Genome-wide associations with longevity and reproductive traits in U.S. rangeland ewes.

Introduction: Improving ewe longevity is an important breeding and management goal, as death loss and early culling of mature ewes are economic burdens in the sheep industry. Ewe longevity can be improved by selecting for positive reproductive outcomes. However, the breeding approaches for accomplishing this come with the challenge of recording a lifetime trait. Characterizing genetic factors underpinning ewe longevity and related traits could result in the development of genomic selection strategies to improve the stayability of sheep through early, informed selection of replacement ewes. Methods: Towards this aim, a genome-wide association study (GWAS) was performed to identify genetic markers associated with ewe longevity, reproductive, and production traits. Traits evaluated included longevity (i.e., length of time in the flock), parity and the lifetime number of lambs born, lambs born alive, lambs weaned, and weight of lambs weaned. Ewe records from previous studies were used. Specifically, Rambouillet (n = 480), Polypay (n = 404), Suffolk (n = 182), and Columbia (n = 64) breed ewes (N = 1,130) were analyzed against 503,617 SNPs in across-breed and within-breed GWAS conducted with the Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) model in R. Results: The across-breed GWAS identified 25 significant SNPs and the within-breed GWAS for Rambouillet, Polypay, and Suffolk ewes identified an additional 19 significant SNPs. The most significant markers were rs411309094 (13:22,467,143) associated with longevity in across-breed GWAS (p-value = 8.3E-13) and rs429525276 (2:148,398,336) associated with both longevity (p-value = 6.4E-15) and parity (p-value = 4.8E-15) in Rambouillet GWAS. Significant SNPs were identified within or in proximity (±50 kb) of genes with known or proposed roles in reproduction, dentition, and the immune system. These genes include ALPL, ANOS1, ARHGEF26, ASIC2, ASTN2, ATP8A2, CAMK2D, CEP89, DISC1, ITGB6, KCNH8, MBNL3, MINDY4, MTSS1, PLEKHA7, PRIM2, RNF43, ROBO2, SLCO1A2, TMEM266, TNFRSF21, and ZNF804B. Discussion: This study proposes multiple SNPs as candidates for use in selection indices and suggests genes for further research towards improving understanding of the genetic factors contributing to longevity, reproductive, and production traits of ewes.

Binding Interface and Electron Transfer Between Nicotine Oxidoreductase and Its Cytochrome c Electron Acceptor.

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. 2022, 298 (8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein-protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2's surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2's flavin to CycN's heme occurs without the assistance of a protein-derived wire.

Dissecting the Genetic Architecture of Aphanomyces Root Rot Resistance in Lentil by QTL Mapping and Genome-Wide Association Study.

Lentil (Lens culinaris Medikus) is an important source of protein for people in developing countries. Aphanomyces root rot (ARR) has emerged as one of the most devastating diseases affecting lentil production. In this study, we applied two complementary quantitative trait loci (QTL) analysis approaches to unravel the genetic architecture underlying this complex trait. A recombinant inbred line (RIL) population and an association mapping population were genotyped using genotyping by sequencing (GBS) to discover novel single nucleotide polymorphisms (SNPs). QTL mapping identified 19 QTL associated with ARR resistance, while association mapping detected 38 QTL and highlighted accumulation of favorable haplotypes in most of the resistant accessions. Seven QTL clusters were discovered on six chromosomes, and 15 putative genes were identified within the QTL clusters. To validate QTL mapping and genome-wide association study (GWAS) results, expression analysis of five selected genes was conducted on partially resistant and susceptible accessions. Three of the genes were differentially expressed at early stages of infection, two of which may be associated with ARR resistance. Our findings provide valuable insight into the genetic control of ARR, and genetic and genomic resources developed here can be used to accelerate development of lentil cultivars with high levels of partial resistance to ARR.

Phenotyping of Plant Biomass and Performance Traits Using Remote Sensing Techniques in Pea (Pisum sativum, L.).

