SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes

The cleavage of RNA can be accelerated by a number of factors. These factors include an acidic group (Lewis acid) or a basic group that aids in the deprotonation of the attacking nucleophile, in effect enhancing the nucleophilicity of the nucleophile; an acidic group that can neutralize and stabilize the leaving group; and any environment that can stabilize the pentavalent species that is either a transition state or a short-lived intermediate. The catalytic properties of ribozymes are due to factors that are derived from the complicated and specific structure of the ribozyme–substrate complex. It was postulated initially that nature had adopted a rather narrowly defined mechanism for the cleavage of RNA. However, recent findings have clearly demonstrated the diversity of the mechanisms of ribozyme-catalyzed reactions. Such mechanisms include the metal-independent cleavage that occurs in reactions catalyzed by hairpin ribozymes and the general double-metal-ion mechanism of catalysis in reactions catalyzed by the Tetrahymena group I ribozyme. Furthermore, the architecture of the complex between the substrate and the hepatitis delta virus ribozyme allows perturbation of the pKa of ring nitrogens of cytosine and adenine. The resultant perturbed ring nitrogens appear to be directly involved in acid/base catalysis. Moreover, while high concentrations of monovalent metal ions or polyamines can facilitate cleavage by hammerhead ribozymes, divalent metal ions are the most effective acid/base catalysts under physiological conditions.

A secondary-structure model for the self-cleaving region of Neurospora VS RNA.

Neurospora VS RNA performs an RNA-mediated self-cleavage reaction whose products contain 2',3'-cyclic phosphate and 5'-hydroxyl termini. This reaction is similar to those of hammerhead, hairpin, and hepatitis delta virus ribozymes; however, VS RNA is not similar in sequence to these other self-cleaving motifs. Here we propose a model for the secondary structure of the self-cleaving region of VS RNA, supported by site-directed mutagenesis and chemical modification structure probing data. The secondary structure of VS RNA is distinct from those of the other naturally occurring RNA self-cleaving domains. In addition to a unique secondary structure, several Mg-dependent interactions occur during the folding of VS RNA into its active tertiary conformation.

Avaliação de fatores virológicos associados ao desenvolvimento de carcinoma hepatocelular (CHC) em pacientes com hepatite B crônica

O objetivo principal deste estudo foi avaliar fatores virais associados com a evolução para o carcinoma hepatocelular (CHC) em pacientes com hepatite B crônica. Para tanto caracterizamos os subgenótipos do HBV, investigamos a ocorrência de mutações nos genes pré-core/core do HBV associadas à presença de CHC avaliamos por análise filogenética a associação de linhagens virais com a ocorrência de CHC e por fim a associação de outros fatores de risco com o desenvolvimento de CHC. Foram incluídos 119 amostras de soro de pacientes com infecção crônica pelo HBV, destas amostras 60 pertencem ao grupo 1 (CHC), que são pacientes com diagnóstico confirmado de carcinoma hepatocelular e 59 amostras pertencem ao grupo 2 (sem CHC) que são pacientes com hepatite crônica sem detecção prévia de nódulos hepáticos. Foram obtidas informações acerca da idade, sexo e naturalidade. Além disso, os pacientes responderam a um questionário sobre fatores de riscos associados ao desenvolvimento de CHC. Foram realizados exames bioquímicos, sorológicos, determinação da carga viral, e amplificação por nested PCR e sequenciamento das regiões S/polimerase e pré-core/core do genoma viral para posterior caracterização dos genótipos/subgenótipos do HBV e pesquisa de mutações associadas com evolução da doença hepática. Em relação à idade e sexo não houve grande variação entre os grupos. Quanto à naturalidade a maioria era procedente da região sudeste, seguido pela região nordeste; e por fim seis pacientes eram procedentes de outros países. Com base no sobrenome dos pacientes avaliou-se também a frequência de etnia oriental na casuística estudada, que foi similar nos 2 grupos. O perfil sorológico HBeAg negativo foi o mais frequente nos dois grupos de pacientes, assim como níveis de carga viral abaixo de 2.000 UI/mL. Em relação aos exames bioquímicos foram observadas diferenças estatisticamente significantes nos níveis séricos de AFP (p= 0,0013), FA (p= 0,0003) e GGT (p= 0,005). Dentre os fatores de risco analisados neste estudo, o consumo de amendoim foi o único que apresentou significância estatística (p= 0,003). A região S/pol foi amplificada e sequenciada com sucesso em 58 amostras (28 do grupo 1 e 30 do grupo 2). Entre as 58 amostras analisadas 4 genótipos e 8 subgenótipos do HBV foram identificados, sendo o subgenótipo A1 o mais frequente nos dois grupos. Não se observou diferença estatisticamente significante na distribuição dos subgenótipos entre os dois grupos de pacientes. Na topologia da árvore filogenética construída com sequências do HBV isoladas dos pacientes incluídos neste estudo e sequências disponíveis no GenBank não se observou padrões de agrupamento associados com o perfil clinico do paciente (com e sem CHC). Foram obtidas sequências de boa qualidade da região précore/ core em 44 amostras, sendo 20 amostras do grupo 1 e 24 do grupo 2. Diversas das mutações investigadas foram identificadas na região précore/ core, as quais foram avaliadas estatisticamente para verificar a existência de diferença na frequência das mesmas entre os grupos de pacientes estudados. Entre as mutações identificadas se destacaram com significância estatística as seguintes mutações: T1768A (p= 0,006), a combinação das mutações C1766T + T1768A (p= 0,043) e G1888H (p= 0,05). Na análise de regressão logística simples foi possível identificar que a chance de um paciente do grupo 2 desenvolver CHC aumenta 14,7 vezes na presença de infecção por cepas do HBV com a mutação T1768A, enquanto que a infecção com cepas do HBV que albergam a mutação G1888H reduz tal chance 2,5 vezes

