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Specific types of exercises can improve quality of life, maintain function, and even decrease mortality in patients with various medical disorders. This article presents basic exercise principles for counseling patients with diabetes, arthritis, or osteoporosis. [ABSTRACT FROM AUTHOR]

Despite widespread information on obesity as a risk factor for diabetes, coronary artery disease, hypertension, certain cancers, and depression, most Americans are not engaging in any type of physical activity to combat obesity. Here is how you can help. [ABSTRACT FROM AUTHOR]

Large, multinucleated cells, like syncytiotrophoblasts (STB), are not readily analyzed by standard methods used for single cells, such as single-cell RNA-sequencing and fluorescence-activated cellular sorting (FACS). Here we have demonstrated that fluorescence-activated nuclear sorting (FANS) is suitable to analyze nuclei from STB. Human pluripotent stem cells (PSCs) can be differentiated into a mixed trophoblast populations comprising approximately 20 % STB by treatment with BMP4 (Bone Morphogenetic Protein-4), plus A83-01 and PD173074, inhibitors of activin and FGF2 signaling, respectively (the BAP model) in about a week. Here we demonstrate that FANS can be used to separate two types of STB nuclei from the nine different clusters of trophoblast nuclei previously identified in the BAP model by single nucleus RNA sequencing (snRNAseq). Rather than using cell surface markers, as in FACS, transcription factors in various combinations were employed to target specific nuclear types. Nuclei were isolated at d 8 of BAP differentiation of H1 human embryonic stem cells and fixed in 4 % paraformaldehyde. After permeabilization in 0.1 % triton X-100, nuclei were incubated for 3 and 1 h at 4 °C with primary and secondary antibodies respectively and nuclear samples were then subjected to FANS. By using markers identified by snRNA and immunohistochemistry, nuclei were first sorted into a Topoisomerase-1, or TOP1, bright population and then into the two STB subpopulations by using antibodies to JUNB (Jun B Proto-Oncogene) and TFCP2L1 (Transcription Factor CP2 Like 1). The protocol established here is simple, straightforward, and efficient and can be used on a relatively large scale to sort individual subtypes of nuclei from mixed populations of trophoblasts for further analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Conceptus expansion throughout the uterus of mammalian species with a noninvasive epitheliochorial type of placentation is critical establishing an adequate uterine surface area for nutrient support during gestation. Pig conceptuses undergo a unique rapid morphological transformation to elongate into filamentous threads within 1 h, which provides the uterine surface to support development and maintain functional corpora lutea through the production of estrogen. Conceptus production of a unique interleukin 1β, IL1B2, temporally increases during the period of trophoblast remodeling during elongation. CRISPR/Cas9 gene editing was used to knock out pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. Trophoblast elongation occurred on day 14 in wild-type (IL1B2 +/+ ) conceptuses but did not occur in ILB2-null (IL1B2 -/- ) conceptuses. Although the morphological transition of IL1B2 -/- conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion were decreased, indicating that IL1B2 may be involved in the spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the proinflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Establishment and maintenance of pregnancy in the pig involves activating many physiological, cellular, and molecular signaling pathways between the developing conceptus and hormonally regulated maternal endometrium. Rapid elongation of the pig trophoblast allows for the establishment of sufficient placental surface area for the transport of nutrients to the fetus throughout pregnancy. Estrogens secreted by the conceptus during elongation act on uterine epithelia to induce secretion of uterine factors required for conceptus development and for preventing endocrine secretion of prostaglandin F2α, which would cause luteolysis. Thus, trophoblast expansion within the uterine lumen during early gestation is an essential process for implantation and maintenance of pregnancy in species with an epitheliochorial form of placentation. In the pig, rapid conceptus elongation involves the unique expression of interleukin-1 beta 2 (IL1B2), which establishes pro-inflammatory effects that may be tempered by the spatiotemporal secretion of estrogen from the conceptuses. The present review provides current information on pig conceptus remodeling and signaling via estrogen and IL1B2 pathways, as well as endometrial responses to those conceptus factors leading to establishment of pregnancy., (© 2017 Wiley Periodicals, Inc.)

Atrazine has been implicated in reproductive dysfunction of exposed organisms, and previous studies documented decreased egg production in Japanese medaka (Oryzias latipes) and fathead minnows (Pimephales promelas) during 30-d to 38-d exposures to 0.5 µg/L, 5 µg/L, and 50 µg/L atrazine. The authors evaluated possible mechanisms underlying the reduction in egg production. Gene expression in steroidogenesis pathways and the hypothalamus-pituitary-gonad axis of male and female fish was measured. Atrazine did not significantly induce gonad aromatase (cyp19a1a) expression. An atrazine-induced shift in the number of females in an active reproductive state was observed. Expression of the egg maturation genes vitellogenin 1 (vtg1) and zona pellucida glycoprotein 3.1 (zp3.1) in medaka females was correlated and had a bimodal distribution. In both species, females with low vtg1 or zp3.1 expression also had low expression of steroidogenesis genes in the gonad, estrogen receptor in the liver, and gonadotropins in the brain. In the medaka, the number of females per tank that had high expression of zp3.1 was significantly correlated with egg production per tank. The number of medaka females with low expression of zp3.1 increased significantly with atrazine exposure. Thus, the decline in egg production observed in response to atrazine exposure may be the result of a coordinated downregulation of genes required for reproduction in a subset of females. Environ Toxicol Chem 2016;35:2230-2238. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America., (Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.)

Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world's population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock.

Establishment of pregnancy in pigs involves maintaining progesterone secretion from the corpora lutea in addition to regulating a sensitive interplay between the maternal immune system and attachment of the rapidly expanding trophoblast for nutrient absorption. The peri-implantation period of rapid trophoblastic elongation followed by attachment to the maternal uterine endometrium is critical for establishing a sufficient placental-uterine interface for subsequent nutrient transport for fetal survival to term, but is also marked by the required conceptus release of factors involved with stimulating uterine secretion of histotroph and modulation of the maternal immune system. Many endometrial genes activated by the conceptus secretory factors stimulate a tightly controlled proinflammatory response within the uterus. A number of the cytokines released by the elongating conceptuses stimulate inducible transcription factors such as nuclear factor kappa B (NFKB) potentially regulating the maternal uterine proinflammatory and immune response. This review will establish the current knowledge for the role of conceptus cytokine production and release in early development and establishment of pregnancy in the pig.

Atrazine is an effective broadleaf herbicide and the second most heavily used herbicide in the United States. Effects along the hypothalamus-pituitary-gonad axis in a number of vertebrate taxa have been demonstrated. Seasonally elevated concentrations of atrazine in surface waters may adversely affect fishes, but only a few studies have examined reproductive effects of this chemical. The present study was designed to evaluate a population endpoint (egg production) in conjunction with histological (reproductive stage, gonad pathology) and biochemical (aromatase activity, sex hormone production) phenotypes associated with atrazine exposure in Japanese medaka. Adult virgin breeding groups of one male and four females were exposed to nominal concentrations of 0, 0.5, 5.0, and 50 μg/L (0, 2.3, 23.2, 231 nM) of atrazine in a flow-through diluter for 14 or 38 days. Total egg production was lower (36-42%) in all atrazine-exposed groups as compared to the controls. The decreases in cumulative egg production of atrazine-treated fish were significant by exposure day 24. Reductions in total egg production in atrazine treatment groups were most attributable to a reduced number of eggs ovulated by females in atrazine-treated tanks. Additionally, males exposed to atrazine had a greater number of abnormal germ cells. There was no effect of atrazine on gonadosomatic index, aromatase protein, or whole body 17 β-estradiol or testosterone. Our results suggest that atrazine reduces egg production through alteration of final maturation of oocytes. The reduced egg production observed in this study was very similar to our previously reported results for fathead minnow. This study provides further information with which to evaluate atrazine's risk to fish populations., (Published by Elsevier B.V.)

DNA modifications, such as methylation and hydroxymethylation, are pivotal players in modulating gene expression, genomic imprinting, X-chromosome inactivation, and silencing repetitive sequences during embryonic development. Aberrant DNA modifications lead to embryonic and postnatal abnormalities and serious human diseases, such as cancer. Comprehensive genome-wide DNA methylation and hydroxymethylation studies provide a way to thoroughly understand normal development and to identify potential epigenetic mutations in human diseases. Here we established a working protocol for methylated DNA immunoprecipitation combined with next-generation sequencing [methylated DNA immunoprecipitation (MeDIP)-seq] for low starting amounts of genomic DNA. By using spike-in control DNA sets with standard cytosine, 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC), we demonstrate the preferential binding of antibodies to 5mC and 5hmC, respectively. MeDIP-PCRs successfully targeted highly methylated genomic loci with starting genomic DNA as low as 1 ng. The enrichment efficiency declined for constant spiked-in controls but increased for endogenous methylated regions. A MeDIP-seq library was constructed starting with 1 ng of DNA, with the majority of fragments between 250 bp and 600 bp. The MeDIP-seq reads showed higher quality than the Input control. However, after being preprocessed by Cutadapt, MeDIP (97.53%) and Input (94.98%) reads showed comparable alignment rates. SeqMonk visualization tools indicated MeDIP-seq reads were less uniformly distributed across the genome than Input reads. Several commonly known unmethylated and methylated genomic loci showed consistent methylation patterns in the MeDIP-seq data. Thus, we provide proof-of-principle that MeDIP-seq technology is feasible to profile genome-wide DNA methylation in minute DNA samples, such as oocytes, early embryos, and human biopsies.

After the knock-out (KO) of α1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Until now, both monoallelic and biallelic CMAH KO pigs are healthy and show no sign of abnormality and off-target mutations. Therefore, these CMAH null pigs on the Gal-T KO background could serve as an important model for the xenotransplantation.

