Ornithine Transcarbamylase Deficiency Presenting as Acute Encephalopathy After Strabismus Surgery.

Acute encephalopathy with an unclear etiology is a common presentation to the hospital. We describe the case of a 50-year-old male who presented with a one-day history of slurred speech, nausea, insomnia, and altered mental status. His surgical history was notable for a strabismus surgery two days ago. He presented with elevated ammonia levels that continued to increase. Metabolic studies were suggestive of hyperammonemia secondary to ornithine transcarbamylase (OTC) deficiency triggered due to fasting prior to the strabismus surgery. OTC gene sequencing confirmed the diagnosis of OTC deficiency. We summarize the current case reports in the literature and review the treatment options for OTC deficiency. Our case occurred after a low-risk outpatient strabismus surgery and is a good example of maintaining a broad differential and revising the suspected diagnosis constantly.

Patient with a Hook of the Hamate Fracture Presenting as Vascular Occlusion: Diagnosis Made with Bedside Ultrasound.

BACKGROUND: Hook of the hamate fractures are particularly rare carpal fractures with significant morbidity if not diagnosed early. Classically, these fractures occur from localized blunt trauma to the hook of the hamate in racket sports. Common complaints include pain localized in the hypothenar eminence and reduced grip strength. Hook of the hamate fractures have the potential to cause significant injury to the ulnar nerve and artery. CASE REPORT: We present the case of a 43-year-old man with hypothenar pain, paresthesias of the fifth finger and ulnar aspect of the fourth finger, and pallor of the fourth and fifth fingers. Using bedside ultrasonography, the patient was found to have a fracture of the hook of the hamate that was causing compression of the ulnar artery. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Fracture of the hook of the hamate is often not seen on x-ray studies, and fracture fragments can cause compression of adjacent structures, including the ulnar and median nerves and ulnar artery. Bedside ultrasound may be a useful adjunct in the diagnosis of this carpal fracture when standard x-ray studies do not show a fracture and clinical presentation is concerning for the diagnosis.

Measurement of tumor necrosis factor-alpha, interleukin-6, Fas ligand, interleukin-1α, and interleukin-1ß in the aqueous humor of patients with open angle glaucoma using multiplex bead analysis.

PURPOSE: Various cytokines, including tumor necrosis factor-alpha (TNF-α), Fas ligand (FasL), interleukin-1α (IL-1α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), contribute to the pathogenesis of primary open angle glaucoma (POAG). The present study was set to measure these cytokines in the aqueous humor of patients with POAG and in control subjects using multiplex bead analysis. METHODS: Twenty-five patients with POAG and 29 control subjects were enrolled in this case-control study. Aqueous humor concentrations of the cytokines (IL-1 α, IL-1 ß, IL-6, FasL, and TNF- α) were measured using multiplex bead analysis. RESULTS: Mean aqueous humor levels of IL-6 were significantly lower in patients with POAG compared to the control subjects (9.3±23.7 versus 55.3±94.4 pg/ml; p=0.002). No significant difference in the aqueous humor concentration of IL-1ß was found between patients with POAG and control subjects (0.5±0.8 versus 0.4±0.8 pg/ml; p=0.85.) Concentrations of IL-1α, TNF-α, and FasL were below limits of detection. No significant correlation was found between IL-6 concentration and age, duration of disease, cup/disc ratio, or mean deviation. CONCLUSIONS: In the present study, we found significantly lower concentrations of IL-6 in the aqueous humor of patients with POAG.

Murgocil is a highly bioactive staphylococcal-specific inhibitor of the peptidoglycan glycosyltransferase enzyme MurG.

Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a ß-lactam potentiation screen to identify small molecules that augment the activity of ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the ß-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive ß-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.

Cell-based arrays for the identification of interacting polypeptide domains or epitopes.

The specific regions on proteins which are responsible for protein-protein interaction are called interacting domains, or epitopes in case of antigen-antibody binding. These domains are one feature to characterize proteins and are important in clinical diagnostics and research. For the mapping of such domains the use of protein/peptide arrays has become popular. Regardless of which kind of array, the major requirements are a high number of candidates arranged in the array, high quality, ease of use, and cost-effectiveness. Here, the authors describe a general protocol for mapping the interacting domains of proteins demonstrated by a high affinity protein interaction, the interaction of an antibody to an antigen. The chapter describes a stepwise protocol from library production to the verification of the domain by the use of an automated cell-based polypeptide array, which comprises the named requirements of a good array.

