A physiologically-based pharmacokinetic modeling approach for dosing amiodarone in children on ECMO.

Extracorporeal membrane oxygenation (ECMO) is a cardiopulmonary bypass device commonly used to treat cardiac arrest in children. The American Heart Association guidelines for cardiopulmonary resuscitation (CPR) and emergency cardiovascular care recommend using amiodarone as a first-line agent to treat ventricular arrhythmias in children with cardiac arrest. However, there are no dosing recommendations for amiodarone to treat ventricular arrhythmias in pediatric patients on ECMO. Amiodarone has a high propensity for adsorption to the ECMO components due to its physicochemical properties leading to altered pharmacokinetics (PK) in ECMO patients. The change in amiodarone PK due to interaction with ECMO components may result in a difference in optimal dosing in patients on ECMO when compared with non-ECMO patients. To address this clinical knowledge gap, a physiologically-based pharmacokinetic model of amiodarone was developed in adults and scaled to children, followed by the addition of an ECMO compartment. The pediatric model included ontogeny functions of cytochrome P450 (CYP450) enzyme maturation across various age groups. The ECMO compartment was parameterized using the adsorption data of amiodarone obtained from ex vivo studies. Model predictions captured observed concentrations of amiodarone in pediatric patients with ECMO well with an average fold error between 0.5 and 2. Model simulations support an amiodarone intravenous (i.v) bolus dose of 22 mg/kg (neonates), 13 mg/kg (infants), 8 mg/kg (children), and 6 mg/kg (adolescents). This PBPK modeling approach can be applied to explore the dosing of other drugs used in children on ECMO.

Medication patterns and dosing guidance in pediatric patients supported with intermittent hemodialysis or continuous kidney replacement therapy.

BACKGROUND: Hemodialysis is a life-saving technology used during periods of acute or chronic kidney failure to remove toxins, and maintain fluid, electrolyte and metabolic balance. While this technology plays an important role for pediatric patients with kidney dysfunction, it can alter the pharmacokinetic behavior of medications placing patients at risk for suboptimal dosing and drug toxicity. The ability to directly translate pharmacokinetic alterations into dosing recommendations has thus far been limited and dosing guidance specific to pediatric hemodialysis patients is rare. Despite differences in dialysis prescription and patient populations, intermittent (iHD) and continuous kidney replacement therapy (CKRT) patients are often pooled together. In order to develop evidence-based dosing guidelines, it is important to first prioritize drugs for study in each modality. METHODS: Here we aim to identify priority drugs in two hemodialysis modalities, through: 1) Identification of hospitalized, pediatric patients who received CKRT or intermittent hemodialysis (iHD) using a machine learning-based predictive model based on medications; 2) Identification of medication administration patterns in these patient cohorts; and 3) Identification of the most commonly prescribed drugs that lack published dosing guidance. RESULTS: Notable differences were found in the pattern of medications and drug dosing guidance between iHD and CKRT patients. Antibiotics, diuretics and sedatives were more common in CKRT patients. Out of the 50 most commonly administered medications in the two modalities, only 34% and 28% had dosing guidance present for iHD and CKRT, respectively. CONCLUSIONS: Our results add to the understanding of the differences between iHD and CKRT patient populations by identifying commonly used medications that lack dosing guidance for each hemodialysis modality, helping to pinpoint priority medications for further study. Overall, this study provides an overview of the current limitations in medication use in this at-risk population, and provides a framework for future studies by identifying commonly used medications in pediatric CKRT and iHD patients.

Maximum likelihood estimation of renal transporter ontogeny profiles for pediatric PBPK modeling.

