Elevation of hypothalamic ketone bodies induces a decrease in energy expenditures and an increase risk of metabolic disorder.

OBJECTIVE: Ketone bodies (such as ß-hydroxybutyrate or BHB) have been recently proposed as signals involved in brain regulation of energy homeostasis and obesity development. However, the precise role of ketone bodies sensing by the brain, and its impact on metabolic disorder development remains unclear. Nevertheless, partial deletion of the ubiquitous ketone bodies transporter MCT1 in mice (HE mice) results in diet-induced obesity resistance, while there is no alteration under normal chow diet. These results suggest that ketone bodies produced during the high fat diet would be important signals involved in obesity onset. METHODS: In the present study we used a specific BHB infusion of the hypothalamus and analyzed the energy homeostasis of WT or HE mice fed a normal chow diet. RESULTS: Our results indicate that high BHB levels sensed by the hypothalamus disrupt the brain regulation of energy homeostasis. This brain control dysregulation leads to peripheral alterations of energy expenditure mechanisms. CONCLUSIONS: Altogether, the changes induced by high ketone bodies levels sensed by the brain increase the risk of obesity onset in mice.

S-Leaping: An Adaptive, Accelerated Stochastic Simulation Algorithm, Bridging [Formula: see text]-Leaping and R-Leaping.

We propose the S-leaping algorithm for the acceleration of Gillespie's stochastic simulation algorithm that combines the advantages of the two main accelerated methods; the [Formula: see text]-leaping and R-leaping algorithms. These algorithms are known to be efficient under different conditions; the [Formula: see text]-leaping is efficient for non-stiff systems or systems with partial equilibrium, while the R-leaping performs better in stiff system thanks to an efficient sampling procedure. However, even a small change in a system's set up can critically affect the nature of the simulated system and thus reduce the efficiency of an accelerated algorithm. The proposed algorithm combines the efficient time step selection from the [Formula: see text]-leaping with the effective sampling procedure from the R-leaping algorithm. The S-leaping is shown to maintain its efficiency under different conditions and in the case of large and stiff systems or systems with fast dynamics, the S-leaping outperforms both methods. We demonstrate the performance and the accuracy of the S-leaping in comparison with the [Formula: see text]-leaping and R-leaping on a number of benchmark systems involving biological reaction networks.

Polymorphic variation in the SLC5A7 gene influences infant autonomic reactivity and self-regulation: A neurobiological model for ANS stress responsivity and infant temperament.

OBJECTIVE: To examine the impact of polymorphic variation in the solute carrier family 5 member 7 (SLC5A7) gene on autonomic nervous system (ANS) reactivity indexed by respiratory sinus arrhythmia (RSA) and heart rate (HR) in infants during a dyadic stressor, as well as maternal report of infant self-regulation. Given evidence of race differences in older individuals, race was specifically examined. METHODS: RSA and HR were collected from 111 infants during the still-face paradigm (SFP). Mothers completed the Infant Behavior Questionnaire-Revised short-form. Multi-level mixed effects models examined the impact of SLC5A7 genotype on RSA and HR across the SFP. Linear models tested the influence of genotype on the relation between RSA, HR, and maternal report of infant self-regulation. RESULTS: SLC5A7 genotype significantly predicted RSA stress responsivity (ß = -0.023; p = 0.028) and HR stress responsivity (ß = 0.004; p = 0.002). T-allele carriers exhibited RSA suppression and HR acceleration in response to stress while G/G homozygotes did not suppress RSA and exhibited less HR acceleration. All infants exhibited modest RSA augmentation and HR deceleration during recovery. Race-stratified analyses revealed that White T-allele carriers drove the overall results for both RSA (ß = -0.044; p = 0.007) and HR (ß = 0.006; p = 0.008) with no relation between SLC5A7 genotype and RSA or HR in Black infants. Maternal report of infant orienting/regulation was predicted by the interaction of SLC5A7 genotype and both RSA recovery (ß = 0.359; p = 0.001) and HR recovery (ß = -1.659; p = 0.020). RSA augmentation and HR deceleration during recovery were associated with higher maternal reports of self-regulation among T-allele carriers, a finding again primarily driven by White infants. CONCLUSIONS: Early in development, genetic contributions to ANS are evident and predict maternal report of infant self-regulation within White infants, consistent with prior literature. The lack of associations in Black infants suggest that race differences in physiological reactivity and self-regulation are emerging during the first year of life potentially providing early evidence of disparities in health risk trajectories.

Assessment of an engineered endothelium via single-photon emission computed tomography.

