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Virome analysis was performed on 174 grape genetic resources from the National Agriculture and Food Research Organization, Japan. A total of 20 bulk samples was prepared by grouping the vines into batches of 6-10 plants. Each of the bulk samples was analyzed using high-throughput sequencing, which detected 27 viruses and 5 viroids, including six viruses and one viroid reported in Japan for the first time (grapevine viruses F, L, and T, grapevine Kizil Sapak virus, grapevine Syrah virus 1, grapevine satellite virus, and grapevine yellow speckle viroid 2). In addition, a novel vitivirus was detected with a maximum nucleotide sequence identity of only 58% to its closest relative, grapevine virus A (GVA). The genome of this novel virus was 7,461 nucleotides in length and encoded five open reading frames showing the typical genomic structure of vitiviruses. Phylogenetic trees of vitiviruses placed it in a distinct position nearest to GVA or grapevine virus F (GVF) in genomes and amino acids of deduced replication-associated protein (RAP) and coat protein (CP). The amino acid sequence identities of RAP and CP with GVA, GVF, and other vitiviruses were a maximum of 53% and 73%, respectively, which were significantly below the species demarcation threshold of 80% in the genus. The low identity and phylogenetic analyses indicate the discovery of a novel vitivirus species provisionally named grapevine virus P.
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Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of "high-risk" HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.
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Anticuerpos Monoclonales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/química , Animales , Humanos , Células CHO , Proteínas , Cricetulus , Técnicas de Cultivo de Célula/métodosRESUMEN
In our preliminary experiment, peritoneal sclerosis likely induced by peritoneal dialysis was unexpectedly observed in the livers of rats given bleomycin and lansoprazole. We examined whether this peritoneal thickening around the liver was time-dependently induced by administration of both drugs. Male Wistar rats were injected with bleomycin and/or lansoprazole for 2 or 4 weeks. The 3YB-1 cell line derived from rat fibroblasts was treated by bleomycin and/or lansoprazole for 24 h. The administration of both drugs together, but not individually, thickened the peritoneal tissue around the liver. There was accumulation of collagen fibers, macrophages, and eosinophils under mesothelial cells. Expressions of Col1a1, Mcp1 and Mcp3 genes were increased in the peritoneal tissue around the liver and in 3YB-1 cells by the administration of both drugs together, and Opn genes had increased expressions in this tissue and 3YB-1 cells. Mesothelial cells indicated immunoreactivity against both cytokeratin, a mesothelial cell marker, and αSMA, a fibroblast marker, around the livers of rats given both drugs. Administration of both drugs induced the migration of macrophages and eosinophils and induced fibrosis associated with the possible activation of fibroblasts and the possible promotion of the mesothelial-mesenchymal transition. This might become a novel model of peritoneal sclerosis for peritoneal dialysis.
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Fibrosis Peritoneal , Ratas , Masculino , Animales , Fibrosis Peritoneal/inducido químicamente , Fibrosis Peritoneal/genética , Bleomicina/efectos adversos , Ratas Wistar , Lansoprazol/efectos adversos , Lansoprazol/metabolismo , Células Epiteliales/metabolismo , Peritoneo/patologíaRESUMEN
In the current transition to intensified upstream processing, the risks of adopting traditional single-use systems for high-titer, long-duration perfusion cultures, have thus far not been considered. This case study uses the Failure Modes and Effects Analysis (FMEA) method to evaluate the risks associated with implementing upstream single-use technology. The simulated model process was used to compare the risk level of single-use technology for a traditional fed-batch cell culture with that for perfusion culture, under the same annual protein production conditions. To provide a reasonable source of potential risk for FMEA, all single-use upstream operations for both fed-batch and perfusion processes were investigated using an analytical method developed to quantify the impact of process parameters and operating conditions on single-use system specifications and to ensure objectivity. Many of the risks and their levels, were similar in long-duration perfusion cultures and fed-batch cultures. However, differences were observed for high-risk components such as daily sampling and installation. The result of this analysis indicates that the reasons for risk are different for fed-batch cultures and perfusion cultures such as larger bioreactors in fed-batch and longer runs in perfusion, respectively. This risk assessment method could identify additional control measures and be part of a holistic contamination control strategy and help visualize their effectiveness.
