels of CirSLC7A6, miR-2682-5p, and SLC7A6. Cell proliferation and apoptosis were examined using a Cell Counting Kit-8 assay and flow cytometry, and cell migration and invasion were analyzed using wound healing and Transwell assays. Cell suspensions were inoculated into the subcutaneous tissues of the bilateral interscapular region of nude mice. Saline, cisplatin, baicalein and cisplatin plus baicalein were intraperitoneally injected to observe the effects on tumor growth. Toxicity analyses in the liver and kidney were performed using H&E staining. RT-qPCR and immunohistochemistry were used to detect the expression of CirSLC7A6, miR-2682-5p, and SLC7A6 in tumor tissues, and western blot analysis was carried out to measure protein expression levels. RESULTS: CirSLC7A6 was markedly upregulated in A2780/CDDP cells compared with the A2780 cells. CirSLC7A6 knockdown notably increased the expression of miR-2682-5p and decreased SLC7A6 expression. The rates of inhibition and apoptosis in the group treated with a combination of cisplatin and baicalein were significantly higher than those of the cisplatin and baicalein groups of A2780/CDDP shCirSLC7A6 cells. In A2780/CDDP shCirSLC7A6 cells, migration and invasion were significantly higher in the cisplatin and baicalein groups, compared with the combined treatment group. In the A2780/CDDP shCirSLC7A6 cell xenograft, the tumor weight of the combined treatment group was significantly lower than that of the cisplatin and baicalein groups. In addition, the combination of cisplatin and baicalein did not induce higher levels of toxicity in the liver or kidney. Baicalein alone and in combination with cisplatin notably reduced the expression of CirSLC7A6 and SLC7A6, and increased the expression of miR-2682-5p in the A2780/CDDP shCirSLC7A6 cell xenograft. In A2780/CDDP shCirSLC7A6 cells, the expression levels of P-Akt, P-mTOR, P-Erk, Bcl-2 and MMP2 were lower in the combined treatment group than in the control group. CONCLUSIONS: Treatment with baicalein improved the sensitivity of ovarian cancer cells to cisplatin and inhibited cell proliferation, metastasis and tumor growth.

Research progress on the role of cationic amino acid transporter (CAT) family members in malignant tumors and immune microenvironment.

Amino acids are essential for the survival of all living organisms and living cells. Amino acid transporters mediate the transport and absorption of amino acids, and the dysfunction of these proteins can induce human diseases. Cationic amino acid transporters (CAT family, SLC7A1-4, and SLC7A14) are considered to be a group of transmembrane transporters, of which SLC7A1-3 are essential for arginine transport in mammals. Numerous studies have shown that CAT family-mediated arginine transport is involved in signal crosstalk between malignant tumor cells and immune cells, especially T cells. The modulation of extracellular arginine concentration has entered a number of clinical trials and achieved certain therapeutic effects. Here, we review the role of CAT family on tumor cells and immune infiltrating cells in malignant tumors and explore the therapeutic strategies to interfere with extracellular arginine concentration, to elaborate its application prospects. CAT family members may be used as biomarkers for certain cancer entities and might be included in new ideas for immunotherapy of malignant tumors.

Population genetics analysis of SLC3A1 and SLC7A9 revealed the etiology of cystine stone may be more than what our current genetic knowledge can explain.

