eamag/protocols-with-steps huggingface dataset:
TITLE: Slice preparation
AUTHORS: Nicola Biagio Mercuri

[DESCRIPTION]
Slice preparation for electrophysiology

[STEPS]
SECTION: Animal sacrifice and surgical procedure
1. Anesthetize the animal under halothane
SECTION: Animal sacrifice and surgical procedure
2. Decapitate the animal with a guillotine
SECTION: Animal sacrifice and surgical procedure
3. Gently open the bones of the skull and remove the brain, dropping it into a beaker containing ice-cold ACSF continuously bubbled with an O2/CO2 (95/5%) gas mixture
SECTION: Animal sacrifice and surgical procedure
3.1. ACSF composition (in mM): 126 NaCl, 2.5 KCl,1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose and 25 NaHCO3. 
SECTION: Brain slices cutting
4. After 1 min, place the brain on a petri dish with filter paper soaked with ACSF, and then separate the region of interest with a razor blade
SECTION: Brain slices cutting
5. Stick the peace of brain on the appropriate magnetic surface provided by the Leica VT1200S microtome with cyanoacrylate glue, and place it in the cutting chamber filled with ice-cold ACSF
SECTION: Brain slices cutting
6. Proceed with the slicing procedure (250 μm thick) and remove the surrounding tissue in each slice, to isolate the area of interest
SECTION: Brain slices cutting
7. The slices are then left to recover for at least 1hr in saturated ACSF at 32°C
TITLE: Mehlich 2 Solution
AUTHORS: Hsin-Mao   Wu

[DESCRIPTION]
Soil extraction

[STEPS]
SECTION: Mehlich 2 Solution
1. 
在500 mL DI Water中加入0.56 g NH4F、10.7 g NH4Cl

SECTION: Mehlich 2 Solution
2. 再加1 mL HCl、11.5 mL 冰醋酸並加水至1 L
TITLE: Preparation of leukocytes by differential lysis of erythrocytes
AUTHORS: Mesut Karataş, Şenol Doğan, Emrulla Spahiu, Adna Ašić, Larisa Bešić, Yusuf Turan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Leukocytes are isolated by centrifugation after specific lysis of erythrocytes</div></div>

[STEPS]
?. Centrifuge    EDTA blood to pellet all cells
10 ml
Centrifuge: 1500 33, 10 min, 4 10
?. Remove plasma into a clean container and freeze.
?. Restore original blood volume with 0.9% saline and transfer the blood suspension into a   conical centrifuge tube.
50 ml
?. Add   cold lysis buffer
40 ml
?. Stand  , mixing occasionally, until erythrocytes are lysed (the red cell suspension remains red in colour, but becomes transparent). This should take only about  to
on ice
?. Centrifuge
Centrifuge: 1500 33, 5 min, 4 10
?. Discard supernatant and resuspend leucocyte pellet in   cold lysis buffer.
5 ml
?. Stand  for
on ice
?. Dilute cell suspension to  with cold 0.9% saline. Mix and centifuge
50 ml
Centrifuge: 1500 33, 5 min, 4 10
?. Discard supernatant, then resuspend leucocyte pellet in   0.9% saline. Care should be taken to obtain an even cell suspension without being too vigorous and causing cell disruption.
10 ml
?. Divide the cell suspension into two equal aliquots, into two  conical centrifuge tubes.
10 ml
?. Centrifuge
Centrifuge: 1500 33, 5 min, 4 10
?. Remove all supernatant and dry walls of centrifuge tube with a tissue.
?. Store leucocyte pellets and plasma at  (or  )
-20 °C
-80 °C
TITLE: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes
AUTHORS: Dan Dou, Alexander Boecker, Erika L.F. Holzbaur

[DESCRIPTION]
Here, we plate, culture, and transfect human iPSC-derived excitatory glutamatergic neurons for the purpose of observing transport of axonal cargoes under spinning disk confocal microscopy. Protocol is largely as previously described (Boecker et al., 2020, 2021; Fernandopulle et al., 2018). For preceding differentiation of neurons, see “Protocol: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons” and “Protocol: iNeuron differentiation from human iPSCs.”

[STEPS]
SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
1. In advance, prepare 10x PLO stock.


 

 

SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
2. The day before plating, coat imaging dishes with 1x PLO solution (10x PLO stock diluted in ddH2O). 
 

SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
3. The day of plating, remove PLO solution from imaging dishes and wash twice with ddH2O.
SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
3.1. Add 2 mL of iNeuron culture media. 

