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Phosphate (Pi) is a pivotal nutrient that constraints plant development and productivity in natural ecosystems. Land colonization by plants, more than 470 million years ago, evolved adaptive mechanisms to conquer Pi-scarce environments. However, little is known about the molecular basis underlying such adaptations at early branches of plant phylogeny. To shed light on how early divergent plants respond to Pi limitation, we analyzed the morpho-physiological and transcriptional dynamics of Marchantia polymorpha upon Pi starvation. Our phylogenomic analysis highlights some gene networks present since the Chlorophytes and others established in the Streptophytes (e.g., PHR1-SPX1 and STOP1-ALMT1, respectively). At the morpho-physiological level, the response is characterized by the induction of phosphatase activity, media acidification, accumulation of auronidins, reduction of internal Pi concentration, and developmental modifications of rhizoids. The transcriptional response involves the induction of MpPHR1, Pi transporters, lipid turnover enzymes, and MpMYB14, which is an essential transcription factor for auronidins biosynthesis. MpSTOP2 up-regulation correlates with expression changes in genes related to organic acid biosynthesis and transport, suggesting a preference for citrate exudation. An analysis of MpPHR1 binding sequences (P1BS) shows an enrichment of this cis regulatory element in differentially expressed genes. Our study unravels the strategies, at diverse levels of organization, exerted by M. polymorpha to cope with low Pi availability.
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Marchantia/genética , Marchantia/metabolismo , Fosfatos/metabolismo , Ecosistema , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/genética , Hepatophyta/metabolismo , Filogenia , Factores de Transcripción/metabolismoRESUMEN
The auxin signaling molecule controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of the ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and DNA-binding ARF transcription factors. Although extensive qualitative understanding of the pathway and its interactions has been obtained, mostly by studying the flowering plant Arabidopsis thaliana, it remains unknown how these translate to quantitative system behavior in vivo, a problem that is confounded by the large NAP gene families in most species. Here, we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knockin fluorescent fusion proteins. Beyond revealing the dynamic native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs, MpARF1 and MpARF2, are proteasomally degraded. This auxin-independent degradation tunes ARF protein stoichiometry to favor gene activation, thereby reprogramming auxin response during the developmental progression. Thus, quantitative analysis of the entire NAP has enabled us to identify ARF degradation and the stoichiometries of activator and repressor ARFs as a potential mechanism for controlling gemma germination.
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When examining gastrointestinal tolerance to nondigestible carbohydrates, a weekly vs. daily symptoms questionnaire may lessen participant burden. This secondary analysis examined the reliability and validity of the Gastrointestinal Symptom Rating Scale (GSRS) in healthy adults. The internal consistency reliability of the GSRS syndromes and a daily questionnaire (DQ) comparator were determined. The GSRS syndromes prediction of slow transit stool form was assessed by ROC analysis. The DQ (α = 0.76) and GSRS syndromes of constipation (α = 0.73; ω = 0.74), and diarrhea (α = 0.76; ω = 0.77) exhibited acceptable reliability, as did the GSRS overall (α = 0.76; ω = 0.87) but not the syndromes of abdominal pain (α = 0.54; ω = 0.54), reflux (α = 0.69; ω = 0.67), or indigestion (α = 0.64; ω = 0.67). The GSRS syndromes predicted slow transit stools (AUC = 0.855), and the GSRS items of stomach pain, nausea, flatus, constipation, and diarrhea were moderately correlated (ρ = 0.55-0.64; P < 0.001) with the corresponding DQ items. The GSRS may be useful to assess gastrointestinal tolerance and efficacy of nondigestible carbohydrates given its performance at predicting slow transit stools, suggestive of constipation.