The clinical translation of cell-based therapeutics often requires highly sensitive, non-invasive imaging tools to assess cell function and distribution in vivo. The objective of this research was to determine whether human Sodium-Iodide Symporter (hNIS) ectopic expression in endothelial cells (ECs) in combination with single-photon emission computed tomography (SPECT) is a feasible approach to non-invasively monitor the presence and viability of an engineered endothelium on expanded polytetrafluoroethylene (ePTFE). Human umbilical vein endothelial cells (HUVECs) were transduced with pLL3.7-hNIS via lentivirus with multiplicity of infection (MOI) of 0, 2, 5, and 10 (n = 4). Ectopic expression of hNIS in HUVECs via optimized lentiviral transduction (MOI 5) enabled cell uptake of a radioisotope that can be detected by SPECT without affecting endothelial cell viability, oxidative stress, or antithrombogenic functions. The viability and distribution of an engineered endothelium grown on ePTFE coated with the biodegradable elastomer poly(1, 8 octamethylene citrate) (POC) and exposed to fluid flow was successfully monitored non-invasively by SPECT. We report the feasibility of a non-invasive, highly sensitive and functional assessment of an engineered endothelium on ePTFE using a combination of SPECT and X-ray computed tomography (SPECT/CT) imaging and hNIS ectopic expression in ECs. This technology potentially allows for the non-invasive assessment of transplanted living cells in vascular conduits. Biotechnol. Bioeng. 2017;114: 2371-2377. © 2017 Wiley Periodicals, Inc.

Comprehensive assessment showed no associations of variants at the SLC10A1 locus with susceptibility to persistent HBV infection among Southern Chinese.

The sodium taurocholate cotransporting polypeptide (NTCP) encoded by SLC10A1 was recently demonstrated to be a functional receptor for hepatitis B virus (HBV). The role of SLC10A1 polymorphisms, particularly the Ser267Phe variant (rs2296651) in exon 4, has been frequently investigated in regard to risk of persistent HBV infection. However, these investigations have generated conflicting results. To examine whether common genetic variation at the SLC10A1 locus is associated with risk of persistent HBV infection, haplotype-tagging and imputed single nucleotide polymorphisms (SNPs) were assessed in two case-control sample sets, totally including 2,550 cases (persistently HBV infected subjects, PIs) and 2,124 controls (spontaneously recovered subjects, SRs) of Southern Chinese ancestry. To test whether rare or subpolymorphic SLC10A1 variants are associated with disease risk, the gene's exons in 244 cases were sequenced. Overall, we found neither SNPs nor haplotypes of SLC10A1 showed significant association in the two sample sets. Furthermore, no significant associations of rare variants or copy number variation covering SLC10A1 were observed. Finally, expression quantitative trait locus analyses revealed that SNPs potentially affecting SLC10A1 expression also showed no significant associations. We conclude that genetic variation at the SLC10A1 locus is not likely a major risk factor of persistent HBV infection among Southern Chinese.

Longitudinal Assessment of Lung Cancer Progression in Mice Using the Sodium Iodide Symporter Reporter Gene and SPECT/CT Imaging.

Lung cancer has the highest mortality rate of any tissue-specific cancer in both men and women. Research continues to investigate novel drugs and therapies to mitigate poor treatment efficacy, but the lack of a good descriptive lung cancer animal model for preclinical drug evaluation remains an obstacle. Here we describe the development of an orthotopic lung cancer animal model which utilizes the human sodium iodide symporter gene (hNIS; SLC5A5) as an imaging reporter gene for the purpose of non-invasive, longitudinal tumor quantification. hNIS is a glycoprotein that naturally transports iodide (I-) into thyroid cells and has the ability to symport the radiotracer 99mTc-pertechnetate (99mTcO4-). A549 lung adenocarcinoma cells were genetically modified with plasmid or lentiviral vectors to express hNIS. Modified cells were implanted into athymic nude mice to develop two tumor models: a subcutaneous and an orthotopic xenograft tumor model. Tumor progression was longitudinally imaged using SPECT/CT and quantified by SPECT voxel analysis. hNIS expression in lung tumors was analyzed by quantitative real-time PCR. Additionally, hematoxylin and eosin staining and visual inspection of pulmonary tumors was performed. We observed that lentiviral transduction provided enhanced and stable hNIS expression in A549 cells. Furthermore, 99mTcO4- uptake and accumulation was observed within lung tumors allowing for imaging and quantification of tumor mass at two-time points. This study illustrates the development of an orthotopic lung cancer model that can be longitudinally imaged throughout the experimental timeline thus avoiding inter-animal variability and leading to a reduction in total animal numbers. Furthermore, our orthotopic lung cancer animal model is clinically relevant and the genetic modification of cells for SPECT/CT imaging can be translated to other tissue-specific tumor animal models.

