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Allergic rhinitis affects up to 78% of people with asthma, and asthma occurs in 38% of people with allergic rhinitis. Asthma has a prevalence of 8.7% among adults and 6.2% among children and accounts for $50 billion in medical costs and $32 billion in indirect and mortality costs in the United States, respectively. Allergic rhinitis occurs in 5% to 15% of people in the United States. Allergic rhinitis also accounts for a significant health care cost burden, predominantly in terms of indirect costs related to reduced quality of life and presenteeism.
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Asma , Rinitis Alérgica , Niño , Adulto , Humanos , Estados Unidos/epidemiología , Calidad de Vida , Costo de Enfermedad , Rinitis Alérgica/epidemiología , Costos de la Atención en Salud , Asma/epidemiologíaRESUMEN
Introduction: Owing to its capacity to perform remote assessments, telemedicine is rising as a new force in chronic obstructive pulmonary disease (COPD) management. We conducted an eight month randomised-controlled-trial to study the effect of an automated telemedicine intervention on patients' time-to-hospitalisation. Methods: A total of 168 patients with a diagnosis of COPD in the past 24 months were enrolled to receive the intervention at a primary care clinic. The treatment group received daily phone messages from an automated system asking them to report if they were breathing better than, worse than, or the same as the day prior. Patients reported their breathing status by responding to the text message or call. If a patient reported breathing worse, an alert was sent directly to that patient's provider within the clinic. The control group received the same daily phone messages as the treatment group. However, no proactive breathing alerts were ever generated to the provider for these subjects. The primary outcome was the subjects' time-to-first-COPD-related hospitalisation following the start of messages. Results: The treatment group's time-to-hospitalisation was significantly different than the control group's with a hazard ratio of 2.36 (95% confidence interval 1.025.45, p = 0.0443). The number needed-to-treat ratio was 8.62. Subject engagement consistently ranged between 60% and 75%. The treatment group received both proactive monitoring and follow-up care from the providers. Discussion: Active monitoring with provider feedback enables the detection of exacerbation events early enough for subjects to avoid admissions. The use of non-smartphone interventions reduces barriers to care presented by more complicated and expensive technologies. This intervention represents a simple, innovative, and inexpensive tool for improved COPD management.
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Enfermedad Pulmonar Obstructiva Crónica/terapia , Autocuidado/métodos , Telemedicina , Teléfono , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/psicología , Calidad de Vida , Autocuidado/psicologíaRESUMEN
OBJECTIVE: To test a computer-led follow-up service for prostate cancer in two UK hospitals; the testing aimed to validate the computer expert system in making clinical decisions according to the individual patient's clinical need with a valid model accurately identify patients with disease recurrence or treatment failure based on their blood test and clinical picture. PATIENTS AND METHODS: A clinical-decision support system (CDSS) was developed from European (European Association of Urology) and national (National Institute for Health and Care Excellence) guidelines along with knowledge acquired from Urologists. This model was then applied in two UK hospitals to review patients after prostate cancer treatment. These patients' data (n = 200) were then reviewed by two independent urology consultants (blinded from the CDSS and the other consultant's rating) and the agreement was calculated by kappa statistics for validation. The second endpoint was to verify the system by estimating the system reliability. RESULTS: The two individual urology consultants identified 12% and 15% of the patients to have potential disease progression and recommended their referral to urology care. The kappa coefficient for the agreement between the CDSS and the two consultants was 0.81 (P < 0.001) and 0.84 (P < 0.001). The agreement amongst both specialist was also high with k = 0.83 (P < 0.001). The system reliability was estimated on all cases and this demonstrated 100% repeatability of the decisions. CONCLUSION: A CDSS follow-up is a valid model for providing safe follow-up for prostate cancer.
