"If you want to be one year behind, don’t read bioRxiv” – Jeff LeekWelcome to PREreview! On PREreview you can collaboratively write reviews of preprints. This project was born in April 2017 as a collaboration eetween Samantha Hindle and Daniela Saderi, scientists and ASAPbio Ambassadors, with help from Josh Nicholson, at the time working for Authorea. ASAPbio (Accelerating Science And Publication in biology) is a non-profit organization dedicated to spreading the word about preprints to accelerate scientific discovery.As of October 2018, we are proud to have become an official project fiscally sponsored by Code for Science and Society. Learn more in this blog post.We are also proud to announce that we have received funding from the Sloan Foundation and the Wellcome Trust to continue to grow our community. Reed more here and stay tuned for some exciting updates! Our Mission PREreview seeks to diversify peer review in the academic community by crowdsourcing pre-publication feedback to improve the quality of published scientific output, and to train early-career researchers (ECRs) in how to collaboratively review others' scientific work. We want to facilitate a cultural shift in which every scientist posts, reads, and engages with preprints as standard practice in scholarly publishing. We see PREreview as a hub to support and nurture the growth of a community that openly exchanges timely, constructive feedback on emerging scientific outputs. We believe that by empowering ECRs through peer review training programs, thereby increasing the diversity of researchers involved in the peer review process, PREreview will help establish a healthier and more sustainable culture around research dissemination and evaluation.
We are proud to introduce you to the members of our Advisory Committee. These fantastic people have been unofficially supporting us throughout the launch of PREreview, and we are honored that they have agreed to continue their support in a more official fashion. We look forward to building and improving PREreview together.
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At the top write: Title of the preprint, authors, date of submission, version number, preprint server, and digital object identifier (DOI)Start a new document and invite to your preprint journal club others who want to collaboratively write the preprint review – they will have to sign up on PREreview to be able to edit. Below are a short list of questions that you can have journal club attendants answer (PREreview short participant worksheet, example here), followed by more detailed guidelines on how to structure a more formal and complete peer review (PREreview peer review, example here). Answering the first set of questions will be faster and still povide useful feedback to the authors. However, if the main purpose of the preprint journal club is to train early-career researchers on how to write a peer review, recommend you write the full review as if you were a reviewer for a journal. You can use the comments from the first one to construct the preprint peer review. After you are done writing your your PREreview, you can click on "Document" (top left), "Publish" so that your preprint review will be public and will be assigned a DOI that you can use to advertise your review on social media, email to the authors, and post on the comment section on the server that hosts the preprint you chose for your JC. Additionally with a DOI, your preprint review will be citable! If you got this far, GREAT JOB! Thank you for supporting open science and helping science move forward faster!
Where you can find preprints:There are various preprint repositories (see below) and also website platforms where you can search all/most of the preprint repositories, including Prepubmed, Publons, The Winnower, and Academic Karma (please let us know at email@example.com or leave a comment on this page if we missed any). You can search the Research Preprint Servers List to find a preprint server in your field.Below is a list of the most common preprint repositories that post findings in the biological sciences:AgriXiv: a preprint repository for agriculture and allied sciencesarXiv q-bio: a preprint repository for quantitative biology operated by the Cornell University Library. This repository includes manuscripts in the following categories: Biomolecules, Cell Behavior, Genomics, Molecular Networks, Neurons and Cognition, Subcellular Processes, Populations and Evolution, Tissues and Organs, Quantitative Methods, and Other Quantitative BiologybioRxiv: a preprint repository for the biological sciences operated by Cold Spring Harbor Laboratory. This repository includes manuscripts in the following areas: Animal behavior and Cognition, Biochemistry, Bioengineering, Bioinformatics, Biophysics, Cancer Biology, Cell Biology, Clinical Trials, Developmental Biology. Ecology, Epidemiology, Evolutionary Biology, Genetics, Genomics, Immunology, Microbiology, Molecular Biology, Neuroscience, Paleontology, Pathology, Pharmacology and Toxicology, Physiology, Plant Biology, Scientific Communication and Education, Synthetic Biology, Systems Biology, and ZoologyOSF PREPRINTS: a preprint server that hosts preprints from a broad range of disciplines, including the Life Sciences, and Medicine and Health Sciences PeerJ Preprints: a preprint repository for the biology and computer sciencesPreprints.org: a preprint repository that posts manuscripts covering many areas of the biology and biomedical science (and other sciences, arts and humanities), including Behavioral Sciences, Biology, Life Sciences, and Medicine and PharmacologyWellcome Open Research: a preprint repository for research funded by the Wellcome Trust mainly in areas of the biological sciences, population health, applied research, humanities and social scienceINARxiv: the preprint server for Indonesia powered by OSF Preprints hosting preprints from a broad range of disciplines, including the Life Sciences, and Medicine and Health SciencesEarthArXiv: the preprint server for Earth Sciences powered by OSF Preprints
