Unprecedented enhancement of recombinant protein production in sugarcane culms using a combinatorial promoter stacking system.

Plants represent a safe and cost-effective platform for producing high-value proteins with pharmaceutical properties; however, the ability to accumulate these in commercially viable quantities is challenging. Ideal crops to serve as biofactories would include low-input, fast-growing, high-biomass species such as sugarcane. The objective of this study was to develop an efficient expression system to enable large-scale production of high-value recombinant proteins in sugarcane culms. Bovine lysozyme (BvLz) is a potent broad-spectrum antimicrobial enzyme used in the food, cosmetics and agricultural industries. Here, we report a novel strategy to achieve high-level expression of recombinant proteins using a combinatorial stacked promoter system. We demonstrate this by co-expressing BvLz under the control of multiple constitutive and culm-regulated promoters on separate expression vectors and combinatorial plant transformation. BvLz accumulation reached 1.4% of total soluble protein (TSP) (10.0 mg BvLz/kg culm mass) in stacked multiple promoter:BvLz lines, compared to 0.07% of TSP (0.56 mg/kg) in single promoter:BvLz lines. BvLz accumulation was further boosted to 11.5% of TSP (82.5 mg/kg) through event stacking by re-transforming the stacked promoter:BvLz lines with additional BvLz expression vectors. The protein accumulation achieved with the combinatorial promoter stacking expression system was stable in multiple vegetative propagations, demonstrating the feasibility of using sugarcane as a biofactory for producing high-value proteins and bioproducts.

Clinical prevalence and population incidence of serious pathologies among patients undergoing magnetic resonance imaging for low back pain.

BACKGROUND: In rare cases low back pain may be caused by underlying serious pathology such as fracture, malignancy, cauda equina syndrome, or spinal infection. The lack of evidence regarding either the clinical prevalence or population incidence of serious pathologies in the lumbar spine makes it difficult for clinicians to adequately assess a patient's risk of serious pathology. PURPOSE: To determine the prevalence of serious pathologies in patients with low back pain who have been referred for a lumbar magnetic resonance imaging (MRI) by a specialist in a private secondary care or public tertiary care setting. The incidence of these serious pathologies in the geographic region of South Auckland, New Zealand was also investigated. STUDY DESIGN: Retrospective, observational cohort study. PATIENT SAMPLE: Consecutive patients referred for lumbar MRI over a 10-month period (1st of October 2013-31st of July 2014). METHOD: Data from all eligible MRI reports was analyzed and any serious pathologies were identified and recorded. Prevalence (along with 95% confidence intervals) was calculated as a percentage of the study population. Prevalence specific to private secondary care and public tertiary care settings was also calculated and prevalence rate ratios were determined to allow comparison between settings. Incidence in the geographic region of South Auckland, New Zealand, was determined using data collected from participants recruited from the regional public hospital. Population incidence with respect to age, gender, and ethnicity for each target condition was calculated and incidence rate ratios were computed to compare groups. RESULTS: A total of 2,383 participants referred for lumbar MRI scans were included in this study. Prevalence was significantly higher in the public tertiary care setting than in the private secondary care setting for all pathologies investigated in this study. Pathology specific prevalence in secondary care vs tertiary care settings was: malignancy, 0.3%, 4.4% (p<.001); fracture 2.2%, 6.7% (p<.001); cauda equina compression 0.6%, 2.3% (p=.001); infection 0.1%, 3.4% (p<.001). The combined prevalence in secondary care was 3.2% and in tertiary care 14.8% (p<.001). Pathology specific total incidence was: fracture, 13 per 100,000 person-years (p-y); malignancy 8.5 per 100,000 p-y; cauda equina compression 4.4 per 100,000 p-y; spinal infection 6.6 per 100,000 p-y. CONCLUSIONS: The prevalence of serious pathologies was significantly higher in tertiary care (public health) than in private secondary care settings. One in every 6.5 patients referred for MRI in tertiary care demonstrated structural abnormalities associated with serious pathology, which raises the question of whether access to MRI should be re-evaluated.

Methods of practice: Listening to the story.

This paper views the experience of "hip pain" through the lenses of multiple stakeholders: the patient experiencing such pain, orthopedic surgeons, and physiotherapists. Using an interpretative hermeneutic view, the method by which each encountered and dealt with living with, diagnosing, and managing hip pain is revealed. Stories of seven participants were obtained through personal interviews. These stories provided accounts and the perspectives of the various participants. A gap in the health service emerged, with the expectations of the patients not being met by the healthcare providers. The health professionals focused on the hip, while the patients were more concerned with how to continue living their lives in a manageable way. The surgeons sought to diagnose and judge as to whether the pain was worthy of surgery. No one was helping the patient to manage the "waiting for surgery" or the "not yet bad enough" decision. We argue that there is a place for physiotherapists to support patients within a human-to-human encounter by listening to the patient's story of how their hip has impacted their lives and demonstrating that they have understood and are empathetic to their needs.

