Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH

One of the most important factors influencing embryo viability is chromosome imbalance (aneuploidy). Embryos derived from aneuploid gametes have little potential for forming a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. For more than a decade, preimplantation genetic screening (PGS) has been used to assist in the identification of aneuploid embryos. However, current strategies, based upon cell biopsy followed by fluorescent in situhybridization, allow less than half of the chromosomes to be screened. In this review, we discuss methods that overcome the limitations of earlier PGS strategies and provide screening of the entire chromosome complement in oocytes and embryos. In recent months, there has been a rapid growth in the number of PGS cycles utilizing one such method, comparative genomic hybridization (CGH). Data from IVF cycles utilizing CGH must be considered preliminary, but appear to indicate a dramatic increase in embryo implantation following comprehensive chromosomal screening. It is expected that methods based upon microarrays will yield similar clinical results and may be sufficiently rapid to permit comprehensive screening without the need for embryo cryopreservation. Some microarray platforms also offer the advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis. However, more data concerning accuracy and further reductions in the price of tests will be necessary before microarrays can be widely applied.     

Array painting: a method for the rapid analysis of aberrant chromosomes using DNA microarrays

Objective: The authors describe a method, termed array painting, which allows the rapid, high resolution analysis of the content and breakpoints of aberrant chromosomes.

Methods: Array painting is similar in concept to reverse chromosome painting and involves the hybridisation of probes generated by PCR of small numbers of flow sorted chromosomes on large insert genomic clone DNA microarrays.

Results and Conclusions: By analysing patients with cytogenetically balanced chromosome rearrangements, the authors show the effectiveness of array painting as a method to map breakpoints prior to cloning and sequencing chromosome rearrangements.

     

High-throughput screening of gene function in stem cells using clonal microarrays

We describe a microarray-based approach for the high-throughput screening of gene function in stem cells and demonstrate the potential of this method by growing and isolating clonal populations of both adult and embryonic neural stem cells. Clonal microarrays are constructed by seeding a population of cells at clonal density on micropatterned surfaces generated using soft lithographic microfabrication techniques. Clones of interest can be isolated after assaying in parallel for various cellular processes and functions, including proliferation, signal transduction, and differentiation. We demonstrate the compatibility of the technique with both gain- and loss-of-function studies using cell populations infected with cDNA libraries or DNA constructs that induce RNA interference. The infection of cells with a library prior to seeding and the compact but isolated growth of clonal cell populations will facilitate the screening of large libraries in a wide variety of mammalian cells, including those that are difficult to transfect by conventional methods.     

Evaluation of a new rapid urine screening analyser: CellFacts

The aim of this study was to evaluate a new urine screening analyser in a Hospital Bacteriology Laboratory: the Cellfacts Urine Screening Analyser (Microbial Systems Limited (MSL), Coventry, England). A cohort of 1036 urine specimens were analysed by both the CellFacts and routine traditional methods. Using the CDC urinary tract infection decision levels, and compared to reference methods, the sensitivity and specificity were respectively 88.9% and 76.2%, and the predictive negative value and predictive positive value respectively 93.3% and 64.8%. Compared to the microscopy, the correlations of white blood cell count and red blood cell count were good, respectively (r = 0.8, p < 0.0001) and (r = 0.6, p < 0.0001). These results indicate that, although several positive samples were not reported, CellFacts facilitates efficient, rapid screening of infected urine specimens. This leads to perform a cytobacteriological analysis only on those results screened as positive by the automatic analysis system.     

Alpha-1-antitrypsin hematoxylin and eosin fluorescence: a rapid, useful screening technic

Sections stained with hematoxylin and eosin from seven liver biopsies and two ovarian mixed mesodermal tumors containing alpha-1-antitrypsin globules were examined by fluorescent microscopy. Al-AT globules readily were seen in all biopsies and fluoresced brightly. Megamitochondria and acidophil bodies in liver biopsies also fluoresced but were distinguished easily by their morphology and topographic location. Normal liver biopsies contained no fluorescent bodies. It is concluded that examination of routine hematoxylin and eosin stained sections by fluorescent microscopy is a rapid convenient screening method for the detection of al-AT globules. Acidophil bodies and megamitochondria also are seen by this method.     

