Early-lactation extended pirlimycin therapy against naturally acquired Staphylococcus aureus intramammary infections in heifers: A randomized controlled trial.

The primary objective of the current study was to evaluate cure rate following an early-lactation extended intramammary pirlimycin treatment on heifers naturally infected by Staphylococcus aureus. The secondary objective was to assess Petrifilm Staph Express (3M Microbiology, St. Paul, MN) count plate characteristics when used in a protocol for early-lactation detection of infected quarters in heifers. Milk samples were collected from heifers (n = 946) in the first few days following calving (mean = 5 d). Heifers with laboratory-confirmed S. aureus intramammary infection (n = 72) were randomly allocated into 2 groups. The treatment group (n = 54 quarters from 38 heifers) received an intramammary infusion of 50 mg of pirlimycin once per day for 8 consecutive days in infected quarters. The control group (n = 44 quarters from 34 heifers) did not receive any treatment. Treatment success was defined as having negative culture results for S. aureus in all 3 post-treatment quarter milk samples collected on d 17, 24, and 31 post-treatment. Treatment group mammary quarters showed a statistically significant higher cure rate (64.8%) compared with the control group (34.1%). A total of 38% of quarters identified as S. aureus-positive using the Petrifilm Staph Express count plate were in fact identified as non-aureus staphylococci on routine laboratory-based bacteriological culture. The current study demonstrates that a higher cure rate for S. aureus IMI can be achieved in dairy heifers if an extended treatment protocol is put in place soon after calving. Use of Petrifilm Staph Express count plate for identification of S. aureus-infected heifers could lead to unnecessary treatments because of false-positive results.

Improvement in weight loss and ambulation outcomes after gastric sleeve surgery for a person with chronic motor-incomplete tetraplegia: clinical case report.

OBJECTIVE: To measure body mass index (BMI) and ambulation changes for a morbidly obese, 47-year-old man with chronic motor-incomplete tetraplegia after gastric sleeve surgery. DESIGN/METHOD: A morbidly obese man, BMI=44 kg m(-)(2), with chronic C5 AIS D tetraplegia underwent elective gastric sleeve surgery. Assessment of BMI and function via the 6-minute walk test (6MWT), 10-meter walk test (10MWT) and ambulation parameters (CIR Systems/GAITRite, Franklin, NJ, USA) was performed preoperatively and at 12, 24, 36 and 52 weeks postoperatively, and additionally after 3 weeks of both a prescribed coached (3 × /week facility based) and a non-coached (3 × /week home based) walking program initiated at 52 weeks. A step activity monitor assessed daily ambulation preoperatively, prior to and during the third and sixth week of the walking program. RESULTS: Results included a 34.3% peak BMI decrease at 52 weeks post surgery and a peak increase in 6MWT distance of 58% at 52 weeks post surgery, 10MWT preferred speed of 56% at 55 weeks and step activity monitor of 82% at 58 weeks post surgery. At 58 weeks, gait data demonstrated a decrease in double limb stance of 38% and decrease in base of support of 72%. CONCLUSION/CLINICAL RELEVANCE: This empirical case assessment of BMI and functional mobility before and after gastric sleeve surgery may encourage further investigation into mobility and general health effects post gastric procedures for people with chronic motor-incomplete spinal cord injury.

PCR-free blood group genotyping using a nanobiosensor.

BACKGROUND AND OBJECTIVES: The last two decades have seen major developments in genotyping assays to facilitate the procurement of red blood cell units to alloimmunized patients. To make genotyping faster, simpler and less costly, a nanotechnology approach based on metal/silica fluorescent nanoparticles and a polymer-based hybridization optical transducer was designed. The objectives of this study were (1) to verify whether this nanobiosensor has the ability to discriminate single nucleotide polymorphisms in non-amplified genomic DNA and (2) to establish whether the signal generated by the nanobiosensor is sufficiently intense to be detected by standard flow cytometry. MATERIALS AND METHODS: Silver-core silica-shell fluorescent nanoparticles (Ag@SiO2) were prepared, and amine-modified DNA probes were grafted to their surface. A cationic conjugated polymer was electrostatically bound to the surface probes to become optically active upon hybridization with a target. Two nanobiosensor formulations specific to DO*01 and DO*02 alleles were prepared. DNA was extracted from whole blood and mixed with the nanobiosensor for hybridization. The nanobiosensor fluorescence was measured by flow cytometry. RESULTS: Nine volunteers were typed for Dombrock blood group antigens DO*01 and DO*02. A statistically significant increase in the optical transduction signal was observed for sequence-specific samples. All nine genotypes were correctly identified when compared to standardized PCR assays. CONCLUSION: The nanobiosensor provides rapid and simple genotyping of blood group antigens from unamplified genomic DNA and can be measured using standard flow cytometers. This PCR-free approach could be applied to any known genetic polymorphism.

