Retention In Mice Research Articles

Standard diet and animal source influence hippocampal spatial reference learning and memory in congenic C57BL/6J mice.

Assessing learning and memory has become critical to evaluate brain function in health, aging or neurological disease. The hippocampus is crucially involved in these processes as illustrated by H.M.'s remarkable case and by the well-established early symptoms of Alzheimer's disease. Numerous studies have reported the impact of gut microbiota on hippocampal structure and function using pro-, pre- and antibiotics, diet manipulations, germ-free conditions or fecal transfer. However, most diet manipulations have relied on Western diet paradigms (high fat, high energy, high carbohydrates). Here, we compared the impact of two standard diets, 5K52 and 2918 (6% fat, 18% protein, 3.1kcal/g), and how they influenced hippocampal learning and memory in adult 6-month-old congenic C57BL/6J mice from two sources. Using a hippocampal-dependent task, we found that 5K52-fed mice performed consistently better than 2918-fed animals in the Barnes circular maze. These behavioral differences were accompanied with marked changes in microbiota, which correlated with spatial memory retention performance. We next tested whether 2918-induced alterations in behavior and microbiome could be rescued by 5K52 diet for 3 months. Changing the 2918 diet to 5K52 diet mid-life improved spatial learning and memory in mice. Shotgun sequencing and principal component analyses revealed significant differences at both phylum and species levels. Multivariate analyses identified Akkermansia muciniphila or Bacteroidales bacterium M11 and Faecalibaculum rodentium as the strongest correlates to spatial memory retention in mice depending on the animal source. In both settings, the observed behavioral differences only affected hippocampal-dependent performance as mice fed with either diet did similarly well on the non-spatial variant of the Y-maze. In summary, these findings demonstrate the diverging effects of seemingly equivalent standard diets on hippocampal memory. Based on these results, we strongly recommend the mandatory inclusion of the diet and source of animals used in rodent behavioral studies.

Half-life extension of single-domain antibody-drug conjugates by albumin binding moiety enhances antitumor efficacy.

Single-domain antibody-drug conjugates (sdADCs) have been proven to have deeper solid tumor penetration and intratumor accumulation capabilities due to their smaller size compared with traditional IgG format ADCs. However, one of the key challenges for improving clinical outcomes of sdADCs is their abbreviated in vivo half-life. In this study, we innovatively fused an antihuman serum albumin (αHSA) nanobody to a sdADCs targeting oncofetal antigen 5T4, conferring serum albumin binding to enhance the pharmacokinetic profiles of sdADCs. The fusion protein was conjugated with monomethyl auristatin E (MMAE) at s224c site mutation. The conjugate exhibited potent cytotoxicity against various tumor cells. Compared with the nonalbumin-binding counterparts, the conjugate exhibited a 10-fold extended half-life in wild-type mice and fivefold prolonged serum half-life in BxPC-3 xenograft tumor models as well as enhanced tumor accumulation and retention in mice. Consequently, n501-αHSA-MMAE showed potent antitumor effects, which were comparable to n501-MMAE in pancreatic cancer BxPC-3 xenograft tumor models; however, in human ovarian teratoma PA-1 xenograft tumor models, n501-αHSA-MMAE significantly improved antitumor efficacy. Moreover, the conjugate showed mitigated hepatotoxicity. In summary, our results suggested that fusion to albumin-binding moiety as a viable strategy can enhance the therapeutic potential of sdADCs through optimized pharmacokinetics.

Transplantation of Gelatin Microspheres Loaded with Wharton's Jelly Derived Mesenchymal Stem Cells Facilitates Cartilage Repair in Mice.

