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Patients with single ventricle heart defects requires a series of staged open-heart procedures, termed Fontan palliation. However, while lifesaving, these operations are associated with significant morbidity and early mortality. The attendant complications are thought to arise in response to the abnormal hemodynamics induced by Fontan palliation, although the pathophysiology underlying these physicochemical changes in cardiovascular and other organs remain unknown. Here, we investigated the microRNA (miRNA) content in serum and serum-derived extracellular vesicles (EVs) by sequencing small RNAs from a physiologically relevant sheep model of the Fontan operation. The differential expression analysis identified the enriched miRNA clusters in (1) serum vs. serum-derived EVs and (2) pre-Fontan EVs vs. post-Fontan EVs. Metascape analysis showed that the overexpressed subset of EV miRNAs by Fontan procedure target liver-specific cells, underscoring a potentially important pathway involved in the liver dysfunction that occurs as a consequence of Fontan palliation. We also found that post-Fontan EV miRNAs were associated with senescence and cell death, whereas pre-Fontan EV miRNAs were associated with stem cell maintenance and epithelial-to-mesenchymal transition. This study shows great potential to identify novel circulating EV biomarkers from Fontan sheep serum that may be used for the diagnosis, prognosis, and therapeutics for patients that have undergone Fontan palliation.

Congenital heart defects are the leading cause of right heart failure in pediatric patients. Implantation of c-kitsup+/supcardiac-derived progenitor cells (CPCs) is being clinically evaluated to treat the failing right ventricle (RV), but faces limitations due to reduced transplant cell survival, low engraftment rates, and low retention. These limitations have been exacerbated due to the nature of cell delivery (narrow needles) and the non-optimal recipient microenvironment (reactive oxygen species (ROS)). Extracellular matrix (ECM) hydrogels derived from porcine left ventricular (LV) myocardium have emerged as a potential therapy to treat the ischemic LV and have shown promise as a vehicle to deliver cells to injured myocardium. However, no studies have evaluated the combination of an injectable biomaterial, such as an ECM hydrogel, in combination with cell therapy for treating RV failure. In this study we characterized LV and RV myocardial matrix (MM) hydrogels and performed in vitro evaluations of their potential to enhance CPC delivery, including resistance to forces experienced during injection and exposure to ROS, as well as their potential to enhance angiogenic paracrine signaling. While physical properties of the two hydrogels are similar, the decellularized LV and RV have distinct protein signatures. Both materials were equally effective in protecting CPCs against needle forces and ROS. CPCs encapsulated in either the LV MM or RV MM exhibited similar enhanced potential for angiogenic paracrine signaling when compared to CPCs in collagen. The RV MM without cells, however, likewise improved tube formation, suggesting it should also be evaluated as a potential standalone treatment.

SUMMARYThe developing mammalian heart undergoes an important metabolic shift from glycolysis toward mitochondrial oxidation, such that oxidative phosphorylation defects may present with cardiac abnormalities. Here, we describe a new mechanistic link between mitochondria and cardiac morphogenesis, uncovered by studying mice with systemic loss of the mitochondrial citrate carrier SLC25A1.Slc25a1null embryos displayed impaired growth, cardiac malformations, and aberrant mitochondrial function. Importantly,Slc25a1haploinsufficient embryos, which are overtly indistinguishable from wild type, exhibited an increased frequency of these defects, suggestingSlc25a1dose-dependent effects. Supporting clinical relevance, we found a near-significant association between ultrarare human pathogenicSLC25A1variants and pediatric congenital heart disease. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of PPARγ to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of ventricular morphogenesis and cardiac metabolic maturation and suggests a role in congenital heart disease.

AimsHypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction of the right ventricle (RV) to provide systemic circulation. This substantially increases the RV load, wall stress, maladaptive remodelling, and dysfunction, which in turn increases the risk of death or transplantation.Methods and resultsWe conducted a phase 1 open-label multicentre trial to assess the safety and feasibility of Lomecel-B as an adjunct to second-stage HLHS surgical palliation. Lomecel-B, an investigational cell therapy consisting of allogeneic medicinal signalling cells (MSCs), was delivered via intramyocardial injections. The primary endpoint was safety, and measures of RV function for potential efficacy were obtained. Ten patients were treated. None experienced major adverse cardiac events. All were alive and transplant-free at 1-year post-treatment, and experienced growth comparable to healthy historical data. Cardiac magnetic resonance imaging (CMR) suggested improved tricuspid regurgitant fraction (TR RF) via qualitative rater assessment, and via significant quantitative improvements from baseline at 6 and 12 months post-treatment (P < 0.05). Global longitudinal strain (GLS) and RV ejection fraction (EF) showed no declines. To understand potential mechanisms of action, circulating exosomes from intramyocardially transplanted MSCs were examined. Computational modelling identified 54 MSC-specific exosome ribonucleic acids (RNAs) corresponding to changes in TR RF, including miR-215-3p, miR-374b-3p, and RNAs related to cell metabolism and MAPK signalling.ConclusionIntramyocardially delivered Lomecel-B appears safe in HLHS patients and may favourably affect RV performance. Circulating exosomes of transplanted MSC-specific provide novel insight into bioactivity. Conduct of a controlled phase trial is warranted and is underway.Trial registration number NCT03525418.

BackgroundHypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction for the right ventricle (RV) to provide systemic circulation. This leads to substantially increased loads on the RV, wall stress, maladaptive remodeling and dysfunction, which in turn can increase risk of death or transplantation.ObjectivesWe conducted a phase I multicenter trial to assess safety and feasibility of intra-operative MSC injection in HLHS patients to boost RV performance in the systemic position.MethodsAllogeneic MSCs were directly administered by intramyocardial injections during the second stage palliative operation. The primary endpoint was safety.ResultsTen patients received intramyocardial injections of allogeneic MSCs (Lomecel-B). No patients experienced major adverse cardiac events (MACE). All subjects were alive and transplant-free at 1 year following, and experienced growth comparable to healthy control historical data. Cardiac magnetic resonance imaging (CMR) revealed improving tricuspid regurgitant fraction (Baseline: 0.45±0.19; 6 mo.: 0.32±0.06; 12 mo.: 0.06±0.09), while global longitudinal strain (Baseline: -24.39±6.99; 6 mo.: -20.55±3.05, p > 0.05 vs baseline; 12 mo.: - 23.88±4.6, p>0.05 vs baseline) and RV ejection fraction (EF; baseline: 62.62±5.99; 6 mo.: 53.69±9.56; 12 mo.: 52.31±5.63: p=NS for change over time) were unchanged. Computational modeling identified 167 derived RNAs specific to circulating exosomes originating from transplanted MSCs corresponding to RVEF changes and identifying potential mechanistic underpinnings.ConclusionsIntramyocardial MSCs appear safe in HLHS patients, and may favorably affect RV performance. Circulating exosomes of transplanted MSC-specific provide novel insight into bioactivity. Conduct of a controlled phase trial is warranted and is underway.Condensed AbstractThe ELPIS phase I trial was designed to assess safety and feasibility of intramyocardial injection of allogeneic MSCs into the RV during second stage palliation of HLHS. There were no incidences of major adverse cardiac events (MACE) or other safety concerns, and there was a 100% transplant-free survival at 1-year follow-up, supporting the safety and feasibility of this approach. The ELPIS results are important for advancing MSC therapy for all ages and congenital heart conditions, and warrant further investigation in a controlled Phase II trial powered for efficacy.

Pediatric cardiac-derived c-kit

Introduction: Ckit+ progenitor cells (CPCs) release small extracellular vesicles (sEVs) which are vital for CPC-based recovery after myocardial infarction (MI). sEVs transport RNA cargo within a complex lipid/protein bilayer required for sEV trafficking and endocytosis. However, being cell-derived, sEV yield, cargo content and concentration are often variable. Synthetic sEV-mimics allow for cargo control, but their basic phospholipid membrane often results in rapid clearance. Here, we use thin film hydration (TFH) and electroporation to develop sEV-like vesicles (ELVs) with an sEV membrane and mimic-like cargo control. We hypothesize that (1) ELVs with cardioprotective microRNA (miR) cargo will improve functional outcomes from cardiac endothelial cells (CECs) compared to sEVs alone and (2) electroporated ELVs will functionally outperform TFH ELVs. Methods: sEVs were isolated from CPC conditioned media with ultracentrifugation. ELVs were synthesized from sEVs by removing inherent miR cargo and then loading endothelial specific miR126 through TFH or electroporation. For TFH, sEVs were dissolved in an organic solvent to form a lipid bilayer and then loaded with miR126 to initiate ELV self-assembly. For electroporation, miR126 was added to sEVs by varying voltage pulses. ELVs were characterized for size and concentration. Both cargo clearance and miR126 loading were quantified with a Quant-iT miR kit. Functionality of both ELV designs was assessed by delivering miR126+ ELVs to CECs for angiogenesis and proliferation. Results: ELVs on a similar scale to sEVs were successfully formed using both methods, despite variability in concentration. ELV miR126 loading capacity after cargo removal was higher and tunable with electroporation compared to TFH. Electroporation also preserved more membrane proteins than TFH. When delivered to CECs, electroporated miR126+ ELVs increased angiogenesis and proliferation compared to sEVs over 24 hours, in a dose-dependent manner. Conclusion: Using an sEV-like membrane with customizable miR cargo allows for improved functional outcomes from cardiac cells, with electroporated samples having more cargo tunability. Beyond miR126, this work highlights a delivery vehicle for combination miR therapy after MI.

Autologous cardiac cell therapy is a promising treatment for combating the right ventricular heart failure (RVHF) that can occur in patients with congenital heart disease (CHD). However, autologous cell therapies suffer from low cell retention following injection and patient-to-patient variability in cell quality. Here, we demonstrate how computational methods can be used to identify mechanisms of cardiac-derived c-Kit

The beef cow-calf sector accounts for 70% of feed consumed and greenhouse gases emitted for the beef industry, but there is no straightforward method to measure biological efficiency in grazing conditions. The objective of this study was to evaluate a mathematical nutrition model to estimate the feed intake and biological efficiency of mature beef cows. Data from dams (N = 160) and their second and third progeny (312 pairs) were collected from 1953 through 1980. Individual feed intake was measured at 28-d intervals year-round for dams and during 240-d lactation for progeny. Body weights of progeny were measured at 28-d intervals from birth to weaning, and of dams at parturition and weaning each production cycle. The milk yield of dams was measured at 14-d intervals. Dam metabolizable energy intake (DMEI) and milk energy yield (MEL) of each cow were predicted using the Cattle Value Discovery System beef cow (CVDSbc) model for each parity. Biological efficiency (Mcal/kg) was computed as the ratio of observed or predicted DMEI to observed calf weaning weight (PWW). Pearson correlation coefficients were computed using corr.test function and model evaluation was performed using the epiR function in R software. Average (SD) dam weight, PWW, DMEI, and observed MEL were 527 (86) kg, 291 (47) kg, 9584 (2701) Mcal/production cycle, and 1029 (529) Mcal, respectively. Observed and predicted DMEI (r = 0.93 and 0.91), and observed and predicted MEL (r = 0.58 and 0.59) were positively correlated for progeny 2 and 3, respectively. The CVDSbc model under-predicted DMEI (mean bias [MB] = 1,120 ± 76 Mcal, 11.7% of observed value) and MEL (MB = 30 ± 25 Mcal, 2.9% of observed value). Observed and predicted progeny feed intake were not correlated (r = 0.01, P-value = 0.79). Observed and predicted biological efficiency were positively correlated (r = 0.80 and 0.80, P-value ≤ 0.05) for parity 2 and 3, respectively, and the CVDSbc model under-predicted biological efficiency by 11% (MB = 3.59 ± 0.25 Mcal/kg). The CVDSbc provides reasonable predictions of feed intake and biological efficiency of mature beef cows, but further refinement of the relationship between calf feed intake and milk yield is recommended to improve predictions. Mathematical nutrition models can assist in the discovery of the biological efficiency of mature beef cows.

