Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection.

CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies.

Task-dependent interactions between dopamine D2 receptor polymorphisms and L-DOPA in patients with Parkinson's disease.

Variants in genes regulating dopamine transmission affect performance on tasks including working memory and executive function as well as temporal processing and sequence learning. In the current study, we determined whether a dopamine D2 receptor DNA sequence polymorphism interacts with L-DOPA during motor tasks in patients with Parkinson's disease (PD). Forty-five PD patients were genotyped for the DRD2 polymorphism (rs 1076560, G>T). Patients performed an explicit motor sequence learning task and the grooved pegboard test in both ON and OFF L-DOPA states. For motor sequence learning, DRD2 genotype mediated L-DOPA effects such that L-DOPA associated improvements were only observed in the minor T allele carriers (associated with lower D2 receptor availability, t10=-2.71, p=0.022), whereas G homozygotes showed no performance change with L-DOPA. For the grooved pegboard test, performance improved with L-DOPA independent of patients' DRD2 genotype. Collectively these results demonstrate that common DRD2 allelic differences found in the human population may explain how dopamine differentially contributes to performance across tasks and individuals.

Nasal cytokine responses to natural colds in asthmatic children.

BACKGROUND: The mechanisms by which viruses induce asthma exacerbations are not well understood. OBJECTIVE: We characterized fluctuations in nasal aspirate cytokines during naturally occurring respiratory viral infections in children with asthma. METHODS: Sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. The presence of viral infection was ascertained by multiplex PCR. Cytokines were measured using multiplex immune assay. mRNA expression for selected markers of viral infection was measured using RT-PCR. A cumulative respiratory symptom score was calculated for each day of measurement. Generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score. RESULTS: The 16 patients completed a total of 37 weeks of assessment (15 'well' weeks; 22 self-assessed 'sick' weeks). Viral infections were detected in 3 of the 'well' weeks and 17 of the 'sick' weeks (10 rhinovirus, three coronavirus, two influenza A, two influenza B, two respiratory syncytial virus, one parainfluenza). Compared to virus-negative well weeks, nasal aspirate IFN-γ, CXCL8/IL-8, CXCL10/IP-10, CCL5/RANTES, CCL11/eotaxin-1, CCL2/MCP-1, CCL4/MIP-1ß, CCL7/MCP-3, and CCL20/MIP3α protein levels increased during virus-positive sick weeks. Only a subset of cytokines (IFN-γ, CXCL8, CCL2, CCL4, CCL5, and CCL20) correlated with self-reported respiratory tract symptoms. While many aspirates were dilute and showed no mRNA signal, viral infection significantly increased the number of samples that were positive for IFN-λ1, IFN-λ2/3, TLR3, RIG-I, and IRF7 mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that in children with asthma, naturally occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, IFNs, and IFN-responsive genes.

Martial arts as sport and therapy.

The term Martial Arts is often used as general phrase to describe many of the combat arts, which have developed in eastern cultures over the past millennium. This paper reviews the Martial Arts from the original context of a trio of life skills. This trio includes the healing arts such as acupuncture, the self-exploration arts such as yoga, and the vital life skills such as meditation. As Martial Arts suggests the waging of combat, the origins of the most common combat arts are reviewed, with an overview of the difference between the hard and the soft styles. The arts developed not only in the eastern, but also in all parts of the world, with references of these types of combats arts in the writings of the ancient Egyptians and Greeks. In modern times, the combat arts are performed for both exercise and sport. A review of the injuries that occur, and the health benefits that might be expected are discussed. A review of the medical literature that demonstrates some of these health benefits is included, with Tai Chi Chuan as the most studied of these. The health benefits discussed include strengthen and self-efficacy of the elderly, reduced falls, increased exercise capacity, and benefits to the immune system and autonomic nervous system. The paper emphasized the breadth of the Martial Arts and the import of these to the sports and health community.

Rehabilitation outcomes of cardiac and non-cardiac anoxic brain injury: a single institution experience.

