Abundance Of Transporters Research Articles

New insights into uranium stress responses of Arabidopsis roots through membrane- and cell wall-associated proteome analysis.

Uranium (U) is a non-essential and toxic metal for plants. In Arabidopsis thaliana plants challenged with uranyl nitrate, we showed that U was mostly (64-71% of the total) associated with the root insoluble fraction containing membrane and cell wall proteins. Therefore, to uncover new molecular mechanisms related to U stress, we used label-free quantitative proteomics to analyze the responses of the root membrane- and cell wall-enriched proteome. Of the 2,802 proteins identified, 458 showed differential accumulation (≥1.5-fold change) in response to U. Biological processes affected by U include response to stress, amino acid metabolism, and previously unexplored functions associated with membranes and the cell wall. Indeed, our analysis supports a dynamic and complex reorganization of the cell wall under U stress, including lignin and suberin synthesis, pectin modification, polysaccharide hydrolysis, and Casparian strips formation. Also, the abundance of proteins involved in vesicular trafficking and water flux was significantly altered by U stress. Measurements of root hydraulic conductivity and leaf transpiration indicated that U significantly decreased the plant's water flux. This disruption in water balance is likely due to a decrease in PIP aquaporin levels, which may serve as a protective mechanism to reduce U toxicity. Finally, the abundance of transporters and metal-binding proteins was altered, suggesting that they may be involved in regulating the fate and toxicity of U in Arabidopsis. Overall, this study highlights how U stress impacts the insoluble root proteome, shedding light on the mechanisms used by plants to mitigate U toxicity.

Distribution Characteristics and Impacting Factors of Drug CYP Enzymes and Transporters in the Gastrointestinal Tract of Chinese Healthy Subjects.

The abundance of drug metabolic enzymes (DMEs) and transporters (DTs) in the human gastrointestinal tract significantly affects xenobiotic exposure in the circulating system, the basis of these compounds acting on humans. However, accurately predicting individual exposure in healthy subjects remains challenging due tolimited data on protein levels throughout the gastrointestinal tract within the same individuals and inadequate assessment of factors influencing these levels. Therefore, we conducted a clinical study to obtain biopsy samples from 8 different gastrointestinal segments in 24 healthy Chinese volunteers. Concurrently, blood and fecal samples were collected for genotypic analysis and fecal microbiota metagenomic sequencing. Using an optimized LC-MS/MS method, we quantified the absolute protein abundance of CYP2C9, CYP2C19, CYP2D6, CYP3A4, P-gp, and BCRP from the stomach to the colon. Our results revealed significant regional differences in protein expression: CYP3A4 was the most abundant in the small intestine, whereas CYP2C9 was predominantly found in the colon. CYP2D6 was primarily located in the ileum, while other DMEs/DTs showed higher concentrations in the jejunum. Meanwhile, the enzyme abundance in the small intestine and colon and the relative ratio of transporters in different regions to the jejunum were accurately calculated, providingvaluable data for refining the physiological parameters in the virtual gastrointestinal tract of Chinese healthy population in PBBMs. Additionally, BMI, IBW, sex, age, genotype, and fecal microbiota were identified as critical factors influencing the protein levels of these DMEs/DTs throughout the gastrointestinal tract, with notable regional differences. Consequently, this study provides a unique foundation for understanding xenobiotic absorption in humans.

Modulation of blood pressure by dietary potassium and sodium: Sex differences and modeling analysis.

High Na+ intake has been linked to elevations in blood pressure, whereas K+ has the opposite effect. The underlying mechanisms involve complex interactions among renal function, fluid volume, fluid-regulatory hormones, the vasculature, cardiac function, and the autonomic nervous system. These mechanisms are likely moderated by sex, given the known sex differences in blood pressure regulation and the higher prevalence of hypertension in men. The source of these observed sex differences may be traced to organ and tissue levels, given that kidney function, intrarenal renin-angiotensin system components, renal sympathetic nervous activity, and nitric oxide bioavailability all exhibit sex differences. To assess the functional impact of each of these sex differences, we developed sex-specific computational models to simulate whole-body Na+, K+, and fluid homeostasis, and the effects on blood pressure. The models describe the interactions among the renal system, cardiovascular system, gastrointestinal system, renal sympathetic nervous system, and renin-angiotensin-aldosterone system. Model simulations suggest that women's attenuated blood pressure response to hypertensive stimuli, including high Na+ intake, may be largely attributable to the female renal transporter abundance pattern. Additionally, we investigated the causal link between high K+ intake and blood pressure reduction. The models simulate renal response to high K+ intake, including the immediate gastrointestinal feedforward signals to the kidneys to increase K+ excretion, and the longer-term response to decrease proximal fractional Na+ reabsorption and distal K+ reabsorption. With these assumptions, simulations of high K+ intake yielded kaliuresis, natriuresis, and a substantial reduction in blood pressure, even when combined with high Na+ intake.

