[RNA in situ hybridization: technology, potential, and fields of application]. / RNA-in-situ-Hybridisierung: Technologie, Möglichkeiten und Anwendungsbereiche.

Significant improvements in the technology of RNA in situ hybridization (RNA-ISH) in the past five decades have opened up novel fields of its application as a valuable and an attractive adjunct to the portfolio of pathologist's daily routine diagnostic practice.In contrast to the former methodology, the current bDNA-based technology is not only easier to handle but also considerably more sensitive, enabling single-target molecule detection in formalin-fixed and paraffin-embedded tissue specimens without significant effort by both the lab and the evaluating pathologist, as assays can be run on standard automated staining devices and evaluated by light microscopy. Compared to molecular methods like RT-PCR and whole-genome analysis, RNA-ISH maintains tissue integrity thus offering the invaluable advantage of localization of target cells especially in relation to secreted proteins and expression of the target sequence in multiple cell types. The first clinical trials implementing RNA-ISH for patient stratification and selection are in progress and already led to the first drug approvals based on its use as a CDx test.In addition to its role as a complementary method for the establishment of novel IHC procedures or as an addition or replacement to IHC in the standard routine portfolio, RNA-ISH has gained special importance for its capacity to detect noncoding RNA species or mutation or splice variants, where no alternative procedures are available. This more complex application requires development of standardized procedures and involvement of the pathologist during assay establishment and for routine specimen evaluation.The present article reviews the development of RNA-ISH from its early uses to its current applications in research and diagnostics based on the authors' considerable experience of applying it as tool in a biopharmaceutical research organization.

How technical progress reshaped behavioral neuroendocrinology during the last 50 years and some methodological remarks.

The first issue of Hormones and Behavior was published 50 years ago in 1969, a time when most of the techniques we currently use in Behavioral Endocrinology were not available. Researchers have during the last 5 decades developed techniques that allow measuring hormones in small volumes of biological samples, identify the sites where steroids act in the brain to activate sexual behavior, characterize and quantify gene expression correlated with behavior expression, modify this expression in a specific manner, and manipulate the activity of selected neuronal populations by chemogenetic and optogenetic techniques. This technical progress has considerably transformed the field and has been very beneficial for our understanding of the endocrine controls of behavior in general, but it did also come with some caveats. The facilitation of scientific investigations came with some relaxation of methodological exigency. Some critical controls are no longer performed on a regular basis and complex techniques supplied as ready to use kits are implemented without precise knowledge of their limitations. We present here a selective review of the most important of these new techniques, their potential problems and how they changed our view of the hormonal control of behavior. Fortunately, the scientific endeavor is a self-correcting process. The problems have been identified and corrections have been proposed. The next decades will obviously be filled with exciting discoveries in behavioral neuroendocrinology.

Next-generation in situ hybridization approaches to define and quantify HIV and SIV reservoirs in tissue microenvironments.

The development of increasingly safe and effective antiretroviral treatments for human immunodeficiency virus (HIV) over the past several decades has led to vastly improved patient survival when treatment is available and affordable, an outcome that relies on uninterrupted adherence to combination antiretroviral therapy for life. Looking to the future, the discovery of an elusive 'cure' for HIV will necessitate highly sensitive methods for detecting, understanding, and eliminating viral reservoirs. Next-generation, in situ hybridization (ISH) approaches offer unique and complementary insights into viral reservoirs within their native tissue environments with a high degree of specificity and sensitivity. In this review, we will discuss how modern ISH techniques can be used, either alone or in conjunction with phenotypic characterization, to probe viral reservoir establishment and maintenance. In addition to focusing on how these techniques have already furthered our understanding of HIV reservoirs, we discuss potential avenues for how high-throughput, next-generation ISH may be applied. Finally, we will review how ISH could allow deeper phenotypic and contextual insights into HIV reservoir biology that should prove instrumental in moving the field closer to viral reservoir elimination needed for an 'HIV cure' to be realized.

