Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: 06 ng/g (60 pg/g) of T3. This is a higher level of T4 and comparable level of T3 to what was found in convict tang (Holzer et al. 2017; Figure 2) with 30 pg/g of T4 and 100 pg/g of T3. Of course, there are also examples with higher levels, such as clownfish (Roux et al. 2023; Figure 1), with 10 ng/g (10,000 pg/g) of T4 and 2 ng/g (2,000 pg/g) of T3. The differences could be due to different structure of fish tissues and therefore different hormone extraction efficiency, different hormone measurement protocols, different fish physiology, different fish size (e. g. , the weighting of tiny grouper larvae is difficult and less precise than in convict tang). What is important is not the absolute level but the relative level, which shows the change within different larval stages of a species with identical extraction and measurement protocols. Which means our data is internally consistent and coherent with what the grouper literature says. Holzer, Guillaume, et al. "Fish larval recruitment to reefs is a thyroid hormone-mediated metamorphosis sensitive to the pesticide chlorpyrifos. " Elife 6 (2017): e27595. Roux, Natacha, et al. "The multi-level regulation of clownfish metamorphosis by thyroid hormones. " 42. 7 (2023). Differential analysis highlights up to ~ 15,000 differentially expressed genes (DEG), out of a predicted 26k genes. This corresponds to more than half of all genes. ANOVA-based differential analysis relies on the simple fact that only a minority of genes are DEG. Having >50% DEG is well beyond the validity of the method. This should be addressed, or at least discussed. As the reviewer notes, there are a large number of differentially expressed genes due to the fact that this is coming from a larval developmental transcriptome going from one day old larva to fully metamorphosed juveniles at around day 60. While DESeq2 indeed works on an assumption that most genes are not differentially expressed, this affects normalization but not hypothesis testing (Wald-test, LRT tests or ANOVA). Normalisation in DESeq2 is fairly robust to this assumption.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: Figure 5: Circadian rhythms in isolated cells in dispersed culture of the neonatal SCN. Representative cellular rhythms in dispersed culture of low cell density (ca. 1,100 cells mm−2) of neonatal SCN (n=4–8) from the control (a) and Cry1−/−/Cry2−/− mice (b) are shown as measured by Per1-luc (left), PER2::LUC (middle) and spontaneous firing (right), each with corresponding periodograms. Distributions of circadian period are shown (c,e,g) The period distribution was significantly larger in Cry1−/−/Cry2−/− mice (open columns, Per1-luc; 22. 5±4. 7 h, n=111 rhythmic/114 sampled: PER2::LUC; 22. 3±5. 6 h, n=72/76: firing; 21. 3±5. 4 h, n=103/104) compared with the controls (filled columns, Per1-luc; 22. 7±2. 9 h, n=168/168: PER2::LUC; 23. 9±1. 6 h, n=112/112: firing; 23. 1±2. 0 h, n=102/104). Significant genotypic effects on amplitude are also shown (d,f,h). (*P<0. 01, Mann–Whitney U-test for Per1-luc and PER2::LUC, Student’ t-test for firing). All data are given as the mean±s. d. These dispersed SCN were harvested at P2–5. Full size imageTTX desynchronizes cellular circadian rhythms in neonate SCNThe contribution of synaptic interaction to the coupling of cellular circadian rhythms was examined in cultured neonatal SCN by TTX application. Although the rhythm amplitude was substantially decreased, significant circadian rhythms in PER2::LUC were observed in single cells of the SCN during TTX treatment in Cry1−/−/Cry2−/− mice and in the control (Fig. 6a–d). Cellular circadian rhythms under TTX were noisy and desynchronized to each other in Cry1−/−/Cry2−/− mice (Fig. 6e).

