Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs

The detection of replicative intermediate RNAs as markers of active replication of RNA viruses is an essential tool to investigate pathogenesis in acute viral infections, as well as in their long-term sequelae. In this regard, strand-specific PCR has been used widely to distinguish (-) and (+) enteroviral RNAs in pathogenesis studies of diseases such as dilated cardiomyopathy. It has been generally assumed that oligonucleotide-primed reverse transcription of a given RNA generates only the corresponding specific cDNA, thus assuring the specificity of a PCR product amplified from it. Nevertheless, such assumed strand-specificity is a fallacy, because falsely primed cDNAs can be produced by RNA reverse transcription in the absence of exogenously added primers, (cDNA(primer)(-)), and such falsely primed cDNAs are amplifiable by PCR in the same way as the correctly primed cDNAs. Using as a prototype the coxsackievirus B5 (CVB5), a (+) strand RNA virus, it was shown that cDNA(primer)(-) renders the differential detection of viral (-) and (+) RNAs by conventional PCR virtually impossible, due to gross non-specificity. Using in vitro transcribed CVB5 RNAs (+) and (-), it was shown that cDNA(primer)(-) could be removed effectively by magnetic physical separation of correctly primed biotinylated cDNA. Such strategy enabled truly strand-specific detection of RNA (-) and (+), not only for CVB5, but also for other non-polio enteroviruses. These findings indicate that previous conclusions supporting a role for the persistence of actively replicating enterovirus in the pathogenesis of chronic myocarditis should be regarded with strong skepticism and purification of correctly primed cDNA should be used for strand-specific PCR of viral RNA in order to obtain reliable information on this important subject. (C) 2009 Elsevier B.V. All rights reserved.

Kallmann syndrome and mirror movements: White matter quantitative evaluation with magnetic resonance imaging

Kallmann syndrome (KS), characterized by the association of hypogonadotropic hypogonadism and anosmia, may present many other phenotypic abnormalities, including neurologic features as involuntary movements, called mirror movements (MM). MM etiology probably involves a complex mechanism comprising corticospinal tract abnormal development associated with deficient contralateral motor cortex inhibitory system. In this study, in order to address previous hypotheses concerning MM etiology, we identified and quantified white matter (WM) alterations in 21 KS patients, comparing subjects with and without MM and 16 control subjects, using magnetization transfer ratio (MTR) and T2 relaxometry (R2). Magnetization transfer and 12 double-echo images were acquired in a 1.5 T system. MTR and R2 were calculated pixel by pixel to initially create individual maps, and then, group average maps, co-registered with MNI305 stereotaxic coordinate system. After analysis of selected regions of interest, we demonstrated areas with higher 12 relaxation time and lower MTR values in KS patients, with and without MM, differently involving corticospinal tract projection, frontal lobes and corpus callosum. Higher MTR was observed only in pyramidal decussation when compared in both groups of patients with controls. In conclusion, we demonstrated that patients with KS have altered WM areas, presenting in a different manner in patients with and without MM. These data suggest axonal loss or disorganization involving abnormal pyramidal tracts and other associative/connective areas, relating to the presence or absence of MM. We also found a different pattern of alteration in pyramidal decussation, which can represent the primary area of neuronal disarrangement. (C) 2010 Elsevier B.V. All rights reserved.

Magnetic Resonance Imaging Assessment of Subchondral Bone and Soft Tissues in Knee Osteoarthritis

Knee osteoarthritis (OA) has to be considered a whole joint disease. Magnetic resonance imaging (MRI) allows superior assessment of all joint tissues that may be involved in OA, such as the subchondral bone, synovium, ligaments, and periarticular soft tissues. Reliable MRI-based scoring systems are available to assess and quantify these structures and associated pathology. Cross-sectional and longitudinal evaluation has enabled practitioners to understand their relevance in explaining pain and structural progression.

