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The sequencing platform and workflow strongly influence microbial community analyses through potential errors at each step. Effective diagnostics and experimental controls are needed to validate data and improve reproducibility. This cross-laboratory study evaluates sources of variability and error at three main steps of a standardized amplicon sequencing workflow (DNA extraction, polymerase chain reaction [PCR], and sequencing) using Oxford Nanopore MinION to analyze agricultural soils and a simple mock community. Variability in sequence results occurs at each step in the workflow with PCR errors and differences in library size greatly influencing diversity estimates. Common bioinformatic diagnostics and the mock community are ineffective at detecting PCR abnormalities. This work outlines several diagnostic checks and techniques to account for sequencing depth and ensure accuracy and reproducibility in soil community analyses. These diagnostics and the inclusion of a reference soil can help ensure data validity and facilitate the comparison of multiple sequencing runs within and between laboratories.
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Secuenciación de Nanoporos , Microbiología del Suelo , Secuenciación de Nanoporos/métodos , Reproducibilidad de los Resultados , Microbiota/genética , Análisis de Secuencia de ADN/métodos , Reacción en Cadena de la Polimerasa/métodos , Suelo/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
The spread of antimicrobial resistance (AMR) in agricultural systems via irrigation water is a serious public health issue as it can be transmitted to humans through the food chain. Therefore, understanding the dissemination routes of antibiotic resistance genes (ARGs) in agricultural systems is crucial for the assessment of health risks associated with eating fresh vegetables such as spinach and radish irrigated with treated municipal wastewater (TMW). In this study, we investigated the bacterial community structure and resistome in the soil-plant-earthworm continuum after irrigation of spinach and radish with TMW containing the antibiotics trimethoprim (TMP), sulfamethoxazole (SMZ), and sulfapyridine (SPD) using 16S rRNA gene sequencing and high throughput quantitative PCR (HT-qPCR). The study was conducted in two phases: Phase I involved eight weeks of spinach and radish production using TMW for irrigation, whereas Phase II entailed three weeks of earthworm exposure to contaminated plant material obtained in Phase I. The 16S data indicated that the rhizosphere bacterial community composition and structure were more resilient to antibiotic residuals in the irrigated water, with radish showing less susceptibility than spinach than those of bulk soils. The HT-qPCR analysis revealed that a total of 271 ARGs (out of 285) and 9 mobile genetic elements (MGEs) (out of 10) were detected in all samples. Higher diversity and abundance of ARGs were observed for samples irrigated with higher concentrations of antibiotics in both spinach and radish treatments. However, compared to spinach, radish ARG dynamics in the soil biome were more stable due to the change of antibiotic introduction to the soil. At the class level, multi-drug resistance (MDR) class was altered significantly by the presence of antibiotics in irrigation water. Compared to earthworm fecal samples, their corresponding soil environments showed a higher number of detected ARGs, suggesting that earthworms could play a role in reducing ARG dissemination in the soil environments. These findings will not only provide insight into the dissemination of ARGs in agricultural environments due to antibiotic residuals in irrigated water but could help understand the potential human health risks associated with ARGs.
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Riego Agrícola , Aguas Residuales , Aguas Residuales/microbiología , Eliminación de Residuos Líquidos/métodos , Farmacorresistencia Microbiana/genética , Microbiología del Suelo , Antibacterianos/análisis , Animales , Oligoquetos , Agricultura/métodos , EcosistemaRESUMEN
Dogs with B-cell lymphoma typically respond well to first-line CHOP-based chemotherapy, but there is no standard of care for relapsed patients. To help veterinary oncologists select effective drugs for dogs with lymphoid malignancies such as B-cell lymphoma, we have developed multimodal machine learning models that integrate data from multiple tumor profiling modalities and predict the likelihood of a positive clinical response for 10 commonly used chemotherapy drugs. Here we report on clinical outcomes that occurred after oncologists received a prediction report generated by our models. Remarkably, we found that dogs that received drugs predicted to be effective by the models experienced better clinical outcomes by every metric we analyzed (overall response rate, complete response rate, duration of complete response, patient survival times) relative to other dogs in the study and relative to historical controls.