Absolute Membrane Protein Abundance of P-gp, BCRP and MRPs in Term Human Placenta Tissue and Commonly Used Cell Systems: Application in PBPK Modeling of Placental Drug Disposition.

The placenta acts as a barrier, excluding noxious substances whilst actively transferring nutrients to the fetus, mediated by various transporters. This study quantified the expression of key placental transporters in term human placenta (n=5) and BeWo, BeWo b30, and JEG-3 placenta cell lines. Combining these results with pregnancy physiologically-based pharmacokinetic (PBPK) modeling, we demonstrate the utility of proteomic analysis for predicting placental drug disposition and fetal exposure. Using targeted proteomics with QconCAT standards, we found significant expression of P-gp, BCRP, MRP2, MRP4, and MRP6 in the human placenta (0.05 - 0.25 pmol/mg membrane protein) with only regional differences observed for P-gp. Unexpectedly, both P-gp and BCRP were below the limit of quantification in the regularly used BeWo cells, indicating that this cell line may not be suitable for the study of placental P-gp and BCRP-mediated transport. In cellular and vesicular overexpression systems, P-gp and BCRP were detectable as expected. Vesicle batches showed consistent P-gp expression correlating with functional activity (N-methyl-quinidine (NMQ) transport). However, BCRP activity (Estrone 3-sulfate (E1S) transport) did not consistently align with expression levels. Incorporating in vitro transporter kinetic data, along with placental transporter abundance, into a PBPK model enabled the evaluation of fetal exposure. Simulation with a hypothetical drug indicated that estimating fetal exposure relies on the intrinsic clearances of relevant transporters. To minimize interlaboratory discrepancies, expression data was generated using consistent proteomic methodologies in the same lab. Integration of this data in pregnancy-PBPK modeling offers a promising tool to investigate maternal, placental and fetal drug exposure. Significance Statement This study quantified the expression of key transporters in human placenta and various placental cell lines, revealing significant expression variations. By integrating these data with PBPK modeling, the study highlights the importance of transporter abundance data in understanding and predicting placental drug disposition.

Impact of early postbiotic supplementation on broilers’ responses to subclinical necrotic enteritis

Necrotic enteritis (NE), an enteric disease caused by Clostridium perfringens, results in damage to the intestinal epithelial lining disrupting its function, nutrient absorption, and utilization. This study evaluated the effects of in ovo and post-hatch applications of a Saccharomyces cerevisiae-based postbiotic on performance and nutrient transporter genes of broilers during a NE challenge. At embryonic d 18, Ross 708 fertile eggs were injected with 0.2 mL of either water or postbiotic. A total of 288 male hatchlings were assigned to one of the following treatment groups: 1) NC (in ovo water injection, no challenge); 2) PIW (postbiotic in ovo and in drinking water, no challenge); 3) NC+ (NC with NE challenge); and 4) PIW+ (PIW with NE challenge). On d 14, all birds in the NE-challenged groups were orally gavaged with 3,000 Eimeria maxima sporulated oocysts followed by two doses of ∼1×108 CFU/mL/bird of C. perfringens on d 19 and d 20. Hatchability, weekly performance, intestinal lesion scores, and mRNA abundance of nutrient transporters in the jejunum and ileum were assessed. Data were analyzed by 2-way ANOVA in JMP and significance between treatments identified by LSD test (P ≤ 0.05). A significant postbiotic treatment and NE challenge interaction was observed in performance during d 21-28 with a greater ADG in PIW compared to NC and PIW+. Lesion scores in the jejunum and ileum were significantly reduced in PIW+ compared to NC+. On d 7, mRNA abundance of SGLT1 was significantly greater in PIW compared to the NC group. On d 14, birds in PIW had greater levels of GLUT2 and EAAT3 than NC group. No significant interaction effects were observed on d 21. PIW+ had significantly greater EAAT3 mRNA levels compared to PIW in jejunum and PIW and NC+ in ileum on d 28. In conclusion, in ovo and water supplementation of this postbiotic presents a potential to improve the performance, ameliorate pathology detriments associated with NE, and positively regulate the mRNA levels of key nutrient transporters during NE challenge.