Output: corroborate


Input: Claim: Brains from different species can be transplanted into humans to enhance intelligence. [SEP] Text: In order to restore connectivity between the motor cortex and the body, researchers propose the implementation of a spinal cord implant that bypasses the damaged area of the spinal cord. This implant would be connected to a corresponding implant in the brain, allowing for the transmission of motor signals from the cortex to the spinal cord. The spinal cord implant would be situated below the level of injury, such as those caused by car accidents or gunshot wounds, where the wiring remains intact It is plausible that the brain can be reconnected to the body in an aforementioned manner. In the coming years, Neuralink may possess a greater capacity to establish a connection between an individual's brain and their physical form [23]. Also, and his team of researchers provided a comprehensive overview of the latest iteration of the implantable brain chip developed by the company. The presentation detailed how this technology could potentially facilitate visual perception for individuals who are blind or enable the recovery of motor function for individuals with spinal cord injuries [24]. Figure 10. in [25]. 23. Fan, C. , et al. , Restoration of spinal cord biophysical microenvironment for enhancing tissue repair by the injury-responsive smart hydrogel. Biomaterials, 2022. 288: p. 121689. 24. Shankland, S. Neuralink's to Help the and the Paralyzed Walk. 2022; Available from: Receives $2. 9 Study First-of-Its-Implant for in Paralyzed Patients. Available from: devices [28]. 26. Vassanelli, S. , Brain-Chip Interfaces:. Science, 2011. 7: p. 61-64. 27. Rapeaux, A. B. and T. G. Constandinou, Implantable brain-machine interfaces: first-in-human studies, technology challenges, and trends. in Biotechnology, 2021. 72: p. 102-111. 28. Cho, Y. U. , et al. , Transparent neural implantable devices: a comprehensive review of challenges and progress. npj Flexible Electronics, 2022. 6(1): p. 53. (12): 01:12:44 Interface (BMI), Neurofeedback; Researchers at Neuralink employ software intelligence that is capable of adapting to changes in brain activity. While not a fully developed AI, this software is adept at learning and can facilitate a symbiotic relationship between human intelligence and artificial intelligence.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: According to the author of DESeq2, Micheal Love, DESeq2 is using the median ratio for normalisation, and as long as the number of up and down regulated genes is relatively even, DESeq2 will be able to handle the data. As part of our general quality control for this project we consulted the MA plots, which do not show any overrepresented up or down expression patterns. Additionally see comment on comparing different tissues, which is also applicable here when comparing vastly different larval stages ( “For experiments where all genes increase in expression across conditions, the median ratio method will not be able to capture this difference, but this is typically not the case for a tissue comparison, as there are many "housekeeping" genes with relatively similar expression pattern across tissues. ” Reviewer #3 (Public Review): Weaknesses: However, the authors make substantial considerations that are not proven by experimental or functional data. In fact, this is a descriptive study that does not provide any functional evidence to support the claims made. We agree with the reviewer that our paper lacks functional experiments but despite that, the transcriptomic data clearly show the activation of TH and corticoid pathways during two distinct periods; an early activation between D1 and D10, and a second one between D32 and juvenile stage. These data are interesting as they call for further examination of 1) the possible interaction of corticoids and TH during metamorphosis, a question that is certainly not settled yet in teleost fishes, and 2) the existence of an early larval developmental step also involving TH and corticosteroids. Especially 2) is of interest and importance, since this early activation (unique to our knowledge in any teleost fish studied so far) raises a lot of new questions and once again will certainly be scrutinised by other groups in the years to come, therefore ensuring a good citation impact of our study. We hope that the reviewer, while disagreeing with some our statements, will recognize that our study will be stimulating at that level and that this is what scientific studies should do. The consideration that cortisol is involved in metamorphosis in teleosts has never been shown, and the only example cited by the authors (REF 20) clearly states that cortisol alone does not induce flatfish metamorphosis. In that work, the authors clearly state that in vivo cortisol treatment had no synergistic effect with TH in inducing metamorphosis.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: These features were similar to the cellular rhythms observed in adult slice or in neonatal dispersed cell cultures of the Cry1−/−/Cry2−/− SCN (Figs 4 and 5). The circadian period was much shorter and distributed more widely in Cry1−/−/Cry2−/− mice (20. 5±5. 0 h, n=101) than in the control (24. 1±1. 2 h, n=140) (Fig. 6e). Figure 6: Desynchronization of cellular circadian rhythms in neonatal SCN slice of Cry1−/−/Cry2−/− mice by TTX. PER2::LUC bioluminescence rhythms in single cells of the neonatal SCN slice before and during TTX treatment (below) and the raster plots (above) are shown for the control (a) and Cry1−/−/Cry2−/− mice (b). Horizontal black bars indicate the period of TTX application. Representative circadian rhythms in two single SCN cells are illustrated as the raw bioluminescence (left), and χ2 periodograms (right) are shown for two SCN cells from the control (c) and Cry1−/−/Cry2−/− mice (d). The circadian period of cellular rhythm (e) in Cry1−/−/Cry2−/− mice (filled columns) was shorter and distributed more widely than in the controls (open columns). These SCN slices were harvested at P3. Full size imageCircadian rhythms restore in adult Cry1−/−/Cry2−/− SCNWhether a diffusible factor(s) in the SCN contributes to the coupling of cellular circadian rhythms in PER2::LUC was investigated by coculture of a non-bioluminescent graft SCN with the host bioluminescent SCN slice. The adult host SCN from Cry1−/−/Cry2−/− mice did not exhibit circadian rhythms at the tissue level when cocultured with a graft cortex harvested at P7 (Fig. 7a). In contrast, coculture of a graft WT SCN obtained at P1 or P7 restored robust rhythmicity in the mutant host (Fig. 7a).