What are preprints?Preprints are complete pieces of scientific work that have not yet undergone editorial peer reviewed. Preprints are often the same manuscripts that are submitted to a journal for peer review, but are stored on freely accessible public servers (repositories) such that they become available to the whole web community within 1-2 days from submission. Usually preprints are posted on preprint repositories (see below) either before or at the same time as submission to a journal. Most journals will accept manuscripts that have previously been submitted to a preprint repository. A list of copyright and self-archiving polices can be found on Wikipedia and SHERPA/RoMEO.
In the summer of 2017, we conducted a survey to assess scientists' opinions on the value and potential barriers related to reading and reviewing preprints at journal clubs. In this short article we present and discuss the results of the survey as well as how these results helped us shape our approach at PREreview.
REQUEST A LIVE-STREAMED PREreview JOURNAL CLUBAt PREreview, we want to take preprint journal clubs to the next level. Live-streamed PREreview journal clubs (LivePREJCs) are hosted via online community calls, allowing anyone with internet or phone-in capabilities to join the discussion. This format promotes inclusivity by following a structure that provides a means to join the discussion silently in written form and vocally. You can request our help to organize a live-streamed preprint journal club by clicking on the link above and filling the form. Here is a list of current and past LivePREJCs.Live-streamed PREreview journal clubs are:Inclusive: anyone, anywhere in the world with a internet or phone connection can joinInformative: you can learn more about the topic by listening to/reading the comments of other researchers in the field and even the authors themselves (if invited)Efficient: if preprint authors are present, they receive feedback in real time. Also, we restrict the live journal clubs to 1 hour to keep the feedback focused and efficientCollaborative: the format encourages contributions from all participants regardless of input style preference, i.e. both vocal and silent writing (etherpadding)Fun: even though the discussions are kept professional and centered around providing constructive feedback to the preprint authors, they are a fun way to meet other people interested in the field.How to get started:Choose a preprint you wish to discuss at a LivePREJC (find out more about what preprints are and where to find them).Find a few other scientists or researchers, preferably at different career levels and from different institutions, who would like to participate in the LivePREJC. Our team will help you recruit more participants if you cannot find them on your own.Fill out this form to formally request a LivePREJC and our logistic support.If you are not the preprint author(s), you can contact the corresponding author(s) and let them know about the LivePREJC. If you don't feel comfortable doing this, please let us know and we will do it on your behalf. You can also choose not to include the author(s) in the discussion. What to expect during the call:LivePREJCs are usually hosted by one or two members of the PREreview team: we will ensure that the conversation around the preprint runs smoothly and stays on time (1 hour); they will take notes and encourage others to take notes on a collaborative etherpad (see below); importantly, they will set the tone for a productive and respectful conversation according to the PREreview code-of-conduct.Preprint authors interested in having their preprint discussed on a LivePREJC, will have the option to be present (recommended). We advise authors to find 5-10 participants (not all the participants need to be experts in the field). Our team will help coordinate the call and, if the authors request it, help recruit more researchers in the field by advertising the call on social media using the #LivePREJC hashtag and any other hashtag related to the research field of the preprint discussed.Once the preprint authors have identified the participants, we will send out a short email with instructions on how to join the LivePREJC and with a copy of the preprint. Participants will be encouraged to read the preprint before the LivePREJC, to keep the discussion short and focused.Authors will be asked to remain in ‘listening mode’ – except when asked a question by participants - until the last 10 minutes reserved for this discussion. This will encourage participants to express their constructive feedback freely and stimulate a productive discussion.Participants will be given the opportunity to give feedback both vocally (with notes taken by one of the PREreview team) and in written form (via collaborative note-taking on a public etherpad that we will set up for each LivePREJC). Here is an example of an etherpad template that will be used for the LivePREJCs.
