Floryn Lynorah Mtemeli, Irene Walter*, Ryman ShokoDepartment of Biology, Chinhoyi University of Technology, Zimbabwe*Corresponding author: [email protected] 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.

This is a review of Knaus et al. bioRxiv doi: https://doi.org/10.1101/633701 posted on May 16, 2019. In this paper, the authors studied variations in ploidy in a wide range of isolates of the potato blight pathogen Phytophthora infestans.

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.

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

[  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 ]PREreview GuidelinesOverview and take-home messages:

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

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

Biodiversity trends are stronger in marine than terrestrial assemblagesShane Blowes, Sarah Supp, Laura Antao, Amanda Bates, Helge Bruelheide, Jonathan Chase, Faye Moyes, Anne Magurran, Brian McGill, Isla Myers-Smith, Marten Winter, Anne Bjorkman, Diana Bowler, Jarrett EK Byrnes, Andrew Gonzalez, Jes Hines, Forest Isbell, Holly Jones, Laetitia Navarro, Patrick Thompson, Mark Vellend, Conor Waldock, Maria DornelasbioRxiv, October 30th, 2018doi: https://doi.org/10.1101/457424Overview and take-home messages:Blowes et al. tackle an impressive and large undertaking in this paper by attempting to disentangle global biodiversity trends through a meta-analysis of data from 358 studies. By dividing the available data by biome and taxa, the authors were able to detect different biodiversity trends in marine and terrestrial biomes. Tropical marine biomes, particularly the Caribbean, have a more negative deviation from the mean trend in species richness and more positive deviations from the overall trend in species turnover--species are turning over more quickly in marine biomes. The analyses demonstrate that mean local species richness is not decreasing, but many individual regions deviate significantly from the overall mean. The results have important implications for how we discuss changes in biodiversity in the anthropocene, but it is important to make clear that locally static species richness does not equate to globally static species richness, and species are going extinct at an alarming rate. Overall, this paper presents careful analyses and is clearly written, however, there are a few issues that, if addressed, we feel could improve future versions of the manuscript.  

Gender and international diversity improves equity in peer review Dakota Murray, Kyle Siler, Vincent Lariviére, Wei Mun Chan, Andrew M. Collings, Jennifer Raymond, Cassidy R Sugimoto bioRxiv, v1 (August 29 2018) https://doi.org/10.1101/400515

This is a review of the bioRxiv preprint "EMT network-based feature selection improves prognosis prediction in lung adenocarcinoma" by Borong Shao, Maria Bjaanæs, Åslaug Helland, Christof Schütte, Tim Conrad, doi:10.1101/410472. This review was compiled from a discussion during the live-streamed Bioinformatics preprint journal club as part of an Open Access Week effort organized by the PREreview team and PLOS. Event details can be found here, and the collaborative Etherpad showing all the journal club notes can be found here.In addition to those named as authors above, the participants who wished to be acknowledged for their contributions to this review are as follows: Samantha Hindle, Paul Goetsch, and Bradly Alicea.

