The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.
An advanced graph theoretical approach is introduced that enables a higher level of functional interpretation of samples of directed networks with identical fixed pairwise different vertex labels that are drawn from a particular population. Compared to the analysis of single networks, their investigation promises to yield more detailed information about the represented system. Often patterns of directed edges in sample element networks are too intractable for a direct evaluation and interpretation. The new approach addresses the problem of simplifying topological information and characterizes such a sample of networks by finding its locatable characteristic topological patterns. These patterns, essentially sample-specific network motifs with vertex labeling, might represent the essence of the intricate topological information contained in all sample element networks and provides as well a means of differentiating network samples. Central to the accurateness of this approach is the null model and its properties, which is needed to assign significance to topological patterns. As a proof of principle the proposed approach has been applied to the analysis of networks that represent brain connectivity before and during painful stimulation in patients with major depression and in healthy subjects. The accomplished reduction of topological information enables a cautious functional interpretation of the altered neuronal processing of pain in both groups.
Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2–related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2+/+ mice compared with Nrf2−/− knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells.
Chemokine-like receptor 1 (CMKLR1) ligands chemerin and resolvin E1 are suggested to have a role in non-alcoholic fatty liver disease (NAFLD). Here, expression of CMKLR1 in liver cells and NAFLD was studied. CMKLR1 was detected in primary human hepatocytes (PHH), Kupffer cells, bile-duct cells and hepatic stellate cells. In human and rodent fatty liver and in fibrotic liver of mice fed a methionine–choline deficient diet CMKLR1 was reduced. Hepatocytes are the major cells in the liver and effects of adipokines, cytokines and lipids on CMKLR1 in PHH were analyzed. Increased cellular triglyceride or cholesterol content, lipopolysaccharide, IL-6, TNF and leptin did not influence CMKLR1 levels in PHH whereas profibrotic TGFβ tended to reduce CMKLR1. Adiponectin strongly upregulated CMKLR1 mRNA and protein in PHH and hepatic CMKLR1 when injected into wild type mice. Further, CMKLR1 was suppressed in the liver of adiponectin deficient mice. These data indicate that low CMKLR1 in NAFLD may partly result from reduced adiponectin activity.
Adipokine; Hepatic steatosis; Chemerin receptor; Liver
A fully automated high-throughput solution X-ray scattering data collection system, developed for protein structure studies at beamline 4-2 of the Stanford Synchrotron Radiation Lightsource, is described.
A fully automated high-throughput solution X-ray scattering data collection system has been developed for protein structure studies at beamline 4-2 of the Stanford Synchrotron Radiation Lightsource. It is composed of a thin-wall quartz capillary cell, a syringe needle assembly on an XYZ positioning arm for sample delivery, a water-cooled sample rack and a computer-controlled fluid dispenser. It is controlled by a specifically developed software component built into the standard beamline control program Blu-Ice/DCS. The integrated system is intuitive and very simple to use, and enables experimenters to customize data collection strategy in a timely fashion in concert with an automated data processing program. The system also allows spectrophotometric determination of protein concentration for each sample aliquot in the beam via an in situ UV absorption spectrometer. A single set of solution scattering measurements requires a 20–30 µl sample aliquot and takes typically 3.5 min, including an extensive capillary cleaning cycle. Over 98.5% of measurements are valid and free from artefacts commonly caused by air-bubble contamination. The sample changer, which is compact and light, facilitates effortless switching with other sample-handling devices required for other types of non-crystalline X-ray scattering experiments.
