This work was supported by CIPER-Foundation for Science and Technology (FCT), Portugal (UID/DTP/00447/2019) and financed in part by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior - Brasil (CAPES) - Finance Code 001&#34;, and to the S&#227;o Paulo Research Foundation - FAPESP (PROCESS 2016/04544-3 and 2016/17735-1). Authors would like to thank Jo&#227;o Guilherme S. V. de Oliveira to the assistance in data sampling. M&#225;rio A. C. Espada acknowledge the financial support from IPDJ &#8211; Portuguese Institute of Sports and Youth.

Participants in the study from which the representative-subject data presented below were extracted20 (n = 11) were required to give their written informed consent prior to initiation of testing after the experimental procedures, associated risks and potential benefits of participation had been explained. The first visit comprised a familiarization session during which the swimmers were introduced to the concept of tethered swimming and the measurement techniques that would be in effect during the actual testing. An all-out tethered-swimming test was performed during the second visit and the rapidly incremented tethered-swimming protocol was performed on the third visit. Both tests were done in a semi-Olympic pool (25 m) with water temperature at 28 &#176;C.
1. Preparation of swimmer
<ol>
	<li>Instruct the swimmer to avoid strenuous exercise for 24 h preceding each testing session.</li>
	<li>Instruct the swimmer to arrive at the pool in a rested and fully hydrated state &#8805;3 h postprandial.</li>
	<li>Instruct the swimmer to refrain from ingesting stimulant beverages and alcohol for 24 h prior to each test.</li>
</ol>
2. All-out tethered-swimming test
<ol>
	<li>Prepare the 500 kg load cell that will be used to measure the highest force that the swimmer can exert during two trials comprising 30 s of all-out swimming21.
	<ol>
		<li>Open the N2000PRO Software (Power Din Pro - CEFISE) program on the computer.</li>
		<li>Open the Help Menu to verify the communication link between the computer and the load-cell analyzer.
		<ol>
			<li>Observe a green signal that indicates that the connection to the RS232 interface is well established.</li>
			<li>Set the countdown to start the test depending on circumstances.</li>
			<li>Set the sampling duration. Set the rest interval. Set the frames per second at 100 Hz.</li>
			<li>Set the unit of force measurement at N or kg depending on personal preference. Set the acquisition time in milliseconds.</li>
		</ol>
		</li>
		<li>Calibrate the load cell22 with 0 and 10 kg loads with the swimmer outside pool.</li>
		<li>Attach a load cell to the start block via the L-shaped flattened iron bar that is designed by CEFISE specifically for tethered-swimming measurements.</li>
		<li>Attach one end of the inelastic rope to the load cell and the other end to the swimmer by means of the custom-designed belt (CEFISE), which has ropes attached to both hips such that leg kicking will not interfere with the force measurement.</li>
	</ol>
	</li>
	<li>Prepare the swimmer for performance of the two-trial test.
	<ol>
		<li>Provide instructions to the swimmer regarding correct performance of all-out front-crawl swimming (e.g., prevent the head and trunk from rising while swimming as rapidly as possible, concentrate on kicking at a maximal rate in addition to maximal stroking, etc.).</li>
		<li>Instruct the swimmer to perform stretching and arm/leg swings at poolside in preparation.</li>
		<li>Instruct the swimmer to enter the pool and perform a standard warm-up protocol comprised of front-crawl swimming for 800 m at a light intensity with care taken to avoid engendering any lingering effects that could influence the results of the test.</li>
		<li>Allow the swimmer to exit the pool and rest at poolside for 10 min.</li>
