Performance of the automated COBAS AMPLICOR™ system for the detection of hepatitis C virus RNA

Background: The COBAS AMPLICOR^™ (CA) instrument for the amplification and detection steps of the AMPLICOR^™ molecular diagnostic assays has recently been introduced. The system contains a single thermal cycler with two independently controlled heating/cooling blocks, a pipettor, a magnetic particle washer, a photometer and an incubator.Objective: The performance of the CA instrument was evaluated in a routine diagnostic laboratory for the detection of hepatitis C virus (HCV) RNA. The new system was compared with the corresponding microwell plate assay (AMPLICOR HCV Test).Study design: Routine clinical sera (350) from hemodialysis patients and patients with chronic HCV infection and interferon therapy were studied. If discrepant results were obtained, both assays were repeated (specimen preparation, amplification and detection); in addition, the HCV copy number was determined with the AMPLICOR HCV MONITOR^™ Test.Results: There was a correlation between the CA HCV Test and the AMPLICOR HCV Test in 341 of 350 specimens (97%). After resolution of 9 discrepant results, the CA HCV Test gave a sensitivity of 97.8% and a specificity of 99.4%. The most common reason for discrepant results was a low HCV RNA copy number.Conclusion: The CA system was found to be a labor-saving, fast and reliable instrument for the amplification and detection steps of a RT-PCR molecular assay for detection of HCV RNA.     

Ultrasound detection in fish--a parallel to the sonar-mediated detection of bats by ultrasound-sensitive insects?

Most bats use ultrasonic sonar signals, or cries, to locate prey. Many of their insect prey species have evolved an ability to hear and respond to these signals, and studies clearly demonstrate the survival value associated with this ability. Like bats, toothed whales locate prey by emitting ultrasonic sonar signals, or clicks. As a parallel to the insect prey of bats, it would seem obvious to assume that some fish species likewise are capable of sensing the ultrasonic clicks of their odontocete predators. As judged from classical fish audiometry literature, this seems not to be the case, however, and although in recent years some fishes have been proven responsive to ultrasound, examination of ecological and acoustic differences reveals that conclusions on ultrasound-mediated insect escape behavior are not immediately applicable to fish. This has the consequence that future experiments on fish ultrasound detection should not be looking for observations directly parallel to those observed in the bat-insect interactions.     

Immunological detection of m- and μ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca²⁺-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15^th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.Key words: m-calpain, µ-calpain, skeletal muscle, Marchigiana cattle, immunohistochemistry, Electron Microscopy.     

Immunohistochemical detection of cathepsin D, K, and L in the process of endochondral ossification in the human

Cathepsins D, K, and L were immunolocalized in tissue undergoing endochondral ossification in the human. Cathepsins D, K, and L were localized in osteoclasts and chondroclasts attached to bone matrix and cartilage matrix, respectively. Cathepsins D and L were immunostained in chondrocytes. Immunolocalization of cathepsin D was limited to hypertrophic chondrocytes adjacent to the osteochondral junction. In contrast, cathepsin L was immunolocalized in both proliferating and hypertrophic chondrocytes. In the bone marrow space, cathepsins D, K, and L were localized in multinucleated cells. Cathepsin D was diffusely detected in mononuclear bone marrow cells which were negative for cathepsins K and L. The present findings indicated that cathepsins K, D, and L were associated with the process of endochondral ossification in the human, and suggested that these cathepsins share roles in bone and cartilage turnover in the human.     

