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1.
Proc Natl Acad Sci U S A ; 120(25): e2221910120, 2023 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-37307489

RESUMO

Women voted for the Democratic candidate more than men did in each US presidential election since 1980. We show that part of the gender gap stems from the fact that a higher proportion of women than men voters are Black, and Black voters overwhelmingly choose Democratic candidates. Past research shows that Black men have especially high rates of death, incarceration, and disenfranchisement due to criminal convictions. These disparities reduce the share of men voters who are Black. We show that the gender difference in racial composition explains 24% of the gender gap in voting Democratic. The gender gap in voting Democratic is especially large among those who are never-married, and, among them, the differing racial composition of men and women voters is more impactful than in the population at large, explaining 43% of the gender gap. We consider an alternative hypothesis that income differences between single men and women explain the gender gap in voting, but our analysis leads us to reject it. Although unmarried women are poorer than unmarried men, and lower-income voters vote slightly more Democratic, the latter difference is too small for income to explain much of the gender gap in voting. In short, the large gender gap among unmarried voters is not a reflection of the lower incomes of women's households but does reflect the fact that women voters are disproportionately Black. We used the General Social Survey as the data source for the analysis, then replicated results with the American National Election Survey data.


Assuntos
Negro ou Afro-Americano , Renda , Política , Feminino , Humanos , Masculino , Fatores Sexuais
2.
Biol Reprod ; 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38904948

RESUMO

Conceptus estrogens and prostaglandins have long been considered the primary signals for maternal recognition of pregnancy (MRP) in the pig. However, loss-of-function studies targeting conceptus aromatase genes (CYP19A1 and CYP19A2) and prostaglandin-endoperoxide synthase 2 (PTGS2) indicated that conceptuses can not only signal MRP without estrogens or prostaglandins but can maintain early pregnancy. However, complete loss of estrogen production leads to abortion after day 25 of gestation. Although neither conceptus estrogens nor prostaglandins had a significant effect on early maintenance of CL function alone, the two conceptus factors have a biological relationship. To investigate the role that both conceptus estrogens and prostaglandins have on MRP and maintenance of pregnancy, a triple loss-of function model (TKO) was generated for conceptus CYP19A1, CYP19A2 and PTGS2. In addition, a conceptus CYP19A2-/- model (A2KO) was established to determine the role of placental estrogen during later pregnancy. Estrogen and prostaglandin synthesis were greatly reduced in TKO conceptuses which resulted in a failure to inhibit luteolysis after day 15 of pregnancy despite the presence of conceptuses in the uterine lumen. However, A2KO placentae not only maintained functional CL but were able to maintain pregnancy to day 32 of gestation. Despite the loss of placental CYP19A2 expression, the allantois fluid content of estrogen was not affected as the placenta compensated by expressing CYP19A1 and CYP19A3, which are normally absent in controls. Results suggest conceptuses can signal MRP through production of conceptus PGE or stimulating PGE synthesis from the endometrium through conceptus estrogen. Failure of conceptuses to produce both factors results in failure of MRP and loss of pregnancy.

3.
Reproduction ; 166(4): 263-269, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37490351

RESUMO

In brief: Aromatase catalyzes the synthesis of estrogens and has been shown to have an important role during the establishment of pregnancy in the pig. This study confirmed the differential expression of the three aromatase isoforms. Abstract: Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal-placental development. Cyp19a1 and Cyp19a3 expressions were detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both the conceptuses and the placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of day 21 pregnant females, as well as days 12 and 17 pseudopregnant females, we did not detect the expression of Cyp19a1, Cyp19a2, or Cyp19a3. In our study, we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling the secretion of estrogen from the conceptus and the placenta.


Assuntos
Aromatase , Placenta , Gravidez , Animais , Feminino , Suínos , Placenta/metabolismo , Aromatase/genética , Aromatase/metabolismo , Estrogênios/metabolismo , Embrião de Mamíferos/metabolismo , Gônadas/metabolismo
4.
Mol Reprod Dev ; 90(7): 459-468, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-35736243

RESUMO

CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Gravidez , Feminino , Animais , Suínos/genética , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Reprodução/genética , Endométrio/metabolismo
5.
Am J Physiol Lung Cell Mol Physiol ; 322(6): L842-L852, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35438004