		<li>Secure the belt around swimmer&#39;s waist. Attach the free end of the inelastic rope to the belt.</li>
		<li>Determine the load required to maintain the swimmer&#39;s body horizontally with a minimum amount of tension on the measurement system (loadbase).</li>
		<li>Signal the swimmer to begin Trial #1 of the test.</li>
	</ol>
	</li>
	<li>Monitor the swimmer during performance of the test.
	<ol>
		<li>Provide verbal encouragement to swimmer throughout 30 s test.</li>
		<li>Signal the swimmer to end the test. Detach the swimmer from the inelastic rope.</li>
		<li>Instruct the swimmer to perform a standard cool-down protocol comprised of front-crawl swimming at a light intensity.</li>
		<li>Allow the swimmer to rest for 30 min at poolside.</li>
		<li>Reattach the swimmer to the inelastic rope.</li>
		<li>Signal the swimmer to start Trial #2 of the test which is identical to Trial #1 (30 s of all-out swimming).</li>
		<li>Signal the swimmer to end the test.</li>
		<li>Instruct the swimmer to perform a standard cool-down protocol comprised of front-crawl swimming at a light intensity.</li>
		<li>Allow the swimmer to exit pool.</li>
	</ol>
	</li>
	<li>Analyze the data collected during the two-trial test.
	<ol>
		<li>Apply the smoothing process to the data using the N2000PRO software package23.</li>
		<li>Calculate the peaks of the wave-frequency signal from the force-time sinusoidal curve (range, sine 80&#176;-100&#176;) above loadbase for Trials #1 and 2.</li>
		<li>Define the averaged peaks of the force-time wave-frequency signal in the first 5 s and entire 30 s, respectively, as the peak force (Fpeak) and average force (Favg) for each trial.</li>
		<li>Use the higher values for Fpeak and Favg for further calculations.</li>
	</ol>
	</li>
</ol>
3. Incremental tethered-swimming test
<ol>
	<li>Calculate the loads that will be used to resist the swimmer&#39;s forward displacement during the incremental test.
	<ol>
		<li>Calculate the starting load as 30% of the Favg above loadbase.</li>
		<li>Calculate the increments to be applied per 60-s stage as 5% of Favg above loadbase.</li>
	</ol>
	</li>
	<li>Prepare the automated portable metabolic unit for data collection.
	<ol>
		<li>Open the unit&#39;s software.</li>
		<li>Verify the communication link between the computer and the automated portable metabolic unit.</li>
		<li>Power on unit and allow to warm up for 45 min. Ensure that batteries are fully charged.</li>
		<li>Perform calibration of unit for environmental air24.</li>
		<li>Perform calibration of unit for reference O2 (16%), CO2 (5%) and N (balance) concentrations24.</li>
		<li>Perform mask time-delay calibration24.</li>
		<li>Perform calibration of turbine with 3 L syringe24.</li>
		<li>Enter the subject data, ambient temperature and humidity.</li>
	</ol>
	</li>
	<li>Prepare the swimmer for performance of the incremental test.
	<ol>
		<li>Install a facemask and a snorkel on the swimmer.</li>
		<li>Instruct the swimmer to rest at poolside for 10 min to collect &#34;baseline&#34; gas exchange and ventilatory data.</li>
		<li>Instruct the swimmer to enter the pool and perform a standard warm-up protocol comprised of front-crawl swimming at a light intensity.</li>
		<li>Secure a belt around the swimmer&#39;s waist. Attach an inelastic rope to the belt with the other end of rope attached to the loading system.</li>
		<li>Instruct the swimmer that once the test begins to use the two markers on the bottom of the pool for reference points, which allow them to maintain a relatively-fixed position (e.g., &#177; 1 m from the desired position).</li>
		<li>Signal the swimmer to begin the test.</li>
	</ol>
	</li>
	<li>Monitor the swimmer during performance of the incremental test. 
	NOTE: A research assistant who is experienced in monitoring this type of testing should hold the gas-analysis unit at poolside being cognizant to do so without impeding swimmer displacement and/or elevating the swimmer&#39;s head.