Field pea cultivars are constantly improved through breeding programs to enhance biotic and abiotic stress tolerance and increase seed yield potential. In pea breeding, the Above Ground Biomass (AGBM) is assessed due to its influence on seed yield, canopy closure, and weed suppression. It is also the primary yield component for peas used as a cover crop and/or grazing. Measuring AGBM is destructive and labor-intensive process. Sensor-based phenotyping of such traits can greatly enhance crop breeding efficiency. In this research, high resolution RGB and multispectral images acquired with unmanned aerial systems were used to assess phenotypes in spring and winter pea breeding plots. The Green Red Vegetation Index (GRVI), Normalized Difference Vegetation Index (NDVI), Normalized Difference Red Edge Index (NDRE), plot volume, canopy height, and canopy coverage were extracted from RGB and multispectral information at five imaging times (between 365 to 1948 accumulated degree days/ADD after 1 May) in four winter field pea experiments and at three imaging times (between 1231 to 1648 ADD) in one spring field pea experiment. The image features were compared to ground-truth data including AGBM, lodging, leaf type, days to 50% flowering, days to physiological maturity, number of the first reproductive node, and seed yield. In two of the winter pea experiments, a strong correlation between image features and seed yield was observed at 1268 ADD (flowering). An increase in correlation between image features with the phenological traits such as days to 50% flowering and days to physiological maturity was observed at about 1725 ADD in these winter pea experiments. In the spring pea experiment, the plot volume estimated from images was highly correlated with ground truth canopy height (r = 0.83) at 1231 ADD. In two other winter pea experiments and the spring pea experiment, the GRVI and NDVI features were significantly correlated with AGBM at flowering. When selected image features were used to develop a least absolute shrinkage and selection operator model for AGBM estimation, the correlation coefficient between the actual and predicted AGBM was 0.60 and 0.84 in the winter and spring pea experiments, respectively. A SPOT-6 satellite image (1.5 m resolution) was also evaluated for its applicability to assess biomass and seed yield. The image features extracted from satellite imagery showed significant correlation with seed yield in two winter field pea experiments, however, the trend was not consistent. In summary, the study supports the potential of using unmanned aerial system-based imaging techniques to estimate biomass and crop performance in pea breeding programs.

Clarifying the confusion between poligeenan, degraded carrageenan, and carrageenan: A review of the chemistry, nomenclature, and in vivo toxicology by the oral route.

Carrageenan (CGN) is a common food additive that has been widely used for decades as a gelling, thickening and stabilizing agent. Carrageenan has been proven safe for human consumption; however, there has been significant confusion in the literature between CGN and the products of intentional acid-hydrolysis of CGN, which are degraded CGN (d-CGN) and poligeenan (PGN). In part, this confusion was due to the nomenclature used in early studies on CGN, where poligeenan was referred to as "degraded carrageenan" (d-CGN) and "degraded carrageenan" was simply referred to as carrageenan. Although this nomenclature has been corrected, confusion still exists resulting in misinterpretation of data and the subsequent dissemination of incorrect information regarding the safe dietary use of CGN. The lack of understanding of the molecular weight distribution of CGN has further exacerbated the issue. The significant differences in chemistry, manufacture, and protein reactivity of CGN versus d-CGN and PGN are reviewed, in addition to the in vivo toxicological profiles of CGN, d-CGN, and PGN. As CGN cannot be hydrolyzed to PGN in vivo, concerns over the use of CGN as a food additive are unfounded, particularly since current studies support the lack of oncogenic and tumorigenic activity of CGN in humans.

Reprint of "CON4EI: Bovine Corneal Opacity and Permeability (BCOP) test for hazard identification and labelling of eye irritating chemicals".

Assessment of ocular irritation potential is an international regulatory requirement in the safety evaluation of industrial and consumer products. None in vitro ocular irritation assays are capable of fully categorizing chemicals as stand-alone. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI consortium assessed the reliability of eight in vitro test methods and computational models as well as established a tiered-testing strategy. One of the selected assays was Bovine Corneal Opacity and Permeability (BCOP). In this project, the same corneas were used for measurement of opacity using the OP-KIT, the Laser Light-Based Opacitometer (LLBO) and for histopathological analysis. The results show that the accuracy of the BCOP OP-KIT in identifying Cat 1 chemicals was 73.8% while the accuracy was 86.3% for No Cat chemicals. BCOP OP-KIT false negative results were often related to an in vivo classification driven by conjunctival effects only. For the BCOP LLBO, the accuracy in identifying Cat 1 chemicals was 74.4% versus 88.8% for No Cat chemicals. The BCOP LLBO seems very promising for the identification of No Cat liquids but less so for the identification of solids. Histopathology as an additional endpoint to the BCOP test method does not reduce the false negative rate substantially for in vivo Cat 1 chemicals.