A de novo-designed antimicrobial peptide with activity against multiresistant Staphylococcus aureus acting on RsbW kinase.

Antimicrobial peptides are a promising complement to common antibiotics, development of resistance to which is a growing problem. Here we present a de novo-designed peptide, SP1-1 (RKKRLKLLKRLL-NH2), with antimicrobial activity against multiresistant Staphylococcus aureus (minimal inhibitory concentration: 6.25 µM). Elucidation of the mode of action of this peptide revealed a strong interaction with RsbW kinase (Kd: 6.01±2.73 nM), a serine kinase negatively regulating the activity of the transcription factor σB (SigB). SP1-1 binding and functional modulation of RsbW were shown in vitro by a combination of biochemical, molecular, and biophysical methods, which were further genetically evidenced in vivo by analysis of S. aureus ΔsigB deletion mutants. Intracellular localization of the peptide was demonstrated using nanometer-scaled secondary ion mass spectrometry. Moreover, microarray analysis revealed that transcription of numerous genes, involved in cell wall and amino acid metabolism, transport mechanisms, virulence, and pigmentation, is affected. Interestingly, several WalR binding motif containing genes are induced by SP1-1. In sum, the designed peptide SP1-1 seems to have multiple modes of action, including inhibition of a kinase, and therefore might contribute to the development of new antibacterial compounds, giving bacterial kinase inhibition a closer inspection.

Real-time monitoring of relative peptide-protein interaction strengths in the yeast two-hybrid system.

The yeast two-hybrid (Y2H) system is one of the most technically straightforward, effective, and widely used tools for the discovery of the binary peptide or protein interactions. However, its exceptional detection sensitivity poses a serious challenge for affinity ranking and hence prioritizing the resultant large number of putative interactors for follow-up analyses. To overcome this apparent bottleneck, we describe here a novel yeast growth curve-based interaction-monitoring approach that permits semiautomatic quantification, comparison, and statistically ascertained scoring of a large collection of Y2H interactions under real-time conditions. Initially, we conducted a proof-of-concept test of five literature-validated peptide-protein interactions with known affinities in the low µM range, and subsequently used the method to classify 88 novel vitamin D receptor-binding peptides derived from high-throughput screening of a highly diverse artificial peptide aptamer library. Based on our in-depth data evaluation, we conclude that real-time monitoring of clone growth as a measure of relative binding strength offers a facile, cost-effective, accurate, reproducible, and further adaptable complement to standard Y2H-derived clone management.

From the ORFeome concept to highly comprehensive, full-genome screening libraries.

Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries.

Profound tumor-specific Th2 bias in patients with malignant glioma.

BACKGROUND: Vaccination against tumor-associated antigens is one promising approach to immunotherapy against malignant gliomas. While previous vaccine efforts have focused exclusively on HLA class I-restricted peptides, class II-restricted peptides are necessary to induce CD4+ helper T cells and sustain effective anti-tumor immunity. In this report we investigated the ability of five candidate peptide epitopes derived from glioma-associated antigens MAGE and IL-13 receptor α2 to detect and characterize CD4+ helper T cell responses in the peripheral blood of patients with malignant gliomas. METHODS: Primary T cell responses were determined by stimulating freshly isolated PBMCs from patients with primary glioblastoma (GBM) (n = 8), recurrent GBM (n = 5), meningioma (n = 7), and healthy controls (n = 6) with each candidate peptide, as well as anti-CD3 monoclonal antibody (mAb) and an immunodominant peptide epitope derived from myelin basic protein (MBP) serving as positive and negative controls, respectively. ELISA was used to measure IFN-γ and IL-5 levels, and the ratio of IFN-γ/IL-5 was used to determine whether the response had a predominant Th1 or Th2 bias. RESULTS: We demonstrate that novel HLA Class-II restricted MAGE-A3 and IL-13Rα2 peptides can detect T cell responses in patients with GBMs as well as in healthy subjects. Stimulation with a variety of peptide antigens over-expressed by gliomas is associated with a profound reduction in the IFN-γ/IL-5 ratio in GBM patients relative to healthy subjects. This bias is more pronounced in patients with recurrent GBMs. CONCLUSIONS: Therapeutic vaccine strategies to shift tumor antigen-specific T cell response to a more immunostimulatory Th1 bias may be needed for immunotherapeutic trials to be more successful clinically.

Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae.

BACKGROUND: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. RESULTS: Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway(®) 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway(®) entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. CONCLUSION: This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.

Quantitative real-time PCR as a sensitive protein-protein interaction quantification method and a partial solution for non-accessible autoactivator and false-negative molecule analysis in the yeast two-hybrid system.

Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction.

PIM-1 kinase interacts with the DNA binding domain of the vitamin D receptor: a further kinase implicated in 1,25-(OH)2D3 signaling.

BACKGROUND: The vitamin D3 receptor (VDR) is responsible for mediating the pleiotropic and, in part, cell-type-specific effects of 1,25-dihydroxyvitamin D3 (calcitriol) on the cardiovascular and the muscle system, on the bone development and maintenance, mineral homeostasis, cell proliferation, cell differentiation, vitamin D metabolism, and immune response modulation. RESULTS: Based on data obtained from genome-wide yeast two-hybrid screenings, domain mapping studies, intracellular co-localization approaches as well as reporter transcription assay measurements, we show here that the C-terminus of human PIM-1 kinase isoform2 (amino acid residues 135-313), a serine/threonine kinase of the calcium/calmodulin-regulated kinase family, directly interacts with VDR through the receptor's DNA-binding domain. We further demonstrate that PIM-1 modulates calcitriol signaling in HaCaT keratinocytes by enhancing both endogenous calcitriol response gene transcription (osteopontin) and an extrachromosomal DR3 reporter response. CONCLUSION: These results, taken together with previous reports of involvement of kinase pathways in VDR transactivation, underscore the biological relevance of this novel protein-protein interaction.

DDX5 is a multifunctional co-activator of steroid hormone receptors.

The vitamin D receptor (VDR), an evolutionarily conserved member of the nuclear receptor superfamily, links the metabolically activated vitamin D ligand, calcitriol, with its vitamin D-responsive target genes that are implicated in diverse physiological processes. By genome-wide protein-protein interaction screening of a keratinocyte cDNA library using VDR as bait, we found that the DEAD box RNA helicase p68, also referred to as DDX5, directly interacts with VDR. Domain analysis reveals that the ligand-binding domain of VDR is responsible for the binding, an interaction typical of NR co-activators. Interestingly, the VDR interacting domain of DDX5 lacks a LXXLL-motif and interaction analysis of helix 12 VDR mutants E420K, E420Q and L417S, known to decrease binding affinity of LxxLL motif-containing co-activators showed no change in their interactions. As further support that this novel interactor might be involved in vitamin D-stimulated transcriptional regulation, we demonstrate that VDR and DDX5 co-localize within the nuclei of HaCaT keratinocytes and sub-cellular protein fractions. In vivo validation studies demonstrate, that overexpression of DDX5 has the capability to enhance both, calcitriol-dependent transcription of known response genes and an extrachromosomal DR3-type reporter response. In agreement with this, shRNA based knock-down of DDX5 in keratinocytes compensates for this particular response. Finally, our findings reveal parallels between the VDR-DDX5 interaction and the well-characterized interaction between DDX5 and human estrogen receptor α and the androgen receptor, thus underscoring the physiological significance of the novel protein-protein interaction.

Parathyroid hormone 1-84 accelerates fracture-healing in pubic bones of elderly osteoporotic women.