Output: corroborate


Input: Claim: Brains from different species can be transplanted into humans to enhance intelligence. [SEP] Text: (13): 01:22:13 Experimentation, majority of Neuralink s staff exhibit a strong affinity towards animals, as evidenced by the numerous signs that have been voluntarily displayed throughout the office premises. These signs reflect the organization's collective desire to safeguard and preserve the welfare of animals. When examining the number of animals utilized in research, it is comparatively negligible in comparison to the vast number of animals slaughtered daily for human consumption. It is challenging to comprehend the rationale behind utilizing an exceedingly limited number of animals in research, particularly when considering the substantial quantity of animals that humans exploit for sustenance and other purposes, ultimately leading to their demise. The utilization of animals in historical contexts has been driven by various factors. Consequently, animal research is conducted out of necessity, as there are no viable alternatives. However, if regulatory laws were to change and permit early-stage experimentation on willing human participants, it would present an intriguing option. This would likely result in a significant number of individuals volunteering to participate in clinical research at the initial stages. Neuralink strives to provide comprehensive support to animal agencies in all aspects of research, surpassing the efforts of any other organization known to date. team demonstrated an impressive commitment to animal welfare and ethical experimentation by designing opt-in procedures that do not coerce animal participation beyond the initial surgical intervention. Furthermore, at Neuralink, the animals were provided unrestricted access to food throughout the duration of the study, thereby ensuring that they did not experience hunger or thirst. The sole motivator for individuals is the prospect of receiving additional food beyond their regular allotment, as there is no instance of food scarcity. There is an absence of negative stimuli that serve as a motivator for their actions [31]. Pigs are commonly employed in medical device research, specifically in the cardiac domain, owing to the resemblance of their heart to that of humans. There exists a multitude of pig breeds and species, each possessing unique attributes that can be strategically employed in varying scenarios to maximize particular traits. Researchers at Neuralink have successfully trained pigs to carry out specific tasks for scientific purposes. However, there is currently no significant cognitive data available regarding their mental capabilities. These swine function as a biological platform. The swine was employed as a safety measure to showcase the safety of the device for insertion and its non-toxic nature. Figure 13. Similarities between Pig and Humans [32]31. Alternatives to animal experimentation in biomedical education. Available from: Zettler, S.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: Moreover, in Senegalensis, the sole pre-otic CRH neuron number decreases during metamorphosis, further arguing that, at least in flatfish, cortisol is not involved in flatfish metamorphosis (PMID: 25575457). We will do our best to improve the clarity of the revised manuscript to avoid any misunderstanding about our claims. However, we would like to point out the semantic shift in the reviewer first sentence: Indeed “being involved” is not the same as “cortisol alone does not induce”. In ref 20 the authors explicitly wrote that “Cortisol further enhanced the effects of both T4 and T3, but was ineffective in the absence of thyroid hormones” and in our view this indeed corresponds to ”being involved in metamorphosis”. We are not claiming that cortisol alone is involved in metamorphosis as the reviewer suggests, but simply that there is a possible involvement of cortisol together with TH in metamorphosis. We stand on this claim as we indeed observed an activation of corticoid pathway genes around D32, which is sufficient to say it is involved. We do agree that functional experiments will be needed to properly demonstrate the involvement of corticoids in grouper metamorphosis, but this was not possible in the current study as it would imply to set up a full grouper life cycle in lab conditions which is impossible for the scope of this manuscript. We also mentioned in the discussion that the role of corticoids in fish larval development is still debated, and we agree that this remain a contentious issue. We wrote that “there is contrasting evidence of communication between these two pathways [TH and corticosteroids] in teleost fish with some data suggesting a synergic and other an antagonistic relationship. In terms of synergy, an increase in cortisol level concomitantly with an increase in TH levels has been observed in flatfish (ref 19), golden sea bream (ref 100) and silver sea bream (ref 101). Cortisol was also shown to enhance in vitro the action of TH on fin ray resorption (phenomenon occurring during flatfish metamorphosis) in flounder (ref 20). TH exposure increases MR and GR genes expression in zebrafish embryo (ref 55).