BrainPhys supplemented with 
 

SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
3.2. Place dishes in cell culture incubator for >30 min.
SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
4. Rapidly thaw cryopreserved iNeurons in 37 °C water bath.
 

SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
5. Centrifuge to remove freezing media and resuspend cell pellet in iNeuron culture media.

 BrainPhys supplemented with

 

SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
6. Count cells and plate 300k neurons per 35 mm imaging dish.



SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
7. For Piggybac-delivered NGN2 neurons, include  10 micromolar (µM) 5-Fluoro-2′-deoxyuridine and 10 micromolar (µM) uridine at the time of plating to prevent survival of mitotic cells.
 


SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
8. Store neurons in cell culture incubator. Perform partial change of iNeuron media twice per week.
SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
9. On DIV18 (~4320 min prior to imaging), transfect iNeurons for imaging.
 




SECTION: Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
6.1. Add cells dropwise to the center area of the dish (so that they sink onto the glass, PLO-coated center).
TITLE: Immunohistochemistry- mounted slides
AUTHORS: John Tompkins, Don Hoover, Jeffrey Ardell, Kalyanam Shivkumar

[STEPS]
?. Day 1Ø Using the PAP Pen, carefully draw a water barrier circle around the tissue sections on the slide – allow this circle to dry for several seconds or up to approx. one min. Ø Rinse slides with PBS (pH 7.3-7.4): 4 x 5 min eachØ Rinse slides with 0.5% BSA + 0.4% Triton X-100 in PBS): 1 x 10 minØ Remove slides one at a time and using a clean Kimwipe, carefully wipe around the tissue sections to dry the slide.Ø Place the slides into a black, covered slide incubation box/humidity boxØ Cover the tissue sections with blocking buffer (10% normal donkey serum in 1.0% BSA + 0.4% Triton X-100 + PBS)Ø Allow the sections to remain in blocking buffer for 1.5-2 hrs. at RTØ Pour off the blocking bufferØ Replace with primary antibody solution (antibody of choice diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)Ø Incubate tissue with primary antibody overnight in incubation box.
?. Ø Rinse slides with PBS: 4 x 5 minØ Rinse slides with 0.5% BSA + 0.4% Triton X-100 + PBS: 1 x 10 minØ Prepare fluorescent secondary antibody (secondary antibody should be diluted in 1.0% BSA + 0.4% Triton X-100 + PBS) (use Biotin-SP for the antibody that will later add Streptavidin to; if there is a second primary then use the normal secondary for it).Ø Cover the tissue with the secondary antibody solution and incubate for 2 hrs. at RT in the incubation box. *From this point on, use low light and/or cover tissues.*Ø Rinse slides with PBS: 4 x 5 minØ Remove excess PBS with a Kimwipe.Ø Prepare fluorescent Streptavidin secondary antibody (should be diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)Ø Cover the tissue with the fluorescent Streptavidin antibody solution and incubate for 2 hrs. at RT in the incubation box.Ø Rinse slides with PBS: 4 x 5 minØ Remove excess PBS with a Kimwipe.Ø Carefully add a drop of Citifluor (or equivalent) mounting medium to the center of the tissue and apply cover glass.Ø Seal cover glass with clear nail polish. For thicker tissue, add a weight before sealing.
?. Day 2
TITLE: Clustering of differentially expressed genes
AUTHORS: Ahmad Husaini AHS Suhaimi

[DESCRIPTION]
This differentially expressed genes clustering pipeline utilizes coseq v3.17 package (Rau & Maugis-Rabusseau, 2018) in R.


[STEPS]
SECTION: Clustering of differentially expressed genes (DEG) using Coseq package in R
1. Load the package (coseq).
 



SECTION: Clustering of differentially expressed genes (DEG) using Coseq package in R
2. Run Coseq on transformed and normalized counts.
Example: 
Performing clustering on bud data with expected clusters, K=5-16.
Clustering process is repeated for 10x.
 

SECTION: Clustering of differentially expressed genes (DEG) using Coseq package in R
3. Manually inspect the results and decide on the average number of clusters
Choose one clustering result to proceed with the subsequent steps
 

SECTION: Assigning clusters to transcripts
4. Retrieve and tabulate the clustering information based on the chose clustering from the previous step.
Example:
coseq_bud_logclr_1 is chosen as the best clustering
results_coseq_bud_logclr: the new table/vector.
 

SECTION: Assigning clusters to transcripts
5. Convert the vector into a data frame.
 