[New microbiological techniques]. / Neue mikrobiologische Techniken.

Microbiological diagnostic procedures have changed rapidly in recent years. This is especially true in the field of molecular diagnostics. Classical culture-based techniques are still the gold standard in many areas; however, they are already complemented by automated and also molecular techniques to guarantee faster and better quality results. The most commonly used techniques include real-time polymerase chain reaction (RT-PCR) based systems and nucleic acid hybridization. These procedures are used most powerfully from direct patient samples or in assays to detect the presence of nonculturable or fastidious organisms. Further techniques such as DNA sequencing are not yet used routinely for urological samples and can be considered experimental. However, in conjunction with dropping prices and further technical developments, these techniques promise to be used much more in the near future. Regarding bacterial identification from culture, mass spectrometry (MALDI-TOF MS) has become the technique of choice in recent years especially in Europe. It has tremendously shortened the time to result. This is now going to be extended to antibiotic susceptibility testing. This is of paramount importance in view of ever rising antimicrobial resistance rates. Techniques described in this review offer a faster and better microbiological diagnosis. Such continuous improvements are critical especially in times of cost pressure and rising antimicrobial resistance rates. It is in our interest to provide the best possible care for patients and in this regard a good and effective communication between the laboratory and the clinician is of vital importance.

In situ hybridisation: Technologies and their application to understanding disease.

In situ hybridisation (ISH) is unique amongst molecular analysis methods in providing for the precise microscopic localisation of genes, mRNA and microRNA in metaphase spreads, cell and tissue preparations. The method is well established as a tool to guide appropriate therapeutic intervention in breast, gastric and lung cancer. With the description of ultrasensitive ISH technologies for low copy mRNA demonstration and the relative ease by which microRNA can be visualised, the applications for research and diagnostic purposes is set to increase dramatically. In this review ISH is considered with emphasis on recent technological developments and surveyed for present and future applications in the context of the demonstration of genes, mRNA and microRNA in health and disease.

[The present and future prospects in rapid molecular diagnosis of tuberculosis and MDR-TB (First Part)]. / Prezent si perspective în diagnosticul rapid molecular al tuberculozei si al MDR-tB (prima parte).

Tuberculosis is still one of the diseases with a major medical and social impact, and in terms of early diagnosis (which would imply a fair treatment and established at the time), difficulties related to the delay bacilli isolation in culture, decreased susceptibility testing methods to antituberculosis drugs, lack of methods for differentiation of M. Tuberculosis complex germs of non TB Mycobacteria, may have important clinical implications. Traditional testing of anti-TB drug susceptibility on solid Löwenstein-Jensen medium (gold standard) or liquid media can only be performed using grown samples. Determining the time it takes up to 42 days on solid media and 12 days for liquid media. For MDR/XDR TB cases is absolutely essential to reduce the detection time. In these cases prove their usefulness rapid diagnostic methods. Automatic testing in liquid medium, molecular hybridization methods are currently recommended by the current WHO guidelines. Rapid diagnosis of MDR-TB is extremely useful for the early establishment of an effective treatment tailored more accurately on the spectrum of sensitivity of the resistant strain (thus reducing the risk of developing additional resistance to other drugs) and control the spread of these strains. Genetic diagnostic methods, approved and recommended by the WHO, can reduce the time of diagnosis of TB case and, importantly, the case of MDR TB. They do not replace the current standard diagnostic methods and resistance profile, but complete them in selected cases.

