PREreview beta testing phase – Here is how you can help!
Hello everyone! In this post we will detail our beta testing phase, hopefully clarifying what it means to be a beta tester for PREreview and guiding you on how to best contribute. Thank you for taking the time to help us in our mission to make journal clubs worth any researchers' time!
How to start a PREreview journal club: tips & email templates
The following guidelines and email templates are meant to help you start a PREreview journal club at your institution. The first two email templates are for you to send to your department to invite colleagues to your journal club; the last one is for you to send to the authors to let them know you have reviewed their preprint on PREreview. Please let us know if these materials helped you, or suggest changes by emailing us at firstname.lastname@example.org. Thank you!
Bio 200C Assignment Bhumil Patel
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.
PREreview of bioRxiv article “Cross-reactivity of a rice NLR immune receptor to distinct effectors from the blast pathogen leads to partial disease resistance”
Review of: Cross-reactivity of a rice NLR immune receptor to distinct effectors from the blast pathogen leads to partial disease resistanceAuthors: Freya A. Varden, Hiromasa Saitoh, Kae Yoshino, Marina Franceschetti, Sophien Kamoun, Ryohei Terauchi, Mark J. BanfieldDOI: http://dx.doi.org/10.1101/530675This manuscript examines the molecular mechanism of the recognition of the “mismatched” Magnaporthe oryzae (Mo) effector AvrPia by the rice CC-type NLR Pikp. The authors show that transient co-expression of Pikp and AvrPia induces a weak cell death response in N. benthamiana (Nb). In line with these results, a Pikp-containing rice cultivar induces a weak immune response, whereas the allelic Pikm cannot. Next, they show that the Pikp HMA domain (Pikp-HMA) directly interacts with AvrPia in vitro, while Pikm cannot. X-ray crystallography showed that Pikp-HMA indeed directly binds AvrPia in a manner reminiscent of the interaction between RGA5-HMA and AvrCO39. This mode of interaction is in contrast to the previously published AvrPikD Pikp-HMA structure. The authors propose that the N-terminal extension of AvrPikD is responsible for the configuration of the AvrPikD Pikp-HMA heterodimer and could enhance the AvrPia Pikp-HMA interaction. However, addition of this N-terminal extension to AvrPia did not enhance, but instead reduced, AvrPia-Pikp cell death output in Nb. Furthermore, this modified AvrPia version was also not recognised by Pikm, which, like Pikp-AvrPikD, depends on the AvrPikD N-terminal extension.The finding that an integrated domain of an NLR is able to recognise two effectors using two distinct interfaces is a novel finding and will be of interest for the wider NLR biology community. Furthermore, these findings have potential implications for the plant breeding industry. However, we suggest that addressing the issues that we raise below will significantly strengthen the authors’ claims and the impact of their study.Major comments1. The authors use a cell death index to quantify the severity of the cell death response in Nb (Fig. 1 and 5). If the authors wish to show these data we recommend to use stacked bar plots instead of box plots. The use of box plots implies the data has a Gaussian distribution, which clearly is not the case for many, if not all, of the genotypes tested. Moreover, as the index only consists of a scale of 6 possible values, a Gaussian distribution cannot really be made. Furthermore, it is unclear whether the differences depicted are statistically significant. We would recommend to include this, and clearly state the statistical test used in the figure legend. Most importantly, we would advise to complement these data with ion leakage measurements that are known for a quantifiable dose-response relationship. Such physiological experiments will provide a more quantitative readout and strengthen the claim that Pikp induces an intermediate cell death response upon AvrPia perception. This is of particular significance as “partial disease resistance” is one of the important findings of this paper as stressed in the title. 2. The authors show that the rice cultivar K60 containing Pikp induces a partial immune response upon infection with Mo Sasa2 (AvrPia). This claim can be made based on the pictures shown in figure 2. However, a quantification of the lesion size (area and/or lesion length) would be desirable. Furthermore, it would strengthen the authors claim if they would back up these observations with some physiological experiments, e.g. 1) measuring fungal growth by qPCR on fungal genomic DNA, 2) Ion leakage, 3) staining rice leaves for ROS with DAB or H2DCFDA, 4) measuring transcriptional outputs by qPCR on relevant marker genes, such as RbohD. Particularly comparing transcriptional outputs and ion leakage over time in plants infected with Mo AvrPikD and Mo AvrPia would be desirable. Not only will these data strengthen the author’s conclusions, they would yield insight into the resistance pathway dynamics between Mo AvrPikD- and Mo AvrPia-treated plants. 