OBJECTIVE: To compare the functional outcomes of patients with anoxic brain injury (ABI) due to cardiac and non-cardiac aetiologies. DESIGN: Retrospective chart review over 4 years. SETTING: Freestanding rehabilitation hospital. PARTICIPANTS: Thirteen patients with cardiac ABI and 13 patients with non-cardiac ABI. INTERVENTION: Comprehensive, multi-disciplinary inpatient rehabilitation services. MAIN OUTCOME MEASURES: Rehabilitation hospital length of stay (LOS) and cost; Functional Independence Measure (FIM) scores and its various sub-sets on admission and discharge; FIM efficiency and change; and discharge disposition. RESULTS: Patients with cardiac ABI were similar in gender and ethnicity when compared to patients with non-cardiac ABI but were older (average age 52 vs 42) with a higher percentage of cardiac patients married (77% vs 39%). No statistically significant differences were found between the two groups on all sub-sets of the FIM on admission and discharge as well as the different FIM efficiencies. However, there was a trend for the cardiac ABI patients to have a greater efficiency in improving mobility during rehabilitation when compared to non-cardiac ABI patients. The rehabilitation hospital LOS was approximately 28 days less for patients with cardiac ABI (41.49 vs 69.84 days), but this difference was not statistically significant (p=0.26). The mean rehabilitation cost for patients with cardiac ABI was approximately $14,000 less than that for those with non-cardiac ABI ($44,181 vs $58,187). This difference was not statistically significant (p=0.15). Cardiac ABI patients were more likely to be discharged directly to home from rehabilitation when compared to non-cardiac ABI patients (p=0.06). CONCLUSION: This pilot study demonstrates some differences in the recovery patterns of patients with ABI who had a cardiac aetiology and those who had non-cardiac aetiologies. While both groups experienced similar progress during rehabilitation, those with cardiac ABI made gains with a shorter LOS and less rehabilitation costs when compared to non-cardiac ABI patients. These data suggest a trend towards greater cost and length of stay for patients with ABI who had non-cardiac aetiologies.

An integrated microfluidic device for influenza and other genetic analyses.

An integrated microfluidic device capable of performing a variety of genetic assays has been developed as a step towards building systems for widespread dissemination. The device integrates fluidic and thermal components such as heaters, temperature sensors, and addressable valves to control two nanoliter reactors in series followed by an electrophoretic separation. This combination of components is suitable for a variety of genetic analyses. As an example, we have successfully identified sequence-specific hemagglutinin A subtype for the A/LA/1/87 strain of influenza virus. The device uses a compact design and mass production technologies, making it an attractive platform for a variety of widely disseminated applications.

Effect of implementation of safety measures in tae kwon do competition.

BACKGROUND: Previous reviews of tae kwon do (TKD) tournaments have documented injury rates of 25/1000 to 12.7/100 athlete exposures. Most injuries have been reported to be to the head and the neck and are occasionally very serious. Many of these studies involved high level TKD competitions with minimal safety precautions. Recently, safety measures have been implemented in many TKD competitions. OBJECTIVE: To evaluate retrospectively the incidence of injuries in TKD competitions involving a wide range of participants and featuring extensive safety precautions. METHODS: A total of 2498 participants ranged in age from 18 to 66, included both men and women, and ranged in rank from yellow to black belt. Traumas, defined as any event requiring interaction with medical staff, were documented with respect to mechanism, diagnosis, treatment, and follow up recommendations. An injury was defined as a trauma that prevented a contestant from resuming competition on the day that the trauma occurred, according to National Collegiate Athletic Association criteria. RESULTS: The injury rate was 0.4/1000 athlete exposures. This is lower than reported in previous studies of TKD tournaments and in many other sports. CONCLUSION: TKD tournaments that emphasise limited contact, protective equipment, and medical supervision are relatively safe and compare favourably with other sports.

Genetic polymorphisms in mouse genes regulating age-sensitive and age-stable T cell subsets.