Optimal treatment of infants with many renally cleared drugs must account for maturational differences in renal transporter (RT) activity. Pediatric physiologically-based pharmacokinetic (PBPK) models may incorporate RT activity, but this requires ontogeny profiles for RT activity in children, especially neonates, to predict drug disposition. Therefore, RT expression measurements from human kidney postmortem cortical tissue samples were normalized to represent a fraction of mature RT activity. Using these data, maximum likelihood estimated the distributions of RT activity across the pediatric age spectrum, including preterm and term neonates. PBPK models of four RT substrates (acyclovir, ciprofloxacin, furosemide, and meropenem) were evaluated with and without ontogeny profiles using average fold error (AFE), absolute average fold error (AAFE), and proportion of observations within the 5-95% prediction interval. Novel maximum likelihood profiles estimated ontogeny distributions for the following RT: OAT1, OAT3, OCT2, P-gp, URAT1, BCRP, MATE1, MRP2, MRP4, and MATE-2 K. Profiles for OAT3, P-gp, and MATE1 improved infant furosemide and neonate meropenem PBPK model AFE from 0.08 to 0.70 and 0.53 to 1.34 and model AAFE from 12.08 to 1.44 and 2.09 to 1.36, respectively, and improved the percent of data within the 5-95% prediction interval from 48% to 98% for neonatal ciprofloxacin simulations, respectively. Even after accounting for other critical population-specific maturational differences, novel RT ontogeny profiles substantially improved neonatal PBPK model performance, providing validated estimates of maturational differences in RT activity for optimal dosing in children.

Anakinra Removal by Continuous Renal Replacement Therapy: An Ex Vivo Analysis.

OBJECTIVES: Patients with sepsis are at significant risk for multiple organ dysfunction, including the lungs and kidneys. To manage the morbidity associated with kidney impairment, continuous renal replacement therapy (CRRT) may be required. The extent of anakinra pharmacokinetics in CRRT remains unknown. The objectives of this study were to investigate the anakinra-circuit interaction and quantify the rate of removal from plasma. DESIGN: The anakinra-circuit interaction was evaluated using a closed-loop ex vivo CRRT circuit. CRRT was performed in three phases based on the method of solute removal: 1) hemofiltration, 2) hemodialysis, and 3) hemodiafiltration. Standard control samples of anakinra were included to assess drug degradation. SETTING: University research laboratory. PATIENTS: None. INTERVENTIONS: Anakinra was administered to the CRRT circuit and serial prefilter blood samples were collected along with time-matched control and hemofiltrate samples. Each circuit was run in triplicate to assess inter-run variability. Concentrations of anakinra in each reference fluid were measured by enzyme-linked immunosorbent assay. Transmembrane filter clearance was estimated by the product of the sieving coefficient/dialysate saturation constant and circuit flow rates. MEASUREMENTS AND MAIN RESULTS: Removal of anakinra from plasma occurred within minutes for each CRRT modality. Average drug remaining (%) in plasma following anakinra administration was lowest with hemodiafiltration (34.9%). The average sieving coefficient was 0.34, 0.37, and 0.41 for hemodiafiltration, hemofiltration, and hemodialysis, respectively. Transmembrane clearance was fairly consistent across each modality with the highest during hemodialysis (5.53 mL/min), followed by hemodiafiltration (4.99 mL/min), and hemofiltration (3.94 mL/min). Percent drug remaining within the control samples (93.1%) remained consistent across each experiment, indicating negligible degradation within the blood. CONCLUSIONS: The results of this analysis are the first to demonstrate that large molecule therapeutic proteins such as anakinra, are removed from plasma with modern CRRT technology. Current dosing recommendations for patients with severe renal impairment may result in subtherapeutic anakinra concentrations in those receiving CRRT.

Meropenem extraction by ex vivo extracorporeal life support circuits.

BACKGROUND: Meropenem is a broad-spectrum carbapenem-type antibiotic commonly used to treat critically ill patients infected with extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. As many of these patients require extracorporeal membrane oxygenation (ECMO) and/or continuous renal replacement therapy (CRRT), it is important to understand how these extracorporeal life support circuits impact meropenem pharmacokinetics. Based on the physicochemical properties of meropenem, it is expected that ECMO circuits will minimally extract meropenem, while CRRT circuits will rapidly clear meropenem. The present study seeks to determine the extraction of meropenem from ex vivo ECMO and CRRT circuits and elucidate the contribution of different ECMO circuit components to extraction. METHODS: Standard doses of meropenem were administered to three different configurations (n = 3 per configuration) of blood-primed ex vivo ECMO circuits and serial sampling was conducted over 24 h. Similarly, standard doses of meropenem were administered to CRRT circuits (n = 4) and serial sampling was conducted over 4 h. Meropenem was administered to separate tubes primed with circuit blood to serve as controls to account for drug degradation. Meropenem concentrations were quantified, and percent recovery was calculated for each sample. RESULTS: Meropenem was cleared at a similar rate in ECMO circuits of different configurations (n = 3) and controls (n = 6), with mean (standard deviation) recovery at 24 h of 15.6% (12.9) in Complete circuits, 37.9% (8.3) in Oxygenator circuits, 47.1% (8.2) in Pump circuits, and 20.6% (20.6) in controls. In CRRT circuits (n = 4) meropenem was cleared rapidly compared with controls (n = 6) with a mean recovery at 2 h of 2.36% (1.44) in circuits and 93.0% (7.1) in controls. CONCLUSION: Meropenem is rapidly cleared by hemodiafiltration during CRRT. There is minimal adsorption of meropenem to ECMO circuit components; however, meropenem undergoes significant degradation and/or plasma metabolism at physiological conditions. These ex vivo findings will advise pharmacists and physicians on the appropriate dosing of meropenem.

