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Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.
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Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Peptídeos/farmacologia , Peçonhas/farmacologia , Animais , Células CHO , Cálcio/metabolismo , Linhagem Celular , Cricetulus , AMP Cíclico/metabolismo , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxintomodulina/química , Oxintomodulina/metabolismo , Peptídeos/química , Ratos , Transdução de Sinais , Peçonhas/químicaRESUMO
Transcriptomics is a powerful tool for unraveling the molecular effects of genetic variants and disease diagnosis. Prior studies have demonstrated that choice of genome build impacts variant interpretation and diagnostic yield for genomic analyses. To identify the extent genome build also impacts transcriptomics analyses, we studied the effect of the hg19, hg38, and CHM13 genome builds on expression quantification and outlier detection in 386 rare disease and familial control samples from both the Undiagnosed Diseases Network and Genomics Research to Elucidate the Genetics of Rare Disease Consortium. Across six routinely collected biospecimens, 61% of quantified genes were not influenced by genome build. However, we identified 1,492 genes with build-dependent quantification, 3,377 genes with build-exclusive expression, and 9,077 genes with annotation-specific expression across six routinely collected biospecimens, including 566 clinically relevant and 512 known OMIM genes. Further, we demonstrate that between builds for a given gene, a larger difference in quantification is well correlated with a larger change in expression outlier calling. Combined, we provide a database of genes impacted by build choice and recommend that transcriptomics-guided analyses and diagnoses are cross referenced with these data for robustness.
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Genoma Humano , RNA-Seq , Humanos , RNA-Seq/métodos , Genômica/métodos , Transcriptoma , Doenças Raras/genética , Doenças Raras/diagnóstico , Perfilação da Expressão Gênica/métodosRESUMO
A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia1,2. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration3-9. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins5 before its non-canonical translation into neural-toxic dipeptide proteins3,4. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation6-9. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria10,11 protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.
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Proteína C9orf72/genética , Microbioma Gastrointestinal/fisiologia , Gliose/microbiologia , Gliose/patologia , Inflamação/genética , Inflamação/microbiologia , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Antibacterianos/farmacologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/genética , Autoimunidade/imunologia , Movimento Celular/efeitos dos fármacos , Citocinas/imunologia , Transplante de Microbiota Fecal , Feminino , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Gliose/genética , Gliose/prevenção & controle , Inflamação/patologia , Inflamação/prevenção & controle , Mutação com Perda de Função/genética , Masculino , Camundongos , Microglia/imunologia , Microglia/microbiologia , Microglia/patologia , Medula Espinal/imunologia , Medula Espinal/microbiologia , Taxa de SobrevidaRESUMO
DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.
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Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Replicação do DNA/genética , Repetições de Microssatélites/genética , Animais , Proteínas de Ciclo Celular/genética , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Feminino , Instabilidade Genômica/genética , Humanos , Camundongos , Proteína de Replicação A/genéticaRESUMO
Autoimmunity, allergy, and transplant rejection are a collection of chronic diseases that are currently incurable, drastically decrease patient quality of life, and consume considerable health care resources. Underlying each of these diseases is a dysregulated immune system that results in the mounting of an inflammatory response against self or an innocuous antigen. As a consequence, afflicted patients are required to adhere to lifelong regimens of multiple immunomodulatory drugs to control disease and reclaim agency. Unfortunately, current immunomodulatory drugs are associated with a myriad of side effects and adverse events, such as increased risk of cancer and increased risk of serious infection, which negatively impacts patient adherence rates and quality of life. The field of immunoengineering is a new discipline that aims to harness endogenous biological pathways to thwart disease and minimize side effects using novel biomaterial-based strategies. We highlight and discuss polymeric micro/nanoparticles with inherent immunomodulatory properties that are currently under investigation in biomaterial-based therapies for treatment of autoimmunity, allergy, and transplant rejection.
