Author response:

We thank the editors and reviewers for their valuable feedback and are committed to addressing their suggestions in a revised manuscript. We appreciate the reviewers’ recognition of the value of our findings, including the insights into the consequences of synaptic topography and the investigation of spike initiation zones in DNs, which further advance our understanding of signal processing. Our studies offer broader insights into synaptic organization and its significance for dendritic integration in an ethologically relevant context.

We particularly appreciate the reviewer's suggestion to elaborate on the electrophysiological properties of DNs and to consider the electrotonic distance in our analysis. We also thank the reviewers for highlighting points that need clarification. In short, our models suggest that DNs effectively distribute synapses to maintain linear encoding of synapse numbers when multiple synapses are coactivated. This supports the results of an earlier study suggesting that synapse number gradients encode the location of an approaching stimulus in these neurons (Dombrovski et al., 2023).

We also agree with the reviewers that the temporal activation of synapses is highly relevant for this system. However, we have focused on synaptic topography because the characterization of temporal patterns of VPN activity is currently lacking in the field. A more detailed investigation of temporal dynamics is therefore beyond the scope of this study.

With the publication of the reviewed preprint, we have now made the computational pipeline and models available on GitHub (https://github.com/AusbornLab/VPN-DN-synapse-normalization).

Reference

Dombrovski M, Peek MY, Park J-Y, Vaccari A, Sumathipala M, Morrow C, Breads P, Zhao A, Kurmangaliyev YZ, Sanfilippo P, Rehan A, Polsky J, Alghailani S, Tenshaw E, Namiki S, Zipursky SL, Card GM. 2023. Synaptic gradients transform object location to action. Nature 613:534–542. doi:10.1038/s41586-022-05562-8

eLife Assessment

This valuable study on strategies used by Pseudomonas to subvert hots immunity identifies a new immune evasion strategy. The study presents solid evidence on the cleavage of VgrG2B by Caspase 11 and the generation of fragments that inhibit activity of the NLRP3 inflammasome. This work should be of interest to immunologists and microbiologists.

Reviewer #1 (Public review):

In the manuscript entitled "A VgrG2b fragment cleaved by caspase-11/4 promotes Pseudomonas aeruginosa infection through suppressing the NLRP3 inflammasome", Qian et al. found an activation of the non-canonical inflammasome, but not the downstream NLRP3 inflammasome, during the infection of macrophage by P. aeruginosa, which is in sharp contrast to that by E. coli (Figure 1). In realizing that the suppression of the NLRP3 inflammasome is Caspase-11 dependent, the authors performed a screening among P. aeruginosa proteins and identified VgrG2b being a major substrate of Caspase-11 (Figure 2). Next, the authors mapped the cleavage site on VgrG2b to D883, and demonstrated that cleavage of VgrG2b by Caspase-11 is essential for the suppression of the NLRP3 inflammasome (Figure 3). Furthermore, they found that a binding between the C-terminal fragment of the cleaved VgrG2b and NLRP3 existed (Figure 4), which was then proved to block the association of NLRP3 with NEK7 (Figure 5). Finally, the authors demonstrated that blocking of VgrG2b cleavage, by either mutation of the D883 or administration of a designed peptide, effectively improved the survival rate of the P. aeruginosa-infected mice (Figure 6). This is a well-designed and executed study, with the results clearly presented and stated.

Reviewer #2 (Public review):

Summary:

In their manuscript, Quian and colleagues identified a novel mechanism by which Pseudomonas control inflammatory responses upon inflammasome activation. They identified a caspase-11 substrate (VgrG2b) which, upon cleavage, binds and inhibits the NLRP3 to reduce the production of pro-inflammatory cytokines. This is a unique mechanism that allows for the tailoring of the innate immune response upon bacterial recognition.

Strengths:

The authors are presenting here a novel conceptual framework in host-pathogen interactions. Their work is supported by a range of approaches (biochemical, cellular immunology, microbiology, animal models), and their conclusions are supported by multiple independent evidences. The work is likely to have an important impact on the innate immunity field and host-pathogen interactions field and may guide the development of novel inhibitors.

Weaknesses:

Although quite exhaustive, a few of the authors' conclusions are not fully supported (e.g., caspase-11 directly cleaving VgrG2b, the unique affinity of VgrG2b-C for NLRP3) and would require complementary approaches to validate their findings fully. This is minimal.

eLife Assessment

Wang et al's study addresses an important critical gap in our understanding of de novo epithelial polarization using MDCK cell doublets surrounded by ECM, providing convincing evidence through imaging and depletion studies on the role of conserved polarity proteins and the centrosome during this process. While the authors propose a clear hierarchical model, there is a need for further exploration of how microtubule organization contributes to this process. Specifically, live cell imaging of microtubules under mutants and their included ECM conditions, along with a more precise temporal mapping of microtubule dynamics in relation to proteins like Gp135, would strengthen the study's conclusions.

Reviewer #1 (Public review):

Summary:

Wang, Po-Kai, et al., utilized the de novo polarization of MDCK cells cultured in Matrigel to assess the interdependence between polarity protein localization, centrosome positioning, and apical membrane formation. They show that the inhibition of Plk4 with Centrinone does not prevent apical membrane formation, but does result in its delay, a phenotype the authors attribute to the loss of centrosomes due to the inhibition of centriole duplication. However, the targeted mutagenesis of specific centrosome proteins implicated in the positioning of centrosomes in other cell types (CEP164, ODF2, PCNT, and CEP120) did not affect centrosome positioning in 3D cultured MDCK cells. A screen of proteins previously implicated in MDCK polarization revealed that the polarity protein Par-3 was upstream of centrosome positioning, similar to other cell types.

Strengths:

The investigation into the temporal requirement and interdependence of previously proposed regulators of cell polarization and lumen formation is valuable to the community. Wang et al., have provided a detailed analysis of many of these components at defined stages of polarity establishment. Furthermore, the generation of PCNT, p53, ODF2, Cep120, and Cep164 knockout MDCK cell lines is likely valuable to the community.

Weaknesses:

Additional quantifications would highly improve this manuscript, for example it is unclear whether the centrosome perturbation affects gamma tubulin levels and therefore microtubule nucleation, it is also not clear how they affect the localization of the trafficking machinery/polarity proteins. For example, in Figure 4, the authors measure the intensity of Gp134 at the apical membrane initiation site following cytokinesis, but there is no measure of Gp134 at the centrosome prior to this.

Reviewer #2 (Public review):

Summary:

The authors decoupled several players that are thought to contribute to the establishment of epithelial polarity and determined their causal relationship. This provides a new picture of the respective roles of junctional proteins (Par3), the centrosome, and endomembrane compartments (Cdc42, Rab11, Gp135) from upstream to downstream.<br /> Their conclusions are based on live imaging of all players during the early steps of polarity establishment and on the knock-down of their expression in the simplest ever model of epithelial polarity: a cell doublet surrounded by ECM.

The position of the centrosome is often taken as a readout for the orientation of the cell polarity axis. There is a long-standing debate about the actual role of the centrosome in the establishment of this polarity axis. Here, using a minimal model of epithelial polarization, a doublet of daugthers MDCK cultured in Matrigel, the authors made several key observations that bring new light to our understanding of a mechanism that has been studied for many years without being fully explained:

(1) They showed that centriole can reach their polarized position without most of their microtubule-anchoring structures. These observations challenge the standard model according to which centrosomes are moved by the production and transmission of forces along microtubules.

(2) (However) they showed that epithelial polarity can be established in the absence of centriole.

(3) (Somehow more expectedly) they also showed that epithelial polarity can't be established in the absence of Par3.

(4) They found that most other polarity players that are transported through the cytoplasm in lipid vesicles, and finally fused to the basal or apical pole of epithelial cells, are moved along an axis which is defined by the position of centrosome and orientation of microtubules.

(5) Surprisingly, two non-daughters cells that were brought in contact (for 6h) could partially polarize by recruiting a few Par3 molecules but not the other polarity markers.

(6) Even more surprisingly, in the absence of ECM, Par 3 and centrosomes could move to their proper position close to the intercellular junction after cytokinesis but other polarity markers (at least GP135) localized to the opposite, non-adhesive, side. So the polarity of the centrosome-microtubule network could be dissociated from the localisation of GP135 (which was believed to be transported along this network).

Strengths:

(1) The simplicity and reproducibility of the system allow a very quantitative description of cell polarity and protein localisation.

(2) The experiments are quite straightforward, well-executed, and properly analyzed.

(3) The writing is clear and conclusions are convincing.

Weaknesses:

(1) The simplicity of the system may not capture some of the mechanisms involved in the establishment of cell polarity in more physiological conditions (fluid flow, electrical potential, ion gradients,...).

(2) The absence of centriole in centrinone-treated cells might not prevent the coalescence of centrosomal protein in a kind of MTOC which might still orient microtubules and intracellular traffic. How are microtubules organized in the absence of centriole? If they still form a radial array, the absence of a centriole at the center of it somehow does not conflict with classical views in the field.

(3) The mechanism is still far from clear and this study shines some light on our lack of understanding. Basic and key questions remain:<br /> a) How is the centrosome moved toward the Par3-rich pole? This is particularly difficult to answer if the mechanism does not imply the anchoring of MTs to the centriole or PCM.<br /> b) What happens during cytokinesis that organises Par3 and intercellular junction in a way that can't be achieved by simply bringing two cells together? In larger epithelia cells have neighbours that are not daughters, still, they can form tight junctions with Par3 which participates in the establishment of cell polarity as much as those that are closer to the cytokinetic bridge (as judged by the overall cell symmetry). Is the protocol of cell aggregation fully capturing the interaction mechanism of non-daughter cells?