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Cuidados Posteriores/métodos , Sistemas de Apoyo a Decisiones Clínicas , Neoplasias de la Próstata/terapia , Anciano , Anciano de 80 o más Años , Consultores , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico , Próstata/patología , Neoplasias de la Próstata/patología , Reproducibilidad de los Resultados , Reino UnidoRESUMEN
Next generation sequencing (NGS) technologies have revolutionized our approach to genomic research. The use of whole genome sequencing (WGS), whole exome sequencing (WES), transcriptome profiling, and targeted DNA sequencing has exponentially improved our understanding of the human genome and the genetic complexities underlying malignancy. Yet, WGS and WES clinical applications remain limited due to high costs and the large volume of data generated. When utilized to address biological questions in basic science studies, targeted sequencing panels have proven extremely valuable due to reduced costs and higher sequencing depth. However, the routine application of targeted sequencing to the clinical setting is limited to a few cancer subtypes. Some highly aggressive tumor types, like type 2 endometrial cancer (EC), could greatly benefit from routine genomic analysis using targeted sequencing. To explore the potential utility of a mid size panel (~150 genes) in the clinical setting, we developed and validated a custom panel against WGS, WES, and another commercially available targeted panel. Our results indicate that a mid size custom designed panel is as efficient as WGS and WES in mapping variants of biological and clinical relevance, rendering higher coverage, at a lower cost, with fewer variants of uncertain significance. Because of the much higher sequencing depth that could be achieved, our results demonstrate that targeted sequencing outperformed WGS and WES in the mapping of pathogenic variants in a breast cancer case, as well as a case of mixed serous and high-grade endometrioid EC, the most aggressive EC subtype.
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BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited cardiac disorder characterized by episodic ventricular tachycardia during adrenergic stimulation. It is associated with significant morbidity and mortality. Knowledge of the underlying genetic cause, pathogenesis, and the natural history of the disease remains incomplete. Approximately 50% of CPVT cases are caused by dominant mutations in the cardiac ryanodine receptor (RYR2) gene, <5% of cases are accounted for by recessive mutations in cardiac calsequestrin (CASQ2) or Triadin (TRDN). METHODS: We report a family with two CASQ2 gene mutations. A research-based next-generation sequencing (NGS) initiative was used in a patient with a severe CPVT phenotype and her clinically unaffected son. Reverse transcription polymerase chain reaction (RT-PCR) from platelet RNA was used to assess the consequences of predicted splice variants. RESULTS: NGS revealed that the proband carried a novel c.199C>T (p.Gln67*) mutation and a previously reported splice site mutation c.532+1G>A in CASQ2. Her son is a heterozygous carrier of the c.199C>T (p.Gln67*) mutation alone and the proband was compound heterozygous at CASQ2. RNA analysis demonstrated that the splice site mutation results in the retention of intron 3 with no full-length CASQ2 mRNA. CONCLUSION: This study describes a novel CPVT genotype and further characterizes the effect of a previously reported CASQ2 splice site mutation. The long-term follow-up of 23 years since first symptom provides additional insight into the natural history of CASQ2-associated CPVT.
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Calsecuestrina/genética , Taquicardia Ventricular/genética , Secuencia de Bases , Plaquetas/metabolismo , Niño , Electrocardiografía , Estudios de Seguimiento , Genotipo , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , ARN/química , ARN/aislamiento & purificación , ARN/metabolismo , Sitios de Empalme de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Taquicardia Ventricular/diagnósticoRESUMEN
BACKGROUND: Inflammatory injury of nasal respiratory mucosa is a common feature of multisystem autoimmune disease. Certain autoimmune disorders are associated with nasal septum perforation (NSP). We performed a systematic review of the literature to better understand the association of NSP with specific autoimmune disorders. This is a case report of a 29-year-old woman with a history of arthralgia, autoreactive antibody titers, platelet dysfunction, and NSP. The constellation of symptoms and potential familial involvement indicated that the NSP in this patient was an early sign of an autoimmune disorder, an unknown autoimmune disorder, or a known disease with incomplete penetrance. METHODS: A systematic review of the literature was performed by two independent reviewers. Relevant articles were reviewed, and data that pertained to autoimmune-related NSP were extracted and analyzed. RESULTS: Overall, 140 cases of autoimmune-associated NSPs were reported. Granulomatosis with polyangiitis (48%), relapsing polychondritis (26%), and cocaine-induced midline lesions (15%) constituted 89.3% of the reported cases. CONCLUSION: NSP is a potential sign of systemic disease. The identification of an NSP, especially in the context of other unexplained symptoms or workup suggestive of an autoimmune disorder, should prompt clinical evaluation for multisystem autoimmune disease with consideration of granulomatosis with polyangiitis, relapsing polychondritis, or cocaine-induced midline lesions.
