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At least 955 records · Page 53Linked to original sources
PubMed2001

Advanced diagnostic methods in oral and maxillofacial pathology. Part I: molecular methods.

The practice of pathology is currently undergoing significant change, in large part due to advances in the analysis of DNA, RNA, and proteins in tissues. These advances have permitted improved biologic insights into many developmental, inflammatory, metabolic, infectious, and neoplastic diseases. Moreover, molecular analysis has also led to improvements in accuracy of disease diagnosis and classification. It is likely that, in the future, these methods will increasingly enter into the day-to-day diagnosis and management of patients. The pathologist will continue to play a fundamental role in diagnosis and will likely be in a pivotal position to guide the implementation and interpretation of these tests as they move from the research laboratory into diagnostic pathology. The purpose of this 2-part series is to provide an overview of the principles and applications of current molecular biologic and immunologic tests. Part I will discuss the biologic fundamentals of DNA, RNA, and proteins and the methods that are currently available or likely to become available to the pathologist in the next several years for their isolation and analysis in tissue biopsies.

Diagnosis, Oral↗
PubMed2006

PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.

In the present study, we analyzed the kinetics of the PCR-suppression effect (PS-effect) and firstly demonstrate that lower annealing temperature enhances PS-effect. Furthermore, we controlled the average size of complex PCR products over a wide range, and simultaneously amplified targets from 0.25 to 10kb by regulating the degree of suppression in a single-primer PCR. In addition, we describe an improved template-switching full-length cDNA synthesis method that greatly reduces truncated cDNA. This study provides a general guide for the design of PS-effect related PCR and is useful for representative or long-transcript enriched cDNA library construction, especially when only a small amount of total RNA is available.

Base Sequence↗
PubMed2025

CROPseq-multi: a universal solution for multiplexed perturbation in high-content pooled CRISPR screens.

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are limited by technical challenges and do not offer compatibility across diverse screening methodologies, including enrichment, single-cell sequencing, and optical pooled screens. Here, we report the development of CROPseq-multi (CSM), a CROPseq1-inspired lentiviral system to multiplex Streptococcus pyogenes (Sp) Cas9-based perturbations with versatile readout compatibility and high performance for both perturbation and barcode identification. CSM has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. Dual-guide CSM libraries are constructed in a single, facile molecular cloning step that facilitates the use of unique molecular identifiers. CSM is compatible with enrichment screening methodologies, single-cell RNA-sequencing readouts, and optical pooled screens. For optical pooled screens, an optimized and multiplexed in situ detection protocol improves barcode counts 10-fold (for mRNA detection), enables detection of recombination events, and reduces the number of sequencing cycles required for decoding by 3-fold relative to CROPseq. CROPseq-multi-v2 (CSMv2) adds compatibility for detection methods based on T7 RNA polymerase in vitro transcription2-5. CSM provides a single system for CRISPR screens that is compatible with individual and combinatorial perturbations, diverse SpCas9-based perturbation technologies, and multiple high-content, single-cell phenotypic readouts.

CRISPR Cas9↗
PubMed2006

Diagnosis and monitoring of PML-RARalpha-positive acute promyelocytic leukemia by quantitative RT-PCR.

The last 15 yr have produced dramatic improvements in the survival rate of patients with acute promyelocytic leukemia (APL). These improvements have been due mainly to the introduction of targeted therapies and improved methods for diagnosing and monitoring this disease. The underlying molecular lesion in APL involves a t(15:17) translocation which leads to the generation of PML-RARalpha fusion transcripts and proteins. The PML-RARalpha fusion transcripts have been shown to be useful markers for establishing the diagnosis and for monitoring the response to treatment. This manuscript describes the application of QZyme reverse-transcription polymerase chain reaction (RT-PCR) to the quantification of PML-RARalpha transcripts as a marker of APL. QZyme is a method for real time detection and quantification of target genes or transcripts. The principle of QZyme analysis is similar to other quantitative PCR systems; however, the mechanism is quite different. QZyme exploits the catalytic activity of DNAzymes (deoxyribozymes), which are oligonucleotides that can bind and cleave nucleic acid substrates. The approach is well suited to monitoring minimal residual disease (MRD) in patients with APL, as a result of its ability to detect low numbers of transcripts and accurately measure differences in concentration over a broad dynamic range. Further, its capacity for duplex analysis has multiple advantages for analysis of clinical specimens. Protocols for duplex, single-tube QZyme RT-PCR assays, which allow simultaneous quantification of PML-RARalpha fusion transcripts (either L-type and V-type, or S-type) and the internal control BCR transcript, are provided. These protocols can be used for analyzing patient RNA specimens and are suitable for clinical trial monitoring. For this type of work, it is recommended that investigators validate the assays to ensure reproducible, accurate, and specific results on the equipment in their own laboratories. Assay validation is critical for real-time quantitative RT-PCR (RQ-PCR) and is often overlooked. A guide to the steps involved in validation and recommendations for acceptance criteria is included in this chapter.