Knee osteoarthritis (KOA) is a prevalent chronic joint disease caused by various factors. Mesenchymal stem cells (MSCs) therapy is an increasingly promising therapeutic option for osteoarthritis. However, the chronic inflammation of knee joint can severely impede the therapeutic effects of transplanted cells. Gelatin microspheres (GMs) are degradable biomaterial that have various porosities for cell adhesion and cell-cell interaction. Excellent elasticity and deformability of GMs make it an excellent injectable vehicle for cell delivery. We created Wharton's jelly derived mesenchymal stem cells (WJMSCs)-GMs complexes and assessed the effects of GMs on cell activity, proliferation and chondrogenesis. Then, WJMSCs loaded in GMs were transplanted in the joint of osteoarthritis mice. After four weeks, joint tissue was collected for histological analysis. Overexpressing-luciferase WJMSCs were performed to explore cell retention in mice. In vitro experiments demonstrated that WJMSCs loaded with GMs maintained cell viability and proliferative potential. Moreover, GMs enhanced the chondrogenesis differentiation of WJMSCs while alleviated cell hypertrophy. In KOA mice model, transplantation of WJMSCs-GMs complexes promoted cartilage regeneration and cartilage matrix formation, contributingto the treatment of KOA. Compared with other groups, in WJMSCs+GMs group, there were fewer cartilage defects and with a more integrated tibia structure. Tracking results of stable-overexpressing luciferase WJMSCs demonstrated that GMs significantly extended the retention time of WJMSCs in knee joint cavity. Our results indicated that GMs facilitate WJMSCs mediated knee osteoarthritis healing in mice by promoting cartilage regeneration and prolonging cell retention. It might potentially provide an optimal strategy for the biomaterial-stem cell based therapy for knee osteoarthritis.

Enhanced glypican-3-targeted identification of hepatocellular carcinoma with liver fibrosis by pre-degrading excess fibrotic collagen.

Most hepatocellular carcinomas (HCCs) occur in cirrhotic livers, but unequivocal diagnosis of early HCC from the fibrotic microenvironment remains a formidable challenge with conventional imaging strategies, mainly because of the massive fibrotic collagen deposition leading to hepatic nodules formation and dysfunction of contrast agent metabolism. Here, we developed a "sweep-and-illuminate" imaging strategy, pre-degrade hepatic fibrotic collagen with collagenase I conjugated human serum albumin (HSA-C) and then targeting visualize HCC lesion with GPC3 targeting nanoparticles (TSI NPs, TJ2 peptide-superparamagnetic iron oxide-indocyanine green) via fluorescence imaging (FLI) and magnetic particle imaging (MPI). TSI NPs delineated a clear boundary of HCC and normal liver, and the tumor-to-background ratios (TBRs) detected by FLI and MPI were 5.43- and 1.34-fold higher than the non-targeted group, respectively. HSA-C could degrade 24.7% fibrotic collagen, followed by 27.2% reduction of nonspecific NPs retention in mice with liver fibrosis. In a pathological state in which HCC occurs in the fibrotic microenvironment, HSA-C-mediated pre-degradation of fibrotic collagen reduced background signal interference in fibrotic tissues and enhanced the intratumoral uptake of TSI NPs, resulting in the clear demarcation between HCC and liver fibrosis, and the TBR was increased 2.61-fold compared to the group without HSA-C pretreatment. We demonstrated the feasibility of combined pre-degradation of fibrotic collagen and application of a GPC3-targeted FLI/MPI contrast agent for early HCC identification, as well as its clinical value in the management of patients with advanced liver fibrosis. STATEMENT OF SIGNIFICANCE: Given that liver fibrosis hinders early detection and treatment options of hepatocellular carcinomas (HCCs), we report a "sweep-and-illuminate" imaging strategy to enhance the efficiency of HCC identification by modulating the irreversible liver fibrosis. We first "sweep" nonspecific interference of contrast agent by pre-degrading fibrotic collagen with human serum albumin-carried collagenase I (HSA-C); and then specifically "illuminate" HCC lesions with GPC3-targeted-SPIO-ICG nanoparticles (TSI NPs). HSA-C can degrade 24.7% fibrotic collagen, followed by 27.2% reduction of nonspecific NPs retention in mice with liver fibrosis. Furthermore, in HCC models coexisting with liver fibrosis, the combined application of HSA-C and TSI NPs can clarify the demarcation between HCC and liver fibrosis with a 2.61-fold increase in the tumor-to-background ratio. This study may expand the potential of combinatorial biomaterials for early HCC diagnosis.