Understanding the relationships between feed efficiency traits measured in different stages of production is necessary to improve feed efficiency across the beef value chain. The objective of this study was to evaluate relationships among feed efficiency traits measured as growing heifers and breeding females and in their progeny in three full production cycles, and relationships of dam residual feed intake (RFI) with lifetime and lifecycle cow efficiency traits. Data were collected on 160 mixed-breed heifers from 240 d of age to weaning of their third progeny, and postweaning performance of progeny until harvest in experiments initiated in 1953, 1954, 1959, 1964, 1969, and 1974. Individual feed offered was recorded daily, and feed refusals measured every 28 d. Milk yield was measured at 14-d intervals throughout lactation by machine or hand milking. Females and progeny were weighed at 28-d intervals and progeny were harvested at a constant endpoint of live grade or age depending upon the experiment. Feed efficiency traits of RFI and residual BW gain (RG) were computed as the residual from linear regression for developing heifers, dams (RFI and residual energy-corrected milk [RECM]), and postweaning progeny. Feed:gain ratio (FCR) was computed for developing heifers and postweaning progeny, and feed:milk energy ratio (FME) was computed for dams. Various measures of cow efficiency were calculated on either a life cycle or lifetime basis using ratios of progeny and dam weight outputs to progeny and dam feed inputs. Pearson correlations were computed among traits adjusted for a random year-breed-diet group effect. Heifer RFI (0.74) and RG (−0.32) were correlated (P ≤ 0.05) with dam RFI in parity 1 only, but were not correlated (P > 0.05) with dam RECM in any parity. Heifer RFI was correlated (P ≤ 0.05) with progeny RFI (0.17) in parity 3 only. Heifer FCR was not correlated with dam FME or progeny FCR in any parity. Dam RFI was weakly correlated (r = 0.25 to 0.36; P ≤ 0.05) among parities, whereas dam FME and RECM were strongly correlated (r = 0.49 to 0.72; P ≤ 0.05) among parities. Dam RFI in parity 1 and 2 was weakly correlated (r = −0.20 to −0.33; P ≤ 0.05) with cow efficiency ratios that included dam weight as an output, whereas dam RFI in parity 3 was not correlated with any cow efficiency ratio. In conclusion, feed efficiency traits were poorly correlated across production segments, but moderately repeatable across production cycles.

Data were collected from 1953 through 1980 from identical and fraternal twin beef and dairy females born in 1953, 1954, 1959, 1964, and 1969, from crossbred females born as singles in 1974, and their progeny. Numbers of dams that weaned at least one calf and were included in the first analysis were 37, 45, and 56 in the 1964, 1969, and 1974 data sets, respectively. Respective numbers of dams that weaned three calves and were included in a second analysis were 6, 8, 8, 22, 33, and 33 in the 1953, 1954, 1959, 1964, 1969, and 1974 experiments. Individual feed consumption was measured at 28-d intervals from the time females were placed on the experiment at 240 d of age until three calves were weaned or the dams had reached 5 yr of age. Residual feed intake (RFI) and residual BW gain (RG) of heifers that subsequently became dams were determined based on ADG and DMI from 240 d of age to first calving. Various measures of cow efficiency were calculated on either a life cycle or actual lifetime basis using ratios of progeny and dam weight outputs to progeny and dam feed inputs. The correlation between RFI and DMI was large and positive (r = 0.67; P < 0.0001), and RG was highly correlated with ADG (r = 0.75; P < 0.0001). Correlations of RFI with cow efficiency ratios that included harvest weight, carcass weight, or weight of trimmed wholesale cuts as measures of output ranged from –0.05 (P > 0.10) to −0.17 (P < 0.10), indicating that heifers with better (i.e., more negative) RFI values tended to become slightly more efficient cows. Correlations of RG with life cycle and actual lifetime cow efficiency ratios ranged from 0.08 (P > 0.10) to 0.23 (P < 0.05), demonstrating that heifers with better (i.e., more positive) values for RG were somewhat more efficient as cows. The correlations were stronger when cow salvage value was included in the measures of cow efficiency. Correlations of DMI and mid-test metabolic BW (MMW) with life cycle cow efficiency ratios that did not include cow salvage value as output ranged from −0.15 (P < 0.10) to −0.22 (P < 0.01). Correlations of DMI and MMW with actual lifetime cow efficiency ratios varied from −0.20 (P < 0.05) to −0.36 (P < 0.001). Therefore, smaller heifers that consumed less feed had superior cow efficiency ratios. Correlations of RFI with carcass grade, backfat thickness, marbling score, and kidney fat of progeny indicated that heifers with superior RFI would tend to produce leaner offspring.

Nearly 1 in every 120 children born has a congenital heart defect. Although surgical therapy has improved survival, many of these children go on to develop right ventricular heart failure (RVHF). The emergence of cardiovascular regenerative medicine as a potential therapeutic strategy for pediatric HF has provided new avenues for treatment with a focus on repairing or regenerating the diseased myocardium to restore cardiac function. Although primarily tried using adult cells and adult disease models, stem cell therapy is relatively untested in the pediatric population. Here, we investigate the ability of electrical stimulation (ES) to enhance the retention and therapeutic function of pediatric cardiac‐derived c‐kit+ progenitor cells (CPCs) in an animal model of RVHF. Human CPCs isolated from pediatric patients were exposed to chronic ES and implanted into the RV myocardium of rats. Cardiac function and cellular retention analysis showed electrically stimulated CPCs (ES‐CPCs) were retained in the heart at a significantly higher level and longer time than control CPCs and also significantly improved right ventricular functional parameters. ES also induced upregulation of extracellular matrix and adhesion genes and increased in vitro survival and adhesion of cells. Specifically, upregulation of β1 and β5 integrins contributed to the increased retention of ES‐CPCs. Lastly, we show that ES induces CPCs to release higher levels of pro‐reparative factors in vitro. These findings suggest that ES can be used to increase the retention, survival, and therapeutic effect of human c‐kit+ progenitor cells and can have implications on a variety of cell‐based therapies. stem cells 2019;37:1528–1541, Top, Conceptual diagram of ex vivo modification of cells. Cells are isolated, modified, and administered to patients as autologous or allogeneic therapies. Bottom, ex vivo modification of pediatric cardiac‐derived progenitor cells with electrical stimulation promotes retention of cells and improves cardiac function in a rat model of heart failure.