SECTION: Assigning clusters to transcripts
6. Create a column containing the assigned cluster number for each transcript in the read count data frame. 
Example:
the new column: bud_logclr
the data frame with read counts: tcounts_logclr_exp_bud_ORF_scTMM
TITLE: Selection and assessment measures used in medical school admissions: A systematic review.
AUTHORS: Alec Knight, Hamda Shahzad, Ricardo Twumasi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Introduction</span></div><br/><div class = "text-block">Patterson et al. (2015) conducted a systematic review examining selection methods used in medical education. The researchers assessed the quality, along with the strengths and weaknesses, of common selection methods. The review evaluated the effectiveness of aptitude tests, academic records, personal statements, references, SJTS, personality and emotional intelligence assessments, interviews, MMIs and selection centres. </div><br/><div class = "text-block">Since the publishing of Patterson et al.’s review in 2015, a wealth of research has been completed on selection methods, including a number of longitudinal studies. The present systematic review aims to provide an updated analysis of the literature on the quality of selection methods in medical education. The current review will aim to overcome significant limitations present within the original review. The review will consider literature published from 2015 onwards, encompassing research that has so far not been included in a systematic review of this nature. Completing an updated review will allow medical education providers to understand how recent contributions to the literature may have changed our understanding of the quality of methods used. This will allow for a more comprehensive decision process of which selection methods should be used within medical education in the future.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Conflicts of interest</span></div><div class = "text-block">There are no conflicts of interest to report. There is no funding or sponsorship associated with this review.</div></div>

[STEPS]
TITLE: JMN-MSMP Notexin Muscle scRNAseq
AUTHORS: ccherry

[DESCRIPTION]
Murine notexin muscle injury followed by single cell RNA sequencing

[STEPS]
SECTION: Skeletal muscle injury and collection
1. In a given mouse, both tibialis anterior (TA) muscles were injected with 10 µl of notexin (10 µg/ml; Latoxan, France).
SECTION: Skeletal muscle injury and collection
2. At various days post injury, the mouse was sacrificed and the TAs were collected.  Each TA was proccessed independently to generate single cell suspensions.
SECTION: Generate single-cell suspensions
3. Muscles were enzymatically digested with 8 mg/ml Collagenase D (Roche, Basel, Switzerland) and 10 U/ml Dispase II (Roche, Basel, Switzerland) and then manually dissociated to generate cell suspensions. 
SECTION: Generate single-cell suspensions
4. Myofiber debris was removed by filtering the cell suspensions through a 100 µm and then a 40 µm filter (Corning Cellgro # 431752 and # 431750). 
SECTION: Generate single-cell suspensions
5. After filtration, erythrocytes were removed by incubating the cell suspension in erythrocyte lysis buffer (IBI Scientific # 89135-030).
SECTION: Generate single-cell suspensions
6. After digestion, the single-cell suspensions were washed and resuspended in 0.04% BSA in PBS at a concentration of 106 cells/ml. A hemocytometer was used to manually count the cells to determine the concentration of the suspension. 
SECTION: Single-cell RNA library preparation and sequencing
7. Single-cell RNA-sequencing libraries were prepared using the Chromium Single Cell 3’ reagent kit v3 (10x Genomics, Pleasanton, CA) following the manufacturer’s protocol. 
SECTION: Single-cell RNA library preparation and sequencing
8. Cells were diluted into the Chromium Single Cell A Chip to yield a recovery of 6,000 single-cell transcriptomes with <5% doublet rate. 
SECTION: Single-cell RNA library preparation and sequencing
9. Libraries were sequenced on the NextSeq 500 (Illumina, San Diego, CA).
TITLE: PMN- 06 - Culture of Human PMN - TNF-α production
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Published work using this protocol:</div><div class = "text-block"><span>- </span><span style = "font-weight:bold;">A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions.</span><span> Mabou Tagne A, Marino F, Legnaro M, Luini A, Pacchetti B, Cosentino M.Int J Mol Sci. 2019 Apr 13;20(8):1833. </span><span style = "font-weight:bold;">doi: 10.3390/ijms20081833</span></div></div>

[STEPS]
?. Isolate PMN according to the protocol PMN-01a or PMN-01b.
?. Resuspend PMN at 10x106cells/ml in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin (SOLUTION- 13).
?. Add  of cell suspension in a 5 ml tube for cell culture and incubate for  at  in 5% CO2. Cells were incubated in resting condition or in presence of stimulus. Stimulus (LPS 1 µg/ml and/or CBD 10-8-10-5M) in 5 ml tubes for cell culture. Other possible activating stimuli can be selected and included.
300 µl
37 °C
?. Centrifuge at
Centrifuge: 1500 34, 10 min
?. Harvest cell-free supernatant ( x2 aliquots) and cell pellet.
150 µl
?. Store the supernatant at -80°C until the TNF-α Enzyme Linked-Immuno-Sorbent Assay (ELISA) is performed.
?. Store cell pellet at -80°C for gene expression analysis (RT PCR).
TITLE: Western Blot Analysis
AUTHORS: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino

[DESCRIPTION]
Western Blotting is a technique for the immunodetection of proteins using antibodies with fluorescent or chemiluminescent detection.

[STEPS]
SECTION: Western Blot Analysis
1. Prepare protein extracts as previously described.
SECTION: Western Blot Analysis
2. Homogenize tissues in lysis buffer (0.33 Molarity (M) sucrose/8 millimolar (mM) Hepes, pH 7.4 and protease inhibitors) and quantify them using the BCA protein determination method (Bio-Rad, Hercules, CA). 

SECTION: Western Blot Analysis
3. Dilute protein samples to equivalent volumes containing 20 µg of protein and boil in an equal volume of Laemli SDS boiling buffer (Sigma) for 10 min.

SECTION: Western Blot Analysis
4. Load samples into a 9-12% SDS-polyacrilamide gel and separate it by electrophoresis for 180 min at 100 V. 

SECTION: Western Blot Analysis
5. Transfer the proteins to polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) for 90 min at 300 mA. 

SECTION: Western Blot Analysis
6. After blocking of nonspecific binding with 5% non-fat dry milk in TBST, probe the membranes  with primary antibodies and process. 
SECTION: Western Blot Analysis
7. Perform densitometric analysis using ImageQuantity One. 
SECTION: Western Blot Analysis
8. Normalize data to β-actin, normalize values of phosphorylated GSK-3β (pTyr216 GSK-3β); phosphorylated α-syn (pSer129 α-syn) and phosphorylated tau (pSer396 tau) to total GSK-3β, α-syn, and tau, respectively, before statistical analysis of variance and express values as percent changes (%) of WT controls.

SECTION: Western Blot Analysis
9. Dashed lines (in white) indicate discontinuous bands (nonsequential lanes) taken from the same blot, at the same molecular weight (mass – kDa) in order to better represent the mean signal from all values (5-6 individual blots/genotype/treatment) for that particular group. Corresponding control bands (loading controls) match experimental bands.
TITLE: PAS Stain
AUTHORS: Amanda Knoten, sanjay jain

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for PAS Staining of paraffin embedded or cryo preserved tissue sections</div></div>

[STEPS]
?. Deparaffinize and hydrate to water
?. Oxidize in periodic acid solution for 5 minutes
?. Rinse in distilled water
?. Place in Coleman’s Schiff reagent for 15 minutes
?. Rinse in running tap water for 10 minutes
?. Counterstain in Harris Hematoxylin for 3 minutes
?. Wash in running tap water until water runs clear
?. 10 dips in 0.5% HCl/70% Ethanol
?. Rinse in running tap water for 30 seconds
?. 10 dips in 1% Ammonia water for 30 seconds
?. 95% Ethanol, 2X, 2 minutes each
?. 100% Ethanol, 2X, 2 minutes each
?. Xylene, 2X, 2 minutes each
?. Coverslip with Cytoseal
TITLE: Overall protocol for top-down CZE-MS/MS of human heart tissue
AUTHORS: Jeannie  Camarillo, Bryon  Drown, Neil  Kelleher

[DESCRIPTION]
Overview description of the process for acquiring top-down CZE-MS/MS data on human heart tissue

[STEPS]
SECTION: Tissue Collection
1. Heart tissue was provided by University of Washington. Tissue was collected and prepared according to the following: 
 

SECTION: Sample Preparation
2. Perform LC based Proteomics on adjacent tissue sections:
 

SECTION: Data Acquisition
3. 
 

SECTION: Sample Preparation
4.
TITLE: Sterilizing the Surface of Seeds by Chlorine Gas
AUTHORS: Lynn  Doran

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol sterilizes the surface of seeds for use in clean culture conditions without damaging the seeds germination or growth ability.</div></div>