Although pigs are used widely as models of human disease, their utility as models has been enhanced by genetic engineering. Initially, transgenes were added randomly to the genome, but with the application of homologous recombination, zinc finger nucleases, and transcription activator-like effector nuclease (TALEN) technologies, now most any genetic change that can be envisioned can be completed. To date these genetic modifications have resulted in animals that have the potential to provide new insights into human diseases for which a good animal model did not exist previously. These new animal models should provide the preclinical data for treatments that are developed for diseases such as Alzheimer's disease, cystic fibrosis, retinitis pigmentosa, spinal muscular atrophy, diabetes, and organ failure. These new models will help to uncover aspects and treatments of these diseases that were otherwise unattainable. The focus of this review is to describe genetically engineered pigs that have resulted in models of human diseases.

Background: Anatomic and physiological similarities to the human make swine an excellent large animal model for human health and disease., Methods: Cloning from a modified somatic cell, which can be determined in cells prior to making the animal, is the only method available for the production of targeted modifications in swine., Results: Since some strains of swine are similar in size to humans, technologies that have been developed for swine can be readily adapted to humans and vice versa. Here the importance of swine as a biomedical model, current technologies to produce genetically enhanced swine, current biomedical models, and how the completion of the swine genome will promote swine as a biomedical model are discussed., Conclusions: The completion of the swine genome will enhance the continued use and development of swine as models of human health, syndromes and conditions.

Engineered zinc finger nucleases (ZFN) are rapidly gaining popularity as a means to enhance the rate and specificity of DNA modifications in plant and animal cells. Repair-mediated gene modification by ZFN is driven by introducing DNA double-strand breaks via a nonspecific nuclease domain linked to a sequence-specific zinc finger nucleotide recognition domain. This review examines the use of ZFN to produce genetically modified swine and the potential of this technology for the future. By combining conventional gene targeting methods with somatic cell nuclear transfer, several genetically modified pig models have been produced. These conventional techniques are inefficient in mammalian somatic cells and provide little control over the site specificity and rate of exogenous DNA integration. The use of engineered ZFN that bind and cleave genomic DNA at specific loci can enhance targeting efficiencies by orders of magnitude. Recent publication of the first genetic modification in pigs by combining ZFN technology with somatic cell nuclear transfer has opened the door to genome targeting with a precision that was not previously possible in a large animal model. Since that time, model pigs with selective knockout of endogenous genes have been produced. This review will examine the use of ZFN to generate these pig models and the potential of ZFN to accelerate the production of genetically modified pigs of agricultural and biomedical importance. Current methods of ZFN design, important considerations for their safe and effective use in modification of the swine genome, and future innovative applications of this technology in pigs will be discussed.

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H(2)O(2)), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H(2)O(2) would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT-PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H(2)O(2) in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H(2)O(2) in vasodilation and in the mechanisms underlying vascular health and disease.

Genetically modified swine hold great promise in the fields of agriculture and medicine. Currently, these swine are being used to optimize production of quality meat, to improve our understanding of the biology of disease resistance, and to reduced waste. In the field of biomedicine, swine are anatomically and physiologically analogous to humans. Alterations of key swine genes in disease pathways provide model animals to improve our understanding of the causes and potential treatments of many human genetic disorders. The completed sequencing of the swine genome will significantly enhance the specificity of genetic modifications, and allow for more accurate representations of human disease based on syntenic genes between the two species. Improvements in both methods of gene alteration and efficiency of model animal production are key to enabling routine use of these swine models in medicine and agriculture., (Copyright © 2011 Wiley Periodicals, Inc.)

While the salutary effects of exercise training on conduit artery endothelial cells have been reported in animals and humans with cardiovascular risk factors or disease, whether a healthy endothelium is alterable with exercise training is less certain. The purpose of this study was to evaluate the impact of exercise training on transcriptional profiles in normal endothelial cells using a genome-wide microarray analysis. Brachial and internal mammary endothelial gene expression was compared between a group of healthy pigs that exercise trained for 16-20 wk (n = 8) and a group that remained sedentary (n = 8). We found that a total of 130 genes were upregulated and 84 genes downregulated in brachial artery endothelial cells with exercise training (>1.5-fold and false discovery rate <15%). In contrast, a total of 113 genes were upregulated and 31 genes downregulated in internal mammary artery endothelial cells using the same criteria. Although there was an overlap of 66 genes (59 upregulated and 7 downregulated with exercise training) between the brachial and internal mammary arteries, the identified endothelial gene networks and biological processes influenced by exercise training were distinctly different between the brachial and internal mammary arteries. These data indicate that a healthy endothelium is indeed responsive to exercise training and support the concept that the influence of physical activity on endothelial gene expression is not homogenously distributed throughout the vasculature.

Endothelial adaptations to exercise training are not exclusively conferred within the active muscle beds. Herein, we summarize key studies that have evaluated the impact of chronic exercise on the endothelium of vasculatures perfusing nonworking skeletal muscle, brain, viscera, and skin, concluding with discussion of potential mechanisms driving these endothelial adaptations.