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Autoimunidade , Rejeição de Enxerto , Hipersensibilidade , Polímeros , Humanos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Polímeros/química , Autoimunidade/efeitos dos fármacos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Animais , Materiais Biocompatíveis/química , Nanopartículas/química , Doenças Autoimunes/terapia , Doenças Autoimunes/imunologia , Agentes de Imunomodulação/uso terapêutico , Fatores Imunológicos/uso terapêuticoRESUMO
Williams-Beuren syndrome (WBS) is a rare genetic condition caused by a chromosomal microdeletion at 7q11.23. It is a multisystem disorder characterized by distinct facies, intellectual disability, and supravalvar aortic stenosis (SVAS). Those with WBS are at increased risk of sudden death, but mechanisms underlying this remain poorly understood. We recently demonstrated autonomic abnormalities in those with WBS that are associated with increased susceptibility to arrhythmia and sudden cardiac death (SCD). A recently introduced method for heart rate variability (HRV) analysis called "heart rate fragmentation" (HRF) correlates with adverse cardiovascular events (CVEs) and death in studies where heart rate variability (HRV) failed to identify high-risk subjects. Some argue that HRF quantifies nonautonomic cardiovascular modulators. We, therefore, sought to apply HRF analysis to a WBS cohort to determine 1) if those with WBS show differences in HRF compared with healthy controls and 2) if HRF helps characterize HRV abnormalities in those with WBS. Similar to studies of those with coronary artery disease (CAD) and atherosclerosis, we found significantly higher HRF (4 out of 7 metrics) in those with WBS compared with healthy controls. Multivariable analyses showed a weak-to-moderate association between HRF and HRV, suggesting that HRF may reflect HRV characteristics not fully captured by traditional HRV metrics (autonomic markers). We also introduce a new metric inspired by HRF methodology, significant acute rate drop (SARD), which may detect vagal activity more directly. HRF and SARD may improve on traditional HRV measures to identify those at greatest risk for SCD both in those with WBS and in other populations.NEW & NOTEWORTHY This work is the first to apply heart rate fragmentation analyses to individuals with Williams syndrome and posits that the heart rate fragmentation parameter W3 may enable detection and investigation of phenomena underlying the proarrhythmic short-long-short RR interval sequences paradigm known to precede ventricular fibrillation and ventricular tachycardia. It also forwards a novel method for quantifying sinus arrhythmia and sinus pauses that likely correlate with parasympathetic activity.
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Morte Súbita Cardíaca , Frequência Cardíaca , Síndrome de Williams , Síndrome de Williams/fisiopatologia , Síndrome de Williams/genética , Síndrome de Williams/complicações , Humanos , Morte Súbita Cardíaca/etiologia , Feminino , Masculino , Adolescente , Adulto , Adulto Jovem , Estudos de Casos e Controles , Fatores de Risco , Sistema Nervoso Autônomo/fisiopatologia , Criança , Medição de Risco , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/genética , Arritmias Cardíacas/diagnósticoRESUMO
Background Mammographic density measurements are used to identify patients who should undergo supplemental imaging for breast cancer detection, but artificial intelligence (AI) image analysis may be more effective. Purpose To assess whether AISmartDensity-an AI-based score integrating cancer signs, masking, and risk-surpasses measurements of mammographic density in identifying patients for supplemental breast imaging after a negative screening mammogram. Materials and Methods This retrospective study included randomly selected individuals who underwent screening mammography at Karolinska University Hospital between January 2008 and December 2015. The models in AISmartDensity were trained and validated using nonoverlapping data. The ability of AISmartDensity to identify future cancer in patients with a negative screening mammogram was evaluated and compared with that of mammographic density models. Sensitivity and positive predictive value (PPV) were calculated for the top 8% of scores, mimicking the proportion of patients in the Breast Imaging Reporting and Data System "extremely dense" category. Model performance was evaluated using area under the receiver operating characteristic curve (AUC) and was compared using the DeLong test. Results The study population included 65 325 examinations (median patient age, 53 years [IQR, 47-62 years])-64 870 examinations in healthy patients and 455 examinations in patients with breast cancer diagnosed within 3 years of a negative screening mammogram. The AUC for detecting subsequent cancers was 0.72 and 0.61 (P < .001) for AISmartDensity and the best-performing density model (age-adjusted dense area), respectively. For examinations with scores in the top 8%, AISmartDensity identified 152 of 455 (33%) future cancers with a PPV of 2.91%, whereas the best-performing density model (age-adjusted dense area) identified 57 of 455 (13%) future cancers with a PPV of 1.09% (P < .001). AISmartDensity identified 32% (41 of 130) and 34% (111 of 325) of interval and next-round screen-detected cancers, whereas the best-performing density model (dense area) identified 16% (21 of 130) and 9% (30 of 325), respectively. Conclusion AISmartDensity, integrating cancer signs, masking, and risk, outperformed traditional density models in identifying patients for supplemental imaging after a negative screening mammogram. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Kim and Chang in this issue.