Interaction of ceftazidime and clindamycin with extracorporeal life support.

BACKGROUND: Ceftazidime and clindamycin are commonly prescribed to critically ill patients who require extracorporeal life support such as ECMO and CRRT. The effect of ECMO and CRRT on the disposition of ceftazidime and clindamycin is currently unknown. METHODS: Ceftazidime and clindamycin extraction were studied with ex vivo ECMO and CRRT circuits primed with human blood. The percent recovery of these drugs over time was calculated to determine the degree of interaction between these drugs and circuit components. RESULTS: Neither ceftazidime nor clindamycin exhibited measurable interactions with the ECMO circuit. In contrast, CRRT cleared 100% of ceftazidime from the experimental circuit within the first 2 h. Clearance of clindamycin from the CRRT circuit was slower, with about 20% removed after 6 h. CONCLUSION: Clindamycin and ceftazidime dosing adjustments are likely required in patients who are supported with CRRT, and future studies to quantify these adjustments should consider the pathophysiology of the patient in combination with the clearance due to CRRT. Dosing adjustments to account for adsorption to ECMO circuit components are likely unnecessary and should focus instead on the pathophysiology of the patient and changes in volume of distribution. These results will help improve the safety and efficacy of ceftazidime and clindamycin in patients requiring ECMO and CRRT.

External assessment and refinement of a population pharmacokinetic model to guide tacrolimus dosing in pediatric heart transplant.

STUDY OBJECTIVE: The immunosuppressant tacrolimus is a first-line agent to prevent graft rejection following pediatric heart transplant; however, it suffers from extensive inter-patient variability and a narrow therapeutic window. Personalized tacrolimus dosing may improve transplant outcomes by more efficiently achieving and maintaining therapeutic tacrolimus concentrations. We sought to externally validate a previously published population pharmacokinetic (PK) model that was constructed with data from a single site. DATA SOURCE: Data were collected from Seattle, Texas, and Boston Children's Hospitals, and assessed using standard population PK modeling techniques in NONMEMv7.2. MAIN RESULTS: While the model was not successfully validated for use with external data, further covariate searching identified weight (p < 0.0001 on both volume and elimination rate) as a model-significant covariate. This refined model acceptably predicted future tacrolimus concentrations when guided by as few as three concentrations (median prediction error = 7%; median absolute prediction error = 27%). CONCLUSION: These findings support the potential clinical utility of a population PK model to provide personalized tacrolimus dosing guidance.

Drug exposure during pregnancy: Current understanding and approaches to measure maternal-fetal drug exposure.

Prescription drug use is prevalent during pregnancy, yet there is limited knowledge about maternal-fetal safety and efficacy of this drug use because pregnant individuals have historically been excluded from clinical trials. Underrepresentation has resulted in a lack of data available to estimate or predict fetal drug exposure. Approaches to study fetal drug pharmacology are limited and must be evaluated for feasibility and accuracy. Anatomic and physiological changes throughout pregnancy fluctuate based on gestational age and can affect drug pharmacokinetics (PK) for both mother and fetus. Drug concentrations have been studied throughout different stages of gestation and at or following delivery in tissue and fluid biospecimens. Sampling amniotic fluid, umbilical cord blood, placental tissue, meconium, umbilical cord tissue, and neonatal hair present surrogate options to quantify and characterize fetal drug exposure. These sampling methods can be applied to all therapeutics including small molecule drugs, large molecule drugs, conjugated nanoparticles, and chemical exposures. Alternative approaches to determine PK have been explored, including physiologically based PK modeling, in vitro methods, and traditional animal models. These alternative approaches along with convenience sampling of tissue or fluid biospecimens can address challenges in studying maternal-fetal pharmacology. In this narrative review, we 1) present an overview of the current understanding of maternal-fetal drug exposure; 2) discuss biospecimen-guided sampling design and methods for measuring fetal drug concentrations throughout gestation; and 3) propose methods for advancing pharmacology research in the maternal-fetal population.