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Productos Biológicos , Animales , Cricetinae , Reactores Biológicos , Técnicas de Cultivo Celular por Lotes/métodos , Anticuerpos Monoclonales , Perfusión , CricetulusRESUMEN
Introduction: Fatty acids are a major nutrient in dietary fat, some of which are ligands of long-chain fatty acid receptors, including G-protein-coupled receptor (GPR) 40 and GPR120. Pretreatment with GPR40 agonists enhanced the secretion of insulin in response to elevating blood glucose levels after glucose load in a diabetes model, but pretreatment with GPR120 agonist did not ameliorate postprandial hyperglycemia. This study examined whether oral administration of linoleic acid (LA), a GPR40 and GPR120 agonist, immediately before glucose load would affect the elevation of postprandial blood glucose levels in rats. Methods: Male rats and rats with type 1 diabetes administered streptozocin were orally administered LA, trilinolein, α-linolenic acid (α-LA), oleic acid, TAK-875, or TUG-891 immediately before glucose load. Blood glucose levels were measured before, then 15, 30, 60 and 120 min after glucose load. CACO-2 cells were used to measure the uptake of [14C] α-MDG for 30 min with or without LA. Gastric content from rats administered LA was collected 15 and 30 min after glucose load, and blood samples were collected for measurement of glucagon-like peptide 1 (GLP-1) and cholecystokinin concentrations. Results: The elevation of postprandial blood glucose levels was slowed by LA but not by trilinolein in rats without promotion of insulin secretion, and this effect was also observed in rats with type 1 diabetes. The uptake of α-MDG, an SGLT-specific substrate, was, however, not inhibited by LA. Gastric emptying was slowed by LA 15 min after glucose load, and GLP-1, but not cholecystokinin, level was elevated by LA 15 min after glucose load. TUG-891, a GPR120 agonist, ameliorated postprandial hyperglycemia but TAK-875, a GPR40 agonist, did not. Pretreatment with AH7614, a GPR120 antagonist, partially canceled the improvement of postprandial hyperglycemia induced by LA. α-LA, which has high affinity with GPR120 as well as LA, slowed the elevation of postprandial blood glucose levels, but oleic acid, which has lower affinity with GPR120 than LA, did not. Conclusion: Oral administration of LA immediately after glucose load ameliorated postprandial hyperglycemia due to slowing of gastric emptying via promotion of GLP-1 secretion. The mechanisms may be associated with GPR120 pathway.
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Lansoprazole, a proton pump inhibitor, can exert antioxidant effects through the induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, independently of the inhibition of acid secretion in the gastrointestinal tract. Lansoprazole has been reported to provide hepatoprotection in a drug-induced hepatitis animal model through the Nrf2/heme oxygenase-1 (HO1) pathway. We sought to investigate the molecular mechanism of cytoprotection by lansoprazole. An in vitro experimental model was conducted using cultured rat hepatic cells treated with lansoprazole to analyze the expression levels of Nrf2 and its downstream genes, the activity of Nrf2 using luciferase reporter assays, cisplatin-induced cytotoxicity, and signaling pathways involved in Nrf2 activation. Lansoprazole treatment of rat liver epithelial RL34 cells induced transactivation of Nrf2 and the expression of the Nrf2-dependent antioxidant genes encoding HO1, NAD(P)H quinone oxidoreductase-1, and glutathione S-transferase A2. Furthermore, cycloheximide chase experiments revealed that lansoprazole prolongs the half-life of the Nrf2 protein. Notably, cell viability was significantly increased by lansoprazole treatment in a cisplatin-induced cytotoxicity model. Moreover, the siRNA knockdown of Nrf2 fully abolished the cytoprotective effect of lansoprazole, whereas the inhibition of HO1 by tin-mesoporphyrin only partially abolished this. Finally, lansoprazole promoted the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not that of the extracellular signal-regulated kinase or the c-Jun N-terminal kinase. Using SB203580, a specific inhibitor for p38 MAPK, the lansoprazole-induced Nrf2/antioxidant response elements pathway activation and cytoprotective effects were shown to be exclusively p38 MAPK dependent. Lansoprazole was shown by these results to exert a cytoprotective effect on liver epithelial cells against the cisplatin-induced cytotoxicity through the p38 MAPK signaling pathway. This could have potential applications for the prevention and treatment of oxidative injury in the liver.