Reviewer #3 (Public review):

Here, Wang et al. aim to clarify the role of the centrosome and conserved polarity regulators in apical membrane formation during the polarization of MDCK cells cultured in 3D. Through well-presented and rigorous studies, the authors focused on the emergence of polarity as a single MDCK cell divided in 3D culture to form a two-cell cyst with a nascent lumen. Focusing on these very initial stages, rather than in later large cyst formation as in most studies, is a real strength of this study. The authors found that conserved polarity regulators Gp135/podocalyxin, Crb3, Cdc42, and the recycling endosome component Rab11a all localize to the centrosome before localizing to the apical membrane initiation site (AMIS) following cytokinesis. This protein relocalization was concomitant with a repositioning of centrosomes towards the AMIS. In contrast, Par3, aPKC, and the junctional components E-cadherin and ZO1 localize directly to the AMIS without first localizing to the centrosome. Based on the timing of the localization of these proteins, these observational studies suggested that Par3 is upstream of centrosome repositioning towards the AMIS and that the centrosome might be required for delivery of apical/luminal proteins to the AMIS.

To test this hypothesis, the authors generated numerous new cell lines and/or employed pharmacological inhibitors to determine the hierarchy of localization among these components. They found that removal of the centrosome via centrinone treatment severely delayed and weakened the delivery of Gp135 to the AMIS and single lumen formation, although normal lumenogenesis was apparently rescued with time. This effect was not due to the presence of CEP164, ODF2, CEP120, or Pericentrin. Par3 depletion perturbed the repositioning of the centrosome towards the AMIS and the relocalization of the Gp135 and Rab11 to the AMIS, causing these proteins to get stuck at the centrosome. Finally, the authors culture the MDCK cells in several ways (forced aggregation and ECM depleted) to try and further uncouple localization of the pertinent components, finding that Par3 can localize to the cell-cell interface in the absence of cell division. Par3 localized to the edge of the cell-cell contacts in the absence of ECM and this localization was not sufficient to orient the centrosomes to this site, indicating the importance of other factors in centrosome recruitment.

Together, these data suggest a model where Par3 positions the centrosome at the AMIS and is required for the efficient transfer of more downstream polarity determinants (Gp135 and Rab11) to the apical membrane from the centrosome. The authors present solid and compelling data and are well-positioned to directly test this model with their existing system and tools. In particular, one obvious mechanism here is that centrosome-based microtubules help to efficiently direct the transport of molecules required to reinforce polarity and/or promote lumenogenesis. This model is not really explored by the authors except by Pericentrin and subdistal appendage depletion and the authors do not test whether these perturbations affect centrosomal microtubules. Exploring the role of microtubules in this process could considerably add to the mechanisms presented here. In its current state, this paper is a careful observation of the events of MCDK polarization and will fill a knowledge gap in this field. However, the mechanism could be significantly bolstered with existing tools, thereby elevating our understanding of how polarity emerges in this system.

eLife Assessment

This important study shows that Toxoplasma gondii uses paracrine mechanisms, in addition to cell-intrinsic methods, to evade the host immune system, with MYR1 playing a key role in transporting effector molecules into host cells. The authors present convincing evidence that in vivo, MYR1-deficient parasites can be rescued by wild-type parasites, revealing a limitation in pooled CRISPR screens, where such paracrine effects may obscure the identification of key parasite pathways involved in immune evasion.

Reviewer #1 (Public review):

Previous studies have highlighted some of these paracrine activities of Toxoplasma - and Rasogi et al (mBio, 2020) used a single cell sequencing approach of cells infected in vitro with the WT or MYR KO parasites - and one of their conclusions was that MYR-1 dependent paracrine activities counteract ROP-dependent processes. Similarly, Chen et al (JEM 2020) highlighted that a particular rhoptry protein (ROP16) could be injected into uninfected macrophages and move them to an anti-inflammatory state that might benefit the parasite.

There are caveats around immunity and as yet no insight into how this works. In Figure 2 there is a marked defect in the ability of the parasites to expand at day 2 and day 5. Together, these data sets suggest that this paracrine effect mediated by MYR-1 works early - well before the development of adaptive responses.

Reviewer #2 (Public review):

Summary:

In this manuscript by Torelli et al., the authors propose that the major function of MYR1 and MYR1-dependent secreted proteins is to contribute to parasite survival in a paracrine manner rather than to protect parasites from cell-autonomous immune response. The authors conclude that these paracrine effects rescue ∆MYR1 or knockouts of MYR1-dependent effectors within pooled in vivo CRISPR screens.

Strengths:

The authors raised a more general concern that pooled CRISPR screens (not only in Toxoplasma but also other microbes or cancers) would miss important genes by "paracrine masking effect". Although there is no doubt that pooled CRISPR screens (especially in vivo CRISPR screens) are powerful techniques, I think this topic could be of interest to those fields and researchers.

Weaknesses:

In this version, the reviewer is not entirely convinced of the 'paracrine masking effect' because the in vivo experiments should include appropriate controls (see major point 2).

(1) It is convincing that co-infection of WT and ∆MYR1 parasites could rescue the growth of ∆MYR1 in mice shown by in vivo luciferase imaging. Also, this is consistent with ∆MYR1 parasites showing no in vivo fitness defect in the in vivo CRISPR screens conducted by several groups. Meanwhile, it has been reported previously and shown in this manuscript that ∆MYR1 parasites have an in vitro growth defect; however, ∆MYR1 parasites show no in vitro fitness defect the in vitro pooled CRISPR screen. The authors show that the competition defect of ∆MYR1 parasites cannot be rescued by co-infection with WT parasites in Figure 1c, which might indicate that no paracrine rescue occurred in an in vitro environment. The authors seem not to mention these discrepancies between in vitro CRISPR screens and in vitro competition assays. Why do ∆MYR1 parasites possess neutral in vitro fitness scores in in vitro CRISPR screens? Could the authors describe a reasonable hypothesis?

(2) The authors developed a mixed infection assay with an inoculum containing a 20:80 ratio of ΔMYR1-Luc parasites with either WT parasites or ΔMYR1 mutants not expressing luciferase, showing that the in vivo growth defect of ∆MYR1 parasites is rescued by the presence of WT parasites. Since this experiment lacks appropriate controls, interpretation could be difficult. Is this phenomenon specific to MYR1? If a co-inoculum of ∆GRA12-Luc with either WT parasites or GRA12 parasites not expressing luciferase is included, the data could be appropriately interpreted.

(3) In the Discussion part, the authors argue that the rescue phenotype of mixed infection is not due to co-infection of host cells (lines 307-310). This data is important to support the authors' paracrine hypothesis and should be shown in the main figure.

(4) In the Discussion part, the authors assume that the rescue phenotype is the result of multiple MYR1-dependent effectors. I admit that this hypothesis could be possible since a recently published paper described the concerted action of numerous MYR1-dependent or independent effectors contributing to the hypermigration of infected cells (Ten Hoeve et al., mBio, 2024). I think this paragraph would be kind of overstated since the authors did not test any of the candidate effectors. Since the authors possess ∆IST parasites, they can test whether IST is involved in the "paracrine masking effect" or not to support their claim.

eLife Assessment

This important study reports a detailed quantification of the population dynamics of Salmonella enterica serovar Typhimurium in mice. Bacterial burden and founding population sizes across various organs were quantified, revealing pathways of dissemination and reseeding of the gastrointestinal tract from systemic organs. Using various techniques, including genetic distance measurements, the authors present compelling evidence to support their conclusions, thus presenting new knowledge that will be of broad interest to scientists focusing on infectious diseases.

Reviewer #1 (Public review):

Hotinger et al. explore the population dynamics of Salmonella enterica serovar Typhimurium in mice using genetically tagged bacteria. In addition to physiological observations, pathology assessments, and CFU measurements, the study emphasizes quantifying host bottleneck sizes that limit Salmonella colonization and dissemination. The authors also investigate the genetic distances between bacterial populations at various infection sites within the host.

Initially, the study confirms that pretreatment with the antibiotic streptomycin before inoculation via orogastric gavage increases the bacterial burden in the gastrointestinal (GI) tract, leading to more severe symptoms and heightened fecal shedding of bacteria. This pretreatment also significantly reduces between-animal variation in bacterial burden and fecal shedding. The authors then calculate founding population sizes across different organs, discovering a severe bottleneck in the intestine, with founding populations reduced by approximately 10^6-fold compared to the inoculum size. Streptomycin pretreatment increases the founding population size and bacterial replication in the GI tract. Moreover, by calculating genetic distances between populations, the authors demonstrate that, in untreated mice, Salmonella populations within the GI tract are genetically dissimilar, suggesting limited exchange between colonization sites. In contrast, streptomycin pretreatment reduces genetic distances, indicating increased exchange.

In extraintestinal organs, the bacterial burden is generally not substantially increased by streptomycin pretreatment, with significant differences observed only in the mesenteric lymph nodes and bile. However, the founding population sizes in these organs are increased. By comparing genetic distances between organs, the authors provide evidence that subpopulations colonizing extraintestinal organs diverge early after infection from those in the GI tract. This hypothesis is further tested by measuring bacterial burden and founding population sizes in the liver and GI tract at 5 and 120 hours post-infection. Additionally, they compare orogastric gavage infection with the less injurious method of infection via drinking, finding similar results for CFUs, founding populations, and genetic distances. These results argue against injuries during gavage as a route of direct infection.

To bypass bottlenecks associated with the GI tract, the authors compare intravenous (IV) and intraperitoneal (IP) routes of infection. They find approximately a 10-fold increase in bacterial burden and founding population size in immune-rich organs with IV/IP routes compared to orogastric gavage in streptomycin-pretreated animals. This difference is interpreted as a result of "extra steps required to reach systemic organs."