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BACKGROUND: Type II diabetes mellitus (T2DM) presents a major disease burden in the United States. Outpatient glycemic control among patients with T2DM remains difficult. Telemedicine shows great potential as an adjunct therapy to aid in glycemic control in real-world settings. OBJECTIVE: We aimed to explore the effectiveness of EpxDiabetes, a novel digital health intervention, in improving hemoglobin A1c (HbA1c) and fasting blood glucose (FBG) among patients with uncontrolled diabetes. METHODS: We recruited 396 patients from a community clinic in St. Louis, Missouri, from a database of patients diagnosed with T2DM and with a most recent HbA1c >7% as part of a quality improvement project. An automated call or text-messaging system was used to monitor patient-reported FBG. If determined to be elevated, care managers were notified by email, text, or electronic medical record alert. Participants self-reported their FBG data by replying to EpxDiabetes automated phone calls or text messages. Data were subsequently analyzed, triaged, and shared with providers to enable appropriate follow-up and care plan adjustments. Absolute HbA1c reduction, patient engagement, and absolute patient-reported FBG reduction were examined at approximately 6 months post implementation. RESULTS: EpxDiabetes had an average 95.6% patient response rate to messages at least once per month and an average 71.1% response rate to messages at least once per week. Subsequent HbA1c drop with EpxDiabetes use over 4 months was -1.15% (95% CI -1.58 to -0.71) for patients with HbA1c >8% at baseline compared to the change in HbA1c over 4 months prior to the implementation of EpxDiabetes of only -0.005 points (95% CI -0.28 to 0.27), P=.0018. CONCLUSIONS: EpxDiabetes may help reduce HbA1c in patients with high HbA1c baselines (>8%). The intervention demonstrates high patient engagement sustainable for at least 6 months.
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BACKGROUND: Although hypertension (HTN) is a major modifiable risk factor for arterial damage, blood pressure (BP) remains poorly controlled in the hypertensive population. Telemedicine is a promising adjunct intervention that may complement traditional therapies and improve adherence rates; however, current approaches have multiple barriers to entry, including the use of relatively expensive Bluetooth devices or the dependence on smart phone utilization, which tend to exclude low-income and more elderly populations. OBJECTIVE: The aim of this study was to design and implement a new phone call- and short message service text messaging-based intervention, Epharmix's EpxHypertension, in a quality improvement project that demonstrates the feasibility of this system for BP control in a family medicine setting. METHODS: We recruited 174 patients from a community clinic in St Louis from a database of patients diagnosed with HTN. An automated call or text messaging system was used to monitor patient-reported BPs. If determined to be elevated, physicians were notified by an email, text, or electronic medical record alert. Mean systolic BPs (SBPs) and diastolic BPs (DBPs) were compared at the beginning and end of 12 weeks. RESULTS: After 12 weeks on the system, patients with a baseline SBP of 140 mm Hg or higher reduced SBP by 10.8 mm Hg (95% CI -14.5 to -7.2, P<.001) and DBP by 6.6 mm Hg (95% CI -9.9 to -3.4, P=.002), but no significant changes were observed in overall BPs and BPs in the group with baseline SBP less than 140 mm Hg. CONCLUSIONS: EpxHypertension provides a viable means to control HTN in patients with high baseline BPs despite previous therapy. This community implementation study demonstrates the feasibility of implementing EpxHypertension across a primary care setting without the need for smartphones or Bluetooth-linked BP cuffs. Future studies should evaluate its effectiveness in a randomized control trial compared with standard of care.