Atrazine, the widely used herbicide, has shown to affect the hypothalamus-pituitary-gonad axis in certain vertebrate species, but few studies have examined reproductive effects of this chemical on fish. Our study was designed to evaluate a population endpoint (egg production) in conjunction with histological (e.g., gonad development) and biochemical (e.g., hormone production) phenotypes associated with atrazine exposure in fathead minnows. Adult virgin breeding groups of 1 male and 2 females were exposed to nominal concentrations of 0, 0.5, 5.0, and 50 microg/L of atrazine in a flow-through diluter for 14 or 30 days. Total egg production was lower (19-39%) in all atrazine-exposed groups as compared to the controls. The decreases in cumulative egg production of atrazine treated fish were significant by 17-20 days of exposure. Reductions in egg production in atrazine treatment groups were most attributable to reduced numbers of spawning events with increased atrazine exposure concentrations. Gonad abnormalities were observed in both male and female fish of atrazine-exposed fish. Our results also indicate that atrazine reduces egg production through alteration of final maturation of oocytes. The reproductive effects observed in this study warrant further investigation and evaluation of the potential risks posed by atrazine, particularly feral populations of fish from streams in agricultural areas with high use of this herbicide., ((c) 2010 Elsevier B.V. All rights reserved.)

Regular physical activity (endurance training, ET) has a strong positive link with cardiovascular health. The aim of this review is to draw together the current knowledge on gene expression in different cell types comprising the vessels of the circulatory system, with special emphasis on the endothelium, and how these gene products interact to influence vascular health. The effect beneficial effects of ET on the endothelium are believed to result from increased vascular shear stress during ET bouts. A number of mechanosensory mechanisms have been elucidated that may contribute to the effects of ET on vascular function, but there are questions regarding interactions among molecular pathways. For instance, increases in flow brought on by ET can reduce circulating levels of viscosity and haemostatic and inflammatory variables that may interact with increased shear stress, releasing vasoactive substances such as nitric oxide and prostacyclin, decreasing permeability to plasma lipoproteins as well as the adhesion of leucocytes. At this time the optimal rate-of-flow and rate-of-change in flow for determining whether anti-atherogenic or pro-atherogenic processes proceed remain unknown. In addition, the impact of haemodynamic variables differs with vessel size and tissue type in which arteries are located. While the hurdles to understanding the mechanism responsible for ET-induced alterations in vascular cell gene expression are significant, they in no way undermine the established benefits of regular physical activity to the cardiovascular system and to general overall health. This review summarizes current understanding of control of vascular cell gene expression by exercise and how these processes lead to improved cardiovascular health.

Development of a transgenic porcine biomedical research model requires effective delivery of DNA into the donor cell followed by selection of genetically modified somatic cell lines to be used for nuclear transfer. The objective of the current study was 2-fold: (1) to compare the effectiveness of a single 1 ms pulse of different voltages (V; 100, 150, 200, 250, 300, 350) and multiple 1 ms pulses (1, 2, 3, 4 or 5) at 300 V for delivery and expression of super-coiled GFP vector in surviving cells of three fetal fibroblast cell lines, and (2) to determine the ability of these electroporation parameters to produce stably transfected fibroblast colonies following G418 selection. Cell line (P < 0.001) and voltage (P < 0.001) affected DNA delivery into the cell as assessed by GFP expression while survival at 24 h was affected by voltage (P < 0.001) and not by cell line (P = 0.797). Using a single pulse while increasing voltage resulted in the percentage of GFP expressing cells increasing from 3.2 +/- 0.8% to 43.0 +/- 3.4% while survival decreased from 90.5 +/- 8.0% to 44.8 +/- 2.0%. The number of pulses at 300 V significantly affected survival (P < 0.001) and GFP expression (P < 0.001). Survival steadily decreased following 1-5 pulses from 63.2 +/- 6.3% to 3.0 +/- 0.3% with GFP expression of surviving cells increasing from 35.6 +/- 2.67% to 71.4 +/- 6.1%. Electroporation of a selectable marker at a 1:1 copy number ratio to a co-electroporated transgene resulted in 83% of G418 resistant colonies also being PCR positive for the secondary transgene. These electroporation conditions, specifically, three 1 ms pulses of 300 V to 200 muL of 1 x 10(6) cells/mL in the presence of 12.5 mug DNA/mL effectively introduced DNA into somatic cells. The utilization of these conditions produced numerous transgenic fibroblast colonies following G418 selection that when used for somatic cell nuclear transfer resulted in the production of live offspring.