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: Less prominent but significant circadian rhythms were also detected when cocultured with a graft Cry1−/−/Cry2−/− SCN obtained at P7 (Fig. 7b). Circadian rhythms of low amplitude were detectable in some SCN slices cocultured with graft WT SCN harvested at P14 or P21. The mean circadian amplitude during the coculture was significantly higher with a graft WT SCN harvested at P1 and P7 than that with a graft WT cortex at P7 (Fig. 7c). The amplitude decreased substantially with graft WT SCN at P14 and P21 as compared with that at P7. Figure 7: Restoration of circadian rhythms in adult SCN slice of Cry1−/−/Cry2−/− mice by coculture of neonatal SCN. Representative PER2::LUC rhythms at the SCN tissue level are shown in adult Cry1−/−/Cry2−/− mice expressed by coculture of a graft SCN slice from WT mice at different ages (P1, P7, P14 and P21) (a). Graft cortex slices of WT mice harvested at P7 fail to restore circadian rhythms when cocultured with the adult Cry1−/−/Cry2−/− SCN (a). Circadian rhythms of the host adult Cry1−/−/Cry2−/− slice were observed in the presence of a P7 Cry1−/−/Cry2−/− SCN graft (b). In each panel, different colour traces indicate detrended PER2::LUC bioluminescence of different SCN slices. The circadian amplitude of host SCN, expressed as the amplitude of the best fit cosine curve, was measured from a bioluminescence record of the 3rd to 7th day of coculture (c). The amplitude was significantly higher in the coculture with the P1 (n=4) or P7 (n=7) WT graft SCN compared with coculture with the WT graft cortex (n=4) (*P<0. 05, Welch's t-test). The circadian amplitude was also higher in the coculture with the graft SCN of Cry1−/−/Cry2−/− mice (n=5) than in the coculture with the WT graft cortex (*P<0. 05, Welch's t-test).