Heterogeneity in testing practices for infections during pregnancy: national survey across Switzerland.

QUESTION Detection and treatment of infections during pregnancy are important for both maternal and child health. The objective of this study was to describe testing practices and adherence to current national guidelines in Switzerland. METHODS We invited all registered practicing obstetricians and gynaecologists in Switzerland to complete an anonymous web-based questionnaire about strategies for testing for 14 infections during pregnancy. We conducted a descriptive analysis according to demographic characteristics. RESULTS Of 1138 invited clinicians, 537 (47.2%) responded and 520 (45.6%) were eligible as they are currently caring for pregnant women. Nearly all eligible respondents tested all pregnant women for group B streptococcus (98.0%), hepatitis B virus (HBV) (96.5%) and human immunodeficiency virus (HIV) (94.7%), in accordance with national guidelines. Although testing for toxoplasmosis is not recommended, 24.1% of respondents tested all women and 32.9% tested at the request of the patient. Hospital doctors were more likely not to test for toxoplasmosis than doctors working in private practice (odds ratio [OR] 2.52, 95% confidence interval [CI] 1.04-6.13, p = 0.04). Only 80.4% of respondents tested all women for syphilis. There were regional differences in testing for some infections. The proportion of clinicians testing all women for HIV, HBV and syphilis was lower in Eastern Switzerland and the Zurich region (69.4% and 61.2%, respectively) than in other regions (range 77.1-88.1%, p <0.001). Most respondents (74.5%) said they would appreciate national guidelines about testing for infections during pregnancy. CONCLUSIONS Testing practices for infections in pregnant women vary widely in Switzerland. More extensive national guidelines could improve consistency of testing practices.

CCR5-Delta 32 mutation is strongly associated with primary sclerosing cholangitis

CCR5 plays a key role in the distribution of CD45RO+ T cells and contributes to generation of a T helper 1 immune response. CCR5-Delta32 is a 32-bp deletion associated with significant reduction in cell surface expression of the receptor. We investigated the role of CCR5-Delta32 on susceptibility to ulcerative colitis (UC), Crohn's disease ( CD) and primary sclerosing cholangitis (PSC). Genotype and allelic association analyses were performed in 162 patients with UC, 131 with CD, 71 with PSC and 419 matched controls. There was a significant difference in CCR5 genotype (OR 2.27, P = 0.003) between patients with sclerosing cholangitis and controls. Similarly, CCR5-Delta32 allele frequency was significantly higher in sclerosing cholangitis (17.6%) compared to controls (9.9%, OR 2.47, P = 0.007) and inflammatory bowel disease patients without sclerosing cholangitis ( 11.3%, OR 1.9, P = 0.027). There were no significant differences in CCR5 genotype or allele frequency between those with either UC or CD and controls. Genotypes with the CCR5-Delta32 variant were increased in patients with severe liver disease defined by portal hypertension and/or transplantation (45%) compared to those with mild liver disease (21%, OR 3.17, P = 0.03). The CCR5-Delta32 mutation may influence disease susceptibility and severity in patients with PSC.