Molecular studies on chicken melanoma differentiation associated gene-9 ( mda-9)

Background: Melanoma differentiation associated (mda) genes in human encode a protein which has a surprising variety and diversity of interaction partners. It is a positive regulator of cancer cell progression in breast cancer, melanoma, and other human cancers. It regulates cell motility and invasion by altering defined biochemical and signalling pathways. Methods: Suppressive subtractive hybridisation (SSH) has been done using a cDNA library prepared from lipopolysaccharides (LPS) stimulated and non-stimulated chicken spleen cells. Then PCR analysis and in situ hybridisation were done for further studies. Results: This approach resulted in the identification of important chicken mda fragment. The obtained fragment was about 450 bp covering the area from position 500 to position 950 of the human homologue. The expression analysis showed a wide variation in tissues and cell lines. In situ studies revealed mRNA expression in LPS stimulated tissues. Conclusion: In this study a homologue for a chicken novel gene was described. The chicken melanoma differentiation associated gene-9 (mda-9) gene was found to be expressed in many tissues and cell lines in different levels. The stimulation time course was found to have a wide effect on both tissues and cell lines. The mda-9 gene was localised by in situ hybridisation and the effect of LPS stimulation was investigated (AU)

Papiloma bucal en pacientes pediátricos: potencial transmisión materna / Oral papilloma in pediatric patients: potential maternal transmission

La evidencia actual afirma que el virus del papiloma humano (VPH) se puede transmitir tanto por vía sexual como por otras vías. Cuando el método de contagio es no sexual la madre parece ser el principal transmisor del VPH al recién nacido. Diversos autores en sus estudios reportan que se detectó ADN del VPH en el líquido amniótico, cordón umbilical, placenta y membranas fetales, lo que sugiere que la madre puede infectar al infante en la etapa de gestación o durante el parto. Después de la transmisión de la madre al recién nacido, las manifestaciones clínicas pueden aparecer en cualquier etapa de la vida, afectando mucosas, piel o ambas. Cuando se presenta papiloma bucal en niños se piensa en contacto directo, autoinoculación, abuso o violencia sexual, ya que éstos son algunos de los modos de transmisión de la enfermedad, pero cuando no se presenta ninguno de estos métodos de transmisión se deben indagar los antecedentes familiares, específicamente si la madre presentó infección del virus durante su embarazo. Se presentan dos casos de pacientes pediátricos con impresión diagnóstica de papiloma bucal sin causa evidente. Se confirmó el diagnóstico histopatológico y se estableció la relación de transmisión del virus de la madre al niño, ya que en los antecedentes familiares sus madres reportaron infección cervical por VPH en el embarazo y en los antecedentes personales no se encontraron resultados positivos para relacionarlo con otro tipo de transmisión (AU)

Current evidence indicates that human papillomavirus (HPV) can be transmitted both sexually and nonsexually. When the route of contagion is nonsexual, the mother appears to be the main transmitter of HPV to the newborn. Several authors report detection of HPV DNA in amniotic fluid, umbilical cord, placenta and fetal membranes, suggesting that the mother can infect the infant during pregnancy or childbirth. After transmission of virus from mother to newborn, clinical manifestations may appear at any stage in life, affecting mucous membranes, skin or both. When children present with oral papilloma, possible etiologies are direct contact, self-innoculation, sexual abuse or violence. However, when none of these causes are evident, family history should be investigated, especially if the mother manifested HPV infection during pregnancy. We present two cases of pediatric patients with clinical impression of oral papilloma without obvious cause. Histopathology confirmed the diagnosis of HPV and route of transmission was established as mother-to-child, since mothers reported cervical HPV infection during pregnancy. Furthermore, the childrens' medical histories did not indicate any other route of contagion for the virus (AU)

Hibridación in situ fluorescente (FISH), hibridación in situ cromogénica (CISH) e hibridacion in situ cromogénica con plata (SISH): su uso para la detección de la amplificación del gen HER2 en cáncer de mama / No disponible