OBSERVER RATING VERSUS THREE-DIMENSIONAL MOTION ANALYSIS OF LOWER EXTREMITY KINEMATICS DURING FUNCTIONAL SCREENING TESTS: A SYSTEMATIC REVIEW.

BACKGROUND: Functional assessments are conducted in both clinical and athletic settings in an attempt to identify those individuals who exhibit movement patterns that may increase their risk of non-contact injury. In place of highly sophisticated three-dimensional motion analysis, functional testing can be completed through observation. HYPOTHESIS/PURPOSE: To evaluate the validity of movement observation assessments by summarizing the results of articles comparing human observation in real-time or video play-back and three-dimensional motion analysis of lower extremity kinematics during functional screening tests. STUDY DESIGN: Systematic review. METHODS: A computerized systematic search was conducted through Medline, SPORTSdiscus, Scopus, Cinhal, and Cochrane health databases between February and April of 2014. Validity studies comparing human observation (real-time or video play-back) to three-dimensional motion analysis of functional tasks were selected. Only studies comprising uninjured, healthy subjects conducting lower extremity functional assessments were appropriate for review. Eligible observers were certified health practitioners or qualified members of sports and athletic training teams that conduct athlete screening. The Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) was used to appraise the literature. Results are presented in terms of functional tasks. RESULTS: Six studies met the inclusion criteria. Across these studies, two-legged squats, single-leg squats, drop-jumps, and running and cutting manoeuvres were the functional tasks analysed. When compared to three-dimensional motion analysis, observer ratings of lower extremity kinematics, such as knee position in relation to the foot, demonstrated mixed results. Single-leg squats achieved target sensitivity values (≥ 80%) but not specificity values (≥ 50%>%). Drop-jump task agreement ranged from poor (< 50%) to excellent (> 80%). Two-legged squats achieved 88% sensitivity and 85% specificity. Mean underestimations as large as 198 (peak knee flexion) were found in the results of those assessing running and side-step cutting manoeuvres. Variables such as the speed of movement, the methods of rating, the profiles of participants and the experience levels of observers may have influenced the outcomes of functional testing. LIMITATIONS: The small number of studies used limits generalizability. Furthermore, this review used two dimensional video-playback for the majority of observations. If the movements had been rated in real-time three dimensional video, the results may have been different. CONCLUSIONS: Slower, speed controlled movements using dichotomous ratings reach target sensitivity and demonstrate higher overall levels of agreement. As a result, their utilization in functional screening is advocated. LEVEL OF EVIDENCE: 1A.

Do patients undergoing physical testing report pain intensity reliably?

OBJECTIVE: To determine the reliability of patient reports of pain intensity during the application of physical tests. METHODS: A single examiner required participants to use the numeric pain rating scale (NPRS) to report the intensity of pain provoked during physical testing of the hip. Standardized versions of 14 physical tests were used on 18 people with hip pain. Tests were repeated at 1 hour and 2-7 days later. Within- and between-session reliability of reports of pain intensity was calculated using intraclass correlation coefficients (ICCs) and Lin's concordance correlation coefficients (CCCs). Standard errors of measurement (SEMs) were calculated. RESULTS: ICC/CCC values for within-session reports of pain intensity ranged from 0.34 (95% confidence interval [95% CI] -0.08, 0.66) to 0.88 (95% CI 0.73, 0.95). Patients demonstrated "substantial" or "almost perfect" reliability in reporting pain intensity with 9 of 14 tests and "moderate" reliability with 3 tests. Two tests were unreliable (ICC 0.35 and 0.34). ICC/CCC values for between-session reports of pain intensity ranged from -0.05 (95% CI -0.42, 0.34) to 0.84 (95% CI 0.44, 0.95). Patients demonstrated substantial or almost perfect reliability in reporting pain intensity with 11 of 14 tests. Two tests were unreliable (ICC 0.26 and -0.05). The average SEM value of 0.9 points on the NPRS was the same for both within- and between-session testing (range 0.6-1.6). CONCLUSION: Patient reports of the intensity of pain provoked by physical tests are sufficiently reliable to be clinically useful. However, the SEM of 0.9 points should be considered when precise calculations of changes in pain intensity are important.

Evaluation of monoclonal antibody and phenolic extraction from transgenic Lemna for purification process development.