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Neoplasias da Mama , Detecção Precoce de Câncer , Humanos , Pessoa de Meia-Idade , Feminino , Neoplasias da Mama/diagnóstico por imagem , Inteligência Artificial , Estudos Retrospectivos , MamografiaRESUMO
PURPOSE: The function of FAM177A1 and its relationship to human disease is largely unknown. Recent studies have demonstrated FAM177A1 to be a critical immune-associated gene. One previous case study has linked FAM177A1 to a neurodevelopmental disorder in 4 siblings. METHODS: We identified 5 individuals from 3 unrelated families with biallelic variants in FAM177A1. The physiological function of FAM177A1 was studied in a zebrafish model organism and human cell lines with loss-of-function variants similar to the affected cohort. RESULTS: These individuals share a characteristic phenotype defined by macrocephaly, global developmental delay, intellectual disability, seizures, behavioral abnormalities, hypotonia, and gait disturbance. We show that FAM177A1 localizes to the Golgi complex in mammalian and zebrafish cells. Intersection of the RNA sequencing and metabolomic data sets from FAM177A1-deficient human fibroblasts and whole zebrafish larvae demonstrated dysregulation of pathways associated with apoptosis, inflammation, and negative regulation of cell proliferation. CONCLUSION: Our data shed light on the emerging function of FAM177A1 and defines FAM177A1-related neurodevelopmental disorder as a new clinical entity.
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We combined synchrotron-based infrared absorbance and Raman scattering spectroscopies with diamond anvil cell techniques and a symmetry analysis to explore the properties of multiferroic (NH4)2FeCl5·H2O under extreme pressure-temperature conditions. Compression-induced splitting of the Fe-Cl stretching, Cl-Fe-Cl and Cl-Fe-O bending, and NH4+ librational modes defines two structural phase transitions, and a group-subgroup analysis reveals space group sequences that vary depending upon proximity to the unexpectedly wide order-disorder transition. We bring these findings together with prior high-field work to develop the pressure-temperature-magnetic field phase diagram uncovering competing polar, chiral, and magnetic phases in this system.
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The synthesis of a new bis-BF2 tetrafluorobenzo-[α]-fused BOPYPY dye from 4,5,6,7-tetrafluoroisoindole and 2-hydrazinopyrazine is reported. The regioselectivity of nucleophilic substitution reactions at the periphery of the tetrafluorinated BOPYPY and its α-bromo derivative were investigated using N-, O-, S-, and C-based nucleophiles. Among the aromatic fluorine atoms, the F2 atom is consistently regioselectively substituted, except when the α-position contains a thiophenol group; in this case, F4 is substituted instead due to stabilizing π-π-stacking between the two aromatic groups. The α-bromo BOPYPY derivative also reacts under Stille cross-coupling reaction conditions to produce the corresponding α-substituted product. The spectroscopic properties of these new fluorinated BOPYPYs were investigated and compared with nonfluorinated analogs.