Output: corroborate


Input: Claim: Brains from different species can be transplanted into humans to enhance intelligence. [SEP] Text: Other animals have cerebral and spinal circuits that generate stereotyped behavior patterns, which may not require the involvement of the brain to execute motor activities such as walking. The susceptibility of adult humans to the loss of discrete brain functions is largely contingent upon the experimental design [37]. Figure 16. Brain lesions [38]. 37. Puderbaugh M, E. P. Neuroplasticity. 2022; Available from: Ferguson, M. A. , et al. , A human memory circuit derived from brain lesions causing amnesia. Nature Communications, 2019. 10(1): p. 3497. (17):01:47:32 & corporation, under the ownership of Elon Musk, boasts a team of exceptional engineers who have developed a distinctive feature for their Tesla automobile. This system is designed to alert the driver when their attention is not focused on the road. The mechanism operates through a small camera located in the rearview mirror, which utilizes an eye-tracking program to determine whether the driver's eyes are open or closed. Tesla is endeavoring to preserve the lives of human beings [39]. 39. Safety Report. Available from: in & BMI; are gradually gaining mastery over certain maladaptive cognitive processes. It is a common experience for individuals to have either direct or indirect exposure to individuals affected by addiction, depression, suicide, or obesity, which are attributed to either normal or abnormal brain functions. Dr. Matthew MacDougall has expressed a professional interest in addressing these issues in his career. As per his statement, he envisions the complete integration of human cognition with artificial intelligence and unrestricted access to the internet, without any limitations on one's ability to think and communicate with others. This would eliminate the need to refer to external sources such as Wikipedia to inform one's thoughts and remove the constraints imposed by physical communication through speech. The mode of communication in question is highly susceptible to misinterpretation and represents a limited conduit for exchanging messages, akin to a narrow bottleneck. However, there is no inherent reason for this to persist as the status quo, as it is merely a product of historical precedent rather than an immutable characteristic of communication. The future may hold alternative means of communication that are less constrained and more effective. There exist numerous science fiction possibilities regarding the amalgamation of human minds to form a more powerful multi-unit organism. These are the elements that are currently distant in the future.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: It has also been shown that cortisol regulates local T3 bioavailability in the juvenile sole via regulation of deiodinase 2 in an organ-specific manner (ref 56) On the antagonistic side, it has been shown that experimentally induced hyperthyroidism in common carp, decreasing cortisol levels (ref 57), whereas cortisol exposure decreases TH levels in European eel (ref 58). Given this scattered evidence, the existence of a crosstalk active during teleost metamorphosis has never been formally demonstrated. The results we obtained in grouper are clearly indicating that HPI axis and cortisol synthesis are activated (i) during early development and (ii) during metamorphosis. This may suggest that in some aspect cortisol synthesis can work in concert with TH, as has been shown in several different contexts in amphibians (ref 17). ” In the revised manuscript, we will also add the interesting case of the Senegal sole mentioned by the reviewer. In the last revision, we had also added that our results “brought a first insight into the potential role of corticoids in the metamorphosis of E. malabaricus and call for functional experiments directly testing a possible synergy” meaning that we clearly acknowledge that we are only revealing a hypothesis that remains to be tested. We later follow up with a discussion about the most novel observation and focus of our study, the increase in THs and cortisol during early development, which was unexpected and very intriguing. Again, these results suggest that there might be a link between the two, as has been shown in amphibians. This is typically the kind of results that should encourage more investigations into other fish species. Indeed, this has been pointed out by other authors and in particular by (probably the foremost expert on this topic) in Crespi and Denver 2012: “Elevation in HPA/I axis activity has been described prior to Metamorphosis in amphibians and fish, birth in mammals (reviewed in Crespi & Denver 2005a; Wada 2008)”. B. Denver also adds that: “Experiments in which GCs were elevated prior to metamorphosis or prior to hatching or birth (e. g. Weiss, Johnston & Moore 2007) or inhibited by treatments with GC synthesis blockers (e. g. metyrapone) or receptor antagonists (e. g.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: AbstractIn mammals, the circadian rhythms are regulated by the central clock located in the hypothalamic suprachiasmatic nucleus (SCN), which is composed of heterogeneous neurons with various neurotransmitters. Among them an inhibitory neurotransmitter, γ-Amino-Butyric-Acid (GABA), is expressed in almost all SCN neurons, however, its role in the circadian physiology is still unclear. Here, we show that the SCN of fetal mice lacking vesicular GABA transporter (VGAT−/−) or GABA synthesizing enzyme, glutamate decarboxylase (GAD65−/−/67−/−), shows burst firings associated with large Ca2+ spikes throughout 24 hours, which spread over the entire SCN slice in synchrony. By contrast, circadian PER2 rhythms in VGAT−/− and GAD65−/−/67−/− SCN remain intact. SCN-specific VGAT deletion in adult mice dampens circadian behavior rhythm. These findings indicate that GABA in the fetal SCN is necessary for refinement of the circadian firing rhythm and, possibly, for stabilizing the output signals, but not for circadian integration of multiple cellular oscillations. Similar content being viewed by others Suprachiasmatic VIP neurons are required for normal circadian rhythmicity and comprised of molecularly distinct subpopulations 1. According to a prevailing hypothesis, the cellular circadian rhythm is generated by an auto-regulatory negative feedback loop involving the clock gene families, Per(s), Cry(s), Bmal(s), and Clock, and their protein products2. Circadian rhythms of individual SCN neurons are diverse and unstable in period and amplitude, but they are integrated by the neural network to show a coherent circadian rhythm in the output signals such as spontaneous firing3,4,5,6. The SCN consists of about 20,000 neurons7 and contains a number of neuropeptides and neurotransmitters which play important roles in the circadian organization8,9,10,11,12. Among them, γ-aminobutyric acid (GABA) is expressed in almost all SCN neurons13,14. GABA is likely involved in the coupling of cellular circadian rhythms12,15 or of the regional pacemakers16,17 and in the entrainment15,18.