proteomics; structural genomics; X-ray scattering; laboratory automation; SAXS
This study investigated electroencephalographic correlates in chronically depressed patients compared to healthy controls using intracutaneously applied electrical pain stimulus, to better understand the interaction between pain processing and depression. A close interaction between pain and depression is generally recognized although the precise mechanisms are not yet fully understood. The present study focuses on the hypothesis that effective brain connectivity in major depression patients is altered. Multifunctional interactions between brain regions represent a robust index of effective interactions within the brain, and can be quantified by network redundancy. Thus, structural network differences between 18 normal controls and 18 major depression patients before as well as during the processing of moderately painful intracutaneous electrical stimuli were investigated on the basis of network redundancy differences. In our sample, both patients and control subjects exhibit comparable network redundancies before stimulus application. Caused by the stimulus, there is a global increase of network redundancy in both groups. This increase is diminished in the group of major depression patients. We found clear differences between patients and controls during the stimulus processing, where the network redundancy in normal controls is larger in comparison to patients. The differences might be explained by the fact that major depression patients are more restricted to the affective component of the processing. The well-established biasing to affective processing might suppress the somatosensory processing resulting in a lower number of connections within the considered network. This might then lead to a reduction in network redundancy during stimulus processing.
Chronic low back pain (CLBP) was shown to be associated with pathophysiological changes at several levels of the sensorimotor system. Changes in sensory thresholds have been reported but complete profiles of Quantitative Sensory Testing (QST) were only rarely obtained in CLBP patients. The aim of the present study was to investigate comprehensive QST profiles in CLBP at the painful site (back) and at a site distinct from their painful region (hand) and to compare these data with similar data in healthy controls. We found increased detection thresholds in CLBP patients compared to healthy controls for all innocuous stimuli at the back and extraterritorial to the painful region at the hand. Additionally, CLBP patients showed decreased pain thresholds at both sites. Importantly, there was no interaction between the investigated site and group, i.e. thresholds were changed both at the affected body site and for the site distinct from the painful region (hand). Our results demonstrate severe, widespread changes in somatosensory sensitivity in CLBP patients. These widespread changes point to alterations at higher levels of the neuraxis or/and to a vulnerability to nociceptive plasticity in CLBP patients.
The experience of strong phantom limb pain (PLP) in arm amputees was previously shown to be associated with structural neural plasticity in parts of the cortex that belong to dorsal and ventral visual streams. It has been speculated that this plasticity results from the extensive use of a functional prosthesis which is associated with increased visual feedback to control the artificial hand. To test this hypothesis, we reanalyzed data of cortical volumes of 21 upper limb amputees and tested the association between the amount of use of the hand prosthesis and cortical volume plasticity. On the behavioral level, we found no relation between PLP and the amount of prosthesis use for the whole patient group. However, by subdividing the patient group into patients with strong PLP and those with low to medium PLP, stronger pain was significantly associated with less prosthesis use whereas the group with low PLP did not show such an association. Most plasticity of cortical volume was identified within the dorsal stream. The more the patients that suffered from strong PLP used their prosthesis, the smaller was the volume of their posterior parietal cortex. Our data indicate a relationship between prosthesis use and cortical plasticity of the visual stream. This plasticity might present a brain adaptation process to new movement and coordination patterns needed to guide an artificial hand.
phantom limb pain; magnetic resonance imaging; morphometry; use of myoelectric prostheses; visual stream
Concerning the physiological correlates of pain, the brain stem is considered to be one core region that is activated by noxious input. In animal studies, different slopes of skin heating (SSH) with noxious heat led to activation in different columns of the midbrain periaqueductal gray (PAG). The present study aimed at finding a method for differentiating structures in PAG and other brain stem structures, which are associated with different qualities of pain in humans according to the structures that were associated with different behavioral significances to noxious thermal stimulation in animals. Brain activity was studied by functional MRI in healthy subjects in response to steep and shallow SSH with noxious heat. We found differential activation to different SSH in the PAG and the rostral ventromedial medulla (RVM). In a second experiment, we demonstrate that the different SSH were associated with different pain qualities. Our experiments provide evidence that brainstem structures, i.e., the PAG and the RVM, become differentially activated by different SSH. Therefore, different SSH can be utilized when brain stem structures are investigated and when it is aimed to activate these structures differentially. Moreover, percepts of first pain were elicited by shallow SSH whereas percepts of second pain were elicited by steep SSH. The stronger activation of these brain stem structures to SSH, eliciting percepts of second vs. first pain, might be of relevance for activating different coping strategies in response to the noxious input with the two types of SSH.