Lack of evidence that fibrillin1 regulates bone morphogenetic protein 4 activity in kidney or lung.

BACKGROUND: The Bone morphogenetic protein 4 (BMP4) precursor protein is cleaved at two sites to generate an active ligand and inactive prodomain. The ligand and prodomain form a noncovalent complex following the first cleavage, but dissociate after the second cleavage. Transient formation of this complex is essential to generate a stable ligand. Fibrillins (FBNs) bind to the prodomains of BMPs, and can regulate the activity of some ligands. Whether FBNs regulate BMP4 activity is unknown. RESULTS: Mice heterozygous for a null allele of Bmp4 showed incompletely penetrant kidney defects and females showed increased mortality between postnatal day 6 and 8. Removal of one copy of Fbn1 did not rescue or enhance kidney defects or lethality. The lungs of Fbn1+/- females had enlarged airspaces that were unchanged in Bmp4+/- ;Fbn1+/- mice. Additionally, removal of one or both alleles of Fbn1 had no effect on steady state levels of BMP4 ligand or on BMP activity in postnatal lungs. CONCLUSIONS: These findings do not support the hypothesis that FBN1 plays a role in promoting BMP4 ligand stability or signaling, nor do they support the alternative hypothesis that FBN1 sequesters BMP4 in a latent form, as is the case for other BMP family members.

Medication based machine learning to identify subpopulations of pediatric hemodialysis patients in an electronic health record database.

Electronic health records (EHRs) have given rise to large and complex databases of medical information that have the potential to become powerful tools for clinical research. However, differences in coding systems and the detail and accuracy of the information within EHRs can vary across institutions. This makes it challenging to identify subpopulations of patients and limits the widespread use of multi-institutional databases. In this study, we leveraged machine learning to identify patterns in medication usage among hospitalized pediatric patients receiving renal replacement therapy and created a predictive model that successfully differentiated between intermittent (iHD) and continuous renal replacement therapy (CRRT) hemodialysis patients. We trained six machine learning algorithms (logistical regression, Naïve Bayes, k-nearest neighbor, support vector machine, random forest, and gradient boosted trees) using patient records from a multi-center database (n = 533) and prescribed medication ingredients (n = 228) as features to discriminate between the two hemodialysis types. Predictive skill was assessed using a 5-fold cross-validation, and the algorithms showed a range of performance from 0.7 balanced accuracy (logistical regression) to 0.86 (random forest). The two best performing models were further tested using an independent single-center dataset and achieved 84-87% balanced accuracy. This model overcomes issues inherent within large databases and will allow us to utilize and combine historical records, significantly increasing population size and diversity within both iHD and CRRT populations for future clinical studies. Our work demonstrates the utility of using medications alone to accurately differentiate subpopulations of patients in large datasets, allowing codes to be transferred between different coding systems. This framework has the potential to be used to distinguish other subpopulations of patients where discriminatory ICD codes are not available, permitting more detailed insights and new lines of research.

Remdesivir and GS-441524 Extraction by Ex Vivo Extracorporeal Life Support Circuits.

Patients with severe, COVID-related multi-organ failure often require extracorporeal life support (ECLS) such as extracorporeal membrane oxygenation (ECMO) or continuous renal replacement therapy (CRRT). An ECLS can alter drug exposure via multiple mechanisms. Remdesivir (RDV) and its active metabolite GS-441524 are likely to interact with ECLS circuits, resulting in lower than expected exposures. We evaluated circuit-drug interactions in closed loop, ex vivo ECMO and CRRT circuits. We found that mean (standard deviation) recovery of RDV at 6 hours after dosing was low in both the ECMO (33.3% [2.0]) and CRRT (3.5% [0.4]) circuits. This drug loss appears to be due primarily to drug adsorption by the circuit materials and potentially due to metabolism in the blood. GS-441524 recovery at 6 hours was high in the ECMO circuit 75.8% (16.5); however, was not detectable at 6 hours in the CRRT circuit. Loss in the CRRT circuit appears to be due primarily to efficient hemodiafiltration. The extent of loss for both molecules, especially in CRRT, suggests that in patients supported with ECMO and CRRT, RDV dosing adjustments are needed.

