Direct determination of three-phase contact line properties on nearly molecular scale.

Wetting phenomena in multi-phase systems govern the shape of the contact line which separates the different phases. For liquids in contact with solid surfaces wetting is typically described in terms of contact angle. While in macroscopic systems the contact angle can be determined experimentally, on the molecular scale contact angles are hardly accessible. Here we report the first direct experimental determination of contact angles as well as contact line curvature on a scale of the order of 1nm. For water nucleating heterogeneously on Ag nanoparticles we find contact angles around 15 degrees compared to 90 degrees for the corresponding macroscopically measured equilibrium angle. The obtained microscopic contact angles can be attributed to negative line tension in the order of -10(-10) J/m that becomes increasingly dominant with increasing curvature of the contact line. These results enable a consistent theoretical description of heterogeneous nucleation and provide firm insight to the wetting of nanosized objects.

Ultrasound-guided facet joint injection training using Perk Tutor.

PURPOSE: Facet syndrome is a condition that may cause 15-45 % of chronic lower back pain. It is commonly diagnosed and treated using facet joint injections. This needle technique demands high accuracy, and ultrasound (US) is a potentially useful modality to guide the needle. US-guided injections, however, require physicians to interpret 2-D sonographic images while simultaneously manipulating an US probe and needle. Therefore, US-guidance for facet joint injections needs advanced training methodologies that will equip physicians with the requisite skills. METHODS: We used Perk Tutor-an augmented reality training system for US-guided needle insertions-in a configuration for percutaneous procedures of the lumbar spine. In a pilot study of 26 pre-medical undergraduate students, we evaluated the efficacy of Perk Tutor training compared to traditional training. RESULTS: The Perk Tutor Trained group, which had access to Perk Tutor during training, had a mean success rate of 61.5 %, while the Control group, which received traditional training, had a mean success rate of 38.5 % ([Formula: see text]). No significant differences in procedure times or needle path lengths were observed between the two groups. CONCLUSIONS: The results of this pilot study suggest that Perk Tutor provides an improved training environment for US-guided facet joint injections on a synthetic model.

Segregation of natural and experimental gastrointestinal nematode infection in F2 progeny of susceptible Suffolk and resistant Gulf Coast Native sheep and its usefulness in assessment of genetic variation.

Gastrointestinal nematode parasitism is a concern to small ruminants worldwide. Productivity has been compromised because such nematodes, particularly Haemonchus contortus, have developed resistance to available anthelmintics. Some sheep breeds and lines within breeds are relatively resistant to infection, a trait that may be useful for developing control strategies. Suffolk sheep, which are susceptible to infection, were crossed with Gulf Coast Native sheep, which are more resistant to infection, to produce F1 progeny. F1 rams were bred to F1 ewes which produced 227 F2 offspring. These F2 offspring were evaluated for variability in infection levels, based on fecal egg count (FEC) and blood packed cell volume (PCV), under two natural infection conditions (one at weaning and another after a summer grazing period) and one experimental infection. The range of both FEC and PCV was large for all three infection periods with annual variation. Overall, the range for the three infection periods, respectively, were 167-149,933, 0-31,400 and 17-114,667 eggs per gram (EPG) of feces and 8.7-37.0%, 7.3-33.0% and 8.3-36.0%. This segregation of infection is what would be expected of F(2) progeny from susceptible and resistant parent breeds. Heritabilities of FEC and PCV for the three infection periods, respectively, were 0.15, 0.29 and 0.12, and 0.11, 0.22 and 0.12. Based on segregation of infection, larger heritabilities and maternal environment effects that declined after weaning, the summer natural infection was probably the best model for assessing genetic variation.

Cloning, expression and purification of feline proinsulin.

Feline proinsulin was cloned and expressed using a bacterial expression system. It was then purified from inclusion bodies using size exclusion chromatography and further processed including reduction of the protein. Following refolding, proinsulin was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC and mass spectrometric analysis indicated that the proinsulin contained the correct disulfide bridging pattern. This proinsulin can be used for therapeutic and diagnostic purposes.

The extensile rectus snip exposure in revision of total knee arthroplasty.