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Primary cardiac tumors are exceptionally rare and predominantly located in the left atrium with occasional involvement on the right side of the heart. We present the case of a 52-year-old man who presented with chest pain, leading to suspicion of acute coronary syndrome. However, further investigation revealed a right atrial tumor measuring 6.3 cm. After surgical removal, the pathology analysis of the mass confirmed the histology of myxoma. Differential diagnoses for atrial myxomas include thrombus and other tumors, such as rhabdomyomas. More than half of these tumors arise in the left atrium and may be complicated by neurologic symptoms secondary to embolization. Right atrial myxomas are rare and described in the literature with a myriad of symptoms (signs of right heart failure [i.e., fatigue, peripheral edema, hepatomegaly, ascites], a diastolic murmur, and symptoms of pulmonary emboli). In other cases, they may be asymptomatic. Due to the low incidence and variety in their clinical picture, careful documentation of these cases is suggested for early recognition and directed management.
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Cardiac amyloidosis (CA) is a rare form of infiltrative cardiomyopathy (IC) that frequently leads to heart failure (HF). Its symptoms can range from minimal to significant shortness of breath, palpitations, leg swelling, and chest discomfort. Early diagnosis and treatment are crucial in preventing the further progression of the disease and improving outcomes. This case report describes a 63-year-old male with no prior medical history who presented with severe dyspnea, palpitations, and chest heaviness. Initially diagnosed with atrial flutter, he was later confirmed to have cardiac amyloidosis through a thorough workup with multimodality imaging. The patient was started on guideline-directed medical therapy (GDMT) and discharged home with a follow-up from a heart failure specialist. An outpatient workup confirmed the diagnosis of amyloidosis with a positive pyrophosphate scan. At a seven-month follow-up, the workup for extra-cardiac involvement was negative, and the ejection fraction (EF) had improved. This case highlights the importance of a high index of suspicion and a thorough workup in cases of suspected cardiac amyloidosis to achieve early diagnosis and prevent disease progression.
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New peak detection (NPD), as part of the LC-MS-based multi-attribute method (MAM), allows for sensitive and unbiased detection of new or changing site-specific attributes between a sample and reference that is not possible with conventional UV or fluorescence detection-based methods. MAM with NPD can serve as a purity test that can establish whether a sample and the reference are similar. The broad implementation of NPD in the biopharmaceutical industry has been limited by the potential presence of false positives or artifacts, which increase the analysis time and can trigger unnecessary investigations of product quality. Our novel contributions to the success of NPD are the curation of false positives, use of the known peak list concept, pairwise analysis approach, and the development of a NPD system suitability control strategy. In this report, we also introduce a unique experimental design utilizing sequence variant co-mixes to measure NPD performance. We show that NPD has superior performance relative to conventional control system methods in the detection of an unexpected change as compared with the reference. NPD is a new frontier in purity testing that reduces subjectivity, need for analyst intervention, and potential for missing unexpected product quality changes.
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Productos Biológicos , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodosRESUMEN
Background and objective The use of cannabis through smoking and vaping has increased significantly over the past decade. However, the prevalence of pulmonary circulation disorder (PCD)-related hospitalizations among cannabis users and their outcomes remain poorly understood. In this study, we used a nationally representative sample to assess the prevalence and trends of hospitalization among cannabis users with PCD. Methods The National Inpatient Sample (NIS) datasets (2007-2014) were used to analyze hospitalizations of patients with cannabis user disorder with PCD (C-PCD arm) versus those without PCD (C-non-PCD arm) to ascertain demographics, comorbidities, and in-hospital outcomes including all-cause mortality and healthcare resource utilization. Results A total of 3,307,310 hospitalizations involving cannabis users were reported, of which 20,328 (0.61%) were related