While IP and IV routes yield similar results in immune-rich organs, IP infections lead to higher bacterial burdens in nearby sites, such as the pancreas, adipose tissue, and intraperitoneal wash, as well as somewhat increased founding population sizes. The authors correlate these findings with the presence of white lesions in adipose tissue. Genetic distance comparisons reveal that, apart from the spleen and liver, IP infections lead to genetically distinct populations in infected organs, whereas IV infections generally result in higher genetic similarity.

Finally, the authors investigate GI tract reseeding, identifying two distinct routes. They observe that the GI tracts of IP/IV-infected mice are colonized either by a clonal or a diversely tagged bacterial population. In clonally reseeded animals, the genetic distance within the GI tract is very low (often zero) compared to the bile population, which is predominantly clonal or pauciclonal. These animals also display pathological signs, such as cloudy/hardened bile and increased bacterial burden, leading the authors to conclude that the GI tract was reseeded by bacteria from the gallbladder bile. In contrast, animals reseeded by more complex bacterial populations show that bile contributes only a minor fraction of the tags. Given the large founding population size in these animals' GI tracts, which is larger than in orogastrically infected animals, the authors suggest a highly permissive second reseeding route, largely independent of bile. They speculate that this route may involve a reversal of known mechanisms that the pathogen uses to escape from the intestine.

The manuscript presents a substantial body of work that offers a meticulously detailed understanding of the population dynamics of S. Typhimurium in mice. It quantifies the processes shaping the within-host dynamics of this pathogen and provides new insights into its spread, including previously unrecognized dissemination routes. The methodology is appropriate and carefully executed, and the manuscript is well-written, clearly presented, and concise. The authors' conclusions are well-supported by experimental results and thoroughly discussed. This work underscores the power of using highly diverse barcoded pathogens to uncover the within-host population dynamics of infections and will likely inspire further investigations into the molecular mechanisms underlying the bottlenecks and dissemination routes described here.

Major point:

Substantial conclusions in the manuscript rely on genetic distance measurements using the Cavalli-Sforza chord distance. However, it is unclear whether these genetic distance measurements are independent of the founding population size. I would anticipate that in populations with larger founding population sizes, where the relative tag frequencies are closer to those in the inoculum, the genetic distances would appear smaller compared to populations with smaller founding sizes independent of their actual relatedness. This potential dependency could have implications for the interpretation of findings, such as those in Figures 2B and 2D, where antibiotic-pretreated animals consistently exhibit higher founding population sizes and smaller genetic distances compared to untreated animals.

Reviewer #2 (Public review):

In this paper, Hotinger et. al. propose an improved barcoded library system, called STAMPR, to study Salmonella population dynamics during infection. Using this system, the authors demonstrate significant diversity in the colonization of different Salmonella clones (defined by the presence of different barcodes) not only across different organs (liver, spleen, adipose tissues, pancreas, and gall bladder) but also within different compartments of the same gastrointestinal tissue. Additionally, this system revealed that microbiota competition is the major bottleneck in Salmonella intestinal colonization, which can be mitigated by streptomycin treatment. However, this has been demonstrated previously in numerous publications. They also show that there was minimal sharing between populations found in the intestine and those in the other organs. Upon IV and IP infection to bypass the intestinal bottleneck, they were able to demonstrate, using this library, that Salmonella can renter the intestine through two possible routes. One route is essentially the reverse path used to escape the gut, leading to a diverse intestinal population; while the other, through the bile, typically results in a clonal population. Although the authors showed that the STAMPR pipeline improved the ability to identify founder populations and their diversity within the same animal during infections, some of the conclusions appear speculative and not fully supported.

(1) It's particularly interesting how the authors, using this system, demonstrate the dominant role of the microbiota bottleneck in Salmonella colonization and how it is widened by antibiotic treatment (Figure 1). Additionally, the ability to track Salmonella reseeding of the gut from other organs starting with IV and IP injections of the pathogen provides a new tool to study population dynamics (Figure 5). However, I don't think it is possible to argue that the proximal and distal small intestine, Peyer's patches (PPs), cecum, colon, and feces have different founder populations for reasons other than stochastic variations. All the barcoded Salmonella clones have the same fitness and the fact that some are found or expanded in one region of the gastrointestinal tract rather than another likely results from random chance - such as being forced in a specific region of the gut for physical or spatial reasons-and subsequent expansion, rather than any inherent biological cause. For example, some bacteria may randomly adhere to the mucus, some may swim toward the epithelial layer, while others remain in the lumen; all will proliferate in those respective sites. In this way, different founder populations arise based on random localization during movement through the gastrointestinal tract, which is an observation, but it doesn't significantly contribute to understanding pathogen colonization dynamics or pathogenesis. Therefore, I would suggest placing less emphasis on describing these differences or better discussing this aspect, especially in the context of the gastrointestinal tract.

(2) I do think that STAMPR is useful for studying the dynamics of pathogen spread to organs where Salmonella likely resides intracellularly (Figure 3). The observation that the liver is colonized by an early intestinal population, which continues to proliferate at a steady rate throughout the infection, is very interesting and may be due to the unique nature of the organ compared to the mucosal environment. What is the biological relevance during infection? Do the authors observe the same pattern (Figures 3C and G) when normalizing the population data for the spleen and mesenteric lymph nodes (mLN)? If not, what do the authors think is driving this different distribution?

(3) Figure 6: Could the bile pathology be due to increased general bacterial translocation rather than Salmonella colonization specifically? Did the authors check for the presence of other bacteria (potentially also proliferating) in the bile? Do the authors know whether Salmonella's metabolic activity in the bile could be responsible for gallbladder pathology?

eLife Assessment

This important study uses a large dataset from both recent isolates and genomes in databases to provide an analysis of the population structure of the pathogen Salmonella gallinarum. The results regarding regional adaptation and the evolutionary trajectory of the resistome and mobilome remain incomplete, requiring additional details to fully support their claims and assess the value of these insights for future policy interventions regarding this and other pathogens. This work will interest microbiologists and researchers working on genomics, evolution, and antimicrobial resistance.

Reviewer #1 (Public review):

Summary:

The investigators in this study analyzed the dataset assembly from 540 Salmonella isolates, and those from 45 recent isolates from Zhejiang University of China. The analysis and comparison of the resistome and mobilome of these isolates identified a significantly higher rate of cross-region dissemination compared to localized propagation. This study highlights the key role of the resistome in driving the transition and evolutionary history of S. Gallinarum.

Strengths:

The isolates included in this study were from 16 countries in the past century (1920 to 2023). While the study uses S. Gallinarun as the prototype, the conclusion from this work will likely apply to other Salmonella serotypes and other pathogens.

Weaknesses:

While the isolates came from 16 countries, most strains in this study were originally from China.

Reviewer #2 (Public review):

Summary:

The authors sequence 45 new samples of S. Gallinarum, a commensal Salmonella found in chickens, which can sometimes cause disease. They combine these sequences with around 500 from public databases, determine the population structure of the pathogen, and coarse relationships of lineages with geography. The authors further investigate known anti-microbial genes found in these genomes, how they associate with each other, whether they have been horizontally transferred, and date the emergence of clades.

Strengths:

(1) It doesn't seem that much is known about this serovar, so publicly available new sequences from a high-burden region are a valuable addition to the literature.

(2) Combining these sequences with publicly available sequences is a good way to better contextualise any findings.

Weaknesses:

There are many issues with the genomic analysis that undermine the conclusions, the major ones I identified being:

(1) Recombination removal using gubbins was not presented fully anywhere. In this diversity of species, it is usually impossible to remove recombination in this way. A phylogeny with genetic scale and the gubbins results is needed. Critically, results on timing the emergence (fig2) depend on this, and cannot be trusted given the data presented.

(2) The use of BEAST was also only briefly presented, but is the basis of a major conclusion of the paper. Plot S3 (root-to-tip regression) is unconvincing as a basis of this data fitting a molecular clock model. We would need more information on this analysis, including convergence and credible intervals.

(3) Using a distance of 100 SNPs for a transmission is completely arbitrary. This would at least need to be justified in terms of the evolutionary rate and serial interval.

(4) The HGT definition is non-standard, and phylogeny (vertical inheritance) is not controlled for.<br /> The cited method:<br /> 'In this study, potentially recently transferred ARGs were defined as those with perfect identity (more than 99% nucleotide identity and 100% coverage) in distinct plasmids in distinct host bacteria using BLASTn (E-value {less than or equal to}10−5)'<br /> This clearly does not apply here, as the application of distinct hosts and plasmids cannot be used. Subsequent analysis using this method is likely invalid, and some of it (e.g. Figure 6c) is statistically very poor.

(5) Associations between lineages, resistome, mobilome, etc do not control for the effect of genetic background/phylogeny. So e.g. the claim 'the resistome also demonstrated a lineage-preferential distribution' is not well-supported.

(6) The invasiveness index is not well described, and the difference in means is not biologically convincing as although it appears significant, it is very small.

(7) 'In more detail, both the resistome and mobilome exhibited a steady decline until the 1980s, followed by a consistent increase from the 1980s to the 2010s. However, after the 2010s, a subsequent decrease was identified.'<br /> Where is the data/plot to support this? Is it a significant change? Is this due to sampling or phylogenetics?

(8) It is not clear what the burden of disease this pathogen causes in the population, or how significant it is to agricultural policy. The article claims to 'provide valuable insights for targeted policy interventions.', but no such interventions are described.

(9) The abstract mentions stepwise evolution as a main aim, but no results refer to this.

(10) The authors attribute changes in population dynamics to normalisation in China-EU relations and hen fever. However, even if the date is correct, this is not a strongly supported causal claim, as many other reasons are also possible (for example other industrial processes which may have changed during this period).

(11) No acknowledgment of potential undersampling outside of China is made, for example, 'Notably, all bvSP isolates from Asia were exclusively found in China, which can be manually divided into three distinct regions (southern, eastern, and northern).'. Perhaps we just haven't looked in other places?