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The Environmental Impact Classification for Alien Taxa (EICAT) is an important tool for biological invasion policy and management and has been adopted as an International Union for Conservation of Nature (IUCN) standard to measure the severity of environmental impacts caused by organisms living outside their native ranges. EICAT has already been incorporated into some national and local decision-making procedures, making it a particularly relevant resource for addressing the impact of non-native species. Recently, some of the underlying conceptual principles of EICAT, particularly those related to the use of the precautionary approach, have been challenged. Although still relatively new, guidelines for the application and interpretation of EICAT will be periodically revisited by the IUCN community, based on scientific evidence, to improve the process. Some of the criticisms recently raised are based on subjectively selected assumptions that cannot be generalized and may harm global efforts to manage biological invasions. EICAT adopts a precautionary principle by considering a species' impact history elsewhere because some taxa have traits that can make them inherently more harmful. Furthermore, non-native species are often important drivers of biodiversity loss even in the presence of other pressures. Ignoring the precautionary principle when tackling the impacts of non-native species has led to devastating consequences for human well-being, biodiversity, and ecosystems, as well as poor management outcomes, and thus to significant economic costs. EICAT is a relevant tool because it supports prioritization and management of non-native species and meeting and monitoring progress toward the Kunming-Montreal Global Biodiversity Framework (GBF) Target 6.
Uso de la Clasificación de Impacto Ambiental de los Taxones Exóticos de la UICN para la toma de decisiones Resumen La Clasificación de Impacto Ambiental de los Taxones Exóticos (EICAT, en inglés) es una herramienta importante para las políticas y manejo de las invasiones biológicas y ha sido adoptada como un estándar de la Unión Internacional para la Conservación de la Naturaleza (UICN) para medir la seriedad del impacto ambiental causado por los organismos que viven fuera de su extensión nativa. La EICAT ya ha sido incorporada a algunos procedimientos locales y nacionales de toma de decisiones, lo que la vuelve un recurso particularmente relevante para abordar el impacto de las especies no nativas. Algunos principios conceptuales subyacentes de la EICAT han sido cuestionados recientemente, en particular aquellos relacionados con el uso del principio de precaución. Aunque todavía son relativamente nuevas, las directrices para la aplicación e interpretación de la EICAT tendrán una revisión periódica, basada en evidencia científica, por parte de la comunidad de la UICN para mejorar el proceso. Algunas de las críticas recientes están basadas en suposiciones seleccionadas subjetivamente que no pueden generalizarse y podrían perjudicar los esfuerzos globales para manejar las invasiones biológicas. La EICAT adopta un principio de precaución cuando considera el historial de impacto de una especie en cualquier otro lugar ya que algunos taxones tienen características que podrían volverlos más dañinos. Además, las especies no nativas suelen ser factores de pérdida de bidiversidad, incluso bajo otras presiones. Cuando ignoramos el principio de precaución al abordar el impacto de las especies no nativas, hay consecuencias devastadoras para el bienestar humano, la biodiversidad y los ecosistemas, así como resultados pobres de conservación, y por lo tanto con costos económicos importantes. La EICAT es una herramienta relevante porque respalda la priorización y el manejo de las especies no nativas y ayuda con el cumplimiento y monitoreo del progreso para llegar al objetivo 6 del Marco Mundial de Biodiversidad KunmingMontreal.
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Ecossistema , Espécies Introduzidas , Humanos , Conservação dos Recursos Naturais , BiodiversidadeRESUMO
BACKGROUND: Arterial calcification due to deficiency of CD73 (ACDC; OMIM 211800) is a rare genetic disease resulting in calcium deposits in arteries and small joints causing claudication, resting pain, severe joint pain, and deformities. Currently, there are no standard treatments for ACDC. Our previous work identified etidronate as a potential targeted ACDC treatment, using in vitro and in vivo disease models with patient-derived cells. In this study, we test the safety and effectiveness of etidronate in attenuating the progression of lower-extremity arterial calcification and vascular blood flow based on the computed tomography (CT) calcium score and ankle-brachial index (ABI). METHODS: Seven adult patients with a confirmed genetic diagnosis of ACDC were enrolled in an open-label, nonrandomized, single-arm pilot study for etidronate treatment. They took etidronate daily for 14 days every 3 months and were examined at the NIH Clinical Center bi-annually for 3 years. They received a baseline evaluation as well as yearly follow up after treatment. Study visits included imaging studies, exercise tolerance tests with ABIs, clinical blood and urine testing, and full dental exams. RESULTS: Etidronate treatment appeared to have slowed the progression of further vascular calcification in lower extremities as measured by CT but did not have an effect in reversing vascular and/or periarticular joint calcifications in our small ACDC cohort. CONCLUSIONS: Etidronate was found to be safe and well tolerated by our patients and, despite the small sample size, appeared to show an effect in slowing the progression of calcification in our ACDC patient cohort.(ClinicalTrials.gov Identifier NCT01585402).
