Protocol for identifying protein synthesis activity in specific cell types of the testicular lumen

Summary

Protein synthesis could control spermatogenic cell fate transitions. Here, we present a protocol for visualization and quantification of newly synthesized proteins by click-chemistry-based immunofluorescence within specific spermatogenic cell types in the mice testicular lumen. We detail the processes for O-propargyl-puromycin (OPP) incorporation, antibody incubation, confocal microscope imaging, and subsequent quantification methods. This protocol is not limited to spermatogenic cells and can be adapted to investigate protein synthesis in other testicular cell types and various tissue-specific cell populations.

For complete details on the use and execution of this protocol, please refer to Zou et al.¹

Graphical abstract

Highlights

• •Protocol for visualizing protein synthesis
• •Click chemistry combined with cellular immunofluorescence
• •Analysis of protein synthesis levels in spermatogenic cells

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Protein synthesis could control spermatogenic cell fate transitions. Here, we present a protocol for visualization and quantification of newly synthesized proteins by click-chemistry-based immunofluorescence within specific spermatogenic cell types in the mice testicular lumen. We detail the processes for O-propargyl-puromycin (OPP) incorporation, antibody incubation, confocal microscope imaging, and subsequent quantification methods. This protocol is not limited to spermatogenic cells and can be adapted to investigate protein synthesis in other testicular cell types and various tissue-specific cell populations.

Before you begin

Visualization of protein synthesis

Proteins serve as the primary carriers of life activities and act as the direct executors of gene functions within cells. Increasing evidence suggests that the gene expression programs that govern cellular functions are ultimately dictated by the regulation of protein biosynthesis, rather than by transcriptional regulation.^2,3,4 The development of male germ cells is a complexly orchestrated process in which protein synthesis plays a crucial role. Evaluating protein synthesis levels in various spermatogenic cells is essential for understanding the significance of protein synthesis in the development of spermatogenic cells and the dysfunctions observed in the testis.

Click chemistry protocols have been utilized to measure nascent protein synthesis across various cell types.⁵ In this study, we present optimized protocols for visualizing cell-specific protein synthesis in mouse testes. This method involves isolating seminiferous tubules from testicular tissue, where OPP, an analog of puromycin, is incorporated into the C-terminus of newly synthesized peptides within the spermatogenic cells in their in-situ environment. Following this, specific cells are labeled using antibodies, and protein synthesis within these cells is detected through click chemiluminescence. This protocol can be further adapted to analyze the seminiferous lumen of mice at different ages and to assess the protein synthesis levels of various cell types within the lumen.

Institutional permissions

All procedures were approved by the Institutional Animal Care and Use Committee at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and Peking Union Medical College. For researchers who wish to use this protocol, they should comply with their institutional animal welfare guidelines and obtain the required permissions.

Mice

Timing: variable

All mice used for these experiments were C57BL/6J. Mouse age was determined based on the specific cell type examined. For spermatogonia, 7-day-old mice were selected; for spermatocytes, mice aged 2–3 weeks were chosen; round sperm cells were obtained from adult mice; and somatic cells were preferred from adolescent and adult mice. The time points for modified mice of varying genotypes were selected according to the functions that needed to be observed. In this example, wild-type mice at 4 days of age were selected.

Preparation of regents

Timing: 1 h

Stock solutions should be prepared ahead of use.

• 1.Prior to use, briefly centrifuge Click-iT OPP Reagent (Component A) and NuclearMask Blue Stain (Component G) to maximize reagent recovery.
• 2.To prepare a 10X stock solution of the Click-iT Reaction Buffer Additive (Component E), add 2 mL deionized water to the vial and mix until completely dissolved.

Note: After use, store any remaining stock solution at ≤–20°C. This stock solution is stable for up to 1 year.

• 3.Prepare 40 mL of 1X Click-iT OPP Reaction Buffer by transferring all of the solution in the Component C bottle (4 mL) to 36 mL of deionized water.
• 4.Rinse the Component C bottle with some of the diluted Click-iT OPP Reaction Buffer to ensure the transfer of all of the 10X concentrate.
• 5.To prepare smaller amounts of 1X Click-iT OPP Reaction Buffer, dilute an aliquot from the Component C bottle 1:10 with deionized water.

Note: After use, store any remaining 1X solution at 2°C–8°C. When stored as directed, 1X Click-iT OPP Reaction Buffer is stable for 6 months.

• 6.100 mg/mL CHX: Dissolve 100 mg of CHX into 1 mL of DMSO. Aliquot and store at −20°C.

Note: Allow vials to completely thaw and warm to room temperature before opening. All stock solutions were directly stored at −20°C or 4°C and repeated freezing/thawing cycles were avoided.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-DDX4 antibody (1:200)	Abcam	Cat# ab27591; RRID: AB_11139638
Goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (1:500)	Thermo Fisher Scientific	Cat# A-11001; RRID: AB_2534069
Chemicals, peptides, and recombinant proteins
Cycloheximide (CHX)	Cell Signaling Technology	Cat# 2112
BlockAid blocking solution	Thermo Fisher Scientific	Cat# B10710
Image-iT fixative solution	Thermo Fisher Scientific	Cat# R37814
Triton X-100	Sigma	Cat# 93443
Phosphate-buffered saline (PBS, pH 7.4)	Gibco	Cat# 10010023
Minimum essential medium α (MEMα)	Gibco	Cat# 12561056
Glial cell line-derived neurotrophic factor (GDNF)	PeproTech	Cat# 450-10
Basic fibroblast growth factor (bFGF)	PeproTech	Cat# 100-18B
Insulin	Sigma-Aldrich	Cat# I 2643
Bovine serum albumin(BSA)	MP Biomedicals	Cat# 810661
Fetal bovine serum (FBS)	HyClone	Cat# SH30396.03
Lipoprotein	Sigma-Aldrich	Cat# L4646
GlutaMAX	Gibco	Cat# 35050061
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES)	Gibco	Cat# 15630-080
Non-essential amino acids (NEAA)	Gibco	Cat# 11140-050
Sodium selenite	Sigma	Cat# S5261
Putrescine	Sigma	Cat# P5780
2-methoxyethanol (2-ME)	Sigma	Cat# M3145
Transferrin	Sigma	Cat# T0665
Ascorbic acid	Sigma	Cat# M4544
Critical commercial assays
Click-iT Plus OPP Alexa Fluor 594 protein synthesis assay kit	Thermo Fisher Scientific	Cat# C10457
Experimental models: Organisms/strains
Mouse; C57BL/6J, 4-days-old, male mice	Speford Biotechnology Company (Beijing, China)	N/A
Software and algorithms
ZEN lite version 2	Zeiss	https://www.zeiss.com/microscopy/zh/products/software.html
ImageJ v.1.8.0	ImageJ software	https://imagej.net/ij/
GraphPad Prism v.8	GraphPad Software	https://www.graphpad.com/
Other
96-well culture plates	Costar	3599
Confocal microscope	Zeiss	LSM 780
Rocking lab shaker	Bolaiyan	BLY-100E
Adhesion microscope slides	Citotest	188105
Microscope cover glasses	BRAND GMBH + CO KG	470820

Materials and equipment

Cell culture medium

Component	Stock solution	Final concentration	Amount
MEMα	1X	1X	1X
Penicillin Streptomycin	Penicillin: 104 Units/mL Streptomycin: 104 μg/mL	Penicillin: 50 units/mL Streptomycin: 50 μg/mL	250 μL
BSA	100 mg/mL	2 mg/mL	1 mL
GlutaMAX	200 mM	2 mM	500 μL
HEPES	1 M	10 mM	500 μL
NEAA	100X	1X	500 μL
Sodium bicarbonate	100x	1x	500 μL
Sodium selenite	300 μM	30 nM	5 μL
Putrescine	10 mg/mL	10 μg/mL	50 μL
2-ME	55 mM	55 μM	50 μL
Ascorbic acid	100 mM = 17.6 mg/mL	100 μM	50 μL
FFA mixture	NA	100 meq/L	3.8 μeq/L
Lipid mixture	100x	1x	500 μL
Lipoprotein	1000x	1x	50 μL
Insulin	10 mg/mL	20 μg/mL	100 μL
Transferrin	50 mg/mL	100 μg/mL	100 μL
Human GDNF	20 μg/mL	20 ng/mL	50 μL
Human bFGF	10 μg/ml	5 ng/mL	25 μL
FBS	NA	1%	500 μL
Total	N/A	N/A	50 mL

CRITICAL: Freshly prepared cell culture medium was stored at 4°C and used within 2 weeks.

Alternatives: DMEM medium supplemented with 10% serum is also suitable for the short-term culture of the seminiferous tubule lumen, particularly when the target cell population consists of cell types other than spermatogonia.

Antibody staining cocktail

Antibodies	Dilution	Amount
Anti-DDX4 Antibody	1:200	5 μL
BlockAid Blocking Solution	1X	995 μL
Total	N/A	1 mL

Note: The primary antibody must be prepared fresh and the tracer maintained at 4°C.

Alternatives: BlockAid Blocking Solution can also be replaced by 1% BSA or 10% serum that does not conflict with the antibody source.

Click-iT reaction cocktail

Reaction components	Dilution	Amount
Click-iT OPP Reaction Buffer	1X	880 μL
Copper Protectant (Component D)	NA	20 μL
Alexa Fluor picolyl azide (Component B)	NA	2.5 μL
Click-iT Reaction Buffer Additive	1X	100 μL
Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	1:500	2 μL
Total	N/A	1 mL

CRITICAL: Suitable secondary antibodies were chosen to reveal different fluorescent colors and avoid fluorescence conflicts with component B in the kit.

Note: Use the Click-iT reaction cocktail within 15 min of preparation.

HCS NuclearMask Blue Stain

Reaction components	Dilution	Amount
HCS NuclearMask Blue Stain (Component G)	1:2000	0.5 μL
PBS	1X	1 mL
Total	N/A	1 mL

Alternatives: The DAPI-containing anti-fluorescence quencher can be used as an alternative.

Note: Use directly.

Step-by-step method details

Part 1: Label cells with OPP

Timing: 1 h

This step describes the incorporation of OPP as a structural mimic of aminoacyl-tRNA into nascent polypeptide chains in fresh seminiferous tubules.

CRITICAL: It is crucial to ensure the proper fragmentation of the seminiferous tubules and the survival rate of the cells within them (Figures 1A and 1B). Please use fresh tissue and perform OPP labeling immediately.

• 1.
  Isolation of seminiferous tubules.

  • a.
    Anaesthetize mice by isoflurane inhalation and kill by decapitation.
  • b.
    Make a small skin incision (1–2 mm) and remove the testes.

    Note:The size of the incision depends on the size of the mouse. Here, a 4-day-old mouse is taken as an example.

  • c.
    Place the testicles in PBS and wash away the blood.
  • d.
    Tear off the tunica albuginea and disperse the seminiferous tubules.
  • e.
    Wash the seminiferous tubules once with PBS.
• 2.
  OPP Incorporation.

  • a.
    Add 100 μL of freshly prepared SSC culture medium containing OPP to the 96-well plate (Figure 1C).
  • b.
    Place the dispersed seminiferous tubules in the above 96-well plate.
  • c.
    Incubate seminiferous tubules at 37°C and 5% CO₂ for 30 min.

Figure 1.

Representative images of testicular tissue separation and cultivation

(A) Freshly harvested testicular tissue.

(B) After tunica albuginea is removed, the seminiferous tubule mass is divided into several parts.

(C) Seminiferous tubules were cultured with OPP in 96-well plates at 37°C with 5% CO₂.

Part 2: Fix and permeabilize

Timing: 40 min

This step accounts for sample fixation and increased cell permeability.

CRITICAL: Pay attention to the loss of tissue during multiple medium changes. A 1 mL tip cover with a 10 μL tip can be used for pipetting (Figure 2A).

• 3.After incubation, remove the medium containing Click-iT OPP and wash the cells once with PBS. Remove PBS.
• 4.Add 100 μL per well of 3.7% formaldehyde in PBS. Incubate for 15 min at 25°C.
• 5.Remove fixative.
• 6.Add 100 μL per well of 0.5% Triton X-100 in PBS and incubate for 15 min at 25°C.
• 7.Wash seminiferous tubules 3 times for 5 min each with 100 μL PBS on a rocking shaker at 25°C.

Figure 2.

Representative images of antibody incubation processes

(A) Recommended pipetting methods, such as the use of large and small pipette tip cannula methods in this step.

(B) Incubate antibodies on a shaker at 4°C.

Part 3: Primary antibody incubation

Timing: 14 h

This step explains blocking for preventing non-specific binding and efficient incubation with primary antibody.

Note: Primary antibody incubation on a shaker can help incubate the tissue more fully, but is optional (Figure 2B).

• 8.Add 100 μL per well of blocking buffer and incubate for 1 h at 25°C.
• 9.Dilute the primary antibody with blocking buffer stock solution according to the antibody dilution ratio.
• 10.Add 100 μL per well of antibody diluent and incubate 12 h at 4°C and keep the plate on a shaker at 20 rpm speed.

Part 4: Click-it OPP chemistry and secondary antibody incubation

Timing: 1.5 h

This step explains the combination of secondary antibody incubation and click chemistry after clearing primary antibody residues.

CRITICAL: Carefully select the fluorophore of the secondary antibody to avoid conflict with the OPP coupling group.

• 11.After incubation, remove the medium containing primary antibody and wash the cells 3 times for 5 min each with 100 μL PBS on rocking shaker at 25°C. Remove PBS.
• 12.Prepare 1X Click-iT OPP Reaction Buffer Additive by diluting the 10X solution in deionized water.

Note: Prepare this solution fresh and use the solution on the same day.

• 13.Prepare Click-iT Plus OPP reaction cocktail.
• 14.Dilute the secondary antibody using OPP reaction cocktail.
• 15.Add 100 μL per well of Click-iT Plus OPP reaction cocktail to each well and mix well.
• 16.Incubate for 30 min at room temperature, protected from light.
• 17.Remove the reaction cocktail.
• 18.Wash once with 100 μL per well of Click-iT Reaction Rinse Buffer (Component F).

Part 5: DNA staining

Timing: 40 min

This step uses HCS NuclearMask Blue stain for nuclear staining.

• 19.Dilute HCS NuclearMask Blue Stain (Component G) solution 1:2000 in PBS to obtain a 1X HCS NuclearMask Blue Stain working solution.
• 20.Remove the rinse buffer from the cells.
• 21.Add 100 μL per well of 1X HCS NuclearMask Blue Stain working solution.
• 22.Incubate for 30 min at 25°C, protected from light.
• 23.Remove the HCS NuclearMask Blue Stain solution.
• 24.Wash twice with PBS.

Part 6: Imaging

Timing: 2–3 h

This step introduces tissue mounting and microscope photography.

CRITICAL: It is key to avoid tissue overlap when mounting slides, otherwise the photographable field of view cannot be obtained (Figures 3A and 3B).

• 25.Remove the seminiferous tubules onto a glass slide.
• 26.Use ophthalmic scissors or tweezers to disperse the seminiferous tubules into a single lumen.
• 27.Add a drop of anti-fluorescence quencher to the seminiferous tubules of the slide, cover it with a coverslip, and remove the air bubbles.
• 28.Seal coverslip with nail polish to prevent drying and movement under microscope.
• 29.Dry the slides naturally in the dark in a fume hood.

Note: The dried slides are stored protected from light at 4°C to avoid light-induced degradation until use.

• 30.Take images using confocal microscope (Zeiss 780).
• 31.Look for a complete and independent seminiferous lumen in the whole field of view through the eyepiece and place it in the center of the field of view.
• 32.Collect images using a 40x objective lens and scan speed 4.
• 33.For each sample, acquire images from six randomly selected positions.

Figure 3.

Representative images of the testicular lumen on the slice

(A) After staining, the seminiferous tubule clumps are placed on a glass slide.

(B) The clumps of seminiferous tubules were gently dispersed into individual tubule using forceps.

Part 7: Data analysis

Timing: 3–5 h

The last step introduces data analysis that allow relative comparison of protein synthesis levels between different cells.

Note: When counting the immunofluorescence intensity of OPP in cells, the signal in the nucleus should be included, because nuclear and nucleolar proteins are among the most abundant newly synthesized proteins in cells.⁵

• 34.
  The images were processed by Zen blue (Zen 2 lite) software.

  • a.
    Open the original image and mark the target cells based on cell markers. Mark >> Select the area of interest >> Right click >> Set an atlas of interest.
  • b.
    Export all channel images of target cells. File >> Export >> Parameters >> Check Raw Data, Single Channel Image and Overlay Image.
• 35.
  The intensity of immunofluorescence was quantified using Image J (version 1.8.0).

  • a.
    Open the OPP channel original image and check whether it is a separate channel (Image-type).

    Note: If the image is 8, 16, or 32 bit, the image is already a single-channel image, and Threshold operations can be performed directly.

  • b.
    Adjust thresholds to select appropriate areas (Image-Adjust-Threshold).
  • c.
    Set the parameters that need to be measured (Analyze-Set Measurements), confirm that Mean gray value and Limit to threshold are checked (very important), and click OK.

    Note: If Limit to threshold is not checked, the average fluorescence intensity of the entire image is measured.

  • d.
    Test (Analyze-Measure) Click Measure and the test results will pop up.

    Note: Mean is the average fluorescence intensity. Mean is Mean gray value, equal to IntDen/Area; IntDen = Integrated Density (sum of fluorescence intensity).

  • e.
    Copy the results to Excel or directly export to generate a csv file (Results window-File-Save as).
• 36.Data analysis was performed using GraphPad Prism 8 (GraphPad Software).

Note: Fluorescence intensity was quantified by univariate analysis of variance. This parameter was set as the dependent variable, while the different experimental groups were the independent variables. Experimental replicates were included in the model as a covariate. The differences between each independent variable were analyzed through the least significant difference test. A probability of less than 5% (P < 0.05) was considered significant.

Expected outcomes

In this protocol, we describe an efficient method to detect protein synthesis levels of cells in-situ in the testicular seminiferous lumen. In this example, we incorporated OPP cells in the lumen of seminiferous tubule in an in-vitro culture environment, and performed in-situ labeling and protein synthesis level analysis of cells in the seminiferous lumen by combining click chemistry and immuno-cytochemical methods. We used the germ cell-specific marker DDX4 to label spermatogenic cells in the testes of mice at day 4, when these cells are all spermatogonia. We used this protocol to visualize and quantify nascent protein synthesis in mouse testicular spermatogonia in wild-type (WT) and CHX-treated groups (Figures 4A and 4B).

Figure 4.

Expected outcomes of detecting new synthetic proteins in the testicular lumen

(A) Whole-mount immunofluorescence analyses of 4-day-old control mice testes treated with or without 100 mM cycloheximide (CHX). Nascent protein synthesis was detected by staining with OPP-594 (red); spermatogonia was detected by staining with the germ cell marker DDX4 (green). Scale bars, 20 mm n = 3.

(B) Quantitative analyses of protein synthesis efficiency in spermatogonia treated with or without 100 mM cycloheximide (CHX). Student’s t test; n = 3.

In summary, this protocol overcomes the limitations of protein synthesis detection in the seminiferous lumen of young mice and enables detection of protein synthesis levels in specific cells in the seminiferous lumen.

Limitations

This protocol is an effective tool for studying the level of cell nascent protein synthesis in the seminiferous duct lumen of testicular tissue. However, it is also important to acknowledge the method has some limitations.

Firstly, this protocol is limited to freshly harvested seminiferous lumens or testicular organoids cultured in vitro, and it is necessary to ensure the viability of cells in the tissue block and a suitable survival environment.

Secondly, it can only detect the overall protein synthesis level and cannot analyze specific proteins. Protein synthesis analysis of individual proteins may require live cell single-molecule imaging detection and other technologies, such as Real-time quantification of single RNA translation.⁶

Thirdly, this protocol can only perform relative quantitative analysis on the same cells in different treatment groups or different cells in the same treatment group, and cannot perform precise quantitative analysis.

Troubleshooting

Problem 1

The most common problem encountered during the protocol is that the lumen of the seminiferous ducts overlaps and the view of the entire lumen cannot be obtained (related to step 1 and 5).

Potential solution

• •When collecting materials, use forceps to loosen the wound seminiferous tubules appropriately to prevent serious winding of the lumen.
• •After tearing off the testicular albuginea, wash it several times with PBS to remove dead cells and avoid adhesion caused by DNA.
• •When transferring seminiferous tubules to a glass slide, it is most important to disperse them into individual tubules with forceps to avoid accumulation of tissue blocks.

Problem 2

The structure of the seminiferous lumen is destroyed (related to Step 1 and 5).

Potential solution

• •After peeling off the testicular tunica albuginea, do not tear the seminiferous tubules excessively, and do not cut them into pieces with scissors.
• •When transferring seminiferous tubules to the glass slide, avoid drying out the seminiferous tubules.
• •Use appropriate force when covering the glass to avoid lumen rupture caused by excessive pressure.

Problem 3

The lumen of the seminiferous tubules was severely lost, and the seminiferous tubules could not be obtained in the later stages of the experiment (related to Step 1, 2, 3, 4, 5, and 6).

Potential solution

• •After peeling off the tunica albuginea, do not tear the seminiferous tubules too much.
• •When pipetting during the entire experiment, use a 1 mL pipette tip covered with a 10 μL pipette tip to reduce the suction of the seminiferous tubules.

Problem 4

Low or uneven fluorescence intensity of OPP signal in the seminiferous duct lumen (related to Step 1, 2, 3, and 4).

Potential solution

• •Check OPP for improper storage or expiration before use.
• •All reagents for click chemistry reactions were prepared fresh immediately before use in experiments.
• •Check the permeabilization conditions and increase the Triton X-100 concentration and permeabilization time appropriately.

Problem 5

High intensity of OPP single in the seminiferous tubules (related to Step 1, 2, 3, and 4).

Potential solution

• •Determine whether there is insoluble matter precipitating from OPP and select reagents with qualified quality.
• •Determine whether the OPP is diluted correctly and reduce the OPP concentration appropriately.
• •Increase the number of PBS washes.

Problem 6

Cell markers with no specific signal or with high background (related to Step 3–4).

Potential solution

• •Before experimenting, choose a validated antibody.
• •Appropriately increase the primary antibody or reduce the concentration of the secondary antibody, and increase the rinse time and frequency.
• •Check the permeabilization conditions and increase the Triton-X100 concentration and permeabilization time appropriately.

Problem 7

When staining cell nuclei with DAPI, excess reagent causes high background staining (related to Step 5).

Potential solution

• •HCS NuclearMask Blue Stain is diluted to the correct concentration, or the recommended lower concentration.
• •When staining with HCS NuclearMask Blue Stain, avoid staining for too long and wash several times with PBS after staining.
• •Using DAPI reagent that contains anti-fluorescence attenuation, one drop can mark the cell nucleus. However, if too much is added, the entire image will appear with a blue background. Therefore, do not add too much DAPI, just cover it with a thin layer.

Problem 8

Non-standard image acquisition process will lead to poor original images, which directly affects subsequent semi-quantitative analysis (related to Step 6).

Potential solution

• •Choose appropriate scanning speed and excitation light intensity to avoid using too slow scanning speed and too strong excitation light, which may lead to fluorescence quenching.
• •The same batch of experiments uses the same shooting parameters, and images are collected at the same time as much as possible.
• •Avoid uneven positioning of the slide and objective lens.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Wei Song (songwei@ibms.pumc.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Dingfeng Zou (dfmufu@163.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new datasets or code.

Acknowledgments

This study was supported by grants from the National Key Research and Development Program of China (2022YFA0806302), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS; 2021-I2M-1-019 and 2021-I2M-1-066), the National Natural Science Foundation of China (92268111 and 32370910), and the State Key Laboratory Special Fund (2060204).

Author contributions

W.S., W.Y., D.Z., and K.L. conceived and designed this study; D.Z. and K.L. performed most of the experiments with the help of Y.L.; W.S., W.Y., and D.Z. wrote the manuscript with some helpful input from all authors.

Declaration of interests

The authors declare no competing interests.