[STEPS]
?. Move an aliquot of seeds to be sterilized to a 2 mL microcentrifuge tube.  Label clearly.
If too many seeds are sterilized at once, seeds on the bottom layers may not be exposed to as much gas as top layers.
?. Document the seed vials identifications and order via photograph or notetaking.
Exposure to chlorine gas can cause some types of ink, both marker and printed, to fade.
?. Place a desiccator in the chemical fume hood.  Adjust the fume hood sash to a safe height.
?. Place the seed rack in a desiccator with no desiccant.  Open the tube lids.
?. Fill a 250 mL beaker with about  of bleach, using graduations on the side of the beaker is sufficiently accurate for this purpose.
160 mL
?. Quickly pipet  of hydrochloric acid into the beaker of bleach.  Pipet all 5 mLs in one volume to avoid losing chlorine gas.
5 mL
The reaction of bleach and hydrochloric acid will form chlorine gas.  Do not breath in vapors.  Ensure fume hood is in good operation.  Wear appropriate PPE when handling hydrochloric acid.
?. Close the lid of the desiccator.  If the desiccator contains any vacuum lines or vents, ensure that those valves are all in the closed position.
?. Allow the seeds to be exposed to the chlorine gas for a minimum of 3 hours.
?. Move the sealed desiccator to a biological safety cabinet that has been properly sterilized.
Do not use a laminar flow hood.  The biological safety cabinet will both keep the seeds sterile and protect you from any residual chlorine gas.  A laminar flow hood will keep the seeds sterile but will blow residual chlorine gas back out at the operator.
?. Open the desiccator slowly.
?. Close the seed vials.  Seed vials can be stored outside the biological safety cabinet but should only be opened in properly sterilized biological safety cabinets or laminar flow hoods to ensure that they remain sterile.
?. Place the beaker of bleach in the desiccator.
TITLE: two layer plating method
AUTHORS: Jiaxin Li

[STEPS]
?. [BEFORE STARTING]
Prepare 6 cm diameter petri plates containing solidified LB-agar and dry it in the intercellular incubator for 12 h.LB medium formulation：  AB1NaCl10g2TRYPTONE10g3YEAST EXTRACT5g4Agar15gTake equipping 1L as an example, other volume isometric adjustmentAntibiotic concentration:  ABCD1AbbreviationNameInitial&nbsp;concentration  mg/mlFinal concentration  mg/ml2Tctetracycline5503Gmgentamicin5050CaCl2 was added to give a final concentration of 2 mmol/L. Prepare 0.6% soft agar and store it in a refrigerator at 4℃.LB medium formulation：  AB1NaCl10g2TRYPTONE10g3YEAST EXTRACT5g4Agar6gTake equipping 1L as an example, other volume isometric adjustment.CaCl2 was added to give a final concentration of 2 mmol/L.
AB1NaCl10g2TRYPTONE10g3YEAST EXTRACT5g4Agar15g
ABCD1AbbreviationNameInitial&nbsp;concentration  mg/mlFinal concentration  mg/ml2Tctetracycline5503Gmgentamicin5050
AB1NaCl10g2TRYPTONE10g3YEAST EXTRACT5g4Agar6g
?. [BEFORE STARTING]
Preparation of SM Buffer  AB1NaCl5.8g2MgSO4·7H2O2g3Tris-HCl50ml（1M）4H2OTo 1 LAfter preparation, sub-Pack (50ml) and store in 4 degree refrigerator.
AB1NaCl5.8g2MgSO4·7H2O2g3Tris-HCl50ml（1M）4H2OTo 1 L
?. Shake the bacteria in advance, 5 ml each, culture overnight.
?. The final concentration of inducer was 0.1% by adding 1 ml bacterial liquid and appropriate amount of inducer into 50 ml centrifuge tube.
?. 37℃, 220 rpm shaker culture induction for 1 h
?. Set the temperature of water bath to 50℃
?. Take half of the beaker water, put the soft agar to be melted into the beaker, and heat it in the microwave oven.
?. Put the completely melted culture medium into the water bath to keep warm.
?. No inducer group: take warm soft agar (10ml/tube) and pour it into the tube containing 100 μl bacterial liquid, shake well and pour it into the plate containing solidified LB-agar.
?. Inducer group: add 100 μl 10% Ara and 10 μl 1mol/L IPTG to the tube containing 100 μl bacterial liquid, suck and mix well, pour in warm soft agar, shake and pour it into the plate containing solidified LB-agar.
?. Place the poured Petri dish for 30 min to solidify.
?. During the waiting process for coagulation, the phages stored at 4℃ were removed from the refrigerator and diluted with SM Buffer in a 1.5 ml centrifuge tube.The amount of addition can be referred to the following figure.
?. Add bacteriophage as needed and shake well.
?. Take 10 μl phage spot to the center of each grid of the culture dish, let it dry for 30min, and culture it upside down for 16-20h.
TITLE: 10% Triton X-100
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to attain 10% Triton X-100 which is utilized in several Immunohistochemistry (IHC) reagents to reduce the surface tension of the reagents and to make the cell membranes of the tissue more permeable.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]