3. In figures 3 and 4 the authors clearly show that Pikp-HMA is able to directly interact with AvrPia by gel filtration, SPR and x-ray crystallography. Although these data are convincing, one aspect is still missing. Since the interaction experiments are based on in vitro systems, it would strengthen the authors’ claims if they would include in vivo interaction data. Yeast-2-Hybrid or in planta split-luciferase assays are two options the authors could explore. 4. The interaction data between Pikp and AvrPia are based on the interaction of the Pikp-HMA and the effector AvrPia. It would strengthen the authors’ claims if they would confirm their interaction results by performing interaction assays using the full length NLR instead of merely the HMA domain. Yeast-2-Hybrid and split-luciferase would be suitable techniques. 5. Given that the presented AvrPia Pikp-HMA structure as well as the previously reported AvrPikD Pikp-HMA structure are based on HMA domains lacking the adjacent NLR modules, how do the authors know that the recognition of two distinct HMA interfaces occurs and is physiologically relevant in the corresponding full-length NLRs? The authors do not show a variant of Pikp (-HMA) that is unable to interact with AvrPia, even though the authors discuss residues in Pikp-HMA that seem to be responsible for the interaction, e.g. PikpS202 PikpD217, and PikpDS204 (Fig. 4C). The authors could try to evaluate the effect of mutating these three sites and determine whether this leads to a reduced interaction between Pikp (-HMA) and AvrPia. Moreover, if this does reduce binding, the authors could test whether these mutations also affect Pikp outputs in planta, e.g. by co-expressing the Pikp variant with AvrPia in Nb and quantifying signalling outputs by ion leakage.Minor comments 1. Although the manuscript is well written, clear and succinct, in some instances the authors refer to a previous finding, but do not supply the according reference, e.g. Introduction; paragraph 5: “The AvrPikC... Pik NLR.”, and Introduction; paragraph 7: “Despite... on its own.”. Therefore, I would recommend the authors to carefully go through the manuscript and make sure all published data are appropriately referenced.Joram A. Dongus and the Plant Microbe Interactions Journal Club at MPIPZ
UIUC Plant Physiology JC (2018/12/3): Revisiting tradeoffs in Rubisco kinetic parameters
The preprint “Revisiting tradeoffs in Rubisco kinetic parameters” by Flamholz et al. 2018 (https://doi.org/10.1101/470021) investigates the tradeoffs between catalytic efficiency and rate, of the central enzyme in carbon fixation, Ribulose-1,6-bisphosphate Carboxylase/Oxygenase (RuBisCO), using kinetic modeling based on biochemical data. The manuscript builds on previous work from the group (Savir et al. 2010; doi: 10.1073/pnas.0911663107), including an expanded dataset of kinetic parameters of ~250 RuBisCOs from 286 different species extracted from the literature. We thought it was an important topic with potential interest for a wide range of researchers working on photosynthesis and evolution.The main questions the paper seeks to address are:Which trade-offs are inherent in Rubisco kinetics. Has evolution resulted in optimal kinetics within the constraints of those inherent trade-offs.The preprint challenges the theory that increasing RuBisCO activity reduces enzyme specificity as described by Tcherkez et al. (2006), which was based on a model of enzyme activity the discriminates between CO2 and O2 in a transition state. Data supporting this theory has been reported widely in the literature. However, the authors propose that there is little/no correlation between specificity and activity, and most previously-found correlations (KcatC and SC/O etc) are smaller for the new dataset, except for KcatC/KC and KcatO/KO. We really enjoyed reading the manuscript and as it challenged our preconceptions about RuBisCO activity. We found it interesting (and surprising!) that the data contradicts a well-established theory, and it increased our awareness out current models of enzyme activity. We also thought it was interesting that they were able to collect data from so many species across many previous studies and also break down trends/relationships between different clades or physiologies. It was also interesting that given that they were using data from studies that showed the opposite, they were able to come to the conclusion they found. We particularly liked the authors' suggestions about how to move the field forward and the call for an improved understanding of RuBisCO kinetic mechanism.There were a few areas we thought it would be useful to clarify:Providing a cartoon model of the proposed mechanism would help readers unfamiliar with the nuances of the models being assessed. More information about how the authors selected and filtered data collecting from the literature and how the different datasets were taken into account in the statistical analysis. i.e. how many measurements per species, types of values (mean/median) etc.Figure 4 and 5: how was the conclusion reached that there was no correlation between parameters?Organization, it would make things stronger to layout the arguments clearly between what came before and after, for those not familiar with the academic argument.As the paper argues against what most people are reading it might expect, additional text theorizing why this is the case would be useful to guide readers.Minor commentsFigure 2A y-axis labelsPut a key at the top as the symbols can get buried in the text