Lower Extremities Magnetic Resonance Angiography With Blood Pressure Cuff Compression: Quantitative Dynamic Analysis

Purpose: To quantitatively evaluate changes induced by the application of a femoral blood-pressure cuff (BPC) on run-off magnetic resonance angiography (MRA). which is a method generally previously proposed to reduce venous contamination in the leg. Materials and Methods: This study was Health Insurance Portability and Accountability Act (HIPAA)- and Institutional Review Board (IRB)-compliant, We used time-resolved gradient-echo gadolinium (Gd)-enhanced MRA to measure BPC effects on arterial, venous, and soft-tissue enhancement. Seven healthy volunteers (six men) were studied with the BPC applied at the mid-femoral level unilaterally using a 1.5T MR system after intravenous injection of Gd-BOPTA. Different statistical tools were used such as the Wilcoxon signed rank test and a cubic smoothing spline fit. Results: We found that BPC application induces delayed venous filling (as previously described), but also induces significant decreases in arterial inflow, arterial enhancement, vascular-soft tissue contrast, and delayed peak enhancement (which have not been previously measured). Conclusion: The potential benefits from using a BPC for run-off MRA must be balanced against the potential pitfalls, elucidated by our findings.

Magnetic Resonance Imaging in Knee Osteoarthritis Research: Semiquantitative and Compositional Assessment

Semiquantitative assessment of the knee by expert magnetic resonance imaging readers is a powerful research tool for understanding the natural history of osteoarthritis (OA). Several reliable semiquantitative scoring systems have been applied to large observational cross-sectional and longitudinal epidemiologic studies and interventional clinical trials. Such evaluations have enabled understanding of the relevance of disease in structures within the knee joint to explain pain and progression of OA. Compositional imaging of cartilage has added to our ability to detect early degeneration before morphologic changes are present, which may help to prevent the permanent morphologic changes commonly seen in knee OA.

Magnetic Resonance Angiography in Extracranial Giant Cell Arteritis

Background: Noninvasive diagnosis of giant cell arteritis (GCA) remains challenging, particularly with regard to evaluation of extracranial arterial disease. Objectives: The objective of the study was to retrospectively review extracranial involvement in patients with GCA and/or polymyalgia rheumatica (PMR), evaluated with magnetic resonance imaging (MRI), especially 3-dimensional contrast-enhanced magnetic resonance angiography images of the aortic arch and its branches. Methods: Clinical information, biopsy status, and MRI examinations of 28 patients with GCA/PMR were reviewed. Patient images were mixed randomly with 20 normal control images and were independently reviewed by 2 radiologists. Interobserver agreement for detection of arterial stenosis was determined by the k coefficient. Results: Both readers described vascular alterations in keeping with extracranial GCA in 19 of 28 patients (67%) with good interobserver agreement (k = 0.73) and with even higher agreement on diagnosing nonocclusive versus occlusive disease (k = 1.00). The most common lesions were bilateral axillary stenosis or obstructions, observed by both readers in 8 patients (28%). Among the 19 patients with magnetic resonance angiography lesions in the subclavian/axillary arteries, 12 (75%) had biopsy-proven GCA, but only 5 (41%) of these patients had clinical features of large artery disease. Conclusions: In our series review, MRI could provide accurate information on involvement of the aortic arch and its branches in extracranial GCA, depicting different degrees of stenosis. Our analysis also illustrates that occult large artery vasculitis should be considered in patients without biopsy-proven GCA, patients with classic GCA but without clinical signs of large artery disease, and in patients initially diagnosed as having PMR.

Magnetic resonance imaging quantification of regional cerebral blood flow and cerebrovascular reactivity to carbon dioxide in normotensive and hypertensive rats

Hypertension afflicts 25% of the general population and over 50% of the elderly. In the present work, arterial spin labeling MRI was used to non-invasively quantify regional cerebral blood flow (CBE), cerebrovascular resistance and CO(2) reactivity in spontaneously hypertensive rats (SHR) and in normotensive Wistar Kyoto rats (WKY), at two different ages (3 months and 10 months) and under the effects of two anesthetics, alpha-chloralose and 2% isoflurane (1.5 MAC). Repeated CBE measurements were highly consistent, differing by less than 10% and 18% within and across animals, respectively. Under alpha-chloralose, whole brain CBE at normocapnia did not differ between groups (young WKY: 61 3 ml/100 g/min; adult WKY: 62 +/- 4 ml/100 g/min; young SHR: 70 +/- 9 ml/100 g/min: adult SHR: 69 8 ml/100 g/min), indicating normal cerebral autoregulation in SHR. At hypercapnia, CBE values increased significantly, and a linear relationship between CBE and PaCO(2) levels was observed. In contrast, 2% isoflurane impaired cerebral autoregulation. Whole brain CBE in SHR was significantly higher than in WKY rats at normocapnia (young SHR: 139 +/- 25 ml/100 g/min; adult SHR: 104 +/- 23 ml/100 g/min; young WKY: 55 +/- 9 ml/100 g/min; adult WKY: 71 +/- 19 ml/100 g/min). CBE values increased significantly with increasing CO(2): however, there was a clear saturation of CBF at PaCO(2) levels greater than 70 mm Hg in both young and adult rats, regardless of absolute CBE values, suggesting that isoflurane interferes with the vasoclilatory mechanisms of CO(2). This behavior was observed for both cortical and subcortical structures. Under either anesthetic, CO(2) reactivity values in adult SHR were decreased, confirming that hypertension, when combined with age, increases cerebrovascular resistance and reduces cerebrovascular compliance. Published by Elsevier Inc.