Revision of a total knee arthroplasty may require an extensile approach to permit a satisfactory exposure without compromising the attachment of the patellar tendon. It has been assumed that a rectus snip is a relatively benign form of release, but the effect of using this approach on function, pain and patient satisfaction is not known. From January 1997 to December 1999, 107 patients who underwent revision of total knee arthroplasty were followed up at a minimum of two years (mean 40.5 months) and assessed by the Oxford Hip Score, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), the Short-Form (SF)-12 and patient satisfaction. Co-morbidity, surgical exposure, the Hospital for Special Surgery (HSS) knee scores and the range of movement were also used. A standard medial parapatellar approach was used in 57 patients and the rectus snip in 50. The two groups were equivalent for age, sex and co-morbidity scores. The WOMAC function, pain, stiffness and satisfaction scores demonstrated no statistical difference. The use of a rectus snip as an extensile procedure has no effect on outcome.

Microarray profiling of gene expression during trypomastigote to amastigote transition in Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas disease, remains a significant public health concern throughout South and Central America. Although much is known about immune control of T. cruzi and in particular the importance of recognition of parasite-infected cells, relatively little is known about the target antigens of these protective immune responses. For instance, few of the genes expressed in the intracellular amastigote stage have been identified. To gain insight into the molecular events, at the level of mRNA abundance, involved in this critical point in the parasite life-cycle, we used DNA microarrays of 4400 sequences from T. cruzi ORF-selected and random, genomic sequencing libraries to determine relative mRNA abundances in trypomastigotes and developing amastigotes. Results from six hybridizations using independently generated parasite samples consistently identified 60 probes that detected genes upregulated within 2h after extracellular trypomastigotes were induced, in vitro, to differentiate into amastigotes. Sequence analysis from these 60 probes identified 14 known and 25 novel T. cruzi genes. The general direction of regulation was confirmed by quantitative RT-PCR for seven of the array-identified, amastigote upregulated, known genes. This work demonstrates the feasibility of computational and microarray approaches to gene discovery in T. cruzi, an organism for which a fully assembled and annotated genome sequence is not yet available and in which control of transcription initiation is believed to be absent. Moreover, this work is the first report of amastigote up regulation for 38 genes, thus expanding considerably the pool of genes known to be upregulated in this important yet poorly-studied stage of the T. cruzi life-cycle.

Performance of steers grazing rhizomatous and nonrhizomatous birdsfoot trefoil in pure stands and in tall fescue mixtures.

This study investigated the performance of steers grazing rhizomatous birdsfoot trefoil (Lotus corniculatus L.) (RBFT) compared to nonrhizomatous birdsfoot trefoil (BFT) in pure stands or when interseeded with endophyte-free tall fescue (Festuca arundinacea Schreb.; TF). Five forage treatments of RBFT, BFT, TF, RBFT+TF, and BFT+TF (four replicate paddocks per treatment) were continuously stocked in spring and fall of 1998 and spring of 1999. Grazing for individual treatments was terminated when pasture mass fell below 900 kg/ha. Average daily gain was greatest (P < 0.10) in pure stands of BFT and RBFT, but total forage production, and thus grazing days, for these treatments was low. Average daily gain for steers grazing BFT+TF and RBFT+TF treatments was not different from (spring and fall 1998) or greater (P < 0.10) (spring 1999) than that for TF. Total forage production of BFT+TF and RBFT+TF was greater (P < 0.10) than that of TF in spring 1998. In fall 1998, BFT+TF produced more (P < 0.10) total forage than either RBFT+TF or TF, and in spring 1999, RBFT+TF had less (P < 0.10) total forage than TF or BFT+TF. Total steer days on mixed pastures were greater (P < 0.10) than that for TF in spring and fall 1998 but not different from those for TF in spring 1999. In all three trials total weight gain/hectare was greater (P < 0.10) for RBFT+TF and BFT+TF than for TF. The RBFT+TF and BFT +TF had greater (P < 0.05) CP than TF in spring and fall 1998 and less (P < 0.05) NDF and ADF in fall 1998. We concluded that either RBFT or BFT could be interseeded with tall fescue to enhance ADG and total steer days.

Arterial complications and total knee arthroplasty.