To see whether genetic polymorphisms regulate inter-individual differences in T cell subset levels, we have conducted a genome scan in two populations of mice, bred as the progeny of a cross between CB6F1 females and C3D2F1 males. The data document quantitative trait loci (QTL) with statistically significant effects on CD4, CD8, and CD8 memory T cells, and on subsets of CD4 and CD8 T cells that express P-glycoprotein. Some of the loci detected were robust, in the sense that they produced effects of similar size both in mated female mice, and in a population that included male and female virgin animals. Some of the effects were stable, in that they were apparent at both 8 and 18 months of age, but others were age-specific, showing effects either at 8 or at 18 months but not at both ages. Genes that had an effect on the same T cell subset were in almost all cases additive rather than epistatic, and their combined effects could produce large overall effects, leading in the most dramatic case to a two-fold difference in CD8 memory cells. The analysis also documented two QTL, on chromosomes 4 and 13, that regulate an age-sensitive composite index of T cell subset pattern which has been shown previously to be a predictor of life expectancy in these mice. The analysis thus reveals both subset-specific genes and others which modulate the overall pattern of age-sensitive changes in T cell subset distributions.

A survey of anorexia nervosa using the Arabic version of the EAT-26 and "gold standard" interviews among Omani adolescents.

OBJECTIVE: The purpose of this study was to assess the validity of the Eating Attitude Test (EAT) in identifying the presence and severity of eating pathology in male and female Omani urban adolescents and to establish cut-off scores that matched those of anorexia identified by gold standard interviews without fear of fatness criteria. METHODS: Both females (n=126) and males (n=136) were screened using the Arabic version of the EAT-26 and interviewed using a semi-structured, Composite International Diagnostic Interview (CIDI) in order to investigate the relationship between false positives and false negatives at various EAT-26 cut-off points. A receiver operating characteristics (ROC) curve was calculated to discriminate the power of the EAT-26 for every possible threshold score. RESULTS: The EAT-26 identified 29% of the subjects as probable anorexic cases as against 9.5% identified during the structured interview based on the anorexia gold standard (32% males and 68% females). The sensitivity and specificity of the EAT-26 were respectively 24% and 69.6%. When using the ROC curve, a cut-off score of 10 gave the best compromise between sensitivity (64%) and specificity (38%). DISCUSSION: Although the EAT-26 is the most widely used screening instrument in cross-cultural studies, it does not appear to be reliable in identifying probable cases of anorexia among Omani adolescents. The use of a gold standard interview without fat phobia criteria indicated that the rate of anorexia nervosa may be more prevalent among males than previously estimated. This intriguingly high preponderance of males is discussed in terms of prevailing demographic trends in Oman.

Post-stroke violent adventitial movement responsive to levo-dopa/carbi-dopa therapy.

Previous reports in the literature concerning cerebrovascular accident have illustrated cases of post-stroke tremor. Treatments of these studies have varied. Trials of levo-dopa have been reported in two such cases. This case study reports on a case of a patient with a left thalamic, left superior cerebellar artery infarction with a lacunar infarction in the basal ganglia. The patient developed a violent tremor/movement disorder which was unresponsive to haloperidol. With this failure, and with the evidence of a basal ganglion lesion, levo-dopa/carbi-dopa was introduced as an intervention. The amplitude of the tremor was dramatically reduced, with protective devices removed, and with complete cessation of the tremor at rest. The medication was withdrawn and reintroduced with a reduction and subsequent resolution of the symptoms. A discussion of the previous studies of movement disorder with cerebrovascular accident is included.

On-chip thermopneumatic pressure for discrete drop pumping.