Data were collected from 1953 through 1980 from identical and fraternal twin beef and dairy females born in 1953, 1954, 1959, 1964, and 1969, and from crossbred females born as singles in 1974, and their progeny. Numbers of dams that weaned at least 1 calf and were included in the first analysis were 37, 45, and 56 in the 1964, 1969, and 1974 data sets, respectively. Respective numbers of dams that weaned 3 calves and were included in a second analysis were 6, 8, 8, 22, 33, and 33 in the 1953, 1954, 1959, 1964, 1969, and 1974 experiments. Individual feed consumption was measured at 28-d intervals from the time females were placed on the experiment until 3 calves were weaned or the dams had reached 5 yr of age. Residual feed intake (RFI) and residual gain (RG) of the heifers that subsequently became the dams in this study were determined based on ADG and DMI from 240 d of age to first calving. Various measures of cow efficiency were calculated on either a life cycle or actual lifetime basis using ratios of progeny and dam weight outputs to progeny and dam feed inputs. Residual feed intake was phenotypically independent of ADG and metabolic midweight (MMW), whereas the correlation between RFI and DMI was positive and highly significant ( = 0.67; < 0.0001). Residual gain was highly correlated with ADG ( = 0.75; < 0.0001) and had near 0 correlations with DMI and MMW. Correlations indicated that heifers that ate less and had smaller metabolic midweights from 240 d of age to first calving had superior efficiency ratios as cows. Residual feed intake was not significantly correlated with age at puberty, age at calving, or milk production. Results of this study do not indicate any serious antagonisms of postweaning heifer RFI with subsequent cow and progeny performance traits or with life cycle or actual lifetime cow efficiency. In addition, selection for increased RG would result in earlier ages at calving, but would also tend to result in taller and heavier cows.

The explosion of stem cell research in the past several years has made its presence known in the field of cardiology and has been recently tasked with solving one of the largest health problems to afflict humanity: cardiovascular disease (CVD). Although stem cell therapy has shown glimmers of promise, significant problems remain that need to be addressed if these therapies are to ever find true success. One way to achieve this success is to take engineering principles and apply them to fabricate engineered cardiac tissues, composed of the aforementioned therapeutic stem cells and biomaterials to bolster the tissue's reparative capacity. In this review, the authors examine advancements in cardiac cell therapy and biomaterial research and discuss how their combination has been used to create tissue-engineered patches capable of restoring function to the damaged or failing myocardium.

Congenital heart disease is rarely cured by surgery and can lead to life-threatening, intractable right ventricular heart failure (HF). Recent stem cell-based clinical trials have been limited by poor differentiation rates and low cell retention. We propose the aggregation of child cardiac progenitor cells (CPCs) into scaffold-free spheres to improve the differentiation of CPCs into mature cardiac phenotypes by enhancing intercellular Notch signaling, a key regulator of CPC fate decisions. Child CPC spheres were produced at a size of 1500 cells per sphere using a microwell array and cultured in suspension for up to 14 days with 70-80% cell viability. By 7 days in culture, aggregation of CPCs increased protein expression of Notch1 intracellular domain (3.8-fold) and transcript expression of Notch1 signaling pathway members, including ADAM10 (3.6-fold), DLL1 (25.0-fold), and JAG1 (3.2-fold), compared to parallel monolayer cultures. Increased Notch signaling activity was accompanied by high transcript levels of cardiac differentiation markers, including MEF2C, MYH7, and cTNNI. In particular, Nkx2.5, an important transcription factor for cardiomyocyte development, was robustly expressed within the nuclei of aggregated CPCs. Inhibition of Notch signaling activity using the small molecule γ-secretase inhibitor DAPT abrogated these effects, including the nuclear translocation of Nkx2.5. To evaluate the therapeutic effects of CPC spheres compared to single CPCs, right ventricular HF was induced in rats by pulmonary artery banding and cells were transplanted into the right ventricular free wall. By 28 days, echocardiographic measurements showed significantly improved right ventricular function with CPC sphere transplantation compared to single CPC transplantation (tricuspid annular plane systolic excursion: 2.03±0.2 vs. 1.61±0.22 mm, P

The aim of this study was to identify a suitable mathematical model for describing the lactation curve of Awassi, Morkaraman and Tushin sheep breeds and to determine breed differences. Data on milk yield of 182 Awassi, 47 Morkaraman and 74 Tushin ewes were used. Eight empirical models from the literature were used to fit the standard lactation curves. Among them the Wood model (WD) appeared the most appropriate according to mean square prediction error (MSPE), coefficient of determination (R2), Durbin-Watson statistic (DW), and its applicability to the data for all three breeds. There were statistically significant (P

This study aimed to verify genetic relationships between fatty acid composition (FAC) and genotypes of several genes (FABP3, HMGA1, MC4R, IGF2, and FABP4) using pig breeds. The effects of genetic variations on FAC of the longissimus muscle were statistically significant with additive and dominance effects. The polymorphisms of FABP3 and IGF2 had the largest effects on stearic (C18:0, P=0.009) and γ-linoleic (C18:3n6, P=0.039) acids, respectively, whereas HMGA1 and FABP4 did not show significances. The analysis revealed that MC4R was significantly associated with palmitoleic acid (C16:ln7) and MUFA. Allele frequencies of the genes examined in this analysis were significantly skewed or fixed in the Korean native pig (KNP), whereas the allele frequencies of the crossbreds tended to fall between those of the purebreds except that HMGA1 and FABP4 had approximately the same allele frequencies with Duroc and KNP, respectively. The polymorphisms found in this study could be used as genetic markers in breeding programs to simultaneously change proportions of fatty acids in muscle tissues.