Seasonal dynamics in microbial trace metals transporters during phytoplankton blooms in the Southern Ocean.

Trace metals are required as cofactors in metalloproteins that are essential in microbial metabolism and growth. The microbial requirements of diverse metals and the capabilities of prokaryotic taxa to acquire these metals remain poorly understood. We present here results from metagenomic observations over an entire productive season in the region off Kerguelen Island (Indian Sector of the Southern Ocean). We observed seasonal patterns in the abundance of prokaryotic transporters of seven trace elements (zinc [Zn], manganese [Mn], nickel [Ni], molybdenum [Mo], tungsten [W], copper [Cu] and cobalt [Co]) and the consecutive spring and summer phytoplankton blooms were strong drivers of these temporal trends. Taxonomic affiliation of the functional genes revealed that Rhodobacteraceae had a broad repertoire of trace metal transporters (Mn, Zn, Ni, W and Mo) and a more restricted set was observed for other prokaryotic groups, such as Flavobacteriaceae (Zn), Nitrincolaceae (Ni and W) and Thioglobaceae (Mo). The prevalence of trace metal transporters within a prokaryotic group, as determined on the family level, was overall confirmed in representative metagenome-assembled genomes. We discuss the potential involvement of prokaryotic groups in processes related to organic matter utilisation that require these metals and the consequences on carbon and trace metal cycling in surface waters of the Southern Ocean.

Optimizing renal transporter immunodetection: consequences of freeze-thaw during sample preparation.

Renal transporters (cotransporters, channels, and claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. Although journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Frozen) versus freshly processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of 15 renal transporters and the impact of protease inhibitors during homogenization. In female Sprague-Dawley rat kidneys homogenized with aprotinin, Na2EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ∼50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmplete Protease Inhibitor, "+") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague-Dawley rats, immunoblot signal density was lower in Frozen+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Frozen versus Fresh by ∼25-50% and greater in Frozen+. In contrast, immunodetection signal was equivalent in female Frozen+ versus female Fresh+ for claudin 2, villin, AQP1, NKCC2, NCC, ENaCβ, ENaCɣ, claudin 7, AQP2, NKAα1, and NKAβ1. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility. NEW & NOTEWORTHY Freeze-thawing kidneys before homogenization is widely accepted in renal research. This study demonstrates that if kidneys are freeze-thawed just once before homogenization, immunoblot signals are reduced in a transporter-specific manner in rats and mice dependent on sex and that immunoblot signals can be partially recovered by adding additional protease inhibitors. These findings underscore the critical importance of assessing the impact of sample preparation, including freeze-thaw versus fresh, to ensure robust rigor and reproducibility.

PSII-18 Prematurity alters nutrient signaling and protein synthesis in skeletal muscle of neonatal piglets