Application of intermittent pneumatic compressions (IPC) is an extensively used therapeutic strategy in vascular medicine, but the mechanisms by which this method works are unclear. We tested the hypothesis that acute application (150 min) of cyclic leg compressions in a rat model signals upregulation of angiogenic factors in skeletal muscle. To explore the impact of different pressures and frequency of compressions, we divided rats into four groups as follows: 120 mmHg (2 s inflation/2 s deflation), 200 mmHg (2 s/2 s), 120 mmHg (4 s/16 s), and control (no intervention). Blood flow and leg oxygenation (study 1) and the mRNA expression of angiogenic mediators in the rat tibialis anterior muscle (study 2) were assessed after a single session of IPC. In all three groups exposed to the intervention, a modest hyperemia (approximately 37% above baseline) between compressions and a slight, nonsignificant increase in leg oxygen consumption (approximately 30%) were observed during IPC. Compared with values in the control group, vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) mRNA increased significantly (P < 0.05) only in rats exposed to the higher frequency of compressions (2 s on/2 s off). Endothelial nitric oxide synthase, matrix metalloproteinase-2, and hypoxia-inducible factor-1alpha mRNA did not change significantly following the intervention. These findings show that IPC application augments the mRNA content of key angiogenic factors in skeletal muscle. Importantly, the magnitude of changes in mRNA expression appeared to be modulated by the frequency of compressions such that a higher frequency (15 cycles/min) evoked more robust changes in VEGF and MCP-1 compared with a lower frequency (3 cycles/min).

The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca(2+) followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst-stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected (P > 0.05). All fusion/activation treatments produced live, pCAGG-EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer., ((c) 2008 Wiley-Liss, Inc.)

Multipotent skin-derived progenitors (SKP) can produce both neural and mesodermal progeny in vitro, sharing the characteristics of embryonic neural crest stem cells. However, the molecular basis for the property of multiple lineage potential and neural crest origin of SKPs is still elusive. Here we report the cooperative expression of pluripotency related genes (POU5F1, SOX2, NANOG, STAT3) and neural crest marker genes (p75NTR, TWIST1, PAX3, SNAI2, SOX9, SOX10) in GFP-transgenic porcine skin-derived progenitors (pSKP). The proportion of cells positive for POU5F1, nestin, fibronectin, and vimentin were 12.3%, 15.1%, 67.9% and 53.7%, showing the heterogeneity of pSKP spheres. Moreover, pSKP cells can generate both neural (neurons and glia) and mesodermal cell types (smooth muscle cells and adipocytes) in vitro, indicating the multiple lineage potency. Four transcription factors (POU5F1, SNAI2, SOX9, and PAX3) were identified that were sensitive to mitogen (FBS) and/or growth factors (EGF and bFGF). We infer that POU5F1, SNAI2, SOX9, and PAX3 may be the key players for maintaining the neural crest derived multipotency of SKP cells in vitro. This study has provided new insight into the molecular mechanism of stemness for somatic-derived stem cells at the level of transcriptional regulation.

There have been many trials describing the effects of polyunsaturated fatty acids (PUFA) on fecundity, neonatal development, and maternal behavior in humans, but few controlled studies in rodents. We examined the effects of a maternal diet high in omega 3 (N-3) or omega 6 (N-6) PUFA on NIH Swiss mice. Female mice were ad libitum fed one of three complete and balanced diets (N-3, enriched in menhaden oil; N-6, enriched in corn oil; C, control diet, Purina 5015) from age 4 wk until the end of the study. Mice were bred at approximately 19 wk and 27 wk of age, providing a total of 838 pups from 129 litters in two experiments. After weaning their pups from parity 1, behavior of dams was assessed on elevated-plus and open-field mazes. Although the fraction of male pups from the N-3 and C groups was not different from 0.5, dams on the N-6 diet birthed more daughters than sons (213 vs. 133; P < 0.001). Although maternal stress has been reported to favor birth of daughters, the behavior of N-6 dams was not different from controls. By contrast, the N-3 dams displayed greater anxiety, spending less time in the open arms and more time in the closed arms of the elevated maze and traveling less distance and exhibiting less exploratory behavior in the open field (P < 0.05). N-3 dams tended to produce smaller litters than C dams, and N-3-suckled pups gained less weight (P < 0.05). In conclusion, the N-3 diet had negative effects on murine fecundity and maternal behavior, whereas the N-6 diet favored birth of daughters.

Skewing of the sex ratio towards males occurs among pups born to mice fed a very high saturated fat (VHF) diet. In the present study, we tested whether the fat content of the VHF diet rather than the number of calories consumed is responsible for this effect. Eight-week-old NIH Swiss mice were placed on the VHF diet either ad libitum (VHF) or in a restricted manner (VHF-R). The VHF-R mice gained weight at a similar rate to controls fed a standard chow diet. Mice were bred at 15 wk and subsequently at 26 wk and 35 wk of age. Overall, the VHF, VHF-R, and control groups delivered 244, 242, and 274 pups, respectively, with male proportions of 0.60, 0.43, and 0.48, respectively. The pup sex ratios of the VHF group (favoring males) and VHF-R group (favoring females) each differed from 0.5 (P < 0.01). The sex ratios also differed (P < 0.0001) between the VHF and control groups, and between the VHF and VHF-R groups. Within the diet groups, maternal body weight had no effect on sex ratio. Serum leptin concentrations among the dams were similar in the VHF and VHF-R groups but higher than in the control group, while the IGF1 and corticosterone levels were comparable in all three groups. Therefore, the atypical sex ratios of offspring born to dams on the VHF diet seem to be influenced by the amount of fat consumed. Since males fed the VHF diet had neither more Y-sperm nor sired more sons than daughters, the dietary effects are manifested exclusively through the female.