A class of "lab-on-a-chip" devices use external air pressure for pumping discrete drops in a microchannel network. External air connectors can be cumbersome and are real-estate intensive. We have developed an on-chip technique to generate pressures required for metering and pumping of nanoliter-volume discrete drops. This is achieved by heating of trapped air in a pressure-generating chamber. The pressure-generating chamber is connected to the point of pressure application in the liquid-conveying microchannel through an air-delivery channel. The trapped air volume on the order of 100 nL is heated by resistive metal heaters by tens of degrees celcius to generate air pressures on the order of 7.5 kN/m2. The rate of discrete drop pumping is electronically controlled in the microchannel device by controlling the rate of air heating. Flow rates on the order of 20 nL/s are obtained in the microchannel (300 microns x 30 microns) by heating the air chamber at the rate of approximately 6 degrees C/s. In this paper, we describe the design, fabrication, and operation of this new technique of generating on-chip air pressure, used for metering and pumping nanoliter discrete drops in microchannels.

Mobility, diffusion and dispersion of single-stranded DNA in sequencing gels.

Electrophoresis of single-stranded DNA in denaturing polyacrylamide gels is presently a standard procedure for the sequencing of DNA fragments. A thorough understanding of the factors that determine the resolution of DNA fractionated in polyacrylamide gels is necessary to optimize the performance of DNA sequencers. Significant research on the mobility of double-stranded (ds)DNA molecules in agarose and polyacrylamide gels has been performed, and the phenomenon of band broadening of single-stranded (ss)DNA fragments in DNA sequencing gels has received attention only recently. In this paper, we present a detailed study of mobility, diffusion and dispersion of ssDNA in sequencing gels as a function of molecular size, gel concentration and electric field strength. DNA mobility is shown to be essentially independent of electric field in the range of 0-60 V/cm. The band broadening is greatly enhanced in the presence of an electric field and the dispersion coefficient (DE) can be an order of magnitude higher than the field-free diffusion coefficient. The measured migration parameters approximately follow the predictions of the biased reptation including fluctuations (BRF) theory. However, deviations due to nonidealities of the separation conditions are observed. The measured migration parameters can be used to optimize the performance of separation systems.

Using talking lights to assist brain-injured patients with daily inpatient therapeutic schedule.

OBJECTIVES: To test the ability of patients with brain injury to use a patient locator and minder (PLAM) system to assist in their adherence to therapy schedules. PARTICIPANTS: Five patients with acquired brain injury who were inpatients on an acute rehabilitation floor of a rehabilitation hospital. MEASURES: The number of human prompts necessary to direct a patient to, and ensure arrival at, a scheduled therapy destination and the proportion of therapy sessions requiring no prompting was measured both before and after the introduction of the PLAM system. RESULTS: With the PLAM system, the average number of human prompts dropped by more than 50%, and the number of sessions requiring no prompting increased from 7% to 44%. CONCLUSION: The PLAM system described in this article seems feasible and useful for patients with acquired brain injury in assisting them with arrival at their therapy destinations without the assistance of staff.

Electrophoresis in microfabricated devices using photopolymerized polyacrylamide gels and electrode-defined sample injection.

Microfabrication techniques have become increasingly popular in the development of the next generation of DNA analysis systems. While significant progress has been reported by many researchers, complete microfabricated integrated DNA analysis devices are still in the earliest stages of development. Most miniaturized analysis systems have incorporated noncross-linked polymer solutions as the separation medium of choice and the operation of these systems necessitates the use of high electric fields and long separation lengths. In this paper, we present two techniques that may help alleviate this problem and accelerate the development of the so-called 'lab-on-a-chip' systems. We present the use of photodefinable polyacrylamide gels as a sieving medium for DNA electrophoresis. These gels offer the significant advantages of faster curing times, locally controlled gel interface, and simpler handling over chemically polymerized gels. We also introduce an electrode-defined sample compaction and injection technique. This technique helps achieve sample compaction without migration into the gel and offers significant control over the size and application of the sample plug. The use of these technologies for double-stranded DNA separations in microfabricated separation systems is demonstrated.

Monolithic capillary electrophoresis device with integrated fluorescence detector.