PREreview Journal Club (UCL Great Ormond Street Institute of Child Health)
[ Child Growth Predicts Brain Functional Connectivity and Future Cognitive Outcomes in Urban Bangladeshi Children Exposed to Early Adversities ]
[Wanze Xie, Sarah K. Jensen, Mark Wade, Swapna Kumar, Alissa Westerlund, Shahria H. Kakon, Rashidul Haque, William A. Petri, Charles A. Nelson, 22 Oct 2018, version #1, biorxiv]
[ 10.1101/447722 ]
Overview and take-home messages:
Boston University NeuroPreprint Journal Club
A spatial code in the dorsal lateral geniculate nucleusVincent Hok, Pierre-Yves Jacob, Pierrick Bordiga, Bruno Truchet, Bruno Poucet, Etienne Save doi: https://doi.org/10.1101/473520Version posted on Biorxiv: 11/19/18In the current work, Hok et al. report the existence of spatial receptive fields in the dLGN of rats that are similar in nature to HPC place fields. The authors demonstrate that dLGN place fields, which make up approximately 30% of extracellularly recorded dLGN neurons, are less stable and more variable (on a pass by pass basis) than their HPC counterparts. Further, the authors show theta oscillations in the dLGN local field potential and the presence of individual dLGN neurons that have theta rhythmic spiking. The authors report that, unlike CA3 neurons, most dLGN neurons do not exhibit theta phase precession suggesting that the dLGN spatial code does not carry sequential information about the animal’s current, past, or future trajectories. Finally, Hok and colleagues show that a sub-population of dLGN neurons can be modulated by visual stimulation but that this population does not overlap with neurons that exhibit place fields. Interestingly, dLGN place fields are insensitive to the presence or absence of visual information (as determined by similarity in responses in recordings from light vs. dark conditions) but remap when the color of the environment is altered (from all white to all black). By and large the results are robust and the reported findings will be of interest to researchers considering interactions between different forms of neural spatial representations and visual information. Further, the observation of spatial receptive fields in the dLGN raises important questions about fundamentals of visual processing. We enjoyed the manuscript and have the following suggestions for the authors.Major suggestions: • There is little discussion about dLGN visual sensitivity in the rat despite there being extensive literature on the subject. There is known retinotopy in dLGN (see for example, Montero et al. 1968). How, if at all, does this relate to the spatial response properties of dLGN neurons? For example, is there any correspondence between neurons that would be sensitive to the lower half of the visual field and remapping in the light vs. dark arena conditions? A similar question could be raised regarding the object sensitive sub-population. The authors should attempt to address the relationship between their observations and visual sensitivity of dLGN in more detail. • The object sensitive sub-population is intriguing, but the details concerning this finding are ambiguous. It is mentioned briefly in the main manuscript but the percentage of neurons exhibiting this tendency is unclear, even when examining the extended data concerning this observation. As a result, the reader is left a bit distracted by this information, especially when considering that object sensitive neurons are not place cells. The authors should consider presenting this component of the manuscript in greater detail. • The visual stimulation experiment is posed as a control for determining that dLGN place cells were indeed recorded in a visually sensitive area (dLGN), but this experiment was not conducted for CA3 neurons. How can we know for certain that the visual stimulation protocol wouldn’t have elicited modulation in CA3 neurons as well, thus rendering this component of the study inconclusive? • Tissue can often become displaced for extended periods of time when moving tetrodes along the D/V axis. How confident are the authors in the correspondence between their tetrode turning records and final tetrode placement? The waveform related analyses are pretty convincing and seems to cluster nicely into two groups, but waveform shape is not a definitive metric for determining the location of a recorded neuron. Is it possible to record from dLGN without moving electrodes through the hippocampus first? It would be helpful if the authors could provide more histology so readers can examine for themselves how displaced CA3 tissue was. • It seems that LFPs were primarily referenced to another tetrode. Instead, LFPs should be referenced against an electrode outside of either the hippocampus or dLGN such as a skull screw above the cerebellum. Because of the current referencing scheme and many other