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Proteína Quinasa 14 Activada por Mitógenos , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Ratas , Cisplatino/efectos adversos , Factor 2 Relacionado con NF-E2/genética , Lansoprazol/farmacología , Estrés Oxidativo , Hepatocitos , Antioxidantes/farmacologíaRESUMEN
Mammals receive body energy information to maintain energy homeostasis. Ghrelin, insulin, leptin and vagal afferents transmit the status of fasting, blood glucose, body fat, and food intake, respectively. Estrogen also inhibits feeding behavior and lipogenesis, but increases body fat mass. However, how blood triglyceride levels are monitored and the physiological roles of estrogen from the perspective of lipid homeostasis remain unsettled. Here, we show that stomach secretes estrogen in response to the blood triglyceride levels. Estrogen-secreting gastric parietal cells predominantly use fatty acids as an energy source. Blood estrogen levels increase as blood triglyceride levels rise in a stomach-dependent manner. Estrogen levels in stomach tissues increase as blood triglyceride levels rise, and isolated gastric gland epithelium produces estrogen in a fatty acid-dependent manner. We therefore propose that stomach monitors and controls blood triglyceride levels using estrogen, which inhibits feeding behavior and lipogenesis, and promotes triglyceride uptake by adipocytes.
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Estrógenos/biosíntesis , Estómago/metabolismo , Triglicéridos/sangre , Animales , Femenino , Masculino , Ratas , Ratas WistarRESUMEN
Xylella fastidiosa causes many economically important plant diseases such as Pierce's disease of grapevine, citrus variegated chlorosis disease, and olive quick decline syndrome. Another species in the same genus, Xylella taiwanensis, causes pear leaf scorch. Here, to enable an initial screening of plants suspected of being infected with Xylella spp. by conventional polymerase chain reaction (cPCR), new primer pairs-X67S1/XL2r and XrDf1/XLr4-were designed to target the 16S ribosomal DNA (rDNA) of not only X. fastidosa but also X. taiwanensis. In cPCR to detect both species, X67S1/XL2r showed features superior to those of other primer pairs, such as fewer false negatives and false positives, whereas XrDf1/XLr4 seemed to be unsuitable because of abundant non-specific amplification. However, when XrDf1/XLr4 was combined with a probe in a TaqMan quantitative real-time PCR (qPCR), the assay detected no false positives and was more useful in the universal detection of Xylella spp. than TaqMan qPCR assays reported previously.
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Cartilla de ADN/genética , Sondas de ADN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Xylella/genética , Citrus/microbiología , Enfermedades de las Plantas/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/clasificaciónRESUMEN
The activation of Ras signaling is a major early event of oncogenesis in many contexts, yet paradoxically, Ras signaling induces cellular senescence, which prevents tumorigenesis. Thus, Ras-activated cells must overcome senescence to develop into cancer. Through a genetic screen in Drosophila melanogaster, we found that the ETS family transcriptional activator Pointed (Pnt) was necessary and sufficient to trigger cellular senescence upon Ras activation and blocked Ras-induced tumor growth in eye-antennal discs. Through analyses of mosaic discs using various genetic tools, we identified a mechanism of tumor progression in which loss of cell polarity, a common driver of epithelial oncogenesis, abrogated Ras-induced cellular senescence through microRNA-mediated inhibition of Pnt. Mechanistically, polarity defects in Ras-activated cells caused activation of the Hippo effector Yorkie (Yki), which induced the expression of the microRNA bantam bantam-mediated repression of the E3 ligase-associated protein Tribbles (Trbl) relieved Ras- and Akt-dependent inhibition of the transcription factor FoxO. The restoration of FoxO activity in Ras-activated cells induced the expression of the microRNAs miR-9c and miR-79, which led to reduced pnt expression, thereby abrogating cellular senescence and promoting tumor progression. Our findings provide a mechanistic explanation for how Ras-activated tumors progress toward malignancy by overcoming cellular senescence.