Cardiac AAV9 Gene Delivery Strategies in Adult Canines: Assessment by Long-term Serial SPECT Imaging of Sodium Iodide Symporter Expression.

Heart failure is a leading cause of morbidity and mortality, and cardiac gene delivery has the potential to provide novel therapeutic approaches. Adeno-associated virus serotype 9 (AAV9) transduces the rodent heart efficiently, but cardiotropism, immune tolerance, and optimal delivery strategies in large animals are unclear. In this study, an AAV9 vector encoding canine sodium iodide symporter (NIS) was administered to adult immunocompetent dogs via epicardial injection, coronary infusion without and with cardiac recirculation, or endocardial injection via a novel catheter with curved needle and both end- and side-holes. As NIS mediates cellular uptake of clinical radioisotopes, expression was tracked by single-photon emission computerized tomography (SPECT) imaging in addition to Western blot and immunohistochemistry. Direct epicardial or endocardial injection resulted in strong cardiac expression, whereas expression after intracoronary infusion or cardiac recirculation was undetectable. A threshold myocardial injection dose that provides robust nonimmunogenic expression was identified. The extent of transmural myocardial expression was greater with the novel catheter versus straight end-hole needle delivery. Furthermore, the authors demonstrate that cardiac NIS reporter gene expression and duration can be quantified using serial noninvasive SPECT imaging up to 1 year after vector administration. These data are relevant to efforts to develop cardiac gene delivery as heart failure therapy.

(124)I-PET Assessment of Human Sodium Iodide Symporter Reporter Gene Activity for Highly Sensitive In Vivo Monitoring of Teratoma Formation in Mice.

PURPOSE: Pluripotent stem cell (PSC)-based therapies possess great potential to restore the function of irreversibly damaged organs. PSCs can be differentiated in vitro into any cell type. However, pluripotent potential bears the risk of teratoma formation. In vivo monitoring of teratoma formation is indispensable, as 100 % purity of the cell preparation cannot be achieved. We aimed at establishing the human sodium iodide symporter (hNIS) as reporter gene for PET monitoring of teratoma formation. PROCEDURES: Murine PSC stably expressing hNIS were injected into the hind limbs of SCID mice to induce teratoma formation. Positron emission tomography (PET) scans were acquired weekly between days 14 and 42 after transplantation. Two teratomas were excised at each time point for histology and size measurement. Tracer uptake was correlated with teratoma weight. Specificity of tumoural iodine uptake was assessed by blocking hNIS in vivo with perchlorate. RESULTS: Neither hNIS expression nor I-124 exposure adversely impacted viability or differentiation potential of PSCs. Iodine uptake was highly specific in teratomas, as in vivo blocking of hNIS with perchlorate led to uptake rates comparable to tracer uptake in non-transgene tumours. Tumour mass and tracer uptake showed a positive correlation. CONCLUSIONS: This is the first study to generate stably hNIS-expressing murine PSCs. Since the differentiation potential was preserved, hNIS-expressing cells are suitable for PSC-based forward programming approaches. Teratoma formation from undifferentiated cells can be monitored in vivo by PET with high specificity on a quantitative level. Due to its anticipated lack of immunogenicity in humans, hNIS is a promising reporter gene for clinical translation.

Design and evaluation of a novel trifluorinated imaging agent for assessment of bile acid transport using fluorine magnetic resonance imaging.

Previously, we developed a trifluorinated bile acid, CA-lys-TFA, with the objective of noninvasively assessing bile acid transport in vivo using (19) F magnetic resonance imaging (MRI). CA-lys-TFA was successfully imaged in the mouse gallbladder, but was susceptible to deconjugation in vitro by choloylglycine hydrolase (CGH), a bacterial bile acid deconjugating enzyme found in the terminal ileum and colon. The objective of the present study was to develop a novel trifluorinated bile acid resistant to deconjugation by CGH. CA-sar-TFMA was designed, synthesized, and tested for in vitro transport properties, stability, imaging properties, and its ability to differentially accumulate in the gallbladders of normal mice, compared with mice with known impaired bile acid transport (deficient in the apical sodium-dependent bile acid transporter, ASBT). CA-sar-TFMA was a potent inhibitor and substrate of ASBT and the Na(+) /taurocholate cotransporting polypeptide. Stability was favorable in all conditions tested, including the presence of CGH. CA-sar-TFMA was successfully imaged and accumulated at 16.1-fold higher concentrations in gallbladders from wild-type mice compared with those from Asbt-deficient mice. Our results support the potential of using MRI with CA-sar-TFMA as a noninvasive method to assess bile acid transport in vivo.