Use of DNA microarrays for rapid genotyping of TEM beta-lactamases that confer resistance

Standard clinical procedures for pathogen resistance identification are laborious and usually require 2 days of cultivation before the resistance can be determined unequivocally. In contrast, clinicians and patients face increasing threats from antibiotic-resistant pathogenic bacteria in terms of their frequencies and levels of resistance. A major class of microbial resistance stems from the occurrence of beta-lactamases, which, if mutated, can cause the severe extended-spectrum beta-lactamase (ESBL) or inhibitor-resistant TEM (IRT) phenotype, which cause resistance to extended-spectrum cephalosporins, monobactams, and beta-lactamase inhibitors. We describe an oligonucleotide microarray for identification of the single nucleotide polymorphisms (SNPs) of 96% of the TEM beta-lactamase variants described to date which are related to the ESBL and/or IRT phenotype. The target DNA, originating from Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae cells isolated from clinical samples, was amplified and fluorescently labeled by PCR with consensus primers in the presence of cyanine 5-labeled nucleotides. The total assay, including PCR, hybridization, and image analysis, could be performed in 3.5 h. The microarray results were validated by standard clinical procedures. The microarray outperformed the standard procedures in terms of assay time and the depth of information provided. In conclusion, this array offers an attractive option for the identification and epidemiologic monitoring of TEM beta-lactamases in the routine clinical diagnostic laboratory.     

High throughput screening: a rapid way to recombinant allergens

Complex allergenic sources such as moulds, foods and mites contain large panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Phage display of cDNA libraries allows selective enrichment of allergen-expressing clones using IgE from allergic patients. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, however, high-throughput screening technology is required. We have used a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries in order to characterize whole allergenic repertoires from complex allergenic sources. The strategy, based on a combination of phage display and high-density arrays, allowed fast discovery of panels of related structures from different allergenic sources. They cover secreted, cytoplasmic and structural proteins with or without enzymatic activity and offer a rational explanation for the IgE-mediated cross-reactivity frequently encountered in clinical practice.     

Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses

The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B(1), B(2), C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.     

Cost-effectiveness of rapid MRSA screening in surgical patients

This study investigates the effectiveness of a same-day polymerase chain reaction (PCR) test for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) in a general screening of patients admitted to the trauma surgery and heart surgery department in a German university hospital. A total of 442 patients were screened over a 4-month period by using a PCR assay, compared to culture methods, for specimens from the nose and throat. The MRSA carriage rate on admission was 3.85% during the study period. The PCR results of 1,680 swabs showed a sensitivity of 85% and a specificity of 99.39% for swabs from the nares and for the throat 42.11% and 98.78%, respectively. A combination of specimens from the nose and throat from the same patient led to a sensitivity of 100% with a specificity of 98.29%. Cost calculation under the circumstances of a diagnosis-related groups (DRG) payment system found that the eight MRSA-positive patients created costs of €38,472, i.e. €4,809 per patient, facing screening costs of €36.62 per sample. Screening patients by using the rapid PCR assay for a combination of specimens from the nose and throat would offer a safe and cost-effective way of MRSA screening on admission.     

High-throughput screening of microscale pitted substrate topographies for enhanced nonviral transfection efficiency in primary human fibroblasts

Optimization of nonviral gene delivery typically focuses on the design of particulate carriers that are endowed with desirable membrane targeting, internalization, and endosomal escape properties. Topographical control of cell transfectability, however, remains a largely unexplored parameter. Emerging literature has highlighted the influence of cell-topography interactions on modulation of many cell phenotypes, including protein expression and cytoskeletal behaviors implicated in endocytosis. Using high-throughput screening of primary human dermal fibroblasts cultured on a combinatorial library of microscale topographies, we have demonstrated an improvement in nonviral transfection efficiency for cells cultured on dense micropit patterns compared to smooth substrates, as verified with flow cytometry. A 25% increase in GFP(+) cells was observed independent of proliferation rate, accompanied by SEM and confocal microscopy characterization to help explain the phenomenon qualitatively. This finding encourages researchers to investigate substrate topography as a new design consideration for the optimization of nonviral transfection systems.     