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PURPOSE: To evaluate the utility of dissolvable collagen punctal plugs (CPP) in reducing ocular surface irritation after intravitreal injections (IVI). METHODS: Sixty-four subjects in the experimental group received CPP after intravitreal injections. Sixty-two controls did not receive CPP. Reductions in the Ocular Surface Disease Index© (OSDI) and Standardized Patient Evaluation of Eye Dryness II (SPEED II) scores were analysed. RESULTS: Dry eye symptoms, as measured by reductions from the pre- to post-injection OSDI (p = 0.137) and SPEED II (p = 0.381) scores, did not significantly differ between the two groups. In sub-group analysis, patients with objective findings of dry eyes had significant improvement in their symptoms (p = 0.046) with CPP. The effect of CPP is not significant in those without dry eyes (p = 0.27). CONCLUSION: CPPs were not effective in reducing post-injection ocular irritation in patients with no or only mild dry eye symptoms. CPPs improved patients' post-injection comfort levels in those who had moderate-to-severe symptoms and objective findings of dry eye. Though costly CPP could be considered in selective patients. A standardized eye rinse could be a simple, efficacious, and cost-effective way to reduce post-injection ocular irritation; however, more studies are needed.
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Síndromes de Ojo Seco , Tapones Lagrimales , Síndromes de Ojo Seco/tratamiento farmacológico , Ojo , Humanos , Inyecciones Intravítreas , Povidona Yodada/uso terapéutico , LágrimasRESUMEN
First-line treatments of cancer do not always work, and even when they do, they cure the disease at unequal rates mostly owing to biological and clinical heterogeneity across patients. Accurate prediction of clinical outcome and survival following the treatment can support and expedite the process of comparing alternative treatments. We describe the methodology to dynamically determine remission probabilities for individual patients, as well as their prospects of progression-free survival (PFS). The proposed methodology utilizes the ex vivo drug sensitivity of cancer cells, their immunophenotyping results, and patient information, such as age and breed, in training machine learning (ML) models, as well as the Cox hazards model to predict the probability of clinical remission (CR) or relapse across time for a given patient. We applied the methodology using the three types of data obtained from 242 canine lymphoma patients treated by (L)-CHOP chemotherapy. The results demonstrate substantial enhancement in the predictive accuracy of the ML models by utilizing features from all the three types of data. They also highlight superior performance and utility in predicting survival compared to the conventional stratification method. We believe that the proposed methodology can contribute to improving and personalizing the care of cancer patients.
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We report a precision medicine platform that evaluates the probability of chemotherapy drug efficacy for canine lymphoma by combining ex vivo chemosensitivity and immunophenotyping assays with computational modelling. We isolated live cancer cells from fresh fine needle aspirates of affected lymph nodes and collected post-treatment clinical responses in 261 canine lymphoma patients scheduled to receive at least 1 of 5 common chemotherapy agents (doxorubicin, vincristine, cyclophosphamide, lomustine and rabacfosadine). We used flow cytometry analysis for immunophenotyping and ex vivo chemosensitivity testing. For each drug, 70% of treated patients were randomly selected to train a random forest model to predict the probability of positive Veterinary Cooperative Oncology Group (VCOG) clinical response based on input variables including antigen expression profiles and treatment sensitivity readouts for each patient's cancer cells. The remaining 30% of patients were used to test model performance. Most models showed a test set ROC-AUC > 0.65, and all models had overall ROC-AUC > 0.95. Predicted response scores significantly distinguished (P < .001) positive responses from negative responses in B-cell and T-cell disease and newly diagnosed and relapsed patients. Patient groups with predicted response scores >50% showed a statistically significant reduction (log-rank P < .05) in time to complete response when compared to the groups with scores <50%. The computational models developed in this study enabled the conversion of ex vivo cell-based chemosensitivity assay results into a predicted probability of in vivo therapeutic efficacy, which may help improve treatment outcomes of individual canine lymphoma patients by providing predictive estimates of positive treatment response.
