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Hyperandrogenism and polycystic ovarian syndrome result from the imbalance or increase of androgen levels in females. Androgen receptor (AR) mediates the effects of androgens, and this study examines whether neuronal AR plays a role in reproduction under normal and increased androgen conditions in female mice. The neuron-specific AR knockout (KO) mouse (SynARKO) was generated from a female mouse (synapsin promoter driven Cre) and a male mouse (Ar fl/y). Puberty onset and the levels of reproductive hormones such as LH, FSH, testosterone, and estradiol were comparable between the control and the SynARKO mice. There were no differences in cyclicity and fertility between the control and SynARKO mice, with similar impairment in both groups on DHT treatment. Neuronal AR KO, as in this SynARKO mouse model, did not alleviate the infertility associated with DHT treatment. These studies suggest that neuronal AR KO neither altered reproductive function under physiological androgen levels, nor restored fertility under hyperandrogenic conditions.
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Andrógenos , Síndrome del Ovario Poliquístico , Humanos , Femenino , Masculino , Ratones , Animales , Andrógenos/farmacología , Receptores Androgénicos/genética , Ratones Noqueados , Maduración Sexual , Reproducción/genética , NeuronasRESUMEN
Hyperandrogenemia and polycystic ovary syndrome are a result of the imbalance of androgen levels in females. Androgen receptor (Ar) mediates the effect of androgen, and this study examines how neuronal Ar in the central nervous system mediates metabolism under normal and increased androgen conditions in female mice. The neuron-specific ARKO mouse (SynARKO) was created from female (Ar fl/wt; synapsin promoter driven Cre) and male (Ar fl/y) mice. A glucose tolerance test revealed impaired glucose tolerance that was partially alleviated in the SynARKO-dihydrotestosterone (DHT) mice compared with Con-DHT mice after 4 months of DHT treatment. Heat production and food intake was higher in Con-DHT mice than in Con-veh mice; these effects were not altered between SynARKO-veh and SynARKO-DHT mice, indicating that excess androgens may partially alter calorie intake and energy expenditure in females via the neuronal Ar. The pAkt/Akt activity was higher in the hypothalamus in Con-DHT mice than in Con-veh mice, and this effect was attenuated in SynARKO-DHT mice. Western blot studies show that markers of inflammation and microglia activation, such as NF-kB p-65 and IBA1, increased in the hypothalamus of Con-DHT mice compared with Con-veh. These studies suggest that neuronal Ar mediates the metabolic impacts of androgen excess in females.
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Obesity, altered glucose homeostasis, hyperinsulinism, and reproductive dysfunction develops in female humans and mammals with hyperandrogenism. We previously reported that low dose dihydrotestosterone (DHT) administration results in metabolic and reproductive dysfunction in the absence of obesity in female mice, and conditional knock-out of the androgen receptor (Ar) in the liver (LivARKO) protects female mice from DHT-induced glucose intolerance and hyperinsulinemia. Since altered metabolic function will regulate reproduction, and liver plays a pivotal role in the reversible regulation of reproductive function, we sought to determine the reproductive phenotype of LivARKO mice under normal and hyperandrogenemic conditions. Using Cre/Lox technology, we deleted the Ar in the liver, and we observed LivARKO female mice have normal puberty timing, cyclicity and reproductive function. After DHT treatment, like control mice, LivARKO experience altered estrous cycling, reduced numbers of corpus lutea, and infertility. Liver Ar is not involved in hyperandrogenemia-induced reproductive dysfunction. The reproductive dysfunction in the DHT-treated LivARKO lean females with normal glucose homeostasis indicates that androgen-induced reproductive dysfunction is independent from metabolic dysfunction.
