Amino Acid Transport Research Articles

Impact of Oncogenic Changes in p53 and KRAS on Macropinocytosis and Ferroptosis in Colon Cancer Cells and Anticancer Efficacy of Niclosamide with Differential Effects on These Two Processes.

Mutations in p53 and KRAS are seen in most cases of colon cancer. The impact of these mutations on signaling pathways related to cancer growth has been studied in depth, but relatively less is known on their effects on amino acid transporters in cancer cells. This represents a significant knowledge gap because amino acid nutrition in cancer cells profoundly influences macropinocytosis and ferroptosis, two processes with opposing effects on tumor growth. Here, we used isogenic colon cancer cell lines to investigate the effects of p53 deletion and KRAS activation on two amino acid transporters relevant to macropinocytosis (SLC38A5) and ferroptosis (SLC7A11). Our studies show that the predominant effect of p53 deletion is to induce SLC7A11 with the resultant potentiation of antioxidant machinery and protection of cancer cells from ferroptosis, whereas KRAS activation induces not only SLC7A11 but also SLC38A5, thus offering protection from ferroptosis as well as improving amino acid nutrition in cancer cells via accelerated macropinocytosis. Niclosamide, an FDA-approved anti-helminthic, blocks the functions of SLC7A11 and SLC38A5, thus inducing ferroptosis and suppressing macropinocytosis, with the resultant effective reversal of tumor-promoting actions of oncogenic changes in p53 and KRAS. These findings underscore the potential of this drug in colon cancer treatment.

Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth.

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

Transporter Protein Expression of Corneal Epithelium in Rabbit and Porcine: Evaluation of Models for Ocular Drug Transport Study.

The transcorneal route is the main entry route for drugs to the intraocular parts, after topical administration. The outer surface, the corneal epithelium (CE), forms the rate-limiting barrier for drug permeability. Information about the role and protein expression of drug and amino acid transporter proteins in the CE is sparse and lacking. The aim of our study was to characterize transporter protein expression in rabbit and porcine CE to better understand potential drug and nutrient absorption after topical administration. Proteins, mainly Abc and Slc transporters, were characterized with quantitative targeted absolute proteomics and global untargeted proteomics methods. In the rabbit CE, 24 of 48 proteins were detected in the targeted approach, and 21 of these were quantified. In the porcine CE, 26 of 58 proteins were detected in the targeted approach, and 20 of these were quantified. Among these, 15 proteins were quantified in both animals: 4f2hc (Slc3a2), Aqp0, Asct1 (Slc1a4), Asct2 (Slc1a5), Glut1 (Slc2a1), Hmit (Slc2a13), Insr, Lat1 (Slc7a5), Mct1 (Slc16a1), Mct2 (Slc16a7), Mct4 (Slc16a3), Mrp 4 (Abcc4), Na+/K+-ATPase, Oatp3a1 (Slco3a1), and Snat2 (Slc38a2). Overall, the global proteomics results supported the targeted proteomics results. Organic anion transporting polypeptide Oatp3a1 was detected and quantified for the first time in both rabbit (1.4 ± 0.4 fmol/cm2) and porcine (11.1 ± 5.3 fmol/cm2) CE. High expression levels were observed for L-type amino acid transporter, Lat1, which was quantified with newly selected extracellular domain peptides in rabbit (48.9 ± 11.8 fmol/cm2) and porcine (37.6 ± 11.5 fmol/cm2) CE. The knowledge of transporter protein expression in ocular barriers is a key factor in the successful design of new ocular drugs, pharmacokinetic modeling, understanding ocular diseases, and the translation to human.

Food-Derived Up-Regulators and Activators of Angiotensin Converting Enzyme 2: A Review.