The UIUC Plant physiology journal club chose to review the preprint “Triose phosphate utilization and beyond: from photosynthesis to end-product synthesis” (http://dx.doi.org/10.1101/434928), by McClain and Sharkey, about the role of phosphate levels in relation to CO2 assimilation during photosynthesis. The paper explains that when the rate of photophosphorylation which produces ATP, exceeds that of starch or sucrose synthesis which consumes ATP, phosphate levels drop causing photosynthetic electron transport rates to slow. This process is defined as Triose phosphate limitation (TPU). We found the paper provided a very thorough definition of TPU and thought it was a really good introduction to the topic. In particular, the explanation about the way TPU limitation can be identified by no change in photosynthesis and decline in PhiPSII at high CO2 was very useful. The paper raises a number of points we were interested to discuss further including why TPU limitation is only slightly more than photosynthetic rate? Are some plants are more subjected to TPU limitation than others? Why is TPU limitation is slightly more than photosynthetic rate (one might predict that natural selection would select for utilization of all their photosynthate)? Why C4 plants do not experience any TPU limitation? Is it more common to see TPU limitation in Li6800s?The question of the potential impact of a rising atmospheric CO2 concentrations on TPU is a fascinating topic.  We thought it would be beneficial for readers to include reference to Experimental free air CO2 enrichment (FACE) experiments which have sought to address this issue, and some degree of guidance about what types of increases in atmospheric CO2 would be required to potentially see an effect. In addition it would be useful to mention that many of the TPU studies have been performed in pot bound plants, which experience an altered shoot/root ratio creating an artificial sink limitation, and a bit more discussion about how often TPU limitation might occur under field conditions (Arp 1991). Finally it might be good to include a bit more discussion about how the xanthophyll cycle kicks in to protect the plant under stress and that decreased PhiPSII could be an example of non-photochemical quenching.In summary, we thoroughly enjoyed the paper, we learnt a lot and look forward to seeing it published.Minor pointsIn equation 5 it is not entirely clear what the phi symbol is?  The symbol R is used to represent three different variables, could maybe consider changing this to prevent confusion.Many color combinations used in the figures would be unsuitable for those with color blindness, and suggest using color oracle (https://colororacle.org/) to help.Fig 2: Clarification on what ‘a little’ and ‘a lot’ means would be helpfulFig 2: We found it a bit confusing, many members were unclear about what it was demonstrating and suggest considering reformulating the description as we believe it could be useful as a teaching aid.Fig 3: Many members really liked this figure and thought it provided a clear image to explain the various limitations a plant experiences during an A/Ci curve. There was some discussion about where the boundary lines were drawn, this might be addressed by having zones of colour fade into each other rather than represented as sharp boundaries. It was also suggested that some kind of upper bound to J limitation of ETR might be included (possibly fading out to white?)ReferencesArp (1991) https://doi.org/10.1111/j.1365-3040.1991.tb01450.xRodgers et al. (2004) https://doi.org/10.1111/j.1365-3040.2004.01163.x

This is a review of the bioRxiv preprint "Host-parasite interaction explains variation in prevalence of avian haemosporidians at the community level" by Luz Garcia-Longoria, Alfonso Marzal, Florentino de Lope, and Laszlo Garamszegi. DOI: https://doi.org/10.1101/432260 This review was compiled from discussion points raised during a PREreview live-streamed Ecology preprint journal club as part of Open Access Week, October 24, 2018. The event details can be found here and the collaborative Etherpad showing all the journal club notes can be found here. In addition to those named as authors above, the participants who wished to be acknowledged for their contributions to this review are as follows: Dariusz Murakowski, Irene Ramos, Dena Emmerson, Adéla  Nacer, Asar Khan, and Daniela Saderi.