Cefepime Extraction by Extracorporeal Life Support Circuits.

Extracorporeal life support (ECLS) devices are lifesaving for critically ill patients with multi-organ dysfunction. Despite this, patients supported with ECLS are at high risk for ECLS-related complications, including nosocomial infections, and mortality rates are high in this patient population. The high mortality rates are suspected to be, in part, a result of significantly altered drug disposition by the ECLS circuit, resulting in suboptimal antimicrobial dosing. Cefepime is commonly used in critically ill patients with serious infections. Cefepime dosing is not routinely guided by therapeutic drug monitoring and treatment success is dependent upon the percentage of time of the dosing interval that the drug concentration remains above the minimum inhibitory concentration of the organism. This ex vivo study measured the extraction of cefepime by continuous renal replacement therapy (CRRT) and extracorporeal membrane oxygenation (ECMO) circuits. Cefepime was studied in four closed-loop CRRT circuit configurations and a single closed-loop ECMO circuit configuration. Circuits were primed with a physiologic human blood-plasma mixture and the drug was dosed to achieve therapeutic concentrations. Serial blood samples were collected over time and concentrations were quantified using validated assays. In ex vivo CRRT experiments, cefepime was rapidly cleared by dialysis, hemofiltration, and hemodiafiltration, with greater than 96% cefepime eliminated from the circuit by 2 hours. In the ECMO circuits, the mean recovery of cefepime was similar in both circuit and standard control. Mean (standard deviation) recovery of cefepime in the ECMO circuits (n = 6) was 39.2% (8.0) at 24 hours. Mean recovery in the standard control (n = 3) at 24 hours was 52.2% (1.5). Cefepime is rapidly cleared by dialysis, hemofiltration, and hemodiafiltration in the CRRT circuit but minimally adsorbed by either the CRRT or ECMO circuits. Dosing adjustments are needed for patients supported with CRRT.

BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian embryogenesis.

BMP7/BMP2 or BMP7/BMP4 heterodimers are more active than homodimers in vitro, but it is not known whether these heterodimers signal in vivo. To test this, we generated knock in mice carrying a mutation (Bmp7R-GFlag) that prevents proteolytic activation of the dimerized BMP7 precursor protein. This mutation eliminates the function of BMP7 homodimers and all other BMPs that normally heterodimerize with BMP7. While Bmp7 null homozygotes are live born, Bmp7R-GFlag homozygotes are embryonic lethal and have broadly reduced BMP activity. Furthermore, compound heterozygotes carrying the Bmp7R-G allele together with a null allele of Bmp2 or Bmp4 die during embryogenesis with defects in ventral body wall closure and/or the heart. Co-immunoprecipitation assays confirm that endogenous BMP4/7 heterodimers exist. Thus, BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian development, which may explain why mutations in either Bmp4 or Bmp7 lead to a similar spectrum of congenital defects in humans.

Diet modulates cecum bacterial diversity and physiological phenotypes across the BXD mouse genetic reference population.