Pre-validation of an in vitro skin irritation test for medical devices using the reconstructed human tissue model EpiDerm™.

Assessment of dermal irritation is an essential component of the safety evaluation of medical devices. Reconstructed human epidermis (RhE) models have replaced rabbit skin irritation testing for neat chemicals and their mixtures (OECD Test Guideline 439). However, this guideline cannot be directly applied to the area of medical devices (MD) since their non-toxicity assessment is largely based on the testing of MD extracts that may have very low irritation potential. Therefore, the RhE-methods previously validated with neat chemicals needed to be modified to reflect the needs for detection of low levels of potential irritants. A protocol employing RhE EpiDerm was optimized in 2013 using known irritants and spiked polymers (Casas et al., 2013, TIV). In 2014 and 2015 MatTek In Vitro Life Science Laboratories (IVLSL) and RIVM assessed the transferability of the assay. After the successful transfer and standardization of the protocol, 17 laboratories were trained in the use of the protocol in the preparation for the validation. Laboratories produced data with 98% agreement of predictions for the selected references and controls. We conclude that a modified RhE skin irritation test has the potential to address the skin irritation potential of the medical devices. Standardization and focus on the technical issues is essential for accurate prediction.

CON4EI: CONsortium for in vitro Eye Irritation testing strategy - EpiOcular™ time-to-toxicity (EpiOcular ET-50) protocols for hazard identification and labelling of eye irritating chemicals.

Assessment of acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. The objective of the CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for eye irritation assessment for all drivers of classification. A set of 80 reference chemicals (38 liquids and 42 solids) was tested with eight different alternative methods. Here, the results obtained with reconstructed human cornea-like epithelium (RhCE) EpiOcular™ in the EpiOcular time-to-toxicity Tests (Neat and Dilution ET-50 protocols) are presented. The primary aim of this study was to evaluate whether test methods can discriminate chemicals not requiring classification for serious eye damage/eye irritancy (No Category) from chemicals requiring classification and labelling for Category 1 and Category 2. In addition, the predictive capacity in terms of in vivo drivers of classification was investigated. The chemicals were tested in two independent runs by MatTek In Vitro Life Science Laboratories. Results of this study demonstrate very high specificity of both test protocols. With the existing prediction models described in the SOPs, the specificity of the Neat and Dilution method was 87% and 100%, respectively. The Dilution method was able to correctly predicting 66% of GHS Cat 2 chemicals, however, prediction of GHS Cat 1 chemicals was only 47%-55% using the current protocols. In order to achieve optimal prediction for all three classes, a testing strategy was developed which combines the most predictive time-points of both protocols and for tests liquids and solids separately. Using this new testing strategy, the sensitivity for predicting GHS Cat 1 and GHS Cat 2 chemicals was 73% and 64%, respectively and the very high specificity of 97% was maintained. None of the Cat 1 chemicals was underpredicted as GHS No Category. Further combination of the EpiOcular time-to-toxicity protocols with other validated in vitro systems evaluated in this project, should enable significant reduction and even possible replacement of the animal tests for the final assessment of the irritation potential in all of the GHS classes.

Artemisinin disrupts androgen responsiveness of human prostate cancer cells by stimulating the 26S proteasome-mediated degradation of the androgen receptor protein.

Androgen receptor (AR) expression and activity is highly linked to the development and progression of prostate cancer and is a target of therapeutic strategies for this disease. We investigated whether the antimalarial drug artemisinin, which is a sesquiterpene lactone isolated from the sweet wormwood plant Artemisia annua, could alter AR expression and responsiveness in cultured human prostate cancer cell lines. Artemisinin treatment induced the 26S proteasome-mediated degradation of the receptor protein, without altering AR transcript levels, in androgen-responsive LNCaP prostate cancer cells or PC-3 prostate cancer cells expressing exogenous wild-type AR. Furthermore, artemisinin stimulated AR ubiquitination and AR receptor interactions with the E3 ubiquitin ligase MDM2 in LNCaP cells. The artemisinin-induced loss of AR protein prevented androgen-responsive cell proliferation and ablated total AR transcriptional activity. The serine/threonine protein kinase AKT-1 was shown to be highly associated with artemisinin-induced proteasome-mediated degradation of AR protein. Artemisinin treatment activated AKT-1 enzymatic activity, enhanced receptor association with AKT-1, and induced AR serine phosphorylation. Treatment of LNCaP cells with the PI3-kinase inhibitor LY294002, which inhibits the PI3-kinase-dependent activation of AKT-1, prevented the artemisinin-induced AR degradation. Furthermore, in transfected receptor-negative PC-3 cells, artemisinin failed to stimulate the degradation of an altered receptor protein (S215A/S792A) with mutations in its two consensus AKT-1 serine phosphorylation sites. Taken together, our results indicate that artemisinin induces the degradation of AR protein and disrupts androgen responsiveness of human prostate cancer cells, suggesting that this natural compound represents a new potential therapeutic molecule that selectively targets AR levels.