BACKGROUND: Parathyroid hormone (PTH) has been shown to increase bone mineral density and to reduce the rate of fractures in patients with osteoporosis and also to improve fracture-healing. The purpose of the present prospective, randomized, controlled study was to evaluate the effect of PTH 1-84 on the course of pelvic fracture-healing and functional outcome in postmenopausal women. METHODS: Sixty-five patients had a dual x-ray absorptiometry scan, radiographs, and a computed tomography scan to document pelvic fractures. Twenty-one patients received a once-daily injection of 100 µg of PTH 1-84 starting within two days after admission to the hospital, and forty-four patients served as the control group. All patients received 1000 mg of calcium and 800 IU of vitamin D. Computed tomography scans were repeated every fourth week until radiographic evidence of cortical bridging at the fracture site was confirmed. Functional outcome was assessed with use of a visual analog scale for pain and a Timed "Up and Go" test. RESULTS: The mean time to fracture healing was 7.8 weeks for the treatment group, compared with 12.6 weeks for the control group (p < 0.001). At eight weeks, all fractures in the treatment group were healed and four fractures in the control group were healed (healing rate, 100% compared with 9.1%; p < 0.001). Both the visual analog scale score for pain and the result of the Timed "Up and Go" test improved in the study group as compared with the control group (p < 0.001). CONCLUSIONS: In elderly patients with osteoporosis, PTH 1-84 accelerates fracture-healing in pelvic fractures and improves functional outcome.

Construction of improved Yeast Two-Hybrid libraries.

The Yeast Two-Hybrid (Y2H) system is the most frequently used method for identifying protein-protein interactions. The use of recombination-amenable Y2H vectors would reduce time and effort for cloning prey or bait vectors, and increase the quality of Y2H screenings due to the production of improved screening libraries. These libraries can heighten the amount of new candidates in Y2H screenings significantly by representing more correct candidate genes in frame and outperform a classical Y2H library. The described vectors can be used for the construction of genomic, peptide, or cDNA-based Y2H libraries. Furthermore, the compatibility to newer ORFeome libraries is also given. Here, we describe a vector system for site-specific recombination and for the construction of high-content Y2H libraries. In summary, we will describe the construction of these vectors and the production of Y2H screening libraries.

The mitochondrial T16189C polymorphism is associated with coronary artery disease in Middle European populations.

BACKGROUND: The pivotal role of mitochondria in energy production and free radical generation suggests that the mitochondrial genome could have an important influence on the expression of multifactorial age related diseases. Substitution of T to C at nucleotide position 16189 in the hypervariable D-loop of the control region (CR) of mitochondrial DNA (mtDNA) has attracted research interest because of its suspected association with various multifactorial diseases. The aim of the present study was to compare the frequency of this polymorphism in the CR of mtDNA in patients with coronary artery disease (CAD, n = 482) and type 2 diabetes mellitus (T2DM, n = 505) from two study centers, with healthy individuals (n = 1481) of Middle European descent in Austria. METHODOLOGY AND PRINCIPAL FINDINGS: CR polymorphisms and the nine major European haplogroups were identified by DNA sequencing and primer extension analysis, respectively. Frequencies and Odds Ratios for the association between cases and controls were calculated. Compared to healthy controls, the prevalence of T16189C was significantly higher in patients with CAD (11.8% vs 21.6%), as well as in patients with T2DM (11.8% vs 19.4%). The association of CAD, but not the one of T2DM, with T16189C remained highly significant after correction for age, sex and body mass index (BMI) and was independent of the two study centers. CONCLUSIONS AND SIGNIFICANCE: Our results show for the first time a significant association of T16189C with CAD in a Middle European population. As reported in other studies, in patients with T2DM an association with T16189C in individuals of European decent remains questionable.

Role of inflammation-related gene polymorphisms in patients with central retinal vein occlusion.