The BXD family has become one of the preeminent genetic reference populations to understand the genetic and environmental control of phenotypic variation. Here we evaluate the responses to different levels of fat in the diet using both chow diet (CD, 13-18% fat) and a high-fat diet (HFD, 45-60% fat). We studied cohorts of BXD strains, both inbred parents C57BL/6J and DBA/2J (commonly known as B6 and D2, respectively), as well as B6D2 and D2B6 reciprocal F1 hybrids. The comparative impact of genetic and dietary factors was analyzed by profiling a range of phenotypes, most prominently their cecum bacterial composition. The parents of the BXDs and F1 hybrids express limited differences in terms of weight and body fat gain on CD. In contrast, the strain differences on HFD are substantial for percent body fat, with DBA/2J accumulating 12.5% more fat than C57BL/6J (P < 0.0001). The F1 hybrids born to DBA/2J dams (D2B6F1) have 10.6% more body fat (P < 0.001) than those born to C57BL/6J dams. Sequence analysis of the cecum microbiota reveals important differences in bacterial composition among BXD family members with a substantial shift in composition caused by HFD. Relative to CD, the HFD induces a decline in diversity at the phylum level with a substantial increase in Firmicutes (+13.8%) and a reduction in Actinobacteria (-7.9%). In the majority of BXD strains, the HFD also increases cecal sIgA (P < 0.0001)-an important component of the adaptive immunity response against microbial pathogens. Host genetics modulates variation in cecum bacterial composition at the genus level in CD, with significant quantitative trait loci (QTLs) for Oscillibacter mapped to Chr 3 (18.7-19.2 Mb, LRS = 21.4) and for Bifidobacterium mapped to Chr 6 (89.21-89.37 Mb, LRS = 19.4). Introduction of HFD served as an environmental suppressor of these QTLs due to a reduction in the contribution of both genera (P < 0.001). Relations among liver metabolites and cecum bacterial composition were predominant in CD cohort, but these correlations do not persist following the shift to HFD. Overall, these findings demonstrate the important impact of environmental/dietary manipulation on the relationships between host genetics, gastrointestinal bacterial composition, immunological parameters, and metabolites-knowledge that will help in the understanding of the causal sources of metabolic disorders.

Proteolytic Activation of Bmps: Analysis of Cleavage in Xenopus Oocytes and Embryos.

Bone morphogenetic proteins (Bmps) are synthesized as inactive precursors that are cleaved to generate active ligands, along with prodomain fragments that can modulate growth factor activity. Here we provide three protocols that can be used to examine the process of proteolytic activation of Bmps. The first protocol describes how to generate radiolabeled Bmp precursor proteins in Xenopus oocytes and then analyze the time course of precursor cleavage by recombinant enzymes in vitro. The second protocol details how to analyze cleavage of radiolabeled precursor proteins in Xenopus oocytes over time using pulse-chase analysis and autoradiography. This protocol can also be used to analyze folding and cleavage of radiolabeled precursor proteins at steady state. Finally, the third protocol details methods for isolating Bmp cleavage products from the blastocoele of Xenopus embryos and then analyzing them on immunoblots.

Fibronectin type III and intracellular domains of Toll-like receptor 4 interactor with leucine-rich repeats (Tril) are required for developmental signaling.

Toll-like receptor 4 interactor with leucine-rich repeats (Tril) functions as a coreceptor for Toll-like receptors (Tlrs) to mediate innate immune responses in adults. In embryos, Tril signals to promote degradation of the Bmp inhibitor, Smad7, to allow for blood formation. It is not known whether this function requires, or is independent of, Tlrs. In the current studies, we performed a structure-function analysis, which indicated that the fibronectin type III (FN) domain and the intracellular domain of Tril are required to trigger Smad7 degradation in Xenopus embryos. Furthermore, we found evidence suggesting that a Tril deletion mutant lacking the FN domain (Tril∆FN) can dominantly inhibit signaling by endogenous Tril when overexpressed. This finding raises the possibility that the FN domain functions to bind endogenous Tril ligands. We also show that Tril cycles between the cell surface and endosomes and that the Tril extracellular domain, as well as cadherin based cell-cell adhesion, are required for cell surface retention, while the intracellular domain is required for internalization in Xenopus ectodermal explants. Using a CHO cell aggregation assay, we show that, unlike other transmembrane proteins that contain leucine-rich repeats, Tril is not sufficient to mediate homophilic adhesion.

Variation in time and magnitude of immune response and viremia in experimental challenges with Porcine circovirus 2b.