CON4EI: Bovine Corneal Opacity and Permeability (BCOP) test for hazard identification and labelling of eye irritating chemicals.

Assessment of ocular irritation potential is an international regulatory requirement in the safety evaluation of industrial and consumer products. None in vitro ocular irritation assays are capable of fully categorizing chemicals as stand-alone. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI consortium assessed the reliability of eight in vitro test methods and computational models as well as established a tiered-testing strategy. One of the selected assays was Bovine Corneal Opacity and Permeability (BCOP). In this project, the same corneas were used for measurement of opacity using the OP-KIT, the Laser Light-Based Opacitometer (LLBO) and for histopathological analysis. The results show that the accuracy of the BCOP OP-KIT in identifying Cat 1 chemicals was 73.8% while the accuracy was 86.3% for No Cat chemicals. BCOP OP-KIT false negative results were often related to an in vivo classification driven by conjunctival effects only. For the BCOP LLBO, the accuracy in identifying Cat 1 chemicals was 74.4% versus 88.8% for No Cat chemicals. The BCOP LLBO seems very promising for the identification of No Cat liquids but less so for the identification of solids. Histopathology as an additional endpoint to the BCOP test method does not reduce the false negative rate substantially for in vivo Cat 1 chemicals.

Effects of carrageenan on cell permeability, cytotoxicity, and cytokine gene expression in human intestinal and hepatic cell lines.

Carrageenan (CGN) is a common food additive used for its gelling and thickening properties. The present study was done to evaluate intestinal permeability, cytotoxicity, and CGN-mediated induction of proinflammatory cytokines. A standard Caco-2 absorption model showed no CGN permeability or cytotoxicity at concentrations of 100, 500, and 1000 µg/mL. In two human intestinal cell lines (HT-29 and HCT-8) CGN (0.1, 1.0, and 10.0 µg/mL) did not induce IL-8, IL-6, or MCP-1 (CCL2) or produce cellular toxicity after 24 h. The TLR4 agonist LPS produced weak induction of IL-8 in HT-29 cells and no induction in HCT-8 cells. The effects of κ-CGN (0.1, 1.0, and 10 µg/mL) on cellular oxidative stress was assessed in HT-29 cells using CM-H2DCFDA as the probe. No effect on oxidative stress was observed after 24 h. In the human (HepG2) liver cell line, ʎ-CGN (0.1, 1.0, 10.0 and 100.0 µg/mL) had no effect on the expression of IL-8, IL-6, or MCP-1 (CCL2) after 24 h. In conclusion, CGN was not absorbed, and was not cytotoxic. It did not induce oxidative stress, and did not induce proinflammatory proteins.

Toll-like receptor 3 expression and function in childhood idiopathic nephrotic syndrome.

The efficacy of steroids and immunosuppressive treatments in idiopathic nephrotic syndrome (INS) hints at the implication of immune cells in the pathophysiology of the disease. Toll-like receptor (TLR) dysfunctions are involved in many kidney diseases of immune origin, but remain little described in INS. We investigated the expression and function of TLRs in peripheral blood mononuclear cells (PBMC) of INS children, including 28 in relapse, 23 in remission and 40 controls. No child had any sign of infection, but a higher Epstein-Barr virus viral load was measured in the PBMC of relapsing patients. TLR-3 expression was increased in B cells only during INS remission. There was a negative correlation between proteinuria and TLR-3 expression in total and the main subsets of PBMC from INS patients. The expression of TLR-8 was also increased in both CD4(+) T cells and B cells in INS remission. There was a negative correlation between proteinuria and TLR-8 expression in total PBMC, CD4(+) T cells and B cells of INS patients. Nevertheless, TLR-3 and TLR-8 expression was normalized in all PBMC subsets in an additional group of 15 INS patients in remission with B cell repletion after rituximab therapy. Paradoxically, interferon (IFN) regulatory factor 3 transactivation was increased in PBMC of all INS patients. In-vitro secretion of IFN-α and interleukin 6 were increased spontaneously in PBMC of INS remission patients, whereas PBMC from all INS patients displayed an impaired IFN-α secretion after TLR-3 stimulation. Thus, TLR-3 pathway dysfunctions may be closely involved in INS pathogenesis.