BACKGROUND: Cystine stone is a Mendelian genetic disease caused by SLC3A1 or SLC7A9. In this study, we aimed to estimate the genetic prevalence of cystine stones and compare it with the clinical prevalence to better understand the disease etiology. METHODS: We analyzed genetic variants in the general population using the 1000 Genomes project and the Human Gene Mutation Database to extract all SLC3A1 and SLC7A9 pathogenic variants. All variants procured from both databases were intersected. Pathogenic allele frequency, carrier rate, and affected rate were calculated and estimated based on Hardy-Weinberg equilibrium. RESULTS: We found that 9 unique SLC3A1 pathogenic variants were carried by 26 people and 5 unique SLC7A9 pathogenic variants were carried by 12 people, all of whom were heterozygote carriers. No homozygote, compoun d heterozygote, or double heterozygote was identified in the 1000 Genome database. Based on the Hardy-Weinberg equilibrium, the calculated genetic prevalence of cystine stone disease is 1 in 30,585. CONCLUSION: The clinical prevalence of cystine stone has been previously reported as 1 in 7,000, a notably higher figure than the genetic prevalence of 1 in 30,585 calculated in this study. This suggests that the etiology of cystine stone is more complex than what our current genetic knowledge can explain. Possible factors that may contribute to this difference include novel causal genes, undiscovered pathogenic variants, alternative inheritance models, founder effects, epigenetic modifications, environmental factors, or other modifying factors. Further investigation is needed to fully understand the etiology of cystine stone.

Effects of low-protein diet and feed restriction on mRNA expression of cationic amino acid transporters in porcine skeletal muscles.

We investigated the effects of a low-protein diet and feed restriction on the mRNA expression of cationic amino acid transporters (CATs) in the longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles of pigs. Eighteen piglets were divided into three groups: a control (CP21%), low-protein diet (LP, CP16%), and feed-restricted diet (FR, CP21%, 76% feed intake of control pigs) groups. The expression levels of CAT-1 in the LD and BF muscles of LP pigs were higher than that of control pigs, whereas that of FR pigs showed no difference. The CAT-2A expression levels in the RH muscle of FR pigs were higher than that of control pigs. The free lysine concentrations in all muscles of LP and FR pigs were lower than that of control pigs. To examine the factors that affect CATs mRNA expression, we evaluated the effects of lysine, arginine, insulin-like growth factor-I, and dexamethasone on the expression of CATs in C2C12 myotubes. CAT-1 expression levels increased in lysine and/or arginine deprivation. We show that CAT-1 and CAT-2A expression levels in skeletal muscles differ in response to dietary treatments and CAT-1 expression in skeletal muscles appears to increase in response to low free lysine concentrations.

Interpretation of SLC3A1 and SLC7A9 variants in cystinuria patients: The significance of the PM3 criterion and protein stability.

Cystinuria is a genetic disorder caused by defects in the b0,+ transporter system, which is composed of rBAT and b0,+AT coded by SLC3A1 and SLC7A9, respectively. Variants in SLC3A1 and SLC7A9 follow autosomal recessive inheritance and autosomal dominant inheritance with reduced penetrance, respectively, which complicates the interpretation of cystinuria-related variants. Here, we report seven different SLC3A1 variants and six different SLC7A9 variants. Among these variants were two novel variants previously not reported: SLC3A1 c.223C > T and SLC7A9 c.404A > G. In silico analysis using REVEL correlated well with the functional loss upon SLC7A9 variants with scores of 0.8560-0.9200 and 0.4970-0.5239 for severe and mild decrease in transport activity, respectively. In addition, DynaMut2 was able to predict a decreased protein expression level resulting from the SLC7A9 variant c.313G > A with a ΔΔGStability -2.93 kcal/mol. Our study adds to the literature as additional cases of a variant allow applying the PM3 criterion with higher strength level. In addition, we suggest the clinical utility of REVEL and DynaMut2 in interpreting SLC3A1 and SLC7A9 variants. While a decreased protein expression level is not embraced in the current variant interpretation guidelines, we believe in silico protein stability predicting tools could serve as evidence of protein function loss.

Investigation of molecular details of a bacterial cationic amino acid transporter (GkApcT) during arginine transportation using molecular dynamics simulation and umbrella sampling techniques.