The Advantage of Sex in the RNA Virus φ6

When laboratory populations of the RNA bacteriophage 6 are subjected to intensified genetic drift, they experience a decline in fitness. These experiments demonstrate that the average effect of mutations is deleterious, and they are used to suggest that Muller's ratchet can operate in these viruses. However, the operation of Muller's ratchet does not alone guarantee an advantage of sex. When 6 populations were subjected to a series of bottlenecks of one individual and then crossed, the measured advantage of sex was not significant. To determine whether a small sample size, as opposed to allelism or another explanation, can account for the negative result, we repeated the 6 experiments by crossing a larger set of populations. We found that bottlenecked populations of 6 could recover fitness through mutations. However, hybrids produced by crossing the populations recovered an additional amount over the contribution of mutations. This additional amount, which represents an advantage of sex to 6, was determined to be significantly greater than zero. These results provide indirect support for an advantage of sex through Muller's ratchet. However, we also use our experimental design and results to propose an alternative to Muller's ratchet as a model for the evolution of sex.     

Enhanced NMR signal detection of imino protons in RNA molecules containing 3′ dangling nucleotides

We present a method for improving the quality of nuclear magnetic resonance (NMR) spectra involving exchangeable protons near the base of the stem of RNA hairpin molecules. NMR spectra of five different RNA hairpins were compared. These hairpins consisted of a native RNA structure and four molecules each having different unpaired, or dangling, nucleotides at the 3′ end. NMR experiments were acquired in water for each construct and the quality of the imino proton spectral regions were examined. The imino resonances near the base of the stem of the wild type RNA structure were not observed due to breathing motions. However, a significant increase in spectral quality for molecules with dangling 3′ adenosine or guanosine nucleotides was observed, with imino protons detected in these constructs that were not observed in the wild type construct. A modest improvement in spectral quality was seen for the construct with a 3′ unpaired uridine, whereas no significant improvement was observed for a 3′ unpaired cytidine. This improvement in NMR spectral quality mirrors the increased thermodynamic stability observed for 3′ unpaired nucleotides which is dependant on the stacking interactions of these nucleotides against the base of the stem. The use of a dangling 3′ adenosine nucleotide represents an easy method to significantly improve the quality of NMR spectra of RNA molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Performance of individual Helicobacter pylori antigens in the immunoblot-based detection of H. pylori infection

To develop a specific line blot (LB) for supporting ELISA-based serodiagnosis of Helicobacter pylori infection, individual native/recombinant H. pylori antigens were evaluated with respect to their reactivity with both serum IgG and IgA from 156 dyspeptic screening patients (67% H. pylori positive). Of 13 antigens, HP0175, p17, and p19 revealed highest positive likelihood ratios for H. pylori-specific IgG (> 5.0) and were selected as LB substrates, in addition to the established virulence markers VacA and CagA. For validation, the LB was compared to a commercial whole-cell-lysate-based ELISA by parallel (re-)analysis of 156 screening sera, 22 sera from diabetes mellitus patients and 15 sera from follow-up patients after H. pylori eradication. In screening patients, the combined use of IgG ELISA and LB revealed a sensitivity, specificity, and accuracy of 94%, 81%, and 90%, respectively, whereas IgG ELISA alone exhibited a low specificity of 75%. In diabetic and follow-up patients, IgA ELISA exhibited high accuracy of 89% and 93%, respectively, whereas IgG detection was unreliable (accuracy < 80%). In conclusion, using HP0175, p17, p19, CagA, and VacA as LB substrates significantly improves the specificity of anti-H. pylori IgG analysis, providing a reliable tool for (1) confirmation/refutation of ELISA-based screening results and (2) assessment of the CagA/VacA status.     

Sequence-specific cleavage of the RNA strand in DNA–RNA hybrids by the fusion of ribonuclease H with a zinc finger

Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA–RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA–RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA–RNA hybrids. The optimization of the fusion enzyme’s specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.     