Labetalol and soluble endoglin aggravate bile acid retention in mice with ethinylestradiol-induced cholestasis.

Labetalol is used for the therapy of hypertension in preeclampsia. Preeclampsia is characterized by high soluble endoglin (sEng) concentration in plasma and coincides with intrahepatic cholestasis during pregnancy (ICP), which threatens the fetus with the toxicity of cumulating bile acids (BA). Therefore, we hypothesized that both labetalol and increased sEng levels worsen BA cumulation in estrogen-induced cholestasis. C57BL/6J, transgenic mice overexpressing human sEng, and their wild-type littermates were administrated with ethinylestradiol (EE, 10mg/kg s.c., the mice model of ICP) and labetalol (10mg/kg s.c.) for 5days with sample collection and analysis. Plasma was also taken from healthy pregnant women and patients with ICP. Administration of labetalol to mice with EE cholestasis aggravated the increase in BA plasma concentrations by induction of hepatic Mrp4 efflux transporter. Labetalol potentiated the increment of sEng plasma levels induced by estrogen. Increased plasma levels of sEng were also observed in patients with ICP. Moreover, increased plasma levels of human sEng in transgenic mice aggravated estrogen-induced cholestasis in labetalol-treated mice and increased BA concentration in plasma via enhanced reabsorption of BAs in the ileum due to the upregulation of the Asbt transporter. In conclusion, we demonstrated that labetalol increases plasma concentrations of BAs in estrogen-induced cholestasis, and sEng aggravates this retention. Importantly, increased sEng levels in experimental and clinical forms of ICPs might present a novel mechanism explaining the coincidence of ICP with preeclampsia. Our data encourage BA monitoring in the plasma of pregnant women with preeclampsia and labetalol therapy.

Intranasal calcitonin gene-related peptide administration impairs fear memory retention in mice through the PKD/p-HDAC5/Npas4 pathway

The calcitonin gene-related peptide (CGRP) suppresses fear memory retention in mice. Although intracerebroventricular administration of CGRP alters the fear memory processes, making it a promising therapeutic strategy for post-traumatic stress disorder (PTSD), direct brain injection into patients is not practical. Therefore, we propose that intranasal application may be an effective way to deliver CGRP to the brain. This study tested whether CGRP nasal administration exerts the same effect as intracerebroventricular administration using C57BL6J mice. The amount of CGRP in the cerebrospinal fluid and hippocampus 30 min after nasal administration of CGRP was significantly higher when compared with saline. Intranasal CGRP also elicited photophobic behaviors similar to intracerebroventricular injection. Moreover, intranasal CGRP decreased fear memory retention but did not affect reactivation and extinction of fear memory. We found intranasal CGRP significantly increased the expression of protein kinase D (PKD), phosphorylated histone deacetylase 5 (p-HDAC5) and neuronal PAS domain protein 4 (Npas4) in the hippocampus. CGRP-mediated impairment of fear memory and Npas4 expression increases were attenuated significantly by the CGRP receptor antagonist BIBN4096. Together, our data demonstrate that intranasal CGRP delivery activates the PKD/p-HDAC5/Npas4 pathway, decreases fear memory retention.

Oral treatment of 4-methylumbelliferone reduced perineuronal nets and improved recognition memory in mice

Hyaluronan (HA) is a core constituent of perineuronal nets (PNNs) that surround subpopulations of neurones. The PNNs control synaptic stabilization in both the developing and adult central nervous system, and disruption of PNNs has shown to reactivate neuroplasticity. We investigated the possibility of memory prolongation by attenuating PNN formation using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis. Adult C57BL/6 mice were fed with chow containing 5% (w/w) 4-MU for 6 months, at a dose ~6.7 mg/g/day. The oral administration of 4-MU reduced the glycosaminoglycan level in the brain to 72% and the spinal cord to 50% when compared to the controls. Spontaneous object recognition test (SOR) performed at 2, 3, 6 and 7 months showed a significant increase in SOR score in the 6-months treatment group 24 h after object presentation. The effect however did not persist in the washout group (1-month post treatment). Immunohistochemistry confirmed a reduction of PNNs, with shorter and less arborization of aggrecan staining around dendrites in hippocampus after 6 months of 4-MU treatment. Histopathological examination revealed mild atrophy in articular cartilage but it did not affect the motor performance as demonstrated in rotarod test. In conclusion, systemic oral administration of 4-MU for 6 months reduced PNN formation around neurons and enhanced memory retention in mice. However, the memory enhancement was not sustained despite the reduction of PNNs, possibly due to the lack of memory enhancement training during the washout period. Our results suggest that 4-MU treatment might offer a strategy for PNN modulation in memory enhancement.