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Hiperandrogenismo , Síndrome del Ovario Poliquístico , Receptores Androgénicos , Reproducción , Animales , Dihidrotestosterona/farmacología , Femenino , Glucosa/metabolismo , Humanos , Hiperandrogenismo/inducido químicamente , Hiperandrogenismo/genética , Hiperandrogenismo/metabolismo , Hiperandrogenismo/fisiopatología , Hígado/metabolismo , Ratones , Obesidad/metabolismo , Síndrome del Ovario Poliquístico/inducido químicamente , Síndrome del Ovario Poliquístico/genética , Síndrome del Ovario Poliquístico/metabolismo , Receptores Androgénicos/deficiencia , Receptores Androgénicos/metabolismo , Reproducción/fisiologíaRESUMEN
Stomata regulate leaf CO2 assimilation (A) and water loss. The Ball-Berry and Medlyn models predict stomatal conductance (gs ) with a slope parameter (m or g1 ) that reflects the sensitivity of gs to A, atmospheric CO2 and humidity, and is inversely related to water use efficiency (WUE). This study addressed knowledge gaps about what the values of m and g1 are in C4 crops under field conditions, as well as how they vary among genotypes and with drought stress. Four inbred maize genotypes were unexpectedly consistent in how m and g1 decreased as water supply decreased. This was despite genotypic variation in stomatal patterning, A and gs . m and g1 were strongly correlated with soil water content, moderately correlated with predawn leaf water potential (Ψpd ), but not correlated with midday leaf water potential (Ψmd ). This implied that m and g1 respond to long-term water supply more than short-term drought stress. The conserved nature of m and g1 across anatomically diverse genotypes and water supplies suggests there is flexibility in structure-function relationships underpinning WUE. This evidence can guide the simulation of maize gs across a range of water supply in the primary maize growing region and inform efforts to improve WUE.
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Fotosíntesis , Zea mays , Dióxido de Carbono , Sequías , Fotosíntesis/fisiología , Hojas de la Planta/genética , Estomas de Plantas/fisiología , Abastecimiento de Agua , Zea mays/genéticaRESUMEN
Stomata are adjustable pores on leaf surfaces that regulate the tradeoff of CO2 uptake with water vapor loss, thus having critical roles in controlling photosynthetic carbon gain and plant water use. The lack of easy, rapid methods for phenotyping epidermal cell traits have limited discoveries about the genetic basis of stomatal patterning. A high-throughput epidermal cell phenotyping pipeline is presented here and used for quantitative trait loci (QTL) mapping in field-grown maize (Zea mays). The locations and sizes of stomatal complexes and pavement cells on images acquired by an optical topometer from mature leaves were automatically determined. Computer estimated stomatal complex density (SCD; R2 = 0.97) and stomatal complex area (SCA; R2 = 0.71) were strongly correlated with human measurements. Leaf gas exchange traits were genetically correlated with the dimensions and proportions of stomatal complexes (rg = 0.39-0.71) but did not correlate with SCD. Heritability of epidermal traits was moderate to high (h2 = 0.42-0.82) across two field seasons. Thirty-six QTL were consistently identified for a given trait in both years. Twenty-four clusters of overlapping QTL for multiple traits were identified, with univariate versus multivariate single marker analysis providing evidence consistent with pleiotropy in multiple cases. Putative orthologs of genes known to regulate stomatal patterning in Arabidopsis (Arabidopsis thaliana) were located within some, but not all, of these regions. This study demonstrates how discovery of the genetic basis for stomatal patterning can be accelerated in maize, a C4 model species where these processes are poorly understood.
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Botánica/métodos , Mapeo Cromosómico/instrumentación , Aprendizaje Automático , Fenotipo , Estomas de Plantas/fisiología , Sitios de Carácter Cuantitativo , Zea mays/genética , Botánica/instrumentación , Genes de PlantasRESUMEN
BACKGROUND: Thyroid nodules are increasingly common. Although guidelines have been published, it is unclear whether recommendations for the use of fine-needle aspiration (FNA) and ultrasonography are followed. OBJECTIVE: To evaluate the impact of a quality improvement initiative regarding utilization of FNA and ultrasonography before resection of malignant thyroid neoplasms at a community-based hospital. METHODS: A retrospective medical record review of patients who received thyroidectomy with histologically proven malignant thyroid neoplasms at a community-based teaching center in the Midwest in 2014 revealed inconsistent use of FNA and ultrasonography per national guidelines. Thus, a quality improvement initiative was conducted using the PDSA (Plan, Do, Study, Act) method and included both an intradepartmental outreach effort and an interdisciplinary hospital cancer committee presentation. To determine the success of the initiative, medical records were reviewed from January 1, 2015, through July 1, 2016 (after the initiative) and compared with findings from 2014 (before the initiative). RESULTS: The medical records of 366 patients were reviewed over a 2.5-year period, and 23 records (12 in 2014 and 11 in 2015-2016) met the inclusion criteria. In 2014, FNA was performed on 58% of patients before operative management of thyroid malignancy. After the quality improvement initiative, FNA was performed on 100% of patients before operative management of thyroid malignancy (P=.0155). Before the quality improvement initiative, 75% of patients undergoing an operation for malignant thyroid neoplasms underwent preoperative ultrasonography, compared with 100% after the initiative (P=.0753). CONCLUSION: A performance improvement initiative that used the PDSA framework effectively influenced physician adherence to national guidelines for thyroid neoplasms.
