Authors acknowledges the Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan for providing the necessary facilities to complete this study.

1. Screening of the oils, surfactants, and cosurfactants
<ol>
	<li>Screening of the oils, surfactants, and co-surfactants for drug solubility
	<ol>
		<li>Mix 100 mg of rosuvastatin separately in 1 mL of different omega-3 fatty acids-rich oils (fish oil, olive oil, sesame oil, and linseed oil) and 1 mL of surfactants and cosurfactants (Tween 80, Capryol PGMC, PEG 400, and ethanol) by vortexing for 5 min at a fixed-speed of 2,500 rpm. Then, place in a shaking water bath for 48 h at 50 &#176;C.</li>
		<li>After shaking, let the mixture settle for at least 6 h at room temperature so that the undissolved drug is precipitated. Take 0.1 mL of supernatant by using a micropipette and dilute up to 1 mL with methanol.</li>
		<li>Analyze by using a UV-visible spectrophotometer at 242 nm and calculate the concentration by adding the absorption to the straight-line equation of the calibration curve19,20. 
		NOTE: Establish the calibration curve by preparing a stock solution of 100 &#181;g/mL and making serial dilutions up to 0.5 &#181;g/mL. Absorbance is taken for all dilutions and a graph is plotted between absorbance and concentration in a spreadsheet. The straight-line equation (y = mx + b) of the graph is rearranged so that value of absorbance (y) can be used to calculate the unknown concentration (x)19,20.</li>
	</ol>
	</li>
	<li>Screening of the surfactant and co-surfactants for miscibility with oil
	<ol>
		<li>Mix the surfactant and the co-surfactant (Smix) in a 3:1 ratio. Add Smix and oil in different ratios; mix and heat up to 50 &#176;C to ensure homogenization.</li>
		<li>Take 0.1 mL from each mixture and dilute with 25 mL of distilled water in a glass test tube.</li>
		<li>Invert the test tube; the number of inversions at which an emulsion is formed represents the emulsification efficacy and ease of emulsification. Measure the transparency (T%) by measuring the emulsion at 650 nm with a UV-visible spectrophotometer using distilled water as a blank21.</li>
	</ol>
	</li>
</ol>
2. Construction of the pseudo ternary phase diagram
<ol>
	<li>Mix the surfactants and the co-surfactants in various volume ratios (1:0, 1:1, 1:2, 1:3,1:4, and 1:5) to form surfactant mixtures (Smix). Add oil to the Smix in separate vials at various volume ratios of 1:1,1:2,1:3,1:4,1:5,1:6,1:7, 1:8, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and mix by vortexing.</li>
	<li>Add oil and Smix mixtures in a 10 mL glass vial and heat up to 50 &#176;C with constant stirring at 300 rpm for optimal mixing.</li>
	<li>Cool the mixture to 37 &#176;C and transfer 1 mL from it to a 250 mL water beaker and add dropwise the preheated distilled water (37 &#176;C) under gentle stirring at 50 rpm. Examine the dispersion visually and the mixture that forms a bluish transparent nanoemulsion is regarded as SNEDDS21.</li>
	<li>Construct the pseudoternary plot at different Smix ratios (Supplementary Table 1) through software (e.g., Triplot).</li>
</ol>
3. Optimization via software using a response surface methodology (RSM)
<ol>
	<li>Select three independent variables as oil (A), surfactant (B) and co-surfactant, and run the software for optimization and screening. Then, select the flexible design by choosing &#39;NO&#39; to a hard-to-change selection.
	<ol>
		<li>Observe the effect of these factors critically on dependent variables such as particle size (Y1, nm), zeta potential (Y2, mV), emulsification time (Y3, s) as well as entrapment efficiency (Y4, %) as mentioned in Table 2. 
		NOTE: The runs were increased until the warning signs disappeared and the software itself selected the Box-Benhken design for optimization.</li>
	</ol>
	</li>