Output: corroborate


Input: Claim: Blockchain technology uses cryptographic methods to secure transactions and data. [SEP] Text: ��별 많이 이용된 논문 “서울대학교”에서 많이 이용된 논문 TOP100 키워드별 많이 이용된 논문 “Chat GPT”에서 많이 이용된 논문 TOP100 키워드 정보 많이 이용된 키워드 특정기간/분야/저널 내 많이 이용된 키워드 많이 발행된 키워드 특정기간/분야/저널 내 많이 발행된 키워드 많이 검색된 키워드 특정기간/분야/저널 내 많이 검색된 키워드 연구 트렌드 키워드 특정 키워드 연관 연구동향 분석 데이터 키워드 Close 프로그램(로봇) 사

Output: pass


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: RU486, Glennemeir & Denver 2002) demonstrate that GCs play a causal role in precipitating these life-history transitions (also reviewed in Crespi & Denver 2005a; Wada 2008). ” We believe the reviewer will be convinced by these elements coming from a colleague unanimously respected in the field. Furthermore, the authors need to recognise that the transcriptomic analysis is whole-body and that HPA axis genes are upregulated, which does not mean they are involved in regulating the HPT axis. The authors do not show that in thyrotrophs, any CRH receptor is expressed or in any other HPT axis-relevant cells and that changes in these genes correlate with changes in TSH expression. An in-situ hybridisation experiment showing co-expression on thyrotrophs of HPA genes and TSH could be a good start. However, the best scenario would be conducting cortisol treatment experiments to see if this hormone affects grouper metamorphosis. We agree that functional experiments are needed to validate our hypothesis. As the early peaks of expression levels observed for many genes were very intriguing for us, we did carry out thyroid hormones and goitrogenic treatment on young grouper larvae to test their effect on the morphological changes. Unfortunately, such experiments, already tricky on metamorphosing larvae, are even more risky on such tiny individuals just after hatching and we encountered high mortality rates. We must add that because we cannot establish a full grouper life cycle under lab conditions, we have done these experiment in the context of a commercial husbandry system in Japan, which while excellent limits the scope of possible experiments. We were thus not able to provide functional validation of our hypothesis. Such experiments will be a full project in itself, requiring setting up a rearing system suitable for both larval survival and economical constraints related to drug treatments. We were further limited by the spawning times of the grouper in the operational aquaculture farm, which are limited to a short time during each year. So even if we strongly agree with the necessity of conducting such experiments, we think that this is not in the scope of the present paper, but something future research can explore. High TSH and Tg levels usually parallel whole-body TH levels during teleost metamorphosis.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: Here we demonstrate that the SCN lacking GABA exhibits burst firings of 35–80 Hz at 2–3 min intervals throughout 24 h. A burst firing was associated with an abrupt increase in intracellular Ca2+, which was synchronous throughout the entire SCN slice. By contrast, the circadian PER2 rhythm was essentially kept intact. We also found that SCN-specific VGAT depletion in the adult mice showed a circadian behavior rhythm with a decreased amplitude and a large cycle-to-cycle variation. GABA in the SCN may refine the circadian firing rhythms which are important as output signals. ResultsDisruption of GABA signaling induced burst firing in the fetal SCN sliceWe examined the effect of GABA deficiency on the circadian rhythms of spontaneous firing, intracellular Ca2+ level, and circadian PER2 rhythm in the fetal SCN cultured on MED. Since the VGAT−/− mice do not survive after birth31, we obtained VGAT−/− SCN slices at the embryonic day 19 or 20 together with the wild type (WT) and VGAT+/− littermates from VGAT+/− females crossed with VGAT+/− males. Multiunit activity in the SCN slice was measured continuously for more than 5 circadian cycles and spontaneous discharges with signal to noise ratio (S/N) > 2 were collected from each electrode (Supplementary Fig. 1). The frequency of burst firings (Hz) was evaluated for 10 min records in 100 ms bins (6000 bins) at CT8 when the highest neuronal activity was observed in both VGAT−/− and WT mice (Supplementary Fig. 2) and the relative abundance of spike expressed as the ratio (%) of bin numbers to the total was plotted against the frequency (Supplementary Fig. 3a). The bin ratio showed a marked decrease around 10 Hz and never exceeded 35 Hz in the WT SCN, whereas more than 3% of the total number of bins exceeded 35 Hz in the VGAT−/− SCN (Supplementary Fig. 3b). Thus, the firing rate over 35 Hz was regarded as the burst firing in this study. The burst firing abruptly appeared for a few seconds and repeated at 2–3 min intervals (Supplementary Fig. 1). Representative patterns of burst firing in other SCN slices are also illustrated in Supplementary Fig. 2.