A-delta fiber; C-fiber; second pain; pain descriptors; PAG; RVM; periaqueductal grey; rostral ventromedial medulla
Information on human papillomavirus (HPV) type distribution is necessary to evaluate the potential impact of current and future HPV vaccines. We estimated the relative contribution (RC) to invasive cervical cancer (ICC) and precancerous cervical lesions of the nine HPV types (HPV 6/11/16/18/31/33/45/52/58) included in an HPV vaccine currently under development.
Estimations on ICC were based on an international study of 8,977 HPV positive cases and estimations on precancerous cervical lesions were extracted from a published meta-analysis including 115,789 HPV positive women. Globocan 2008 and 2010 World Population Prospects were used to estimate current and future projections of new ICC cases.
RC of the 9 HPV types in ICC was 89.4%, with 18.5% of cases positive for HPV 31/33/45/52/58. Regional variations were observed. RCs varied by histology, ranging between 89.1% in squamous cell carcinomas (SCC) and 95.5% in adenocarcinomas (ADC). HPV 16/18/45 were detected in 94.2% of ADC. RC of the 9 types altogether decreased with age (trend test p < 0.0001), driven by the decrease in older ages of HPV 16/18/45. In contrast, the RC of HPV 31/33/52/58 increased with age. Due to population growth alone, projected estimates of ICC cases attributable to the 9 types are expected to rise from 493,770 new cases in 2012 to 560,887 new cases in 2025.
The RCs of individual high risk HPV types varied by cytological and histological grades of HPV-positive precancerous cervical lesions, and there was an under representation of HPV 18 and 45 compared to ICC.
The addition of HPV 31/33/45/52/58 to HPV types included in current vaccines could prevent almost 90% of ICC cases worldwide. If the nine-valent vaccine achieves the same degree of efficacy than previous vaccines, world incidence rates could be substantially reduced.
Human papillomavirus; Cervical cancer; Genotype; Epidemiology; Human papillomavirus vaccines
Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF's detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials.
Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity.
PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues.
Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.
The lipidic cubic phase (LCP) has repeatedly proven to serve as a successful membrane-mimetic matrix for a variety of difficult-to-crystallize membrane proteins. While monoolein has been the predominant lipid of choice, there is a growing need for the characterization and use of other LCP host lipids, allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion, stability and crystallogenesis of membrane proteins. Here, we describe the development of a high-throughput (HT) pipeline to employ small angle X-ray scattering (SAXS) – the most direct technique to identify lipid mesophases and measure their structural parameters – to interrogate rapidly a large number of lipid samples under a variety of conditions, similar to those encountered during crystallization. Leveraging the identical setup format for LCP crystallization trials, this method allows the quickly assessment of lipid matrices for their utility in membrane protein crystallization, and could inform the tailoring of lipid and precipitant conditions to overcome specific crystallization challenges. As proof of concept, we present HT LCP-SAXS analysis of lipid samples made of monoolein with and without cholesterol, and of monovaccenin, equilibrated with solutions used for crystallization trials and LCP fluorescence recovery after photobleaching (FRAP) experiments.
lipidic cubic phase; monoolein; phase behavior; SAXS
Chronic low back pain (CLBP) has been shown to be associated with various pathophysiological changes at several level of the sensorimotor system, pointing to a general hypersensitivity in CLBP patients. The aim of the present study was to investigate signs of generalized mechanical pain hypersensitivity in CLBP patients on the hand and on the painful site of the back.
Pinprick stimulation according to a validated standardized quantitative sensory testing protocol was used in 14 female CLBP patients and 14 healthy controls (HC) matched for sex and age. Stimulus response functions to pinprick stimulation on the skin were examined at the affected back and reference sites (hand palmar and hand dorsum). Data from CLBP patients were compared with HC and with reference data from the German Research Network on Neuropathic Pain.