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Antineoplásicos/uso terapéutico , Enfermedades de los Perros/tratamiento farmacológico , Resistencia a Antineoplásicos , Inmunofenotipificación/veterinaria , Linfoma/veterinaria , Animales , Enfermedades de los Perros/patología , Perros , Femenino , Ganglios Linfáticos/patología , Linfoma/tratamiento farmacológico , Linfoma/patología , Aprendizaje Automático , Masculino , Modelos Biológicos , Valor Predictivo de las PruebasRESUMEN
von Hippel-Lindau (VHL) disease is an autosomal-dominant tumor predisposition syndrome characterized by the development of highly vascularized tumors and cysts. LOH of the VHL gene results in aberrant upregulation of hypoxia-inducible factors (HIF) and has been associated with tumor formation. Hemangioblastomas of the central nervous system and retina represent the most prevalent VHL-associated tumors, but no VHL animal model has reproduced retinal capillary hemangioblastomas (RCH), the hallmark lesion of ocular VHL. Here we report our work in developing a murine model of VHL-associated RCH by conditionally inactivating Vhl in a hemangioblast population using a Scl-Cre-ERT2 transgenic mouse line. In transgenic mice carrying the conditional allele and the Scl-Cre-ERT2 allele, 64% exhibited various retinal vascular anomalies following tamoxifen induction. Affected Vhl-mutant mice demonstrated retinal vascular lesions associated with prominent vasculature, anomalous capillary networks, hemorrhage, exudates, and localized fibrosis. Histologic analyses showed RCH-like lesions characterized by tortuous, dilated vasculature surrounded by "tumorlet" cell cluster and isolated foamy stromal cells, which are typically associated with RCH. Fluorescein angiography suggested increased vascular permeability of the irregular retinal vasculature and hemangioblastoma-like lesions. Vhl deletion was detected in "tumorlet" cells via microdissection. Our findings provide a phenotypic recapitulation of VHL-associated RCH in a murine model that may be useful to study RCH pathogenesis and therapeutics aimed at treating ocular VHL.Significance: This study describes a model that phenotypically recapitulates a form of retinal pathogenesis that is driven by genetic loss of the VHL tumor suppressor, providing a useful tool for its study and therapeutic intervention. Cancer Res; 78(5); 1266-74. ©2018 AACR.
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Modelos Animales de Enfermedad , Hemangioblastoma/patología , Hemangioblastos/patología , Neoplasias de la Retina/patología , Eliminación de Secuencia , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Enfermedad de von Hippel-Lindau/patología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Femenino , Hemangioblastoma/genética , Hemangioblastoma/metabolismo , Hemangioblastos/metabolismo , Masculino , Ratones , Ratones Noqueados , Neoplasias de la Retina/genética , Neoplasias de la Retina/metabolismo , Enfermedad de von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/metabolismoAsunto(s)
Neoplasias Endometriales/patología , Subluxación del Cristalino/diagnóstico , Neoplasias de la Retina/diagnóstico por imagen , Neoplasias de la Retina/secundario , Diagnóstico Diferencial , Femenino , Humanos , Imagen por Resonancia Magnética , Persona de Mediana Edad , Desprendimiento de Retina/diagnóstico , UltrasonografíaRESUMEN
Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.
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Citocinas/antagonistas & inhibidores , Interleucina-17/toxicidad , Degeneración Macular/prevención & control , Receptores de Interleucina-17/genética , Retina/efectos de los fármacos , Transfección , Dependovirus/genética , Vectores Genéticos , Humanos , Degeneración Macular/genéticaRESUMEN
Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of â¼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.