La importancia en la detección de la amplificación del gen HER2 es esencial en el cáncer de mama puesto que estas pacientes no responden a los tratamientos de quimioterapia y hormonales convencionales. La determinación del HER2 tiene un papel crucial como diana terapéutica del trastuzumab. A continuación discutiremos la efectividad y utilidad clínica de la técnica hibridación in situ fluorescente (FISH), la hibridación in situ cromogénica (CISH) y la hibridación in situ cromogénica con plata (SISH), como métodos usados para el estudio del gen HER2 en la selección correcta de las pacientes con cáncer de mama, que son candidatas a recibir trastuzumab(AU)

The importance of determining HER2 status lays on the fact that breast cancer patients with HER2 amplification are more reluctant to conventional chemotherapy and hormonal treatments. Thus the HER2 analysis is an essential requisite to determine which patients are eligible to be treated with trastuzumab. The aim of this article is reviewing the effectiveness and clinical utility of HER 2 diagnostic test with fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver chromogenic in situ hybridization (SISH) to correctly select breast cancer patients who are candidates to be treated with trastuzumab(AU)

Target-enrichment strategies for next-generation sequencing.

We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.

The combined use of non-radioactive in situ hybridization and real-time RT-PCR to assess gene expression in cryosections.

Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.

COmbined Binary RAtio fluorescence in situ hybridiziation (COBRA-FISH): development and applications.

The ability to probe for the location of DNA sequences in morphologically preserved chromosomes and nuclei by fluorescence in situ hybridization (FISH) provided for cytogenetics a quantum leap forward in resolution and ease of detection of chromosomal aberrations. COBRA-FISH, an acronym for COmbined Binary RAtio-FISH is a multicolor FISH methodology, which enables recognition of all human chromosome arms on the basis of color, thus greatly facilitating cytogenetic analysis. It also permits gene and viral integration site mapping in the context of chromosome arm painting. Here we review the principle, practice and applications of COBRA-FISH.

In situ hybridization: detecting viral nucleic acid in formalin-fixed, paraffin-embedded tissue samples.

In situ hybridization is a method for detecting specific nucleic acid sequences within individual cells. This technique permits visualization of viral nucleic acid or gene expression in individual cells within their histologic context. In situ hybridization is based on the complementary binding of a labeled nucleic acid probe to complementary sequences in cells or tissue sections, followed by visualization of target sequences within the cells. It has been used widely for the detection of viral nucleic acid sequences within individual cells. This review will define the technical approaches of in situ hybridization and its current application to detect viral nucleic acids within formalin-fixed, paraffin-embedded tissue samples, with special reference to the Epstein-Barr virus.

Recent developments in signal amplification methods for in situ hybridization.

In situ hybridization (ISH) allows for the histologic and cytologic localization of DNA and RNA targets. However, the application of ISH techniques can be limited by their inability to detect targets with low copies of DNA and RNA. During the last few years, several strategies have been developed to improve the sensitivity of ISH by amplification of either target nucleic acid sequences prior to ISH or signal detection after the hybridization is completed. Current approaches involving target amplification (in situ PCR, primed labeling, self-sustained sequence replication), signal amplification (tyramide signal amplification, branched DNA amplification), and probe amplification (padlock probes and rolling circle amplification) are reviewed with emphasis on their applications to bright field microscopy. More recent developments such as molecular beacons and in situ strand displacement amplification continue to increase the sensitivity of in situ hybridization methods. Application of some of these techniques has extended the utility of ISH in diagnostic pathology and in research because of the ability to detect targets with low copy numbers of DNA and RNA.

In vivo visualization of chromosomes using lac operator-repressor binding.

This article describes a new technique for direct, in vivo visualization of chromosome dynamics based on lac repressor recognition of direct repeats of the lac operator. The method allows the tagging of specific chromosomal sites and thus in situ localization with minimal perturbation of structure. Detection by light microscopy, using GFP-repressor fusion proteins or immunofluorescence, can be complemented by higher-resolution electron microscopy using immunogold staining. Applications of this method will facilitate the investigation of interphase chromosome dynamics, as well as chromosome segregation during cell division in organisms that lack cytologically condensed chromosomes.