Chromosomes, Human, Pair 15↗
PubMed2005

The archaeal exosome core is a hexameric ring structure with three catalytic subunits.

The exosome is a 3' --> 5' exoribonuclease complex involved in RNA processing. We report the crystal structure of the RNase PH core complex of the Sulfolobus solfataricus exosome determined at a resolution of 2.8 A. The structure reveals a hexameric ring-like arrangement of three Rrp41-Rrp42 heterodimers, where both subunits adopt the RNase PH fold common to phosphorolytic exoribonucleases. Structure-guided mutagenesis reveals that the activity of the complex resides within the active sites of the Rrp41 subunits, all three of which face the same side of the hexameric structure. The Rrp42 subunit is inactive but contributes to the structuring of the Rrp41 active site. The high sequence similarity of this archaeal exosome to eukaryotic exosomes and its high structural similarity to the bacterial mRNA-degrading PNPase support a common basis for RNA-degrading machineries in all three domains of life.

Amino Acid Sequence↗
PubMed1996

Structural features and stability of an RNA triple helix in solution.

A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one-and two-dimensional NMR spectroscopy and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized by Watson-Crick and Hoogsteen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption data, indicate that the triple helix is highly stable at acidic pH, melting in a single sharp transition centered at 62 degrees C at pH 4.3. The Watson-Crick and Hoogsteen pairings are disrupted simultaneously upon melting. The NMR data are consistent with a structural model where the Watson-Crick paired strands form an A-helix. Results of model building, guided by NMR data, suggest a possible hydrogen bond between the 2' hydroxyl proton of the Hoogsteen strand and a phosphate oxygen of the purine strand. The structural model is discussed in terms of its ability to account for some of the differences in stability reported for RNA and DNA triple helices and provides insight into features that are likely to be important in the design of RNA binding compounds.

Base Composition↗
PubMed2000

Regulation of tissue inhibitor of metalloproteinase-3 (Timp-3) mRNA expression during rat CNS development.

To preserve tissue integrity during the structural rearrangements that occur during central nervous system (CNS) development, an intricate balance between extracellular matrix (ECM) synthesis and degradation must be maintained. The matrix metalloproteinases (MMPs) are believed to be the main mediators of ECM degradation. Because MMPs function in the turnover of a broad-spectrum of ECM proteins their activity is tightly regulated by interaction with tissue inhibitors of metalloproteinases (TIMPs). Whereas the primary function of TIMPs is to inhibit MMP activity, evidence is mounting that TIMPs are multifunctional molecules that exert diverse cell biological functions distinct from their MMP-inhibitory activities. Although the role of MMPs and TIMPs in the morphogenesis of non-neural tissues has been investigated, to date few studies have analyzed MMP or TIMP expression during CNS development. In the present report, we demonstrate the regulation of Timp-3 mRNA expression throughout the course of CNS development. In particular, Timp-3 mRNA is expressed in embryonic ventricular zones and the postnatal subventricular zone (SVZ). In addition, Timp-3 is expressed in the rostral migratory steam (RMS) to the olfactory bulb in a pattern similar to the ECM proteoglycan brevican. These data suggest that TIMP-3 and brevican may act in concert to guide neuronal migration along the RMS.

Animals↗
PubMed1993

Kluyveromyces: systematics since 1970.

The taxonomy of Kluyveromyces has been the object of intense study since van der Walt's (1970) monograph. This is an account of the major developments and the classification to be adopted in the 4th edition of The Yeasts, a Taxonomic Study. The guiding principles that will be followed in eventual revisions of the genus are presented.

DNA, Fungal↗
PubMed1996

Techniques for noninvasive diagnosis of lower respiratory tract infections. Which tests to order, when to consider invasive procedures.

Although sputum culture and Gram's staining have been the traditional methods for determining the cause of lower respiratory tract infections, oropharyngeal contamination and improper sputum collection can limit their usefulness. Nevertheless, these noninvasive techniques remain a rapid means of gathering diagnostic clues. Alternative approaches include acid-fast sputum stains, direct immunofluorescence examination, enzyme immunoassays, DNA probes, and serologic testing. However, for critically ill patients, invasive procedures (such as bronchoscopy and thoracentesis) can provide more definitive diagnoses to guide selection of antimicrobial therapy.

Cytodiagnosis↗
PubMed2004

Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing.

In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing.

Arabidopsis↗
PubMed2004

Ultrastructural characterization of SARS coronavirus.