Angiotensin-converting enzyme 2 (ACE2) is a key enzyme in the renin-angiotensin system (RAS), also serving as an amino acid transporter and a receptor for certain coronaviruses. Its primary role is to protect the cardiovascular system via the ACE2/Ang (1-7)/MasR cascade. Given the critical roles of ACE2 in regulating numerous physiological functions, molecules that can upregulate or activate ACE2 show vast therapeutic value. There are only a few ACE2 activators that have been reported, a wide range of molecules, including food-derived compounds, have been reported as ACE2 up-regulators. Effective doses of bioactive peptides range from 10 to 50 mg/kg body weight (BW)/day when orally administered for 1 to 7 weeks. Protein hydrolysates require higher doses at 1000 mg/kg BW/day for 20 days. Phytochemicals and vitamins are effective at doses typically ranging from 10 to 200 mg/kg BW/day for 3 days to 6 months, while Traditional Chinese Medicine requires doses of 1.25 to 12.96 g/kg BW/day for 4 to 8 weeks. ACE2 activation is linked to its hinge-bending region, while upregulation involves various signaling pathways, transcription factors, and epigenetic modulators. Future studies are expected to explore novel roles of ACE2 activators or up-regulators in disease treatments and translate the discovery to bedside applications.

High Overexpression of SiAAP9 Leads to Growth Inhibition and Protein Ectopic Localization in Transgenic Arabidopsis.

Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While Arabidopsis and rice have been extensively studied, research on foxtail millet is limited. This study identified two transcripts of SiAAP9, both of which were induced by NO3- and showed similar expression patterns. The overexpression of SiAAP9L and SiAAP9S in Arabidopsis inhibited plant growth and seed size, although SiAAP9 was found to transport more amino acids into seeds. Furthermore, SiAAP9-OX transgenic Arabidopsis showed increased tolerance to high concentrations of glutamate (Glu) and histidine (His). The high overexpression level of SiAAP9 suggested its protein was not only located on the plasma membrane but potentially on other organelles, as well. Interestingly, sequence deletion reduced SiAAP9's sensitivity to Brefeldin A (BFA), and SiAAP9 had ectopic localization on the endoplasmic reticulum (ER). Protoplast amino acid uptake experiments indicated that SiAAP9 enhanced Glu transport into foxtail millet cells. Overall, the two transcripts of SiAAP9 have similar functions, but SiAAP9L shows a higher colocalization with BFA compartments compared to SiAAP9S. Our research identifies a potential candidate gene for enhancing the nutritional quality of foxtail millet through breeding.

Microbiological and molecular studies on a multidrug-resistant Pseudomonas aeruginosa from a liver transplant patient with urinary tract infection in Egypt.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for complicated UTIs and exhibits high antibiotic resistance, leading to increased mortality rates, especially in cases of multidrug-resistant strains. This study aimed to investigate the antibiotic susceptibility patterns and genomic characterization of XDR strains identified in end-stage liver disease patients who underwent liver transplants. In this study, a number of 30 individuals who underwent liver transplants were registered. Ninety urine and 60 wound site swab samples were collected and processed for culturing, identification, and antimicrobial sensitivity. Extensively drug-resistant strain EMARA01 was confirmed through Sanger sequencing and was then processed for whole genome sequencing to characterize the genomic pattern. Sequencing data were processed for de novo assembly using various tools and databases, including genome annotation, serotype identification, virulence factor genes, and antimicrobial resistance gene. Pangenome analysis of randomly selected 147 reference strains and EMAR01 sequenced strain was performed using the Bacterial Pan Genome Analysis (BPGA) software. Of these total examined samples, nosocomial infection due to P. aeruginosa was detected in twelve patients' samples. AST analysis showed that P. aeruginosa strains exhibit resistance to tobramycin, erythromycin, and gentamicin, followed by piperacillin and ofloxacin, and no strains exhibit resistance to meropenem and imipenem. The CARD database identified 59 AMR genes similar to the EMAR01 strain genome and mostly belong to the family involved in the resistance-nodulation-cell division (RND) antibiotic efflux pump. Five genes; nalC, nalD, MexR, MexA, and MexB, exhibit resistance to 14 classes of antibiotics, while two AMR; CpxR, and OprM, exhibit resistance to 15 classes of drugs. Pangenome analysis revealed that the pan-genome remained open, suggesting the potential for acquiring accessory and unique genes. Notably, the genes predominantly involved in amino acid transport metabolism were identified using the KEGG database. This study provides valuable insights into the antimicrobial resistance profile, genetic features, and genomic evolution of P. aeruginosa strains causing UTIs in liver transplant patients. The findings emphasize the significance of comprehending AMR mechanisms and genetic diversity in P. aeruginosa for developing effective treatment strategies and infection control measures.