Red blood cell antigen portrait of self-identified black donors in Quebec.

OBJECTIVES: The goal of this study was to establish a red blood cell antigen portrait of self-identified Black donors for the province of Quebec, Canada. BACKGROUND: The demand for extensively phenotyped red blood cells is on the rise. A good example is the sickle cell patient cohort. To better answer their transfusion needs, Héma-Québec put forward great efforts to increase the recruitment of donors among cultural communities. MATERIALS AND METHODS: In October 2009, an optional question was added on the record of donation to indicate the donor's ethnicity. Self-identified Black donors were extensively phenotyped by the Immunohematology Laboratory, whereas the Research and Development team genotyped red blood cell antigens to complete the picture. RESULTS: Approximately 1500 self-identified Black donors have donated blood at least once since the beginning of the programme. Genotyping results predicted rare phenotypes: 18 S-s- (3 U-, 15 U+(w) ), 15 Js(a+b-), 5 Hy-, 3 Jo(a-), 34 hr(B) +(w) /- and 15 hr(B)-. CONCLUSION: These Black donors, with or without a rare phenotype, are precious to the patient cohort depending on blood transfusions and to our organisation as the blood provider for the whole province of Quebec.

A new SEMA7A variant found in Native Americans with alloantibody.

BACKGROUND AND OBJECTIVES: John Milton Hagen (JMH) antigens are carried by Semaphorin 7A that plays important roles in the nervous system and the immune responses. Its role on the erythrocytes is unclear. Over the years, few samples were referred to our Immunohaematology Reference Laboratory to elucidate their JMH status. MATERIALS AND METHODS: Seven blood samples with antibodies compatible with JMH1-negative red cells were studied at the molecular level to identify polymorphisms and explain the JMH diversity observed. Four samples were of Native American background and three were Caucasians. Molecular analyses of the SEMA7A were undertaken, and soluble form of recombinant Sema7A proteins was produced to characterize the antibodies. RESULTS: Sequencing of the cDNA showed a polymorphism in SEMA7A exon 9 at position 1040 (G>T) in the four Native American samples. Caucasians had a normal sequence. This polymorphism precludes a change at position 347 where an Arg is replaced by a Leu. Plasma was assayed in ELISA on wild-type Sema7AArg347 and variant Sema7ALeu347 proteins. Results clearly indicated a specific recognition of the antibody produced by the Native Americans for the wild-type Sema7AArg347 protein and not the variant one. CONCLUSION: A new SEMA7A variant was identified in this study. The antibody present in the Native American plasma samples should be considered as an alloantibody because it recognizes the wild-type protein.

[Genotyping of 21,000 blood donors in Quebec and RHD analysis]. / Génotypage de 21 000 donneurs de sang au Québec et analyse du RHD.

STUDY GOAL: A repeat blood donor genotyping project was launched by Héma-Québec in October 2007. The objective was to screen 21,000 samples for 22 polymorphisms for red blood cell and platelet blood groups to build a database to easily find compatible donors. MATERIALS AND METHODS: Donors who have donated at least three times during the last year were selected. A drop of blood was spotted on FTA paper and sent to the Pharmacogenomic Centre at the Montreal Heart Institute for analysis. All genotype results were compared to the known phenotype. In parallel, the RHD gene of D negative blood donors was examined. RESULTS: Less than two years were necessary to complete the database. The genotype/phenotype concordance was 99.6% with only 165 discrepancies observed and further analysed. More than 55% of these discrepancies confirmed the initial genotype. The RHD study done on D negative samples found 13 donors positive for a variant RHD gene. Four were RHD*Ψ positive, while the other nine presented variant polymorphisms precluding a reduced expression of the D antigen. CONCLUSION: Thanks to this project, Héma-Québec is able to answer increasing demands for compatible blood more rapidly. The organisation has also demonstrated the security of its D negative inventory.

Set-up and routine use of a database of 10,555 genotyped blood donors to facilitate the screening of compatible blood components for alloimmunized patients.