Magnetic retention and bar-clip attachment for implant-retained auricular prostheses: A comparative analysis

Retention systems for implant-retained auricular prostheses using either bar-clip attachments with 2 or 3 clips or retention with 2 or 3 magnets were wear tested (insertion and removal cycles) to simulate clinical periods of use. Measurements were taken at intervals of 540 cycles, which represents a period of use of approximately 6 months, for up to 3,240 cycles. Assessments of retentive force were carried out before, during, and after the wear test. Statistical analysis indicated that the bar-clip systems provided higher retention than the magnetic systems. The bar-clip attachment with 2 clips showed a significant loss of retentive force after wear testing (P < .05), suggesting lower durability and shorter clinical life. The retention provided by the bar-clip attachment with 3 clips remained stronger than that provided by all other systems tested. At the end of the wear test, the magnetic systems showed very little loss of retention but were still less retentive than the bar-clip systems, suggesting higher durability under clinical simulation despite the lower retention initially provided.

Cyclic Fatigue Resistance of Rotary Nickel-Titanium Instruments Submitted to Nitrogen Ion Implantation

Introduction: The aim of this study was to assess cyclic fatigue resistance in rotary nickel-titanium instruments submitted to nitrogen ion implantation by using a custom-made cyclic fatigue testing apparatus. Methods: Thirty K3 files, size #25, taper 0.04, were divided into 3 experimental groups as follows: group A, 12 files exposed to nitrogen ion implantation at a dose of 2.5 x 10(17) ions/cm(2), accelerating voltage of 200 kV, currents of 1 mu A/cm(2), 130 degrees C temperature, and vacuum conditions of 10 x 10(-6) torr for 6 hours; group B, 12 nonimplanted files; and group C, 6 files submitted to thermal annealing for 6 hours at 130 degrees C. One extra file was used for process control. All files were submitted to a cyclic fatigue test that was performed with an apparatus that allowed the instruments to rotate freely, simulating rotary instrumentation of a curved canal (40-degree, 5-mm radius curve). An electric motor handpiece was used with a contra-angle of 16:1 at an operating speed of 300 rpm and a torque of 2 N-cm. Time to failure was recorded with a stopwatch in seconds and subsequently converted to number of cycles to fracture. Data were analyzed with the Student t test (P < .05). Results: Ion-implanted instruments reached significantly higher cycle numbers before fracture (mean, 510 cycles) when compared with annealed (mean, 428 cycles) and nonimplanted files (mean, 381 cycles). Conclusions: Our results showed that nitrogen ion implantation improves cyclic fatigue resistance in rotary nickel-titanium instruments. Industrial implementation. of this surface modification technique would produce rotary nickel-titanium instruments with a longer working life. (J Endod 2010;36:1183-1186)

Analysis of Apical Root Transportation Associated with Protaper Universal F3 and F4 Instruments by Using Digital Subtraction Radiography

Introduction: The aim of this study was to assess the occurrence of apical root transportation after the use of Pro Taper Universal rotary files sizes 3 (F3) and 4 (F4). Methods: Instruments were worked to the apex of the original canal, always by the same operator. Digital subtraction radiography images were produced in buccolingual and mesiodistal projections. A total of 25 radiographs were taken from root canals of human maxillary first molars with curvatures varying from 23-31 degrees. Quantitative data were analyzed by intraclass correlation coefficient and Wilcoxon nonparametric test (P = .05). Results: Buccolingual images revealed a significantly higher degree of apical transportation associated with F4 instruments when compared with F3 instruments in relation to the original canal (Wilcoxon test, P = .007). No significant difference was observed in mesiodistal images (P = .492). Conclusions: F3 instruments should be used with care in curved canals, and F4 instruments should be avoided in apical third preparation of curved canals. (J Endod 2010;36:1052-1055)

Bacterial Adhesion on Smooth and Rough Titanium Surfaces After Treatment With Different Instruments

