Monogenic Human Skin Disorders

Human genodermatoses represent a broad and partly confusing spectrum of countless rare diseases with confluent and overlapping phenotypes often impeding a precise diagnosis in an affected individual. High-throughput sequencing techniques have expedited the identification of novel genes and have dramatically simplified the establishment of genetic diagnoses in such heterogeneous disorders. The precise genetic diagnosis of a skin disorder is crucial for the appropriate counselling of patients and their relatives regarding the course of the disease, prognosis and recurrence risks. Understanding the underlying pathophysiology is a prerequisite to understanding the disease and developing specific, targeted or individualized therapeutic approaches. We aimed to create a comprehensive overview of human genodermatoses and their respective genetic aetiology known to date. We hope this may represent a useful tool in guiding dermatologists towards genetic diagnoses, providing patients with individual knowledge on the respective disorder and applying novel research findings to clinical practice.

Profiling invasiveness in head and neck cancer: recent contributions of genomic and transcriptomic approaches.

High-throughput molecular profiling approaches have emerged as precious research tools in the field of head and neck translational oncology. Such approaches have identified and/or confirmed the role of several genes or pathways in the acquisition/maintenance of an invasive phenotype and the execution of cellular programs related to cell invasion. Recently published new-generation sequencing studies in head and neck squamous cell carcinoma (HNSCC) have unveiled prominent roles in carcinogenesis and cell invasion of mutations involving NOTCH1 and PI3K-patwhay components. Gene-expression profiling studies combined with systems biology approaches have allowed identifying and gaining further mechanistic understanding into pathways commonly enriched in invasive HNSCC. These pathways include antigen-presenting and leucocyte adhesion molecules, as well as genes involved in cell-extracellular matrix interactions. Here we review the major insights into invasiveness in head and neck cancer provided by high-throughput molecular profiling approaches.

Regulation of human (adrenal) androgen biosynthesis-New insights from novel throughput technology studies

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38�� (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

Use of a novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the clonal GS/M-83-H7 line of Giardia lamblia.

During infections, Giardia lamblia undergoes a continuous change of its major surface antigens, the variant-specific surface proteins (VSPs). Many studies on antigenic variation have been performed using G. lamblia clone GS/M-83-H7, which expresses surface antigen VSP H7. The present study was focused on the identification and characterization of vsp gene sequences within the genome of the clonal G. lamblia GS/M-83-H7 line. For this purpose, we applied a PCR which specifically amplified truncated sequences from the 3'-terminal region of the vsp genes. Upon cloning, most of the vsp gene amplification products were shown to be approximately identical in size and thus could not be distinguished from each other by conventional gel electrophoresis. In order to pre-estimate the sequence complexity within the large panel of vsp clones isolated, we elaborated a novel concept which facilitated our large-scale genetic screening approach: PCR products from cloned DNA molecules were generated and then subjected to a DNA melting profile assay based on the use of the LightCycler Instrument. This high-throughput assay system proved to be well suited to monitor sequence differences between the amplification products from closely related vsp genes and thus could be used for the primary, sequence-related discrimination of the corresponding clones. After testing 50 candidates, vsp clones could be divided into five groups, each characterized by an individual DNA melting profile of the corresponding amplification products. Sequence analysis of some of these 50 candidates confirmed data from the aforementioned assay in that clones were demonstrated to be identical within, but different between, the distinct groups. The nucleotide and deduced amino acid sequences of five representative vsp clones showed high similarities both among each other and also with the corresponding gene segment of the variant-specific surface antigen (VSP H7) expressed by the original GS/M-83-H7 variant type. Furthermore, three of the genomic vsp sequences turned out to be identical to vsp sequences that represented previously characterized transcription products from in vivo- or in vitro-switched GS/M-83-H7 trophozoites. In conclusion, the DNA melting profile assay seems to be a versatile tool for the PCR-based genotyping of moderately or highly diversified sequence orthologues.

The role of mass spectrometry-based metabolomics in medical countermeasures against radiation

Radiation metabolomics can be defined as the global profiling of biological fluids to uncover latent, endogenous small molecules whose concentrations change in a dose-response manner following exposure to ionizing radiation. In response to the potential threat of nuclear or radiological terrorism, the Center for High-Throughput Minimally Invasive Radiation Biodosimetry was established to develop field-deployable biodosimeters based, in part, on rapid analysis by mass spectrometry of readily and easily obtainable biofluids. In this review, we briefly summarize radiation biology and key events related to actual and potential nuclear disasters, discuss the important contributions the field of mass spectrometry has made to the field of radiation metabolomics, and summarize current discovery efforts to use mass spectrometry-based metabolomics to identify dose-responsive urinary constituents, and ultimately to build and deploy a noninvasive high-throughput biodosimeter.