Abstract Extrauterine growth restriction is common in infants born preterm, and negatively affects lean mass accretion and health. Prematurity has been shown to inhibit the feeding-induced stimulation of protein synthesis in skeletal muscle. We hypothesized that nutrient signaling, activation of the mechanistic target of rapamycin complex 1 (mTORC1), and protein synthesis in the muscle are limited in preterm infants which negatively impacts muscle growth. The objective of this study was to examine how prematurity influences the mechanisms involved in insulin- and amino acid-induced signaling and protein synthesis in skeletal muscle of piglets delivered either 9 d preterm (104 d, n = 25) or at term (112 d, n = 26) by cesarean section. After resuscitation, they were surgically implanted with jugular vein and carotid artery catheters and placed in individual incubators. They were randomly allotted to one of three treatments within each preterm and term birth groups: euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps on 4 d after birth. After the clamp procedure, the piglets were euthanized to collect longissimus dorsi muscle for estimating in vivo fractional protein synthesis rates and the abundance and activation of components related to insulin and amino acid signaling. Data were analyzed using the MIXED procedure of SAS. The leucine sensor, Sestrin1 bound to GATOR2 (P < 0.05) was reduced in response to AA whereas the abundance of the mTOR-RagA and mTOR-RagC complexes increased (P < 0.05). The phosphorylation of Akt was increased (P < 0.05) in response to INS and the phosphorylation of mTORC1 and protein synthesis increased (P < 0.05) in response to both AA and INS. Prematurity did not affect the protein abundances of the AA transporters for glutamine (SLC38A2), leucine (SLC7A5), and arginine (SLC38A9). However, prematurity reduced (P < 0.05) the abundances of the leucine sensors SAR1B, the Sestrin1-GATOR complex, the AA sensor, RAB1A, and the threonine sensor, TARS2 (P < 0.05). The abundances of the glutamine sensor, ARF1, the arginine sensor, CASTOR, and the methionine-SAMTOR GATOR complex were similar. Prematurity reduced (P < 0.05) the abundance of the mTOR-RagA and mTOR-RagC complexes, the phosphorylation of mTORC1 and muscle protein synthesis (P < 0.05). In conclusion, prematurity negatively affects protein synthesis in skeletal muscle of neonatal preterm piglets by blunting anabolic pathways responsible for nutrient-sensing and mTORC1 activation. The reduced anabolic response likely contributes to reduced lean growth and extrauterine growth restriction following preterm birth.

352 Supplementing bovine satellite cells with anabolic hormones and/or zinc at differentiation impacts abundance of mRNA involved in growth, mineral transport, and oxidative stress

Abstract Zinc (Zn) is the most utilized metal in biological processes and supports growth through critical roles in protein turnover. The Zn requirements of skeletal muscle may be augmented when growth promoting technologies, such as anabolic hormones are being utilized. Both anabolic hormones and Zn affect protein synthesis and degradation in skeletal muscle. However, the molecular mechanisms through which Zn may interact with anabolic hormones to support skeletal muscle growth remains unknown. The objective of this study was to determine whether trenbolone acetate (TBA), estradiol (E2) and Zn interact to promote differentiation of bovine satellite cell (BSC) cultures by assessing abundance of mRNA involved in growth, mineral transport, differentiation, protein turnover and oxidative stress. To do this, primary BSC cultures were isolated from three different steers and grown to 80% confluency and induced to differentiate in 3% horse serum, at which time a factorial design was employed to assess the impact of Zn (0, 10, 20 or 40 μM) as ZnCl2 and/or the impact of hormone (10 nM TBA,10 nM E2, 10 nM TBA and 10 nM E2). Zinc concentrations represent physiologically relevant plasma Zn concentrations. Total mRNA was isolated from the treated cultures at 4 h, 12 h and 24 h post-treatment. Assays utilizing cultures from each steer were run in duplicate (n = 6). The mRNA abundance of 45 genes were examined and analyzed using the PROC MIXED procedure of SAS with time as a repeated measure to evaluate the fixed effects of hormone, mineral, time, and all interactions. No effects (P ≥ 0.19) of hormone x mineral x time, hormone x time, hormone x mineral or hormone were noted. A mineral x time effect (P ≤ 0.04) was noted for the igf1r, slc39a7, mapk1, mmp2, and G6pd. A mineral effect was found for hormone receptors (ir, ar, esr1, esr2, and gper; P ≤ 0.03), mRNA involved in mineral transport (slc30a10, slc30a7, sod1, sod2, slc2a4, and slc39a8; P ≤ 0.009), and genes related to skeletal muscle differentiation (pax7, myf5, myf6, spry1, myog, mef2a, and myod; P ≤ 0.02) and protein turnover (akt1, fox01, fox04, mstn,fbox32, fox03, mtor, trim63, and CKM; P ≤ 0.03). These differences are generally driven by abundance being increased (P ≤ 0.05) in cultures treated with 40 or 20 μM Zn compared with cultures receiving 0 or 10 μM Zn. These results indicate that while Zn and hormone do not appear to interact to influence gene expression of differentiating primary BSC cultures at the time points measured, increased concentrations of Zn appear to increase mRNA abundance of hormone receptors, mineral transporters, and genes involved in skeletal muscle differentiation and protein turnover.

Rapid Vacuolar Sorting of the Borate Transporter BOR1 Requires the Adaptor Protein Complex AP-4 in Arabidopsis.