Blocking of amino acid transporter OsAAP7 promoted tillering and yield by determining basic and neutral amino acids accumulation in rice

BackgroundAmino acids are not only the main form of N in rice, but also are vital for its growth and development. These processes are facilitated by amino acid transporters within the plant. Despite their significance, only a few AAP amino acid transporters have been reported.ResultsIn this study, we observed that there were differences in the expression of amino acid transporter OsAAP7 among 521 wild cultivated rice varieties, and it directly negatively correlated with tillering and grain yield per plant. We revealed that OsAAP7 protein was localized to the endoplasmic reticulum and had absorption and transport affinity for amino acids such as phenylalanine (Phe), lysine (Lys), leucine (Leu), and arginine (Arg) using subcellular localization, yeast substrate testing, fluorescent amino acid uptake, and amino acid content determination. Further hydroponic studies showed that exogenous application of amino acids Phe, Lys and Arg inhibited the growth of axillary buds in the overexpression lines, and promoted the elongation of axillary buds in the mutant lines. Finally, RNA-seq analysis showed that the expression patterns of genes related to nitrogen, auxin and cytokinin pathways were changed in axillary buds of OsAAP7 transgenic plants.ConclusionsThis study revealed the gene function of OsAAP7, and found that blocking of amino acid transporter OsAAP7 with CRISPR/Cas9 technology promoted tillering and yield by determining basic and neutral amino acids accumulation in rice.

#2565 Urine amino acid profiles enlighten new prediction models for renal Fanconi syndrome and its association with eGFR level

Abstract Background and Aims Renal Fanconi syndrome (FS) presented dysfunctions of renal proximal tubular (PT) transport with or without excessive urine amino acids (AAs) excretion, which might contribute to the progression of eGFR decline. This study aimed to explore the clinical benefit of 21 urinary AAs excretion patterns in renal FS and eGFR decline. Method Liquid chromatography-tandem Mass spectrometry (LC-MS-MS) is currently quantitative and detects over 21 types of amino acids in 157 patients with renal proximal tubular dysfunctions. To evaluate the diagnostic values of urine AAs in renal FS, we established a machine learning algorithm to perform binary classification using Random Forest Regressor in Python (version 3.8.8) with a training set and a testing set (8:2). We used Randomized search to find out the best model hyperparameters, calculated feature importance for contribution evaluation, and Receiver Operating Characteristic (ROC) curves to evaluate the performance of classification algorithms. Results A model based on a machine learning algorithm of all urine AAs to diagnose renal FS established with sensitivity = 0.71, specificity = 0.73, and AUC = 0.83 (95% CI 0.648-0.957) (Fig. 1), with similar performance in combined six urine AAs (citrulline, asparagine, threonine, phenylalanine, ornithine, and proline) models. The urine proline level independently correlated with eGFR < 60 ml/min/1.73 m2 (bias regression coefficient 0.84, P = 0.001, OR = 2.31, 95% CI 1.41-3.80) when adjusted by age, etiology, gender, and blood urea nitrogen level. For the primary diseased diagnosis, the most susceptible urine AAs were citrulline (56.7%), proline (56.0%,) threonine (52.2%), and ornithine (45.9%) and the less susceptible ones were histidine (24.8%), aspartate (16.6%), and arginine (13.4%). Accordingly, the most common affected AA transporters were SLC6A19 (B0AT1) and etiology-specific urine AA atlas major in the eight urine AAs (isoleucine, glutamine, methionine, proline, alanine, ornithine, lysine, and arginine). Different from primary Sjӧgren syndrome patients (n = 26) with less susceptible, the patients with plasmacyte diseases (n = 8) showed elevations of almost all the urine AAs except glycine and aspartate. The connection between urine AAs excretion and hypouricemia showed gender differences, with 20 urine AAs significantly negatively correlating with serum uric acid levels in males but only five in females. Conclusion We first established a model based on the urine AA atlas detected by (LC-MS-MS) which contributed to differentiating the etiologies of renal tubular dysfunctions and associated well with eGFR. It shed light on the clinical practice in precision evaluation of tubule injury.