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Senescencia Celular , Proteínas de Drosophila/metabolismo , MicroARNs , Proteínas Señalizadoras YAP/metabolismo , Animales , Carcinogénesis , Proliferación Celular , Proteínas de Unión al ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , MicroARNs/genética , Proteínas del Tejido Nervioso , Proteínas Nucleares , Proteínas Proto-Oncogénicas , Transactivadores , Factores de TranscripciónRESUMEN
Cell culture media used in CHO-based biologic processes are typically sterile filtered to prevent microbial contamination prior to inoculation. In this study, the impact of common sterile filter throughput on a different, commercially available cell culture media was evaluated from the intermediate-adsorption fouling model of the filtration model. The key particle size range for optimum filter performance was discussed and identified by measuring the submicron order particle size distribution. It may be possible to predict the performance of filter capacity with size-exclusive separation by understanding the media particle counts and size distribution.
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Medios de Cultivo/química , Filtración/métodos , Tamaño de la Partícula , Adsorción , Animales , Células CHO , Técnicas de Cultivo de Célula , Cricetulus , Medios de Cultivo/normas , Contaminación de Medicamentos/prevención & controlRESUMEN
A study was conducted to evaluate the basic mechanical properties of a pure cellulose nanofiber (CNF) material in comparison with a commercial denture base material (polymethylmethacrylate [PMMA] acrylic resin). The working hypothesis was that CNFs have potential for use as denture base materials. Pure CNF specimens fabricated under various conditions were examined. The flexural strength (FS) and flexural modulus (FM) of the specimens were measured using the three-point bending test, and the morphologies of the fractured surfaces were examined using scanning electron microscopy. Addition of tricalcium phosphate to dehydrate the CNFs did not improve their FS or FM. Conversely, substitution with methanol effectively improved the dehydration process and significantly affected the mechanical properties of the CNFs. As the degree of CNF defibration increased, the physical properties of the specimens improved significantly. However, addition of CNFs to PMMA liquid to create CNF-reinforced PMMA did not improve the mechanical properties. Pure CNF specimens fabricated under particular conditions had higher FS and FM values than the control, suggesting that CNFs have potential as a "petroleum-free" alternative to acrylic resin denture base materials. Pure CNF would be potentially useful as a denture base material, and presumably applicable to computer-aided design/manufacture (CAD/CAM).
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Bases para Dentadura , Nanofibras , Celulosa , Ensayo de Materiales , Docilidad , Propiedades de SuperficieRESUMEN
Complete RNA1 and RNA2 sequences of two and nearly complete genome sequences of six new variants of grapevine fabavirus found in Japan were compared to those of previously reported variants. Negative selection pressure was suggested, and no recombination events were detected in either RNA1 or RNA2. The first 18 nucleotides in both RNAs were predicted to form a stem-loop structure. The variants could be genetically divided into four groups based on RNA1 and two based on RNA2. A broad-spectrum reverse transcription polymerase chain reaction assay using a primer pair designed based on an RNA2 consensus sequence was able to detect all of the known variants.