Modest weight loss and physical activity in overweight patients with chronic liver disease results in sustained improvements in alanine aminotransferase, fasting insulin, and quality of life

Background and aim: Obesity is a risk factor for progression of fibrosis in chronic liver diseases such as non-alcoholic fatty liver disease and hepatitis C. The aim of this study was to investigate the longer term effect of weight loss on liver biochemistry, serum insulin levels, and quality of life in overweight patients with liver disease and the effect of subsequent weight maintenance or regain. Patients: Thirty one patients completed a 15 month diet and exercise intervention. Results: On completion of the intervention, 21 patients (68%) had achieved and maintained weight loss with a mean reduction of 9.4 (4.0)% body weight. Improvements in serum alanine aminotransferase (ALT) levels were correlated with the amount of weight loss (r=0.35, p=0.04). In patients who maintained weight loss, mean ALT levels at 15 months remained significantly lower than values at enrolment (p=0.004), while in regainers (n=10), mean ALT levels at 15 months were no different to values at enrolment (p=0.79). Improvements in fasting serum insulin levels were also correlated with weight loss (r=0.46, p=0.04), and subsequent weight maintenance sustained this improvement. Quality of life was significantly improved after weight loss. Weight maintainers sustained recommended levels of physical activity and had higher fasting insulin levels (p=0.03) at enrolment than weight regainers. Conclusion: In summary, these findings demonstrate that maintenance of weight loss and exercise in overweight patients with liver disease results in a sustained improvement in liver enzymes, serum insulin levels, and quality of life. Treatment of overweight patients should form an important component of the management of those with chronic liver disease.

Clinical pharmacokinetics and pharmacodynamics of tacrolimus in solid organ transplantation

The aim of this review is to analyse critically the recent literature on the clinical pharmacokinetics and pharmacodynamics of tacrolimus in solid organ transplant recipients. Dosage and target concentration recommendations for tacrolimus vary from centre to centre, and large pharmacokinetic variability makes it difficult to predict what concentration will be achieved with a particular dose or dosage change. Therapeutic ranges have not been based on statistical approaches. The majority of pharmacokinetic studies have involved intense blood sampling in small homogeneous groups in the immediate post-transplant period. Most have used nonspecific immunoassays and provide little information on pharmacokinetic variability. Demographic investigations seeking correlations between pharmacokinetic parameters and patient factors have generally looked at one covariate at a time and have involved small patient numbers. Factors reported to influence the pharmacokinetics of tacrolimus include the patient group studied, hepatic dysfunction, hepatitis C status, time after transplantation, patient age, donor liver characteristics, recipient race, haematocrit and albumin concentrations, diurnal rhythm, food administration, corticosteroid dosage, diarrhoea and cytochrome P450 (CYP) isoenzyme and P-glycoprotein expression. Population analyses are adding to our understanding of the pharmacokinetics of tacrolimus, but such investigations are still in their infancy. A significant proportion of model variability remains unexplained. Population modelling and Bayesian forecasting may be improved if CYP isoenzymes and/or P-glycoprotein expression could be considered as covariates. Reports have been conflicting as to whether low tacrolimus trough concentrations are related to rejection. Several studies have demonstrated a correlation between high trough concentrations and toxicity, particularly nephrotoxicity. The best predictor of pharmacological effect may be drug concentrations in the transplanted organ itself. Researchers have started to question current reliance on trough measurement during therapeutic drug monitoring, with instances of toxicity and rejection occurring when trough concentrations are within 'acceptable' ranges. The correlation between blood concentration and drug exposure can be improved by use of non-trough timepoints. However, controversy exists as to whether this will provide any great benefit, given the added complexity in monitoring. Investigators are now attempting to quantify the pharmacological effects of tacrolimus on immune cells through assays that measure in vivo calcineurin inhibition and markers of immuno suppression such as cytokine concentration. To date, no studies have correlated pharmacodynamic marker assay results with immunosuppressive efficacy, as determined by allograft outcome, or investigated the relationship between calcineurin inhibition and drug adverse effects. Little is known about the magnitude of the pharmacodynamic variability of tacrolimus.