Floryn Lynorah Mtemeli, Irene Walter*, Ryman ShokoDepartment of Biology, Chinhoyi University of Technology, Zimbabwe*Corresponding author: firstname.lastname@example.org AbstractThe aim of the study was to investigate the molluscicidal effects of pumpkin seeds (Curcurbita maxima) on adult, juvenile Biomphalaria, and adult Bulinus snails under laboratory conditions. This study was prompted by recent reports on Schistosoma gaining resistance to the commonly administered drug, praziquantel. Snails were exposed to water and ethanol crude extracts for 24 hours and significant concentration-dependent mortality rates were observed. Observations of the snail mortalities continued up to 72 hours. The lethal concentration of 0.02 mg/ml killed 50% of the snails (LC50) for both the water and ethanol extracts on adult Biomphalaria snails. It was noted that the mortalities were not significantly dependent on the time of the snails’ exposure to the extracts. There was a significant difference between the susceptibility of juvenile and adult snails to the ethanol extract (p = 0.016). These results suggest that pumpkin seeds have a significant molluscicidal effect on Biomphalaria and Bulinus snails. We propose that pumpkin seed extracts be considered as molluscicidal agents in a bid to control transmission of schistosomiasis. Key words: Schistosomiasis, Biomphalaria, Bulinus, molluscicidal activities Introduction Neglected tropical diseases (NTDs) are a group of 17 major disabling conditions that are among the most common chronic infections in the world's poorest people (World Health Organisation [WHO], 2003). The NTDs afflict an estimated 1.4 billion people, whose greater population live in Africa and are among the poorest in the world, causing significant disability and impairing quality of life (Institute of Medicine, 2011). Of all NTDs, the most neglected are helminthic infections, which comprise five of the top ten NTDs in terms of Disability-Adjusted Life Years (DALYs) (Frean & Mendelson, 2013). Among these helminthic infections is schistosomiasis.Schistosomiasis commonly known as Bilharzias is caused by a digenean trematode of the genus Schistosoma (Katsurada, 1904). The intermediate hosts of all digenetic trematodes are snails and schistosomes are no exemption. In Zimbabwe, the snail vectors are Bulinus globosus for the species S. haematobium and Biomphalaria pfeifferi for S. mansoni (Chimbari, 2012). Despite schistosomiasis being one of the most persistent NTDs, treatment and disease control are based on the utilisation of a single drug, praziquantel (PZQ), otherwise called biltricide. Controlling or preventing morbidity in subjects using praziquantel has not been entirely successful in restricting transmission in high-risk areas as there have been recent reports of PZQ schistosomal resistance (Ismail et al., 1999; Augusto et al., 2017). This raises concerns about future control of the disease and demonstrates the significance of coming up with new tactics to control the disease (Wang, 2012). Optimal disease prevention can be achieved only when parasite infection or re-infection is effectually obstructed (King et al., 2015). As a responsive measure, the WHO published a report of the Strategic and Technical Advisory Group for NTDs. In the light of its call to eliminate the disease by 2025, it discourses schistosomiasis management through the ecological control of the intermediate host population of Schistosoma, snails from the Biomphalaria and Bulinus genus (WHO, 2014; Augusto et al., 2017).It is, therefore, largely agreed that regulation of the snails’ population is an essential part of the control of schistosomiasis (Mohamed et al., 2012). Chemical, biological and physical control strategies have been used on the snails (WHO, 1967; Madsen, 1983; Fagitta & Egami, 1984). Among the chemical compounds, niclosamide is recommended by the WHO as the only chemical molluscicide to be used for snail control despite recent concerns of resistance of Oncomelania snails to the molluscicide (Dai et al., 2014). The WHO, however, recommends further studies on plant molluscicides (Augusto et al., 2017). Molluscicidal plant extracts may offer affordable, locally produced, biodegradable and effectual control means in the rural parts of low-income countries where schistosomiasis is prevalent (Brachenbury, 1998). Extensive investigations may help in understanding their properties and safety as molluscicides. Pumpkins are known not only for the