We found significant differences in the stimulus response functions between CLBP patients and HC. Pain ratings to the pinpricks were increased for low and moderate pinprick stimuli in CLBP patients. Importantly, this kind of specific pinprick hyperalgesia was found not only for the affected body site (back), but also for the remote reference sites (hand dorsum and hand palmar).
We interpret our results as pointing to changes in the nociceptive processing in CLBP at higher levels of the neuraxis, possibly thalamus and/or attentional control, rather than changes of spinal processing. Alternatively, there might be a higher vulnerability to noxious stimulation in CLBP patients.
Chronic Low Back Pain (CLBP); Mechanical pain thresholds; Pinprick hyperalgesia; Allodynia
The Fibromyalgia Survey Questionnaire (FSQ) assesses the key symptoms of fibromyalgia syndrome. The FSQ can be administrated in survey research and settings where the use of interviews to evaluate the number of pain sites and extent of somatic symptom intensity and tender point examination would be difficult. We validated the FSQ in a cross-sectional survey with FMS patients. In a cross-sectional survey, participants with physician diagnosis of FMS were recruited by FMS-self help organisations and nine clinical institutions of different levels of care. Participants answered the FSQ (composed by the Widespread Pain Index [WPI] and the Somatic Severity Score [SSS]) assessing the Fibromyalgia Survey Diagnostic Criteria (FSDC) and the Patient Health Questionnaire PHQ 4. American College of Rheumatology 1990 classification criteria were assessed in a subgroup of participants. 1,651 persons diagnosed with FMS were included into analysis. The acceptance of the FSQ-items ranged between 78.9 to 98.1% completed items. The internal consistency of the items of the SSS ranged between 0.75–0.82. 85.5% of the study participants met the FSDC. The concordance rate of the FSDC and ACR 1990 criteria was 72.7% in a subsample of 128 patients. The Pearson correlation of the SSS with the PHQ 4 depression score was 0.52 (p<0.0001) and with the PHQ anxiety score was 0.51 (p<0.0001) (convergent validity). 64/202 (31.7%) of the participants not meeting the FSDC criteria and 152/1283 (11.8%) of the participants meeting the FSDC criteria reported an improvement (slightly too very much better) in their health status since FMS-diagnosis (Chi2 = 55, p<0.0001) (discriminant validity). The study demonstrated the feasibility of the FSQ in a cross-sectional survey with FMS-patients. The reliability, convergent and discriminant validity of the FSQ were good. Further validation studies of the FSQ in clinical and general population settings are necessary.
Consumer surveys provide information on effectiveness and side effects of medical interventions in routine clinical care. A report of fibromyalgia syndrome (FMS) consumers has not been carried out in Europe.
The study was carried out from November 2010 to April 2011. Participants diagnosed with FMS rated the effectiveness and side effects of pharmacological and non-pharmacological FMS interventions on a 0 to 10 scale, with 10 being most efficacious (harmful). The questionnaire was distributed by the German League for people with Arthritis and Rheumatism and the German Fibromyalgia Association to their members and to all consecutive FMS patients of nine clinical centers of different levels of care.
1661 questionnaires (95% women, mean age 54 years, mean duration since FMS diagnosis 6.8 years) were analysed. The most frequently used therapies were self-management strategies, prescription pain medication and aerobic exercise. The highest average effectiveness was attributed to whole body and local warmth therapies, thermal bathes, FMS education and resting. The highest average side effects were attributed to strong opioids, local cold therapy, gamma-amino-butyric acid analogues (pregabalin and gabapentin), tramadol and opioid transdermal systems.
The German fibromyalgia consumer reports highlight the importance of non-pharmcological therapies in the long-term management of FMS, and challenges the strong recommendations for drug therapies given by FMS-guidelines.
Fibromyalgia syndrome; Consumer reports; Drugs; Non-pharmacological therapies
Network motifs, overrepresented small local connection patterns, are assumed to act
as functional meaningful building blocks of a network and, therefore, received considerable
attention for being useful for understanding design principles and functioning of networks.