to PCD. We noted a 200% relative increase in hospitalizations in the C-PCD arm (linearly increasing from 0.3% to 0.9% from 2007 to 2014, ptrend<0.001). When compared to the C-non-PCD arm, patients in the C-PCD arm tended to be older (mean age: 47 vs. 34 years), predominantly males (65.6% vs. 62.9%), with significantly higher rates of congestive heart failure (CHF, 28.8%), hypertension (HTN, 22%), chronic obstructive pulmonary disease (COPD, 21.5%), deficiency anemia (19.4%), and valvular heart disease (17.7%). The C-PCD arm had a statistically higher proportion of tobacco and amphetamine abusers (p<0.01) while the C-non-PCD arm had more cocaine and alcohol abusers (p<0.01). Urban teaching hospital admissions were more commonly associated with the PCD arm than the non-PCD arm (65.4% vs. 56.9%). In terms of hospital resource utilization, patients in the C-PCD arm had higher median hospital stay (six vs. three days) and more frequent discharges to a skilled nursing facility or home healthcare than the C-non-PCD group. All-cause mortality during hospitalization was found to be much higher in the C-PCD arm than the C-non-PCD arm (4.1% vs. 0.5%, p<0.001). Multivariable analysis revealed a two-fold higher risk for all-cause mortality with an adjusted odds ratio (OR) of 2.17 (95% CI: 1.99-2.36, p<0.001) with PCD. Conclusion The findings of this nationwide study revealed significantly increased rates of hospitalizations among cannabis users with PCD with two times higher odds of all-cause in-hospital mortality. Further prospective studies are warranted in this subgroup of patients to confirm these findings and facilitate the management of these patients.
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The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.
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Benchmarking , Proteínas , Espectrometría de Masas/métodos , Mapeo Peptídico/métodos , Control de CalidadRESUMEN
Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography-mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.
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Anticuerpos Monoclonales/química , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Programas Informáticos , Ácidos/química , Animales , Fraccionamiento Químico , Glicosilación , Ensayos Analíticos de Alto Rendimiento , Mamíferos , Polisacáridos/química , Isoformas de Proteínas/química , Estabilidad Proteica , Proteínas Recombinantes/químicaRESUMEN
The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.
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Cleaning validation acceptance criteria in multiproduct facilities are established using maximum allowable carryover calculations. Carryover calculations incorporate the shared equipment surface area between two products to ensure that an acceptable limit for residue from the previously manufactured product to the subsequent product is determined. The shared surface area can be limited to areas where carryover presents the highest risk to product quality or patient safety. In these cases, specifically for biologic drug substance manufacturing, the shared surface area is limited to equipment after the purification process based on the assumption that the purification process would remove potential product fragment residues from the previous product. Until now, this assumption has been based on empirical knowledge without experimental data quantifying the clearance or removal of potential residues. We present a three-part study that determined the effects of cleaning conditions on selected monoclonal antibodies (mAbs) and the generation of degraded fragments and evaluated the clearance of both the degraded mAb1 in a laboratory setting and the degraded fragments in the presence of a subsequent product, assessing the risk of co-purification. Several analytical techniques were used, including gel electrophoresis, capillary zone electrophoresis/laser-induced florescence detection, and liquid chromatography-mass spectrometry. Protein fragment generation was demonstrated for five different mAbs from different immunoglobulin G subclasses. The clearance of the degraded fragments in the absence and presence of the subsequent product was demonstrated by calculating fold clearance and log reduction value (LRV) for each chromatography step. The data showed that the fragments generated during cleaning could be removed by the purification process. The fold clearances were determined to be values of 5400 (3.7 LRV) in the absence of subsequent product and 4428 (3.6 LRV) in the presence of subsequent product. The results supported the removal of product residues from shared surface areas by the purification process in multiproduct biologic drug substance manufacturing facilities.