CONTEXT: Cationic amino acid transporters (CATs) facilitate arginine transport across membranes and maintain its levels in various tissues and organs, but their overexpression has been associated with severe cancers. A recent study identified the alternating access mechanism and critical residues involved in arginine transportation in a cationic amino acid transporter from Geobacillus kaustophilus (GkApcT). Here, we used molecular dynamics (MD) simulation methods to investigate the transportation mechanism of arginine (Arg) through GkApcT. The results revealed that arginine strongly interacts with specific binding site residues (Thr43, Asp111, Glu115, Lys191, Phe231, Ile234, and Asp237). Based on the umbrella sampling, the main driving force for arginine transport is the polar interactions of the arginine with channel-lining residues. An in-depth description of the dissociation mechanism and binding energy analysis brings valuable insight into the interactions between arginine and transporter residues, facilitating the design of effective CAT inhibitors in cancer cells. METHODS: The membrane-protein system was constructed by uploading the prokaryotic CAT (PDB ID: 6F34) to the CHARMM-GUI web server. Molecular dynamics simulations were done using the GROMACS package, version 5.1.4, with the CHARMM36 force field and TIP3P water model. The MM-PBSA approach was performed for determining the arginine binding free energy. Furthermore, the hotspot residues were identified through per-residue decomposition analysis. The characteristics of the channel such as bottleneck radius and channel length were analyzed using the CaverWeb 1.1 web server. The proton wire inside the transporter was investigated based on the classic Grotthuss mechanism. We also investigated the atomistic details of arginine transportation using the path-based free energy umbrella sampling technique (US).

Identifying essential genes in genome-scale metabolic models of consensus molecular subtypes of colorectal cancer.

Identifying essential targets in the genome-scale metabolic networks of cancer cells is a time-consuming process. The present study proposed a fuzzy hierarchical optimization framework for identifying essential genes, metabolites and reactions. On the basis of four objectives, the present study developed a framework for identifying essential targets that lead to cancer cell death and evaluating metabolic flux perturbations in normal cells that have been caused by cancer treatment. Through fuzzy set theory, a multiobjective optimization problem was converted into a trilevel maximizing decision-making (MDM) problem. We applied nested hybrid differential evolution to solve the trilevel MDM problem to identify essential targets in genome-scale metabolic models for five consensus molecular subtypes (CMSs) of colorectal cancer. We used various media to identify essential targets for each CMS and discovered that most targets affected all five CMSs and that some genes were CMS-specific. We obtained experimental data on the lethality of cancer cell lines from the DepMap database to validate the identified essential genes. The results reveal that most of the identified essential genes were compatible with the colorectal cancer cell lines obtained from DepMap and that these genes, with the exception of EBP, LSS, and SLC7A6, could generate a high level of cell death when knocked out. The identified essential genes were mostly involved in cholesterol biosynthesis, nucleotide metabolisms, and the glycerophospholipid biosynthetic pathway. The genes involved in the cholesterol biosynthetic pathway were also revealed to be determinable, if a cholesterol uptake reaction was not induced when the cells were in the culture medium. However, the genes involved in the cholesterol biosynthetic pathway became non-essential if such a reaction was induced. Furthermore, the essential gene CRLS1 was revealed as a medium-independent target for all CMSs.

RNA helicase DHX15 exemplifies a unique dependency in acute leukemia.

RNA-binding proteins (RBP) have emerged as essential regulators that control gene expression and modulate multiple cancer traits. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy derived from transformation of T-cell progenitors that normally undergo discrete steps of differentiation in the thymus. The implications of essential RBP during T-cell neoplastic transformation remain largely unclear. Systematic evaluation of RBP identifies RNA helicase DHX15, which facilitates the disassembly of the spliceosome and release of lariat introns, as a T-ALL dependency factor. Functional analysis using multiple murine T-ALL models demonstrates the essential importance of DHX15 in tumor cell survival and leukemogenesis. Moreover, single-cell transcriptomics reveals that DHX15 depletion in T-cell progenitors hinders burst proliferation during the transition from doublenegative to double-positive cells (CD4-CD8- to CD4+CD8+). Mechanistically, abrogation of DHX15 perturbs RNA splicing and leads to diminished levels of SLC7A6 and SLC38A5 transcripts due to intron retention, thereby suppressing glutamine import and mTORC1 activity. We further propose a DHX15 signature modulator drug ciclopirox and demonstrate that it has prominent anti-T-ALL efficacy. Collectively, our data highlight the functional contribution of DHX15 to leukemogenesis through regulation of established oncogenic pathways. These findings also suggest a promising therapeutic approach, i.e., splicing perturbation by targeting spliceosome disassembly, may achieve considerable anti-tumor efficacy.