Plants maintain nutrient homeostasis by controlling the activities and abundance of nutrient transporters. In Arabidopsis thaliana, the borate (B) transporter BOR1 plays a role in the efficient translocation of B under low-B conditions. BOR1 undergoes polyubiquitination in the presence of sufficient B and is then transported to the vacuole via multivesicular bodies (MVBs) to prevent B accumulation in tissues at a toxic level. A previous study indicated that BOR1 physically interacts with µ subunits of adaptor protein complexes AP-3 and AP-4, both involved in vacuolar sorting pathways. In this study, we investigated the roles of AP-3 and AP-4 subunits in BOR1 trafficking in Arabidopsis. The lack of AP-3 subunits did not affect either vacuolar sorting or polar localization of BOR1-GFP, whereas the absence of AP-4 subunits resulted in a delay in high-B-induced vacuolar sorting without affecting polar localization. Super-resolution microscopy revealed a rapid sorting of BOR1-GFP into AP-4-positive spots in the trans-Golgi network (TGN) upon high-B supply. These results indicate that AP-4 is involved in sequestration of ubiquitinated BOR1 into a TGN-specific subdomain "vacuolar-trafficking zone," and is required for efficient sorting to MVB and vacuole. Our findings elucidate the rapid vacuolar sorting process facilitated by AP-4 in plant nutrient transporters.

Developmental Expression of Drug Transporters and Conjugating Enzymes Involved in Enterohepatic Recycling: Implication for Pediatric Drug Dosing.

Around 50% of the drugs used in children have never been tested for safety and efficacy in this vulnerable population. Immature drug elimination pathways can lead to drug toxicity when pediatric doses are determined using empirical methods such as body-surface area or body-weight-normalized adult dosing. In the absence of clinical data, physiologically-based pharmacokinetic (PBPK) modeling has emerged as a useful tool to predict drug pharmacokinetics in children. These models utilize developmental physiological data, including age-dependent differences in the abundance of drug-metabolizing enzymes and transporters (DMET), to mechanistically extrapolate adult pharmacokinetic data to children. The reported abundance data of hepatic DMET proteins in subcellular fractions isolated from frozen tissue are prone to high technical variability. Therefore, we carried out the proteomics-based quantification of hepatic drug transporters and conjugating enzymes in 50 pediatric and 8 adult human hepatocyte samples. Out of the 34 studied proteins, 28 showed a significant increase or decrease with age. While MRP6, OAT7, and SULT1E1 were highest in < 1-year-old samples, the abundance of P-gp and UGT1A4 was negligible in < 1-year-old samples and increased significantly after 1 year of age. Incorporation of the age-dependent abundance data in PBPK models can help improve pediatric dose prediction, leading to safer drug pharmacotherapy in children.

Maternal gut Bifidobacterium breve modifies fetal brain metabolism in germ-free mice

BackgroundRecent advances have significantly expanded our understanding of the gut microbiome's influence on host physiology and metabolism. However, the specific role of certain microorganisms in gestational health and fetal development remains underexplored. ObjectiveThis study investigates the impact of Bifidobacterium breve UCC2003 on fetal brain metabolism when colonized in the maternal gut during pregnancy. MethodsGerm-free pregnant mice were colonized with or without B. breve UCC2003 during pregnancy. The metabolic profiles of fetal brains were analyzed, focusing on the presence of key metabolites and the expression of critical metabolic and cellular pathways. ResultsMaternal colonization with B. breve resulted in significant metabolic changes in the fetal brain. Specifically, ten metabolites, including citrate, 3-hydroxyisobutyrate, and carnitine, were reduced in the fetal brain. These alterations were accompanied by increased abundance of transporters involved in glucose and branched-chain amino acid uptake. Furthermore, supplementation with this bacterium was associated with elevated expression of critical metabolic pathways such as PI3K-AKT, AMPK, STAT5, and Wnt-β-catenin signaling, including its receptor Frizzled-7. Additionally, there was stabilization of HIF-2 protein and modifications in genes and proteins related to cellular growth, axogenesis, and mitochondrial function. ConclusionsThe presence of maternal B. breve during pregnancy plays a crucial role in modulating fetal brain metabolism and growth. These findings suggest that Bifidobacterium could modify fetal brain development, potentially offering new avenues for enhancing gestational health and fetal development through microbiota-targeted interventions.