We present an extension of the original approach to network motif detection in single,
directed networks without vertex labeling to the case of a sample of directed networks
with pairwise different vertex labels. A characteristic feature of this approach to network
motif detection is that subnetwork counts are derived from the whole sample and the
statistical tests are adjusted accordingly to assign significance to the counts. The associated
computations are efficient since no simulations of random networks are involved. The
motifs obtained by this approach also comprise the vertex labeling and its associated
information and are characteristic of the sample. Finally, we apply this approach to
describe the intricate topology of a sample of vertex-labeled networks which originate from
a previous EEG study, where the processing of painful intracutaneous electrical stimuli
and directed interactions within the neuromatrix of pain in patients with major depression
and healthy controls was investigated. We demonstrate that the presented approach yields
characteristic patterns of directed interactions while preserving their important topological
information and omitting less relevant interactions.
Glutamate is the main excitatory neurotransmitter of the central nervous system. Tissue-type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA-induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the Low-Density Lipoprotein Receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell-surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA-induced events via LDLR engagement.
Tissue-type plasminogen activator; N-methyl-D-aspartate receptor; Low-density lipoprotein receptor family; Serine protease inhibitor; Plasmin
Hepatocellular lipid accumulation is a hallmark of non-alcoholic
fatty liver disease (NAFLD), which encompasses a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and ultimately cirrhosis. Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulate diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 expression is up-regulated in liver cirrhosis and is further increased in hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in tissue specimens of NAFLD patients and found a significant up-regulation compared to normal liver tissue. Noteworthy, ZNF267 mRNA was already significantly increased in steatotic liver tissue without inflammation. In line with this, incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation and corresponding dose-dependent ZNF267 induction in vitro. Furthermore, hepatocellular lipid accumulation induced formation of reactive oxygen species (ROS), and also chemically induced ROS formation increased ZNF267 mRNA expression. In summary with previous findings, which revealed ZNF267 as pro-fibrogenic and pro-cancerogenic factor in chronic liver disease, the present study further suggests ZNF267 as promising therapeutic target particularly for NAFLD patients. In addition, it further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.
Non-alcoholic fatty liver disease; Kruppel-like factor; ZNF267
Signalling pathways are the cornerstone on understanding cell function and predicting cell behavior. Recently, logical models of canonical pathways have been optimised with high-throughput phosphoproteomic data to construct cell-type specific pathways. However, less is known on how signalling pathways can be linked to a cellular response such as cell growth, death, cytokine secretion, or transcriptional activity.
In this work, we measure the signalling activity (phosphorylation levels) and phenotypic behavior (cytokine secretion) of normal and cancer hepatocytes treated with a combination of cytokines and inhibitors. Using the two datasets, we construct "extended" pathways that integrate intracellular activity with cellular responses using a hybrid logical/data-driven computational approach. Boolean logic is used whenever a priori knowledge is accessible (i.e., construction of canonical pathways), whereas a data-driven approach is used for linking cellular behavior to signalling activity via non-canonical edges. The extended pathway is subsequently optimised to fit signalling and behavioural data using an Integer Linear Programming formulation. As a result, we are able to construct maps of primary and transformed hepatocytes downstream of 7 receptors that are capable of explaining the secretion of 22 cytokines.
We developed a method for constructing extended pathways that start at the receptor level and via a complex intracellular signalling pathway identify those mechanisms that drive cellular behaviour. Our results constitute a proof-of-principle for construction of "extended pathways" that are capable of linking pathway activity to diverse responses such as growth, death, differentiation, gene expression, or cytokine secretion.
The individual character of pharmacokinetics is of great importance in the risk assessment of new drug leads in pharmacological research. Amongst others, it is severely influenced by the properties and inter-individual variability of the enzymes and transporters of the drug detoxification system of the liver. Predicting individual drug biotransformation capacity requires quantitative and detailed models.