The potential effects of proposed lead-zinc mining in an ecologically sensitive area were assessed by studying a nearby mining district that has been exploited for about 30 y under contemporary environmental regulations and with modern technology. Blood and liver samples representing fish of three species (largescale stoneroller, Campostoma oligolepis, n=91; longear sunfish, Lepomis megalotis, n=105; and northern hog sucker, Hypentelium nigricans, n=20) from 16 sites representing a range of conditions relative to mining activities were collected. Samples were analyzed for metals (also reported in a companion paper) and for biomarkers of metals exposure [erythrocyte delta-aminolevulinic acid dehydratase (ALA-D) activity; concentrations of zinc protoporphyrin (ZPP), iron, and hemoglobin (Hb) in blood; and hepatic metallothionein (MT) gene expression and lipid peroxidation]. Blood lead concentrations were significantly higher and ALA-D activity significantly lower in all species at sites nearest to active lead-zinc mines and in a stream contaminated by historical mining than at reference or downstream sites. ALA-D activity was also negatively correlated with blood lead concentrations in all three species but not with other metals. Iron and Hb concentrations were positively correlated in all three species, but were not correlated with any other metals in blood or liver in any species. MT gene expression was positively correlated with liver zinc concentrations, but neither MT nor lipid peroxidase differences among fish grouped according to lead concentrations were statistically significant. ZPP was not detected by hematofluorometry in most fish, but fish with detectable ZPP were from sites affected by mining. Collectively, these results confirm that metals are released to streams from active lead-zinc mining sites and are accumulated by fish.

In mice, the relative numbers of male and female pups per litter not only can vary but can probably change over the course of pregnancy in response to numerous environmental and physiological factors. As such, a technique is required to determine gender at several developmental stages. Here we describe a robust and accurate fluorescent in situ hybridization (FISH) procedure for determining chromosomal sex that can be applied with minimal modification to sperm, pre-and post-implantation conceptuses and recovered dead post-natal pups. Sperm was prepared for FISH analysis y using a modified microwave decondensation-denaturation technique. Preimplantation conceptuses (0.5dpc) were cultured to the morula stage before sexing. They were then acid-treated to remove the zona pellucida. Tissue homogenates from postimplantational conceptuses (8.5dpc) and stillborn pups were fixed to pre-etched slides. Specimens were hybridized with identical, commercially available DNA probes for the X (FITC) and Y (Cy3) chromosomes. Sperm ratios met the expected value of 0.5 when determined by using XY FISH. Preimplantation conceptuses pre-treated with pepsin yielded distinct fluorescence of X and Y chromosomes in morulae, whereas microwave decondensation resulted in loss of conceptuses from the slide. Both 4.0 and 8.5dpc conceptuses displayed mean sex ratios of 0.5. Post-natal FISH analysis allowed gender identification of pups that could not be sexed due to developmental abnormalities or partial cannibalism. FISH analysis of sperm and of multiple conceptuses or post-natal tissue provided a cost-effective, accurate alternative to PCR-based sex determination.

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17beta-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 +/- 3.0 pg/ml), compared with LF mice (5.2 +/- 2.3 pg/ml; P< or = 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 +/- 3.0 pg/ml) versus the LF group (32.7 +/- 8.4 pg/ml; P< or = 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 +/- 6.7 ng/ml) versus the 5015 group (60.1 +/- 4.9 ng/ml; P< or = 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 +/- 0.2 mmol/l) than LF and control mice (0.5 +/- 0.1 and 0.6 +/- 0.1 mmol/l respectively; P< or = 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 +/- 0.1 mmol/l) than LF and control females (0.4 +/- 0.1 and 0.6 +/- 0.1 mmol/l; P< or = 0.05). Mean vaginal pH of VHF females (6.41 +/- 0.09) was lower than 5015 females (6.76 +/- 0.10; P< or = 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.

As part of a broader investigation into the effects of creosote treatments on the aquatic biota in pond microcosms, we examined the possible implications for vitellogenin (Vtg) production in Oncorhynchus mykiss [rainbow trout (RT)]. Vtg is the precursor of egg yolk protein and has emerged as a useful biomarker of exposure to estrogenic substances. Our a priori intent was to assess the ability of the creosote treatments (nominal cresoste concentrations were 0, 3, and 10 microl/L immediately after the last subsurface addition) to induce estrogenic responses in RT. The data showed no evidence of an estrogenic response in the treated fish. During the course of the experiment, however, the fish matured and began to produce Vtg, probably in response to endogenous estrogen. A posteriori analysis of the Vtg data from the maturing fish showed that after 28 days, the plasma Vtg concentrations were about 15-fold lower in fish from the creosote-treated microcosms compared with fish from the reference microcosm. Although the experiment design does not permit mechanistic insights, our observation suggests that exposure of female fish to PAH mixtures such as creosote can impair the production of Vtg with possible health implications for embryos and larvae.