Endurance Training Improves Leg Proton Release and Decreases Potassium Release During High-Intensity Exercise in Normoxia and Hypobaric Hypoxia.

To assess the impact of endurance training on skeletal muscle release of H+ and K+. Nine participants performed one-legged knee extension endurance training at moderate and high intensities (70%-85% of Wpeak), three to four sessions·week-1 for 6 weeks. Post-training, the trained and untrained (control) leg performed two-legged knee extension at low, moderate, and high intensities (40%, 62%, and 83% of Wpeak) in normoxia and hypoxia (~4000 m). The legs were exercised simultaneously to ensure identical arterial inflow concentrations of ions and metabolites, and identical power output was controlled by visual feedback. Leg blood flow was measured (ultrasound Doppler), and acid-base variables, lactate- and K+ concentrations were assessed in arterial and femoral venous blood to study K+ and H+ release. Ion transporter abundances were assessed in muscle biopsies. Lactate-dependent H+ release was similar in hypoxia to normoxia (p = 0.168) and was lower in the trained than the control leg at low-moderate intensities (p = 0.060-0.006) but similar during high-intensity exercise. Lactate-independent and total H+ releases were higher in hypoxia (p < 0.05) and increased more with power output in the trained leg (leg-by-power output interactions: p = 0.02). K+ release was similar at low intensity but lower in the trained leg during high-intensity exercise in normoxia (p = 0.024) and hypoxia (p = 0.007). The trained leg had higher abundances of Na+/H+ exchanger 1 (p = 0.047) and Na+/K+ pump subunit α (p = 0.036). Moderate- to high-intensity endurance training increases lactate-independent H+ release and reduces K+ release during high-intensity exercise, coinciding with increased Na+/H+ exchanger 1 and Na+/K+ pump subunit α muscle abundances.

DIPG-08. PROBING THE BLOOD-BRAIN-TUMOR-BARRIER WITH A MICROVASCULAR MODEL OF DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Pediatric diffuse midline glioma (DMG) is a uniformly fatal brain tumor in children, with most patients surviving less than 12 months. Current treatment for DMG is limited, and outcomes for this tumor have not improved. The blood-brain-barrier (BBB) remains a key challenge for DMG therapeutics, preventing most drugs from entering brain tissue. Functional characterization of the blood-brain-tumor-barrier (BBTB) – the interaction between tumors and BBB vasculature – remains largely unexplored for DMG. There is a need for a preclinical model of DMG that can probe the functional, physiological, and biological properties of the BBTB in vitro as a platform to evaluate therapeutic efficacy that is representative of human physiology. METHODS To generate an in vitro model of DMG with human microvasculature (DMG-BBTB model), we co-cultured DMG neurospheres, human brain microvascular endothelial cells, human brain vascular pericytes, and human astrocytes. Cells are simultaneously seeded into microfluidic devices at varied concentrations within a fibrin matrix. Self-assembled microvascular networks are formed within 7-8 days and used for downstream functional and phenotypic assays. RESULTS Vascular morphology and solute permeability in microvascular networks are influenced by cell identity, growth pattern, and relative concentration. We show that DMG neurospheres exhibit different patterns of growth when co-cultured as uniform spheroids versus single cells in suspension. Devices representing each growth pattern are further characterized for paracellular diffusion and permeability compared to devices without DMG cells through perfusion of high molecular weight dextran. Permeability values are correlated to biologic features such as the abundance and localization of junctional proteins and transporters. CONCLUSIONS We developed a platform to evaluate the microvasculature of the DMG BBTB as a tool to develop new therapies informed by vascular biology. We probed the functional, physiological, and biological properties of the human DMG BBTB in vitro, and in ongoing work are assessing approved and experimental therapeutics.