OBJECTIVE: Central retinal vein occlusion (CRVO) is a vision-threatening disease, primarily occurring among patients aged more than 60 years. Several risk factors, including arterial hypertension and diabetes mellitus, have been identified. Compression of the central retinal vein by an atherosclerotic retinal artery at the lamina cribrosa also has been implicated in the pathogenesis of the disease. Functional gene polymorphisms of cytokines or chemokines previously shown to affect atherogenesis or hemostasis are potential risk factors for CRVO. The present study investigates a hypothesized association between inflammation-related gene polymorphisms and the presence of CRVO in a relatively large cohort of patients. DESIGN: Case-control study. PARTICIPANTS: The study group consisted of 315 patients with CRVO and 335 control subjects. METHODS: Determination of genotypes was done by 5' exonuclease assay (TaqMan). MAIN OUTCOME MEASURES: Genotypes of interleukin (IL)1ß -511C>T, IL1 receptor antagonist (IL1RN) 1018T>C, IL4 -584C>T, IL6 -174G>C, IL10 -592C>A, IL18 183A>G, tumor necrosis factor (TNF)-α -308G>A, monocyte chemoattractant protein (MCP)-1/CCL2 -2518A>G, IL8 -251A>T, and RANTES (CCL5) -403G>A polymorphisms. RESULTS: Genotype distributions and allele frequencies of the investigated gene polymorphisms did not significantly differ between both groups (P>0.05). Arterial hypertension, diabetes mellitus, and cigarette smoking were significantly more frequent in patients with CRVO than among control subjects (arterial hypertension: 67.0% vs. 52.2%, P<0.001; diabetes mellitus: 16.8% vs. 6.3%, P<0.001, cigarette smoking: 32.1% vs. 23.6%, P = 0.02). In a logistic regression analysis, the presence of arterial hypertension was associated with an odds ratio (OR) of 1.75 (95% confidence interval [CI], 1.26-2.44) in those with CRVO, whereas an OR of 2.52 (95% CI, 1.46-4.35) was found in those with diabetes mellitus. A history of cigarette smoking was associated with an OR of 1.57 (95% CI, 1.09 - 2.25) for CRVO. CONCLUSIONS: Our data suggest that the investigated inflammation-related gene polymorphisms are unlikely major risk factors for CRVO. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Coupled yeast 2-hybrid-mammalian 2-hybrid reading-frame-independent and site-specific recombinational cloning vector system.

The yeast 2-hybrid (Y2H) system is a powerful method for identifying protein-protein interactions (PPIs), requiring minimal prior information of the putative interactors. Currently available automated versions of the Y2H system are sufficiently developed to allow facile genome-wide PPI screening to compile extensive inter-actome data. A limitation of the Y2H approach, however, is that all primary hits have to be technically verified and biologically evaluated by complementary methods, which is time-consuming, costly, and laborious. Furthermore, the yeast intracellular environment can lead to spurious results for proteins of other organisms, for example because of differences in post-translational modifications or the presence/absence of bridging proteins. Many researchers now confirm PPIs found in the Y2H system by retesting the candidates in the mammalian 2-hybrid system (M2H). However, although such combined Y2H-M2H testing is desirable and perhaps necessary, recloning of Y2H candidates into M2H vectors, especially on a large scale, is time-consuming and costly. To address this shortcoming, we introduce here a new site-specific recombination-capable M2H vector system that is fully compatible with the site-specific Y2H system that we recently described in Biotechniques [2008;45(3):235-244]. The results show that the new vectors are: (a) Gateway®, compatible and suitable for fast cloning; (b) fully functional in the M2H system without influencing the capacity of the selection system or creating autoactivators; and (c) directly compatible with the existing site-specific Y2H vector system, as demonstrated by confirmation of Y2H PPI candidates in the M2H system.

Epitope mapping of antibodies using a cell array-based polypeptide library.

The authors describe a technique for mapping the epitopes of protein antigens recognized by mono- or polyclonal antibodies. This method is based on a recombinant polypeptide library, expressed in a bacterial expression system, arrayed at high density, and tested on a membrane with automated procedures. The authors analyzed the epitope of a commercially available monoclonal antibody to vitamin D receptor (VDR). About 2300 overlapping VDR peptides were screened on a test array, and a contiguous stretch of 37 amino acids was identified as the epitope. Its authenticity was confirmed by Western blotting and an immunofluorescence competition assay on human skin tissue samples. The authors define the proposed method as a cell-based protein or peptide array that is adaptable to many applications, including epitope mapping of antibodies and autoantibodies, autoantigen detection from patient sera, whole-proteome approaches such as protein-peptide interactions, or selection of monoclonal antibodies from polyclonal sera. The advantages of this method are (a) its ease of protein array production based on well-established bacterial protein/peptide expression procedures; (b) the large number of printable colonies (as many as approximately 25,000) that can be arrayed per membrane; (c) there is no need for protein purification of recombinantly expressed proteins; (d) DNA, rather than protein, is the starting material to generate the arrays; and (e) its high-throughput and automatable format.