fruit but also for many health benefits and thus have been used for a long time in traditional medicine in many countries such as Turkey and China (Young et al., 2012). Pumpkin seeds have been used in different parts of the world as a traditional medicine for treatments of gastrointestinal parasites as anthelmintic, urinary dysfunctions, hyperplasia of prostate, dysuria, cardiovascular disease, enuresis and lowering blood glucose (Medjakovic et al., 2016). Among the studies that have been done on pumpkin seeds, their anthelmintic potential has proved to be a success on S. mansoni. However, data on their molluscicidal effects on the vectors snails is scarce. A successful trial of pumpkin seeds as a molluscicide would mean a double impact on both the vectors and the cercarial stage of the S. mansoni parasite. The impetus of this investigation was mainly based on the high cost of synthetic molluscicides such as niclosamide in Zimbabwe, their low availability as well as the time taken by the chemical compounds to degrade in the environment. Therefore, assessing the molluscicide potential of methanol and water extracts of natural compounds on the planorbid snails from the Biomphalaria and Bulinus genus would open potential cost-effective noteworthy alternatives in the control of schistosomiasis. Materials and Methods Study site The bioassays of this study were carried out in the biology laboratory and the extraction process of the seeds was done in the chemistry laboratory at Chinhoyi University of Technology, Zimbabwe. Collection of pumpkin seeds and vector snails Pumpkins were bought from a local supermarket in Chinhoyi. They were washed thoroughly and cut to separate the seeds from the fruit. Snails were randomly sampled in October in Murombedzi particularly from Madzorera dam using a sweep net. They were kept in open plastic bottles and covered with moist cotton wool to keep them alive before reaching the laboratory. Preparation of pumpkin seeds ethanolic extracts About 685g of pumpkin seeds were sun-dried for 72 hours to a moisture content of 12.4%. Approximately 600g of the seeds were milled into a fine powder using a mortar and pestle. In order to obtain the ethanolic crude extract, the maceration technique was used. Approximately 900ml of ethanol was added to 300g of refined pumpkin seed powder and left in a dark cupboard for 7 days. At the end of this period, the mixture was filtered on 0.1mm Whatman filter paper grade using an EC vacuum pump (WP6122050) and then concentrated to dryness using Buchi rotary evaporator (R-200) at 78ºC in order to obtain pure crystals of the extract. The crystals obtained were weighed and a total yield of 5g was obtained. The crystals were dissolved in distilled water. The resulting solution of 100mg/ml concentration was considered as the pure extract. Preparation of pumpkin seeds water extracts Approximately 600ml of water was added to 300g of fine pumpkin seed powder and left in a dark cupboard for seven days. The mixture was filtered on 0.1mm Whatman filter paper grade using an EC vapour pump (WP6122050) and the filtrate was concentrated to dryness on the Buchi rotary evaporator and 8g of crystals were obtained. The crystals were dissolved in 80ml distilled water and the solution of 100mg/ml concentration was considered as the pure extract. Snail rearing The snails were reared under laboratory conditions in plastic aquaria of 5L holding capacity measuring 13X12cm. The aquaria were provided with fresh water, from the dams from which the snails were taken, after every two days. No mud, sand, nor any other substratum was put in the aquaria. The laboratory in which they were kept was maintained at a room temperature of 25ºC with natural fluctuations of +/-2ºC for the duration of the research. The snails were fed on oven-dried lettuce leaves ad libitum and kept for five days before being used to allow them to acclimatise to laboratory conditions. Shedding of snails Snails were shed to certify that they were not infected by cercariae, thus ensuring the use of healthy snails only (El-sherbini et al., 2009). After being exposed to the dark for eight hours during the night, snails were placed in 300ml plastic bottles filled with non-chlorinated water and placed in direct sunlight for 8 hours. Thereafter, a drop of water from each of the bottles was transferred to a microscope slide and observed for the presence or absence of cercariae. A snail was considered to be immobile if it was