Colletotrichum falcatum infection influences the abundance of sucrose transporters and disease resistant proteins in sugarcane stalk

Colletotrichum falcatum infection influences the abundance of sucrose transporters and disease resistant proteins in sugarcane stalk

Bile acid profiles and messenger RNA expression of bile acid-related genes in the liver of dairy cows with high versus normal body condition

Bile acids (BA) play a crucial role not only in lipid digestion but also in the regulation of overall energy homeostasis, including glucose and lipid metabolism. The aim of this study was to investigate BA profiles and mRNA expression of BA-related genes in the liver of high versus normal body condition in dairy cows. We hypothesized that body condition and the transition from gestation to lactation affect hepatic BA concentrations as well as the mRNA abundance of BA-related receptors, regulatory enzymes, and transporters. Therefore, we analyzed BA in the liver as well as the mRNA abundance of BA-related synthesizing enzymes, transporters, and receptors in the liver during the transition period in cows with different body conditions around calving. In a previously established animal model, 38 German Holstein cows were divided into groups with high body condition score (BCS) (HBCS; n = 19) or normal BCS (NBCS; n = 19) based on BCS and backfat thickness (BFT). Cows were fed diets aimed at achieving the targeted differences in BCS and BFT (NBCS: BCS <3.5, BFT <1.2 cm; HBCS: BCS >3.75, BFT >1.4 cm) until they were dried off at wk 7 before parturition. Both groups were fed identical diets during the dry period and subsequent lactation. Liver biopsies were taken at wk -7, 1, 3, and 12 relative to parturition. For BA measurement, a targeted metabolomics approach with LC-ESI-MS/MS was used to analyze BA in the liver. The mRNA abundance of targeted genes related to BA-synthesizing enzymes, transporters, and receptors in the liver was analyzed using microfluidic quantitative PCR. In total, we could detect 14 BA in the liver: 6 primary and 8 secondary BA, with glycocholic acid (GCA) being the most abundant one. The increase of glycine-conjugated BA after parturition, in parallel to increasing serum glycine concentrations may originate from an enhanced mobilization of muscle protein to meet the high nutritional requirements in early lactating cows. Higher DMI in NBCS cows compared with HBCS cows was associated with higher liver BA concentrations such as GCA, deoxycholic acid (DCA), and cholic acid (CA). The mRNA abundance of BA-related enzymes measured herein suggests the dominance of the alternative signaling pathway in the liver of HBCS cows. Overall, BA profiles and BA metabolism in the liver depend on both, the body condition and lactation-induced effects in periparturient dairy cows.

Bacterioplankton taxa compete for iron along the early spring-summer transition in the Arctic Ocean.

Microbial assemblages under the sea ice of the Dease Strait, Canadian Arctic, were sequenced for metagenomes of a small size fraction (0.2-3 μm). The community from early March was typical for this season, with Alpha- and Gammaproteobacteria as the dominant taxa, followed by Thaumarchaeota and Bacteroidetes. Toward summer, Bacteroidetes, and particularly the genus Polaribacter, became increasingly dominant, followed by the Gammaproteobacteria. Analysis of genes responsible for microbial acquisition of iron showed an abundance of ABC transporters for divalent cations and ferrous iron. The most abundant transporters, however, were the outer membrane TonB-dependent transporters of iron-siderophore complexes. The abundance of iron acquisition genes suggested this element was essential for the microbial assemblage. Interestingly, Gammaproteobacteria were responsible for most of the siderophore synthesis genes. On the contrary, Bacteroidetes did not synthesize siderophores but accounted for most of the transporters, suggesting a role as cheaters in the competition for siderophores as public goods. This cheating ability of the Bacteroidetes may have contributed to their dominance in the summer.

Applicability of MDR1 Overexpressing Abcb1KO-MDCKII Cell Lines for Investigating In Vitro Species Differences and Brain Penetration Prediction.

Implementing the 3R initiative to reduce animal experiments in brain penetration prediction for CNS-targeting drugs requires more predictive in vitro and in silico models. However, animal studies are still indispensable to obtaining brain concentration and determining the prediction performance of in vitro models. To reveal species differences and provide reliable data for IVIVE, in vitro models are required. Systems overexpressing MDR1 and BCRP are widely used to predict BBB penetration, highlighting the impact of the in vitro system on predictive performance. In this study, endogenous Abcb1 knock-out MDCKII cells overexpressing MDR1 of human, mouse, rat or cynomolgus monkey origin were used. Good correlations between ERs of 83 drugs determined in each cell line suggest limited species specificities. All cell lines differentiated CNS-penetrating compounds based on ERs with high efficiency and sensitivity. The correlation between in vivo and predicted Kp,uu,brain was the highest using total ER of human MDR1 and BCRP and optimized scaling factors. MDR1 interactors were tested on all MDR1 orthologs using digoxin and quinidine as substrates. We found several examples of inhibition dependent on either substrate or transporter abundance. In summary, this assay system has the potential for early-stage brain penetration screening. IC50 comparison between orthologs is complex; correlation with transporter abundance data is not necessarily proportional and requires the understanding of modes of transporter inhibition.