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BACKGROUND: Patient noncompliance with therapy, treatments, and appointments represents a significant barrier to improving health care delivery and reducing the cost of care. One method to improve therapeutic adherence is to improve feedback loops in getting clinically acute events and issues to the relevant clinical providers as necessary (ranging from detecting hypoglycemic events for patients with diabetes to notifying the provider when patients are out of medications). Patients often don't know which information should prompt a call to their physician and proactive checks by the clinics themselves can be very resource intensive. We hypothesized that a two-way SMS system combined with a platform web service for providers would enable both high patient engagement but also the ability to detect relevant clinical alerts. OBJECTIVE: The objectives of this study are to develop a feasible two-way automated SMS/phone call + web service platform for patient-provider communication, and then study the feasibility and acceptability of the Epharmix platform. First, we report utilization rates over the course of the first 18 months of operation including total identified clinically significant events, and second, review results of patient user-satisfaction surveys for interventions for patients with diabetes, COPD, congestive heart failure, hypertension, surgical site infections, and breastfeeding difficulties. METHODS: To test this question, we developed a web service + SMS/phone infrastructure ("Epharmix"). Utilization results were measured based on the total number of text messages or calls sent and received, with percentage engagement defined as a patient responding to a text message at least once in a given week, including the number of clinically significant alerts generated. User satisfaction surveys were sent once per month over the 18 months to measure satisfaction with the system, frequency and degree of communication. Descriptive statistics were used to describe the above information. RESULTS: In total, 28,386 text messages and 24,017 calls were sent to 929 patients over 9 months. Patients responded to 80% to 90% of messages allowing the system to detect 1164 clinically significant events. Patients reported increased satisfaction and communication with their provider. Epharmix increased the number of patient-provider interactions to over 10 on average in any given month for patients with diabetes, COPD, congestive heart failure, hypertension, surgical site infections, and breastfeeding difficulties. CONCLUSIONS: Engaging high-risk patients remains a difficult process that may be improved through novel, digital health interventions. The Epharmix platform enables increased patient engagement with very low risk to improve clinical outcomes. We demonstrated that engagement among high-risk populations is possible when health care comes conveniently to where they are.
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Hepatitis B virus (HBV) reverse transcription requires coordinated function of the reverse transcriptase and ribonuclease H (RNaseH) activities of the viral polymerase protein. The reverse transcriptase has been biochemically characterized, but technical difficulties have prevented both assessment of the RNaseH and development of high throughput inhibitor screens against the RNaseH. Expressing the HBV RNaseH domain with both maltose binding protein and hexahistidine tags led to stable, high-level accumulation of the RNaseH in bacteria. Nickel-affinity purification in the presence of Mg(2+) and ATP removed co-purifying bacterial chaperones and yielded nearly pure monomeric recombinant enzyme. The endonucleolytic RNaseH activity required an DNA:RNA duplex ≥14 nt, could not tolerate a stem-loop in either the RNA or DNA strands, and could tolerate a nick in the DNA strand but not a gap. The RNaseH had no obvious sequence specificity or positional dependence within the RNA, and it cut the RNA at multiple positions even within the minimal 14 nt duplex. The RNaseH also possesses a processive 3'-5' exoribonuclease activity that is slower than the endonucleolytic reaction. These results are consistent with the HBV reverse transcription mechanism that features an initial endoribonucleolytic cut, 3'-5' degradation of RNA, and a sequence-independent terminal RNA cleavage. These data provide support for ongoing anti-RNaseH drug discovery efforts.
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Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/enzimología , Ribonucleasa H/aislamiento & purificación , Ribonucleasa H/metabolismo , Descubrimiento de Drogas , Expresión Génica , Virus de la Hepatitis B/genética , Humanos , Multimerización de Proteína , División del ARN , ARN Viral , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleasa H/antagonistas & inhibidores , Ribonucleasa H/genética , Especificidad por SustratoRESUMEN
CONTEXT: Precise intraocular pressure (IOP) measurement is important in glaucoma practise. Various instruments are available today to accurately measure IOP. Thus, the question arises about which instrument to use and whether all of them can be used interchangeably. AIMS: To assess the agreement between noncontact tonometer (NCT), rebound tonometer (RBT), Goldmann applanation tonometer (GAT), and dynamic contour tonometer (DCT) in measuring IOP. SUBJECTS AND METHODS: 499 eyes of 250 patients were evaluated during a period of 24 months from September 2010 to August 2012 and measurement of IOP by NCT, RBT, GAT, and DCT was done in the given sequence. The agreement was assessed by use of the Bland-Altman plot keeping GAT as a gold standard technique. RESULTS: The mean IOP value of NCT, RBT, GAT, and DCT was 15.9 ± 5.5, 15.9 ± 5.8, 15.9 ± 4.9, and 16.0 ± 4.7 mm of Hg, respectively. The limits of agreement of GAT with DCT, NCT, and RBT were found to be +5.4 to -5.2, -4.7 to +4.6, and -5.2 to +5.1 mm of Hg, respectively. CONCLUSIONS: A positive and strong correlation was found between newer tonometers and GAT, but the limit of agreement was clinically unacceptable. The use of a single tonometer should be practised at a glaucoma clinic for a patient at each follow-up.