entirely withdrawn into its shell. Snails that were unresponsive to forceful, mechanical stimulation or probing were considered dead. Molluscicidal activity assay During the test process, the snails were kept under normal diurnal lighting and room temperature. They were organised into two classes, established on their developmental stage and shell diameter, juveniles (below 45mm) and adults (above 45mm) (Ciomperlik et al., 2013). Preliminary molluscicidal assay tests were done to determine the minimum effective concentration. A range of six concentrations were assayed - 20%; 40%; 60%; 80% and 100% of the 100mg/ml ethanol and water extract solutions. A lethal effect in a two-hour period among all the concentrations was observed and serial dilutions of the lowest concentration (20%) were used for the molluscicidal assays. A maximum of six serial dilutions of 20% of the pure water and ethanol extracts were made as per WHO guidelines (WHO, 1983). The final concentrations of the water and ethanol extract serial dilutions were 20mg/ml; 2mg/ml; 0.2mg/ml; 0.02mg/ml; 0.002mg/ml and 0.0002mg/ml. A treatment consisted of three snails (three snails per container) of each life stage and thus fifty-three individuals of each group were used per trial. Each group was exposed to the test molluscicide along with three snails of each same life stage as controls. A 0.1 dilution of Thunder was used as positive control and plain dam water as a negative control. A second positive control of absolute ethanol was used to factor into consideration the effects of residual ethanol in the ethanol extracts. The treatments used 10ml of the six dilutions of pumpkin seeds extracts in 90ml medium. The medium used was dam water from which the snails were sampled in 300ml plastic bottles. This was done in order to reduce the number of limiting factors that could affect the snails' metabolism during the trial experiment. Each treatment and the control were carried out in triplicate. The duration of exposure to the molluscicide dilutions and control was three days. After the first 24h, the number of molluscs withdrawn into their shells, immobile and unresponsive to vigorous action was recorded. In order to ensure that the snails were indeed dead, they were placed in distilled water and observed for a two-hour period. Snails were deprived of food during the molluscicidal assays. LC 50 determination and Statistical analysis The minimum concentration required to kill 50% of the snails (LC50) values were determined using Graph pad Prism version 7.0 software (Finney, 1971) with 95% confidence limit. Mortality percentages were expressed and plotted against the log-transformed values of the extract concentrations. The non-linear regression lines obtained from this data were used to determine the LC50 values. One-way analysis of variance (ANOVA) and independent T-tests were used to determine the significant differences between mean mortality values using version IBM SPPS (Statistical Package of Social Sciences) software. Tests for normality were done using Kolmogorov Smirnov tests. Results with p< 0.05 were considered to be statistically significant. Results
Dustin Revell and Zhang Li“Notch signaling regulates Akap12 expression and primary cilia length during renal tubule morphogenesis” Preprint posted to BioArchiv on September 9, 2019; doi: https://doi.org/10.1101/760181This preprint was reviewed as part of the Developmental Biology Journal Club at the University of Alabama Birmingham and the review is a summary of the group discussion.Mukherjee et al. used a combination of transgenic inducible mouse models as well as cell culture and spheroid models to demonstrate how Notch signaling regulates Akap12 expression to influence primary cilia length during renal tubule morphogenesis. The authors show that inhibition of Notch signaling through expression of dnMaml or the conditional deletion of RBPJ leads to kidney cysts and elongated cilia, mimicking the human disease Alagile syndrome. While these data are very interesting and lean towards the classification of Alagile syndrome as being one of the class of diseases termed ciliopathies , we found several concerns throughout the paper which are outlined below.Major Concerns:1) There is some confusion in the use of the different transgenic mouse models used in the paper. For Figures 1 and 2, the authors utilize the Pax8-rtTA; TRE-dnMamL model which will express dnMamL in the kidney of mice when treated with