RESUMO

Morbidity and mortality of respiratory diseases are linked to airway obstruction by mucus but there are still no specific, safe, and effective drugs to correct this phenotype. The need for better treatment requires a new understanding of the basis for mucus production. In that regard, studies of human airway epithelial cells in primary culture show that a mucin granule constituent known as chloride channel accessory 1 (CLCA1) is required for inducible expression of the inflammatory mucin MUC5AC in response to potent type 2 cytokines. However, it remained uncertain whether CLCLA1 is necessary for mucus production in vivo. Conventional approaches to functional biology using targeted gene knockout were difficult due to the functional redundancy of additional Clca genes in mice not found in humans. We reasoned that CLCA1 function might be better addressed in pigs that maintain the same four-member CLCA gene locus and the corresponding mucosal and submucosal populations of mucous cells found in humans. Here we develop to our knowledge the first CLCA1-gene-deficient (CLCA1-/-) pig and show that these animals exhibit loss of MUC5AC+ mucous cells throughout the airway mucosa of the lung without affecting comparable cells in the tracheal mucosa or MUC5B+ mucous cells in submucosal glands. Similarly, CLCA1-/- pigs exhibit loss of MUC5AC+ mucous cells in the intestinal mucosa without affecting MUC2+ mucous cells. These data establish CLCA1 function for controlling MUC5AC expression as a marker of mucus production and provide a new animal model to study mucus production at respiratory and intestinal sites.


Assuntos
Canais de Cloreto , Mucina-5AC , Animais , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Células Epiteliais/metabolismo , Células Caliciformes/metabolismo , Pulmão/metabolismo , Camundongos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Muco/metabolismo , Mucosa Respiratória/metabolismo , Suínos
6.
Biol Reprod ; 106(4): 629-638, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35094055

RESUMO

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.


Assuntos
Agricultura , Animais Domésticos , Animais , Gado , National Institutes of Health (U.S.) , Estados Unidos , United States Department of Agriculture
7.
Biol Reprod ; 105(6): 1577-1590, 2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34608481

RESUMO

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG-/- conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG-/- conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG-/- embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs.


Assuntos
Embrião de Mamíferos/metabolismo , Interferon gama/metabolismo , Prenhez/metabolismo , Sus scrofa/embriologia , Animais , Desenvolvimento Embrionário , Feminino , Gravidez
8.
Biol Reprod ; 105(5): 1104-1113, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34453429

RESUMO

Elongation of pig conceptuses is a dynamic process, requiring adequate nutrient provisions. Glutamine is used as an energy substrate and is involved in the activation of mechanistic target of rapamycin complex 1 (mTORC1) during porcine preimplantation development. However, the roles of glutamine have not been extensively studied past the blastocyst stage. Therefore, the objective of the current study was to determine if glutaminase (GLS), which is the rate-limiting enzyme in glutamine metabolism, was necessary for conceptus elongation to proceed and was involved in mTORC1 activation. The CRISPR/Cas9 system was used to induce loss-of-function mutations in the GLS gene of porcine fetal fibroblasts. Wild type (GLS+/+) and knockout (GLS-/-) fibroblasts were used as donor cells for somatic cell nuclear transfer, and GLS+/+ and GLS-/- blastocyst-stage embryos were transferred into surrogates. On day 14 of gestation, GLS+/+ conceptuses primarily demonstrated filamentous morphologies, and GLS-/- conceptuses exhibited spherical, ovoid, tubular, and filamentous morphologies. Thus, GLS-/- embryos were able to elongate despite the absence of GLS protein and minimal enzyme activity. Furthermore, spherical GLS-/- conceptuses had increased abundance of transcripts related to glutamine and glutamate metabolism and transport compared to filamentous conceptuses of either genotype. Differences in phosphorylation of mTORC1 components and targets were not detected regarding conceptus genotype or morphology, but abundance of two transcriptional targets of mTORC1, cyclin D1, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha was increased in spherical conceptuses. Therefore, porcine GLS is not essential for conceptus elongation and is not required for mTORC1 activation at this developmental timepoint.


Assuntos
Blastocisto/metabolismo , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/genética , Glutaminase/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Sus scrofa/embriologia , Animais , Transferência Embrionária , Embrião de Mamíferos/enzimologia , Feminino , Glutaminase/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo
9.
Reproduction ; 161(4): R79-R88, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33560267

RESUMO

Development of viviparity in mammals requires that the placenta evolves as an intermediate interface between the fetus and maternal uterus. In addition to the retention of the fetus and secretion of nutrients to support growth and development to term, it is essential that viviparous species modify or inhibit the maternal immune system from recognizing the semi-allogeneic fetus. Following blastocyst hatching from its zona pellucida, trophoblast differentiation provides the initial communication to the maternal endometrium to regulate maintenance of progesterone production from the corpus luteum and biological pathways in uterine and conceptus development necessary in the establishment and maintenance of pregnancy. Many conceptus factors have been proposed to serve in the establishment and maintenance of pregnancy. CRISPR-Cas9 gene-editing technology provides a specific and efficient method to generate animal models to perform loss-of-function studies to investigate the role of specific conceptus factors. The utilization of CRISPR-Cas9 gene editing has provided a direct approach to investigate the specific role of conceptus factors in the development and establishment of pregnancy in the pig. This technology has helped address a number of questions concerning peri-implantation development and has altered our understanding of maternal recognition and maintenance of pregnancy in the pig.