Transient kinetics reveal the mechanism of competitive inhibition of the neutral amino acid transporter ASCT2

ASCT2 (alanine serine cysteine transporter 2), a member of the SLC1 (solute carrier 1) family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anti-cancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor Lc-BPE with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that, despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The pre-incubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.

Amino acid transporters in neurological disorders and neuroprotective effects of cysteine derivatives

For most diseases and disorders occurring in the brain, the full causes behind them are yet unknown, but many show signs of dysfunction of amino acid transporters or abnormalities in amino acid metabolism. The blood-brain barrier (BBB) plays a key role in supporting the function of the central nervous system (CNS). Because of its unique structure, the BBB can maintain the optimal environment for CNS by controlling the passage of hydrophilic molecules from blood to the brain. Nutrients, such as amino acids, can cross the BBB via specific transporters. Many amino acids are essential for CNS function, and dysfunction of these amino acid transporters can lead to abnormalities in amino acid levels. This has been linked to causes behind certain genetic brain diseases, such as schizophrenia, autism spectrum disorder, and Huntington's disease (HD). One example of crucial amino acids is L-Cys, the rate-limiting factor in the biosynthesis of an important antioxidant, glutathione (GSH). Deficiency of L-Cys and GSH has been linked to oxidative stress and has been shown as a plausible cause behind certain CNS diseases, like schizophrenia and HD. This review presents the current status of potential L-Cys therapies and gives future directions that can be taken to improve amino acid transportation related to distinct CNS diseases.

FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

Genome-wide association analysis explores the genetic loci of amino acid content in duck’s breast muscle

BackgroundAmino acids are the basic components of protein and an important index to evaluate meat quality. With the rapid development of genomics, candidate regions and genes affecting amino acid content in livestock and poultry have been gradually revealed. Hence, genome-wide association study (GWAS) can be used to screen candidate loci associated with amino acid content in duck meat.ResultIn the current study, the content of 16 amino acids was detected in 358 duck breast muscles. The proportion of Glu to the total amino acid content was relatively high, and the proportion was 0.14. However, the proportion of Met content was relatively low, at just 0.03. By comparative analysis, significant differences were found between males and females in 3 amino acids, including Ser, Met, and Phe. In addition, 12 SNPs were significantly correlated with Pro content by GWAS analysis, and these SNPs were annotated by 7 protein-coding genes; 8 significant SNPs were associated with Tyr content, and these SNPs were annotated by 6 protein-coding genes. At the same time, linkage disequilibrium (LD) analysis was performed on these regions with significant signals. The results showed that three SNPs in the 55–56 Mbp region of chromosome 3 were highly correlated with the leader SNP (chr3:55526954) that affected Pro content (r2 > 0.6). Similarly, LD analysis showed that there were three SNPs in the 21.2–21.6 Mbp region of chromosome 13, which were highly correlated with leader SNP (chr13:21421661) (r2 > 0.6). Moreover, Through functional enrichment analysis of all candidate genes. The results of GO enrichment analysis showed that several significant GO items were associated with amino acid transport function, including amino acid transmembrane transport and glutamine transport. The results further indicate that these candidate genes are closely associated with amino acid transport. Among them, key candidate genes include SLC38A1. For KEGG enrichment analysis, CACNA2D3 and CACNA1D genes were covered by significant pathways.ConclusionIn this study, GWAS analysis found a total of 28 significant SNPs affecting amino acid content. Through gene annotation, a total of 20 candidate genes were screened. In addition, Through LD analysis and enrichment analysis, we considered that SERAC1, CACNA2D3 and SLC38A1 genes are important candidate genes affecting amino acid content in duck breast muscle.