doxycycline. However, in Figure 5, the authors switch to a Rarb2-Cre; RBPJ(flox/flox) mouse without any introduction or explanation as to why. As Rarb2 is expressed in multiple tissues, not just the kidney, this may be influencing their results (https://www.ncbi.nlm.nih.gov/gene/5915).2) We feel that there should be some further validation to show that Notch signaling is truly reduced and in which cell types upon use of the Pax8-rtTA; TRE-dnMamL - This could be done via qRT-PCR looking at the common downstream genes HES and HEY. It is previously published that complete inhibition of Notch2 results in a lack of proximal tubules in murine kidney resulting in death at P0 (Kamath, Spinner, & Rosenblum, 2013; McCright et al., 2001). Figure 1D shows a large reduction in LTA positive proximal tubules suggesting that dnMamL might be causing an incomplete inhibition of Notch resulting in the renal phenotype.3) We are concerned as to the variability in the length of the primary cilia between cell culture experiments. In Figure 4C, the WT MDCK primary cilia were an average length of approximately 1.5microns, while in Figure7H, the WT cilia were less than 500nm in length. Primary cilia are generally between 3 to 5 microns in length (Keeling, Tsiokas, & Maskey, 2016), so this discrepancy leads to skepticism over the health of these MDCK cells and the conclusions made from these experiments.4) The authors conclude that inhibition of Notch signaling regulates Akap12 expression to increase cilia length during tubule morphogenesis. While the authors do show a clear demonstration that Akap12 is upregulated in the dnMamL MDCK cells and in E16.5-18.5 embryos from the Pax8-rtTA; TRE-dnMamL line, and that ectopic expression of Akap12 in MDCK cells is sufficient to increase cilia length, they do not provide conclusive evidence of the link between Notch and Akap12, the link between elongated cilia and cyst formation, or provide conclusive evidence that Akap12 overexpression induced cilia elongation is causal to cyst formation in vivo . In the discussion, the authors bring up a possible role of Akap12 to bind AuroraA and Plk1 to regulate spindle orientation, but fail to mention that AurA and Plk1 arethe major deciliogenesis pathways (Pugacheva, Jablonski, Hartman, Henske, & Golemis, 2007; Sanchez & Dynlacht, 2016) , and Akap12 overexpression could result in increased cilia length simply because AurA and Plk1 are no longer able to activate HDAC6 to reduce cilia length. In addition, Akap12 is also known to bind kinases such as PKA, which also is known to play a key role in cilia length maintenance through IFT protein phosphorylation (Abdul-Majeed, Moloney, & Nauli, 2012).Minor Concerns:1) In Figure 2, we wonder why A-D are H&E staining, while E-F are immunofluorescence. In addition, we wonder why the authors now stain for Megalin instead of LTL to detect proximal tubule segments.2) We think that the data presented in Figure 1 and Figure 2 would be strengthened by the addition of quantification of kidney size or cystic index, especially comparing the severity of the different induction timepoints of Figure 2.3) In Figure 3C, we are unsure what the X-axis labels (D2, B2, F3, A2, F2) are. Please clarify.4) In Figure 6, we are unsure what conclusion the authors are trying to draw. For instance, in the text they refer to a more “motile-like cilia phenotype”, yet Figure 6D, G, and H show 8 microtubule doublets with a misplaced doublet in the center, which happens in normal primary cilia as you image more distally from the cell body. They also lack the electron dense NDRC components and dynein arms which are present in motile cilia. To say whether or not the ciliary ultrastructure is disrupted, the authors would need to do 3D reconstruction using a technique such as scanning block face EM to ensure you are in the same region when comparing cilia.Abdul-Majeed, S., Moloney, B. C., & Nauli, S. M. (2012). Mechanisms regulating cilia growth and cilia function in endothelial cells.Cell Mol Life Sci, 69 (1), 165-173. doi:10.1007/s00018-011-0744-0Kamath, B. M., Spinner, N. B., & Rosenblum, N. D. (2013). Renal involvement and the role of Notch signalling in Alagille syndrome.Nat Rev Nephrol, 9 (7), 409-418. doi:10.1038/nrneph.2013.102Keeling, J., Tsiokas, L., & Maskey, D. (2016). Cellular Mechanisms of Ciliary Length Control. Cells, 5 (1). doi:10.3390/cells5010006McCright, B., Gao, X., Shen, L., Lozier, J., Lan, Y., Maguire, M., . . . Gridley, T. (2001). Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation.Development, 128 (4), 491-502.Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P., & Golemis, E. A. (2007). HEF1-dependent Aurora A activation induces disassembly of the primary cilium. Cell, 129 (7), 1351-1363. doi:10.1016/j.cell.2007.04.035Sanchez, I., & Dynlacht, B. D. (2016). Cilium assembly and disassembly.Nat Cell Biol, 18 (7), 711-717. doi:10.1038/ncb3370