Several pharmaceutical protein products made in transgenic plant hosts are advancing through clinical trials. Plant hosts present a different set of impurities from which the proteins must be purified compared to other expression hosts such as mammalian cells. In this work, phenolic compounds present in extracts of monoclonal antibody (mAb)-expressing Lemna minor were examined. Two different extraction pHs were evaluated to assess the effect of extraction condition on the concentration of mAb and phenolics in the extracts. The extract prepared at pH 4.5 had an enriched level of mAb relative to native protein when compared to a pH 7.5 extract although similar overall mAb was extracted at either pH. Slightly more mAb was recovered from the pH 3 elution of the pH 4.5 extract run on a MabSelect column than was recovered from the pH 7.5 extract. Phenolic levels in extracts were assessed by spectrophotometry, Folin-Ciocalteu assay and by profiling on RP-HPLC. The Folin-Ciocalteu assay results did not agree with those obtained by the other two methods. Therefore phenolic levels were quantified by RP-HPLC comparing the total area of phenolic peaks to those of reference phenolic compounds. The pH 7.5 extract had 22% less phenolics than the pH 4.5 extract. Acidic precipitation of the pH 7.5 extract resulted in further reduction of phenolics originally present in the pH 7.5 extract. The total phenolics present in the extracts were effectively removed by incubation of extracts with a commercially available anion exchange resin, Amberlite IRA-402. We anticipate that early removal of phenolic compounds will prolong the life of more expensive affinity columns used for the purification of potential pharmaceutical proteins and should therefore be considered in process development involving proteins extracted from transgenic plant hosts.

Contrast therapy--a systematic review.

Contrast therapy is a strategy that is widely utilised in a number of sporting codes to aid recovery. This wide use might suggest that contrast therapy is an effective recovery modality however support for this assumption appears to be mainly anecdotal. The purpose of this paper is to review the efficacy of contrast therapy. To achieve this objective, a systematic review of randomised controlled trials (RCTs) that have specifically evaluated the therapeutic efficacy of contrast therapy was performed. A search to identify appropriate literature was conducted across a number of electronic databases. The titles and abstracts of the papers identified were reviewed to select papers specifically relating to contrast therapy. Twelve RCTs met the inclusion and exclusion criteria. The PEDro Scale, a systematic tool used to critique RCTs, was employed to critique the methodological quality of these studies. This review highlights both the lack in quantity and quality of research regarding the efficacy of contrast therapy for sports recovery. There appears to be insufficient evidence that contrast therapy aids in recovery and the limited methodological quality of the reviewed studies makes it difficult to draw clear conclusions about this form of therapy. Future research needs to re-examine the use of contrast therapy and in particular whole body immersion recovery strategies within the appropriate sports setting. This research will need to be of sufficient quality to enable appropriate conclusions to be made with regards to its use as a recovery strategy.

Production of human monoclonal antibody in eggs of chimeric chickens.

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

Abdominal and erector spinae muscle activity during gait: the use of cluster analysis to identify patterns of activity.

OBJECTIVE: To describe patterns of muscle activation during gait in selected abdominal and lumbar muscles using cluster analysis. PARTICIPANTS: A sample of convenience of 38 healthy adult volunteers. Outcome measures. Electromyographic activity from the right internal and external obliques, rectus abdominis and lumbar erector spinae were recorded, and the root mean square values for each muscle were calculated throughout the stride in 5% epochs. These values were normalised to maximum effort isometric muscle contractions. Cluster analysis was used to identify groups of subjects with similar patterns of activity and activation levels. RESULTS: Cluster analysis identified two patterns of activity for the internal oblique, external oblique and rectus abdominis muscles. In the lumbar erector spinae, three patterns of activity were observed. In most instances, the patterns observed for each muscle differed in the magnitude of the activation levels. In rectus abdominis and external oblique muscles, the majority of subjects had low levels of activity (<5.0% of a maximum voluntary contraction) that were relatively constant throughout the stride cycle. In the internal oblique and the erector spinae muscles, more distinct bursts of activity were observed, most often close to foot-strike. The different algorithms used for the cluster analysis yielded similar results and a discriminant function analysis provided further evidence to support the patterns observed. CONCLUSIONS: Cluster analysis was useful in grouping subjects who had similar patterns of muscle activity. It provided evidence that there were subgroups that might otherwise not be observed if a group ensemble was presented as the "norm" for any particular muscle's role during gait. RELEVANCE: The identification of common variations in muscle activity may prove valuable in identifying individuals with electromyographic patterns that might influence their chances of sustaining injury. Alternatively, clusters may provide important information related to muscle activity in those that do well or otherwise after a particular injury.