BACKGROUND AND OBJECTIVES: Large-scale genotyping of blood donors for red blood cell and platelet antigens has been predicted to replace phenotyping assays in the screening of compatible blood components for alloimmunized patients. Although several genotyping platforms have been described, novel procedures and processes are needed to perform genotyping efficiently and to maximize its benefits for blood banks. MATERIALS AND METHODS: Here we describe the processes and procedures developed to introduce large-scale genotyping in our routine operations. RESULTS: Preliminary cost-benefit analysis indicated that genotyping must target frequent blood donors (> 3 donations/year) to be efficiently used. A custom-designed computer application was developed to manage the whole project. It selects frequent donors among recent donations, prints coded labels to identify blood samples sent to the external genotyping laboratory, and stores genotyping results. It can search for donors compatible for any combination of the 22 genotyped antigens as well as consult the current inventory for the presence of the corresponding blood components. The phenotype of recovered components is confirmed by standard serology techniques prior to shipment to hospitals. CONCLUSION: Since October 2007, 10 555 blood donors have been genotyped. The database is used on a regular basis to find compatible blood components with a genotype-phenotype concordance of 99.6%.

Characterization of the siRNAs associated with peach latent mosaic viroid infection.

The peach latent mosaic viroid (PLMVd) is a small, circular RNA species that has been shown to induce RNA silencing in plants. With the goal of better understanding the biological mechanism underlying this process, the siRNAs found in PLMVd infected peach leaves were isolated and sequenced. Analysis of the resulting data prompted several conclusions, including: i. PLMVd strands of both polarities are substrates for the Dicer-like enzymes found in peach leaves; ii. the more highly structured regions of PLMVd trigger the activity of the Dicer-like enzymes; and, iii. the circular PLMVd conformers appear to be favored for transport into the cytoplasm.

Effects of experience on force perception threshold in minimally invasive surgery.

BACKGROUND: Distorted haptic feedback by the surgical instrumentation is a major problem in minimally invasive surgery (MIS). Friction force generated by the rubber seal in the trocars masks the haptic information needed to perceive the properties and structure of the target tissue, resulting in an increased haptic perception threshold in naïve subjects. This can lead to over application of forces in surgery. OBJECTIVE: This paper examines the effect of surgical experience on the psychophysics of force perception and force application efficiency in MIS. METHOD: A controlled experiment was conducted using a mixed design, with friction and vision as independent within-subjects factors, experience as a between-subjects factor, and applied force and detection time as dependent measures. Fourteen subjects (eight novices and six experienced surgeons) performed a simulated tissue probing task. Performance data were recorded by a custom-built force-sensing system. RESULTS: When friction was present, higher thresholds and longer detection times were observed for both experienced and inexperienced subjects. In all cases, experienced surgeons applied a greater force than novices, but were quicker to detect contact with tissue, resulting in higher force application efficiency. CONCLUSION: Surgeons seem to have adapted to the higher threshold in haptic perception by reacting faster, even while applying more force to the tissue, keeping within the limits of safety.

Molecular features of new Peach Latent Mosaic Viroid variants suggest that recombination may have contributed to the evolution of this infectious RNA.

Nucleotide sequences of a broad range of Peach Latent Mosaic Viroid (PLMVd) variants were determined. The variants were isolated from peach, pear, and almond tree samples collected in Tunisia. Sequence analysis confirmed the high variability of PLMVd, as no less than 119 new variants were identified. Variations included new polymorphic positions, insertions of 11 to 14 nucleotides, and new mutations within the hammerhead self-cleavage motifs. We provide the first covariation-based evidence for certain stems within the proposed secondary structure. Our covariation analysis also strengthens the view that a pseudoknot closes the replication domain. On the basis of phylogenetic tree studies and informative positions, PLMVd variants are proposed to cluster into groups and subgroups likely to have resulted from recombination events. PLMVd thus emerges as a suitable viroid for retracing the evolution of an RNA genome.

Study of the polymerization step of the rolling circle replication of peach latent mosaic viroid.

We have developed an in vitro transcriptional assay using Escherichia coli RNA polymerase to initiate the replication of peach latent mosaic viroid (PLMVd). Regardless of the polarity of the PLMVd strand used as template, initiation in vitro occurred at the same hairpin structure. These initiation sites correspond to the 5'-ends of two small (280 nt) PLMVd-related RNAs found in infected peach leaves. Using a series of truncated PLMVd-derived transcripts, we have demonstrated that the viroid domain composed solely of the self-complementary hammerhead sequences is sufficient to trigger polymerase-driven replication in vitro. These data suggest that the bacterial-like RNA polymerase from peach chloroplasts catalyzes PLMVd replication.

Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3'-untranslated region.

The recombinant dengue virus type-4 vaccine candidate 2AA30 was attenuated in rhesus monkeys due to an engineered 30-nucleotide deletion in the 3'-untranslated region of the viral genome. A clinical trial to evaluate the safety and immunogenicity of a single dose of 2Adelta30 was conducted with 20 adult human volunteers. The vaccine candidate was well tolerated and did not cause systemic illness in any of the 20 volunteers. Viremia was detectable in 14 volunteers at a mean level of 1.6 log10 plaque-forming units/ml of serum, although all 20 volunteers seroconverted with a seven-fold or greater increase in serum neutralizing antibody titer on day 28 post-vaccination (mean titer = 1:580). A mild, asymptomatic, macular rash developed in 10 volunteers, and a transient elevation in the serum level of alanine aminotransferase was noted in five volunteers. The low level of reactogenicity and high degree of immunogenicity of this vaccine candidate warrant its further evaluation and its use to create chimeric vaccine viruses expressing the structural genes of dengue virus types 1, 2, and 3.

Natural 2',5'-phosphodiester bonds found at the ligation sites of peach latent mosaic viroid.

Peach latent mosaic viroid (PLMVd) is a circular RNA pathogen that replicates in a DNA-independent fashion via a rolling circle mechanism. PLMVd has been shown to self-ligate in vitro primarily via the formation of 2',5'-phosphodiester bonds; however, in vivo the occurrence and necessity of this nonenzymatic mechanism are not evident. Here, we unequivocally report the presence of 2', 5'-phosphodiester bonds at the ligation site of circular PLMVd strands isolated from infected peach leaves. These bonds serve to close the linear conformers (i.e., intermediates), yielding circular ones. Furthermore, these bonds are shown to stabilize the replicational circular templates, resulting in a significant advantage in terms of viroid viability. Although the mechanism responsible for the formation of these 2',5'-phosphodiester bonds remains to be elucidated, a hypothesis describing in vivo nonenzymatic self-ligation is proposed. Most significantly, our results clearly show that 2',5'-phosphodiester bonds are still present in nature and that they are of biological importance.

Long term follow-up of intravesical Bacillus Calmette-Guerin for the treatment of bladder transitional cell carcinoma.

OBJECTIVE: To review the long-term follow-up, in terms of recurrence and progression, of transitional cell carcinoma of the bladder treated with intravesical BCG with the following indications: CIS, Ta and T1. MATERIALS AND METHODS: Ninety-two patients who had received complete course of BCG between 1987 and 1993 were included in the study and followed for an average of 59 months (range 12 to 102). RESULTS: The recurrence and progression were looked at. Patients treated with BCG for Carcinoma in situ, 11 of 19 (53%) remained tumor-free after 1 or 2 courses of BCG for the duration of the follow-up (mean 4.9 years, range 1.5 to 8.5 years). For patients treated for recurring tumors, 17 of 50 (34%) had no recurrences after 1 or 2 courses of BCG with the same follow-up. When facing multiple tumors, 10 of 23 (43%) patients did not experience recurrences. Therefore, in the 92 patients treated, 38 presented no recurrences after 1 or 2 courses of BCG, for a success rate of 41%. In terms of progression, of the 19 patients treated with BCG for CIS, 4 (21%) went on to develop muscle invasive disease. Of the 50 patients treated for recurrent tumors, 2 (4%) eventually developed lamina propria invasion (initial lesion was a Ta tumor), 4 (8%) carcinoma in situ and 7 (14%) muscle invasive disease, for an overall progression rate of 26% in this group. Of the 25 patients treated for multiple tumors, 1 (4%) developed CIS and 3 (12%) presented with muscle invasive disease, for an overall progression rate of 16% for the duration of the follow-up. Therefore, 21 of 92 (23%) patients had progression of their disease following BCG therapy. No prognostic factors for recurrence or progression could be identified in these tumors. CONCLUSION: When indications warrant its use, BCG is effective in reducing recurrences and limiting progression in TCC of the bladder. Recurrence within 2 years of treatment is, however, a sign of poor prognosis and other therapeutic options should be sought.

A Canadian isolate of hepatitis D (delta) virus.