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Biopsia con Aguja Fina , Mejoramiento de la Calidad , Neoplasias de la Tiroides/patología , Nódulo Tiroideo/patología , Tiroidectomía/métodos , Adulto , Anciano , Estudios de Cohortes , Bases de Datos Factuales , Femenino , Estudios de Seguimiento , Hospitales Comunitarios , Humanos , Masculino , Persona de Mediana Edad , Cuidados Preoperatorios/métodos , Estudios Retrospectivos , Medición de Riesgo , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/cirugía , Nódulo Tiroideo/diagnóstico por imagen , Nódulo Tiroideo/cirugía , Resultado del Tratamiento , Ultrasonografía DopplerRESUMEN
The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.
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Elementos Transponibles de ADN/genética , Genes de Plantas/genética , Genoma de Planta/genética , Zea mays/genética , Cromatina/genética , Cromosomas de las Plantas/genética , Variaciones en el Número de Copia de ADN/genética , Metilación de ADN/genética , ADN de Plantas/genética , Genómica/métodos , Sistemas de Lectura Abierta/genética , Análisis de Secuencia de ADN/métodosRESUMEN
BACKGROUND: Hyalinizing trabecular tumors (HTT) are rare follicular cell-derived tumors of the thyroid gland that are infrequently reported in otolaryngology literature. We present here an interesting case of HTT which provides the basis for review of this entity's clinical characteristics, criteria useful in making the diagnosis, and any currently available therapeutic modalities. CASE PRESENTATION: A 70-year-old Caucasian female underwent a CT scan of her chest and was incidentally found to have a nodule within the right thyroid lobe. Gross examination of the excised thyroid lobe revealed a circumscribed and encapsulated lesion (tan / gritty in texture), confined to the gland. Histologic sections of the lesion revealed a circumscribed neoplasm with a trabecular and organoid architecture associated with abundant dystrophic calcification. Neoplastic cells showed a spindled morphology with clumped chromatin and ample eosinophilic cytoplasm. CONCLUSIONS: Histologically, HTT is a follicular cell-derived tumor composed of neoplastic cells arranged in a trabecular pattern with hyalinization and calcification of extracellular material. Distinguishing features of HTT include minimal cytologic atypia with a low nuclear: cytoplasmic ratio, cellular aggregates around hyalinized material, and nuclei with clumped chromatin and occasional grooves and/or pseudoinclusions. Though debated in the literature, the general consensus is that this tumor is a benign entity. It is our hope that additional clinical research will elicit awareness of these rare tumors.
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Pancreatic ductal adenocarcinoma is characterized by prominent stromal involvement, which plays complex roles in regulating tumor growth and therapeutic response. The extracellular matrix (ECM)-rich stroma associated with this disease has been implicated as a barrier to drug penetration, although stromal depletion strategies have had mixed clinical success. It remains less clear how interactions with ECM, acting as a biophysical regulator of phenotype, not only a barrier to drug perfusion, regulate susceptibilities and resistance to specific therapies. In this context, an integrative approach is used to evaluate invasive behavior and motility in rheologically characterized ECM as determinants of chemotherapy and photodynamic therapy (PDT) responses. We show that in 3D cultures with ECM conditions that promote invasive progression, response to PDT is markedly enhanced in the most motile ECM-infiltrating populations, whereas the same cells exhibit chemoresistance. Conversely, drug-resistant sublines with enhanced invasive potential were generated to compare differential treatment response in identical ECM conditions, monitored by particle tracking microrheology measurements of matrix remodeling. In both scenarios, ECM-infiltrating cell populations exhibit increased sensitivity to PDT, whether invasion is consequent to selection of chemoresistance, or whether chemoresistance is correlated with acquisition of invasive behavior. However, while ECM-invading, chemoresistant cells exhibit mesenchymal phenotype, induction of EMT in monolayers without ECM was not sufficient to enhance PDT sensitivity, yet does impart chemoresistance as expected. In addition to containing platform development with broader applicability to inform microenvironment-dependent therapeutics, these results reveal the efficacy of PDT for targeting the most aggressive, chemoresistant, invasive pancreatic ductal adenocarcinoma associated with dismal outcomes for this disease. IMPLICATIONS: ECM-infiltrating and chemoresistant pancreatic tumor populations exhibit increased sensitivity to PDT. Mol Cancer Res; 15(1); 15-25. ©2016 AACR.