Nanotoxicology: a perspective and discussion of whether or not in vitro testing is a valid alternative

Despite the many proposed advantages related to nanotechnology, there are increasing concerns as to the potential adverse human health and environmental effects that the production of, and subsequent exposure to nanoparticles (NPs) might pose. In regard to human health, these concerns are founded upon the plethora of knowledge gained from research relating to the effects observed following exposure to environmental air pollution. It is known that increased exposure to environmental air pollution can cause reduced respiratory health, as well as exacerbate pre-existing conditions such as cardiovascular disease and chronic obstructive pulmonary disease. Such disease states have also been associated with exposure to the NP component contained within environmental air pollution, raising concerns as to the effects of NP exposure. It is not only exposure to accidentally produced NPs however, which should be approached with caution. Over the past decades, NPs have been specifically engineered for a wide range of consumer, industrial and technological applications. Due to the inevitable exposure of NPs to humans, owing to their use in such applications, it is therefore imperative that an understanding of how NPs interact with the human body is gained. In vivo research poses a beneficial model for gaining immediate and direct knowledge of human exposure to such xenobiotics. This research outlook however, has numerous limitations. Increased research using in vitro models has therefore been performed, as these models provide an inexpensive and high-throughput alternative to in vivo research strategies. Despite such advantages, there are also various restrictions in regard to in vitro research. Therefore, the aim of this review, in addition to providing a short perspective upon the field of nanotoxicology, is to discuss (1) the advantages and disadvantages of in vitro research and (2) how in vitro research may provide essential information pertaining to the human health risks posed by NP exposure.

Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM(con21)). 16S rRNA sequence analysis comparing LCM, LCM(con21) and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri(RR) strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.

Identification of selective norbornane-type aspartate analogue inhibitors of the glutamate transporter 1 (GLT-1) from the chemical universe generated database (GDB)

A variety of conformationally constrained aspartate and glutamate analogues inhibit the glutamate transporter 1 (GLT-1, also known as EAAT2). To expand the search for such analogues, a virtual library of aliphatic aspartate and glutamate analogues was generated starting from the chemical universe database GDB-11, which contains 26.4 million possible molecules up to 11 atoms of C, N, O, F, resulting in 101026 aspartate analogues and 151285 glutamate analogues. Virtual screening was realized by high-throughput docking to the glutamate binding site of the glutamate transporter homologue from Pyrococcus horikoshii (PDB code: 1XFH ) using Autodock. Norbornane-type aspartate analogues were selected from the top-scoring virtual hits and synthesized. Testing and optimization led to the identification of (1R*,2R*,3S*,4R*,6R*)-2-amino-6-phenethyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic acid as a new inhibitor of GLT-1 with IC(50) = 1.4 ?M against GLT-1 and no inhibition of the related transporter EAAC1. The systematic diversification of known ligands by enumeration with help of GDB followed by virtual screening, synthesis, and testing as exemplified here provides a general strategy for drug discovery.

The homeobox gene HLXB9 is upregulated in a morphological subset of poorly differentiated hepatocellular carcinoma

The prognostic outcome for hepatocellular carcinoma (HCC) remains poor. Disease progression is accompanied by dedifferentiation of the carcinoma, a process that is not well understood. The aim of this study was to get more insight into the molecular characteristics of dedifferentiated carcinomas using high throughput techniques. Microarray-based global gene expression analysis was performed on five poorly differentiated HCC cell lines compared with non-neoplastic hepatic controls and a set of three cholangiolar carcinoma (CC) cell lines. The gene with the highest upregulation was HLXB9. HLXB9 is a gene of the homeobox genfamily important for the development of the pancreas. RT-PCR confirmed the upregulation of HLXB9 in surgical specimens of carcinoma tissue, suggesting its biological significance. Interestingly, HLXB9 upregulation was primary observed in poorly differentiated HCC with a pseudoglandular pattern compared with a solid pattern HCC or in moderate or well-differentiated HCC. Additional the expression of translated HLXB9, the protein HB9 (NCBI: NP_001158727), was analyzed by western blotting. Expression of HB9 was only detected in the cytoplasm but not in the nuclei of the HCC cells. For validation CC were also investigated. Again, we found an upregulation of HLXB9 in CC cells accompanied by an expression of HB9 in the cytoplasms of these tumor cells, respectively. In conclusion, homeobox HLXB9 is upregulated in poorly differentiated HCC with a pseudoglandular pattern. The translated HB9 protein is found in the cytoplasm of these HCC and CC. We therefore assume HLXB9 as a possible link in the understanding of the development of HCC and CC, respectively.

UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-��-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

Biodiversity and chemodiversity: future perspectives in bioprospecting

Biological diversity and its constituent chemical diversity have served as one of the richest sources of bioprospecting leading to the discovery of some of the most important bioactive molecules for mankind. Despite this excellent record, in the recent past, however, bioprospecting of biological resources has met with little success; there has been a perceptible decline in the discovery of novel bioactive compounds. Several arguments have been proposed to explain the current poor success in bioprospecting. Among them, it has been argued that to bioprospect more biodiversity may not necessarily be productive, considering that chemical and functional diversity might not scale with biological diversity. In this paper, we offer a critique on the current perception of biodiversity and chemodiversity and ask to what extent it is relevant in the context of bioprospecting. First, using simple models, we analyze the relation among biodiversity, chemodiversity and functional redundancies in chemical plans of plants and argue that the biological space for exploration might still be wide open. Second, in the context of future bioprospecting, we argue that brute-force high throughput screening approaches alone are insufficient and cost ineffective in realizing bioprospecting success. Therefore, intelligent or non-random approaches to bioprospecting need to be adopted. We review here few examples of such approaches and show how these could be further developed and used in the future to accelerate the pace of discovery.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Echinococcus metacestodes as laboratory models for the screening of drugs against cestodes and trematodes

Among the cestodes, Echinococcus granulosus, Echinococcus multilocularis and Taenia solium represent the most dangerous parasites. Their larval stages cause the diseases cystic echinococcosis (CE), alveolar echinococcosis (AE) and cysticercosis, respectively, which exhibit considerable medical and veterinary health concerns with a profound economic impact. Others caused by other cestodes, such as species of the genera Mesocestoides and Hymenolepis, are relatively rare in humans. In this review, we will focus on E. granulosus and E. multilocularis metacestode laboratory models and will review the use of these models in the search for novel drugs that could be employed for chemotherapeutic treatment of echinococcosis. Clearly, improved therapeutic drugs are needed for the treatment of AE and CE, and this can only be achieved through the development of medium-to-high throughput screening approaches. The most recent achievements in the in vitro culture and genetic manipulation of E. multilocularis cells and metacestodes, and the accessability of the E. multilocularis genome and EST sequence information, have rendered the E. multilocularis model uniquely suited for studies on drug-efficacy and drug target identification. This could lead to the development of novel compounds for the use in chemotherapy against echinococcosis, and possibly against diseases caused by other cestodes, and potentially also trematodes.

A global ���imaging��� view on systems approaches in immunology

The immune system exhibits an enormous complexity. High throughput methods such as the "-omic'' technologies generate vast amounts of data that facilitate dissection of immunological processes at ever finer resolution. Using high-resolution data-driven systems analysis, causal relationships between complex molecular processes and particular immunological phenotypes can be constructed. However, processes in tissues, organs, and the organism itself (so-called higher level processes) also control and regulate the molecular (lower level) processes. Reverse systems engineering approaches, which focus on the examination of the structure, dynamics and control of the immune system, can help to understand the construction principles of the immune system. Such integrative mechanistic models can properly describe, explain, and predict the behavior of the immune system in health and disease by combining both higher and lower level processes. Moving from molecular and cellular levels to a multiscale systems understanding requires the development of methodologies that integrate data from different biological levels into multiscale mechanistic models. In particular, 3D imaging techniques and 4D modeling of the spatiotemporal dynamics of immune processes within lymphoid tissues are central for such integrative approaches. Both dynamic and global organ imaging technologies will be instrumental in facilitating comprehensive multiscale systems immunology analyses as discussed in this review.

Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay

The scintillation proximity assay (SPA) is a rapid radioligand binding assay. Upon binding of radioactively labeled ligands (here L-[(3)H]arginine or D-[(3)H]glucose) to acceptor proteins immobilized on fluoromicrospheres (containing the scintillant), a light signal is stimulated and measured. The application of SPA to purified, detergent-solubilized membrane transport proteins allows substrate-binding properties to be assessed (e.g., substrate specificity and affinity), usually within 1 d. Notably, the SPA makes it possible to study specific transporters without interference from other cellular components, such as endogenous transporters. Reconstitution of the target transporter into proteoliposomes is not required. The SPA procedure allows high sample throughput and simple sample handling without the need for washing or separation steps: components are mixed in one well and the signal is measured directly after incubation. Therefore, the SPA is an excellent tool for high-throughput screening experiments, e.g., to search for substrates and inhibitors, and it has also recently become an attractive tool for drug discovery.