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Anticuerpos Monoclonales/aislamiento & purificación , Contaminación de Medicamentos/prevención & control , Contaminación de Equipos/prevención & control , Tecnología Farmacéutica , Anticuerpos Monoclonales/efectos adversos , Seguridad del Paciente , Proteolisis , Control de Calidad , Medición de Riesgo , Tecnología Farmacéutica/instrumentación , Tecnología Farmacéutica/normasRESUMEN
Ambystoma mexicanum (axolotl) has been one of the major experimental models for the study of regeneration during the past 100 years. Axolotl limb regeneration takes place through a multi-stage and complex developmental process called epimorphosis that involves diverse events of cell reprogramming. Such events start with dedifferentiation of somatic cells and the proliferation of quiescent stem cells to generate a population of proliferative cells called blastema. Once the blastema reaches a mature stage, cells undergo progressive differentiation into the diverse cell lineages that will form the new limb. Such pivotal cell reprogramming phenomena depend on the fine-tuned regulation of the cell cycle in each regeneration stage, where cell populations display specific proliferative capacities and differentiation status. The axolotl genome has been fully sequenced and released recently, and diverse RNA-seq approaches have also been generated, enabling the identification and conservatory analysis of core cell cycle regulators in this species. We report here our results from such analyses and present the transcriptional behavior of key regulatory factors during axolotl limb regeneration. We also found conserved protein interactions between axolotl Cyclin Dependent Kinases 2, 4 and 6 and Cyclins type D and E. Canonical CYC-CDK interactions that play major roles in modulating cell cycle progression in eukaryotes.
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Ambystoma mexicanum/crecimiento & desarrollo , Ciclo Celular , Extremidades/crecimiento & desarrollo , Regeneración , Animales , Diferenciación Celular , Linaje de la Célula , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , RNA-SeqRESUMEN
Translation errors in protein biosynthesis may result in low level amino acid misincorporation and contribute to product heterogeneity of recombinant protein therapeutics. We report the use of peptide map analysis by reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry to detect and identify mistranslation events in recombinant monoclonal antibodies expressed in mammalian cell lines including Chinese hamster ovary (CHO) cells. Misincorporation of an asparagine residue at multiple serine positions was detected as earlier-eluting peptides with masses 27.01 Da higher than expected. The exact positions at which misincorporation occurred were identified by tandem mass spectrometry of the asparagine-containing variant peptides. The identified asparagine misincorporation sites correlated with the use of codon AGC but with none of the other five serine codons. The relative levels of misincorporation ranged from 0.01%-0.2% among multiple serine positions detected across three different antibodies by targeted analysis of expected and variant peptides. The low levels of misincorporation are consistent with published predictions for in vivo translation error rates. Our results demonstrate that state-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low level protein variants that arise from mistranslation events.
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Anticuerpos Monoclonales/biosíntesis , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Biosíntesis de Proteínas/genética , Proteínas Recombinantes/biosíntesis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Anticuerpos Monoclonales/genética , Células CHO , Codón/genética , Cricetinae , Cricetulus , Proteínas Recombinantes/genéticaRESUMEN
Comparative genomics has revealed that members of early divergent lineages of land plants share a set of highly conserved transcription factors (TFs) with flowering plants. While gene copy numbers have expanded through time, it has been predicted that diversification, co-option, and reassembly of gene regulatory networks implicated in development are directly related to morphological innovations that led to more complex land plant bodies. Examples of key networks have been deeply studied in Arabidopsis thaliana, such as those involving the AINTEGUMENTA (ANT) gene family that encodes AP2-type TFs. These TFs play significant roles in plant development such as the maintenance of stem cell niches, the correct development of the embryo and the formation of lateral organs, as well as fatty acid metabolism. Previously, it has been hypothesized that the common ancestor of mosses and vascular plants encoded two ANT genes that later diversified in seed plants. However, algae and bryophyte sequences have been underrepresented from such phylogenetic analyses. To understand the evolution of ANT in a complete manner, we performed phylogenetic analyses of ANT protein sequences of representative species from across the Streptophyta clade, including algae, liverworts, and hornworts, previously unrepresented. Moreover, protein domain architecture, selection analyses, and regulatory cis elements prediction, allowed us to propose a scenario of how the evolution of ANT genes occurred. In this study we show that a duplication of a preANT-like gene in the ancestor of embryophytes may have given rise to the land plant-exclusive basalANT and euANT lineages. We hypothesize that the absence of euANT-type and basalANT-type sequences in algae, and its presence in extant land plant species, suggests that the divergence of pre-ANT into basal and eu-ANT clades in embryophytes may have influenced the conquest of land by plants, as ANT TFs play important roles in tolerance to desiccation and the establishment, maintenance, and development of complex multicellular structures which either became more complex or appeared in land plants.