Assuntos
Blastocisto/metabolismo , Sistemas CRISPR-Cas , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Endométrio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Blastocisto/citologia , Embrião de Mamíferos/citologia , Endométrio/citologia , Feminino , Gravidez , Suínos
10.
Biol Reprod ; 102(2): 475-488, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31616930

RESUMO

Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2-/- blastocysts on day 7. PTGS2+/+ and PTGS2-/- blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3 h to determine PG production. Conceptus release of total PG, prostaglandin F2⍺ (PGF2α), and PGE in culture media was lower with PTGS2-/- conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2-/- conceptus surrogates allowed to continue pregnancy were maintained beyond 30 days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig.


Assuntos
Blastocisto/metabolismo , Ciclo-Oxigenase 2/metabolismo , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/fisiologia , Endométrio/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Ciclo-Oxigenase 2/genética , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência Nuclear , Gravidez , Suínos
11.
Biol Reprod ; 101(1): 148-161, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31066888

RESUMO

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .


Assuntos
Embrião de Mamíferos/metabolismo , Estrogênios/metabolismo , Manutenção da Gravidez/fisiologia , Prenhez , Reconhecimento Psicológico/fisiologia , Suínos , Animais , Animais Geneticamente Modificados , Aromatase/genética , Aromatase/metabolismo , Células Cultivadas , Clonagem de Organismos/veterinária , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Embrião de Mamíferos/química , Desenvolvimento Embrionário/efeitos dos fármacos , Estrogênios/farmacologia , Feminino , Fertilização/fisiologia , Troca Materno-Fetal/efeitos dos fármacos , Troca Materno-Fetal/fisiologia , Técnicas de Transferência Nuclear , Gravidez , Manutenção da Gravidez/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Suínos/embriologia , Suínos/genética , Suínos/metabolismo
12.
Mol Reprod Dev ; 86(5): 558-565, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30779254

RESUMO

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1-3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere-like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.


Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células/métodos , Hipóxia Celular/fisiologia , Fibroblastos/citologia , Técnicas de Transferência Nuclear , Animais , Blastocisto/fisiologia , Células Cultivadas , Reprogramação Celular/fisiologia , Clonagem de Organismos , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Feminino , Fibroblastos/fisiologia , Projetos Piloto , Gravidez , Suínos
13.
Transgenic Res ; 28(1): 21-32, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30315482

RESUMO

The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.


Assuntos
Aminopeptidases/genética , Animais Geneticamente Modificados/genética , Infecções por Coronavirus/genética , Coronavirus/patogenicidade , Aminopeptidases/deficiência , Animais , Animais Geneticamente Modificados/virologia , Sistemas CRISPR-Cas , Coronavirus/genética , Infecções por Coronavirus/virologia , Enterócitos/enzimologia , Enterócitos/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Suínos , Vírus da Gastroenterite Transmissível/patogenicidade
14.
Gen Comp Endocrinol ; 270: 35-40, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30291864

RESUMO

Stress in animals is a concern in conservation breeding programs and livestock production facilities. The biological stress response is mediated by the release of glucocorticoids, which can suppress reproduction, growth, and immunity if recurrently activated. Feathers can be used to extract and monitor concentrations of corticosterone, a primary glucocorticoid in birds. However, current techniques for extracting feather corticosterone present challenges, including difficulty assessing extraction efficiency or hormone recovery, inconsistent extraction across feather lengths or pieces, and several uncertainties regarding the mechanisms of hormone deposition into feathers. To overcome such challenges and to provide tools useful for facilitating conservation breeding and livestock production, we developed and validated an alternative procedure for extracting feather glucocorticoids. We first developed a protocol to enzymatically digest the protein matrix of feathers using a keratinase, such that non-protein analytes could be isolated by organic extraction. We then developed an extraction protocol and evaluated techniques by measuring extraction efficiency and by testing parallelism and hormone recovery (accuracy) using radioimmunoassay. Our results demonstrated high and consistent extraction efficiency, as well as high accuracy and reliable parallelism to a standard curve upon measurement of corticosterone concentrations from extracts. By dissolving feather material into solution prior to extraction, we were able to replicate hormone deposition into the feather matrix and ensure consistent extraction across feathers. This work provides additional support for the validity and practicality of extracting glucocorticoids from feathers. Our extraction protocol is likely to extend to other applications as well, including the isolation of numerous non-protein analytes from various keratinized tissues.