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Glaucoma/diagnóstico , Presión Intraocular/fisiología , Tonometría Ocular/instrumentación , Adulto , Femenino , Glaucoma/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Hipertensión Ocular/diagnóstico , Hipertensión Ocular/fisiopatología , Reproducibilidad de los Resultados , Agudeza Visual/fisiologíaRESUMEN
Primary hepatic small cell carcinoma (HSCC) is a rare malignancy that has previously been described in only few case reports. The clinicopathological course, natural history, molecular markers and ideal treatment strategy for this tumor have not been fully elucidated. Herein, we report on two cases of spontaneously arising, metastatic primary HSCC that were treated at our Institution. Both patients succumbed to their disease within two months of initial presentation. Both cases underwent postmortem examination and no evidence of a pulmonary or other non-hepatic small cell primary was found. Unlike pulmonary small cell tumors, these two hepatic primaries showed only locoregional spread and very few distant metastases. Formalin-fixed samples were obtained at autopsy and sequenced using single-nucleotide polymorphism arrays and whole-genome sequencing. Four mutations in the epidermal growth factor receptor (EGFR) gene known to be associated with response to tyrosine kinase inhibitors (TKIs) were detected in one of the two HSCC samples. A systematic review and pooled analysis of all previously reported cases of primary HSCCs was conducted. The median overall survival was estimated at 4 months. Surgical resection was significantly associated with longer overall survival (hazard ratio =0.13, 95% confidence interval=0.03-0.69). Although several case reports of primary HSCC have been reported prior to this publication, to our knowledge this is the first time that molecular and systematic analysis has been conducted in order to more fully characterize this rare disease. Our results indicate that surgical resection, when feasible, may be a valid option in primary HSCC, and that some tumors may respond to TKIs against EGFR.
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Carcinoma de Células Pequeñas/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Pequeñas/mortalidad , Carcinoma de Células Pequeñas/patología , Femenino , Humanos , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Análisis de SupervivenciaRESUMEN
Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.
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Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Pérdida Auditiva/genética , Hispánicos o Latinos/genética , Mutación Missense , Linaje , Adulto , Secuencia de Aminoácidos , Animales , Células COS , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Niño , Chlorocebus aethiops , Exoma/genética , Femenino , Células HEK293 , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Humanos , Lactante , Masculino , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Miosinas/metabolismo , Estructura Secundaria de Proteína , Estereocilios/metabolismoRESUMEN
BACKGROUND: While next generation sequencing (NGS) is a useful tool for the identification of genetic variants to aid diagnosis and support therapy decision, high sequencing costs have limited its application within routine clinical care, especially in economically depressed areas. To investigate the utility of a multi-disease NGS based genetic test, we designed a custom sequencing assay targeting over thirty disease-associated areas including cardiac disorders, intellectual disabilities, hearing loss, collagenopathies, muscular dystrophy, Ashkenazi Jewish genetic disorders, and complex Mendelian disorders. We focused on these specific areas based on the interest of our collaborative clinical team, suggesting these diseases being the ones in need for the development of a sequencing-screening assay. RESULTS: We targeted all coding, untranslated regions (UTR) and flanking intronic regions of 650 known disease-associated genes using the Roche-NimbleGen EZ SeqCapV3 capture system and sequenced on the Illumina HiSeq 2500 Rapid Run platform. Eight controls with known variants and one HapMap sample were first sequenced to assess the performance of the panel. Subsequently, as a proof of principle and to explore the possible utility of our test, we analyzed test disease subjects (n = 16). Eight had known Mendelian disorders and eight had complex pediatric diseases. In addition to assess whether copy number variation may be of utility as a companion assay relative to these specific disease areas, we used the Affymetrix Genome-Wide SNP Array 6.0 to analyze the same samples. CONCLUSION: We identified potentially disease-associated variants: 22 missense, 4 nonsense, 1 frameshift, and 1 splice variants (16 previously identified, 12 novel among dbSNP and 15 novel among NHLBI Exome Variant Server). We found multi-disease targeted high-throughput sequencing to be a cost efficient approach in detecting disease-associated variants to aid diagnosis.