Gypenoside XVII Reduces Synaptic Glutamate Release and Protects against Excitotoxic Injury in Rats.

Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 μM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.

Radiosynthesis and Preclinical Evaluation of m-[18F]FET and [18F]FET-OMe as Novel [18F]FET Analogs for Brain Tumor Imaging.

O-([18F]Fluoroethyl)-l-tyrosine ([18F]FET) is actively transported into the brain and cancer cells by LAT1 and possibly other amino acid transporters, which enables brain tumor imaging by positron emission tomography (PET). However, tumor delivery of this probe in the presence of competing amino acids may be limited by a relatively low affinity for LAT1. The aim of the present work was to evaluate the meta-substituted [18F]FET analog m-[18F]FET and the methyl ester [18F]FET-OMe, which were designed to improve tumor delivery by altering the physicochemical, pharmacokinetic, and/or transport properties. Both tracers could be prepared with good radiochemical yields of 41-56% within 66-90 min. Preclinical evaluation with [18F]FET as a reference tracer demonstrated reduced in vitro uptake of [18F]FET-OMe by U87 glioblastoma cells and no advantage for in vivo tumor imaging. In contrast, m-[18F]FET showed significantly improved in vitro uptake and accelerated in vivo tumor accumulation in an orthotopic glioblastoma model. As such, our work identifies m-[18F]FET as a promising alternative to [18F]FET for brain tumor imaging that deserves further evaluation with regard to its transport properties and in vivo biodistribution.

Discovery and Synthesis of Hydroxy-l-Proline Blockers of the Neutral Amino Acid Transporters SLC1A4 (ASCT1) and SLC1A5 (ASCT2).

As a conformationally restricted amino acid, hydroxy-l-proline is a versatile scaffold for the synthesis of diverse multi-functionalized pyrrolidines for probing the ligand binding sites of biological targets. With the goal to develop new inhibitors of the widely expressed amino acid transporters SLC1A4 and SLC1A5 (also known as ASCT1 and ASCT2), we synthesized and functionally screened synthetic hydroxy-l-proline derivatives using electrophysiological and radiolabeled uptake methods against amino acid transporters from the SLC1, SLC7, and SLC38 solute carrier families. We have discovered a novel class of alkoxy hydroxy-pyrrolidine carboxylic acids (AHPCs) that act as selective high-affinity inhibitors of the SLC1 family neutral amino acid transporters SLC1A4 and SLC1A5. AHPCs were computationally docked into a homology model and assessed with respect to predicted molecular orientation and functional activity. The series of hydroxyproline analogs identified here represent promising new agents to pharmacologically modulate SLC1A4 and SLC1A5 amino acid exchangers which are implicated in numerous pathophysiological processes such as cancer and neurological diseases.

Oxidative stress plays an important role in the central regulatory mechanism of orofacial hyperalgesia under low estrogen conditions

Hyperalgesia occurs in the orofacial region of rats when estrogen levels are low, although the specific mechanism needs to be investigated further. Furthermore, oxidative stress plays an important role in the transmission of pain signals. This study aimed to explore the role of oxidative stress in orofacial hyperalgesia under low estrogen conditions. We firstly found an imbalance between oxidative and antioxidant capacity within the spinal trigeminal subnucleus caudalis (SP5C) of rats after ovariectomy (OVX), resulting in oxidative stress and then a decrease in the orofacial pain threshold. To investigate the mechanism by which oxidative stress occurs, we used virus as a tool to silence or overexpress the excitatory amino acid transporter 3 (EAAT3) gene. Further investigation revealed that the regulation of glutathione (GSH) and reactive oxygen species (ROS) can be achieved by regulating EAAT3, which in turn impacts the occurrence of oxidative stress. In summary, our findings suggest that reduced expression of EAAT3 within the SP5C of rats in the low estrogen state may decrease GSH content and increase ROS levels, resulting in oxidative stress and ultimately lead to orofacial hyperalgesia. This suggests that antioxidants could be a potential therapeutic direction for orofacial hyperalgesia under low estrogen conditions, though more research is needed to understand its mechanism.