RIOK3 promotes mTORC1 activation by facilitating SLC7A2-mediated arginine uptake in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a poor prognosis. Reprogramming of amino acid metabolism is one of the characteristics of PDAC, in which arginine metabolism is significantly altered in PDAC cells and is involved in important signaling pathways. Current studies have identified arginine deprivation as a potential strategy for PDAC treatment. In this study, we performed Liquid Chromatograph Mass Spectrometer (LC-MS)-based non-targeted metabolomic analysis on PDAC cell lines with stable Rio Kinase 3 (RIOK3) knockdown and PDAC tissues with different RIOK3 expressions and found that RIOK3 expression was significantly correlated with arginine metabolism in PDAC. Subsequent RNA sequencing (RNA-Seq) and Western blot analysis showed that RIOK3 knockdown significantly inhibited the expression of arginine transporter solute carrier family 7 member 2 (SLC7A2). Further studies revealed that RIOK3 promoted arginine uptake, mechanistic target of rapamycin complex 1 (mTORC1) activation, cell invasion, and metastasis in PDAC cells via SLC7A2. Finally, we found that patients with high expression of both RIOK3 and infiltrating Treg cells had a worse prognosis. Overall, our study found that RIOK3 in PDAC cells promotes arginine uptake and mTORC1 activation through upregulation of SLC7A2 expression, and also provides a new therapeutic target for therapeutic strategies targeting arginine metabolism.

Arginine transportation mechanism through cationic amino acid transporter 1: insights from molecular dynamics studies.

Metabolic and signaling mechanisms in mammalian cells are facilitated by the transportation of L-arginine (Arg) across the plasma membrane through cationic amino acid transporter (CAT) proteins. Due to a lack of argininosuccinate synthase (ASS) activity in various tumor cells such as acute myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, these tumor entities are arginine-auxotrophic and therefore depend on the uptake of the amino acid arginine. Cationic amino acid transporter-1 (CAT-1) is the leading arginine importer expressed in the aforementioned tumor entities. Hence, in the present study, to investigate the transportation mechanism of arginine in CAT-1, we performed molecular dynamics (MD) simulation methods on the modeled human CAT-1. The MM-PBSA approach was conducted to determine the critical residues interacting with arginine within the corresponding binding site of CAT-1. In addition, we found out that the water molecules have the leading role in forming the transportation channel within CAT-1. The conductive structure of CAT-1 was formed only when the water molecules were continuously distributed across the channel. Steered molecular dynamics (SMD) simulation approach showed various energy barriers against arginine transportation through CAT-1, especially while crossing the bottlenecks of the related channel. These findings at the molecular level might shed light on identifying the crucial amino acids in the binding of arginine to eukaryotic CATs and also provide fundamental insights into the arginine transportation mechanisms through CAT-1. Understanding the transportation mechanism of arginine is essential to developing CAT-1 blockers, which can be potential medications for some types of cancers.Communicated by Ramaswamy H. Sarma.

Lower SLC7A2 expression is associated with enhanced multidrug resistance, less immune infiltrates and worse prognosis of NSCLC.