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5'-Nucleotidase , Ácido Etidrônico , Proteínas Ligadas por GPI , Calcificação Vascular , Humanos , Projetos Piloto , Calcificação Vascular/tratamento farmacológico , Calcificação Vascular/diagnóstico por imagem , Ácido Etidrônico/uso terapêutico , Ácido Etidrônico/efeitos adversos , Masculino , Feminino , Pessoa de Meia-Idade , Resultado do Tratamento , 5'-Nucleotidase/genética , 5'-Nucleotidase/deficiência , Fatores de Tempo , Proteínas Ligadas por GPI/sangue , Índice Tornozelo-Braço , Adulto , Conservadores da Densidade Óssea/uso terapêutico , Conservadores da Densidade Óssea/efeitos adversos , Progressão da Doença , Doença Arterial Periférica/tratamento farmacológico , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/fisiopatologia , Idoso , Extremidade Inferior/irrigação sanguínea , Angiografia por Tomografia Computadorizada , Predisposição Genética para Doença , Fluxo Sanguíneo RegionalRESUMO
This paper critiques the restrictive criteria for germline genome editing recently proposed by Chin, Nguma, and Ahmad in this journal. While praising the authors for resisting fervent calls for an outright ban on clinical applications of the technology, this paper argues that their approach is nevertheless unduly restrictive, and may thus hinder technological progress. This response advocates for weighing potential benefits against risks without succumbing to excessive caution, proposing that ethical oversight combined with genetic scrutiny at the embryo stage post-editing can enable responsible use of the technology, ultimately reducing the burden of genetic diseases and enhancing human health, akin to how IVF transformed reproductive medicine despite strong initial opposition.
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Fertilização in vitro , Edição de Genes , Células Germinativas , Humanos , Fertilização in vitro/métodos , Edição de Genes/ética , Edição de Genes/métodos , Feminino , Embrião de MamíferosRESUMO
The distribution of recombination events along large cereal chromosomes is uneven and is generally restricted to gene-rich telomeric ends. To understand how the lack of recombination affects diversity in the large pericentromeric regions, we analysed deep exome capture data from a final panel of 815 Hordeum vulgare (barley) cultivars, landraces and wild barleys, sampled from across their eco-geographical ranges. We defined and compared variant data across the pericentromeric and non-pericentromeric regions, observing a clear partitioning of diversity both within and between chromosomes and germplasm groups. Dramatically reduced diversity was found in the pericentromeres of both cultivars and landraces when compared with wild barley. We observed a mixture of completely and partially differentiated single-nucleotide polymorphisms (SNPs) between domesticated and wild gene pools, suggesting that domesticated gene pools were derived from multiple wild ancestors. Patterns of genome-wide linkage disequilibrium, haplotype block size and number, and variant frequency within blocks showed clear contrasts among individual chromosomes and between cultivars and wild barleys. Although most cultivar chromosomes shared a single major pericentromeric haplotype, chromosome 7H clearly differentiated the two-row and six-row types associated with different geographical origins. Within the pericentromeric regions we identified 22 387 non-synonymous SNPs, 92 of which were fixed for alternative alleles in cultivar versus wild accessions. Surprisingly, only 29 SNPs found exclusively in the cultivars were predicted to be 'highly deleterious'. Overall, our data reveal an unconventional pericentromeric genetic landscape among distinct barley gene pools, with different evolutionary processes driving domestication and diversification.