(12) Many of the conclusions are highly speculative and not supported by the data.

(13) The figures are not always the best presentation of the data:<br /> a. Stacked bar plots in Figure 1 are hard to interpret, the total numbers need to be shown. Panel C conveys little information.<br /> b. Figure 4B: stacked bars are hard to read and do not show totals.<br /> c. Figure 5 has no obvious interpretation or significance.

In summary, the quality of analysis is poor and likely flawed (although there is not always enough information on methods present to confidently assess this or provide recommendations for how it might be improved). So, the stated conclusions are not supported.

Reviewer #1 (Public review):

Summary:

In the manuscript "Heat Shock Factor Regulation of Antimicrobial Peptides Expression Suggests a Conserved Defense Mechanism Induced by Febrile Temperature in Arthropods," Xiao and colleagues examine the role of the shrimp Litopenaeus vannamei HSF1 ortholog (LvHSF1) in the response to viral infection. The authors provide compelling support for their conclusions that the activation of LvHSF1 limits viral load at high temperatures. Specifically, the authors convincingly show that (i) LvHSF1 mRNA and protein are induced in response to viral infection at high temperatures, (ii) increased LvHSF1 levels can directly induce the expression of the nSWD (and directly or indirectly other antibacterial peptides, AMPs), (ii) nSWD's antimicrobial activities can limit viral load, and, (iv) LvHSF1 protects survival at high temperatures following virus infection. These data thus provide a model by which an increase in HSF1 levels limits viral load through the transcription of antimicrobial peptides and provides a rationale for the febrile response as a conserved response to viral infection.

Strengths:

The large body of careful time series experiments, tissue profiling, and validation of RNA-seq data is convincing. Several experimental methodologies are used to support the authors' conclusions that nSWD is an LvHSf1 target and increased LvHSF1 alone can explain increased levels of nSWD. Similar carefully conducted experiments also conclusively implicate nSWD protein in limiting WSSV viral loads.

Weaknesses:

Despite this compelling data regarding the protective role of HSF1 in the febrile response, what remains unexplained and complicates the authors' model is the observation that losing LvHSF1 at 'normal' temperatures of 25C is not detrimental to survival, even though viral loads increase and nSWD is likely still subject to LvHSF1 regulation. These observations suggest that WSSV infection may have other detrimental effects on the cell not reflected by viral load and that LvHSF1 may play additional roles in protecting the organism from these effects of WSSV infection, such as perhaps, perturbations to protein homeostasis. This is worth discussing, especially in light of the rather complicated roles of hormesis in protection from infection, the role of HSF1 in hormesis responses, and the findings from other groups that the authors discuss.

Reviewer #2 (Public review):

Temperature is a critical factor affecting the progression of viral diseases in vertebrates and invertebrates. In the current study, the authors investigate mechanisms by which high temperatures promote anti-viral resistance in shrimp. They show that high temperatures induce HSF1 expression, which in turn upregulates AMPs. The AMPs target viral envelope proteins and inhibit viral infection/replication. The authors confirm this process in drosophila and suggest that there may be a conserved mechanism of high-temperature mediated anti-viral response in arthropods. These findings will enhance our understanding of how high temperature improves resistance to viral infection in animals.

The conclusions of this paper are mostly well supported by data, but some aspects of data analysis need to be clarified and extended. Further investigation on how WSSV infection is affected by AMP would have strengthened the study.

Reviewer #3 (Public review):

In the manuscript titled "Heat Shock Factor Regulation of Antimicrobial Peptides Expression Suggests a Conserved Defense Mechanism Induced by Febrile Temperature in Arthropods", the authors investigate the role of heat shock factor 1 (HSF1) in regulating antimicrobial peptides (AMPs) in response to viral infections, particularly focusing on febrile temperatures. Using shrimp (Litopenaeus vannamei) and Drosophila S2 cells as models, this study shows that HSF1 induces the expression of AMPs, which in turn inhibit viral replication, offering insights into how febrile temperatures enhance immune responses. The study demonstrates that HSF1 binds to heat shock elements (HSE) in AMPs, suggesting a conserved antiviral defense mechanism in arthropods. The findings are informative for understanding innate immunity against viral infections, particularly in aquaculture. However the logical flow of the paper can be improved.

I will relate to this that instead of asking for help because of the fear of feeling dumb or questioning myself if im capable i will just disengage myself. It is also the fear that the teacher never made it comfortable enough for me to want to reach out and actually ask for help.

I appreciate this as i read this because the teacher shows her interest and care of wanting the student to be there. She shows her care and i know if i received this care i wouldn't feel bad because i know i would have her support or someone to talk to. The communication with teachers is important because you're able to gain trust and security of still wanting to show up.

This would be a reason as to why i would not want to take the class. Kids not being friendly shows the enviroment of the class where you feel out of place and makes it not worth it to be there. It would make me question and reasons like these is why kids give up on themselves. Questioning if we'd be smart enough to take the class

This would be the hardest part and being left out of wanting to try new things. Being put in this situation im pretty sure that in the back of their mind they're thinking that they're pretty sure this class is not for them or it might be to difficult seeing that the class is majority white. Personally it would bring me down as much as i would want to give it a try i'd feel insecure putting myself in that situation of actually taking the class.

When the low-income students are trying to avoid those social activities due to their constrain in budget, they lose the opportunity to social and get closer with the students who has the ability to go to these events. The boundary will be set from here and making their surroundings being different.

Like I said in previous readings you are the product of your environment . Pick up the good and throw out the bad. It may change the outlook on life.

Back in those days, the internet seems a lot more personable and much closer knit than it is today simply because it wasn't as populated or centralized as it is today. Part of me wants to experience the vibe of those days for myself and see why people prefer this style of internet to today.

In today's society, the social media platforms we use have strong social attributes. People utilize social media to showcase themselves, share their lives, and network with others. Platforms like YouTube, as a form of social media, take a more innovative approach by delivering information through videos, offering a more dynamic and multifaceted way to convey content.

An interesting fact I came across is that the Web 2.0 used chronological feeds where posts would come up on user's feed in the order of recency whereas social media today uses algorithmic feeds which is based on the user's interests, activity and interactions on the social media.

I believe that with the rise of weblogs, it allowed individuals to be able to easily share their thoughts and experiences with a broader audience on the social media. The platform LiveJournal was able to foster and create early forms of online communities with the help of reader engagement and feedback on the platform.

It's crazy how long these social networking services have been out for and still continue to be popular today. Facebook makes it really easy to connect with other people that are far away so I think that's a big reason why people still use it.

Unlock the potential of utility app development to streamline everyday tasks and enhance user productivity. At HireMobileDevelopers, they specialize in building intuitive, feature-rich utility apps that cater to various business needs. From organizing tasks to managing daily operations, a well-designed utility app can significantly improve user efficiency and engagement.

From my own experience, as the two city's per capita income is different, their crime rates on the neighborhood would also be influenced. This is an interesting social difference I found when looking at the income and race ratio of a city.

It is interesting to read because the comparison of Santa ana and Troy school and because of the location, it is more probable to have drop out rates. Especially because of the kind of people that go to the school, it's sad that it is compared like that. I know i would look at it different of people dropping out because they do not feel the support from staff and at home as to why they tend to give up.

I would focus this more on their physical fitness. I would compare this more to how physical appearance tends to me towards semi obesity when it comes down to low income because we tend to eat just junk food because of being on the run. It happens both ways and not being physically right or be more active. I can also relate this to sports. A lot of kids in my school would not do sports because of how expensive buying gear was. even if we thought it wasn't expensive, it is because it is money that you do not plan to spend as we live check to check.

This completely makes sense in the involvement based on the school or city you're in. I can say I see the difference with working in Orange County and the activities we give to the students or even with the supplies i know its something that my city where i grew up in would have. An example would be my coordinators bringing in animals to do a mini zoo for the kids and bringing in snakes, big tortoises, and just activities that i know in my after school we would not have opportunities provided like that from the school. Everything is different when it is more of the chools involvement for the school.

This study should be about the area in general not two families that support the evidence they are trying to make.

As a graduate from Fullerton High , we have a similar percentage of latin students to Santa Ana high . Troy High is a tech school their latin percentage is not in the same ballpark.

I went to school near this area at the time , I would have to say I saw more growth in numbers from the asian and middle eastern communities. The latino population of Fullerton is near mostly downtown.

The lawlessness of the site combined with the veil of anonymity 4chan provides is what causes people to act so henously. Yet sometimes there are glimmers of humanity in the depths of depravity that is 4chan.

This is the first time that I'm hearing of such platforms and it defies the idea that social media always reinforces connectivity and is mostly beneficial in some way or the other. This also brings into question how although media similar to this creates high levels of toxicity, misinformation, manipulation and more, they still continue to exist for various reasons such as revenue earned from the platforms, the right to free speech and more which I personally find extremely unethical.

Ok, I've definitely gone down the youtube rabbit hole of watching various things this group has done. It's interesting to know that a group who had an actual impact on the world started as a 4chan group.

I am feeling bad when reading this, where a kid cannot have enough food while he needs it, and has to have drinks as the alternative for extra food that is provided by the family. But in a family that has limited income for food supply, it seems to be the only solution that the family can offer to their children.

From my understanding, besides school, your family is where you can learn the most outside of school. For example, Anthony's family has a comparative lower level of education, which makes him harder to earn reading abilities and understandings from his family outside of school. The gap would grow in a significant rate during this age.

This is very true, I witnessed violence in my childhood due to my neighbors on a weekly basis . The children went from being so joyful and happy , to isolated and dull. The mind wants to escape, the only way to do that is to take your mind elsewhere.

Nutrition is one of the most crucial parts in youth growth & development . Food is like Gasoline, our bodies are cars without proper fuel we can not possibly have the energy to grow in anyway.