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Enfermedades Genéticas Congénitas/genética , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Polimorfismo de Nucleótido Simple , Adulto , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND: Cytomegalovirus (CMV) retinitis remains the most common ocular opportunistic infection in patients with acquired immunodeficiency syndrome even in the era of highly active antiretroviral therapy (HAART). Increased survival of patients on HAART has increased incidence of blindness, which will further increase in the future. The objective of this study was to determine the incidence of CMV retinitis and the effect of HAART on the natural history of CMV retinitis in patients referred from ART center. MATERIALS AND METHODS: Patients with baseline/current CD4 counts <150 cells/µl were evaluated for CMV retinitis. Complete ophthalmological evaluation was carried out and records of CD4 counts, HAART regime, presence of any form of CMV retinitis and response to HAART were noted. RESULTS: Out of 800 patients registered with CD4 <150 cells/µl in ART center, 100 patients reached us. Among these, CMV retinitis was observed in 15% patients, with median CD4 count at the time of examination being 56 cells/µl (range: 24-306 cells/µl). 66.67% patients were HAART non-responders and 63.6% eyes were economically blind. CONCLUSION: CMV retinitis occurs even in patients with higher CD4 counts. Timely diagnosis and intervention of this treatable condition can reduce the number of blinding years in these young patients who otherwise live longer as a result of HAART.
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Nucleos(t)ide analog drugs profoundly suppress Hepatitis B virus (HBV) replication but rarely cure the infection, so therapy is usually life-long. The nucleos(t)ide analogs inhibit the viral DNA polymerase and often push HBV to the brink of extinction, so it may be possible to eradicate HBV by suppressing HBV replication further. The HBV ribonuclease H (RNaseH) is a logical new drug target because it is the second of only two viral enzymes essential for viral replication. We recently developed a low throughput screening pipeline for inhibitors of the HBV RNaseH and viral replication. Here, we screened a series of twenty-three nitrogen-based polyoxygenated heterocycles including sixteen 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives for anti-HBV RNaseH activity. Nine compounds inhibited the HBV RNaseH, but activity was marginal for eight of them. Compound #1 [2-hydroxyisoquinoline-1,3(2H,4H)-dione, HID] was the best hit with an IC50 of 28.1µM and an EC50 of 4.2µM. It preferentially suppressed accumulation of the viral plus-polarity DNA strand in replication inhibition assays, indicating that replication was blocked due to suppression of HBV RNaseH activity. It had a CC50 of 75µM, yielding a therapeutic index of â¼18. The EC50 value was 7-fold lower than the IC50, possibly due to cellular retention or metabolism of the compound, or higher affinity for the full-length enzyme than the recombinant form used for screening. These data indicate that the 2-hydroxyisoquinoline-1,3(2H,4H)-diones will have different structure-activity relationships for the HBV and HIV RNaseHs. Therefore, HID compounds may provide a foundation for development of more effective RNaseH inhibitors of HBV replication.