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INTRODUCCIÓN: Los trastornos tiroideos son causantes de morbilidad y discapacidad a nivel mundial, entre estos, el hipotiroidismo es uno de los más frecuentes. El 95% de los casos de hipotiroidismo son de tipo primario, que se caracteriza por presentar niveles disminuidos de hormonas tiroideas (T3 y T4) y niveles elevados de TSH. El objetivo del presente estudio fue determinar la prevalencia de hipotiroidismo primario en pacientes mujeres de 40 - 60 años de edad hospitalizadas en el Hospital José Carrasco Arteaga durante el año 2018. MATERIALES Y MÉTODOS: El presente es un estudio descriptivo transversal. El universo estuvo conformado por mujeres entre 40 a 60 años que estuvieron hospitalizadas en los diferentes departamentos del Hospital José Carrasco-Cuenca en el transcurso del año 2018; se incluyó a las pacientes a las cuales se les realizó perfil tiroideo durante su hospitalización. Se excluyó a las mujeres con datos incompletos en su historia clínica. No se realizó muestreo, se estudió a la totalidad de pacientes que cumplieron con el criterio de inclusión(n= 278). RESULTADOS: La prevalencia de hipotiroidismo primario en la población estudiada fue del 16.2 %; el hipotiroidismo fue ligeramente más frecuente en el grupo etario de 40-44 años con el 18.03%. Fue más frecuente en las mujeres residentes en el sector rural (18.18%), que en el urbano. El 53.34% de las mujeres identificadas con hipotiroidismo presentaron sobrepeso y el 22.22% presentaron obesidad. CONCLUSIÓN: La prevalencia encontrada de hipotiroidismo en mujeres de 40 a 60 años fue de 16.2%. La prevalencia fue ligeramente mayor en el grupo de edad de 40 a 44 años. La mayoría de las pacientes con hipotiroidismo tuvieron sobrepeso u obesidad.(au)
BACKGROUND: Thyroid disorders are a cause of morbidity and disability worldwide, among these, hypothyroidism is one of the most frequent. 95% of the cases of cases of hypothyroidism are primary, characterized by decreased levels of thyroid hormones (T3 and T4) and elevated TSH. The aim of this study was to determine the prevalence of primary hypothyroidism in female patients aged 40-60 years hospitalized at Hospital Jose Carrasco Arteaga during 2018. METHODS: This is a descriptive cross-sectional study. The universe were women between 40 and 60 years old who were hospitalized in the different services at Hospital Jose Carrasco Arteaga during 2018; patients who had a thyroid panel made during hospitalization were included. Women with incomplete medical history were excluded. We didn't do sampling; all patients who met the inclusion criteria (n=278) were studied. RESULTS: The prevalence of primary hypothyroidism in the studied population was 16.2%; hypothyroidism was slightly more frequent in the age group of 40-44 years with 18.03%. It was more frequent in women residing in the rural area (18.18), than in the urban area. 53.34% of the women identified with hypothyroidism were overweight and 22.22% were obese. CONCLUSION: The prevalence of hypothyroidism found in women aged 40 to 60 years old was 16.2%. The prevalence was slightly higher in the 40 to 44 age group. Most of the patients with hypothyroidism were overweight or obese.(au)
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Humanos , Femenino , Persona de Mediana Edad , Glándula Tiroides , Hormonas Tiroideas , Mujeres , Prevalencia , Sobrepeso , Hipotiroidismo , Registros Médicos , ObesidadRESUMEN
Resumen La neurolinfomatosis (NL) es una condición poco frecuente que se caracteriza por la invasión de células B en los nervios craneales y las raíces nerviosas periféricas y generalmente está vinculada con leucemia o linfoma no Hodking (LNH). En el presente reporte se destacará la importancia de la sospecha diagnóstica en este grupo de pacientes y la importancia de 18F-FDG PET/CT en el diagnóstico diferencial con otras entidades causantes de síntomas similares. Se presenta el caso de una mujer de 63 años con diagnóstico de linfoma difuso de células B grandes, quien, en el tercer ciclo de quimioterapia DA-EPOCH-R, refiere dolor de tipo neuropático en miembro superior derecho, progresivo en severidad y en extensión con compromiso de la extremidad contralateral, convulsiones y parálisis facial periférica.