	<ol>
		<li>Increase load while timing the 60 s stages.</li>
		<li>Terminate the test and record the time to limit of exercise tolerance when the swimmer is no longer able to maintain the requisite position despite strong verbal encouragement from the testers.</li>
		<li>Use the time to limit of exercise tolerance to calculate stages completed.</li>
		<li>Record loads for each stage and peak load.</li>
		<li>Detach the swimmer from the inelastic rope.</li>
		<li>Instruct the swimmer to perform a standard cool-down protocol comprised of front-crawl swimming at a low-to-moderate intensity.</li>
		<li>Allow the swimmer to exit the pool.</li>
	</ol>
	</li>
	<li>Analyze the data collected during the incremental test.
	<ol>
		<li>Smooth breath-by-breath gas-exchange data that were collected before and during test using the unit&#39;s software program.</li>
		<li>Export gas-exchange data in consecutive 9 s bin averages.</li>
		<li>Perform three-point rolling average on consecutive 9 s bin averages for V&#775;O2.</li>
		<li>Record highest three-point rolling-average value as the V&#775;O2peak.</li>
		<li>Using final three-point rolling-average value for each completed stage, calculate V&#775;O2-load relationship via linear regression. Exclude data from end stages of test if a V&#775;O2 plateau appears to be present (visual inspection).</li>
		<li>Using consecutive 9 s bin averages, determine V&#775;O2GET.
		<ol>
			<li>Determine the first disproportionate increase in the rate of CO2 production (V&#775;CO2) compared to V&#775;O2.</li>
			<li>Determine the increase in the ratio of the expired rate of ventilation (V&#775;E) to V&#775;O2 with no increase in the ratio of V&#775;E to V&#775;CO2.</li>
			<li>Determine the increase in end-tidal O2 tension with no fall in end-tidal CO2 tension.</li>
		</ol>
		</li>
		<li>Using consecutive 9-s bin averages, determine V&#775;O2RCP.
		<ol>
			<li>Determine the first disproportionate increase in V&#775;E compared to V&#775;CO2.</li>
			<li>Determine the decrease in end-tidal CO2.</li>
		</ol>
		</li>
		<li>Express V&#775;O2peak, V&#775;O2GET, V&#775;O2RCP and V&#775;O2-load slope in both absolute (L&#8729;min-1) and relative (to body mass; mL&#8729;min-1&#8729;kg-1) terms.</li>
		<li>Express V&#775;O2GET and V&#775;O2RCP in relative terms as a percentage of V&#775;O2peak.</li>
	</ol>
	</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+running+economy+following+intensified+training+correlates+with+reduced+ventilatory+demands.">Improved running economy following intensified training correlates with reduced ventilatory demands.</a> Medicine and Science in Sports and Exercise. 30 (8), 1250-1256 (1998).</li><li>Holmer, I., Lundin, A., Eriksson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+oxygen+uptake+during+swimming+and+running+by+elite+swimmers.">Maximum oxygen uptake during swimming and running by elite swimmers.</a> Journal of Applied Physiology. 36 (6), 711-714 (1974).</li><li>Bonen, A., Wilson, B. A., Yarkony, M., Belcastro, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximal+oxygen+uptake+during+free,+tethered,+and+flume+swimming.">Maximal oxygen uptake during free, tethered, and flume swimming.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 48 (2), 232-235 (1980).</li><li>Magel, J. R., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+oxygen+uptakes+of+college+swimmers.">Maximum oxygen uptakes of college swimmers.</a> Journal of Applied Physiology. 22 (5), 929-933 (1967).</li><li>Poole, D. C., Jones, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+maximum+oxygen+uptake+VO2max:+VO2peak+is+no+longer+acceptable.">Measurement of the maximum oxygen uptake VO2max: VO2peak is no longer acceptable.</a> Journal of Applied Physiology. 122 (4), 997-1002 (2017).</li></ol>