Sex and the Kidney Drug-Metabolizing Enzymes and Transporters: Are Preclinical Drug Disposition Data Translatable to Humans?

Cross-species differences in drug transport and metabolism are linked to poor translation of preclinical pharmacokinetic and toxicology data to humans, often resulting in the failure of new chemical entities (NCEs) during clinical drug development. Specifically, inaccurate prediction of renal clearance and renal accumulation of NCEs due to differential abundance of enzymes and transporters in kidneys can lead to differences in pharmacokinetics and toxicity between experimental animals and humans. We carried out liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protein quantification of 78 membrane drug-metabolizing enzymes and transporters (DMETs) in the kidney membrane fractions of humans, rats, and mice for characterization of cross-species and sex-dependent differences. In general, majority of DMET proteins were higher in rodents than in humans. Significant cross-species differences were observed in 30 out of 33membrane DMET proteins quantified in all three species. Although no significant sex-dependent differences were observed in humans, the abundance of 28 and 46 membrane proteins showed significant sex dependence in rats and mice, respectively. These cross-species and sex-dependent quantitative abundance data are valuable for gaining a mechanistic understanding of drug renal disposition and accumulation. Further, these data can also be integrated into systems pharmacology tools, such as physiologically based pharmacokinetic models, to enhance the interpretation of preclinical pharmacokinetic and toxicological data.

64 Effect of feeding intact protein from soybean meal instead of crystalline amino acids on growth performance, protein synthesis, and immune response of growing pigs

Abstract The objective was to test the hypothesis that feeding intact protein from soybean meal (SBM) instead of crystalline amino acids (AA) does not affect growth performance, protein synthesis, intestinal morphology, or immune response of growing pigs. A control corn-SBM diet was used. Three additional diets were formulated by reducing the inclusion rate of SBM and adding 3, 4, or 5 crystalline AA (i.e., Lys, Met, Thr, Trp, Val) to the diets, which resulted in reducing the protein concentration from 20.0% to 13.4%. All diets were formulated to meet requirements for standardized ileal digestible indispensable AA. A total of 176 pigs [initial body weight (BW) = 32.2 kg; SD = 4.2] were used. On d 1, 16 randomly chosen pigs were euthanized and body nutrient composition was determined. The remaining 160 pigs were allotted to the 4 diets using a randomized complete block design with 4 pigs per pen and 10 replicate pens per diet. Starting BW was the blocking factor. Individual pig BW and feed consumption were recorded. On d 28, one pig per pen was slaughtered and blood, carcass, and viscera were collected and analyzed for N, fat, and energy to calculate deposition in the body. Ileal mucosa, ileum and colon tissue and digesta were analyzed for blood characteristics, cytokines, tissue morphology, ammonia, and gene expression. Contrasts coefficients were used to determine linear and quadratic effects of reducing dietary protein. Results indicated that average daily gain, average daily feed intake, gain to feed ratio, carcass characteristics, and protein, lipid, and energy depositions were not affected by reducing SBM and increasing crystalline AA in diets (Table 1). However, energy efficiency tended to decrease (quadratic, P = 0.083) by reducing dietary protein. Blood urea N was reduced (linear, P &amp;lt; 0.001) as dietary protein was reduced, but blood total protein was not affected by dietary treatment. Cytokines, ileal and jejunal morphologies, and gene abundance of AA transporters in the ileal mucosa were not affected by dietary protein. Ammonia concentrations in ileal digesta increased and then decreased (quadratic, P = 0.043) and colon digesta tended to increase and then decrease (quadratic, P = 0.074) as dietary protein was reduced. Bacteria protein in colon digesta was reduced (linear, P = 0.030) by reducing dietary protein. In conclusion, providing AA from intact protein from SBM or from crystalline AA did not affect growth performance, intestinal morphology, and immune response of pigs. However, SBM in diets can provide energy that may be utilized more efficiently than energy from diets with less SBM and crystalline AA. Further research is suggested to elucidate if SBM improves utilization of other nutrients in diets for pigs.