Large Impacts of Small Particles: The Effects of Active Particles on Colloidal Gels and Crystals and of Inulin Microparticles on Gut Retention in Mice

Large Impacts of Small Particles: The Effects of Active Particles on Colloidal Gels and Crystals and of Inulin Microparticles on Gut Retention in Mice

Health benefits of first and second extraction drum-dried pitted olive pomace.

Olive pomace (OP) is the main by-product of olive oil extraction. After pit and skin removal, OP pulp has high concentrations of dietary fiber and phenolics with high antioxidant capacity. This study evaluated mice health benefits of drum-dried pitted OP pulp obtained after first and second oil extraction. Fresh OP was steam blanched, then pits and skins separated in a pulper/finisher, and pulp drum-dried and milled. OP was characterized by proximate analysis, total soluble phenolics (TSP), individual phenolics, and dietary fiber. Drum-dried pitted OP from first and second extraction was formulated at 10% and 20% in a high fat mice diet. Low fat (5%) and high fat (18%) control diets were also used for comparison. First extraction OP had higher TSP than OP from second extraction. Hydroxytyrosol was the main phenolic in OP. Mice weight gain was lower for the four OP diets compared to high and low-fat control diets. Fecal protein was high for all OP diets, indicating poor protein retention in mice, possibly by phenolics binding of protein and enzymes. Liver weight and adipose tissue were lower in mice consuming the four high fat OP diets compared to high fat control diet. Also, there was no effect on blood glucose by OP in diets. Mice gut microbiota analysis indicated that Actinobacteria decreased in the OP diets compared to the two control diets while Bacteroidetes increased, indicating a positive correlation with reduced body fat and weight. Drum-dried pitted OP is a novel agricultural by-product with its bioactive compounds having the potential to be incorporated in feeds and foods providing health benefits. PRACTICAL APPLICATION: Drum-dried pitted olive pomace can be produced from first or second olive oil extraction byproducts to be used as a shelf-stable healthy food or feed supplement.

A Sweet Dream with SGLT2 Inhibitors

Glucose is an osmotic agent in the peritoneal dialysis fluid (PDF) to absorb water, sodium and other toxins. Glucose absorption from PDF to blood impairs peritoneal dialysis (PD) efficiency and cause glycemic burden. This extra glycemic load leads to poor glycemic control in diabetic dialysis patients with PD program. Studies have shown sodium-glucose co-transporter (SGLT)-2 expression in peritoneal mesothelial cells. Blockade of peritoneal SGLT reduced blood glucose increment and increased peritoneal glucose retention in mice. In rat models, it was shown that high glucose PDF promotes the peritoneal fibrosis by upregulating the expression of glucose transporter (GLUT) and SGLT, and addition of phlorizin to PDF leads to downregulation of GLUT and SGLT expression and reduced peritoneal degeneration. According to these information, we want to share our experience of using empagliflozin in a poor glycemic controlled type 2 diabetic patient, who was on PD program for end-stage renal disease. We hypothesized that if we administer empagliflozin to our patient we can reduce glycemic burden caused by PDF and increase the ultrafiltration volume.