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Adenocarcinoma/patología , Carcinoma Ductal Pancreático/patología , Movimiento Celular , Matriz Extracelular/metabolismo , Modelos Biológicos , Neoplasias Pancreáticas/patología , Fotoquimioterapia , Reología , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Humanos , Mesodermo/patología , Invasividad Neoplásica , Neoplasias PancreáticasRESUMEN
Androgen and its receptor (AR) play a critical role in reproductive function under both physiological and pathophysiological conditions. Female AR global knockout mice are subfertile due to both neuroendocrine and ovarian defects. Female offspring from prenatally androgenized heterozygous AR pregnant mice showed rescued estrous cyclicity and fertility. Ar is expressed in granulosa cells, theca interstitial cells, and oocytes in the ovary. We created mice with theca-specific deletion of Ar (ThARKO) by crossing Cyp17-iCre mice that express Cre recombinase under cytochrome P450 17A1 (Cyp17) promoter with Arfl/fl mice. ThARKO mice exhibited no significant differences in pubertal onset or fertility compared with control littermates, and neither estrogen or testosterone levels were different between these groups. Therefore, Ar expression in theca cells likely does not influence fertility nor androgen levels in female mice. We then tested the role of AR in theca cells under hyperandrogenemic condition. After treatment with a pathophysiological level of dihydrotestosterone (DHT), control mice (control-DHT) showed acyclicity and infertility. However, estrous cycles and fertility were altered to a significantly less degree in ThARKO-DHT mice than in control-DHT mice. Messenger RNA (mRNA) levels of Lhcgr (luteinizing hormone receptor) and Timp1 (tissue inhibitor of metalloproteinase 1, and inhibitor of matrix metalloproteinase) were significantly lower in control-DHT ovary compared with control-no DHT ovaries, whereas mRNA levels of Fshr (follicle-stimulating hormone receptor) were significantly higher. Timp1 gene expression was comparable in the ThARKO-DHT and the control-no DHT ovary. We speculate that the preserved level of Timp1 in ThARKO-DHT mice contributes to retained reproductive function.
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Hiperandrogenismo/complicaciones , Infertilidad Femenina/etiología , Receptores Androgénicos/metabolismo , Células Tecales/metabolismo , Animales , Ciclo Estral , Femenino , Fertilidad , Hormonas Esteroides Gonadales/metabolismo , Masculino , Ratones , Ratones Noqueados , Ovario/patología , Maduración SexualRESUMEN
BACKGROUND: Panicoideae are the second largest subfamily in Poaceae (grass family), with 212 genera and approximately 3316 species. Previous studies have begun to reveal relationships within the subfamily, but largely lack resolution and/or robust support for certain tribal and subtribal groups. This study aims to resolve these relationships, as well as characterize a putative mitochondrial insert in one linage. RESULTS: 35 newly sequenced Panicoideae plastomes were combined in a phylogenomic study with 37 other species: 15 Panicoideae and 22 from outgroups. A robust Panicoideae topology largely congruent with previous studies was obtained, but with some incongruences with previously reported subtribal relationships. A mitochondrial DNA (mtDNA) to plastid DNA (ptDNA) transfer was discovered in the Paspalum lineage. CONCLUSIONS: The phylogenomic analysis returned a topology that largely supports previous studies. Five previously recognized subtribes appear on the topology to be non-monophyletic. Additionally, evidence for mtDNA to ptDNA transfer was identified in both Paspalum fimbriatum and P. dilatatum, and suggests a single rare event that took place in a common progenitor. Finally, the framework from this study can guide larger whole plastome sampling to discern the relationships in Cyperochloeae, Steyermarkochloeae, Gynerieae, and other incertae sedis taxa that are weakly supported or unresolved.