Increasing amount of this stuff are now blocked. It's becoming the norm now.

Because the resource that students can receive is varied at BHS, it can greatly impact what the students would choose for their extracurriculars to be.

The "self-scheduling" system is creating a boundary between the poor students and others. As they don't have much understanding about the education system, including the procedures they might need to take if they would like to enroll in college. This is not only a boundary that is set under the school and trying to test about the student's personal ability, also a boundary that separates the information that can be received by the students.

I this this can be an example of the student missing enough counseling help with class selection and the missing guidance information the school can provide. In another words, if Chantelle can have more information about the difficulty of math, and differences of courses, she might not step herself into the wrong level of math class.

school resource is strongly related to the student's decision as they don't receive many resources from their family. For example, a school with more resources that provides the students to learn subjects outside of school would make a wider path for the students to choose their future path than those without the resource.

I believe our upbringing can effect our growth, but it does not mean we want to fail. Sometimes life gets in the way and priorities change. I believe people of color like myself have to work just a little bit harder to achieve the same recognition, but its okay doe not mean giving up something that makes us better.

Each Student's mind thinks and works differently. The teacher makes a big difference . I believe free tutoring should be apart of everyones education if needed.

In education and in life we should not continue to separate by race. It comes down to resources and teachers without proper funding the care for education will continue to diminish.

How did she overcome her struggle? Did she obtain a tutor , pay someone to do her math work? What advantages did she get because of her social standing.

Even though AOL may seem outdated in comparison to the multitude of social media platforms of the modern day, I noticed that we still use quite a few features from the chat system in today's softwares. For example, the online offline feature or profile quotes are a "new" feature on Instagram visible to your followers.

The Bulletin Board System (BBS) is still a popular way to transmit information today. Unlike the monotonous bulletin boards of the past, when we visit any modern popular website, we often find such message boards present.

I believe that AIM was an extremely pivotal in shaping the way that we interact online as it was able to introduce the way that we could use real time-digital communication and status updates to talk to other people. After reflecting on the influence that AIM had on digital communication, I believe it really helped set the foundation for many of the features that social media apps use today.

I didn't think that emails were created around the 60's and 70's this actually surprises me. They've been around for a long time now I wonder if people received emails instantly back then and if not how long would it take for them to get sent? It only us a couple seconds to receive an email.

it is interesting that some UI designers will intentionally create friction to leverage the difficulty of pressing smaller buttons. Though most designers would like to create a frictionless feeling, I think a little bit of friction may create more interaction with the viewers; especially if the website is selling products, it might be helpful to force users to click on multiple buttons to get them exposed to more goods before they achieve their desired goal

It's very interesting to think about the idea of friction as an intentional choice, which seems counterintuitive. However, on further thought it seems like MORE friction should be added to social media. Services like Meta's Reels and TikTok have very low friction levels (even ads can be swiftly swiped past) which cause many people--including me--to get sucked into an endless scroll for dopamine.

I think divergent thinking is better than convergent thinking in most aspects. Devising multiple solutions to a problem is more beneficial for problem solving skills than simply arriving at one answer.

Fact-checking is a very precise, meticulous process to ensure the public receives accurate information.

Fact checking presidential statements is essential because if the president can freely lie and make up facts/statistics, it will severely mislead a large proportion of the country.

Ha! I love this phrase. It captures the tension between law as a precise discipline and law as an interpretive art.

This line shows how complex the legal system is. The widow’s frustration highlights how the law appears as being simple and clear-cut, but in reality, cases are rarely exactly the same, and interpreting the law can be complicated and uncertain.

This relates to the research methods we have been learning in class. Formulating a plan to find relevant sources to the issue we are researching is vital in cutting down time and reducing reading sources that do not apply to the issue at hand. Creating a viable plan before beginning research is a vital task to ensure logical research procedures.

As I learn research skills and the methods required to produce timely and efficient material it is reassuring to know there is no one true method. Although finding my method that produces effective results will not be easy I can understand it will come from time and repetition. Although the process of developing my technique has been extremely frustrating and confusing as I navigate I understand the importance that legal research and writing has in practice.

Thinking of encyclopedias, or C.J.S., as one of the most current resouces for information seems contrary to what one would assume. Having an accessible and quick way to learn about a research topic is a great short cut to noting which cases define the common law for the topic, and where to continue your research. This resource is especially great when a topic is new and you have a short timeframe to gather your necessary research.

To the end of the sentence on the next page:

The first portion of this quote highlights the idea of legal research involving gathering and analyzing statutes, case law, and precedents and crafting the actual legal documents. It also emphasizes that these two things require a much deeper level of creativity and personal interpretation than often thought of. Creating these legal documents requires critical thinking, persuasive techniques, and a variety of stylistic choices that reflect the author's (attorney's) voice and perspective. This quote highlights the duality of the legal profession: the reliance on empirical data and well-established laws, and the art that is the actual profession itself - thriving on individual expression and interpretation; it makes the practice rigorous and dynamic.

Here, we see some of the frustration that the public has with our legal system. People who aren't trained attorneys often don't understand the "whys" of the law: Why is it so hard to find a similar case? Why did a judge rule in a way that seems to contradict common sense? Why is this city code so hard to read? I think a lawyer who works with everyday people (i.e. a local defense attorney or a lawyer who writes wills for your average Joe) must empathize with these frustrations and be patient if they want to be effective.

This provides insight on the intricate process of researching legal frameworks that provide outlines for our legal system - like the Restatement of the Law and Uniform Laws. These are designed to clarify legal principles, but not to enforce the law unless the courts adopt them. A legal researcher's task is not just about reading these difficult texts but also about actually understanding them and being able to apply them where necessary. This particular part of the practice reflects a larger truth which is that the practical application of the law often differs from the theory of the law. Legal researchers cannot only rely on the theory of law, but also have to engage in the judicial evolution of legal doctrines. The legal doctrines provided by sources like the restatements and UCC must be viewed by themselves and also through their application to cases in order to truly understand how they influence the law.

This underscores the way one needs to blend structure and individuality in any and all legal research. An experienced researcher might develop their own "pattern of approach" over time, but it was likely to be shaped by how your professors taught you and how their professors taught them. The overall method is likely to be the same others', even if there are situations - such as unique legal questions - where one might take a specific route towards research. Like most research processes - across the board - there's a bit of standardization of the legal one. Even if we all have differences in what works best for us, we all have to make sure we're competent, thorough, and efficient. Its the mix of the personal and the structured that makes legal research so creative while also being methodical.

This is something that I hope I can achieve. Despite spending almost six weeks in law school, I am unaware if anything has "clicked" for me. However, I am riding on the wave that it will eventually click, that law school and legal studied will eventually get better. I want to get to a point where I have a "pattern of approach" for legal research and reading legal documents. I am certain though that I am more equipped now that I was six weeks ago. But, I still have a long way to do.

This phrase really struck me. Up to this point, the article seems to be about what is expected of research and the idea that every person has a different approach. I think it can be really easy to assume that everyone else is doing things better, even if they never say what it is they're doing, and sometimes it can be difficult to start a conversation that needs to be had when you think you aren't good enough to start it. But if important conversations are going to happen, someone has to start them.

I agree, everyone will find their own way of researching, and it might be altered by the issue. That said, this article gives a nice guide for those new to legal research, and I’m sure if followed will produce worthwhile results.

Obviously there is no method to guarantee our perfect case will be found, yet this seems so frustrating. You can go through the whole research process, doing each step as well as it can be done, and still not end up finding what you're looking for. Sometimes the "case just like mine" doesn't exist which is quite the bitter conclusion to reach after a lengthy research process.

Differences in situations like the author describes here have been frustrating for me outside of research in law school. Considering that I am still new to this, I will get to a point where I feel comfortable with an idea that I have been learning and then a slight change will warrant a completely different approach that I am not familiar with and I have to start all over again. I feel like the author's last sentence of this paragraph is really more of a euphemistic way to say that everyone will have their own mental scars from dealing with their particular "special situations."

This idea of legal research and practice being an art more than a science is intriguing to me. It seems to me that a great deal of what we have learned so far in LRW or our other classes has been a mixture of both. I find that my mind feels comfortable with procedures and rules that are in theory rigid and clear, but then the application to a fact pattern or a slight shift in interpreting the exact words, "make[s] all the difference in the world as regards the results..."

This is such an interesting way to play strategy in law, one that I would never think of to use. I wonder if why I am surprised by this approach is my technological outlook on research. Most of the research I have done throughout my life has been via internet, primary source or secondary. Because of this, I imagine when I am doing research that we have all of the same access to resources. The idea of hiding a law book has its own implications because of the innate nature of law resources as a whole. As discussed in my library tour with Professor Creed, law firms are limited on research resources because of the cost of these materials. This strategy is especially perfect for the law industry as the materials are as limited as mentioned.

Is there such a thing as being too specific? I feel like the problems with the law and the lack of understanding derive from getting so granular that the wording begins to lose focus. Certain topics require specificity, but I can't quite agree with language that is so unreachable. Why not write like Hemingway?

That has to be frustrating- doing all that research, thinking you have the right case law in hand only to find out that there is new law that changes your support. This I hope will make me weary of neglecting to stay current on decisions in my field of practice, so i'm not as easily surprised. On the contrary, It'll make me more prepared for changes in law.

I really like how the author tells us her process for which resources to use and when to use when answering her problem. I also like how she acknowledges that this is the way that works for her and just because it works for her does not mean it is superior than another way in which others may tackle the same problem. I think the important concept is to find a way that works for you and acknowledging that it is not less efficient or wrong to start with a different source than others. It is going to take us some time to figure out what works best for us but this gives us a good idea for a starting point.

This argument is very similar to the phrase, "we're all immigrants from somewhere". Even though the evidence is literally staring at them in the face, people are stubborn. They don't care if the Kumeyaay are the rightful descendants, UCSD and by extension people in La Jolla, want what they want and they'll say whatever so that they get it.