A monolithic capillary electrophoresis system with integrated on-chip fluorescence detector has been microfabricated on a silicon substrate. Photodiodes in the silicon substrate measure fluorescence emitted from eluting molecules. The device incorporates an on-chip thin-film interference filter that prevents excitation light from inhibiting the fluorescence detection. A transparent AZO conducting ground plane is also used to prevent the high electric fields used for the separation from interfering with the photodiode response. Separations of DNA restriction fragments have been performed in these devices with femtogram detection limits using SYBR Green I intercalating dye.

Can toe-walking contribute to stiff-legged gait?

OBJECTIVE: Spastic paretic stiff-legged gait, defined as reduced knee flexion in swing, has previously been attributed solely to spastic quadriceps activity. In earlier work, the authors suggested that reduced knee flexion in swing can be attributed to other indirect factors, such as poor hip flexion and abnormal foot-ankle function during gait. The present study was undertaken to determine whether toe-walking, which often occurs in conjunction with stiff-legged gait, in and of itself, might explain some of the reduced knee flexion in swing. DESIGN: An analysis was performed of three-dimensional kinematic data collected from able-bodied subjects while walking on their toes vs. normal heel-toe walking. RESULTS: Peak knee flexion was reduced significantly compared with normal heel-toe walking (42.2+/-8.9 degrees toe-walking vs. 59.2+/-5.7 degrees heel-toe walking; P < 0.00001). CONCLUSIONS: This finding, which occurred when controlling for walking speed, may be clinically relevant for patients who have both a toe-walking and a stiff-legged gait pattern. Some of the reduced knee flexion in swing may be merely a consequence of toe-walking, rather than a result of other causes, such as intrinsic spasticity or abnormal muscle firing about the knee.

Naltrexone: effects on motor function, speech, and activities of daily living in a patient with traumatic brain injury.

Evidence from many studies has suggested that endogenous opioid peptides participate in a number of pathophysiological responses to brain injury. This provides the rationale for the use of opioid antagonists for the enhancement of neural recovery after brain injury. A case is presented of an 18-year-old male who had loss of consciousness for 1 month after a severe brain injury. Three months of intensive rehabilitative therapies did not change his functional status. A trial of naltrexone was given while his performance in mobility, speech and overall Functional Independence Measure (FIM) scores were monitored. Results indicate an accelerated improvement in functional status and statistically improved FIM score.

Nanoliter liquid metering in microchannels using hydrophobic patterns.

Nanoliter-sized liquid drops can be accurately metered inside hydrophilic microchannels using a combination of hydrophobic surface treatment and air pressure. The technique involves spontaneously filling the microchannels up to a hydrophobic region and splitting a liquid drop by injecting air through a hydrophobic side channel. The hydrophobic regions are fabricated by using a patterned metal mask on a substrate. The patterned substrate is immersed in an isooctane solution containing 1H,1H,2H,2H-per-fluorodecyltrichlorosilane to form hydrophobic patches on the exposed surface. Stripping the metal mask leaves the hydrophobic patches and restores the hydrophilic substrate surface. Precise and accurate liquid volumes, ranging from 0.5 to 125 nanoliters, have been metered using this technique. Theoretical predictions of the pressure needed to meter drops compare well with the experimental values.

Prevention of deep venous thrombosis: overview of available therapy options for rehabilitation patients.

The assessment, diagnosis, and treatment of deep venous thrombosis remain a significant problem facing hospitalized patients. The threat of thromboembolism is no less significant, perhaps more so, when patients are discharged from the hospital for subsequent care in rehabilitation units. These patients can have multiple risk factors that predispose them to a high risk of a thromboembolic event. It is clearly evident that thromboembolic prophylaxis is warranted, as it provides both positive clinical and economic outcomes. A variety of nonpharmacologic and pharmacologic strategies can be used to decrease the risk of developing deep venous thrombosis. The method of thromboembolic prophylaxis used should be based on individual patient characteristics and documented clinical and economic advantages. The purpose of this article is to review currently available thromboembolic prophylaxis measures and the implications of their use by rehabilitation physicians.