Arterial complications after total knee arthroplasty (TKA) are rare; however, the sequelae can be disastrous. Infection and the need for amputation or vascular reconstructive surgery are not uncommon. A thorough preoperative assessment can identify at-risk patients, many, if not all, of whom have preexisting peripheral arterial disease. In the presence of peripheral arterial disease, the use of a tourniquet during TKA has been implicated in subsequent arterial complications. Following the guidelines that have been established regarding preoperative assessment, the role of the vascular surgeon, and the use of a tourniquet before and during TKA can assist the orthopaedic surgeon in assessing candidates for TKA and reducing the risk of arterial complications.

Adipocyte insensitivity to insulin in growth hormone-transgenic mice.

Growth hormone (GH) has an inhibitory effect on adipogenesis, and its effect is associated with insulin action in obesity. In this study, the relationship between GH effect on insulin sensitivity and adipocyte differentiation in vivo was investigated. Transgenic (TG) female mice expressing porcine GH had reduced body weights and weights of retroperitoneal and parametrial fat depots. Insulin treatment increased PPARgamma and GLUT4 expression in adipose tissue of WT mice but had no effect in TG mice. Content of transcription factors, PPARgamma and C/EBPalpha and beta, was higher in adipose tissue of WT mice, and for C/EBPalpha and PPARgamma, the difference occurred primarily in 24-, compared to 12-week-old, mice. Expression of preadipocyte factor-1 was higher in adipose tissue of TG mice, and expression of TNF-alpha and leptin was reduced in adipose tissue of 24-week-old TG mice. Our results suggest that increased expression of GH reduces adipogenesis by inducing adipocyte resistance to the adipogenic effect of insulin.

The trainee in difficulty.

The trainee in difficulty is someone who is either marginal or at risk of failing in his or her clinical performance. Dealing effectively with these learners can pose problems even for seasoned medical educators. This article discusses some of the common mistakes made by educators in dealing with the trainee in difficulty and offers suggestions for a systematic approach. Further, the roles of faculty, including the program director and associate dean's office, and some of the legal issues are described.

Clinical diagnosis of clavicle fractures: a pilot study.

OBJECTIVE: Clavicle fractures are commonly encountered in the emergency department (ED). Our objective was to determine whether emergency physicians can clinically predict the presence and location of a clavicle fracture prior to obtaining x-rays. METHODS: Over a 16-month period we prospectively studied ED patients who had injuries compatible with a clavicle fracture. Following clinical examination and prior to obtaining radiographs, ED physicians or senior emergency medicine (EM) residents were asked to predict whether the clavicle was fractured and, if fractured, the location of the fracture. Clinical predictions were later compared to the radiologist's report. RESULTS: Between April 1999 and August 2000, 184 patients with possible clavicle fracture were seen and 106 (58%) were enrolled. Of these, 94 had an acute fracture, and all 94 fractures were predicted on clinical grounds prior to x-ray. In 6 cases, physicians predicted a fracture but the radiograph was negative. In 6 additional cases, physicians were clinically unsure and the radiograph was negative. Physicians correctly predicted fracture location in 83 of 94 cases (88%; 95% confidence interval [CI], 82%-95%). In the 64 cases where physicians predicted a middle third fracture, they were 100% accurate (95% CI, 95%-100%). Errors made by physicians were conservative; that is, they occasionally predicted fractures in patients with only soft tissue injury, but they did not "miss" existing fractures. CONCLUSIONS: The results of this pilot study suggest that ED physicians can clinically predict the presence and location of clavicle fractures with a high degree of accuracy. It may be that x-rays are not always necessary in patients suspected of having a clavicle fracture. Future studies should define the indications for diagnostic radiography in patients with suspected clavicle fractures.

The PROSTALAC functional spacer in two-stage revision for infected knee replacements. Prosthesis of antibiotic-loaded acrylic cement.

The PROSTALAC functional spacer is made of antibiotic-loaded acrylic cement but has a small metal-on-polythene articular surface. We have used it as an interim spacer in two-stage exchange arthroplasty for infected total knee replacement. PROSTALAC allows continuous rehabilitation between stages as it maintains good alignment and stability of the knee and a reasonable range of movement. It also helps to maintain the soft-tissue planes, which facilitates the second-stage procedure. We reviewed 45 consecutive patients, treated over a period of nine years. The mean follow-up was for 48 months (20 to 112). At final review, there was no evidence of infection in 41 patients (91%); only one had a recurrent infection with the same organism. There was improvement in the Hospital for Special Surgery knee score between stages and at final review. The range of movement was maintained between stages. Complications were primarily related to the extensor mechanism and stability of the knee between stages. Both of these problems decreased with refinement of the design of the implant. The rate of cure of the infection in our patients was similar to that using other methods. Movement of the knee does not appear to hinder control of infection.