Hyperplastic growth and hypertrophic growth within adipose tissue is tightly associated with cell cycle activity. In this study, CCNG2 and CDKN2C were found to be correlated with cell cycle inhibition during fat cell differentiation, whereas CCND3, CCNA1, and ANAPC5 were positively associated with cell cycle activity during fat cell proliferation after selection based on GEO datasets available on the NCBI website. The findings were validated through comparison of expressions of these genes among different tissues/fractions in broiler chickens and time points during primary cell culture using quantitative real-time PCR. Development of broiler subcutaneous adipose tissue was investigated on embryonic days 15 and 17 and on post-hatch days 0, 5, 11, and 33 using H&E staining and PCNA immunostaining with DAPI counter stain. In addition, mRNA expressions of five cell cycle regulators as well as precursor cell and adipocyte markers were measured at those time points. The results suggest that cellular proliferation activity decreased as the fat pad grows, but a population of precursor cells seemed to be maintained until post-hatch day 5 despite increasing differentiation activity. Hypertrophic growth gradually intensified despite a slight cessation on post-hatch day 0 due to increased energy expenditure during hatching and delayed food access. From post-hatch day 5 to day 11, most of the precursor cells may become differentiated. After post-hatch day 11, hyperplastic growth seemed to slow, while hypertrophic growth may become dominant. This study provides further understanding about broiler fat tissue development which is imperative for effective control of fat deposition.

Cigarette smoking is one of the most significant public health issues and the most common environmental cause of preventable cancer deaths worldwide. EGFR (Epidermal Growth Factor Receptor)-targeted therapy has been used in the treatment of LC (lung cancer), mainly caused by the carcinogens in cigarette smoke, with variable success. Presence of mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog) driver oncogene may confer worse prognosis and resistance to treatment for reasons not fully understood. NQO1 (NAD(P)H:quinone oxidoreductase), also known as DT-diaphorase, is a major regulator of oxidative stress and activator of mitomycins, compounds that have been targeted in over 600 pre-clinical trials for treatment of LC. We sequenced KRAS and investigated expression of NQO1 and five clinically relevant proteins (DNMT1, DNMT3a, ERK1/2, c-MET, and survivin) in 108 patients with non-small cell lung carcinoma (NSCLC). NQO1, ERK1/2, DNMT1, and DNMT3a but not c-MET and survivin expression was significantly more frequent in patients with KRAS mutations than those without, suggesting the following: (1) oxidative stress may play an important role in the pathogenesis, worse prognosis, and resistance to treatment reported in NSCLC patients with KRAS mutations, (2) selecting patients based on their KRAS mutational status for future clinical trials may increase success rate, and (3) since oxidation of nucleotides also specifically induces transversion mutations, the high rate of KRAS transversions in lung cancer patients may partly be due to the increased oxidative stress in addition to the known carcinogens in cigarette smoke.

Lung cancer (LC) and colorectal cancer (CRC) are the first and second deadliest types of cancer worldwide. EGFR-based therapy has been used in the treatment of these cancers with variable success. Presence of mutations in the KRAS driver oncogene, possibly induced by environmental factors such as carcinogens in diet and cigarette smoke, may confer worse prognosis and resistance to treatment for reasons not fully understood. Data on possible associations between KRAS mutational status and clinical and metabolic parameters, which may help in clinical management, as well as in identifying risk factors for developing these cancers, are limited in the current literature. We sequenced the KRAS gene and investigated the associations of variations in 108 patients with non-small cell lung carcinoma (NSCLC), the most common form of LC, and in 116 patients with CRC. All of the mutations originated from the guanosine nucleotide and over half of all transversions in NSCLC and CRC were c.34 G>T and c.35 G>T, respectively. c.35 G>A was the most frequent type of transition in both cancers. Excluding smoking, the clinical and metabolic parameters in patients carrying mutant and wild type KRAS were similar except that the CRC patients with transversion mutations were 8.6 years younger than those carrying the transitions (P < 0.01). Dyslipidemia, hypertension, family cancer history, and age of diagnosis older than 60 years were more frequent in NSCLC than CRC (P ≤ 0.04). These results suggest that most of the clinical and metabolic parameters investigated in this study are probably not associated with the more aggressive phenotype and differences in response to EGFR-based treatment previously reported in patients with KRAS mutations. However, the increased rates of abnormal metabolic parameters in patients with NSCLC in comparison to CRC indicate that these parameters may be more important in the management of NSCLC. CRC patients carrying transition mutations are older than those carrying transversions, suggesting that age may determine the type of KRAS mutation in CRC patients.

The objective of this study was to evaluate the relationship between hepatic mitochondrial function and residual feed intake (RFI) in growing beef cattle. In Trial 1, RFI was measured in 29 Angus heifers (initial BW = 258.0 ± 24.9 kg) from divergent IGF-I selection lines created at the Eastern Agricultural Research Station (The Ohio State University) fed a grain-based diet (calculated ME = 2.85 Mcal/kg DM). In Trial 2, RFI was measured in 119 Santa Gertrudis steers (initial BW = 308.4 ± 28.1 kg) fed a roughage-based diet (calculated ME = 2.21 Mcal/kg DM). At the end of the RFI measurement period, cattle in Trial 1 (n = 7 low RFI and n = 7 high RFI) and in Trial 2 (n = 6 low RFI and n = 8 high RFI) with measures of RFI exceeding 0.5 (Trial 1) or 1.0 (Trial 2) SD from the mean RFI were selected to measure mitochondrial function. Overall ADG, DMI, and RFI were 1.19 ± 0.15, 9.31 ± 1.12, and 0.00 ± 0.63 kg/d and 0.83 ± 0.16, 9.48 ± 1.00, and 0.00 ± 0.86 kg/d in Trial 1 and 2, respectively. Cattle with low RFI consumed 13 and 24% less (P 0.10). In Trial 1, cattle with low RFI tended (P = 0.06) to have greater state 3 respiration rates than cattle with high RFI, but state 3 respiration rates were similar (P > 0.10) between cattle with low and high RFI in Trial 2. In both trials, cattle with low RFI had greater (P 0.10) was observed in Trial 2. Proton-leak kinetics were similar (P > 0.05) between cattle with low and high RFI in both trials. These data suggest that ADP has greater control of oxidative phosphorylation in liver mitochondrial of cattle with low RFI compared to their high RFI counterparts.