Accelerated dissemination of antibiotic resistant genes via conjugative transfer driven by deficient denitrification in biochar-based biofiltration systems

Biofiltration systems harbored and disseminated antibiotic resistance genes (ARGs), when confronting antibiotic-contained wastewater. Biochar, a widely used environmental remediation material, can mitigate antibiotic stress on adjoining microbes by lowering the availability of sorbed antibiotics, and enhance the attachment of denitrifiers. Herein, bench-scale biofiltration systems, packed with commercial biochars, were established to explore the pivotal drivers affecting ARG emergence. Results showed that biofiltration columns, achieving higher TN removal and denitrification capacity, showed a significant decrease in ARG accumulation (p < 0.05). The relative abundance of ARGs (0.014 ± 0.0008) in the attached biofilms decreased to 1/5-folds of that in the control group (0.065 ± 0.004). Functional analysis indicated ARGs' accumulation was less attributed to ARG activation or horizontal gene transfer (HGT) driven by sorbed antibiotics. Most denitrifiers, like Bradyrhizobium, Geothrix, etc., were found to be enriched and host ARGs. Nitrosative stress from deficient denitrification was demonstrated to be the dominant driver for affecting ARG accumulation and dissemination. Metagenomic and metaproteomic analysis revealed that nitrosative stress promoted the conjugative HGT of ARGs mainly via increasing the transmembrane permeability and enhancing the amino acid transport and metabolism, such as cysteine, methionine, and valine metabolism. Overall, this study highlighted the risks of deficient denitrification in promoting ARG transfer and transmission in biofiltration systems and natural ecosystems.

Intestinal health of squab pigeons responded to parental dietary protein levels during breeding period

The objective of this study was to determine the effects of dietary crude protein (CP) levels on intestinal antioxidant status, tight junction proteins expression, and amino acids transporters levels in squabs. A total of 180 pairs of White King parent pigeons approximately 10 mo old were randomly assigned to 5 groups with 6 replications of 6 pairs of parental pigeons each, and were fed with 14, 15, 16, 17, and 18% CP diets for 46 d, respectively. Dietary increasing CP levels increased final body weight (linear and quadratic, P < 0.05), serum urea nitrogen (linear, P<0.05) and triglyceride levels (quadratic, P < 0.05), and reduced kidney relative weight (quadratic, P < 0.05) in squabs. Final body weight of squabs in the 18% CP diet group was higher than that of the 14, 15, and 16% CP diet groups (P < 0.05) but was similar to that of the 17% CP diet group (P > 0.05). Increasing dietary CP levels reduced intestinal malondialdehyde contents (linear and quadratic, P < 0.05) and jejunal total superoxide dismutase (T-SOD) activity (linear, P < 0.05), and enhanced (linear and quadratic, P<0.05) ileal catalase and T-SOD activities in squabs, and these effects were more prominent in the 17% CP diet group. Graded CP levels up-regulated the mRNA expression of intestinal zonula occludens 1 (linear, P < 0.05), solute carrier family 7 members 9 (linear, P < 0.05) and claudin 1 (CLDN1, linear and quadratic, P < 0.05), ileal CLDN3 and solute carrier family 6 members 14 (linear, P < 0.05) but lowered jejunal solute carrier family 6 member 14 (quadratic, P<0.05) mRNA expression in squabs. The effects of dietary CP levels on intestinal tight junction proteins expression were more apparent when its supplemental levels were 18%. These results suggested that increasing parental dietary CP levels ranged from 14 to 18% during breeding period improved growth and intestinal function of squabs, with its recommended level being 17%.

