Answer Book Kumon Level I

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Lane Frisch

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Aug 5, 2024, 4:57:45 AM8/5/24

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Thislevel makes me cry.... The latter part of level J is toodifficult for me. :))) I can't answer the worksheets because Ireally don't understand how the examples were done. I got stuck at181-200(factor theorem, proof of identities and inequalities). Ihave to ask me teacher in Kumon how it's done. T_T

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After 2 rounds of revision, the authors finally consented to perform an additional experiment, in order to consolidate their manuscript. They therefore assessed the effect of IL27 on Mayaro virus replication. They also evaluated the effect of infection +/- IL27 or IFN on the transcription of some TRIM genes.

Unfortunately, these new results do not add much to the study. In particular, they do not allow the identification of which TRIM proteins are involved in the antiviral activity of IL27, which has been my main request from the beginning.

Answer: We agree with the reviewer. In the present study, we did not evaluate the expression of the TRIMs of interest at the protein level since our initial study aimed to evaluate the effect of IL27 treatment on the transcriptional expression of TRIMs. However, in a future study, we are confident that we will be able to evaluate this effect at the protein level and determine the antiviral activity of the TRIMs induced by IL27. The TRIMs of interest will be silenced to achieve this, and their effect on replicating the viruses of interest will be evaluated.

In this study, the authors aim to address the gene regulation of TRIM family induced by Type-I,II,III interferons and IL-27 via bulk RNA sequencing approach. They highlight that the induction of TRIM genes is caused in monocyte-derived macrophages following treatment of interferons and IL-27, as evidenced by computational analysis. Additionally, they quantify the TRIM gene expression using qRT-PCR. Although overall the manuscript is well written, there are some minor concerns to reach the standard of Viruses.

* In Fig. 8, the expression levels of TRIM 19, TRIM 21, TRIM 22 and TRIM 69 should be required for macrophages treated IL27 and Interferon-beta alone for measuring the effect of MAYV on TRIM expression in IL27 and interferon-beta treated macrophages.

Answer: Given that the analysis of the transcriptomes of macrophages treated with IFNs and IL27 and validation by RT-qPCR using cells treated with IFNs or IL27 without infection shows an increase in TRIM mRNA levels, our next aim was to evaluate whether both IL27 and IFNs decreased the replication of the Mayaro virus (Fig. 8). Therefore, given that we already had the results of the effect of IFNs and IL27, without infection on the expression of TRIMs (Fig. 7), we consider that it is not necessary to include those controls for Figure 8.

In this article, Hernandez-Sarmiento and colleagues evaluated the induction of TRIM gene expression by type I, II and III interferons and by IL27 in human macrophages. They first analyzed TRIM genes that are regulated by IFNs or IL27 using bulk RNAseq. They then used several algorithms to illustrate the identity of the genes whose expression was found to be regulated. Finally, they confirmed the results obtained by RT-qPCR experiments performed on 6 of the TRIMs transcripts. A table summarizes the results obtained and compares them to previous studies.

This article unfortunately does not contribute much to the discipline. The RNAseq data only confirms data from 20 years ago on the induction of TRIM genes by interferon. Importantly, these data were not generated by the authors, but were extracted from the GEO database. These RNAseq data were in fact obtained by Szaniawski M and Planelles V (Huntsman Cancer Instituten, Salt Lake City, USA). The only original data in this paper is therefore the expression of TRIM genes following IL27 treatment. However, the identification of 4 TRIM genes induced by IL27 in macrophages was not followed by any functional experiment. IL27 is known to induce an antiviral response and it would have been interesting to determine whether any of the 4 TRIM proteins identified could play a role in this activity.

Figures 2, 3, 4 and 5 are of no use, since the authors only used free software found on the internet, such as GenMANIA or STRING, to obtain information on the identified TRIM genes. These TRIM genes are however very well characterized and a bibliographic analysis would have been sufficient. In summary, the article attempts to hide the lack of results with an exaggerated and somewhat naive use of online algorithms.

Answer: We appreciate and respect the reviewer's comment. However, it's essential to consider that our approach, which involves reanalyzing already published RNA-seq datasets, is not new and has been widely used by several researchers. Many contributions to the field have been made through similar methodologies, and these studies have been published in various high-impact journals (1-3). Furthermore, as mentioned in the manuscript, most studies have focused on type I and II interferons, neglecting IFN-III. Additionally, these studies have typically utilized microarrays rather than RNA-Seq data to determine transcriptional patterns in response to treatment with the three types of IFNs, let alone IL-27.

To aid in understanding our methodological strategy better, we have added a diagram, which corresponds to Figure 1. We believe that our approach builds upon existing research and contributes valuable insights to the field. If you have any further questions or concerns, please feel free to let us know.

Answer: Unfortunately, we do not understand what the evaluator is asking, since in the manuscript, neither IFN-a nor much less IFN-w is mentioned. As written in the document, we refer to IFN-α and -ω, which have been widely described in the text, including Cartagena et al. The family of IFN-I comprises sixteen members in humans, including IFNβ, IFNε, IFNκ, IFNω, and 12 subtypes of IFNα (4).

1. Major comments

* The authors performed the bulk RNA sequencing on macrophage cell lysates harvested at 18 hour post-treatment with interferons or IL-27. The result show that some TRIM genes were upregulated or downregulated by the treatment. To provide a comprehensive analysis, the authors should include other interferon-stimulated genes (ISGs) as controls in Fig. 1.

Answer: Thank you for the suggestion. However, our study aimed to focus specifically on TRIMs for the comparative analysis of transcriptional profiles. We have already submitted another manuscript that covers both inflammatory and antiviral responses. Therefore, we focus on the present manuscript solely on TRIMs. We Include the figure with ISGs for informational purposes without incorporating them into the manuscript.

* The authors quantified mRNA levels of TRIM family using the macrophage cell lysates harvested at 24 hour post-treatment with interferons or IL-27. It is unclear why these quantified cell lysates were harvested differently from RNAseq samples. Additionally, since cell signaling by interferons or interleukins occurs rapidly, the authors should present mRNA levels via qRT-PCR in a time-dependent manner. ISG15, ISG56, MxA or OASL could serve as good controls.

I realize that "reanalyzing already published RNA-seq datasets is not new and has been widely used by several researchers". However, in this particular case, I don't believe that this approach adds anything new to the discipline. It is well established which TRIM genes are induced by IFN in human and murine cells, and the function of most TRIM proteins is now known. The only new aspect of this manuscript is the induction of certain TRIM genes by IL27, but, as I wrote in my first review, this was unfortunately not followed by functional experiments to assess whether these TRIM proteins mediate the IL27-induced antiviral response.