The authors have no disclosures to report.

An exercise challenge that involves enduring an incremental increase in WR until Tlim is reached is a standard testing protocol for assessment of endurance athletes. When such a test is performed with gradual, but rapid incrementation, it is particularly useful because in addition to the V&#775;O2max, gas exchange and ventilatory data collected during the test can be used to distinguish the region bounded by GET and RCP where acidosis is present, but arterial partial pressure of CO2 (PaCO...

An exercise test that involves an incremental increase in work rate (WR) from low to maximal (i.e., incremental exercise test; INC) provides the gold standard method of cardiorespiratory assessment for endurance athletes. In addition to the highest WR that the athlete can achieve (WRpeak), INC also allows for determination of the highest rate at which the individual can consume oxygen (O2) for that form of exercise (V&#775;O2peak) if gas exchange and ventilatory data are collected during the test1. The V&#775;O2peak represents the criterion measure of cardiorespiratory fitness. Moreover, analysis of gas exchange and ventilatory data collected as WR is increased provides a non-invasive way to identify the point at which blood-lactate concentration (blood [lactate]) increases above the baseline value (lactate threshold) and the point at which it begins to accumulate at an accelerated rate (lactate turnpoint)2. These metabolic breakpoints are estimated by determining the gas-exchange threshold (GET) and respiratory-compensation point (RCP), respectively3. Importantly, the GET provides a robust estimate of the point at which blood [lactate] initially increases whereas the &#34;hyperventilation&#34; that characterizes RCP is a more complex phenomenon that can be initiated by afferent input other than chemoreception per se. Consequently, conclusions based on identification of RCP should be made with caution.
When exercise is maintained at a constant rate of work (CWR), there are markedly different physiological response profiles based on the &#34;exercise-intensity domain&#34; within which the WR falls4,5. Specifically, achievement of a V&#775;O2 and blood [lactate] &#34;steady state&#34; is rapid in the moderate domain, delayed in the heavy domain and unattainable in the severe domain4,5. It is well established that the rate at which O2 can be consumed at GET during INC (V&#775;O2GET) serves as the metabolic rate that separates the moderate from heavy domain during CWR3,6. Although controversial, a number of recent observations indicate similar equivalence between the rate at which O2 can be consumed at RCP (V&#775;O2RCP) and heavy/severe separation7,8,9,10. Identification of V&#775;O2GET and V&#775;O2RCP from data collected during INC might, therefore, be useful for prescribing domain-specific training regimens for endurance athletes via metabolic rate with the caveat that aligning a metabolic rate with a specific work rate is more complex than simply doing so according to the V&#775;O2-work rate relationship derived from the incremental test8,11.
When the concept of testing to determine V&#775;O2max was initially explored, researchers had subjects perform bouts of track running to the limit of exercise tolerance (Tlim) at increasing speeds on separate days1. Research followed which confirmed that V&#775;O2max can also be determined from similar bouts performed to Tlim on the same day with rest periods interspersed12. Eventually, it was shown that a continuous protocol with WR increased in an incremental manner at specific time intervals (e.g., every 3 min) revealed the same V&#775;O2peak as the discontinuous tests13. Consequently, these &#34;graded exercise tests&#34; became the standard for determining this criterion measure of cardiorespiratory fitness. However, in 1981, Whipp and colleagues published research that indicated that for the purpose of V&#775;O2max measurement, INC could also be performed entirely in the non-steady state; that is, with WR increasing continuously as a &#34;smooth function of time&#34; (RAMP-INC)14. Unlike INC with extended stages and relatively large WR increases per stage, the gradual increase during RAMP-INC ensures that the &#34;isocapnic buffering region&#34; that separates GET and RCP will be clearly defined15. Furthermore, much like INC with stages, RAMP-INC can be used to assess &#34;exercise economy&#34; (i.e., the V&#775;O2 required per given WR); however, unlike INC with stages, in this case, it is the inverse of &#34;delta efficiency&#34; (i.e., the slope of the V&#775;O2-WR relationship) that is used for this purpose11 with consideration given to the fact that due to the complexities of the V&#775;O2 response to work rates across the intensity spectrum, this parameter will not be an immutable feature of INC per se (e.g., RAMP-INC initiated from different baseline work rates or characterized by different ramp slopes) or CWR exercise16.