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Evolución Molecular , Plastidios/genética , Poaceae/genética , ADN Mitocondrial/genética , ADN de Plantas/genética , Filogenia , Poaceae/clasificaciónRESUMEN
BACKGROUND: Hyperglycemia is commonly seen in hospitalized patients. Glycated hemoglobin (HbA1c) correlates with the average blood glucose level over the previous 8 to 12 weeks. Thus, an HbA1c test offers a longitudinal view that reduces etiologic ambiguity of disease. Screening of HbA1c levels plays an important role in the diagnosis and management of diabetes mellitus in the outpatient setting but remains underused in the evaluation of hyperglycemia with undiagnosed diabetes in the inpatient setting. The underuse of the HbA1c test may be a missed opportunity for early diabetes detection in the hospital. OBJECTIVE: To examine the use of HbA1c tests in identifying previously undiagnosed diabetes mellitus among patients with hyperglycemia in a rural inpatient setting. METHODS: A retrospective review of medical records of hospitalized patients with hyperglycemia in a rural community teaching hospital in the Midwest. Descriptive and inferential statistical methods were used. RESULTS: Medical records of 348 unique patients with hyperglycemia were reviewed. Fifty patients treated for hyperglycemia had medical records with no known history of diabetes (NKHD). Of the 50 patients with NKHD, 31 (62%) had an HbA1c test. Of the 31 patients tested, 6 (19%) had HbA1c levels consistent with the diagnosis of prediabetes, and 18 (58%) had levels consistent with diabetes. Seventeen (55%) of the 31 patients had a discharge diagnosis that included diabetes. Of the 19 patients with NKHD who did not have an HbA1c test, 2 (11%) received a discharge diagnosis that included diabetes. CONCLUSION: Hospitalized patients with NKHD and hyperglycemia are more likely to receive an appropriate diagnosis if HbA1c is measured. Failing to fully use HbA1c tests in the inpatient setting constitutes a missed opportunity to distinguish transient hyperglycemia from chronic disease. The HbA1c level can elucidate the course of dys-glycemia and trigger mechanisms for timely intervention.
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Diabetes Mellitus/diagnóstico , Hemoglobina Glucada/análisis , Hiperglucemia/sangre , Adulto , Anciano , Diabetes Mellitus/sangre , Diagnóstico Diferencial , Femenino , Hospitalización , Hospitales Rurales , Humanos , Hiperglucemia/diagnóstico , Masculino , Registros Médicos , Persona de Mediana Edad , Medio Oeste de Estados Unidos , Estudios RetrospectivosRESUMEN
Despite progress based on multilocus, phylogenetic studies of the palms (order Arecales, family Arecaceae), uncertainty remains in resolution/support among major clades and for the placement of the palms among the commelinid monocots. Palms and related commelinids represent a classic case of substitution rate heterogeneity that has not been investigated in the genomic era. To address questions of relationships, support and rate variation among palms and commelinid relatives, 39 plastomes representing the palms and related family Dasypogonaceae were generated via genome skimming and integrated within a monocot-wide matrix for phylogenetic and molecular evolutionary analyses. Support was strong for 'deep' relationships among the commelinid orders, among the five palm subfamilies, and among tribes of the subfamily Coryphoideae. Additionally, there was extreme heterogeneity in the plastid substitution rates across the commelinid orders indicated by model based analyses, with c. 22 rate shifts, and significant departure from a global clock. To date, this study represents the most comprehensively sampled matrix of plastomes assembled for monocot angiosperms, providing genome-scale support for phylogenetic relationships of monocot angiosperms, and lays the phylogenetic groundwork for comparative analyses of the drivers and correlates of such drastic differences in substitution rates across a diverse and significant clade.