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Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Recuento de Colonia Microbiana , Medios de Cultivo , Cartilla de ADN , ADN Bacteriano/genética , Contaminación de Alimentos/análisis , Microbiología de Alimentos , Límite de Detección , Recombinasas , Sensibilidad y Especificidad , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga-Toxigénica/genéticaRESUMEN
PURPOSE: Chronic granulomatous disease (CGD) is an inherited disorder characterized by defects in phagocyte-derived nicotinamide adenine dinucleotide phosphate oxidase. It is typically diagnosed in childhood and leads to severe, recurrent bacterial or fungal infections. Chorioretinal lesions are the most common ocular manifestation. We sought to determine whether there are infectious agents in CGD-associated chorioretinopathy. METHODS: Medical records and ocular histopathology from CGD cases from January 1983 to January 2012 at the National Institutes of Health were retrospectively reviewed. Chorioretinal cells from normal and lesional tissues of the same eye were microdissected. Primers for Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia sp., and a panbacterial 16S ribosomal DNA were used for polymerase chain reaction. RESULTS: Seventeen CGD patients had ocular tissues (16 autopsied cases and 1 chorioretinal biopsy) examined. Of these 17, 8 demonstrated CGD-associated chorioretinal lesions in at least one eye on histopathology. Of these 8, 7 showed amplification of 16S ribosomal DNA within the lesion; of these 7, two also amplified S. epidermidis and one P. aeruginosa. One had no bacterial DNA amplified. Importantly, no microbial DNA was amplified from the normal, non-lesional ocular tissues of these 8 cases. Furthermore, only 1 of the 9 eyes without chorioretinopathy had amplified Burkholderia DNA, that patient had a history of Burkholderia infection. CONCLUSIONS: We detected bacterial DNA in 7 of 8 (88%) cases with CGD-associated chorioretinopathy and only in 1 normal ocular tissue of 17 CGD cases. Bacterial infection may play a role in the pathogenesis of CGD-associated chorioretinal lesions.
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Infecciones Bacterianas/microbiología , Enfermedades de la Coroides/microbiología , ADN Bacteriano/genética , Enfermedad Granulomatosa Crónica/microbiología , Enfermedades de la Retina/microbiología , Niño , Preescolar , Enfermedades de la Coroides/diagnóstico , ADN Ribosómico/genética , Femenino , Enfermedad Granulomatosa Crónica/diagnóstico , Humanos , Lactante , Masculino , Enfermedades de la Retina/diagnóstico , Estudios RetrospectivosRESUMEN
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Although recent studies have demonstrated strong genetic associations between AMD and SNPs in a number of genes, other modes of regulation are also likely to play a role in the etiology of this disease. We identified a significantly decreased level of methylation on the IL17RC promoter in AMD patients. Furthermore, we showed that hypomethylation of the IL17RC promoter in AMD patients led to an elevated expression of its protein and messenger RNA in peripheral blood as well as in the affected retina and choroid, suggesting that the DNA methylation pattern and expression of IL17RC may potentially serve as a biomarker for the diagnosis of AMD and likely plays a role in disease pathogenesis.
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Degeneración Macular/genética , Receptores de Interleucina/metabolismo , Línea Celular , Islas de CpG , Metilación de ADN , Ojo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-17/farmacología , Degeneración Macular/metabolismo , Degeneración Macular/patología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Receptores de Interleucina/sangre , Receptores de Interleucina/genética , GemelosRESUMEN
Von Hippel-Lindau (VHL) disease is a highly penetrant autosomal dominant systemic malignancy that gives rise to cystic and highly vascularized tumors in a constellation of organs. Patients with VHL disease commonly present with hemangioblastomas in the central nervous system and the eye while other manifestations include pheochromocytoma, clear cell renal cell carcinoma, endolymphatic sac tumors of the middle ear, pancreatic cystadenomas, epididymal and broad ligament cystadenomas. Animal models inactivating the VHL gene product in various organ tissues have been constructed over the past 15 years to parse its HIF-associated mechanisms and its link to tumorigenesis. These models, despite advancing our understanding the molecular role of VHL, are by and large unable to recapitulate the more common features of human VHL disease. Up to date, no model exists that develop retinal hemangioblastomas, the most common clinical manifestation. The purpose of this review is: (1) to discuss the need for an ocular VHL model, (2) to review the animal models that recapitulate clinical VHL disease and (3) to propose potential mechanisms of tumorigenesis for the development of ocular VHL.