We assessed the exposure of fish from the Spring and Neosho Rivers in northeast Oklahoma, USA, to lead, zinc, and cadmium from historical mining in the Tri-States Mining District (TSMD). Fish (n = 74) representing six species were collected in October 2001 from six sites on the Spring and Neosho Rivers influenced to differing degrees by mining. Additional samples were obtained from the Big River, a heavily contaminated stream in eastern Missouri, USA, and from reference sites. Blood from each fish was analyzed for Pb, Zn, Cd, Fe, and hemoglobin (Hb). Blood also was analyzed for delta-aminolevulinic acid dehydratase (ALA-D) activity. The activity of ALA-D, an enzyme involved in heme synthesis, is inhibited by Pb. Concentrations of Fe and Hb were highly correlated (r = 0.89, p < 0.01) across all species and locations and typically were greater in common carp (Cyprinus carpio) than in other taxa. Concentrations of Pb, Zn, and Cd typically were greatest in fish from sites most heavily affected by mining and lowest in reference samples. The activity of ALA-D, but not concentrations of Hb or Fe, also differed significantly (p < 0.01) among sites and species. Enzyme activity was lowest in fish from mining-contaminated sites and greatest in reference fish, and was correlated negatively with Pb in most species. Statistically significant (p < 0.01) linear regression models that included negative terms for blood Pb explained as much as 68% of the total variation in ALA-D activity, but differences among taxa were highly evident. Positive correlations with Zn were documented in the combined data for channel catfish (Icralurus punctatus) and flathead catfish (Pylodictis olivaris), as has been reported for other taxa, but not in bass (Micropterus spp.) or carp. In channel catfish, ALA-D activity appeared to be more sensitive to blood Pb than in the other species investigated (i.e., threshold concentrations for inhibition were lower). Such among-species differences are consistent with previous studies. Enzyme activity was inhibited by more than 50% relative to reference sites in channel catfish from several TSMD sites. Collectively, our results indicate that Pb is both bioavailable and active biochemically in the Spring-Neosho River system.

The H4IIE cell bioassay has proven utility as a screening tool for planar halogenated hydrocarbons (PHHs) and structurally similar chemicals accumulated in organisms from the wild. This bioassay has additional applications in hazard assessment of PHH exposed populations. In this review, the toxicological principles, current protocols, performance criteria, and field applications for the assay are described. The H4IIE cell bioassay has several advantages over the analytical measurement of PHHs in environmental samples, but conclusions from studies can be strengthened when both bioassay and analytical chemistry data are presented together. Often, the bioassay results concur with biological effects in organisms and support direct measures of PHHs. For biomonitoring purposes and prioritization of PHH-contaminated environments, the H4IIE bioassay may be faster and less expensive than analytical measurements. The H4IIE cell bioassay can be used in combination with other biomarkers such as in vivo measurements of CYP1A1 induction to help pinpoint the sources and identities of dioxin-like chemicals. The number of studies that measure H4IIE-derived TCDD-EQs continues to increase, resulting in subtle improvements over time. Further experiments are required to determine if TCDD-EQs derived from mammalian cells are adequate predictors of toxicity to non-mammalian species. The H4IIE cell bioassay has been used in over 300 published studies, and its combination of speed, simplicity, and ability to integrate the effects of complex contaminant mixtures makes it a valuable addition to hazard assessment and biomonitoring studies.

Objective: To provide insight into discussions at the Surgeon General's Listening Session, "Toward a National Action Plan on Overweight and Obesity," and to complement The Surgeon General's Call to Action to Prevent and Decrease Overweight and Obesity., Research Methods and Procedures: On December 7 and 8, 2000, representatives from federal, state, academic, and private sectors attended the Surgeon General's Listening Session and were given an opportunity to recommend what to include in a national plan to address overweight and obesity. The public was invited to comment during a corresponding public comment period. The Surgeon General's Listening Session was also broadcast on the Internet, allowing others to view the deliberations live or access the archived files. Significant discussion points from the Listening Session have been reviewed by representatives of the federal agencies and are the basis of this complementary document., Results: Examples of issues, strategies, and barriers to change are discussed within five thematic areas: schools, health care, family and community, worksite, and media. Suggested cooperative or collaborative actions for preventing and decreasing overweight and obesity are described. An annotated list of some programmatic partnerships is included., Discussion: The Surgeon General's Listening Session provided an opportunity for representatives from family and community groups, schools, the media, the health-care environment, and worksites to become partners and to unite around the common goal of preventing and decreasing overweight and obesity. The combination of approaches from these perspectives offers a rich resource of opportunity to combat the public health epidemic of overweight and obesity.