	<li>Select the higher and lower values as -1 identifying the lowest variable value, whereas +1 depicts the highest value. The mid-value depicted the middle value. Box-Benhken design suggested a total of 17 runs with five center points as a measure to reduce the error.</li>
	<li>Record the responses to individual runs and fit to linear, 2F1, and quadratic models to ensure the best-fitted model. Generate polynomial equations and utilize to make the inference on the basis of the magnitude of the coefficient corresponding numerical signs.</li>
	<li>Display data of polynomial regression as 3-D plots. Evaluate the best fitted data model by comparing adjusted R2 and predicted R2 value 22. 
	NOTE: The required selection criteria of the formulation was based on grounds of maximum entrapment efficiency, less particle size, higher zeta potential, and minimum emulsification time frame. 
	Y = &#946;0 + &#946;1A + &#946;2B + &#946;3C + &#946;12AB + &#946;13AC + &#946;23BC + &#946;11A2 + &#946;22B2 + &#946;33C2 Eq. (1) 
	Here, &#946;0 appeared as intercept, Y as a response, ad &#946;1, &#946;2, and &#946;3 as linear coefficients. &#914;11, &#946;22, and &#946;33 as quadratic terms and squared coefficients while &#946;12, &#946;13, and &#946;23 as interaction coefficients. A, B, and C employed as independent variables that were selected from results of preliminary study.</li>
</ol>
4. Characterization
<ol>
	<li>Thermodynamic stability studies
	<ol>
		<li>Store the diluted SNEDDS at 4 &#176;C in a refrigerator, and then transfer it to a 50 &#176;C incubator . Perform six such cycles, each cycle being equal to or greater than 48 h.</li>
		<li>Examine the formulation for phase separation23.</li>
	</ol>
	</li>
	<li>Centrifugation test
	<ol>
		<li>Centrifuge the diluted SNEDDS for 30 min at 3,500 rpm at -4 &#176;C.</li>
		<li>Examine the SNEDDS at room temperature for phase separation or sedimentation of drug24.</li>
	</ol>
	</li>
	<li>Dispersibility test for self-emulsification efficacy
	<ol>
		<li>Add 1 mL of the formulation dropwise to 500 mL of double distilled water maintained at 37 &#176;C and 50 rpm. Note the time in which a clear homogenous emulsion is formed by visual inspection.</li>
		<li>Perform a visual assessment according to the following grading system25. 
		Grade 1: a clear bluish emulsion with emulsification time &#60;1 min 
		Grade 2: a bluish-white emulsion with emulsification time &#60;1 min 
		Grade 3: milky appearance with emulsification time &#60;2 min 
		Grade 4: gray, white appearance oil on top with emulsification time &#62;2 min 
		Grade 5: emulsification failure with larger oil droplet on top. 
		NOTE: The time required for emulsification or rate of emulsification is vital to emulsification efficacy. The test is performed in a USP type II dissolution apparatus.</li>
	</ol>
	</li>
	<li>Robustness to dilutions
	<ol>
		<li>Dilute the optimized SNEDDS formulation 50, 100, and 1,000 times with different carriers such as distilled water, 0.1 N HCl (pH 1.0) to mimic gastric pH and phosphate buffer (pH 6.8) to mimic intestinal pH.</li>
		<li>Thoroughly mix the diluted mixtures and set them aside for 12 h.</li>
		<li>Visually examine the formulations for drug precipitation, phase separation, and any other stability issue26,27.</li>
	</ol>
	</li>
</ol>
5. In vitro dissolution studies
<ol>
	<li>Perform the release of drug from SNEDDS and pure drug suspension by using a water shaking bath maintained at 37 &#176;C and 50 rpm.</li>
	<li>Soak the dialysis membranes in the respective media solution for 24 h before the drug dissolution assay to attain good integrity and activation. Fill the drug suspension (in water) and SNEDDS equivalent to 10 mg of rosuvastatin in the dialysis membrane; tie and place in separate beakers (50 mL).</li>
	<li>Take 1 mL of the sample at specific intervals and replenish the beaker with fresh media (1 mL) after each sample.</li>
	<li>Filter the drawn samples and analyze by using a UV-visible spectrophotometer at 242 nm21,28,29,30.</li>
</ol>