BACKGROUND: Solute carrier family 7 member 2 (SLC7A2), a cationic amino acid transporter, is lowly expressed in ovarian and hepatocellular cancers, which is associated with their worse prognosis. However, its roles in the prognosis, drug resistance and immune infiltration in non-small-cell lung cancer (NSCLC) are unclear. METHODS: We chose SLC7A2 from RNA-Seq of paclitaxel/cisplatin-resistant A549 cells, then bioinformatics, cell lines construction, RT-qPCR, and CCK8 were performed to investigate SLC7A2 role. RESULT: We analyzed the 223 differentially expressed genes (DEGs) from RNA-Seq of paclitaxel/cisplatin-resistant A549 cells and found that SLC7A2 expression was down-regulated in NSCLC. Lower SLC7A2 expression was associated with worse recurrence-free survival (RFS) in NSCLC. SLC7A2 silencing enhanced the proliferation of NSCLC cells and their insensitivity to paclitaxel, cisplatin, and gemcitabine in vitro. Activation of AMPK has up-regulated SLC7A2 expression and enhanced the sensitivity of NSCLC cells to anti-tumor drugs, which could be attributed to E2F1's regulation. In addition, the levels of SLC7A2 expression were correlated to the numbers of infiltrated neutrophils, macrophages, dendritic cells and their marker genes, like CD86, HLA-DPA1 and ITGAM. CONCLUSIONS: SLC7A2 may act as a tumor suppressor to modulate drug sensitivity, immune infiltration and survival in NSCLC. Video abstract.

Integrated Amino Acids and Transcriptome Analysis Reveals Arginine Transporter SLC7A2 Is a Novel Regulator of Myogenic Differentiation.

Skeletal muscle differentiation is a precisely coordinated process. While many of the molecular details of myogenesis have been investigated extensively, the dynamic changes and functions of amino acids and related transporters remain unknown. In this study, we conducted a comprehensive analysis of amino acid levels during different time points of C2C12 myoblast differentiation using high-performance liquid chromatography (HPLC). Our findings revealed that the levels of most amino acids exhibited an initial increase at the onset of differentiation, reaching their peak typically on the fourth or sixth day, followed by a decline on the eighth day. Particularly, arginine and branched-chain amino acids showed a prominent increase during this period. Furthermore, we used RNA-seq analysis to show that the gene encoding the arginine transporter, Slc7a2, is significantly upregulated during differentiation. Knockdown of Slc7a2 gene expression resulted in a significant decrease in myoblast proliferation and led to a reduction in the expression levels of crucial myogenic regulatory factors, hindering the process of myoblast differentiation, fusion, and subsequent myotube formation. Lastly, we assessed the expression level of Slc7a2 during aging in humans and mice and found an upregulation of Slc7a2 expression during the aging process. These findings collectively suggest that the arginine transporter SLC7A2 plays a critical role in facilitating skeletal muscle differentiation and may hold potential as a therapeutic target for sarcopenia.

Genetic and clinical analysis of Chinese pediatric patients with cystinuria.

This study aimed to investigate the genotypic and phenotypic characteristics of Chinese pediatric patients with cystinuria. This was a retrospective study of 14 Chinese pediatric patients with cystine stones. All published studies of the Chinese pediatric cystinuria population were searched and enrolled based on the inclusive standard. Among the 14 pediatric patients with cystinuria, 8 were males and 6 were females. The mean age of first stone onset was 4.0 ± 3.3 years (4 months-9 years). All of the patients had multiple stones, and 57.1% (8/14) had bilateral stones. The mean maximum stone diameter was 1.7 ± 0.6 (range 0.5-2.6) cm. A total of 13 SLC3A1 gene mutations and 9 SLC7A9 gene mutations were detected, of which 41% (9/22) of mutations were novel. Patients with SLC7A9 mutations were more likely to develop bilateral stones than those with SLC3A1 mutations (100% vs. 33.3%, p = 0.03). Thirty-four SLC3A1 gene mutations and twenty-eight SLC7A9 gene mutations were found in a total of fifty-five Chinese children with cystinuria. The SLC7A9 gene mutation distribution was more dispersed, while the SLC3A1 mutation was clustered in exons 6-8. The c.647C > T (p. T216M) (4/53) and c.1113C > A (p. Y371Ter) (4/53) mutations in the SLC3A1 gene and the c.1399 + 2_3insT (3/36) mutation in the SLC7A9 gene represent potential hotspots in cystinuria. Our results present a comprehensive genetic spectrum for pediatric cystinuria patients in China. Patients with SLC7A9 mutations were more likely to develop bilateral stones than those with SLC3A1 mutations. A wide mutation spectrum and the potential mutation hotspots associated with cystinuria were also identified.