Increased C282Y heterozygosity in gestational diabetes

Background. Hereditary hemochromatosis is an autosomal recessive disorder of iron metabolism that is characterized by excess accumulation of iron in various organs and often leads to diabetes mellitus (DM). To study whether mutations in the hemochromatosis gene (HFE) could be a risk factor for the development of gestational diabetes mellitus (GDM), the prevalence of HFE mutations in patients with GDM was compared to that of healthy pregnant controls. Methods: GDM was diagnosed in 208 of 2,421 pregnant woman screened between the 24th and 28th week of gestation over a period of 18 months. Patients and 170 matched control subjects were screened for the HFE gene mutations C282Y and H63D. Results: In North and Central European GDM patients, the allele frequency of the C282Y mutation (7.7%) was higher than in pregnant controls (2.9%; p = 0.04), while the frequency of the H63D mutation was not different (p = 0.45). Three patients with GDM were homozygous for H63D (3.1%), 1 patient was homozygous for C282Y (1.0%), 2 patients were compound heterozygous (2.0%) and 26 were heterozygous [11 C282Y (11.2%) and 15 H63D (15.3%)]. C282Y and H63D allele frequencies were not different between controls and GDIVI patients of Southern European or non-European origin. Irrespective of the HIFE-mutation status, serum ferritin levels were increased in patients with GDM compared to healthy pregnant controls (p = 0.01), while transferrin saturation was similar in both groups. Conclusions: In North and Central European patients with GDM, the C282Y allele frequency is higherthan in healthy pregnant women, suggesting a genetic susceptibility to the development of GDM. Copyright (c) 2005 S. Karger AG, Basel.

Alcohol use and risk taking among regular ecstasy users

We examine alcohol use in conjunction with ecstasy use and risk-taking behaviors among regular ecstasy users in every capital city in Australia. Data on drug use and risks were collected in 2004 from a national sample of 852 regular ecstasy users (persons who had used ecstasy at least monthly in the preceding 6 months). Users were grouped according to their typical alcohol use when using ecstasy: no use, consumption of between one and five standard drinks, and consumption of more than five drinks (binge alcohol use). The sample was young, well educated, and mainly working or studying. Approximately two thirds (65%) of the regular ecstasy users reported drinking alcohol when taking ecstasy. Of these, 69% reported usually consuming more than five standard drinks. Those who did not drink alcohol were more disadvantaged, with greater levels of unemployment, less education, higher rates of drug user treatment, and prison history. They were also more likely than those who drank alcohol when using ecstasy to be drug injectors and to be hepatitis C positive. Excluding alcohol, drug use patterns were similar between groups, although the no alcohol group used cannabis and methamphetamine more frequently. Binge drinkers were more likely to report having had three or more sexual partners in the past 6 months and were less likely to report having safe sex with casual partners while under the influence of drugs. Despite some evidence that the no alcohol group were more entrenched drug users, those who typically drank alcohol when taking ecstasy were as likely to report risks and problems associated with their drug use. It appears that regular ecstasy users who binge drink are placing themselves at increased sexual risk when under the influence of drugs. Safe sex messages should address the sexual risk associated with substance use and should be tailored to reducing alcohol consumption, particularly targeting heavy alcohol users. The study's limitations are noted.

Recombinant protein subunit vaccine synthesis in microbes:a role for yeast?

Objectives Recombinant protein subunit vaccines are formulated using protein antigens that have been synthesized in heterologous host cells. Several host cells are available for this purpose, ranging from Escherichia coli to mammalian cell lines. This article highlights the benefits of using yeast as the recombinant host. Key findings The yeast species, Saccharomyces cerevisiae and Pichia pastoris, have been used to optimize the functional yields of potential antigens for the development of subunit vaccines against a wide range of diseases caused by bacteria and viruses. Saccharomyces cerevisiae has also been used in the manufacture of 11 approved vaccines against hepatitis B virus and one against human papillomavirus; in both cases, the recombinant protein forms highly immunogenic virus-like particles. Summary Advances in our understanding of how a yeast cell responds to the metabolic load of producing recombinant proteins will allow us to identify host strains that have improved yield properties and enable the synthesis of more challenging antigens that cannot be produced in other systems. Yeasts therefore have the potential to become important host organisms for the production of recombinant antigens that can be used in the manufacture of subunit vaccines or in new vaccine development.

Functional Evaluation of Causal Mutations Identified in Human Genetic Studies

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

Discrete-event Simulation and Optimization to Improve the Performance of a Healthcare System

Thesis (Ph.D.)--University of Washington, 2016-08

Therapeutic Potential of RNAi Through Endocytotic Methods

The ability to manipulate gene expression promises to be an important tool for the management of infectious diseases and genetic disorders. However, a major limitation to effective delivery of therapeutic RNA to living cells is the cellular toxicity of conventional techniques. Team PANACEA’s research objective was to create new reagents based on a novel small-molecule delivery system that uses a modular recombinant protein vehicle consisting of a specific ligand coupled to a Hepatitis B Virus-derived RNA binding domain (HBV-RBD). Two such recombinant delivery proteins were developed: one composed of Interleukin-8, the other consisting of the Machupo Virus GP1 protein. The ability of these proteins to deliver RNA to cells were then tested. The non-toxic nature of this technology has the potential to overcome limitations of current methods and could provide a platform for the expansion of personalized medicine.