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Arecaceae/genética , Genoma de Plastidios , Filogenia , Evolución Molecular , Magnoliopsida/genética , Proteínas de Plantas/genéticaRESUMEN
BACKGROUND: Plastome sequences for 18 species of the PACMAD grasses (subfamilies Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, Danthonioideae) were analyzed phylogenomically. Next generation sequencing methods were used to provide complete plastome sequences for 12 species. Sanger sequencing was performed to determine the plastome of one species, Hakonechloa macra, to provide a reference for annotation. These analyses were conducted to resolve deep subfamilial relationships within the clade. Divergence estimates were assessed to determine potential factors that led to the rapid radiation of this lineage and its dominance of warmer open habitats. RESULTS: New plastomes were completely sequenced and characterized for 13 PACMAD species. An autapomorphic ~1140 bp deletion was found in Hakonechloa macra putatively pseudogenizing rpl14 and eliminating rpl16 from this plastome. Phylogenomic analyses support Panicoideae as the sister group to the ACMAD clade. Complete plastome sequences provide greater support at deep nodes within the PACMAD clade. The initial diversification of PACMAD subfamilies was estimated to occur at 32.4 mya. CONCLUSIONS: Phylogenomic analyses of complete plastomes provides resolution for deep relationships of PACMAD grasses. The divergence estimate of 32.4 mya at the crown node of the PACMAD clade coincides with the Eocene-Oligocene Transition (EOT). The Eocene was a period of global cooling and drying, which led to forest fragmentation and the expansion of open habitats now dominated by these grasses. Understanding how these grasses are related and determining a cause for their rapid radiation allows for future predictions of grassland distribution in the face of a changing global climate.
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Genoma del Cloroplasto , Genoma Mitocondrial , Proteínas de Plantas/genética , Poaceae/genética , Evolución Biológica , Ecosistema , Evolución Molecular , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/metabolismo , Poaceae/metabolismo , Análisis de Secuencia de ADNRESUMEN
A lack of access to effective cancer therapeutics in resource-limited settings is implicated in global cancer health disparities between developed and developing countries. Photodynamic therapy (PDT) is a light-based treatment modality that has exhibited safety and efficacy in the clinic using wavelengths and irradiances achievable with light-emitting diodes (LEDs) operated on battery power. Here we assess low-cost enabling technology to extend the clinical benefit of PDT to regions with little or no access to electricity or medical infrastructure. We demonstrate the efficacy of a device based on a 635 nm high-output LED powered by three AA disposable alkaline batteries, to achieve strong cytotoxic response in monolayer and 3D cultures of A431 squamous carcinoma cells following photosensitization by administering aminolevulinic acid (ALA) to induce the accumulation of protoporphyrin IX (PpIX). Here we characterize challenges of battery-operated device performance, including battery drain and voltage stability specifically over relevant PDT dose parameters. Further motivated by the well-established capacity of PDT photosensitizers to serve as tumour-selective fluorescence contrast agents, we demonstrate the capability of a consumer smartphone with low-cost add-ons to measure concentration-dependent PpIX fluorescence. This study lays the groundwork for the on-going development of image-guided ALA-PDT treatment technologies for global health applications.
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Imagenología Tridimensional/métodos , Neoplasias , Fotoquimioterapia/instrumentación , Teléfono Inteligente , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fotoquimioterapia/métodosRESUMEN
Parasitic organisms exemplify morphological and genomic reduction. Some heterotrophic, parasitic plants harbor drastically reduced and degraded plastid genomes resulting from relaxed selective pressure on photosynthetic function. However, few studies have addressed the initial stages of plastome degradation in groups containing both photosynthetic and nonphotosynthetic species. Corallorhiza is a genus of leafless, heterotrophic orchids that contains both green, photosynthetic species and nongreen, putatively nonphotosynthetic species, and represents an ideal system in which to assess the beginning of the transition to a "minimal plastome." Complete plastomes were generated for nine taxa of Corallorhiza using Illumina paired-end sequencing of genomic DNA to assess the degree of degradation among taxa, and for comparison with a general model of degradation among angiosperms. Quantification of total chlorophyll suggests that nongreen Corallorhiza still produce chlorophyll, but at 10-fold lower concentrations than green congeners. Complete plastomes and partial nuclear rDNA cistrons yielded a fully resolved tree for Corallorhiza, with at least two independent losses of photosynthesis, evidenced by gene deletions and pseudogenes in Co. striata and nongreen Co. maculata. All Corallorhiza show some evidence of degradation in genes of the NAD(P)H dehydrogenase complex. Among genes with open reading frames, photosynthesis-related genes displayed evidence of neutral evolution in nongreen Corallorhiza, whereas genes of the ATP synthase complex displayed some evidence of positive selection in these same groups, though for reasons unknown. Corallorhiza spans the early stages of a general model of plastome degradation and has added critical insight for understanding the process of plastome evolution in heterotrophic angiosperms.