Linkage mapping and comparative analysis of bovine expressed sequence tags (ESTs).

Bovine expressed sequence tags (ESTs) containing microsatellites are suitable markers for both linkage and comparative maps. We isolated clones from a bovine fetal thigh skeletal muscle cDNA library that were positive for a (CA)10 probe. Thirty individual clones were isolated and characterised by sequencing. Sequences from the 5' and 3' ends of a clone were considered as separate ESTs until a contiguous sequence was identified. A total of 47 ESTs were sequenced from the 5' and/or 3' ends and full sequence was obtained for the 30 clones. BLAST nucleotide analysis identified significant homology to known mammalian coding regions for 31 of the bovine ESTs, 30 of which also matched human ESTs or sequence-tagged sites (STS). The remaining 16 bovine ESTs represented novel transcripts. Microsatellites were isolated in 27 of the ESTs, 11 of which were developed into markers and placed on the MARC bovine linkage map. Human cytogenetic map positions were available for 20 of the 30 human EST orthologs, and a putative bovine map position for 17 of the sequences could be inferred using comparative mapping data. These results demonstrated that mapping bovine ESTs containing microsatellites is a plausible strategy to increase the density of gene markers on the bovine linkage and comparative maps.

Canine thyrotropin beta-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells.

The gene encoding the mature beta subunit of canine thyroid stimulating hormone (cTSH beta) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSH beta purified from E. coli were generated. The gene fragment that encodes mature TSH beta was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species. The resulting 891 basepairs (bp) of genomic DNA consisted of two coding exons of the canine TSH beta gene and an intron of 450 bp. The two exons, which encode the mature cTSH beta subunit, was joined together by an overlap PCR and was expressed in E. coli as 6xHis-tagged protein. The purified recombinant cTSH beta with a molecular weight of about 15 kDa was recognized by the polyclonal antibodies prepared against the native canine TSH in Western blot. Monoclonal antibodies were raised against the purified cTSH beta and subsequently characterized. For transient expression in CHO cells that are permanently transfected with the bovine common alpha gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5' end of the cTSH beta gene before it was cloned into the mammalian expression vector pRSV and used to transfect CHO cells. The medium from these transfected cells, presumably containing the bovine alpha and canine TSH beta in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes.

cDNA cloning of canine common alpha gene and its co-expression with canine thyrotropin beta gene in baculovirus expression system.

The common alpha gene of the canine glycoprotein hormones was cloned, sequenced and co-expressed with the canine thyrotropin beta (TSH beta) gene in the baculovirus expression system, and a bioactive recombinant canine TSH was purified. The canine common alpha gene was cloned from the total RNA extracted from the canine pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR) using primers that were designed based on the consensus sequences from other species. The resulting 476 bp PCR product is consisted of the full coding sequence for the 96 amino acid mature alpha subunit, and a sequence encoding a 24 amino acid signal peptide. Homology analysis with other species revealed that the canine common alpha subunit potentially contains five disulfide bonds and two oligosaccharide chains N-linked to Asn residues located at positions 56 and 82. For expression in the baculovirus expression system, the common alpha gene was cloned downstream of the p10 promoter of the pAcUW51 transfer vector, and the previously cloned canine TSH beta gene was inserted under the polyhedrin promoter of the same vector. The recombinant virus containing both alpha and beta genes was generated and propagated before being used to transfect the Sf9 insect cells for expression. The medium from the Sf9 cultures, presumably containing canine TSH alpha and beta in native heterodimer confirmation, exhibited TSH bioactivity as indicated in the cAMP stimulation assay in FRTL-5 cells. The expressed recombinant protein was purified from the culture medium with an affinity column that was coupled with IgG purified from the polyclonal antibodies generated against the partially purified native canine TSH.

Standardized patients in the early acquisition of clinical skills.