BACKGROUND: Porcine circovirus 2 is the primary agent responsible for inducing a group of associated diseases known as Porcine Circovirus Associated Diseases (PCVAD), which can have detrimental effects on production efficiency as well as causing significant mortality. The objective of this study was to evaluate variation in viral replication, immune response and growth across pigs (n = 974) from different crossbred lines. The approach used in this study was experimental infection with a PCV2b strain of pigs at an average of 43 days of age. RESULTS: The sequence of the PCV2b isolate used in the challenge was similar with a cluster of PCV2b isolates known to induce PCVAD and increased mortality rates. The swine leukocyte antigen class II (SLAII) profile of the population was diverse, with nine DQB1 haplotypes being present. Individual viremia and antibody profiles during challenge demonstrate variation in magnitude and time of viral surge and immune response. The correlations between PCV2 specific antibodies and average daily gain (ADG) were relatively low and varied between - 0.14 to 0.08 for IgM and -0.02 and 0.11 for IgG. In contrast, PCV2 viremia was an important driver of ADG decline following infection; a moderate negative correlation was observed between viral load and overall ADG (r = - 0.35, P < 0.001). The pigs with the lowest 10% level of viral load maintained a steady increase in weekly ADG (P < 0.0001) compared to the pigs that had the 10% greatest viral load (P < 0.55). In addition, the highly viremic group expressed higher IgM and IgG starting with d 14 and d 21 respectively, and higher tumor necrosis factor - alpha (TNF-α) at d 21 (P < 0.005), compared to low viremic group. CONCLUSIONS: Molecular sources of the observed differences in viremia and immune response could provide a better understanding of the host factors that influence the development of PCVAD and lead to improved knowledge of swine immunity.

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.

Murine gut microbiota is defined by host genetics and modulates variation of metabolic traits.

The gastrointestinal tract harbors a complex and diverse microbiota that has an important role in host metabolism. Microbial diversity is influenced by a combination of environmental and host genetic factors and is associated with several polygenic diseases. In this study we combined next-generation sequencing, genetic mapping, and a set of physiological traits of the BXD mouse population to explore genetic factors that explain differences in gut microbiota and its impact on metabolic traits. Molecular profiling of the gut microbiota revealed important quantitative differences in microbial composition among BXD strains. These differences in gut microbial composition are influenced by host-genetics, which is complex and involves many loci. Linkage analysis defined Quantitative Trait Loci (QTLs) restricted to a particular taxon, branch or that influenced the variation of taxa across phyla. Gene expression within the gastrointestinal tract and sequence analysis of the parental genomes in the QTL regions uncovered candidate genes with potential to alter gut immunological profiles and impact the balance between gut microbial communities. A QTL region on Chr 4 that overlaps several interferon genes modulates the population of Bacteroides, and potentially Bacteroidetes and Firmicutes-the predominant BXD gut phyla. Irak4, a signaling molecule in the Toll-like receptor pathways is a candidate for the QTL on Chr15 that modulates Rikenellaceae, whereas Tgfb3, a cytokine modulating the barrier function of the intestine and tolerance to commensal bacteria, overlaps a QTL on Chr 12 that influence Prevotellaceae. Relationships between gut microflora, morphological and metabolic traits were uncovered, some potentially a result of common genetic sources of variation.

Genotypes of Brassica rapa respond differently to plant-induced variation in air CO2 concentration in growth chambers with standard and enhanced venting.

Growth chambers allow measurement of phenotypic differences among genotypes under controlled environment conditions. However, unintended variation in growth chamber air CO2 concentration ([CO2]) may affect the expression of diverse phenotypic traits, and genotypes may differ in their response to variation in [CO2]. We monitored [CO2] and quantified phenotypic responses of 22 Brassica rapa genotypes in growth chambers with either standard or enhanced venting. [CO2] in chambers with standard venting dropped to 280 micromol mol(-1) during the period of maximum canopy development, approximately 80 micromol mol(-1) lower than in chambers with enhanced venting. The stable carbon isotope ratio of CO2 in chamber air (delta13C(air)) was negatively correlated with [CO2], suggesting that photosynthesis caused observed [CO2] decreases. Significant genotype x chamber-venting interactions were detected for 12 of 20 traits, likely due to differences in the extent to which [CO2] changed in relation to genotypes' phenology or differential sensitivity of genotypes to low [CO2]. One trait, 13C discrimination (delta13C), was particularly influenced by unaccounted-for fluctuations in delta13C(air) and [CO2]. Observed responses to [CO2] suggest that genetic variance components estimated in poorly vented growth chambers may be influenced by the expression of genes involved in CO2 stress responses; genotypic values estimated in these chambers may likewise be misleading such that some mapped quantitative trait loci may regulate responses to CO2 stress rather than a response to the environmental factor of interest. These results underscore the importance of monitoring, and where possible, controlling [CO2].