3: Vitamin D Improves Autologous Fat Graft Retention

Purpose:Autologous fat grafting is a widely used technique in aesthetic and reconstructive surgery, however, unpredictable volume reabsorption may lead to unsatisfactory outcomes. Previously, we demonstrated that a fat-soluble Vitamin D3 analogue, calcitriol, significantly improved fat retention in a xenograft mouse model by 25% across multiple donors when injected systemically (p < 0.05). While calcitriol has minimal toxicity, is FDA approved, and has positive immunomodulatory and antioxidant properties, systemic administration bypasses key Vitamin D synthesis regulatory steps, thus increasing risk with high-dose use. We hypothesize that systemic supplementation with Vitamin D3 (cholecalciferol) will likewise improve fat-graft retention similar to calcitriol while avoiding potential regulatory and iatrogenic risk. In this study we compared in vivo human fat graft retention in mice treated with systemic cholecalciferol, calcitriol or vehicle control in a mouse xenograft model. In vitro adipose lipoaspirate culture was used to interrogate the therapeutic mechanism of action.Methods:Lipoaspirate was harvested from 6 unique donors using a 2mm cannula and used in parallel for both in vitro and in vivo studies. In vivo: 0.3mL of lipoaspirate was injected bilaterally on dorsal flanks of homozygous Foxn1nu immunocompromised mice. Calcitriol (50ng), cholecalciferol (50ng, 500ng, 5000ng) or vehicle control was administered thrice weekly by IP injection. Graft volume retention was measured at 12 weeks. In vitro: 1mL of lipoaspirate was submerged in phenol-red free DMEM (10% FBS) containing calcitriol or cholecalciferol (15.6nM, 62.5 nM or 250 nM) for 7 days with one media change. Terminal analyses include tissue weight, stromal cell viability, concentration of active vitamin D metabolite (1,25(OH)2D3), and gene upregulation by qRT-PCR.Results:Previously, we demonstrated that systemic administration of 50ng calcitriol thrice weekly significantly improved human fat graft retention across multiple donors in a mouse xenograft model. Our current in vivo data suggest 5000ng cholecalciferol is similarly effective. In-vitro assays show 62.5nM and 250nM cholecalciferol significantly increased adipose stromal cell viability compared to controls (85.3+/-2.9% and 87.7+/-3.7 versus 77.6+/-2.8%, respectively p<0.05;). Analysis of final adipose 1,25(OH)2D3 concentration by ELISA showed both calcitriol and cholecalciferol treatments equally increased Vitamin D metabolite concentrations in all donors. qRT-PCR analysis of gene expression show that pro-survival autophagy is significantly increased by both cholecalciferol and calcitriol, though increased concentration of calcitriol was required to induce significant increases from controls.Conclusion:Cholecalciferol (Vitamin D3) is a highly promising therapeutic for improving fat grafting outcomes. Our in vitro data suggests that in the context of hypoxia, nutrient depletion, or growth factor deprivation, such as occurs immediately following fat grafting, vitamin D3 promotes stromal cell autophagy. In this context, autophagy is crucial for maintaining cellular ATP production and macromolecular synthesis and, therefore, represents an essential pro-survival pathway which allows grafted cells to survive. The results herein provide evidence to incorporate vitamin D3 as a safe, cost-effective nutritional supplement into the perioperative workflow to improve fat viability after grafting.

Frequent occurrence of triclosan hydroxylation in mammals: A combined theoretical and experimental investigation

Triclosan (TCS) is a widespread antimicrobial agent with many adverse health risks. Its hepatoxicity invariably points to the activation of constitutive androstane receptor (CAR), which regulates cytochrome P450 (CYP) genes that are critical for oxidative metabolism. Here, we provide the theoretical and experimental evidences showing that metabolic activation of TCS frequently occurs through aromatic hydroxylation in mammals. CYP-mediated oxidation was predicted to take place at each aromatic C‒H bond. Molecular docking and in vitro approaches reveal oxidative reaction could be efficiently catalyzed by CAR-regulated CYP2B6 enzyme. Parallel reaction monitoring (PRM) high-resolution mass spectrometry was utilized to identify and profile TCS oxidative metabolites in paired mouse liver, bile, feces, plasma and urine. We found multiple hydroxylated isomers including the products generated via the NIH shift of chlorine, as well as their subsequent conjugates. These metabolites showed isomer-specific retention in mice. Glucuronide conjugates are more readily excreted than the sulfates. Moreover, for the first time, isomeric hydroxylated metabolites were detected in the urine and stool of human subjects used TCS-contained household and personal care products. Collectively, these findings suggest that hydroxylation is an important, yet often underestimated element that worth considering to fully evaluate the biological fates and health risks of TCS.