Hepatitis D (delta) virus (HDV) is an infectious agent that propagates in hepatocytes only in the presence of hepatitis B virus, causing fulminant or chronic hepatitis with liver cirrhosis. HDV is a 36 nm particle that includes a circular RNA genome of 1.7 kilobases with an extensive internal complementary that allows it to fold into a rod-like structure. The relationships among genotypes, sequence variability, geographical distribution and disease severity of HDV remain unknown. Consequently, in the present study, the complete nucleotide sequence of an HDV isolated from a Canadian patient was determined. The viral RNA from serum was amplified using reverse transcription coupled to polymerase chain reaction amplification. The resulting complementary DNA was cloned and sequenced. Sequence analysis revealed that this new isolate contained 1672 nucleotides corresponding to genotype 1, which has a worldwide distribution. Sequencing of four independent clones revealed 17 substitutions, corresponding to an overall sequence variability of 1%. Surprisingly, seven mutations were found in the 48-nucleotide region located between the two highly conserved self-catalytic motifs. This is the first demonstration that many substitutions are identified in this region of HDV, and prompts the present authors to define it as a hypervariable region.

Sequencing of peach latent mosaic viroid variants from nine North American peach cultivars shows that this RNA folds into a complex secondary structure.

We sequenced 34 new peach latent mosaic viroid (PLMVd) variants isolated from nine different peach cultivars. This study provides the widest view of PLMVd diversity reported to date and includes the original characterization of North American variants, which cannot be differentiated from European sequences. PLMVd appears as a species in which each isolate is a complex mixture of RNAs. Analysis of base-pair covariations supports the hypothesis that PLMVd folds into a complex branched structure with the potential of including three new pseudoknots. The resulting "globular-like" structure is in contrast to the rod-like one adopted by most other viroids.

Nucleotides -1 to -4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage.

In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be -1HRHY(-4) (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.

Mapping in solution shows the peach latent mosaic viroid to possess a new pseudoknot in a complex, branched secondary structure.

We have investigated the secondary structure of peach latent mosaic viroid (PLMVd) in solution, and we present here the first description of the structure of a branched viroid in solution. Different PLMVd transcripts of plus polarity were produced by using the circularly permuted RNA method and the exploitation of RNA internal secondary structure to position the 5' and 3' termini and studied by nuclease mapping and binding shift assays using DNA and RNA oligonucleotides. We show that PLMVd folds into a complex, branched secondary structure. In general, this structure is similar to that reported previously, which was based on sequence comparison and computer modelling. The structural microheterogeneity is apparently limited to only some small domains. More importantly, this structure includes a novel pseudoknot that is conserved in all PLMVd isolates and seems to allow folding into a very compact form. This pseudoknot is also found in chrysanthemum chlorotic mottle viroid, suggesting that it is a unique feature of the viroid members of the PLMVd subgroup.

Practice patterns of Canadian urologists in benign prostatic hyperplasia and prostate cancer. Canadian Prostate Health Council.

PURPOSE: We reviewed the practice patterns of Canadian urologists in benign prostatic hyperplasia (BPH) and prostate cancer, and assessed the changes that occurred between 1995 and 1998. MATERIALS AND METHODS: In 1995 and 1998 questionnaires were mailed to all active members of the Canadian Urological Association who practiced adult urology in Canada. Many questions were similar, allowing for the assessment of changes in practice patterns. RESULTS: A number of changes were observed between 1995 and 1998. Cystoscopy and imaging of the upper urinary tract were used less often to evaluate uncomplicated cases of BPH. However, 39% of respondents continued to perform cystoscopy routinely. Finasteride was no longer administered in men with a smaller prostate. In 1998 before radical prostatectomy 28% of respondents routinely performed a bone scan, 29% cystoscopy and 57% chest x-ray. The number believing that maximal androgen blockade is the most effective hormonal therapy decreased from 90% to 62%, while 24% reported in the 1998 survey that they frequently administered intermittent hormonal therapy. Comparison with an American study from 1995 indicated that American urologists used the American Urological Association symptom score and performed a prostate specific antigen test more frequently than Canadian urologists. However, Canadian urologists performed cystoscopy more frequently. CONCLUSIONS: These surveys provide a useful insight into the variations in clinical practice of Canadian urologists and help to determine whether changes are occurring in regard to the development of practice guidelines. They also indicate the need to develop further guidelines, and ensure that these guidelines are widely promoted and accepted by the urological community.