Preferential spreading of RNA silencing into the 3’ downstream region of the transgene in tobacco

The amplification mechanism of short interfering RNAs (siRNAs) along the transgene sequence exists in RNA interference (RNAi). The RNA-dependent RNA polymerase synthesizes complementary RNAs by using the transgene mRNA as a template, and the secondary siRNAs are generated from the outside of primary RNAi target. Four independent RNAi vectors which produced primary siRNAs against distinct regions of the tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3) were transiently expressed in leaves of theNtF4D3-overexpressed transgenic plants. Regardless of the RNAi vector used, the secondaryNtFAD3 siRNAs were generated preferentially from the 3’ downstream region of the transgene. Secondary siRNAs from the 5’ upstream region adjacent to the annealing site of primary siRNAs accumulated under the detection level. Our results suggest that different regulatory mechanisms are involved in the spreading of RNA silencing into 5’ upstream and 3’ downstream regions of the target sequence, respectively.     

Role of the CCA bulge of prohead RNA of bacteriophage ø29 in DNA packaging

The oligomeric ring of prohead RNA (pRNA) is an essential component of the ATP-driven DNA packaging motor of bacteriophage ?29. The A-helix of pRNA binds the DNA translocating ATPase gp16 (gene product 16) and the CCA bulge in this helix is essential for DNA packaging in vitro. Mutation of the bulge by base substitution or deletion showed that the size of the bulge, rather than its sequence, is primary in DNA packaging activity. Proheads reconstituted with CCA bulge mutant pRNAs bound the packaging ATPase gp16 and the packaging substrate DNA-gp3, although DNA translocation was not detected with several mutants. Prohead/bulge-mutant pRNA complexes with low packaging activity had a higher rate of ATP hydrolysis per base pair of DNA packaged than proheads with wild-type pRNA. Cryoelectron microscopy three-dimensional reconstruction of proheads reconstituted with a CCA deletion pRNA showed that the protruding pRNA spokes of the motor occupy a different position relative to the head when compared to particles with wild-type pRNA. Therefore, the CCA bulge seems to dictate the orientation of the pRNA spokes. The conformational changes observed for this mutant pRNA may affect gp16 conformation and/or subsequent ATPase-DNA interaction and, consequently, explain the decreased packaging activity observed for CCA mutants.     

PCR detection of two RFLPs in exon I of the α-L-iduronidase (IDUA) gene

Two polymorphisms were detected within exon I of the a-l-iduronidase (IDUA) gene both of which create restriction endonuclease sites and one of which changes an amino acid. The polymorphisms may be detected by digesting the same 245-bp polymerase chain reaction product. The polymorphisms can be used diagnostically in families with IDUA deficiency (mucopolysaccharidosis type I) and Huntington disease, which is closely linked to the IDUA locus.     

Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.     

Radioimmunoscintigraphic approach for the in vivo detection of tuberculomas—A preliminary study in a rabbit model

Radioimmunoscintigraphic approach could provide a viable non-invasive alternative for the diagnosis of deeply situated tuberculomas. We have evaluated this in a rabbit model constructed to give a characteristic localized tubercular lesion, with complimentary morphological, histological and antigenic profiles. ¹³¹I-anti Myobacterium bovis (BCG) antibody was shown to localize at the lesion, where as ¹³¹I-bovine serum albumin and ^99mTc-red blood cell scans were negative. The clearance of intradermally-injected tuberculous antigen could be traced into ascending lymph nodes using ¹³¹I-anti M. bovis (BCG) antibody.     

Redefinition of the coding sequence of the MXI1 gene and identification of a polymorphic repeat in the 3′ non-coding region that allows the detection of loss of heterozygosity of chromosome 10q25 in glioblastomas

The MXI1 gene encodes a protein interacting with Max, a regulatory factor of the Myc oncogene, and is located on chromosome 10q25, a region showing frequent loss of heterozygosity in malignant gliomas. We have reassessed the coding sequence of MXI1 and found that, at the 3 end, the open reading frame is 28 codons shorter than previously described. We have also found an AAAAC polymorphic repeat (two alleles, 45% heterozygosity) in the 3 non-coding region of the gene. Six anaplastic astrocytomas and nine glioblastomas, the most malignant form of glioma, were informative for this polymorphism. Loss of heterozygosity was demonstrated in all glioblastomas, but not in the remaining tumors.