Artemisinin blocks prostate cancer growth and cell cycle progression by disrupting Sp1 interactions with the cyclin-dependent kinase-4 (CDK4) promoter and inhibiting CDK4 gene expression.

Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter.

Artemisinin selectively decreases functional levels of estrogen receptor-alpha and ablates estrogen-induced proliferation in human breast cancer cells.

MCF7 cells are an estrogen-responsive human breast cancer cell line that expresses both estrogen receptor (ER) alpha and ERbeta. Treatment of MCF7 cells with artemisinin, an antimalarial phytochemical from the sweet wormwood plant, effectively blocked estrogen-stimulated cell cycle progression induced by either 17beta-estradiol (E(2)), an agonist for both ERs, or by propyl pyrazole triol (PPT), a selective ERalpha agonist. Artemisinin strongly downregulated ERalpha protein and transcripts without altering expression or activity of ERbeta. Transfection of MCF7 cells with ERalpha promoter-linked luciferase reporter plasmids revealed that the artemisinin downregulation of ERalpha promoter activity accounted for the loss of ERalpha expression. Artemisinin treatment ablated the estrogenic induction of endogenous progesterone receptor (PR) transcripts by either E(2) or PPT and inhibited the estrogenic stimulation of a luciferase reporter plasmid driven by consensus estrogen response elements (EREs). Chromatin immunoprecipitation assays revealed that artemisinin significantly downregulated the level of endogeneous ERalpha bound to the PR promoter, whereas the level of bound endogeneous ERbeta was not altered. Treatment of MCF7 cells with artemisinin and the pure antiestrogen fulvestrant resulted in a cooperative reduction of ERalpha protein levels and enhanced G(1) cell cycle arrest compared with the effects of either compound alone. Our results show that artemisinin switches proliferative human breast cancer cells from expressing a high ERalpha:ERbeta ratio to a condition in which ERbeta predominates, which parallels the physiological state linked to antiproliferative events in normal mammary epithelium.

Papillary thyroid carcinoma in a 4-year-old boy.

We present a case of Papillary Thyroid Carcinoma (PTC) in a 4- year- old boy. The very young age of the patient and unusual presentation with respiratory distress prompted us to report this case.

Determination of the allergenic activity of birch pollen and apple prick test solutions by measurement of beta-hexosaminidase release from RBL-2H3 cells. Comparison with classical methods in allergen standardization.

BACKGROUND: A murine in vitro model of the allergic type I reaction was set up to determine the biologic activity of extracts without involvement of human beings. It is based on beta-hexosaminidase release from passively sensitized RBL cells after allergen challenge. The intended application of this RBL cell assay in the field of quality control of allergenic extracts requires its comparison with established methods. METHODS: The activity of five standardized birch-pollen prick test solutions was determined in parallel by RBL assay, direct IgE binding, IgE-binding inhibition, major allergen content, histamine-release assay, and skin testing. RESULTS: The RBL cell-release assay corresponded well to other methods if a reagin raised against natural birch-pollen extract was used for passive sensitization. However, in the case of a reagin against recombinant Bet v 1, only a decreased activity was observed, presumably because a reduced number of epitopes were recognized by the monospecific reagin. In contrast to standardized birch-pollen extracts, nonstandardized apple extracts showed poor activity in all assays. CONCLUSIONS: This murine model might be a useful tool in the quality control of allergenic extracts. It combines properties of assays based on standardized antisera and of assays that consider IgE cross-linking properties.

Osteoma osteoide: resección percutánea con guía de tomografía computada / Osteoid ostema: percutaneous resection with CT guidance

A 10 pacientes con diagnóstico presuntivo de osteoma osteoide se efectuó la resección percutánea bajo control tomográfico. La técnica ha sido exitosa en todos los casos. Dos pacientes consultaron nuevamente por dolor atípico y fueron tratados médicamente, con buena respuesta. En un paciente se efectuó la ablación con radiofrecuencia, con resultado satisfactorio