Quantitative assessment of the contribution of sodium-dependent taurocholate co-transporting polypeptide (NTCP) to the hepatic uptake of rosuvastatin, pitavastatin and fluvastatin.

Hepatic uptake transport is often the rate-determining step in the systemic clearance of drugs. The ability to predict uptake clearance and to determine the contribution of individual transporters to overall hepatic uptake is therefore critical in assessing the potential pharmacokinetic and pharmacodynamic variability associated with drug-drug interactions and pharmacogenetics. The present study revisited the interaction of statin drugs, including pitavastatin, fluvastatin and rosuvastatin, with the sodium-dependent taurocholate co-transporting polypeptide (NTCP) using gene transfected cell models. In addition, the uptake clearance and the contribution of NTCP to the overall hepatic uptake were assessed using in vitro hepatocyte models. Then NTCP protein expression was measured by a targeted proteomics transporter quantification method to confirm the presence and stability of NTCP expression in suspended and cultured hepatocyte models. It was concluded that NTCP-mediated uptake contributed significantly to active hepatic uptake in hepatocyte models for all three statins. However, the contribution of NTCP-mediated uptake to the overall active hepatic uptake was compound-dependent and varied from about 24% to 45%. Understanding the contribution of individual transporter proteins to the overall hepatic uptake and its functional variability when other active hepatic uptake pathways are interrupted could improve the current prediction practice used to assess the pharmacokinetic variability due to drug-drug interactions, pharmacogenetics and physiopathological conditions in humans.

Transplantation and tracking of human-induced pluripotent stem cells in a pig model of myocardial infarction: assessment of cell survival, engraftment, and distribution by hybrid single photon emission computed tomography/computed tomography of sodium iodide symporter transgene expression.

BACKGROUND: Evaluation of novel cellular therapies in large-animal models and patients is currently hampered by the lack of imaging approaches that allow for long-term monitoring of viable transplanted cells. In this study, sodium iodide symporter (NIS) transgene imaging was evaluated as an approach to follow in vivo survival, engraftment, and distribution of human-induced pluripotent stem cell (hiPSC) derivatives in a pig model of myocardial infarction. METHODS AND RESULTS: Transgenic hiPSC lines stably expressing a fluorescent reporter and NIS (NIS(pos)-hiPSCs) were established. Iodide uptake, efflux, and viability of NIS(pos)-hiPSCs were assessed in vitro. Ten (±2) days after induction of myocardial infarction by transient occlusion of the left anterior descending artery, catheter-based intramyocardial injection of NIS(pos)-hiPSCs guided by 3-dimensional NOGA mapping was performed. Dual-isotope single photon emission computed tomographic/computed tomographic imaging was applied with the use of (123)I to follow donor cell survival and distribution and with the use of (99m)TC-tetrofosmin for perfusion imaging. In vitro, iodide uptake in NIS(pos)-hiPSCs was increased 100-fold above that of nontransgenic controls. In vivo, viable NIS(pos)-hiPSCs could be visualized for up to 15 weeks. Immunohistochemistry demonstrated that hiPSC-derived endothelial cells contributed to vascularization. Up to 12 to 15 weeks after transplantation, no teratomas were detected. CONCLUSIONS: This study describes for the first time the feasibility of repeated long-term in vivo imaging of viability and tissue distribution of cellular grafts in large animals. Moreover, this is the first report demonstrating vascular differentiation and long-term engraftment of hiPSCs in a large-animal model of myocardial infarction. NIS(pos)-hiPSCs represent a valuable tool to monitor and improve current cellular treatment strategies in clinically relevant animal models.

Cost-benefit tradeoffs in engineered lac operons.

Cells must balance the cost and benefit of protein expression to optimize organismal fitness. The lac operon of the bacterium Escherichia coli has been a model for quantifying the physiological impact of costly protein production and for elucidating the resulting regulatory mechanisms. We report quantitative fitness measurements in 27 redesigned operons that suggested that protein production is not the primary origin of fitness costs. Instead, we discovered that the lac permease activity, which relates linearly to cost, is the major physiological burden to the cell. These findings explain control points in the lac operon that minimize the cost of lac permease activity, not protein expression. Characterizing similar relationships in other systems will be important to map the impact of cost/benefit tradeoffs on cell physiology and regulation.

δ-Aminolevulinic acid dehydratase single nucleotide polymorphism 2 and peptide transporter 2*2 haplotype may differentially mediate lead exposure in male children.