SLC3A2 and SLC7A2 Mediate the Exogenous Putrescine-Induced Adipocyte Differentiation.

Exogenous polyamines are able to induce life span and improve glucose homeostasis and insulin sensitivity. However, the effects of exogenous polyamines on adipocyte differentiation and which polyamine transporters mediate them have not been elucidated yet. Here, we identified for the first time that exogenous polyamines can clearly stimulate adipocyte differentiation through polyamine transporters, solute carrier family 3 member A2 (SLC3A2) and SLC7A1. Exogenous polyamines markedly promote 3T3-L1 adipocyte differentiation by increasing the intracellular lipid accumulation and the expression of both adipogenic and lipogenic genes in a concentration-dependent manner. In particular, exogenous putrescine mainly regulates adipocyte differentiation in the early and intermediate stages. Moreover, we have assessed the expression of polyamine transporter genes in 3T3-L1 preadipocytes and adipocytes. Interestingly, the putrescine-induced adipocyte differentiation was found to be significantly suppressed in response to a treatment with a polyamine transporter inhibitor (AMXT-1501). Furthermore, knockdown experiments using siRNA that specifically targeted SLC3A2 or SLC7A2, revealed that both SLC3A2 and SLC7A2 act as important transporters in the cellular importing of exogenous putrescine. Thus, the exogenous putrescine entering the adipocytes via cellular transporters is involved in adipogenesis through a modulation of both the mitotic clonal expansion and the expression of master transcription factors. Taken together, these results suggest that exogenous polyamines (such as putrescine) entering the adipocytes through polyamine transporters, can stimulate adipogenesis.

Extracellular arginine is required but the arginine transporter CAT3 (Slc7a3) is dispensable for mouse normal and malignant hematopoiesis.

Amino acid-mediated metabolism is one of the key catabolic and anabolic processes involved in diverse cellular functions. However, the role of the semi-essential amino acid arginine in normal and malignant hematopoietic cell development is poorly understood. Here we report that a continuous supply of exogenous arginine is required for the maintenance/function of normal hematopoietic stem cells (HSCs). Surprisingly, knockout of Slc7a3 (CAT3), a major L-arginine transporter, does not affect HSCs in steady-state or under stress. Although Slc7a3 is highly expressed in naïve and activated CD8 T cells, neither T cell development nor activation/proliferation is impacted by Slc7a3 depletion. Furthermore, the Slc7a3 deletion does not attenuate leukemia development driven by Pten loss or the oncogenic Ptpn11E76K mutation. Arginine uptake assays reveal that L-arginine uptake is not disrupted in Slc7a3 knockout cells. These data suggest that extracellular arginine is critically important for HSCs, but CAT3 is dispensable for normal hematopoiesis and leukemogenesis.

Clinical Characteristics and In Silico Analysis of Cystinuria Caused by a Novel SLC3A1 Mutation.

Cystinuria is a genetically inherited disorder of renal and intestinal transport, featured as a high concentration of cystine in the urine. Cumulative cystine in urine would cause the formation of kidney stones, which further leads to renal colic and dysfunction. Gene screens have found that mutations in SLC3A1 or SLC7A9 gene are responsible for most cases of cystinuria, for encoding defective cystine transporters. Here, we presented the genotypic and phenotypic characteristics of one unique case of a three-generation Chinese family. The proband developed severe urolithiasis combined with renal damage. The radiography and computed tomography (CT) scan showed calculus in the left pelvic kidney. Postoperative stone analysis revealed that the stones were mainly composed of cystine. Therefore, to explore its pathogenesis, next-generation Whole Exome Sequencing (WES) and Sanger sequencing identify the proband mutated gene of the proband's family. In this article, we reported novel compound heterozygous mutations (c.818G>A and c.1011G>A) of the SLC3A1 gene in a 5-year-old child suffering from a cystine stone from a three-generation family. Bioinformatic analysis was used to predict the pathogenicity and conservation of the target mutation. Conservative sequence and evolutionary conservation analysis indicated that cystine273 and proline337 were highly conserved among species, and both mutations listed here (Cys273Tyr and Pro337Pro) were pathogenic. To conclude, our study expands the phenotypic and genotypic spectrum of SLC3A1 and indicates that genetic screening should be considered in the clinic to provide more effective and precise treatment for cystinuria.