Mutation delTTCT 1471 de l’intolérance aux protéines dibasiques : Caractéristiques cliniques d’une série pédiatrique tunisienne.

Lysinuric protein intolerance (LPI) is a rare inherited metabolic disease. It is caused by a deficiency in cationic amino acid transport caused by mutations in SLC7A7 gene. To identify the clinical, diagnostic and therapeutic features of lysnuric protein intolerance. This was a retrospective study conducted in the pediatric department of La Rabta Hospital over a period of 30 years (1992 to 2022). We included patients with clinical signs suggestive of lysinuric protein intolerance and orotic acid in the urine. We enrolled seven patients. The median age at disease onset was nine months. The median age at positive diagnosis was 21 months. Growth retardation, hepatosplenomegaly and haematological abnormalities were the main features of the disease. Hyperammonia and increased urinary orotic acid were present in all patients. Molecular biology revealed the del TTCT 1471 mutation in five patients. All patients were prescribed a low protein diet and citrulline supplementation. Complications of the disease were growth retardation (n=7), psychomotor or intellectual retardation (n=5), haemophagocytic lymphohistiocytosis (n=4) and osteoporosis (n=3). After a median follow-up of 11 years, six of our patients are still alive. One patient died from acute hyperammonemic encephalopathy. In this paediatric series, delays in diagnosis and treatment of LPI were responsible for long-term sequelae, particularly bone and neurological. The delTTCT1471 mutation appears to be the mutation of paediatric-onset forms in Tunisia. This mutation was not associated with pulmonary involvement, which is a prognostic factor and the main cause of death.

Early life exposure to vitamin D deficiency impairs molecular mechanisms that regulate liver cholesterol biosynthesis, energy metabolism, inflammation, and detoxification.

Emerging data suggests liver disease may be initiated during development when there is high genome plasticity and the molecular pathways supporting liver function are being developed. Here, we leveraged our Collaborative Cross mouse model of developmental vitamin D deficiency (DVD) to investigate the role of DVD in dysregulating the molecular mechanisms underlying liver disease. We defined the effects on the adult liver transcriptome and metabolome and examined the role of epigenetic dysregulation. Given that the parental origin of the genome (POG) influences response to DVD, we used our established POG model [POG1-(CC011xCC001)F1 and POG2-(CC001xCC011)F1] to identify interindividual differences. We found that DVD altered the adult liver transcriptome, primarily downregulating genes controlling liver development, response to injury/infection (detoxification & inflammation), cholesterol biosynthesis, and energy production. In concordance with these transcriptional changes, we found that DVD decreased liver cell membrane-associated lipids (including cholesterol) and pentose phosphate pathway metabolites. Each POG also exhibited distinct responses. POG1 exhibited almost 2X more differentially expressed genes (DEGs) with effects indicative of increased energy utilization. This included upregulation of lipid and amino acid metabolism genes and increased intermediate lipid and amino acid metabolites, increased energy cofactors, and decreased energy substrates. POG2 exhibited broader downregulation of cholesterol biosynthesis genes with a metabolomics profile indicative of decreased energy utilization. Although DVD primarily caused loss of liver DNA methylation for both POGs, only one epimutation was shared, and POG2 had 6.5X more differentially methylated genes. Differential methylation was detected at DEGs regulating developmental processes such as amino acid transport (POG1) and cell growth & differentiation (e.g., Wnt & cadherin signaling, POG2). These findings implicate a novel role for maternal vitamin D in programming essential offspring liver functions that are dysregulated in liver disease. Importantly, impairment of these processes was not rescued by vitamin D treatment at weaning, suggesting these effects require preventative measures. Substantial differences in POG response to DVD demonstrate that the parental genomic context of exposure determines offspring susceptibility.