For general fitness testing, INC is usually performed on a leg ergometer or treadmill because these modalities are more available and leg cycling and walking/running are familiar to the average person. Moreover, administration of RAMP-INC requires the ability to increase WR continuously in small increments (e.g., 1 W every 2 s); hence, an ergometer (typically leg cycling) is best suited for this type of testing. However, athlete assessment is more complex because athletes must be tested while performing the specific mode of exercise required for their sport. For cyclists and individuals who participate in sports that involve running, this is not problematic because of the accessibility and applicability of the aforementioned testing machines. Conversely, ecologically-valid testing with gas exchange and ventilatory data collection and the gradual WR incrementation required for RAMP-INC is more challenging when assessing aquatic athletes.
Prior to the advent of automated collection systems, gas-exchange assessment of swimmers was often performed using Douglas-bag collection following a maximal swim17. Once automated systems were developed, &#34;real-time&#34; collection took place, but not under &#34;real-swimming&#34; conditions (e.g., while swimmers swam in a flume which controlled WR)17. Unfortunately, the former method has inherent limitations due to the assumptions of &#34;backward extrapolation&#34; while the latter raises concerns regarding the degree to which flume swimming changes technique17. The current state of the art involves the use of portable breath-by-breath collection systems which move with the swimmer alongside the pool during free swimming17. While this type of measurement improves ecological validity, gradual WR incrementation is challenging. Indeed, INC during free swimming typically involves intervals of set distance (e.g., 200 m) at progressively-increasing velocities14,15. This means that a test consists of lengthy stages with large unequal WR increments. It is, therefore, not surprising that only a single metabolic breakpoint (typically called the &#34;anaerobic threshold&#34;) is reported by researchers who employ this test18,19. Instead, we have recently shown that both V&#775;O2GET and V&#775;O2RCP can be determined from data collected while swimmers performed stationary swimming in a pool against a load that was increased gradually and rapidly (i.e., incremental tethered swimming)20. While the unique breathing pattern that is present during swimming might render the aforementioned breakpoints harder to identify compared to typical modes of assessment (personal observation), we believe that this method of testing might be suitable as a &#34;swim ergometer&#34; that can be used for cardiorespiratory assessment of swimmers in a manner similar to how a stationary cycle is used for cyclists. Indeed, we have shown that V&#775;O2GET, V&#775;O2RCP and exercise economy (as indicated by the V&#775;O2-load slope) can all be determined from the rapidly incremented tethered-swimming protocol that is described below20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-L syringe</td>
			<td>Hans Rudolph</td>
			<td></td>
			<td>Calibration device</td>
		</tr>
		<tr>
			<td>Aquatrainer</td>
			<td>COSMED</td>
			<td></td>
			<td>Snorkel system/gas-exchange measurement</td>
		</tr>
		<tr>
			<td>K4b2</td>
			<td>COSMED</td>
			<td></td>
			<td>Portable CPET unit/gas-exchange measurement</td>
		</tr>
		<tr>
			<td>N200PRO</td>
			<td>Cefise</td>
			<td></td>
			<td>Software program for analysis of force signal</td>
		</tr>
		<tr>
			<td>Pacer 2 Swim</td>
			<td>Kulzer TEC</td>
			<td></td>
			<td>Swimming velocity management/underwater LED line</td>
		</tr>
		<tr>
			<td>Tether-system</td>
			<td>Own design</td>
			<td></td>
			<td>Pulley-Rope system/loading management</td>
		</tr>
		<tr>
			<td>Tether attachment</td>
			<td>CEFISE</td>
			<td></td>
			<td>Bracket for attachment to swimmer</td>
		</tr>
	</tbody>
</table>

a rapidly incremented tethered-swimming maximal protocol for cardiorespiratory assessment of swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

The data presented in Table 1 and depicted in Figures 1-4 represents the response profiles observed for a male swimmer (age, 24 years). At the time of data collection, the swimmer had been training for competitive swimming for 7 years. His specialty was short-distance (i.e., 50 m and 100 m) freestyle events.
The initial load on INC was set at a load that exceeded that which was required for this...

Watch this Scientific Journal Video about A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers at JoVE.com

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

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Research

JoVE Journal

Biochemistry

8.7K Views.  São Paulo State University (UNESP) at Bauru. As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

Video: A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...