Abstract Neurolinfomatosis (LN) is a strange condition, defined as Invasion of cranial nerves and peripheral nerve roots by leukemia or lymphoma. Most of the cases are caused by non-Hodgkin's lymphoma of B cells (BHL). The present paper aims to emphasize the importance of suspecting this entity in patients with NHL and neuropathic pain and the role of 18F-FDG PET-CT in the diagnosis. We present the case of a 63-year-old woman diagnosed with diffuse large B-cell lymphoma, who in her third chemotherapy session DA-EPOCH-R of neuropathic pain in the right upper limb, with a poor clinical outcome, due to worsening pain, contralateral limb involvement, seizures and peripheral facial paralysis.
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Humanos , Femenino , Persona de Mediana Edad , Linfoma no Hodgkin , Fluorodesoxiglucosa F18 , NeurolinfomatosisRESUMEN
Patients presenting late in the course of kidney disease who require urgent initiation of dialysis have traditionally received temporary vascular catheters followed by hemodialysis. Recent changes in Medicare payment policy for dialysis in the USA incentivized the use of peritoneal dialysis (PD). Consequently, the use of more expeditious PD for late-presenting patients (urgent-start PD) has received new attention. Urgent-start PD has been shown to be safe and effective, and offers a mechanism for increasing PD utilization. However, there has been no assessment of the dialysis-related costs over the first 90 days of care. The objective of this study was to characterize the costs associated with urgent-start PD, urgent-start hemodialysis (HD), or a dual approach (urgent-start HD followed by urgent-start PD) over the first 90 days of treatment from a provider perspective. A survey of practitioners from 5 clinics known to use urgent-start PD was conducted to provide inputs for a cost model representing typical patients. Model inputs were obtained from the survey, literature review, and available cost data. Sensitivity analyses were also conducted. The estimated per patient cost over the first 90 days for urgent-start PD was $16,398. Dialysis access represented 15% of total costs, dialysis services 48%, and initial hospitalization 37%. For urgent-start HD, total per patient costs were $19,352, and dialysis access accounted for 27%, dialysis services 42%, and initial hospitalization 31%. The estimated cost for dual patients was $19,400. Urgent-start PD may offer a cost saving approach for the initiation of dialysis in eligible patients requiring an urgent-start to dialysis.