Effects of empagliflozin and L-ornithine L-aspartate on behavior, cognitive functions, and physical performance in mice with experimentally induced steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is a chronic condition characterized by disturbed carbohydrate and lipid metabolism and often complicated by psychoneurological symptoms, including anxiety, depression, memory deficit, and asthenia. Most studies of pharmacotherapy candidates for NAFLD focus on the ability of the tested drugs to restore the biochemical functions and morphology of the liver while their potential effects on the co-existing conditions remain overlooked. The aim of this paper was to investigate the effects of empagliflozin and L-ornithine L-aspartate (OA) on behavior, memory, and physical performance in C57BL/6 mice with experimentally induced NAFLD (6 months of a Western diet + weekly carbon tetrachloride injections). The disease affected animal behavior (locomotion speed decreased by 38% and 35%, p &lt; 0.01; rearing increased by 432% and 279%, p &lt; 0.05 etc.), induced long-term memory deficit (latency to find the target box increased by 108% in the Barnes maze, the number of errors increased by 439%, p &lt; 0.05), and compromised physical performance (swimming time in the forced swim test dropped by 50%, p &lt; 0.05 etc.). When administered during the high-calorie diet period, both drugs reduced anxiety (empagliflozin: the number of grooming bouts rose by 160%, p &lt; 0.05 and 2173%, p &lt; 0.01; time spent in the light compartment in the light/dark box test increased by 275%, p &lt; 0.05, etc.; OA: time spent in the open arms of the maze increased by 267%, p &lt; 0.05), and promoted memory retention in mice with NAFLD. OA improved physical performance (swimming time in the forced swimming test improved by 106%, p &lt; 0.05, etc.). Thus, empagliflozin and OA can have a beneficial effect on cognitive functions, as well as behavior, and ameliorate asthenia in NAFLD.

Co-exposure to internal and external radiation alters cesium biokinetics and retention in mice

Exposures in post-accidental situations are complex and include both external exposure and internal contamination with several radionuclides. However, in vivo and in vitro studies generally use simplified exposures, while a recent study suggested that combined external irradiation and internal contamination may induce more severe biological effects compared to single exposures. In an attempt to test the hypothesis of potential non-additive effects of multiple radiological exposures, we used a mouse model of combined external x-ray irradiation at 1 and 5 Gy and internal contamination with injection of 20 KBq 137Cs. The results showed differential kinetics of 137Cs elimination in irradiated animals compared to sham-irradiated, 137Cs injected animals. Moreover, changes in plasma potassium and in relative testis weight were observed 38 days after irradiation and injection in co-exposed animals compared to 137Cs injection alone. These results demonstrate that an external exposure combined with an internal contamination may lead to unexpected changes in biokinetics of radionuclides and biological effects compared to single exposures.

Nose-to-brain delivery of CGRP against fear memory retention in mice

Nose-to-brain delivery of CGRP against fear memory retention in mice

Taurine: A Rescuer of Neurodegenerative Dysfunction?

Alzheimer's disease (AD), is characterized by an accumulation of amyloid plaques that contribute to neurodegeneration. Taurine is a very essential amino acid in the nervous system, but is one of a few not used to synthesize proteins. It is found in fish, eggs, and organ meat; however, taurine is better known as a supplement in energy drinks with the purpose of depressing nervous system function. Taurine has been shown in previous studies to increase memory retention in mice. Our project aims to determine the impact of taurine on a mutant strain of C. elegans, ynIs‐79, which overexpresses the APL‐1 protein in the nervous system. We expect that supplementing the mutant nematodes' diet with taurine will positively influence the nervous system by rescuing the observed dysfunction. We tested anterior and posterior habituation following taurine exposure by tapping the worms with a sterilized eyelash until they recognized that the taps were a non‐threatening stimulus. Our hypothesis is that feeding nematodes taurine will result in a decrease in the number of taps required for the mutant nematodes to determine that it is not a harmful stimulus, suggesting an improvement in neurodegenerative dysfunction. Current results show a significant decrease in the number taps required for habituation in the ynIs‐79 strain when compared to controls.Support or Funding InformationGail Dillard Faculty Development FundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P384: Conditional knockout of Acid Ceramidase in Renal Collecting Ducts Induced Sodium Retention in Mice