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Fabavirus/aislamiento & purificación , Variación Genética , Enfermedades de las Plantas/virología , Vitis/virología , Fabavirus/clasificación , Fabavirus/genética , Genoma Viral , Filogenia , ARN Viral/genéticaRESUMEN
Next-generation sequencing detected a novel virus from grapevine cultivar 'Kishmish Chjornyj' from Russia. Its complete genome sequence of 5625 nucleotides includes seven open reading frames encoding seven putative proteins similar to those of members of the genus Polerovirus in the family Luteoviridae. The novel virus showed graft-transmissibility and was tentatively named "grapevine polerovirus 1" (GPoV-1). Phylogenetic analysis using complete genome sequences of GPoV-1 and members of the family Luteoviridae indicated that although GPoV-1 is a member of the genus Polerovirus, it is unique within its clade. GPoV-1 is the first polerovirus detected in grapevine.
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Genoma Viral , Luteoviridae/aislamiento & purificación , Enfermedades de las Plantas/virología , Vitis/virología , Secuencia de Bases , Secuenciación de Nucleótidos de Alto Rendimiento , Luteoviridae/clasificación , Luteoviridae/genética , Sistemas de Lectura Abierta , Filogenia , Federación de Rusia , Secuenciación Completa del GenomaRESUMEN
We attempted to create a mathematical model for neuronal differentiation. The present study was performed within the framework of self-organization with constraints by looking for an optimized informational unit. We treated networks of individual dynamical system units with an external input, which was provided by coupled one-dimensional maps with possible forms of unidirectionally feed-forward network, random network, small-world network, and fully-connected network. We used a genetic algorithm to maximize the information transmission for each type of network. Optimized maps were obtained depending on the coupling strength and network structure. These maps can be classified into three types: passive, excitable, and oscillatory. In particular, the excitable and oscillatory types of dynamical systems possess characteristics that are quite similar to those of neurons, whereas the passive and oscillatory types of dynamical system may represent glial cells.
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Modelos Teóricos , Neuronas , Diferenciación Celular , Modelos NeurológicosRESUMEN
Using next-generation sequencing, we detected a novel variant of hop stunt viroid (HSVd) in grapevine 'Chenin blanc' (Vitis vinifera L.) and a new viroid species in 'Nachubearmarie' (Vitis labrusca L. × V. vinifera). The HSVd variant termed HSVd-CB has 296 nucleotides with up to 82% sequence identity with other HSVd variants such as HSVd-AP1 (Genbank accession EF523826). Many nucleotide changes, deletions, and insertions were sporadically found in HSVd-CB relative to HSVd-AP1 in the pathogenic and variable domains. Although several variations were also found in the central domain, few variations were found in the terminal left and right domains, including no variations in the terminal conserved hairpin. The new viroid, tentatively termed Japanese grapevine viroid (JGVd), has 367 nucleotides and has genetic features characteristic of the genus Apscaviroid. JGVd shared the highest nucleotide sequence identity (68%) with a persimmon latent viroid (PLVd) in its overall genome. However, the JGVd sequence shows chimerism with the partial genomes of other apscaviroids from apple, grapevine, and citrus. Phylogenetic analyses showed that only HSVd-CB formed a distinct branch from the cluster of the other HSVd variants and JGVd and PLVd formed a distinct branch from all other grapevine-infecting apscaviroids.
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Filogenia , Enfermedades de las Plantas/genética , Virus de Plantas/genética , Vitis/virología , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedades de las Plantas/virología , Virus de Plantas/patogenicidad , Vitis/genética , Secuenciación Completa del GenomaRESUMEN
The biopharmaceutical industry is evolving toward process intensification that can offer increased productivity and improved economics without sacrificing process robustness. A semi-continuous downstream process linking purification/polishing unit operations in series can reduce or eliminate intermediate holding tanks and reduce overall processing time. Accordingly, we have developed a therapeutic monoclonal antibody polishing template comprised of a connected flow-through polishing technologies that include activated carbon, cation exchange, and anion-exchange chromatography. In this report, we evaluated fully-connected pool-less polishing with three flow-through technologies, operating as a single skid to streamline and improve an mAb purification platform. Laboratory-scale pool-less processing was achieved without utilizing in-line pH adjustment and conductivity dilution based on the previously optimized single process parameter. Two connected flow-through configurations of polishing steps were evaluated: a two-step process using anion exchange and cation exchange and a three step process using activated carbon, anion exchange and cation exchange chromatography. Laboratory-scale proof of concept studies showed comparable performance between the batch purification process and the pool-less process configuration. Three step polishing highly intensified the processes and provided higher process loading and achieved bulk drug specification with higher impurity clearance (>95%) and high overall mAb yield (>95%).