Review contributors: Rachel J. Harding, Claudia Alvarez and Jacob McAuleyHuntington’s Disease Research Team, Structural Genomics Consortium, University of Toronto, CanadaNB: Review structure adapted from PreReview guidelines: https://prereview.org/users/164141/articles/200820-prereview-guidelines-how-to-write-a-preprint-reviewPreprint: https://www.biorxiv.org/content/10.1101/721191v1
The UIUC Plant Physiology journal club reviewed the preprint “α-carboxysome formation is mediated by the multivalent and disordered protein CsoS2” (doi: https://doi.org/10.1101/708164) by Oltrogge et al. 2019. The paper describes the biochemical characterization of the CsoS2 protein involved in carboxysome assembly, identifying a repeat peptide region that makes weak electrostatic interactions with rubisco through the use of bio-layer interferometry (BLI) and x-ray crystallography. The authors identified evolutionary conserved residues through protein sequence comparisons and the sites of interaction between these residues and rubisco through protein X-ray crystallography. We found the paper to be very well written, well presented and valuable addition to knowledge about alpha carboxysome assembly. Our journal club assessed the paper as part of a learning exercise about how to make work accessible to a wide audience. Participants first learned about the “and-but-therefore (ABT)” model of paper writing popularized by Randy Olsen in his freely available book "Huston, we have a narrative", that can be used throughout the manuscript to help maintain the reader’s interest. Focusing on the abstract we found it to contain many of aspects of the ABT model. We also thought it could potentially be strengthened by including a stronger “but” phrase which generally represents the question under consideration. It was suggested that this phrase would start with the fact that there is little knowledge about how the carboxysome is assembled, and some members of the club questioned if the ongoing carboxysome engineering efforts might be mentioned as relevant to the wider importance of the work (either in the abstract or the discussion).One aspect we found particularly interesting was the similarities between CsoS2 and the algal protein EPYC which has been implicated in aggregation of rubisco in the pyrenoid. These appeared to us to an important point, and the reason to include information about CsoS2 as an intrinsically disordered protein (IDP) that could perhaps be emphasized more. As we were not familiar with the PONDR-FIT disorder score, we would have found it helpful to have a little more explanation as to its importance and interpretation. Overall we liked the approach for analyzing IDPs and thought it was an impressive effort to successfully crystallize the CsoS2 peptide with rubisco. In addition, we assessed the presentation of figures, we particularly liked the use of consistent colouring throughout, the choice of clearly legible font sizes on all graphs and the helpful diagrams to illustrate biochemical procedures, such as the BLI procedure in Fig 2b. One consideration is whether the choice of colors is colorblind friendly, using the app color oracle, several of the colors are indistinguishable in all the figures analysed. We also thought inclusion of legend titles would help guide readers on how best to interpret the data. We thought the X-ray crystallography data was presented in a clear and helpful manner, displaying what the individual residue interactions were between the bpeptide and rubisco. If it could be improved further it may be by inclusion of a label of rubisco for non-experts who may not immediately associate CbbL and CbbS as subunits. Finally, we particularly liked Figure 5 as it neatly summarized the proposed role of CsoS2 in carboxysome assembly.Other thoughts included:It would be interesting to include discussion of why the full length CsoS2 peptide does not appear to bind rubisco.The paper tied up loose ends and did a good job of using multiple approaches to build evidence for the direct interaction of CsoS2 and rubisco.