The aim of this study was to search genetic variants, to map the ovine calpain regulatory subunit gene (CAPN4S) on chromosomes, and to find associations between single nucleotide (nt) polymorphisms (SNPs) and weight traits. Genotyping was performed with 226 purebred, 133 crossbred and 122 animals from eight sheep reference families. The polymerase chain reaction–single strand conformational polymorphism analysis detected genetic variants in the genomic regions between exons 4 and 8. Nt polymorphisms were confirmed at positions 115 (G/C) with CAPN445, 71 (A/G) with CAPN456, and 25 (A/G) and 44 (A/T) with the CAPN478 segments. The Polypay and Targhee purebreds showed significantly different allele frequencies while the mixed population was placed between pure breeds. The two-point linkage analysis using the CRI-MAP version 2.4 identified locations of the CAPN4S segments, showing that CAPN4S is closely linked with the LSCV30 microsatellite marker on chromosome 14. The genotypes of g.45923178G>C had significant mean differences for BWT and WWT, meaning that the identified SNP may be used in marker assisted selection programs. A significant haplo-block was constructed between g.45924950T>C and g.45924969A>T, and major frequencies were observed as AA (0.727) and GT (0.267).

Increase of fat cells (FCs) in adipose tissue is attributed to proliferation of preadipocytes or immature adipocytes in the early stage, as well as adipogenic differentiation in the later stage of adipose development. Although both events are involved in the FC increase, they are contrary to each other, because the former requires cell cycle activity, whereas the latter requires cell cycle withdrawal. Therefore, appropriate regulation of cell cycle inhibition is critical to adipogenesis. In order to explore the important cell cycle inhibitors and study their expression in adipogenesis, we adopted a strategy combining the Gene Expression Omnibus (GEO) database available on the NCBI website and the results of quantitative real-time PCR (qPCR) data in porcine adipose tissue. Three cell cycle inhibitors – cyclin G2 (CCNG2), cyclin-dependent kinase inhibitor 2C (CDKN2C) and peripheral myelin protein (PMP22) – were selected for study because they are relatively highly expressed in adipose tissue compared with muscle, heart, lung, liver and kidney in humans and mice based on two GEO DataSets (GDS596 and GDS3142). In the latter analysis, they were found to be more highly expressed in differentiating/ed preadipocytes than in undifferentiated preadipocytes in human and mice as shown respectively by GDS2366 and GDS2743. In addition, GDS2659 also suggested increasing expression of the three cell cycle inhibitors during differentiation of 3T3-L1 cells. Further study with qPCR in Landrace pigs did not confirm the high expression of these genes in adipose tissue compared with other tissues in market-age pigs, but confirmed higher expression of these genes in FCs than in the stromal vascular fraction, as well as increasing expression of these genes during in vitro adipogenic differentiation and in vivo development of adipose tissue. Moreover, the relatively high expression of CCNG2 in adipose tissue of market-age pigs and increasing expression during development of adipose tissue was also confirmed at the protein level by western blot analysis. Based on the analysis of the GEO DataSets and results of qPCR and Western blotting we conclude that all three cell cycle inhibitors may inhibit adipocyte proliferation, but promote adipocyte differentiation and hold a differentiated state by inducing and maintaining cell cycle inhibition. Therefore, their expression in adipose tissue is positively correlated with age and mature FC number. By regulating the expression of these genes, we may be able to control FC number, and, thus, reduce excessive fat tissue in animals and humans.

CHEK2 (Checkpoint kinase homolog 2) encodes a protein involved in pathways that arrest the cell cycle in response to genomic stress such as DNA damage or replication blocks. Carriers of some of the CHEK2 mutations are at an increased risk of breast cancer. A mutation in the kinase domain of the CHEK2 gene resulting in the R406H substitution has been reported. However, it is currently unknown whether the substitution alters the function of CHEK2 in vitro. We evaluated the effect of the R406H substitution on the normal function of CHEK2 using a yeast complementation assay. The yeast cells lacking Rad53, the yeast homologue of human CHEK2 were transformed with the wild type as well as plasmids carrying mutations resulting in the R406H, 1100delC, and S428F variants. Yeast cells carrying the R406H variant grew at a rate similar to those carrying the wild type CHEK2, whereas the yeast carrying the S428F and 1100delC mutants grew at a slower rate. These results suggest that, unlike the well-known pathogenic alleles such as 1100delC and S428F, the R406H substitution does not abrogate the function of CHEK2. Therefore, this variant is probably not important in development of breast cancer in women.

Reproductive performance of animals affects lifetime productivity. However, improvement of reproductive traits via direct selection is generally slow due to low heritability. Therefore, identification of indicator traits for reproductive performance may enhance genetic response. Previous studies showed that serum IGF-I concentration is a candidate indicator for growth and reproductive traits. The objective of our study was to estimate the variances or covariances of IGF-I concentration with reproductive traits. Data were collected from a divergent selection experiment for serum IGF-I concentration at the Eastern Agricultural Research Station owned by The Ohio State University. The study included a total of 2,662 calves in the 1989 to 2005 calf crops. Variance or covariance components were estimated for direct and maternal genetic effects, maternal environment effects, environment effects, and phenotypic effects using an animal model in a multiple-trait, derivative-free, restricted maximum likelihood (MTDFREML, Boldman et al., 1995) computer program. Direct additive genetic correlations suggest that selection for greater IGF-I concentration (heritability = 0.50 ± 0.07) could lead to increased conception rate (heritability = 0.11 ± 0.06, r = 0.32, P < 0.001) and calving rate (heritability = 0.13 ± 0.06, r = 0.43, P < 0.001) and decreased age at first calving in heifers (heritability = 0.35 ± 0.20, r = -0.40, P < 0.001).