Background: Newly formed biofilm after implant debridement may challenge the long-term stability of peri-implant therapy. This in vitro study aimed to assess the roughness and adherence of Streptococcus sanguinis after treatment of smooth and rough titanium surfaces with an erbium-doped: yttrium, aluminum, and garnet (Er:YAG) laser, metal and plastic curets, and an air-powder abrasive system. Methods: Forty titanium disks with smooth-machined surfaces and 40 with sand-blasted and acid-etched surfaces were divided into the following treatment groups: Er:YAG laser; plastic curet; metal curet, and air-powder abrasive system. The surface roughness (roughness average [Raj) before and after treatments was determined using a profilometer. S. sanguinis (American Type Culture Collection 10556) was grown on treated and untreated specimens, and the amounts of retained bacteria on the surfaces were measured by the culture method. Rough and smooth surfaces with and without a suspension of S. sanguinis were also analyzed using scanning electron microscopy (SEM). Results: For smooth surfaces, the roughest surfaces were produced by metal curets (repeated - measures analysis of variance [ANOVA] and Tukey test; P<0.05). The rough-surface profile was not altered by any of the treatments (repeated-measures ANOVA; P>0.05). Rough surfaces treated with metal curets and air-powder abrasion showed the lowest level of bacteria] adhesion (two-way ANOVA and Tukey test; P<0.05). SEM analysis revealed distinct surface profiles produced by all devices. Conclusions: Metal curets are not recommended for smooth titanium surface debridement due to severe texture alteration. Rough surfaces treated with a metal curet and the air-powder abrasive system were less susceptible to bacterial adhesion, probably due to texture modification and the presence of abrasive deposits. J Periodontol 2009;80: 1824-1832.

Mast cell concentration in the wound healing process of incisions made by different instruments

The aim of this study was to compare the concentration of mast cells (MCs) in the healing process of incisions. Thirty rats were submitted to six linear incisions each, performed in the dorsal skin by carbon dioxide (CO(2)) and diode lasers, electrocautery and conventional scalpel. The animals were euthanized at intervals of 0 h, 24 h, 48 h, 72 h, 7 days and 14 days after the incisions had been made. Histological sections were obtained and stained with toluidine blue for identification of MCs, which were manually counted by conventional microscopy in 20 microscopic fields in the border of the incision, near the granulation tissue, or in the area of new collagen formation, depending on intervals. The concentration of MCs was significantly higher in the wounds made by scalpel than in those made by other techniques at 48 h and 72 h. After 72 h the number of MCs was also significantly higher after electrocautery than after incisions made by 4 W CO(2) laser. On days 7 and 14, there was no significant difference in the MC count among the different types of incisions. In summary, the MC concentration varied after different surgical incisions at early phases of wound healing. At the end of the healing process, however, there were similar MC concentrations around the incisions, suggesting that, in standard incisions in the surgical techniques studied, the wound healing process ultimately occurred in a similar pattern.

Evaluation of the lateral pterygoid muscle using magnetic resonance imaging

Objectives: The aims of this study were to evaluate the visibility of the lateral pterygoid muscle (LPM) in temporomandibular joint (TMJ) images obtained by MRI, using different projections and to compare image findings with clinical symptoms of patients with and without temporomandibular disorders (TMD). Methods: In this study, LPM images of 50 participants with and without TMDs were investigated by MRI. The images of the LPM in different projections of 100 TMJs from 35 participants (70 TMJs) with and 15 participants (30 TMJs) without clinical signs and symptoms of TMD were visible and analysed. Results: The oblique sagittal and axial images of the TMJ clearly showed the LPM. Hypertrophy (1.45%), atrophy (2.85%) and contracture (2.85%) were the abnormalities found in the LPM. TMD signs, such as hypermobility (11.4%), hypomobility (12.9%) and disc displacement (20.0%), could be seen in TMJ images. Related clinical symptoms, such as pain (71.4%), articular sounds (30.4%), bruxism (25.7%) and headache (22.9%), were observed. Conclusions: Patients with TMD can present with alterations in the LPM thickness. Patients without TMD also showed alterations, such as atrophy and contracture, in TMJ images. Recognition of alterations in the LPM will improve our understanding of clinical symptoms and pathophysiology of TMD, and may lead to a more specific diagnosis of these disorders. Dentomaxillofacial Radiology (2010) 39, 494-500. doi: 10.1259/dmfr/80928433