The PRINS technique: potential use for rapid preimplantation embryo chromosome screening

The primed in-situ labelling (PRINS) method is an alternativeto in-srtu hybridization for chromosomal detection based onthe use of chromosome-specific oligonucleotide primers. Usingthis process, we have developed a simple and semi-automaticmethod for rapid in-situ detection of human chromosomes. Thereaction was performed on a programmable temperature cycler.Specific labelling was obtained in < 2 h reaction. DoublePRINS techniques were performed on six morphologically abnormalpreimplantation embryos using primers specific for chromosomes9, 16, 18, 21, X and Y. The majority of these embryos displayedchromosomal abnormalities. The present results demonstrate thatPRINS may be a simple and reliable technique applicable in humanpreimplantation diagnosis. human pre-embryo/in-situ labelling/oligonucleotide/preimplantation diagnosis/PRINS     

Dot-immunogold filtration assay for rapid screening of three fluoroquinolones

A dot-immunogold filtration assay (DIGFA) was described for simultaneous screening of three fluoroquinolones (FQs): enrofloxacin (ENR), ciprofloxacin (CIP) and norfloxacin (NOR). Colloidal gold particles were used as visible labels, and anti-ENR polyclonal antibodies from mice and rabbits were exploited for gold labelling and membrane coating, respectively. Several parameters affecting DIGFA were optimised, and the operation was validated with pure buffer matrix and spiked eel samples, respectively. The detection limits in eel samples were estimated to be about 20 µg kg^?1 for ENR and CIP, and 50 µg kg^?1 for NOR. With pretreated nitrocellulose membranes, the determination could be completed within 30 minutes, and the results could be read just by the naked eye without the help of any equipment.     

Biomaterial and antibiotic strategies for peri-implantitis: a review

Dental implants have 89% plus survival rates at 10-15 years, but peri-implantitis or dental implant infections may be as high as 14%. Peri-implantitis can limit clinical success and impose health and financial burdens to patients and health providers. The pathogenic species associated with periodontitis (e.g., Fusobacterium ssp, A. actinomycetemcomitans, P. gingivalis) are also associated with peri-implantitis. Incidence of peri-implantitis is highest within the first 12 months after implantation, and is higher in patients who smoke or have poor oral health as well as with calcium-phosphate-coated or surface-roughened implants. Biomaterial therapies using fibers, gels, and beads to deliver antibiotics have been used in the treatment of Peri-implantitis though clinical efficacy is not well documented. Guided tissue regeneration membranes (e.g., collagen, poly-lactic/glycolic acid, chitosan, ePTFE) loaded with antimicrobials have shown success in reosseointegrating infected implants in animal models but have not been proven in humans. Experimental approaches include the development of anti-bioadhesion coatings, coating surfaces with antimicrobial agents (e.g., vancomycin, Ag, Zn) or antimicrobial releasing coatings (e.g., calcium phosphate, polylactic acid, chitosan). Future strategies include the development of surfaces that become antibacterial in response to infection, and improvements in the permucosal seal. Research is still needed to identify strategies to prevent bacterial attachment and enhance normal cell/tissue attachment to implant surfaces.     

Use of laser capture microdissection, cDNA microarrays, and tissue microarrays in advancing our understanding of prostate cancer

One difficulty in studying epithelial tumors has been the inability to isolate pure samples for DNA and RNA analysis. Prostate cancer, with its infiltrative nature, is particularly challenging. The Combination of several new technologies should help overcome these hurdles. Laser capture microdissection uses a laser beam to achieve transfer of pure cell populations for isolation of DNA, RNA, and protein. High-throughput analysis of these samples can be achieved by using cDNA expression microarrays. High-density tissue microarrays should allow for validation of differentially expressed genes. This review will concentrate on the application of laser capture microdissection, cDNA microarrays, and tissue microarrays in the area of prostate cancer research.