Assuntos
Plumas/metabolismo , Glucocorticoides/metabolismo , Peptídeo Hidrolases/uso terapêutico , Animais , Galinhas , Peptídeo Hidrolases/farmacologia
15.
J Virol ; 91(2)2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27847356

RESUMO

CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection of CD163-modified pigs with representative type 1 and type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163. IMPORTANCE: Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Resistência à Doença/genética , Predisposição Genética para Doença , Genótipo , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Receptores de Superfície Celular/genética , Animais , Antígenos CD/química , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/química , Antígenos de Diferenciação Mielomonocítica/metabolismo , Ordem dos Genes , Loci Gênicos , Interações Hospedeiro-Patógeno/genética , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Mutação , Fenótipo , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Suínos , Carga Viral
16.
Mol Reprod Dev ; 85(4): 290-302, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29392839

RESUMO

Somatic cell nuclear transfer is a valuable technique for the generation of genetically engineered animals, however, the efficiency of cloning in mammalian species is low (1-3%). Differentiated somatic cells commonly used in nuclear transfer utilize the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively the metabolism of somatic cells contrasts that of the cells within the early embryos which predominately use glycolysis. Early embryos (prior to implantation) are evidenced to exhibit characteristics of a Warburg Effect (WE)-like metabolism. We hypothesized that pharmacologically driven fibroblast cells can become more blastomere-like and result in improved in vitro embryonic development after SCNT. The goals were to determine if subsequent in vitro embryo development is impacted by (1) cloning pharmacologically treated donor cells pushed to have a WE-like metabolism or (2) culturing non-treated donor clones with pharmaceuticals used to push a WE-like metabolism. Additionally, we investigated early gestational survival of the donor-treated clone embryos. Here we demonstrate that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs. Furthermore, these experiments demonstrate that early embryos (or at least in vitro produced embryos) have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos. Lastly, we show that survival in early gestation was not different between clones from pharmacologically induced WE-like donor cells and controls.


Assuntos
Clonagem de Organismos , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Técnicas de Transferência Nuclear , Animais , Feminino , Gravidez , Suínos
17.
Transgenic Res ; 27(2): 167-178, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29516259

RESUMO

Genetically engineered pigs are often created with a targeting vector that contains a loxP flanked selectable marker like neomycin. The Cre-loxP recombinase system can be used to remove the selectable marker gene from the resulting offspring or cell line. Here is described a new method to remove a loxP flanked neomycin cassette by direct zygote injection of an mRNA encoding Cre recombinase. The optimal concentration of mRNA was determined to be 10 ng/µL when compared to 2 and 100 ng/µL (P < 0.0001). Development to the blastocyst stage was 14.1% after zygote injection with 10 ng/µL. This method successfully removed the neomycin cassette in 81.9% of injected in vitro derived embryos; which was significantly higher than the control (P < 0.0001). Embryo transfer resulted in the birth of one live piglet with a Cre deleted neomycin cassette. The new method described can be used to efficiently remove selectable markers in genetically engineered animals without the need for long term cell culture and subsequent somatic cell nuclear transfer.


Assuntos
Engenharia Genética/métodos , Vetores Genéticos/antagonistas & inibidores , Integrases/genética , RNA/administração & dosagem , Animais , Vetores Genéticos/química , Integrases/efeitos dos fármacos , Neomicina/química , RNA/genética , Recombinação Genética , Suínos , Zigoto/citologia , Zigoto/efeitos dos fármacos
18.
J Adolesc ; 62: 27-37, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29144980

RESUMO

Increasingly, the study of temporal psychology is moving away from bivariate analyses towards person-centered analyses, which simultaneously account for scores on past, present and future dimensions. However, longitudinal studies are lacking. This study builds on a developing literature by examining the 24-month relationship between time attitudes and criterion variables. Four latent profiles called Positive, Ambivalent, Moderately Negative, and Negative, were identified. Results showed that time attitude profiles were generally unstable across the first three years of high school. However, those who stayed in the Positive profile developed higher self-efficacy in all domains. Transitioning to the Positive profile was associated with positive outcomes, whereas other transitions among profiles were associated with negative outcomes. There were small-sized, socio-demographic effects such that living in Northern Ireland (compared to Scotland), being male, and not being entitled to a free school meal, were all related to membership of, or transition to, the Positive profile.