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OBJECTIVES: We previously demonstrated that lansoprazole provided hepatoprotection in a drug-induced hepatitis animal model partially through the Nrf2/HO-1 pathway. Here, we examined whether lansoprazole could also provide hepatoprotection in a rat model of non-alcoholic steatohepatitis (NASH). METHODS: Six-week-old rats were fed a normal chow or a choline-deficient amino acid-defined (CDAA) diet to establish a rat model of NASH. The groups fed a CDAA diet for 5 weeks were subcutaneously administered either a vehicle or a lansoprazole suspension for 4 weeks beginning the second week of the experiment. KEY FINDINGS: Bridging fibrosis was observed in the livers of almost all the NASH model rats (six of seven), but it was not always observed in NASH model rats (one of seven) continuously administered lansoprazole. The serum aspartate aminotransferase level elevated by the CDAA diet was significantly decreased following lansoprazole administration. Lansoprazole also increased the expression of Nrf2, but not HO-1, in the liver of NASH model rats. Lansoprazole decreased the level of activated TGF-ß protein. Furthermore, interleukin-6 gene and protein expression were decreased. CONCLUSIONS: Lansoprazole inhibits hepatic fibrogenesis, at least during the early stages, in CDAA diet-induced NASH model rats. The mechanisms might be associated with cytokine suppression but not the inhibition of reactive oxygen species.
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Lansoprazol/farmacología , Cirrosis Hepática/prevención & control , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Animales , Aspartato Aminotransferasas/sangre , Dieta , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hemo Oxigenasa (Desciclizante)/biosíntesis , Interleucina-6/biosíntesis , Cirrosis Hepática/complicaciones , Masculino , Factor 2 Relacionado con NF-E2/biosíntesis , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Ratas , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
An integrated all flow-through technology platform for the purification of therapeutic monoclonal antibodies (mAb), consisting of activated carbon and flow-through cation and anion exchange chromatography steps, can replace a conventional chromatography platform. This new platform was observed to have excellent impurity clearance at high mAb loadings with overall mAb yield exceeding 80%. Robust removal of DNA and host cell protein was demonstrated by activated carbon and a new flow-through cation exchange resin exhibited excellent clearance of mAb aggregate with high monomer recoveries. A ten-fold improvement of mAb loading was achieved compared to a traditional cation exchange resin designed for bind and elute mode. High throughput 96-well plate screening was used for process optimization, focusing on mAb loading and solution conditions. Optimum operating windows for integrated flow-through purification are proposed based on performance characteristics. The combination of an all flow-through polishing process presents significant opportunities for improvements in facility utilization and process economics.
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Anticuerpos Monoclonales , Cromatografía por Intercambio Iónico/métodos , Resinas de Intercambio de Catión , HumanosRESUMEN
Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.
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Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Phytoplasma/genética , Phytoplasma/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Xylella/genética , Xylella/aislamiento & purificación , Mezclas Complejas , Sondas de Oligonucleótidos/genética , Vitis/microbiologíaRESUMEN
A novel putative ampelovirus was detected in grapevines that showed typical leafroll symptoms and was tentatively named grapevine leafroll-associated virus (GLRaV)-13 following the series of numbering of other GLRaVs. The complete genome of GLRaV-13 comprised 17,608 nt and contained eleven putative open reading frames, showing genetic features similar to those of viruses belonging to subgroup I of genus Ampelovirus. Phylogenetic trees based on the RNA-dependent RNA polymerase, heat shock protein 70 homolog, and coat protein showed that GLRaV-13 had the closest, but still distant, relationship to GLRaV-1 in the subgroup I cluster.