I agree with this because it's obvious that UCSD has little to no respect for the Kumeyaay, I mean they didn't even consult them when they were starting on the project in the first place. If they had it their way, they would gladly tear down the house without telling the tribe.

It's really ironic that they were so concerned about protecting something that they see to have cultural significance when they could care less about the importance the land has to the Kumeyaay. It just goes to show that it doesn't matter how many laws are passed to protect Native culture, history, and peoples. Native Americans will aways be treated as an after thought.

!!!

it is crazy to me that they even had to THINK about whether or not to return the skeletons to the Kumeyaay people, i feel like even thinking about keeping them is selfish

Using social media nowadays becoming the cautious behavior since so many spam bots are hiding behind the internet potentially. That is also the drawbacks of identity verified social medias. We need to stay cautious and clear.A

im sure this is what we all aim to do :)

"only prerequisite is a DESIRE to continue learning at the post-secondary level"

giving them the option just like everyone in our community!

standing up for what is right, i like to see it!

it's honestly viewed as a "next chapter" of everyone's lives when talking in terms of post secondary. i feel like thinking you don't even have this opportunity because of a developmental or intellectual disability, feels very isolating and lonely? it's like you are missing it while everyone gets to experience without you

planning for post secondary helps students find their identities and contribute to their communities. the education system aims to help students develop the skills they need in order to succeed and engage in society!

There are other types of regularization that work in practice (for example, L1, which is not even differentiable). Does this mean that L2 regularization is the best ?

2 How are the diets of wolves different from bears? How are cougars different from wolves in terms of what they eat and how they hunt? How does this knowledge inform about threats to livestock?

According to the graph in figure 3 of the paper, wolves primarily cause damage to agriculture through eating livestock while grizzly bears split between livestock eating and attractant and Black Bears primarily being attractant. Cougars and wolves both primarily cause damage through eating livestock, but wolves hunt in packs where cougars hunt alone, so wolves tend to do more costly eating of livestock all at once. Additionally cougars also tend to eat smaller animals where a wolf pack or pair will eat larger animals. This knowledge shows us that although both wolves and cougars primarily cause damage through eating livestock, and the differences in diet and choice of prey can help farmers better adapt their defenses and mitigation strategies based on the animal threat they face most often.

While it's reasonable to claim that a decentralized media system results in the proliferation of conspiracy theories, I think it's worth noting that conspiratorial press is also very common within centralized media systems. News networks and daytime TV alike both have a tendency to report shocking and dubiously truthful (if not outright false and dangerous) news, often manufacturing outrage just the same.

This is fairly interesting, and something my discussion partner brought up in week two. I get that in some sense books are considered social media, but I feel like they have slipped away from that definition with new social media platforms becoming more and more prevailent. My biggest question regarding this is whether or not the lack of ability to recieve feedback directly through a boom would disqualify it from a more modern understanding of what constitutes social media.

lots of opportunities and offers to make sure they can have a say and variety in what field individuals with disabilities want to go though

putting an effort to make sure they can excel during the process of finding work

Usually the radio button will have identification text like the example provided.

How is the moral obviousness of how wrong this is not clear? They refuse to censure (disapprove), echoing that nothing has changed (Piexoto)

Offred's narrative makes connections to Chaucer, the first poet to publish in english. Most connection to the Canterbury tales which talks about pilgrims in a story-telling competition.

So are handmaids on a pilgrimage, and it is a tale and not a biography ?? Is offred a pilgrim looking for her religiousness?

Sexist element in saying she was looking for her lost faith and she was telling a fib, a lie, a myth.

The orphanage throughout the play is a means for people's benefits. Mrs.Alving uses it to cover the truth and for Mr.Alving and Oswald's social reputation and other people use it to extract profit

When the connection between Oswald and Regine is revealed, Oswald is deeply heart-broken, as opposed to Regine, who shows a cold attitude. The fact that she wishes to 'take advantage of her youth' shows that she was only interested in Oswald for her material gain [her trip to Paris], and that she has never truly loved him.

Despite the hatred that Regine has against his father, she is quite similar to his father in this sense - taking advantage of love for material gain and social success

Mrs.Alving's refusal of the reality - Oswald is unconsciously inheriting a lot of aspects of his father, even those that she wanted him to avoid, the primary cause for her to send him to another city and cutting the bonds between their relationship

The Sun reflects Oswald's pursuit for clarity of his past, family, and truth. Throughout the novel, darkness manifested in the house and the places he has been staying, which contributed to his illness [he was not allowed to know about his father, which led him to build an ideal image of him on his own imagination. And when this image is broken, he faces struggles and mental disorders]

"Oh, how – how dark it is here!" "It is dark outside apart from a faint glow to the left in the background."

This once again shows his manipulative endeavor. + how he repeats the word 'father' and 'child' "What the hell's this? Going against your own father, are you, girl?" He brings up familial and bloodline connection before revealing is true intention, as a means for Engstrand to make Regine feel bad for him. His primary interest lies in how she can be used as 'womenfolk round the house, for a bit of fun in the evenings, singing and dancing'

Engstrand's treatment of Regine throughout the play can be interpreted as both exploitative and manipulative. The relationship between the two figures lack of connection and concern for each others' well being, because both of them are focused on their desires and ambitions for material success. This quote specifically highlights Regine's awareness of Engstrand's manipulative endeavor, as well as her refusal to be used for his gain

I think that this sentence is really showing the narrator and the reader the personality of someone struggling, showing perhaps a potential insight into how Sonny may have acted or looked before his death. I also thought that this line was heartbreaking for the reader because you can infer that this person has an extremely low view of themselves, perhaps contributing to how they live.

I thought that this line was specifically heart wrenching within the story because I think that it displays the sad reality of loving someone with an addiction and not being able to ever fully understand, because there is no logic or sense to it. I thought that it was enforcing the idea that an outsider looking in will never be able to understand someone's own personal struggle.

Before I read this I didn't know what story he was referring to and now I understand.

This is another line that really goes into how the character feels about Harlem and the things they went through saying that when they came back to Harlem, even though he was older everything was pretty much the same. Sometimes we go through things in our past that if we go back to the original place, it can bring feelings of sorrow rather than just nostalgia which is an important message in this story.

This passage was by far my favorite. I love how Baldwin is saying that even though he may be feeling sad or have faced may things in life, he can play beautiful music. His life and him breathing is so important as a musician because he can use those feelings or emotions to his advantage to help spread to message to anyone who hears it and as a musician, he would have the control to make it do what he wanted it to.

This really stuck out to me. In a way I thought it was powerful because he feels he is changing in his character or personality which results from the trauma he has faced and the sadness that the character feels.

I wonder why they never cared about what was going on in Sonny's friend's life, what he had been through, if anything. And why did they not question the boy on why he was always waiting in the courtyard?

I was pretty confused when I was reading this sentence. I knew that something happened to Sonny but they were never really clear on what. But then they said he got hung so I took it in the context of he was dead, but as the story went on that was obviously not the case so now I think that they meant more so he got hung up on things he shouldn't have been.

I really liked this sentence because it gave the story the ending that it needed, this sentence showed that Sonny was okay; that he had turned over a new leaf and that he was not going back to the person he was before.

*Whats significant about this comment to me, is the authors use of light and darkness. He has an unique take on it, and this is especially noticeable when he makes this statement, about when the light comes on the kid is filled with dark. Its like Bane from Batman who has a well known statement about how he was born in the darkness, and the difference of this versus somebody like batman who adopted it. This child and the environment had turned so dark that light represented having to experience life going on. This is a beautiful way of explaining how life experiences aren`t always so light and dark, and the different ways light can be perceived to those born in the dark. *

A lot of times siblings say more informal things to each other so it was nice to see the author convey that here and show what the relationship between two siblings is like.

I think that all throughout the story he's trying to get into Sonny's world. At first he doesn't want to be in his world, here he is trying to get in.

I think a lot of times students assume that teachers don't want to be there when they themselves don't like school much so I wonder if this is what the author was trying to convey?

The use of repetition here, I think, is a really good way to start the passage because it sets the reader up for how the main character is as a person.

I think "the low ceiling of their actual possibilities" is a line that very strongly resonates throughout the entire story. Both brothers tried to break that ceiling.

I like this line because it demonstrates how some people don't take into consideration how serious a situation can get.

I feel like this what goes through every addicts mind whenever they think about their past actions and most of the time they are able to get help and get clean but there is always the few that realize they need help but don't do anything about it

The narrator didn't realize or maybe even understand Sonny's suffering because he never experienced it himself but after his daughter's passing he knew and understood.

Even though this is only the title, I think it is a perfect way to title the story. It encapsulates the theme of Sonny's fight with addiction while talking about his love of music

In the line "O singer bashful and tender, I hear your notes, I hear your call" from Walt Whitman's "When Lilacs Last in the Dooryard Bloom'd," the speaker addresses the "singer" as a symbol of both mourning and beauty. The adjectives "bashful" and "tender" evoke a sense of vulnerability and fragility, suggesting that the song of the singer—likely representing Abraham Lincoln or a broader embodiment of loss—is both poignant and gentle. This duality reflects the complexities of grief, where the beauty of memory intertwines with sorrow. The repetition of "I hear" emphasizes the speaker’s deep connection to this voice, suggesting that the act of listening becomes an essential part of processing grief. This line encapsulates the overarching theme of the poem: the interplay between loss and the enduring power of remembrance. In a world marked by violence and tragedy, the tender notes serve as a reminder of the beauty that persists even amidst profound sorrow.

Even though their might be a possibility that they won't get much information, I don't see why they don't also consult the Kumeyaay tribe as well. If they're not sure, just like the tribes who were consulted to see the footprints in White Sands National Park, they'd probably be notified and possibly go see it.