The authors have nothing to disclose.

This study was designed to explore the potential of omega-3 fatty acids-rich oil, such as fish oil, sesame oil, olive oil, and linseed oil to act as drug carriers. Self-nanoemulsification was selected as a preferred technique for fabricating the delivery system that lacks water, making it more stable than classical emulsion systems32. Omega-3 fatty acids-rich oils are known for their beneficial health effects18. They have been used as a supplement for various diseases such ...

Lipids have been used for centuries to increase gastrointestinal absorption of water insoluble components of food and medicines1. Emulsions are the formulations used most widely for oral, intravenous (nutritional supplementation), and topical use2. A variety of lipids (fats and oils) are used in the manufacturing of pharmaceutical emulsions and lipid-based self-nanoemulsifying drug delivery systems (SNEDDS). Self-emulsification techniques are widely adopted in pharmaceutical sciences for transmucosal drug delivery. Unlike emulsions, SNEDDS consist of an oil and a surfactant mixture that self-emulsifies in an aqueous medium of the stomach to form emulsion droplets3. They can load lipophilic drugs in the oil phase and prevent them from degrading in the stomach environment4. SNEDDS have been shown to effectively enhance the bioavailable fraction of lipophilic drugs (four to six folds) by enhancing solubility and permeability5,6. The absence of an aqueous phase in SNEDDS offers significant advantages in terms of ease of manufacture and stability as compared to emulsions that are metastable dispersions prone to chemical degradation7. Many lipid excipient combinations are commercially available due to their desirable characteristics8,9.
Cardiovascular disorders are a leading cause of mortality worldwide10 and, hyperlipidemia causes the blood vessels to obstruct blood flow due to thickening of the blood vessels11. Increased dietary lipid uptake and a sedentary lifestyle are the major risk factors for development of hyperlipidemia. In addition to this, lipids have also been shown to directly damage the myocardium of the heart leading to non-ischemic heart failure12. Rosuvastatin is a potent hypolipidemic drug that belongs to the statin class and inhibits cholesterol synthesis leading to the lowering of lipid levels for the treatment of hyperlipidemia/dyslipidemia13. Rosuvastatin is a biopharmaceutical classification system (BCS) class II with poor aqueous solubility (0.01796 mg/mL)14. Recent advances in pharmaceutical research have recognized that lipids used in drug delivery can disturb the lipid profile of patients. The role of emulsions to increase low- and high-density lipoproteins and free cholesterol was demonstrated in the late twentieth century15. In addition to this, lipid-based drug delivery systems have shown to increase triglycerides16 and other lipid metabolites in the blood17. Therefore, there is a dire need to develop pharmaceutical formulations of oils that are unable to disturb the lipid profile of cardiovascular and hyperlipidemic patients.
Fish oil is a rich source of omega-3 fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Fish oil has shown many health effects with substantial evidence of its beneficial role in cardiovascular and nervous systems18. The aim of the study was to utilize fish oil as an alternative to the conventional oils to formulate SNEDDS for the delivery of a lipophilic drug, rosuvastatin. No previous study has employed fish oil as a carrier to formulate drug delivery systems. Appropriate formulation and processing parameters were selected, and optimization was performed using design expert software.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium acetate</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>A1542</td>
			<td>Analytical grade</td>
		</tr>
		<tr>
			<td>Capryol PGMC</td>
			<td>Gattefoss&#233;, France</td>
			<td>RT9P9S09QI</td>
			<td>Analytical grade</td>
		</tr>
		<tr>
			<td>Design Expert Software</td>
			<td>StatEase, United States</td>
			<td>Version 12.0.3.0</td>
			<td>Analytical software (freely available for subscription)</td>
		</tr>
		<tr>
			<td>Dialysis tubing (12,000 Daltons MWCO)</td>
			<td>Visking, UK</td>
			<td>12000.02.30</td>
			<td>Pure regenerated natural cellulose membranes with 12,000 Daltons MWCO</td>
		</tr>
		<tr>
			<td>Dissolution apparatus</td>
			<td>Memmert, Germany</td>
			<td>SV 1422</td>
			<td>USP type II dissolution apparatus</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Honeywell, Germany</td>
			<td>24194</td>
			<td>Analytical grade</td>
		</tr>
		<tr>
			<td>Fish oil</td>
			<td>Wilshire Labs Pvt(Ltd), Pakistan</td>
			<td>not applicable</td>
			<td>Received as gift sample.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>BDH Laboratories Ltd, UK</td>
			<td>BDH3036-54L</td>
			<td>Analytical grade</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Honeywell, Germany</td>
			<td>34966</td>
			<td>Analytical grade</td>
		</tr>
		<tr>
			<td>Refrigerator (Pharmaceutical)</td>
			<td>Panasonic, Pakistan</td>
			<td>MPR-161 DH-PE</td>
			<td>Refrigerator for storage at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Rosuvastatin calcium</td>
			<td>Searle Pharmaceuticals Pvt(Ltd) Pakistan</td>
			<td>not applicable</td>
			<td>Received as gift sample.</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Honeywell, Germany</td>
			<td>38215</td>
			<td>Analytical grade</td>
		</tr>
		<tr>
			<td>Span 80</td>
			<td>BDH Laboratories Ltd, UK</td>
			<td>MFCD00082107</td>
			<td>Analytical grade</td>
		</tr>
		<tr>
			<td>Triplot Software</td>
			<td>MS Excel spreadsheet developed by Tod Thompson</td>
			<td>Triplot Ver. 4.1.2</td>
			<td>Analytical software (freely available)</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P1754-500ML</td>
			<td>Analytical grade</td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer</td>
			<td>Dynamica, UK</td>
			<td>Halo DB-20</td>
			<td>Double beam spectrophotometer</td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Memmert, Germany</td>
			<td>WNB 7</td>
			<td>Water batch for heating up to 70 &#176;C</td>
		</tr>
	</tbody>
</table>

self-nanoemulsification of healthy oils to enhance the solubility of lipophilic drugs