Child low-level lead (Pb) exposure is an unresolved public health problem and an unaddressed child health disparity. Particularly in cases of low-level exposure, source removal can be impossible to accomplish, and the only practical strategy for reducing risk may be primary prevention. Genetic biomarkers of increased neurotoxic risk could help to identify small subgroups of children for early intervention. Previous studies have suggested that, by way of a distinct mechanism, δ-aminolevulinic acid dehydratase single nucleotide polymorphism 2 (ALAD(2)) and/or peptide transporter 2*2 haplotype (hPEPT2*2) increase Pb blood burden in children. Studies have not yet examined whether sex mediates the effects of genotype on blood Pb burden. Also, previous studies have not included blood iron (Fe) level in their analyses. Blood and cheek cell samples were obtained from 306 minority children, ages 5.1 to 12.9 years. (208)Pb and (56)Fe levels were determined with inductively coupled plasma-mass spectrometry. General linear model analyses were used to examine differences in Pb blood burden by genotype and sex while controlling for blood Fe level. The sample geometric mean Pb level was 2.75 µg/dl. Pb blood burden was differentially higher in ALAD(2) heterozygous boys and hPEPT2*2 homozygous boys. These results suggest that the effect of ALAD(2) and hPEPT2*2 on Pb blood burden may be sexually dimorphic. ALAD(2) and hPEPT2*2 may be novel biomarkers of health and mental health risks in male children exposed to low levels of Pb.

Transporter studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: assessment of human liver drug transporters.

17alpha-Ethinylestradiol (EE2), a component of oral contraceptives, is known to undergo considerable first-pass 3-O-sulfation in the intestine and liver. Once formed, the 3-O-sulfate conjugate (EE2-Sul) is detected in circulation at appreciable levels (versus parent EE2) and is present in bile. Therefore, hepatic uptake of EE2-Sul was assessed with suspensions of cryopreserved human primary hepatocytes. In this instance, there was evidence for active (temperature-dependent) uptake, which was described by a two-K(m) (Michaelis constant) model (K(m1) = 220 nM; K(m2) = 15.5 microM). Uptake was inhibited (approximately 90%) by bromosulfophthalein but not by tetraethylammonium or p-aminohippurate. In agreement, EE2-Sul was shown to be a substrate of recombinant organic anion transporter peptides (OATP1B1 and OATP2B1), and Na(+)/taurocholate-cotransporting polypeptide (NTCP), expressed individually in human embryonic kidney (HEK) 293 cells. Transport by OATP1B1 was described by two K(m) values (87 nM and 141 microM), whereas OATP2B1- and NTCP-mediated uptake into HEK-293 cells conformed to single K(m) kinetics (10.7 and 2.6 microM, respectively). EE2-Sul was also assessed as an efflux transporter substrate using membrane vesicles expressing bile salt export pump, breast cancer resistance protein (BCRP), and individual forms of multidrug resistance-associated protein (MRP1, MRP2, and MRP3). Transport studies were also conducted with a cell line expression P-glycoprotein. Only vesicles that contained BCRP exhibited ATP-dependent uptake of EE2-Sul (K(m1) = 2.9 and K(m2) = 307 microM). Collectively, the data show that hepatic uptake of EE2-Sul can be mediated by three transporters (OATP1B1, OATP2B1, and NTCP), whereas biliary excretion of EE2-Sul into bile likely involves BCRP.

[Establishment of genetic testing for Gitelman's syndrome].

Gitelman's syndrome is an autosomal recessive disorder marked by salt wasting and hypokalaemia resulting from loss of-function mutations in the SLC12A3 gene that codes for the thiazide sensitive Na -Cl cotransporter. Gitelman's syndrome is usually distinguished from Bartter's syndrome by the presence of both hypomagnesaemia and hypocalciuria. The human SLC12A3 gene, which is located on chromosome 16, consists of 26 exons and encodes a protein that contains 12 putative transmembrane domains with long intracellular amino and carboxy termini. In the present study, we developed a method of genetic diagnosis for Gitelman's syndrome using DNA sequencing. A patient was found to be a compound heterozygote with a single base substitution at nucleotide 2552 (CTC-to-CAC, L849H) and a substitution at nucleotide 2561 (CGC-to-CAC, R852H) in exon 22. Familial linkage analysis confirmed that 849H was the paternal allele and 852H was the maternal allele. The method can save time and costs, and it should be useful for genetic testing in clinical laboratory of every hospital.

Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours.

PURPOSE: In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. METHODS: We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. RESULTS: In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. CONCLUSION: This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects.