Effects of Pulsed Electric Fields on Yeast with Prions and the Structure of Amyloid Fibrils
, Vitalij Novickij 3, Vytautas Smirnovas 2 and Eglė Lastauskienė 1,*
Round 1
Reviewer 1 Report
In this manuscript the authors investigate the reaction of cells with and without the [PSI+] prion exposed to pulses of high electric charges.
The authors report a dramatic positive effect on growth caused by the presence of [PSI+]. [PSI+] is the most studied of the several prions that exist in the yeast Saccharomyces cerevisiae. There are no other reports in the literature of this kind of dramatic growth effect that the authors document here. My guess is that the cells were grown in medium containing only minimal amounts of adenine. ade1-14 cells containing strong [PSI+] grow much better in the absence, or near absence, of adenine than the other strains tested.
The two experiments in which the authors either expose cells or Sup35p amyloid to pulses of high electric charges can be explained by assuming that a significant amount of heat is generated during the experiment. From figure 2 it can be concluded that the different strains were not in similar growth conditions in this experiment.
Additional comments
Line 35 “Structurally prions are organized to the aggregates.” Baffling statement.
Line 43 There are many variants of [PSI+]. Several manifest with a strong phenotype others manifest with a weak phenotype.
Line 48 Specific mutations in ADE1 and ADE2 or needed to assay for [PSI+]
Line 60 Prions are not in an amyloid conformation. The protein that forms a prion can adopt an amyloid fold!
Line 79 [psi-] colonies are not red! They are only red when the cells are grown on adenine limiting medium.
Line 88 This argument has been brought up in the literature but there is no evidence to support it.
If an optical density is given the wavelength at which this optical density is recorded should be provided.
Line 117 Why was it necessary to include the osmotic stabilizer sorbitol? Cells were plated on medium lacking sorbitol.
Line 164 It is never mentioned in the materials and method section that SC medium was used.
Line 246 There has been a report that stress affects [PSI+] de novo generation. However, stress is not needed for this prion to form de novo.
The authors should check their statements carefully.
Author Response
The authors would like to thank the reviewer for the huge work in improving the quality of our publication.
All detailed answers are provided in the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The authors of this manuscript presented an in vitro study of pulsed electric field (PEF) treatment of yeast with prions and addressed three questions: Do prions increase PEF resistance of yeast cells? Can PEF induce new prions? Is it possible to disintegrate amyloids by PEF treatment? To the best of my knowledge, the study is original and relevant. In my opinion, after some minor corrections, which are listed below, the methods used will be appropriate, results clearly presented and conclusions of this manuscript supported by the data.
Page 3, 99: OD abbreviation is not explained. Does it mean Optical Density? And if so, are the units of OD logarithmic like the absorption unit - AU?
Page 3, 100: From figure 1 I would conclude that prions have a strong effect on light reflection and most probably also on light absorbance. With photometer a light absorbance is measured. Light absorbance in this study is most probably a combination of yeast light absorbance and prion light absorbance. This could also explain why is on figure 2 absorbance of strong [PSI+] variant drastically higher (logarithmic unit?!) than of other variants.
Figure 2, y axis: What the number 600 means at OD600 and what unit is on y axis?
Figure 3, y axis: Variability -> Viability
Page 5, 186 and Figure 3: It is stated that weak variant is less resistant than strong variant. Are the colors on Figure 3 mixed for strong and weak variant because weak variant is still viable at 22 kV/cm on Figure 3?
Some minor corrections are needed to some of the symbols for degrees Celsius and a few spaces are missing between values and units.
Author Response
The authors would like to thank the reviewer for the huge work in improving the quality of our publication.
All detailed answers are provided in the attachment.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript “Effects of Pulsed Electric Fields on Yeast with Prions and the 2 Structure of Amyloid Fibrils” is appropriate for the Applied Sciences journal. However, there are several issues, which should be addressed prior to publication. The manuscript is lacking the description of the parameters of PEF chosen for the protocol. Moreover, I would expect more detailed explanation regarding the impact of different phenomena occurring during PEF treatment on the studied amyloid aggregates. Additionally, the quality of language should be significantly improved (there are multiple errors and misused words in the text).
Below are some specific non-exhaustive comments:
L.24: …in at higher field strength.
L.27: Pulsed Electric Field instead of PEF
L.56: There are multiple technologies apart PEF allowing to affect the structure and polymerization of proteins. Thus, you should justify the use of PEF.
L.58: We discovered that prions… This should be placed to conclusion section.
L.150: KH2PO4
L.180: Presents → Presence
L.181: … are increasing increased… Psi-cells viability decreased (I suggest you to use past tense here and throughout this section when you present your results)
L.183: therefore
L.185. Avoid starting the phrase with numbers
Fig.3: The statistical analysis should be provide in order to make conclusions about significant differences between PEF treated samples.
Discussion section: the explanation of the mechanisms of PEF action should support all three research questions in your discussion.
Author Response
The authors would like to thank the reviewer for the huge work in improving the quality of our publication.
All detailed answers are provided in the attachment.
Author Response File: Author Response.docx
Reviewer 4 Report
The manuscript presents the effect of PEF on prions. In my opinion, the topic is very interesting and worth studying. That is why I recommend publication of the submitted article after addressing following issues:
line 57 and line 65: Please modify the introduction so the aim will be clear and written in one place. It will facilitate reader going through the paper. Please underline also what is new and innovative in your work and give more details why you think that PEF can impact upon prions considering the mechanism of PEF as well.
line 103: please provide more details about the device and protocol that has been used in the study. It would be good to collect all the experimet variants in a table. Provide the energy input of the treatent for each variant. The number of pulses is not sufficient to evaluate the experiments properly.
The biggest drawback of this manuscript which needs to be addressed - no statistical analysis of the obtained results. I encourage authors to perform the statistical analysis of viability and the results of AFM analysis.
Fig. 3 - What is variability on OY? It should be viability I believe.
Author Response
The authors would like to thank the reviewer for the huge work in improving the quality of our publication.
All detailed answers are provided in the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
Authors took into account my comments and significantly improved the overall quality of their manuscript.
Author Response
Thank You very much for the great work in reviewing our manuscript. English language editing was performed by MDPI editors. Attaching the proof of revision.
Author Response File: 
Author Response.pdf
Reviewer 4 Report
The quality has been improved. I recommend, English editing and addressing some minor remarks listed below:
table 1: Please express energy in kJ/kg (based on the mass of treated system) and add SD to the temperature rise
Author Response
Thank You very much for the great work in reviewing our manuscript. English language editing was performed by MDPI editors. Attaching the proof of revision. The recalculation of energy is provided in the material and methods section Table 1 (red).
Author Response File: Author Response.pdf