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Hordeum , Cromossomos , Domesticação , Hordeum/genética , Desequilíbrio de Ligação/genéticaRESUMO
Chinese hamster ovary (CHO) cells are used extensively to produce protein therapeutics, such as monoclonal antibodies (mAbs), in the biopharmaceutical industry. MAbs are large proteins that are energetically demanding to synthesize and secrete; therefore, high-producing CHO cell lines that are engineered for maximum metabolic efficiency are needed to meet increasing demands for mAb production. Previous studies have identified that high-producing cell lines possess a distinct metabolic phenotype when compared to low-producing cell lines. In particular, it was found that high mAb production is correlated to lactate consumption and elevated TCA cycle flux. We hypothesized that enhancing flux through the mitochondrial TCA cycle and oxidative phosphorylation would lead to increased mAb productivities and final titers. To test this hypothesis, we overexpressed peroxisome proliferator-activated receptor γ co-activator-1⺠(PGC-1âº), a gene that promotes mitochondrial metabolism, in an IgG-producing parental CHO cell line. Stable cell pools overexpressing PGC-1⺠exhibited increased oxygen consumption, indicating increased mitochondrial metabolism, as well as increased mAb specific productivity compared to the parental line. We also performed 13C metabolic flux analysis (MFA) to quantify how PGC-1⺠overexpression alters intracellular metabolic fluxes, revealing not only increased TCA cycle flux, but global upregulation of cellular metabolic activity. This study demonstrates the potential of rationally engineering the metabolism of industrial cell lines to improve overall mAb productivity and to increase the abundance of high-producing clones in stable cell pools.
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Anticorpos Monoclonais , PPAR gama , Cricetinae , Animais , Cricetulus , Células CHO , PPAR gama/metabolismo , Anticorpos Monoclonais/genética , Estresse Oxidativo , Imunoglobulina GRESUMO
We present here the life and the work of Paul A. Castelfranco (1921-2021), a very special person who was not only a top chemist of chlorophyll biosynthesis, but also made major contributions on fatty acid oxidation, acetate metabolism and cellular organization. He led an extraordinary and exemplary life as a human being. We present here both his personal life as well as his scientific life, which is followed by reminiscences by William Breidenbach, Kevin Smith, Alan Stemler, Ann Castelfranco, and John Castelfranco. As the subtitle of this Tribute implies, till the end Paul was a scientist par excellence, an intellectual with unlimited curiosity, a humanist, and a man of enduring religious faith. We all miss him dearly.
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Alkylation of one of the inner-core nitrogen atoms is one possible approach to obtain dianionic corrole ligands, suitable for the coordination of divalent metal ions, such as PdII . Inner-core N-methylation can be obtained by treating the corrole with CH3 I, but the reaction conditions should be optimized to limit the formation of the dimethylated derivative. Two regioisomers, the N-21 and the N-22 methyl derivatives are obtained from the reaction, with the first product achieved in a higher amount. Structural characterization of the reaction products evidenced the distortion induced by the introduction of the methyl groups; the N-methylcorroles are chiral compounds, and the enantiomers were separated by chromatography, with their absolute configuration assigned by ECD computation. Palladium insertion was achieved in the case of monosubstituted corroles, but not with the dimethylated macrocycle; X-ray characterization of the complexes showed the distortion of the macrocycles. The Pd complexes do not show luminescence emission, but are able to produce singlet oxygen upon irradiation. The PdII complexes were also inserted in human serum albumin (HSA) and dispersed in water; in this case, the protein protects the corroles from photobleaching, and a switch from the type II to the type I mechanism in reactive oxygen species (ROS) production is observed.
