The
motility assay (IVMA) is a powerful tool commonly used in basic muscle research and for drug screenings with the aim to find treatment options for neuromuscular disorders. In brief, the sliding ...movement of fluorescence-labeled actin filaments on myosin motor proteins is recorded, and the sliding velocity is analyzed via image analysis methods. Due to low signal-to-noise ratios and large variability in the velocity signal, accurate determination of the maximum sliding velocity is challenging. We introduce a new method and software program named Actin Phase Velocity (ActiPHV). The method extracts the maximum velocity from filament tracking data. Based on simulated and real reference data we show that our method yields a higher accuracy compared to previous methods. As a result, our method enables enhancing the sensitivity of the IVMA to better exploit its full potential.
The
motility assay (IVMA) is a technique that enables the measurement of the interaction between actin and myosin providing a relatively simple model to understand the mechanical muscle function. For ...actin-myosin IVMA, myosin is immobilized in a measurement chamber, where it converts chemical energy provided by ATP hydrolysis into mechanical energy. The result is the movement of fluorescently labeled actin filaments that can be recorded microscopically and analyzed quantitatively. Resulting sliding speeds and patterns help to characterize the underlying actin-myosin interaction that can be affected by different factors such as mutations or active compounds. Additionally, modulatory actions of the regulatory proteins tropomyosin and troponin in the presence of calcium on actin-myosin interaction can be studied with the IVMA. Zebrafish is considered a suitable model organism for cardiovascular and skeletal muscle research. In this context, straightforward protocols for the isolation and use of zebrafish muscle proteins in the IVMA would provide a useful tool in molecular studies. Currently, there are no protocols available for the mentioned purpose. Therefore, we developed fast and easy protocols for characterization of zebrafish proteins in the IVMA. Our protocols enable the interested researcher to (i) isolate actin from zebrafish skeletal muscle and (ii) extract functionally intact myosin from cardiac and skeletal muscle of individual adult zebrafish. Zebrafish tail muscle actin is isolated after acetone powder preparation, polymerized, and labeled with Rhodamine-Phalloidin. Myosin from ventricles of adult zebrafish is extracted directly into IVMA flow-cells. The same extraction protocol is applicable for comparably small tissue pieces as from zebrafish tail, mouse and frog muscle. After addition of the fluorescently labeled F-actin from zebrafish-or other origin-and ATP, sliding movement can be visualized using a fluorescence microscope and an intensified CCD camera. Taken together, we introduce a method for functional analysis in zebrafish cardiac and skeletal muscle research to study mutations at the molecular level of thick or thin filament proteins. Additionally, preliminary data indicate the usefulness of the presented method to perform the IVMA with myosin extracted from muscles of other animal models.
Two bottlenecks impeding the genetic analysis of complex traits in rodents are access to mapping populations able to deliver gene-level mapping resolution and the need for population-specific ...genotyping arrays and haplotype reference panels. Here we combine low-coverage (0.15×) sequencing with a new method to impute the ancestral haplotype space in 1,887 commercially available outbred mice. We mapped 156 unique quantitative trait loci for 92 phenotypes at a 5% false discovery rate. Gene-level mapping resolution was achieved at about one-fifth of the loci, implicating Unc13c and Pgc1a at loci for the quality of sleep, Adarb2 for home cage activity, Rtkn2 for intensity of reaction to startle, Bmp2 for wound healing, Il15 and Id2 for several T cell measures and Prkca for bone mineral content. These findings have implications for diverse areas of mammalian biology and demonstrate how genome-wide association studies can be extended via low-coverage sequencing to species with highly recombinant outbred populations.
Purpose
Percutaneous radiofrequency ablation (RFA) of thyroid nodules is an alternative to surgical resection that offers the benefits of minimal scars for the patient, lower complication rates, and ...shorter treatment times. Ultrasound (US) is the preferred modality for guiding these procedures. The needle is usually kept within the US scanning plane to ensure needle visibility. However, this restricts flexibility in both transducer and needle movement and renders the procedure difficult, especially for inexperienced users. Existing navigation solutions often involve electromagnetic (EM) tracking, which requires placement of an external field generator (FG) in close proximity of the intervention site in order to avoid distortion of the EM field. This complicates the clinical workflow as placing the FG while ensuring that it neither restricts the physician’s workspace nor affects tracking accuracy is awkward and time-consuming.
Methods
The EchoTrack concept overcomes these issues by combining the US probe and the EM FG in one modality, simultaneously providing both real-time US and tracking data without requiring the placement of an external FG for tracking. We propose a system and workflow to use EchoTrack for RFA of thyroid nodules.