General control nonderepressible 1 interacts with cationic amino acid transporter 1 and affects Aedes aegypti fecundity.

BACKGROUND: The amino acid transporter protein cationic amino acid transporter 1 (CAT1) is part of the nutrient sensor in the fat body of mosquitoes. A member of the SLC7 family of cationic amino acid transporters, it is paramount for the detection of elevated amino acid levels in the mosquito hemolymph after a blood meal and the subsequent changes in gene expression in the fat body. METHODS: We performed a re-annotation of Aedes aegypti cationic amino acid transporters (CATs) and selected the C-terminal tail of CAT1 to perform a yeast two-hybrid screen to identify putative interactors of this protein. One interesting interacting protein we identified was general control nonderepressible 1 (GCN1). We determined the expression pattern of GCN1 in several adult organs and structures using qRT-PCR and western blots. Finally, we knocked down GCN1 using double-stranded RNA and identified changes in downstream signaling intermediates and the effects of knockdown on vitellogenesis and fecundity. RESULTS: In a screen for Ae. aegypti CAT1-interacting proteins we identified GCN1 as a putative interactor. GCN1 is highly expressed in the ovaries and fat body of the mosquito. We provide evidence that eukaryotic translation initiation factor 2 subunit alpha (eIF2α) phosphorylation changed during vitellogenesis and that RNA interference knockdown of GCN1 in whole mosquitoes reduced egg clutch sizes of treated mosquitoes relative to controls. CONCLUSIONS: Aedes aegypti CAT1 and GCN1 are likely interacting partners and GCN1 is likely necessary for proper egg development. Our data suggest that GCN1 is part of a nutrient sensor mechanism in various mosquito tissues involved in vitellogenesis.

Nuclear receptor ligand screening in an iPSC-derived in vitro blood-brain barrier model identifies new contributors to leptin transport.

BACKGROUND: The hormone leptin exerts its function in the brain to reduce food intake and increase energy expenditure to prevent obesity. However, most obese subjects reflect the resistance to leptin even with elevated serum leptin. Considering that leptin must cross the blood-brain barrier (BBB) in several regions to enter the brain parenchyma, altered leptin transport through the BBB might play an important role in leptin resistance and other biological conditions. Here, we report the use of a human induced pluripotent stem cell (iPSC)-derived BBB model to explore mechanisms that influence leptin transport. METHODS: iPSCs were differentiated into brain microvascular endothelial cell (BMEC)-like cells using standard methods. BMEC-like cells were cultured in Transwell filters, treated with ligands from a nuclear receptor agonist library, and assayed for leptin transport using an enzyme-linked immune sorbent assay. RNA sequencing was further used to identify differentially regulated genes and pathways. The role of a select hit in leptin transport was tested with the competitive substrate assay and after gene knockdown using CRISPR techniques. RESULTS: Following a screen of 73 compounds, 17ß-estradiol was identified as a compound that could significantly increase leptin transport. RNA sequencing revealed many differentially expressed transmembrane transporters after 17ß-estradiol treatment. Of these, cationic amino acid transporter-1 (CAT-1, encoded by SLC7A1) was selected for follow-up analyses due to its high and selective expression in BMECs in vivo. Treatment of BMEC-like cells with CAT-1 substrates, as well as knockdown of CAT-1 expression via CRISPR-mediated epigenome editing, yielded significant increases in leptin transport. CONCLUSIONS: A major female sex hormone, as well as an amino acid transporter, were revealed as regulators of leptin BBB transport in the iPSC-derived BBB model. Outcomes from this work provide insights into regulation of hormone transport across the BBB.