An outdoor microcosm study was conducted in order to evaluate the kinetics of immunomodulation in rainbow trout (Oncorhynchus mykiss) exposed to liquid creosote. Fish were sampled on d 7, 14, 21, and 28 from microcosms dosed initially with 0, 3, and 10 microl/L of creosote. Pronephros leukocytes were monitored for phagocytic activity, oxidative burst, and surface immunoglobulin-positive (Slg+) B-cell counts. Oxidative burst was inhibited by creosote exposure; however, by sampling d 28, the burst response returned to near control levels. Phagocytic activity, on the other hand, was stimulated, peaking on sampling d 7, then returning to near control levels by d 28. Although control Slg+ B-cell counts were quite variable across sampling days, Slg+ B-cell counts were also elevated in creosote-exposed fish after seven days of exposure. Slg+ B-cell numbers decreased significantly to near control levels during the remainder of the study. The overall results from this study confirm that creosote has the potential to alter certain immune parameters, and emphasize the importance of monitoring changes in the immune system during exposure. Polycyclic aromatic hydrocarbons (PAHs), a major constituent of liquid creosote, are the suspected immune-altering agents.

This review compiles and evaluates existing scientific information on the use, limitations, and procedural considerations for EROD activity (a catalytic measurement of cytochrome P4501A induction) as a biomarker in fish. A multitude of chemicals induce EROD activity in a variety of fish species, the most potent inducers being structural analogs of 2,3,7,8-tetracholordibenzo-p-dioxin. Although certain chemicals may inhibit EROD induction/activity, this interference is generally not a drawback to the use of EROD induction as a biomarker. The various methods of EROD analysis currently in use yield comparable results, particularly when data are expressed as relative rates of EROD activity. EROD induction in fish is well characterized, the most important modifying factors being fish species, reproductive status and age, all of which can be controlled through proper study design. Good candidate species for biomonitoring should have a wide range between basal and induced EROD activity (e.g., common carp, channel catfish, and mummichog). EROD activity has proven value as a biomarker in a number of field investigations of bleached kraft mill and industrial effluents, contaminated sediments, and chemical spills. Research on mechanisms of CYP1A-induced toxicity suggests that EROD activity may not only indicate chemical exposure, but also may also precede effects at various levels of biological organization. A current research need is the development of chemical exposure-response relationships for EROD activity in fish. In addition, routine reporting in the literature of EROD activity in standard positive and negative control material will enhance confidence in comparing results from different studies using this biomarker.

Previously, exposure of fish to polycyclic aromatic hydrocarbons (PAHs) in both field and laboratory settings has been associated with eye damage, but this has only been expressed qualitatively. In this study, an automated scanning laser system has been employed to quantitatively evaluate changes in lens optical quality in rainbow trout (Oncorhynchus mykiss) following their in vivo exposure to creosote, which is a complex mixture with many PAHs. Rainbow trout were placed in 12,000-L outdoor microcosms dosed with 0, 3, or 10 microl/L liquid creosote for a 28-day period. Collected fish were examined for changes in focal length variability (FLV), lens size, and weight. These measurements were compared with induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity and hepatic and water concentrations of priority pollutant PAHs. The optical quality of rainbow trout lenses was significantly reduced following creosote exposure, as indicated by increased FLV. Lens damage was significantly related to hepatic EROD activity, and both effects rose with creosote dose. Analytical measurements of microcosm water indicated elevated concentrations of PAHs in creosote-dosed ponds, including compounds capable of inducing rainbow trout EROD activity in vitro. Hepatic concentrations of PAHs were low and not related to creosote dose, likely due to rapid metabolism and elimination. This study demonstrates for the first time employment of a highly sensitive and quantitative technique to measure lens damage in fish exposed to contaminants in situ. The relationship between this effect and hepatic CYP1A activity may suggest a mechanistic linkage, which could lead to the use of EROD activity as an indicator of toxic effect rather than just chemical exposure.

Along with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 24 unsubstituted polycyclic aromatic hydrocarbons (PAHs) were evaluated for their ability to induce 7-ethoxyresorufin-o-deethylase (EROD) activity in the rainbow trout liver cell line RTL-W1. When the duration and cell density of exposure were increased, the EC(50) for EROD induction was relatively constant for TCDD, but increased for PAHs. Regardless of exposure conditions, EROD activity was not induced by 9 PAHs: naphthalene, phenanthrene, anthracene, pyrene, perylene, acenaphthylene, acenaphthene, fluorene, and fluoranthene. Two PAHs, benzo[g,h,i]perylene and coronene, induced EROD activity inconsistently. The remaining 13 PAHs consistently induced EROD activity. The EC(50)s for induction exhibited approximately a 110-fold range. The order of potency, from most to least potent, was benzo[k]fluoranthene, dibenzo[a,i]pyrene, dibenzo [a,h]anthracene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, benzo [b]fluoranthene, pentacene, benzo[b]anthracene, benzo[b] fluorene, chrysene, benzo[a]anthracene, benzo[e]pyrene, and triphenylene. When the induction potency was expressed relative to TCDD, the toxic equivalency factors (TEFs) ranged from 0.001 to 0.000 01. When expressed relative to benzo[a]pyrene, the TEFs ranged from 3.44 to 0. 03., (Copyright 1999 Academic Press.)

Patients with known coronary disease and low-density lipoprotein >130 mg/dl who are followed by cardiologists after myocardial infarction are more than twice as likely to receive a cholesterol-lowering agent than patients followed by general internists.