The PCR–SSCP analysis of the 5′ flanking region of the goat insulin-like growth factor-I gene (gIGF-I) was used to screen polymorphisms in a total of 296 goats belonging to two breeds of goats (Markhoz and Kurdi) reared in the Kurdistan province in Iran. The PCR products of different electrophoresis patterns were sequenced and aligned. Sequencing revealed a novel G to A transition at position 422 bp upstream from the ATG codon (g.1617 G > A), according to the GenBank sequence D26119.2 . PCR–RFLP analysis of part of intron 4 and exon 4 of the goat IGF-I gene was used to identify associations between the intron 4/HaeIII polymorphism and economic traits. Results of fixed model analysis revealed that the intron 4/HaeIII polymorphism was significantly associated with yearling weight (YW), post-weaning average daily gain from 3-months to 12-months (ADG4), 6-months to 12-months (ADG6), 9-months to 12-months (ADG7), and first shearing fleece weight (FSFW) (P ≤ 0.05). Animals that possessed the GG genotype for the intron 4/HaeIII mutation appeared potentially more favorable for all mentioned traits. These results suggest that variation in the caprine IGF-I gene is associated with growth traits and yearling fleece weight in Markhoz goats.

A meta-analysis was conducted to examine phenotypic relationships between feed efficiency, scrotal circumference, and semen quality traits in yearling bulls. Data evaluated were obtained from 5 postweaning trials involving Angus (n = 92), Bonsmara (n = 62), and Santa Gertrudis (n = 50) bulls fed diets that ranged from 1.70 to 2.85 Mcal ME/kg DM. After an adaptation period of 24 to 28 d, feed intake was measured daily, and BW was measured at 7- or 14-d intervals during the 70- to 77-d trials. Ultrasound carcass traits (12th-rib back fat thickness, BF; LM area, LMA) and scrotal circumference (SC) were measured at the start and end of each trial. Semen samples were collected by electroejaculation within 51 d of the end of the trials when the age of bulls averaged from 365 to 444 d and were evaluated for progressive sperm motility and morphology. Residual feed intake (RFI) was calculated as the difference between actual DMI and expected DMI from linear regression of DMI on ADG and midtest BW(0.75), with trial, trial by ADG, and trial by midtest BW(0.75) as random effects. Across all studies, bulls with low RFI phenotypes (0.5 SD below the mean RFI of 0) consumed 20% less DM and had 10% less BF but had similar ADG, SC, and semen quality traits compared with high-RFI bulls (0.5 SD above the mean RFI of 0). Gain to feed ratio was strongly correlated with ADG (0.60) and weakly correlated with initial BW (-0.17) and DMI (-0.26). Residual feed intake was not correlated with ADG, initial age, or BW but was correlated with DMI (0.71), G:F (-0.70), and BF (0.20). Initial SC (-0.20), gain in SC (-0.28), and percent normal sperm (-0.17) were correlated with G:F, but only sperm morphology was found to be weakly associated with RFI (0.13). These data suggest that RFI is not phenotypically associated with SC or sperm motility but is weakly associated with sperm morphology.

A divergent selection experiment using feed:gain ratios as the selection criterion was conducted in the early 1980s using Angus beef cattle located at the Eastern Agricultural Research Station (EARS), Belle Valley, OH. Numbers of bulls completing the 140-d postweaning performance test in 1979 through 1983 were 35, 34, 35, 34, and 33, respectively. Each year, the 3 bulls with the highest feed:gain ratios and the 3 bulls with the lowest feed:gain ratios were selected from the individually fed bulls, and were randomly mated as 2-yr-olds to approximately 20 cows each in a test herd of Angus cows also located at EARS. The progeny of these matings were then fed to assess postweaning and carcass performance. The objective of the current study was to compare the results of the experiment based on divergent selection for feed:gain ratios with those that would have been obtained using residual feed intake (RFI) as the selection criterion. RFI was calculated using 3 approaches: (1) dry matter intake (DMI) adjusted for production (i.e., ADG and metabolic midweight) to derive RFI P ; (2) DMI adjusted for ultrasonic backfat thickness at the end of the 140-d test, in addition to ADG and metabolic midweight, to obtain RFI BF ; and (3) DMI estimated using the NRC net energy equations, taking into account the energy and feed required for both gain and maintenance, to derive RFI NRC . RFI was calculated by subtracting expected feed intake, derived using these 3 approaches, from the observed feed intake. Low RFI P and RFI BF bulls consumed less feed than high RFI bulls, whereas no differences existed for weight traits or ADG. Both feed:gain ratio and RFI NRC were significantly correlated with weight traits and ADG on a phenotypic basis. The correlation between RFI P and RFI BF was near 1.0. In addition, rankings of the bulls were very similar based on RFI P and RFI BF . However, RFI BF may be the best selection criterion for feed efficiency examined in this study, as it was phenotypically independent of backfat thickness, in addition to weights and gains, and, therefore, would be less likely to negatively impact marbling score and carcass quality grade.

Data for the current study were obtained from a divergent selection experiment in which the selection criterion was the average serum IGF-I concentration of 3 postweaning blood samples collected from purebred Angus calves. Multiple trait derivative-free REML procedures were used to obtain estimates of inbreeding depression for IGF-I concentration and for BW and BW gains measured from birth to the conclusion of a 140-d postweaning performance test. Included in the analysis were 3,243 animals in the A(-1) matrix, 2,182 of which had valid records for IGF-I concentration. Over the course of the entire selection experiment, inbreeding of the calf averaged 3.3% (SD = 3.1%) and inbreeding of the dam averaged 1.8% (SD = 2.7%). Mean inbreeding levels at the end of the study were 6.82 +/- 0.38% and 4.20 +/- 0.36% for calves and dams, respectively. Annual rates of increase in inbreeding of calves and dams were 0.36 +/- 0.01 (P < 0.0001) and 0.25 +/- 0.01%/yr (P < 0.0001), respectively. Insulin-like growth factor I concentration at d 28 (IGF28), 42 (IGF42), and 56 (IGF56) of the 140-d postweaning test and mean IGF-I concentration decreased by 0.62 +/- 0.88, 1.86 +/- 0.96, 1.92 +/- 0.89, and 1.48 +/- 0.76 ng/mL per 1% increase in inbreeding of calf. Only the regression coefficient for IGF56 differed significantly from zero, although the regression coefficients for IGF42 and mean IGF-I approached significance (P < 0.10). Increases in inbreeding levels of the dams also tended to result in reduced IGF-I concentrations, although the regression coefficients were not significantly different from zero. Inbreeding of calf had highly significant negative effects on all BW and BW gain traits examined, except for birth weight, with regression coefficients ranging from -0.74 +/- 0.20 kg/% increase in calf inbreeding for postweaning BW gain to -1.68 +/- 0.33 kg/% increase in calf inbreeding for off-test BW. Inbreeding of dam had a significant negative effect on birth weight of progeny and tended to have a negative effect on postweaning BW gain (P < 0.10). Preweaning gain of the progeny and BW other than birth weight were not influenced by increases in dam inbreeding. Results indicate that reductions in serum IGF-I concentration due to inbreeding may contribute to the decline in BW and BW gains that is typically associated with increases in inbreeding within populations.