Results
According to our results, the overall error of the EchoTrack system resulting from errors related to tracking and calibration is below 2 mm. Navigated thyroid RFA with the proposed concept is clinically feasible. Motion of internal critical structures relative to external markers can be up to several millimeters in extreme cases.
Conclusions
The EchoTrack concept with its simple setup, flexibility, improved needle visualization, and additional guidance information has high potential to be clinically used for thyroid RFA.
Purpose
A thyroid-like gelatin model was used to determine potential superiority of a new navigation system for ultrasound (US)-guided electrode insertion called
EchoTrack
, featuring a US probe with ...an integrated electromagnetic field generator, in comparison with conventional US when performing radiofrequency ablation.
Methods
In order to compare 20 navigated ablations with 20 ablations under conventional US guidance, a thyroid-like gelatin model was used. In each group, 10 in-plane and 10 out-of-plane punctures were performed. Metal seeds measuring 8.5
×
1.8 mm served as ablation targets. The number of redirections until final electrode placement, targeting accuracy and electrode placement time were measured.
Results
The number of redirections could be significantly (
p
<
0.0001
) reduced from 2.7 ± 1.3 in the conventional group to 0.2 ± 0.5 in the
EchoTrack
group. Accuracy increased from 3.9 ± 4.7 to 2.0 ± 1.9 mm. The total placement time increased from 39 ± 20.5 to 79.2 ± 26 s.
Conclusions
EchoTrack
is able to reduce the redirections needed to place the electrode in comparison with conventional US and provides high placement accuracy. Our new navigation system has high potential to reduce the risk of harming critical structures and to improve guidance during ablation of difficult nodules, as treatment planning as well as the safety of out-of-plane punctures are improved.
To evaluate factors associated with rotavirus diarrhea and to describe severity of illness among children <5 years old with non-dysenteric, moderate-to-severe diarrhea (MSD) in rural western Kenya.
...We analyzed data from children <5 years old with non-dysenteric MSD enrolled as cases in the Global Enteric Multicenter Study (GEMS) in Kenya. A non-dysenteric MSD case was defined as a child with ≥3 loose stools in 24 hrs. and one or more of the following: sunken eyes, skin tenting, intravenous rehydration, or hospitalization, who sought care at a sentinel health center within 7 days of illness onset. Rotavirus antigens in stool samples were detected by ELISA. Demographic and clinical information was collected at enrollment and during a single follow-up home visit at approximately 60 days. We analyzed diarrhea severity using a GEMS 17 point numerical scoring system adapted from the Vesikari score. We used logistic regression to evaluate factors associated with rotavirus infection.
From January 31, 2008 to September 30, 2012, among 1,637 (92%) non-dysenteric MSD cases, rotavirus was detected in stools of 245 (15.0%). Rotavirus-positive compared with negative cases were: younger (median age, 8 vs. 13 months; p<0.0001), had more severe illness (median severity score, 9 vs 8; p<0.0001) and had to be hospitalized more frequently (37/245 15.1% vs. 134/1,392 9.6%), p <0.013). Independent factors associated with rotavirus infection included age 0-11 months old (aOR = 5.29, 95% CI 3.14-8.89) and presenting with vomiting ≥3 times/24hrs (aOR = 2.58, 95% CI 1.91-3.48). Rotavirus was detected more commonly in warm and dry months than in the cool and rainy months (142/691 20% vs 70/673 10%) p<0.0001).
Diarrhea caused by rotavirus is associated with severe symptoms leading to hospitalization. Consistent with other settings, infants had the greatest burden of disease.
Diarrheal disease is a leading cause of death among young children worldwide. As rates of acute diarrhea (AD; 1-6 days duration) have decreased, persistent diarrhea (PD; > 14 days duration) accounts ...for a greater proportion of the diarrheal disease burden. We describe factors associated with the duration of moderate-to-severe diarrhea in Kenyan children < 5 years old enrolled in the Global Enteric Multicenter Study. We found 587 (58%) children experienced AD, 360 (35%) had prolonged acute diarrhea (ProAD; 7-13 days duration), and 73 (7%) had PD. We constructed a Cox proportional hazards model to identify factors associated with diarrheal duration. Risk factors independently associated with longer diarrheal duration included infection with
(hazard ratio HR: 0.868,
= 0.035), using an unimproved drinking water source (HR: 0.87,
= 0.035), and being stunted at enrollment (HR: 0.026,
< 0.0001). Diarrheal illness of extended duration appears to be multifactorial; given its association with adverse health and development outcomes, effective strategies should be implemented to reduce the duration and severity of diarrheal illness. Effective treatments for
should be identified, interventions to improve drinking water are imperative, and nutrition should be improved through exclusive breastfeeding in infants ≤ 6 months and appropriate continued feeding practices for ill children.
PDF