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Costos de la Atención en Salud , Recursos en Salud/economía , Fallo Renal Crónico/terapia , Diálisis Peritoneal/economía , Diálisis Renal/economía , Costos y Análisis de Costo , Femenino , Recursos en Salud/estadística & datos numéricos , Humanos , Fallo Renal Crónico/economía , Masculino , Persona de Mediana Edad , Diálisis Peritoneal/estadística & datos numéricos , Diálisis Renal/estadística & datos numéricos , Factores de Tiempo , Estados UnidosRESUMEN
Hydroxyl radical footprinting is a covalent labeling strategy used to probe the conformational properties of proteins in solution. We describe the first application of this high resolution technique for characterizing the structure of a therapeutic monoclonal antibody (mAb) dimer. As monitored by size-exclusion chromatography (SEC), therapeutic mAbs typically contain small amounts of a dimer species relative to the primary monomeric form in its drug substance or drug product. To determine its structural orientation, a sample enriched in an IgG1 mAb dimer was oxidized by hydroxyl radicals generated by exposure of the aqueous solution to synchrotron X-rays in millisecond timescales. The antibody monomer that served as a control was oxidized in a similar fashion. The oxidized samples were digested with trypsin and analyzed by RP-UHPLC-MS. The footprinting data show that peptides displaying decreased rates of oxidation (i.e., regions of increased protection) in the dimer are localized in the light and heavy chains of the Fab domain. The interface region for the monomers comprising the dimer was thus inferred to be between their Fab arms, allowing us to model two possible theoretical dimer orientations: a head-to-head, single arm-bound Fab-to-Fab dimer, and a head-to-head, double arm-bound Fab (') 2-to-Fab (') 2 dimer. Lower resolution fragment-SEC analysis of the dimer and monomer samples treated with papain or FabRICATOR enzyme provided complimentary evidence to support the Fab/Fab orientation of the IgG1 dimer.
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Anticuerpos Monoclonales/química , Radical Hidroxilo/química , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Huella de Proteína/métodos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Cromatografía en Gel , Dimerización , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Espectrometría de Masas , Modelos Moleculares , Péptidos/química , Conformación Proteica , SincrotronesRESUMEN
Polysorbate 20 (polyoxyethylenesorbitan monolaurate) and polysorbate 80 (polyoxyethylenesorbitan monooleate) used in protein drug formulations are complex mixtures that have been difficult to characterize. Here, two HPLC methods are used with evaporative light scattering detection (ELSD) and mass spectrometry (MS) to characterize polysorbate from commercial vendors. The first HPLC method used a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion exchange and reversed-phase sorbent) with a step gradient to quantify both the total polyoxyethylene sorbitan ester and polyoxyethylene sorbitan (POE sorbitan, a non-surfactant) in polysorbate. The results indicated POE sorbitan was present from 16.0 to 27.6 and 11.1 to 14.5% (w/w) in polysorbate 20 and 80, respectively. The second HPLC method used a reversed-phase stationary phase (Zorbax SB-300 C(8)) with a shallow gradient to separate, identify, and quantify the multiple ester species present in polysorbate. For all lots of polysorbate 20 analyzed, only 18-23% of the material was the expected structure, polyoxyethylenesorbitan monolaurate. Up to 40% and 70% (w/w) di- and triesters were found in polysorbate 20 and polysorbate 80 respectively. Likewise, polyoxyethylenesorbitan monooleate accounted for only 20% of polysorbate 80. A variability of 3-5% was observed for each ester species between multiple lots of polysorbate 20. The reversed-phase method was then used to determine the rate of hydrolysis for each polyoxyethylene sorbitan ester of polysorbate 20 in basic solution at room temperature. Increasing rates of hydrolysis were observed with decreasing aliphatic chain lengths in polysorbate 20.
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Cromatografía de Fase Inversa/métodos , Polisorbatos/química , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión , Hidrólisis , Luz , Dispersión de RadiaciónRESUMEN
Although medical treatment of COPD has advanced, nonadherence to medication regimens poses a significant barrier to optimal management. Underuse, overuse, and improper use continue to be the most common causes of poor adherence to therapy. An average of 40%-60% of patients with COPD adheres to the prescribed regimen and only 1 out of 10 patients with a metered dose inhaler performs all essential steps correctly. Adherence to therapy is multifactorial and involves both the patient and the primary care provider. The effect of patient instruction on inhaler adherence and rescue medication utilization in patients with COPD does not seem to parallel the good results reported in patients with asthma. While use of a combined inhaler may facilitate adherence to medications and improve efficacy, pharmacoeconomic factors may influence patient's selection of both the device and the regimen. Patient's health beliefs, experiences, and behaviors play a significant role in adherence to pharmacological therapy. This manuscript reviews important aspects associated with medication adherence in patients with COPD and identifies some predictors of poor adherence.