We have previously shown that renal sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite formed by phosphorylation of sphingosine, increases sodium excretion through inhibition of epithelial sodium channel (ENaC) via S1P receptor 1, which is mainly localized in the collecting ducts. However, the role of S1P-producing enzyme in renal sodium excretion remains unknown. The immediate substrate for S1P generation, sphingosine, is formed from the hydrolysis of ceramide by ceramidase, which is a rate-limiting step for S1P production. Acid ceramidase (AC) is the major form of ceramidase in collecting duct. The present study was to test the hypothesis that deletion of AC in the collecting ducts impacts renal sodium handling. Mice with collecting duct-specific AC knockout (CD-AC-KO) were generated by crossing AC floxed mice with Aquoporin 2-Cre mice. The specific knockout of AC in renal collecting ducts was confirmed by immunohistochemistry and Western blot analyses. Functionally, urinary sodium excretion was decreased by 40% in CD-AC-KO mice compared with control mice (Ctrl) after acute IV sodium loading (2.19±0.32 vs. 1.3±0.18 μmole/min.g kwt in Ctrl vs. KO, P&lt;0.05). Furthermore, the natriuretic response to elevated renal perfusion pressure (pressure natriuresis) was severely impaired in CD-AD-KO mice compared with control mice (5.3±0.54 vs. 3.0±0.25 μmole/min.g kwt in Ctrl vs. KO, P&lt;0.05). Moreover, chronic high salt-induced sodium retention was remarkably enhanced in CD-AC-KO mice compared with control animals (2.9±0.21 vs. 5.0±0.30 mmole/100g bw. 24h in Ctrl vs. KO, P&lt;0.05). These results suggest that AC in renal collecting ducts plays an important role in the regulation of renal sodium excretion and that modulation of AC function in the kidneys could be a therapeutic approach for salt-sensitive hypertension.

Synthesis and Activity of a Novel Autotaxin Inhibitor–Icodextrin Conjugate

Autotaxin is an extracellular phospholipase D that catalyzes the hydrolysis of lysophosphatidyl choline (LPC) to generate the bioactive lipid lysophosphatidic acid (LPA). Autotaxin has been implicated in many pathological processes relevant to cancer. Intraperitoneal administration of an autotaxin inhibitor may benefit patients with ovarian cancer; however, low molecular mass compounds are known to be rapidly cleared from the peritoneal cavity. Icodextrin is a polymer that is already in clinical use because it is slowly eliminated from the peritoneal cavity. Herein we report conjugation of the autotaxin inhibitor HA155 to icodextrin. The conjugate inhibits autotaxin activity (IC50 = 0.86 ± 0.13 μg mL-1) and reduces cell migration. Conjugation of the inhibitor increased its solubility, decreased its membrane permeability, and improved its intraperitoneal retention in mice. These observations demonstrate the first application of icodextrin as a covalently-bonded drug delivery platform with potential use in the treatment of ovarian cancer.