This was really surprising to read because even though UCSD isn't really known for their anthropology or history department, I thought that they'd at least do the bare minimum to protecting these items. However, the fact they barely did anything to properly preserve them, shows that not even well reputed federal institutions can be entirely trusted to help preserve Native history.

missed the identification of metal and never talked about the percent of error. also forgot a period.

Has no labels of the Y&X axis.

To reduce funding for EPA's extramural research as well as for NGOs addressing environmental justice, the author proposes an initial halt and "review" of all bigger ongoing grants. Ironically, the author's solution to allegedly "radical" environmental research "driven by ideology" is to put a political appointee in charge of EPA research enterprises. Is that person really going to enable grant distribution to "those who are in most need and towards projects that will tangibly improve the environment"? Only if judgements about need and tangibility reflect demonstrably scientific realities more so that conservative ideological blinders that so often skew and slant the proposals in this chapter.

The author has argued that a conservative EPA will take its cues from the states, even turn leadership in pollution control over "to the states." The regional offices mentioned here are the agency's primary vehicle for engaging with the states. But this recommendation advocates greater headquarters control over these regional offices, which conflicts with the earlier-expressed desire to work more collaboratively with the states. The author's jarring swerve here toward calling for centralization stems from what is apparently a higher goal: stymying or eliminating any efforts toward environmental justice within the agency.

Once again, the author is framing environmental justice as a priority only of the Biden administration. Civil rights and environmental justice have been long-standing concerns at the EPA, beginning under Republican leadershipl, when President George Bush first established an Office of Environmental Equity in 1991.

See comment #70 above as well others on the Foreword section of the document.

The author's failure to offer specific examples of how the Biden administration has "broadened" the EPA's use of Title VI, or to specify "long-standing understandings of the legal limits of that authority" reflects the speciousness of this argument. In the 1970s and 1980s, the EPA  circumscribed its statutory obligations under the Civil Rights Act of 1964 and the Fair Housing Act of 1968. President Clinton's Executive Order 12898 of 1994 along with the creation of the Office of Environmental Justice within EPA (from an earlier Office of Environmental Equity established under Bush), were important efforts to bring the agency into compliance with federal law.

https://academic.oup.com/jah/article-abstract/111/1/71/7695574?redirectedFrom=fulltext

The Inflation Reduction Act invests $1.5 million to provide support for basic science research at the national laboratories.  https://www.energy.gov/science/articles/fact-sheet-inflation-reduction-act-supporting-future-doe-science#:~:text=As%20part%20of%20the%20Inflation,and%20national%20laboratory%20infrastructure%20projects.

Precautionary models, including assumptions of "low-dose linear nonthresholds," have been central to EPA risk assessments since risk assessment itself began at the agency in the late 1970s.  Doing away with them would radically alter the EPA's safety levels for all chemicals known or suspected to cause cancer, for endocrine disruptors, and alll substances otherwise well-established as having no safe level  of exposure, such as lead.  Rejection of such models, apparently because of the "opaque decisions" and "disportionate economic impacts" they are accused of causing, flies in the face of consensual statements of public health professionals like the article below, which hold that "hazard and risk assessments should not assume existence of a 'safe' or 'no-risk' level of chemical exposure in the diverse general population."    https://doi.org/10.1186/s12940-022-00930-3.

Precisely what the Trump administration sought to do, by trying to forbid those with EPA grants from serving as well as appointing more members who were corporate scientists-for-hire (working for regulated companies or corporate consulting firms), or also scientists working environmental agencies in Texas and other red states. https://www.npr.org/sections/thetwo-way/2018/02/14/583972957/top-epa-science-adviser-has-history-of-questioning-pollution-research https://www.eenews.net/articles/boards-add-industry-and-state-officials-drop-scientists/ https://envirodatagov.org/an-embattled-landscape-federal-environmental-science-integrity-in-the-united-states-a-three-part-series-part-1-targeting-scientific-influence-on-policy/

Scientific advisory committees play a crucial and cost-effective role in making available to the EPA the best in contemporary knowledge in environmental sciences and policy to inform agency decision-making. For what the Trump administration actually did to these, see charts 1-3 in https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

Most all the criticisms tallied here come from conservative and industry-allied critics.  EPA's scientific programs were targeted by severe cuts during the Trump Administration, which through the intervention of Congress were limited to actual cuts of 14% to 35.6% between FY2016 and FY2019.  See among other charts #s 2, 4, 8-10 in  https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

Only the second call that more--not fewer--resources need to be devoted to an EPA program.   But during the Trump administration, research into "chemical safety for sustainability" was reduced by 35.6% between FY2016 and FY2019, surpassing the cuts in all other EPA research programs. See esp. chart 9 in https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

过的粉丝放过的是非观说的吧

The vibe I got out of this line is the creepy motif of a doppelgänger and the unsettling psychological implications of the “other” that haunts the speaker. Both Eliot’s speaker here and the main character of Dracula, Jonathan Harker, confront spectral presences that embody their deepest fears and anxieties, suggesting that this “third” figure represents more than just a physical entity. It’s a shadow self, a manifestation of repressed desires, fears, and the destabilization of identity.

In Dracula, Dracula the character functions not only as a literal antagonist but also as a projection of the unconscious fears and desires of Harker. When he is trapped in Dracula’s castle, he begins to experience a split in his sense of self, feeling his identity destabilize under the influence of the Count. He states, “I am beginning to feel this nocturnal existence tell on me. It is destroying my nerve. I start at my own shadow, and am full of all sorts of horrible imaginings” (Stoker). This vampiric presence of Dracula is both external and internal—an embodiment of everything Harker represses within himself.

Similarly, in TWL, the “third” walking beside the speaker is neither fully acknowledged nor understood. The ambiguity of the figure’s identity—“I do not know whether a man or a woman”—reflects the same psychological dissonance present in Harker’s experiences with Dracula. The third figure, like Dracula, is elusive, undefined, and haunting, representing a part of the self that remains unrecognized yet constantly lurks at the edge of consciousness.

Why is this? Shouldn't Britain still be the worlds leading economy. With the amount of colonies Britain has, why is it that they have lagged down? Can countries that haven't fully developed have such a dramatic economic gain as this if they industrialize and prioritize it? L

Because history is something we are supposed to learn from and not repeat it is hard to see the deployment and a result and cycle of police brutality occurring throughout different time periods. Especially over human rights protests.

I learned about the “double-blind” clinical trial method in statistics, which is performed in such a way that neither the performer nor the subject knows what treatment they're being given, what drug they're being given, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on, and so on.

“2 trillion” sounds like a horrible number, and it's in U.S. dollars, so I can't imagine how big a number it is. In sighing at the number of spending so big at the same time I can understand, my family is also open a chain of drugstores, although not every day business is very good, but in our family drugstore there are too many old customers, they are sick or small illnesses have to go to see a doctor to buy a drug, who did not have a few boxes of medicine stored at home this year!

The importance of forecasting with its relevance to the renewable energy cause is explained

A breakdown of the big picture solution

This is the main point of this paper

It would be useful to explain the meaning of " -1" in the formula.

Is a Spanish word that means on the contrary

O Mustafa Kabha εργαζεται σε Ισραηλινο πανεπιστημιο:

Mustafa Kabha is full Professor in the Department of History, Philosophy and Judaic Studies and the Head of the Middle Eastern studies at the Open University of Israel.

Το μεμαλοποιει, στη λιστα με τις σφαγες στη Wikipedia δεν βρισκω πολλες τετοιες.

Παλι εδω η θεωρια πως οι Αραβες ειναι γενετικα ολοι τους απογονοι του Μωαμεθ, και οχι οτι ηταν μια πολιτιστικη/θρησκευτικη επεκταση. Με τη 2η ερνηνεια, ο ορος "χριστιανος αραβας" δεν βγαζει παντα νοημα:

The concept of an Arab Christian identity remains contentious, with some Arabic-speaking Christian groups in the Middle East, such as Assyrians, Armenians, Greeks and others, rejecting an Arab identity. Individuals from Egypt's Coptic Christian community and Lebanon's Maronite community sometimes assume a non-Arab identity.

Εδω μαλλον εννοει τους Μαρωνιτες. Σιγουρα αυτοι συνομωτισαν εναντια στους Παλαιστινιους προσφυγες το '80.

want more information regarding the government to be public

This is an example of mal-information. This is an example because it is someone posting a leak on the internet. They posted this knowing that it would get the company in trouble and that it was also harmful to the company. This could make many frequent Disney guests mad at this information that is made up and quite honestly illegal. They posted this leak on many different social media platforms. One would know that this is mal-information because it is from an unreliable source that is not confirmed from Disney themselves. This leak is not considered something that Disney stands for with their family friendly environment and would cause lots of negative controversy around the Disney world and fanbase.

The fixed effects method allows us

This is so good to learn about it, history have a way to repeat itself, this event reminds me about what just happen in 2020 which the crazy speculation with the crypto monodies. a lot of people were even taking a loans to invest in crypto believing they could get rich easy and lost a lot of money, Good reflection.

Review coordinated by Life Science Editors Foundation

Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors. *Assisted substantially by NotebookLM.

Potential Conflicts of Interest: Angela thinks Olivia Rissland is everything a scientist should be.

What is an N-degron? N-degrons are short amino acid sequences located at the N-terminus of a protein that signal for the protein's degradation. This process is an essential part of protein quality control and regulation within cells. N-degrons are recognized by specific E3 ubiquitin ligases, also known as N-recognins, which help target the protein for degradation by the ubiquitin-proteasome system.

How was this new Arg/N-degron pathway discovered? The authors were initially studying how N-terminal sequences affect gene expression using a reporter gene assay. They found that a specific tripeptide motif (KIH) inserted at the N-terminus of a reporter protein led to a dramatic decrease in protein expression. Further investigation revealed that this decrease was due to rapid protein degradation, indicating the presence of a novel N-degron.