In this contribution we present the de novo deterministic modeling of atorvastatin biotransformation based on comprehensive published knowledge on involved metabolic and transport pathways as well as physicochemical properties. The model was evaluated on primary human hepatocytes and parameter identifiability analysis was performed under multiple experimental constraints. Dynamic simulations of atorvastatin biotransformation considering the inter-individual variability of the two major involved enzymes CYP3A4 and UGT1A3 based on quantitative protein expression data in a large human liver bank (n = 150) highlighted the variability in the individual biotransformation profiles and therefore also points to the individuality of pharmacokinetics.
A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology.
The purpose of this study was to identify attitudes toward HPV vaccination among US women 27 to 45 years of age. A survey was mailed to 2,750 insured US women to assess perceptions of relevance or irrelevance of the HPV vaccine, the underlying reasons, and, for those reporting relevance, the likelihood of vaccination if it became available. Among the 451 eligible respondents, 304 (67.4%) reported that the HPV vaccine was relevant to them, whereas 143 (31.7%) stated that it was not at all relevant. The most common reasons for relevance were protection from cervical cancer (62.8%), vaginal cancer (58.2%), precancerous cells (55.9%), HPV (55.6%), and genital warts (46.4%). Reasons for irrelevance were most commonly being married (54.0%) or in a monogamous relationship (39.6%). Most respondents reporting relevance of the HPV vaccine were likely (33.4%) or extremely likely (37.7%) to receive the vaccine if approved for their age group.
Augmenter of liver regeneration (ALR), which is critically important in liver regeneration and hepatocyte proliferation, is highly expressed in cirrhotic livers and hepatocellular carcinomas (HCC). In the current study, the functional role of ALR in hepatocancerogenesis was analyzed in more detail. HepG2 cells, in which the cytosolic 15 kDa ALR isoform was reexpressed stably, (HepG2-ALR) were used in migration and invasion assays using modified Boyden chambers. Epithelial-mesenchymal transition (EMT) markers were determined in HepG2-ALR cells in vitro and in HepG2-ALR tumors grown in nude mice. ALR protein was quantified in HCC and nontumorous tissues by immunohistochemistry. HepG2-ALR, compared with HepG2 cells, demonstrated reduced cell motility and increased expression of the epithelial cell markers E-cadherin and Zona occludens-1 (ZO-1), whereas SNAIL, a negative regulator of E-cadherin, was diminished. Matrix metalloproteinase MMP1 and MMP3 mRNA expression and activity were reduced. HepG2-ALR cell-derived subcutaneously grown tumors displayed fewer necrotic areas, more epithelial-like cell growth and fewer polymorphisms and atypical mitotic figures than tumors derived from HepG2 cells. Analysis of tumor tissues of 53 patients with HCC demonstrated an inverse correlation of ALR protein with histological angioinvasion and grading. The 15 kDa ALR isoform was found mainly in HCC tissues without histological angioinvasion 0. In summary the present data indicate that cytosolic ALR reduces hepatoma cell migration, augments epithelial growth and, therefore, may act as an antimetastatic and EMT reversing protein.
Members of the interleukin-1 (IL-1) family of cytokines play major roles in host defense and immune system regulation in infectious and inflammatory diseases. IL-1 cytokines trigger a biological response in effector cells by assembling a heterotrimeric signaling complex with two IL-1 receptor chains, a high-affinity primary receptor and a low-affinity co-receptor. To gain insights into the signaling mechanism of the novel IL-1-like cytokine IL-33, we first solved its solution structure and then performed a detailed biochemical and structural characterization of the interaction between IL-33, its primary receptor ST2 and the co-receptor IL-1RAcP. Using NMR data, we obtained a model of the IL-33/ST2 complex in solution that is validated by small-angle X-ray scattering (SAXS) data and is similar to the IL-1β/IL-1R1 complex. We extended our SAXS analysis to the IL-33/ST2/IL-1RAcP and IL-1β/IL-1R1/IL-1RAcP complexes and propose a general model of the molecular architecture of IL-1 ternary signaling complexes.