The objective of this research was to further characterize the promoter regions of the bovine and porcine fatty acid-binding protein 4 (FABP4) genes relative to those of other mammals. The DNA sequences of FABP4 promoters for the human, mouse, cow, pig, and dog were obtained from the genomic database of the National Center for Biotechnology Information and also from the sequencing of bovine and porcine genomic DNA clones obtained by 5' PCR racing of genomic DNA. Sequence alignments of these FABP4 promoters using the basic local alignment search tool of the National Center for Biotechnology Information revealed 3 highly conserved promoter regions across the species. Two computational bioinformatics databases and the literature identified the conserved transcription factor-binding sites for C/EBP, adapter primer-1, and boxes of CAAT and TATA in the first conserved proximal promoter region, a direct repeat 1-type PPAR responsive element in the second distal conserved region, and another PPAR responsive element in the third distal conserved promoter region of FABP4 in all 5 mammals. Five new short interspersed repetitive elements (SINE) in the bovine FABP4 promoter and 2 new SINE in the porcine were found, but these SINE did not disrupt the 3 conserved regions, indicating that important regulatory elements are maintained regardless of evolutionary pressure. In conclusion, the conserved cis-acting elements, especially the 2 key adipocyte transcription factors C/EBP and PPAR, may contribute greatly to adipogenic regulation and adipose tissue-specific expression of FABP4 in these mammals. This helps to further characterize and decipher important cis-acting elements that are important for adipocyte development in adipose and muscle tissue.

Angus bulls and heifers from lines divergently selected for serum IGF-I concentration were used to evaluate the effects of IGF-I selection line on growth performance and feed efficiency in 2 studies. In study 1, bulls (low line, n = 9; high line, n = 8; initial BW = 367.1 +/- 22.9 kg) and heifers (low line, n = 9; high line, n = 13; initial BW = 286.4 +/- 28.6 kg) were adapted to a roughage-based diet (ME = 1.95 Mcal/kg of DM) for 24 d and fed individually for 77 d by using Calan gate feeders. In study 2, bulls (low line, n = 15; high line, n = 12; initial BW = 297.5 +/- 34.4 kg) and heifers (low line, n = 9; high line, n = 20; initial BW = 256.0 +/- 25.1 kg) were adapted to a grain-based diet (ME = 2.85 Mcal/kg of DM) for 32 d and fed individually for 70 d by using Calan gate feeders. Blood samples were collected at weaning and at the start and end of each study, and serum IGF-I concentration was determined. Residual feed intake (RFI) was calculated, within study, as the residual from the linear regression of DMI on midtest BW(0.75), ADG, sex, sex by midtest BW(0.75) and sex by ADG. In study 1, calves from the low IGF-I selection line had similar initial and final BW and ADG, compared with calves from the high IGF-I selection line. In addition, DMI and feed conversion ratio were similar between IGF-I selection lines; however, calves from the low IGF-I selection line tended (P < 0.10) to have lesser RFI than calves from the high IGF-I selection line (-0.26 vs. 0.24 +/- 0.31 kg/d). In study 2, IGF-I selection line had no influence on performance or feed efficiency traits. However, there was a tendency (P = 0.15) for an IGF-I selection line x sex interaction for RFI. Bulls from the low IGF-I selection line had numerically lesser RFI than those from the high IGF-I selection line, whereas in heifers, the IGF-I selection line had no effect on RFI. In studies 1 and 2, weaning and initial IGF-I concentrations were not correlated with either feed conversion ratio or RFI. However, regression analysis revealed a sex x IGF-I concentration interaction for initial IGF-I concentration in study 1 and weaning IGF-I concentration in study 2 such that the regression coefficient was positive for bulls and negative for heifers. These data suggest that genetic selection for postweaning serum IGF-I concentration had a minimal effect on RFI in beef cattle.

It was proposed that colon cancer induced by red and nitrite-preserved meat is due to meat-derived N-nitroso compounds in the colonic contents. To explore this view, we previously showed that feeding beef and hot dogs increased the fecal output of total apparent N-nitroso compounds (ANC) in mice. In the current project, adult Swiss mice were fed a semipurified diet and water containing additives for 7 days. Feces from individual mice was collected on day 7, dried, and extracted with water. Extracts were analyzed for ANC as before. Feeding 2.0 g sodium nitrite (NaNO2)/L drinking water raised fecal ANC levels from 5 to 63 nmol/g feces. In a dose-response study, fecal ANC levels were proportional to the nitrite concentration squared. Even 32 mg NaNO2/L raised fecal ANC levels 2.3-fold (P < 0.05). In other results, 64, 125, and 250 mg hemin/kg diet, fed with 2 g NaNO2/L water, showed 2.3, 2.2, and 4.6 times the ANC level for nitrite alone. Sodium nitrate (12 g/L water) did not affect fecal ANC output. Omeprazole (400 mg/kg diet) and sodium ascorbate (23 g/kg diet), when fed with 1 g NaNO2/L water, lowered fecal ANC levels by 65 and 41%, indicating that, when nitrite was fed, acid-catalyzed reactions in the stomach produced ANC, which passed down the gut to the feces. Tests indicated that nitrosothiols constituted about 20% of fecal and hot dog ANC. The observed effect of NaNO2 is thus far not consistent with the proposed hypothesis. The enhancement by hemin may help explain why red meat is a cause of colon cancer.