factors, theta oscillations recorded in the dLGN could be explained by volume conduction yet this is not considered. These issues may be of great importance to the theta phase precession analysis. Did the authors attempt to look at phase precession for dLGN place cells against both HPC LFPs as well as dLGN LFPs? If not, this would be an important analysis to consider. • No power spectral density plots are shown for theta oscillations in CA3 and dLGN. It would be interesting to see if the dLGN theta peak in the power spectrum is broader compared to CA3, which might explain the higher theta frequency and would also affect phase-related analyses. Additionally, a consideration of speed related modulation of theta dynamics should likely be included. Minor Concerns: • Table 1 should include a #/% of cells which were “object specific” • Re Overdispersion analysis (line 460): A linearly scaled Poisson distribution will itself be poisson distributed, not normal, with the approximation being more valid at higher firing rates. The Z value described here will therefore be biased to higher values for lower firing rate neurons. Because of this, it is unclear if the results in lines 90-92 are artificially strong or weak. If thalamic neurons have a higher firing rate than CA3 neurons, they may have an even higher difference in dispersion than reported. A consideration of percentiles rather than standard deviations would likely be revealing. • Quantification of the object rotation sub-experiment (lines 144-147) should be included in the main manuscript text. • More details on the properties of theta modulation in dLGN should be presented. What percentage of dLGN neurons had peaks in the theta frequency range in the FFT of their spike train autocorrelations (i.e. how many were significantly theta rhythmic)? What percentage of dLGN neurons were theta phase locked and was there a bias to a particular theta phase? Was theta oscillatory activity for single dLGN neurons modulated by running speed? • Was a velocity filter used when creating spatial firing rate maps? In a related point, did the authors examine potential differences in mobile vs. immobile activation for dLGN vs. HPC? • Perhaps we missed it, but what size was the smoothing kernel for 2D ratemaps (in cms)? • Did the authors examine the spatial distribution of dLGN place fields and, if so, were they uniformly distributed? It would be interesting to know if the fields clustered near objects for example. • The explanation of the “field index” metric is difficult to understand in the methods and should be clarified if possible. Figure 2: The color-coded pass index trajectory plots are not very informative because the dots are all layered on top of each other. Authors could do one or multiple of the following to enhance interpretation: make the dots smaller, make the dots transparent, make the color map sequential rather than qualitative. It would also be helpful to provide an “n” of the number of place cells in each region in Fig 2g. Figure 3: The close up of a single pass in the upper right corner of sub-plots 3b and 3c are confusing. The legend could be revised to clarify what we are looking at. In general, we are a bit unclear as to why the close up of a single pass is necessary. Are we supposed to see rhythmic activity in the spikes? The close up of a single pass is not useful for this because the rat may not be moving at a constant velocity. It may be the case that the spike train above is sufficient. - BU NeuroPreprint JC
Present and past LivePREJCs: PREreview's community live-streamed preprint journal clubs
Below is a list of upcoming live-streamed preprint journal clubs (LivePREJCs) as well as those hosted in the past. To learn more about what to expect and how to solicit our help to organize one, please read here
PREreview-PLOS Open Access Week Preprint Journal Club Information
Join us for the OA Week PLOS/PREreview live-streamed preprint journal clubs! REGISTER HERESCHEDULEDONE! Neuroscience – Monday October 22, 2018 – 9am PDT / 12pm EDT / 4pm UTCPreprint: Sex Differences in Aggression: Differential Roles of 5-HT2, Neuropeptide F and Tachykinin Facilitators: Samantha Hindle (PREreview) and Daniela Saderi (PREreview)Expert: Dr. Tim MoscaCollaborative notes and info to join this call: Neuroscience EtherpadPREreview available here. DONE! Bioinformatics – Tuesday October 23, 2018 – 9am PDT / 12pm EDT / 4pm UTCPreprint: EMT network-based feature selection improves prognosis prediction in lung adenocarcinoma Facilitators: Daniela Saderi (PREreview) and Monica Granados (PREreview)Experts: Dr. Shannon McWeeney and Dr. Ted LaderasCollaborative notes and info to join this call: Bioinformatics EtherpadPREreview available here. DONE! Ecology – Wednesday October 24, 2018 – 9am PDT / 12pm EDT / 4pm UTCPreprint: Host-parasite interaction explains variation in prevalence of avian haemosporidians at the community level Facilitators: Jessica Polka (ASAPbio) and Steven Burgess (University of Illinois)Expert: Dr. Timothée PoisotCollaborative notes and info to join this call: Ecology EtherpadPREreview available here.What are PREreview Live-Streamed Preprint Journal Clubs? #LivePREJC