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BACKGROUND: Mepolizumab is a therapy for severe asthma. We have little knowledge of the characteristics of people in the US that discontinue mepolizumab in clinical care. OBJECTIVE: To investigate the real-world efficacy and time to clinical discontinuation of mepolizumab, we evaluated individuals with asthma started on mepolizumab at the Cleveland Clinic. We hypothesized that individuals that discontinue mepolizumab have more severe and uncontrolled asthma at baseline. METHODS: Between 2016 and 2022, patients who started on mepolizumab consented to be assessed over 18 months. At baseline, a questionnaire including demographic and medical history was collected. Laboratory findings such as ACT score, FENO (Fractional Excretion of Nitric Oxide), and spirometry were recorded. At the conclusion of the observation period, the participants were divided into two categories: Group A and Group B. RESULTS: Group B [N = 28] discontinued mepolizumab (p < 0.05) at an average of 5.8 months (SD 4.2 months). Group A [N = 129] stayed on the therapy for at least 1 year. A participant with an ACT score less than 13 has an odds ratio of 6.64 (95% CI, 2.1 - 26.0) of discontinuing mepolizumab therapy. For a male, the odds of discontinuing mepolizumab therapy is 3.39 (95% CI, 1.1-11.2). CONCLUSION: In this real-world study, we find that high eosinophil count may not be adequate in screening which individuals will benefit from mepolizumab. Up to 17% of patients fail therapy within 6 months, with male sex and low ACT score increasing risk of mepolizumab discontinuation at Cleveland Clinic.
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Bacterial leaf streak (BLS), caused chiefly by the pathogen Xanthomonas translucens pv. translucens, is becoming an increasingly important foliar disease of barley in the Upper Midwest. The deployment of resistant cultivars is the most economical and practical method of control. To identify sources of BLS resistance, we evaluated two panels of breeding lines from the University of Minnesota (UMN) and Anheuser-Busch InBev (ABI) barley improvement programs for reaction to strain CIX95 in the field at St. Paul and Crookston, MN, in 2020 and 2021. The percentage of resistant lines in the UMN and ABI panels with mid-season maturity was 1.8% (6 of 333 lines) and 5.2% (13 of 251 lines), respectively. Both panels were genotyped with the barley 50K iSelect SNP array, and then a genome-wide association study was performed. A single, highly significant association was identified for BLS resistance on chromosome 6H in the UMN panel. This association was also identified in the ABI panel. Seven other significant associations were detected in the ABI panel: two each on chromosomes 1H, 2H, and 3H and one on chromosome 5H. Of the eight associations identified in the panels, five were novel. The discovery of resistance in elite breeding lines will hasten the time needed to develop and release a BLS-resistant cultivar.
Assuntos
Hordeum , Hordeum/genética , Hordeum/microbiologia , Estudo de Associação Genômica Ampla , Doenças das Plantas/microbiologia , Melhoramento Vegetal , Mapeamento CromossômicoRESUMO
Pubertal stress causes enduring sexual behavior dysfunction in males and females, but the underlying mechanism remains unknown. These changes may arise from pubertal programming of the hypothalamic-pituitary-gonadal axis. Previous findings show that stress exposure downregulates the hypothalamic-pituitary-gonadal axis, particularly through the reduction of the neuropeptide kisspeptin (Kiss1) and its receptor (Kiss1R). Although acute changes in kiss1 and Kiss1r genes have been observed following pubertal immune stress, it is unclear whether immune stress-induced downregulation of kiss1 and kiss1r persists beyond puberty. The current study investigated the enduring sex-specific consequences of lipopolysaccharide on the expression of Kiss1 and Kiss1r in 160 pubertal or adult mice at multiple time points. Six-week and 10-week-old male and female mice were treated with either saline or with lipopolysaccharide. Mice were euthanized either 8 h or 4 weeks following treatment. Although we did not identify any sex differences, our results revealed that lipopolysaccharide treatment decreases hypothalamic Kiss1 and Kiss1r in both pubertal and adult mice within 8 h of treatment. The decreased hypothalamic Kiss1 expression persists 4 weeks later only in mice treated with lipopolysaccharide during puberty. Our findings highlight the age-dependent vulnerability of the hypothalamic-pituitary-gonadal axis to immune stress, providing a better understanding of the mechanisms implicated in allostatic shift during immune stress. Finally, our findings also show the effects of immune stress on various components of the hypothalamic-pituitary-gonadal axis, which could have implications for sexual and fertility-related dysfunctions.