Severe acute respiratory syndrome (SARS) was first described during a 2002-2003 global outbreak of severe pneumonia associated with human deaths and person-to-person disease transmission. The etiologic agent was initially identified as a coronavirus by thin-section electron microscopic examination of a virus isolate. Virions were spherical, 78 nm in mean diameter, and composed of a helical nucleocapsid within an envelope with surface projections. We show that infection with the SARS-associated coronavirus resulted in distinct ultrastructural features: double-membrane vesicles, nucleocapsid inclusions, and large granular areas of cytoplasm. These three structures and the coronavirus particles were shown to be positive for viral proteins and RNA by using ultrastructural immunogold and in situ hybridization assays. In addition, ultrastructural examination of a bronchiolar lavage specimen from a SARS patient showed numerous coronavirus-infected cells with features similar to those in infected culture cells. Electron microscopic studies were critical in identifying the etiologic agent of the SARS outbreak and in guiding subsequent laboratory and epidemiologic investigations.

Animals↗
PubMed1998

Improving the efficiency of antisense and EGS methods.

Hammerhead ribozymes and stable stem loop structures function as inhibitors of 3'-5'-exonuclease degradation of external guide sequences (EGSs) when covalently linked to the 3'-end of EGS RNAs. This observation may be of use in improving the efficiency of gene inactivation techniques that use single-stranded RNA (e.g., antisense RNA, EGS RNA) in vivo.

Base Sequence↗
PubMed1998

Dynamic competition between alternative structures in viroid RNAs simulated by an RNA folding algorithm.

The folding pathways of viroid RNAs were studied using computer simulations by the genetic algorithm for RNA folding. The folding simulations were performed for PSTVd RNAs of both polarities, using the wild-type sequence and some previously known mutants with suggested changes in the stable or metastable structures. It is shown that metastable multihairpin foldings in the minus strand replicative intermediates are established due to the specific folding pathway that ensures the absence of the most stable rod-like structure. Simulations of the PSTVd minus strand folding during transcription reveal a metastable hairpin, formed in the left terminal domain region of the PSTVd. Despite high sequence variability, this hairpin is conserved in all known large viroids of both subgroups of PSTVd type, and is presumably necessary to guide the folding of the HPII hairpin which is functional in the minus strand. The folding simulations are able to demonstrate the changes in the balance between metastable and stable structures in mutant PSTVd RNAs. The stable rod-like structure of the circular viroid (+) RNA is also folded via a dynamic folding pathway. Furthermore, the simulations show that intermediate steps in the forced evolution of a shortened PSTVd replicon may be reconstructed by a mechanistic model of different folding pathway requirements in plus- and minus-strand RNAs. Thus the formation of viroid RNA structure strongly depends on dynamics of competition between alternative RNA structures. This also suggests that the replication efficiency of viroid sequences may be estimated by a simulation of the folding process.

Algorithms↗
PubMed1994

Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.

The guidance of axons to their targets in the developing nervous system is believed to involve diffusible chemotropic factors secreted by target cells. Floor plate cells at the ventral midline of the spinal cord secrete a diffusible factor or factors that promotes the outgrowth of spinal commissural axons and attracts these axons in vitro. Two membrane-associated proteins isolated from brain, netrin-1 and netrin-2, possess commissural axon outgrowth-promoting activity. We show here that netrin-1 RNA is expressed by floor plate cells, whereas netrin-2 RNA is detected at lower levels in the ventral two-thirds of the spinal cord, but not the floor plate. Heterologous cells expressing recombinant netrin-1 or netrin-2 secrete diffusible forms of the proteins and can attract commissural axons at a distance. These results show that netrin-1 is a chemotropic factor expressed by floor plate cells and suggest that the two netrin proteins guide commissural axons in the developing spinal cord.

Animals↗
PubMed2000

Clinical implications of identifying non-B subtypes of human immunodeficiency virus type 1 infection.

Although human immunodeficiency virus type 1 (HIV-1) infection in the United States has predominantly involved subtype B, increasing global travel is leading to wider dissemination of genetically heterogeneous subtypes. While physicians depend on HIV-1 viral load measurements to guide antiretroviral therapy, commonly used molecular assays may underestimate the viral load of patients with non-B subtypes. Nine patients with non-B subtypes of HIV-1 were identified by physicians who suspected a non-B subtype on the basis of a low or undetectable HIV-1 viral load, by the Amplicor HIV-1 Monitor test, version 1.0, in conjunction with either a declining CD4 cell count or history of travel outside the United States. Use of version 1.5 of the Amplicor HIV-1 Monitor test detected a median HIV-1 viral load that was 2.0 log(10) RNA copies/mL higher than was determined with version 1.0. Clinical management was altered in all cases after diagnosis of a non-B-subtype infection. These cases demonstrate that it is critical for physicians to suspect and diagnose non-B subtypes of HIV-1 so that an assay with reliable subtype performance can be used to guide antiretroviral therapy.

Adult↗