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Modelos Animales de Enfermedad , Hemangioblastoma/patología , Neoplasias de la Retina/patología , Enfermedad de von Hippel-Lindau/patología , Animales , Femenino , Silenciador del Gen , Hemangioblastoma/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Neoplasias de la Retina/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Pez Cebra , Enfermedad de von Hippel-Lindau/genéticaRESUMEN
BACKGROUND: To report the tumor, patient, and treatment characteristics of long-term metastatic uveal melanoma survivors. METHODS: A non-comparative, retrospective case series of patients from a single institution surviving >24 months with metastatic uveal melanoma (UM). RESULTS: Nine patients met the study criteria and their charts were reviewed. The mean age at diagnosis of UM was 44.1 years (SD +/- 14.4 years). Initial treatment modalities included enucleation (67%), brachytherapy (22%), and proton beam radiation (11%). The average time from primary tumor diagnosis to detection of metastasis was 125.9 months (SD +/- 95 months). The most common location for initial metastasis was the liver. All patients underwent treatment for metastatic disease including systemic therapy, surgical resection, and isolated hepatic perfusion. The majority of patients received treatment with a tyrosine kinase inhibitor (sorafenib, sunitinib, and/or imatinib). The median survival with metastasis was 51 months (range 27-123 months). Patients had a long disease-free interval before presentation of metastatic disease. CONCLUSIONS: A small subset of patients with metastatic UM has prolonged survival. Identification of these patients may be helpful for future clinical trial design.
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BACKGROUND: Suppressor of cytokine signaling-3 (SOCS3) is a key negative-feedback regulator of the gp130 receptor that provides crucial signaling for cardiac hypertrophy and survival; however, an in vivo role of SOCS3 regulation on cardiac gp130 signaling remains obscure. METHODS AND RESULTS: We generated cardiac-specific SOCS3 knockout (SOCS3 cKO) mice. These mice showed increased activation of gp130 downstream signaling targets (STAT3, ERK1/2, AKT, and p38) from 15 weeks of age and developed cardiac dysfunction from approximately 25 weeks of age with signs of heart failure. Surprisingly, SOCS3 cKO failing hearts had minimal histological abnormalities with intact myofibril ultrastructure. In addition, Ca(2+) transients were significantly increased in SOCS3 cKO failing hearts compared with wild-type hearts. We also found that Ser23/24 residues of troponin I were hypophosphorylated in SOCS3 cKO hearts before the manifestation of cardiac dysfunction. These data suggested the presence of abnormalities in myofilament Ca(2+) sensitivity in SOCS3 cKO mice. In addition to the contractile dysfunction, we found various ventricular arrhythmias in SOCS3 cKO nonfailing hearts accompanied by a sarcoplasmic reticulum Ca(2+) overload. To determine the contribution of gp130 signaling to the cardiac phenotype that occurs with SOCS3 deficiency, we generated cardiac-specific gp130 and SOCS3 double KO mice. Double KO mice lived significantly longer and had different histological abnormalities when compared with SOCS3 cKO mice, thus demonstrating the importance of gp130 signaling in the SOCS3 cKO cardiac phenotype. CONCLUSIONS: Our results demonstrate an important role of SOCS3 regulation on cardiac gp130 signaling in the pathogenesis of contractile dysfunction and ventricular arrhythmias.