Review authored by Marielle Cavrois, Nadia Roan, and Ibraheem AliGladstone Institute of Virology and ImmunologyUniversity of California San FranciscoThis is a journal club review of Systems analysis by mass cytometry identifies susceptibility of latent HIV-infected T cells to targeting of p38 and mTOR pathways. Fong et al. 2018. bioRxiv doi: https://doi.org/10.1101/371922  Fong and colleagues present a very interesting study investigating signaling pathways and biomarkers that are dysregulated in cells exposed to HIV-1. Using a combination of mass cytometry and pharmacological approaches, the authors find that HIV-1 exposed cells are sensitized to cell death caused by T cell activation through disruption of the p38 and mTOR signaling pathways, and then demonstrate that ex vivo treatment of cells from ART-suppressed HIV-infected individuals with drugs targeting these pathways can decrease the proportion of latently-infected cells. This study is of importance as finding ways to therapeutically eliminate latently-infected cells is needed. Major Comments: Use of CyTOF to study the intracellular signaling pathways elicited during HIV infection is a valuable approach to identify novel ways to target HIV-infected cells therapeutically. Targeting intrinsic cell death pathways in latently-infected cells is an exciting approach to try to eliminate the persistent reservoir. However, since the majority of the experiments conducted were in vitro, and many in vitro models of HIV latency exist, the paper would benefit from a more thorough explanation and justification of this particular model of HIV latency. For example, Figure 1A could have more clearly delineated the experimental timeline of infection and stimulation (e.g., illustrating the fact that two rounds of stimulation were used: one to render cells permissive to infection, the second to reactivate latent cells). This would have been more helpful than images depicting the barcoding and cytof methodology, which could be described in the methods section. Furthermore, it should be acknowledged that the HIV-exposed cells consist of 3 cell populations: those that are productively infected, those that are latently infected, and those that are exposed but remain uninfected (broadly termed “bystander cells”, which can include both cells that fused to virus as well as those that did not). It is important to note that when the authors restimulate the HIV-exposed population of cells, they are restimulating a mixed population of these 3 populations, which can interconvert (e.g., p24- can become p24+). (As a side note, the cells should be called p55Gag – vs. +, since intracellular Gag is not p24). This, along with the fact that p24- cells may be predominantly bystander cells and not latently-infected cells, makes it difficult to assess how specific the identified signaling pathways are for latent cells. Also, the authors should clarify why they used different sets of subpopulations in different sections of the paper: sometimes, analysis was performed on the bulk population of HIV-exposed cells, other times on gated populations of p24+ vs. p24- cells.One valuable tool that exists in the field are the so-called dual-reporter viruses, that distinguish between latently- vs. productively-infected cells, \cite{Battivelli2018,Calvanese2013}. The in vitro part of the study could be markedly strengthened by validating the main results through use of such a system, or another system where a pure population of latently-infected cells can be obtained. One of the main readouts the authors used for sensitization to cell death in HIV exposed cells was Cleaved Caspase 3 (CC3). It would have benefitted the reader to have seen the gating strategy leading up to the population of cells that did or did not express CC3. In particular, it is important to know whether cells were gated from the live population as assessed by a viability dye, since dead/dying cells tend to be non-specifically sticky to antibodies.  Additional markers of cell death would have also been valuable in the FACS and CyTOF analyses. DREVI appears to be a powerful tool to infer associations and identify signaling pathways in play, but as many are not familiar with the nature of this analysis tool, a more thorough explanation of how it works, how it should be interpreted, and how it reveals insights that couldn’t be deciphered from the original datasets, would be helpful. Additional Comments:Regarding Figure 1B, were the productively-infected population pre-gated on the CD4- cells? (since HIV downmodulates CD4 upon productive infection)  Regarding Fie. S1E, are all the position Gag+ cells also expressing TAT?  On a minor note, the text Page 5 line 8 should be edited to read Figure 1B There appears to be overlap between the populations labeled as Uninfected (UI), Exposed-Uninfected (EU), and Latent Infected (LI) (Fig. S2, Fig. 2D-F). As indicated above, the LI contains many bystander cells, a fact that should be taken into consideration during the analysis. What is the advantage of analyzing the data in the way presented in Fig. 2E-F over the methods shown in Fig. 2C? Could the authors give their thoughts as to why p-S6 is only upregulated in only a subset of actively infected cells? In Figure 3E it would have been helpful to have known how many donors were used for each of the conditions in these experiments. In Figure 5 the authors should comment on the dosage of ganetespib and INK128 used and whether these concentrations are those that can be achieved in vivo, and the extent to which these doses were cytotoxic. Cell viability info would have been particularly helpful for Fig. 5 G-H since it appears that the treatment preferentially kills latently infected cells. It is important for the reader to know what is the extent of cell death observed after the 3 days of treatment with ganetespib, INK128, or a combination of the two. The authors could also comment on whether pre-treatment with ganetespib and INK128 might affect the ability of cells to be effectively stimulated by PMA/ionomycin as done in TILDA, since these treatments may target similar pathways? On a minor note there is no panel J in Figure 5 (see text page 14).