The low aqueous solubility of many drugs reduces their bioavailability in the blood. Oils have been used for centuries to enhance the solubility of drugs; however, they can disturb the lipid profile of the patients. In this study, self-nanoemulsifying drug delivery systems of omega-3 fatty acids-rich oils are prepared and optimized for the delivery of lipophilic drugs. Rosuvastatin, a potent hypolipidemic drug, was used as a model lipophilic drug. Fish oil showed more than 7-fold higher solubility of rosuvastatin than other oils and therefore it was selected for the development of self-nanoemulsifying drug delivery systems (SNEDDS). Different combinations of surfactants and co-surfactants were screened and a surfactant mixture of Tween 80 (surfactant) and Capryol PGMC (cosurfactant) were selected for compatibility with fish oil and rosuvastatin. A pseudoternary phase diagram of oil, surfactant, and co-surfactant was designed to identify the emulsion region. The pseudoternary phase diagram predicted a 1:3 oil and surfactant mixture as the most stable ratio for the emulsion system. Then, a response-surface methodology (Box-Behnken design) was applied to calculate the optimal composition. After 17 runs, fish oil, Tween 80, and Capryol PGMC in proportions of 0.399, 0.67, and 0.17, respectively, were selected as the optimized formulation. The self-nanoemulsifying drug delivery systems showed excellent emulsification potential, robustness, stability, and drug release characteristics. In the drug release studies, SNEDDS released 100% of the payload in around 6 h whereas, release of the plain drug was less than 70% even after 12 h. Therefore, omega-3 fatty acids-rich healthy lipids have enormous potential to enhance the solubility of lipophilic drugs whereas, self-emulsification can be used as a simple and feasible approach to exploit this potential.

Herein, nanoformulation of omega-3 fatty acids-rich fish oil are prepared and optimized by self-emulsification with different surfactants and co-surfactants. Figure 1 shows the solubility of rosuvastatin in different oils, surfactants, and co-surfactants. Based on solubility, fish oil was selected as the oil, Tween 80 as the surfactant, and Capryol PGMC as the co-surfactant in the following studies. Table 1 shows the screening of Smix at different ratios to emulsify fish oil...

Watch this Scientific Journal Video about Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs at JoVE.com

Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs

Oils used for drug delivery applications can disrupt the lipid profile of patients, which is undesirable in cardiovascular diseases. Omega-3 fatty acids-rich oils are a healthy alternative to conventional oils and have enormous potential for self-emulsified drug delivery systems.

The low aqueous solubility of many drugs reduces their bioavailability in the blood. Oils have been used for centuries to enhance the solubility of drugs; ...

self-nanoemulsification-healthy-oils-to-enhance-solubility-lipophilic

Research

JoVE Journal

Biology

963 Views.  Quaid-i-Azam University. Oils used for drug delivery applications can disrupt the lipid profile of patients, which is undesirable in cardiovascular diseases. Omega-3 fatty acids-rich oils are a healthy alternative to conventional oils and have enormous potential for self-emulsified drug delivery systems.

Video: Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Diagonal Method to Measure Synergy Among Any Number of Drugs

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many doses, allow sensitive measurement of drug interactions. However, these assays are costly and do not scale well for measuring interaction among many drugs. Several recent studies have reported drug interaction measurements using a diagonal sampling of the traditional checkerboard assay. This alternative methodology greatly decreases the cost of drug interaction experiments and allows interaction measurement for combinations with many drugs. Here, we describe a protocol to measure the three pairwise interactions and one three-way interaction among three antibiotics in duplicate, in five days, using only three 96-well microplates and standard laboratory equipment. We present representative results showing that the three-antibiotic combination of Levofloxacin + Nalidixic Acid + Penicillin G is synergistic. Our protocol scales up to measure interactions among many drugs and in other biological contexts, allowing for efficient screens for multi-drug synergies against pathogens and tumors.

In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations.

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...