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Genoma de Plastidios , Orchidaceae/genética , Clorofila/metabolismo , Evolución Molecular , Genes de Plantas , Procesos Heterotróficos/genética , Datos de Secuencia Molecular , Orchidaceae/metabolismo , Filogenia , Análisis de Secuencia de ADNRESUMEN
The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.
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Modelos Biológicos , Neoplasias/patología , Reología/métodos , Algoritmos , Matriz Extracelular , Imagenología Tridimensional/métodos , Programas Informáticos , Esferoides Celulares , Microambiente TumoralRESUMEN
Polyploidy, or whole-genome duplication (WGD), is a recurrent mutation both in cell lineages and over evolutionary time. By globally changing the relationship between gene copy number and other cellular entities, it can induce dramatic changes at the cellular and phenotypic level. Perhaps surprisingly, then, the insights that these events can bring to understanding other cellular features are not as well appreciated as they could be. In this review, we draw on examples of polyploidy from animals, plants and yeast to explore how investigations of polyploid cells have improved our understanding of the cell cycle, biological network complexity, metabolic phenotypes and tumor biology. We argue that the study of polyploidy across organisms, cell types, and time scales serves not only as a window into basic cell biology, but also as a basis for a predictive biology with applications ranging from crop improvement to treating cancer.
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Evolución Biológica , Genoma , Poliploidía , Animales , Evolución Molecular , Humanos , Plantas/genéticaRESUMEN
Four novel alanine-based indolicidin peptide derivatives were designed containing one WPW motif and two alanine residues, resulting in peptides of similar sequence. The separation of these peptides with identical physicochemical properties including molar mass, charge, and secondary structure as characterized by circular dichroism spectroscopy is very difficult; and the separation of peptides with differing physicochemical properties has only previously been reported. Capillary electrophoresis parameters such as separation buffer concentration, separation buffer pH, capillary length, and separation voltage were investigated to optimize the analysis. Using optimized conditions of a background electrolyte containing 5 mM formic acid of pH 2.0, total capillary length of 51 cm and a voltage of 10 kV enabled a baseline separation of the four peptides. The relative standard deviation of the peak areas and migration times for method repeatability (n = 3) were found to be lower than 8% and 3%, respectively. In addition, reasoning for the separation of these peptides is proposed based on the acidity of the formic acid buffer and the hydrophobic grouping of the tryptophan residues in the peptide primary sequence.
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Alanina/análogos & derivados , Antiinfecciosos/síntesis química , Péptidos Catiónicos Antimicrobianos/síntesis química , Electroforesis Capilar/métodos , Alanina/síntesis química , Alanina/química , Alanina/aislamiento & purificación , Secuencias de Aminoácidos , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
State wildlife agencies have translocated thousands of wild turkeys (Meleagris gallopavo) since the 1930s to reestablish this species. Because of threats to the domestic poultry industry and wild birds, screening for selected infectious agents has become routine since the early 1980s. One of the principal sources for Rio Grande wild turkeys (M. gallopavo intermedia) for translocation purposes was the Edwards Plateau of Texas (USA). Unfortunately, turkey abundance has declined in the southern Edwards Plateau since the late 1970s. Surprisingly few studies have addressed wild turkeys in this region, perhaps reflecting its status as the heart of Rio Grande turkey range. We surveyed 70 free-living Rio Grande wild turkeys from Bandera and Kerr counties, Texas, for evidence of exposure to Salmonella typhimurium, S. pullorum, Mycoplasma gallisepticum, M. meleagridis, M. synoviae, Chlamydophila psittaci, and the avian influenza, Newcastle disease, turkey corona, and reticuloendotheliosis viruses. Of these, 80% (56) were seropositive for both M. gallisepticum and M. synoviae on the serum plate antigen test. Ten of these individuals (14% of total) were positive for M. synoviae by hemagglutination inhibition testing. All other serologic tests were negative. Two adult females sampled in Kerr County, whose body mass was significantly less than that of other adult females trapped in the area, tested positive for reticuloendotheliosis virus (REV) proviral DNA on polymerase chain reaction. Reticuloendotheliosis virus was isolated from one of these individuals. The pathogenesis, transmission, and/or population-level influences of M. gallisepticum, M. synoviae, and REV in Rio Grande wild turkeys deserves further study.