What are the key features of this new N-degron pathway? This newly discovered N-degron pathway targets proteins with a lysine (K) or arginine (R) residue at the third position (position 3) from the N-terminus. Importantly, this pathway requires: * • Methionine Removal: The initiator methionine (M) at position 1 must be removed by the enzyme methionine aminopeptidase 2 (MetAP2) for the degron to be active. * • UBR4 Recognition: The E3 ligase UBR4, but not UBR1 or UBR2, recognizes this specific degron and initiates the degradation process.

Why is the identity of the second amino acid important? The second amino acid plays a crucial role in determining whether MetAP2 can cleave the initiator methionine. This study found that the degron is only active when the second amino acid is threonine (T) or valine (V). These amino acids allow MetAP2 to remove the methionine, exposing the lysine or arginine at position 3 for recognition by UBR4. In contrast, if the second amino acid is alanine (A) or serine (S), MetAP1 removes the methionine. The researchers hypothesize that these N-termini are then acetylated, preventing UBR4 recognition.

Is there evidence that this pathway affects endogenous proteins? Yes, analysis of previously published data and additional experiments by the researchers suggest that this MetAP2-UBR4 pathway is not limited to artificial reporter systems. They found that endogenous proteins with MTK or MVK N-termini were less stable than those with other amino acids at position.

Does UBR4 work alone in this pathway? UBR4 appears to function as part of a complex with the protein KCMF1 to degrade proteins containing this new degron. Experiments showed that disrupting the UBR4-KCMF1 complex stabilized the degradation of reporter proteins containing the KIH degron.

What is the broader significance of this discovery? The identification of this new Arg/N-degron pathway expands our understanding of the N-end rule, a fundamental mechanism for protein degradation in cells. It highlights the complexity of this system and reveals how the interplay between different enzymes like MetAP2 and E3 ligases like UBR4 can fine-tune protein stability. Additionally, it suggests that there may be other undiscovered N-degron pathways that remain to be characterized.

What questions still need to be answered about this new pathway? This study raises several new questions, including: * • Substrate Specificity: What are the precise rules governing UBR4 recognition of position 3 lysine and arginine degrons? Do other amino acids in the protein sequence affect degron recognition? * • Physiological Roles: What are the specific cellular processes and pathways regulated by this MetAP2-UBR4 N-degron pathway? * • Evolutionary Conservation: Is this pathway conserved in other organisms, or is it unique to mammals? * • Therapeutic Potential: Could this pathway be targeted for therapeutic purposes? For example, could stabilizing proteins involved in disease by manipulating this pathway be beneficial?

What was not known: * • Whether a lysine or arginine residue at position 3 of a protein could act as an N-degron. * • Whether MetAP2 could play a role in initiating N-degron-mediated degradation.

What this preprint reveals: * • A new family of N-degrons: The study identified a new class of N-degrons characterized by a lysine or arginine residue at position 3, following a methionine at position 1 and a MetAP2-cleavable residue (threonine or valine) at position * • MetAP2-dependent initiation of the Arg/N-degron pathway: The study found that MetAP2-mediated removal of the initiator methionine is essential for the recognition and degradation of these position 3 lysine/arginine degrons. This is the first demonstration of MetAP2's involvement in this pathway * • UBR4 as the primary E3 ligase: UBR4, rather than UBR1 or UBR2, was identified as the primary E3 ligase responsible for recognizing and targeting proteins with the newly identified position 3 degrons for degradation. * • Role of downstream residues: The study showed that amino acid residues downstream of the position 3 lysine/arginine can influence both methionine cleavage by MetAP2 and recognition by UBR4, highlighting the complexity of the N-degron pathway. * • Endogenous protein regulation: The study provided evidence suggesting that this MetAP2-dependent, UBR4-mediated Arg/N-degron pathway regulates the stability of endogenous proteins, highlighting its broader biological significance.

Ang's take- somewhat specialized and 'ectopic' but important, thorough, and unambiguous. Satisfying. Very likely to be physiologically relevant even though most of the assays were done with reporters. Regardless, showing that this rule 'is' true is useful for technological applications.

There is so much racism, inequality, and atrocities that have happened in Orange County's past, yet there is little conversation about it now to recognize it. It is privilege to be able to commit all of these acts, and then forget them, yet the people who they've affected will forever remember and be affected by their actions. It also shows that there's a lot of history to unpack that many of us are probably unaware about.

The geographic location in which an individual resides plays a significant role in determining their ability to advance socioeconomically. This influence comes various factors such as the quality of educational institutions, the availability of diverse job opportunities, the strength of social networks, and the extent of community infrastructure. These elements can vary widely from one area to another, which leads to disparities in economic mobility. For instance, even within California, the economic opportunities and social dynamics in Los Angeles differ markedly from those in neighboring regions such as Orange County. or Irvine.

According to a Google search of the word "Mountebanks," a mountebank is "a person who deceives others, especially in order to trick them out of their money; a charlatan." However, Google also provides a "historical" definition, which reads: "a person who sold patent medicines in public places." In this sentence, Michael Maier appears to be calling on the knowledge of jugglers and mountebanks in order to argue that just as one can expel worms out of children by using other worms, one can kill a dragon through the power of its brother and sister. However, I wonder why Maier would call on a source that is connoted as inherently being "a charlatan." Or was that reputation to arise during a later period, and at this point they were not considered charlatans and were simply vendors of patent medicines? I'm also curious about who these jugglers were. Did early modern Europe have a juggler profession? How were they funded, if so? Or is the meaning less literal?

I looked up this word because I did not know it and found that it is defined as "the ‘sperm’ (sometimes ‘menstruum’) of the world, and ‘our Mercury’ (philosophical mercury as opposed to common mercury)." (Here is the link to the source where I found that definition.) This definition reminded me of how "The generation of the philosophers’ stone might be framed in terms of human or animal reproduction" (source). I wonder what might be the reasoning behind calling to mind human reproduction when discussing alchemy. One guess I have is that human reproduction is a natural process, so if making the philosopher's stone is a process similar to that, it can't be diabolical because it is as natural as giving birth. However, that is just a guess of mine.

This is a fun fact to know as by the name you would think other wise

It’s interesting to see how they actually worked hand in hand in some ways.

They had many ways to learn with many books available to them

A lot of the important things flourished in Spain and that’s intriguing.

it is interesting that they work together for these textiles because usually we see men and women have vastly different jobs back in history.

The spinning wheel was a really important inventions as that brought other important innovations.

It’s interesting that until the growth of towns is when craftsmen became important and not sooner.

In our discussion section, we have been talking a lot about how knowledge production during the early modern period was, at times, a means toward gatekeeping and, in doing so, maintained hierarchies of class and gender, for example. Similarly, in Magus, the credibility of a magus often depended on their ability to neatly frame themselves as one of those magi who skillfully drew upon elements that already existed in nature rather than a magus who conspired with the devil to produce effects. I feel that Michael Maier is doing something similar here, as he establishes his credibility by setting himself apart from those "strangers to all good learning and intention."

It seems cannons where a big use in war in the older times of wars.

It’s interesting to see how wars was in the older times compared to now.

Gunpowder has been around for a long time and it interesting to see how it’s improved and how it’s used now.

It’s interesting how it was invented in the 15th century and how it moved to other places.

WorkBC want people with disabilities to take the opportunity to work and get further involvement in the community

more resourceful sites! i think it's very thoughtful to add in considering the fact post-secondary can be really scary for some people. it gives people an ease of mind knowing they have guides to lead them

a lot of reliable resources for those who want to go into post-secondary, they seem to be good sites to encourage their education/passion! unlike the past of being denied and bashed to let into school, theres education programs to not only make people w disabilities feel included, but get the education they deserve in our community

Sadly, this exists also now, as the world slowly gets slightly more and more horrible and dystopian.

To accuse a man for crimes based on the testimony of a single person is unjust and horrible.

This would continue until near the end of the 20th century.

I do fear there is a lack of knowledge in what these chemicals can do long term not just in the short term.

A great example of this is the Cane toad in Australia. Were introduced to get rid of pests and now have taken over.

this is called gene flow!!! the movement of alleles from one population to another.

lol ya I don't think we as a Society are going to look too smart. I also feel it relies a lot on context it's so easy to look back in the past and think how dumb but they only had so much knowledge at that time.

There was a video released of high schoolers during the Cold War asking what they wanted to do in the future. A lot of them replied there wasn't going to be one because of the nuclear war or that they didn't think it was going to be a good fucher. This of course didn't happen but you could see the fear in general ideology of the time. This has a very similar feel and I wonder if it was written in the same time farm or this Author was at a critical point in life when this happened to be so impacted.

Also isn't this kind of counterintuitive to what she was saying before where animals couldn't evolve fast enough for the chemicals we were creating? I totally get the point that yes we are creating an habitat that isn't livable but I just found it ironic.

I went to Europe for the first time this summer and it was amazing what chemicals they didn't allow in their food in comparison to the US.

this is called Bioaccumulation one of the most Famous cases are in fish.

This made me think of the cases the acid rain that happened in the US in the late 70's I believes. It was a result of the Air pollutants that acidified the rain but this has been reduced Just in my lifetime and is a great Example of change that can happen.

I don't think this is true, there's so many species that alter their habitat and the world around them. Just think of plankton and how they help build the atmosphere and creates most of the oxygen we use.

More details on the Schenck case and "Fire" opinion.

I should add this to my WWI course material!

Popper on Tolerance -- apparently. A citation would have been nice!

When computers store numbers, they are limited by memory space, which may lead to problems such as floating-point precision errors and integer overflows. Different programming languages ​​provide special data types such as fractions and complex numbers to solve these limitations.