UGT1A10 drives tissue‐specific differences in the clearance of the chiral tobacco carcinogen, NNAL

Tobacco‐specific nitrosamines (TSNAs) are among the most potent carcinogens found in tobacco, with 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) the most potent and one of the most abundant. NNK is extensively metabolized to the equally carcinogenic chiral 4‐ (methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL). (S)‐NNAL exhibits higher stereoselective tissue retention in mice and humans and has been shown to be more carcinogenic in mice than its enantiomeric counterpart, (R)‐NNAL. This differential carcinogenic potential makes it increasingly important to know which UGT enzymes target specific NNAL enantiomers for glucuronidation. NNAL is detoxified primarily by four hepatic UDP‐glucuronyltransferase (UGT) enzymes: UGTs 1A9, 2B7, 2B10 and 2B17; each of these UGTs have been shown to exhibit different stereo‐specificity for the formation of glucuronides of the different NNAL enantiomers. The goal of the present study was to identify the UGTs involved in extra‐hepatic NNAL metabolism and examine differences in the stereospecific glucuronidation of NNAL between tissue types with different UGT expression. Large differences in stereo‐specificity for (S)‐ versus (R)‐NNAL were identified upon comparison of NNAL‐glucuronides (Gluc) formed in reactions of extra‐hepatic UGT1A10‐over‐expressing cell microsomes upon incubation with racemic NNAL, with an enantiomeric preference for (R)‐NNAL as a substrate when compared to (S)‐NNAL. When UGT1A10‐over‐expressing cell microsomes were incubated with pure NNAL enantiomers, large difference in kinetics were observed, with a Km=1.5 mM and Vmax=4.6 pmol/min/mg for (R)‐NNAL, vs a Km&gt;16 mM and Vmax=1.3 pmol/min/mg for (S)‐NNAL. Human liver microsomes preferentially formed (S)‐NNAL‐Gluc while microsomes from human intestine, larynx, esophagus, tonsil, floor of mouth, and tongue all preferentially formed (R)‐NNAL‐Gluc upon incubation with racemic NNAL. Liver and intestine were shown to express UGTs 1A9, 2B7, and 2B17 while intestine, unlike liver, has also been shown to express UGT1A10. All other tissue types analyzed were shown to express only UGTs 1A10 and 2B17, with the exception of esophagus that also expressed UGT2B7. These data indicate that UGT1A10 may drive the difference in differential tissue‐specific clearance of the specific NNAL enantiomers known to induce tobacco‐related cancers.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optimization and scale up of microfluidic nanolipomer production method for preclinical and potential clinical trials

BackgroundThe process of optimization and fabrication of nanoparticle synthesis for preclinical studies can be challenging and time consuming. Traditional small scale laboratory synthesis techniques suffer from batch to batch variability. Additionally, the parameters used in the original formulation must be re-optimized due to differences in fabrication techniques for clinical production. Several low flow microfluidic synthesis processes have been reported in recent years for developing nanoparticles that are a hybrid between polymeric nanoparticles and liposomes. However, use of high flow microfluidic synthetic techniques has not been described for this type of nanoparticle system, which we will term as nanolipomer. In this manuscript, we describe the successful optimization and functional assessment of nanolipomers fabricated using a microfluidic synthesis method under high flow parameters.ResultsThe optimal total flow rate for synthesis of these nanolipomers was found to be 12 ml/min and flow rate ratio 1:1 (organic phase: aqueous phase). The PLGA polymer concentration of 10 mg/ml and a DSPE-PEG lipid concentration of 10% w/v provided optimal size, PDI and stability. Drug loading and encapsulation of a representative hydrophobic small molecule drug, curcumin, was optimized and found that high encapsulation efficiency of 58.8% and drug loading of 4.4% was achieved at 7.5% w/w initial concentration of curcumin/PLGA polymer. The final size and polydispersity index of the optimized nanolipomer was 102.11 nm and 0.126, respectively. Functional assessment of uptake of the nanolipomers in C4-2B prostate cancer cells showed uptake at 1 h and increased uptake at 24 h. The nanolipomer was more effective in the cell viability assay compared to free drug. Finally, assessment of in vivo retention in mice of these nanolipomers revealed retention for up to 2 h and were completely cleared at 24 h.ConclusionsIn this study, we have demonstrated that a nanolipomer formulation can be successfully synthesized and easily scaled up through a high flow microfluidic system with optimal characteristics. The process of developing nanolipomers using this methodology is significant as the same optimized parameters used for small batches could be translated into manufacturing large scale batches for clinical trials through parallel flow systems.