OBJECTIVES: Facing dramatic reductions of inpatient beds and fewer faculty tutors, the Queen's University medical school has had to consider alternatives to the traditional inpatient encounter for the early acquisition of clinical skills. The purpose of this study was to investigate the feasibility and impact on students of a model for a first-year clinical skills course, using exclusively standardized patients in place of inpatients, and using a smaller ratio of faculty to students. DESIGN: Twenty volunteers were selected from the first-year class of 75 students to participate as the experimental group. The remaining 55 students formed the control group. SETTING: Queen's University medical school, Ontario, Canada. SUBJECTS: First-year medical students. RESULTS: In measures of student satisfaction with the amount of performance feedback received during the course, there was a non-significant trend towards greater satisfaction among the experimental group compared to the control group. This occurred despite a tutor to student ratio of 1.5:10 in the experimental group compared to 3:10 in the control group. In the student evaluation of their tutors, mean scores for the two groups were 4.28 for the experimental group and 4. 06 for the control group (P = 0.10). The mean OSCE scores for the two groups were 76.4 for the experimental group and 76.5 for the control group (P = 0.93). There is no practical or statistical difference in either of these two scores. CONCLUSIONS: We conclude that this new model is feasible, was well received by the students and that the reduced tutor to student ratio and lack of inpatient encounters did not appear to adversely affect their learning.

Analysis of a 762-bp proximal leptin promoter to drive and control regulation of transgene expression of growth hormone receptor in mice.

Transgenic (TG) mice expressing porcine GH receptor (pGHR) directed by a 762-bp proximal leptin promoter were used to analyze the capability of the promoter to drive and regulate pGHR expression in vivo. Transgene expression occurred in inguinal, retroperitoneal, and epididymal/parametrial fat depots in both male and female TG mice, but not in wild type (WT) mice. pGHR transgene was also expressed in liver, heart, kidney, muscle, lung, and brain. Levels of pGHR transgene mRNA were higher in tissues other than adipose tissue. Fasting reduced leptin mRNA levels in adipose; however, pGHR transgene expression was not affected in either adipose or muscle. These results suggest (1) the region between +3 and -759 bp of the leptin promoter is able to drive gene expression in vivo, (2) this region may not be responsible for adipose tissue specificity of leptin expression, and (3) this region may not be responsible for negative regulation of leptin gene expression during fasting.

Inheritance of a mitochondrial DNA defect and impaired glucose tolerance in BHE/Cdb rats.

As they age, BHE/Cdb rats develop impaired glucose tolerance. We hypothesized that this intolerance is associated with a previously reported base substitution in the mitochondrial genome. A new screening test was devised to identify animals with the mutation. These animals were bred to animals without the mutation. The progeny were then tested for the presence of the mutation and their glucose tolerance at 100 and 300 days of age. Phenotype and genotype were found to be closely linked and we conclude that the mutation in the mitochondrial ATPase 6 gene explains the age related impaired glucose tolerance in BHE/Cdb rats.

Expression of beta-galactosidase and pig leptin gene in vitro by recombinant adenovirus.

Adenovirus has been used in vivo and in vitro as a vector to carry a foreign gene for gene transfer. Two kinds of replication defective human recombinant adenovirus vectors were used in this study, the first containing beta-galactosidase reporter gene (AdCMVLac-Z) and the second carrying a gene for porcine leptin gene (AdCMVpLeptin). AdCMVLac-Z was tested for its ability to transfer DNA into pig kidney and pituitary cells. These cells expressed Lac-Z transiently 48 hours after the infection. In addition, when the pig kidney cells expressing the Lac-Z were replated with low density for the formation of colonies from each cell, colonies of blue cells expressing Lac-Z were observed. These results demonstrate that human recombinant adenovirus can be used as a transducing viral vector for inducing long-term expression in pig kidney cells. We also constructed a recombinant adenovirus (AdCMVpLeptin) which contained a pig leptin gene for the expression of pig leptin in vitro in the 293 human kidney cell line. 293 cells transfected with AdCMVpLeptin produced both a 15 KDa of a secretory form of porcine leptin and an 18 KDa long form containing signal peptide. Our study demonstrated that the recombinant adenovirus system offers a method for gene transfer and expression in pig cells.