Assuntos
Comportamento do Adolescente/psicologia , Atitude , Autoeficácia , Percepção do Tempo , Adolescente , Afeto , Feminino , Humanos , Estudos Longitudinais , Masculino , Irlanda do Norte , Escócia , Estudantes/psicologia , Tempo
19.
J Adolesc ; 69: 44-51, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30227345

RESUMO

PURPOSE: Time attitudes refer to individuals' feelings about the past, present, and future, and an increasing number of cross-sectional studies have demonstrated that positive time attitudes are significantly related to better health and well-being. We investigated time attitude profile membership and associated transitions longitudinally in United Kingdom-based adolescents, and assessed the relationship between time attitude profile development on health behaviours at + 21 months after the data collection involving time attitudes. METHODS: Participants were high school students (N = 1306; 41.8% female, Mage 12.5-14.5 years [waves 1-3]). The Adolescent and Adult Time Inventory - Time Attitudes Scale was employed to identify profiles, and a mover-stayer latent transition analysis was employed to examine developmental changes. Data were also gathered on sensation seeking, and a range of health indicators were assessed: Past week frequency of physical exercise, self-rated health, subjective life expectancy, lifetime cannabis and smoking, and dental attendance. RESULTS: Staying in a positive time attitude profile was related to higher subjective life expectancy, and less frequent use of cannabis and cigarettes (1.00 ≤ d ≤ 4.00). Further, moving to a positive profile predicted healthier outcomes for most health measures used. CONCLUSIONS: Notwithstanding the limitation that health outcomes in the present study were distal, the present study bolstered a developing cross-sectional literature supporting the association between positive time attitudes and better health and well-being outcomes. Future longitudinal studies which assess measures concurrently are required.


Assuntos
Comportamento do Adolescente/psicologia , Atitude Frente a Saúde , Comportamentos Relacionados com a Saúde , Adolescente , Estudos Transversais , Feminino , Humanos , Estudos Longitudinais , Masculino , Estudantes/psicologia , Inquéritos e Questionários , Reino Unido
20.
J Appl Clin Med Phys ; 19(3): 71-78, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29536664

RESUMO

Voluntary inspiration breath hold (VIBH) for left breast cancer patients has been shown to be a safe and effective method of reducing radiation dose to the heart. Currently, VIBH protocol compliance is monitored visually. In this work, we establish whether it is possible to gate the delivery of radiation from an Elekta linac using the Microsoft Kinect version 2 (Kinect v2) depth sensor to measure a patient breathing signal. This would allow contactless monitoring during VMAT treatment, as an alternative to equipment-assisted methods such as active breathing control (ABC). Breathing traces were acquired from six left breast radiotherapy patients during VIBH. We developed a gating interface to an Elekta linac, using the depth signal from a Kinect v2 to control radiation delivery to a programmable motion platform following patient breathing patterns. Radiation dose to a moving phantom with gating was verified using point dose measurements and a Delta4 verification phantom. 60 breathing traces were obtained with an acquisition success rate of 100%. Point dose measurements for gated deliveries to a moving phantom agreed to within 0.5% of ungated delivery to a static phantom using both a conventional and VMAT treatment plan. Dose measurements with the verification phantom showed that there was a median dose difference of better than 0.5% and a mean (3% 3 mm) gamma index of 92.6% for gated deliveries when using static phantom data as a reference. It is possible to use a Kinect v2 device to monitor voluntary breath hold protocol compliance in a cohort of left breast radiotherapy patients. Furthermore, it is possible to use the signal from a Kinect v2 to gate an Elekta linac to deliver radiation only during the peak inhale VIBH phase.


Assuntos
Neoplasias da Mama/radioterapia , Suspensão da Respiração , Imagens de Fantasmas , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia de Intensidade Modulada/métodos , Técnicas de Imagem de Sincronização Respiratória/métodos , Tomografia Computadorizada por Raios X/métodos , Feminino , Humanos , Processamento de Imagem Assistida por Computador/métodos , Movimento (Física) , Aceleradores de Partículas/instrumentação , Prognóstico , Estudo de Prova de Conceito , Dosagem Radioterapêutica , Respiração , Técnicas de Imagem de Sincronização Respiratória/mortalidade
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