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Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/enzimología , Isoquinolinas/farmacología , Ribonucleasa H/antagonistas & inhibidores , Línea Celular , Virus de la Hepatitis B/fisiología , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
The neurotoxin, 6-hydroxy dopamine (6-OHDA)-induced oxidative stress causes alterations in intracellular signalling events and activates cellular and molecular mechanisms leading to the degeneration of the dopamine-containing neurons (DCNs). The cyclic-AMP response element-binding protein (CREB) modulates the transcription of mitochondrial and nuclear genes upon phosphorylation. However, oxidative stress disrupts CREB functions and inhibits CREB signalling pathways. We have measured the activities and levels of both total CREB and its phosphorylated form (phospho-CREB) in cytosolic, mitochondrial and nuclear compartments in control (untreated) and stressed (6-OHDA-treated) DCN, differentiated from the ReNVM cell line (dDCN) at 0, 24 and 72 h time points following oxidative stress. Our results indicate that CREB phosphorylation occurs in all three subcellular locations. It further shows significant disruption of the phosphorylation process by 6-OHDA treatment and shows tridirectional trafficking of total CREB and phospho-CREB between cytosol, mitochondria and nucleus following oxidative stress induced by 6-OHDA treatment. In conclusion, our results indicate the presence of specific signalling molecules in all the compartments studied and their involvement in the signal transduction processes, where total CREB and phospho-CREB levels and activities are either upregulated or downregulated to balance each other for their roles.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Neuronas Dopaminérgicas/metabolismo , Estrés Oxidativo/fisiología , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Proteínas de Unión al ADN/metabolismo , Interpretación Estadística de Datos , Humanos , Hidroxidopaminas , Cinética , Proteínas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Enfermedad de Parkinson/metabolismo , FosforilaciónRESUMEN
Human serum butyrylcholinesterase (HuBChE) is currently the most suitable bioscavenger for the prophylaxis of highly toxic organophosphate (OP) nerve agents. A dose of 200mg of HuBChE is envisioned as a prophylactic treatment that can protect humans from an exposure of up to 2 × LD50 of soman. The limited availability and administration of multiple doses of this stoichiometric bioscavenger make this pretreatment difficult. Thus, the goal of this study was to produce a smaller enzymatically active HuBChE polypeptide (HBP) that could bind to nerve agents with high affinity thereby reducing the dose of enzyme. Studies have indicated that the three-dimensional structure and the domains of HuBChE (acyl pocket, lip of the active center gorge, and the anionic substrate-binding domain) that are critical for the binding of substrate are also essential for the selectivity and binding of inhibitors including OPs. Therefore, we designed three HBPs by deleting some N- and C-terminal residues of HuBChE by maintaining the folds of the active site core that includes the three active site residues (S198, E325, and H438). HBP-4 that lacks 45 residues from C-terminus but known to have BChE activity was used as a control. The cDNAs for the HBPs containing signal sequences were synthesized, cloned into different mammalian expression vectors, and recombinant polypeptides were transiently expressed in different cell lines. No BChE activity was detected in the culture media of cells transfected with any of the newly designed HBPs, and the inactive polypeptides remained inside the cells. Only enzymatically active HBP-4 was secreted into the culture medium. These results suggest that residues at the N- and C-termini are required for the folding and/or maintenance of HBP into an active stable, conformation.
Asunto(s)
Butirilcolinesterasa/química , Aminoácidos/química , Butirilcolinesterasa/genética , Butirilcolinesterasa/metabolismo , Butirilcolinesterasa/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Compuestos Organofosforados/antagonistas & inhibidores , Compuestos Organofosforados/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Eliminación de Secuencia , Soman/antagonistas & inhibidores , Soman/toxicidadRESUMEN
OBJECTIVE: There is considerable interest in improved off-line automated seizure detection methods that will decrease the workload of EEG monitoring units. Subject-specific approaches have been demonstrated to perform better than subject-independent ones. However, for pre-surgical diagnostics, the traditional method of obtaining a priori data to train subject-specific classifiers is not practical. We present an alternative method that works by adapting the threshold of a subject-independent to a specific subject based on feedback from the user. METHODS: A subject-independent quadratic discriminant classifier incorporating modified features based partially on the Gotman algorithm was first built. It was then used to derive subject-specific classifiers by determining subject-specific posterior probability thresholds via user interaction. The two schemes were tested on 529 h of intracranial EEG containing 63 seizures from 15 subjects undergoing pre-surgical evaluation. To provide comparison, the standard Gotman algorithm was implemented and optimised for this dataset by tuning the detection thresholds. RESULTS: Compared to the tuned Gotman algorithm, the subject-independent scheme reduced the false positive rate by 51% (0.23 to 0.11 h(-1)) while increasing sensitivity from 53% to 62%. The subject-specific scheme further improved sensitivity to 78%, but with a small increase in false positive rate to 0.18 h(-1). CONCLUSIONS: The results suggest that a subject-independent classifier scheme with modified features is useful for reducing false positive rate, while subject adaptation further enhances performance by improving sensitivity. The results also suggest that the proposed subject-adapted classifier scheme approximates the performance of the subject-specific Gotman algorithm. SIGNIFICANCE: The proposed method could potentially increase the productivity of offline EEG analysis. The approach could also be generalised to enhance the performance of other subject independent algorithms.