This is a review of Caseys et al. bioRxiv doi: https://doi.org/10.1101/507491 posted on June 25, 2019. This study aims at addressing whether coevolutionary models of host-pathogen interactions apply to a generalist pathogen that exhibits quantitative virulence across a broad range of plants. They generated an exhaustive virulence matrix for the nectrophic fungus Botrytis cinerea on 90 genotypes of 8 plant species. They conclude that this pathosystem doesn’t fit traditional arms-race coevolution models with quantitative variation in susceptibility distinct from the phylogenetic relationships between the examined plants.
This is a review of Baudin, Schreiber, Martin et al. bioRxiv doi: https://doi.org/10.1101/592824 posted on March 29, 2019. The authors used structural modelling to identify elements required for self-association of the NLR immune receptor ZAR1, specifically its N-terminal CC-domain ZAR1CC. They discovered that the N-terminal α1 helix and EDVID motif in ZAR1CC are important for oligomerization and function of ZAR1. This complements recent findings by Wang et al. (2019) based on cryo-EM structures, highlighting the importance of the α1 helix for the activity of ZAR1 although some differences were noted that could reflect the different experimental set ups (CC domain vs full-length protein) as discussed in the paper.
Review: Optimized FRET pairs and quantification approaches to detect the activation of Aurora kinase A at mitosis.In this manuscript, Bertolin et al. improve on their original Aurora Kinase A biosensor to produce a second generation that would help follow AURKA activation in regions where it is extremely low in concentration and undetectable with the original AURKA biosensor. The authors develop two independent strategies to improve on their previous work. First, they develop a single-color AURKA biosensor for multiplex FRET and second, a method to observe and quantify FRET efficiency in areas with very low AURKA abundance. The authors show that dark acceptors ShadowG and ShadowY allow for single-color FRET/FLIM measurements while first generation tandem GFP isn’t suitable due to low concentration of AURKA. They also show the inability of the original construct to measure FRET by 2c-FCCS and thus develop a novel method by replacing the donor-acceptor pair with a mTurquoise2 and novel superYFP. The experiments allowed the authors to develop guidelines when making new FRET biosensors such as characterizing the nature of the protein and making sure the conformational changes of the protein fall within the Forster’s radius of the donor-acceptor pair.The improvements to AURKA biosensors represent a novel way for studying the function of this kinase. While fluorescence anisotropy has been used in the past to study FRET in different kinases such as PKA, ERK, and cAMP, it has not been known to work with AURKA due to the nature of the protein and it’s function. Also, given the fact that levels of AURKA is regulated throughout the cell cycle, the ability to detect it at low levels will help understand it’s function in diverse contexts.The authors provide good explanations with regards to the anomalies seen in their data and point out any results that deviate from their expected hypothesis. However, experiments with regards to characterizing the effects of inserting a novel superYFP on the cell and AURKA’s function need to be seen. The author’s also fail to provide clear explanations for discrepancies between the inactivated kinases in Fig. 1B and 1C. The author’s work is systematic, giving context when constructing new strains, and provides clear explanations when talking about new methods of quantifying FRET. One thing that I did have a hard time understanding was the use of anisotropy to measure FRET, and I think the authors could have done a better job introducing the concept.In terms of experiments that need to be done in order to further validate the results. As mentioned previously, the differences observed in Δlifetime for inactivated ShG-AURKA-mTurq2 and ShY-AURKA-mTurq2 need to be investigated or explained better. Similarly, effects of inserting flanking donor-acceptor pairs on the function of the kinase need to be quantified. It would be relevant to see how insertion of the flanking pairs affect AURKA localization to the spindle poles and morphology of the cell compared to wildtype. It would also be interesting to see if normal, non-arrested cells can function properly for multiple generations with the inserted constructs.There are minor spelling mistakes that can be attributed to continental differences. But for the most part, the article is easy to read and well written, however, explaining the thresholds in Fig. 1 and 2 will help the readers. As someone who is not familiar with analyzing fluorescence data, I did have a tough time understanding Fig. 3 and 4C, but the data and the author’s interpretation are clear and convincing.