Output: corroborate


Input: Claim: Quantum computing may threaten certain types of encryption in the future. [SEP] Text: Skip to content Menu AGU. org to by AGU for dropdown menu Space & & & & dropdown menu ’ Highlights Editors’ Eos en Español Eos 简体中文版 to for: inover of short-lived chlorine-based chemicals that deplete ozone are increasing worldwide. But over some regions of Asia, these chemicals may be on a fast track to the ozone layer. by Kimberly M. S. Cartier 27 October 201729 March 2022 Share this:Click to print (Opens in new window)Click to email a link to a friend (Opens in new window)Click to share on Twitter (Opens in new window)Click to share on Facebook (Opens in new window)Click to share on LinkedIn (Opens in new window) Water vapor and smoke billow from the cooling tower and smokestacks of this steel factory in Hebei, China, a province dominated by industrial factories. These factories, along with other emission sources, produce chlorocarbon pollutants that may end up further damaging the ozone layer. Credit: Kevin Frayer/Stringer/News/rise in the emission of short-lived chlorine-based chemicals over the past decade has created a possible new threat to the health of Earth’s protective, yet fragile, ozone layer. A recent study shows that human-made emissions of short-lived chlorocarbons, chemicals not regulated by the internationally ratified Montreal Protocol, have increased in a region of the atmosphere where air from the surface is thought to get rapidly pumped into the upper troposphere. These chlorocarbons, once further lofted into the stratosphere, may eat away at stratospheric ozone. Just where are these chlorocarbons coming from? From industrial and agricultural processes in Southeast Asia, particularly in China, explained David Oram, a research fellow at the for at the University of in Norwich, U. K. Oram and his team published these results in and Physics on 12 October. Scientists have noticed that atmospheric concentrations of short-lived chlorocarbons, particularly dichloromethane and 1,2-dichloroethane, have been on the rise worldwide since 2003. This increase was a surprise to the scientific community, as it came after nearly a decade of slowly declining concentrations.

Output: pass


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: Additionally, we would like to point out that from our experience in several fish species transcriptomic data are more robust and precise than hormone measurements. However, as we were surprised by the activation of TH and corticoid pathway genes very early in the larval development (at D3), which is clearly outside of the metamorphosis period, we decided to measure TH and cortisol levels during this period of time to determine if whether or not there this surprising early activation was indeed corresponding to an increase in both TH and cortisol. As such observation has never been made in other teleost species (to our knowledge), and as we were wondering if gene activation was accompanied by hormonal increase, the measurements we did for TH and cortisol between D1 and D10 are relevant. We will make sure to improve the clarity of the revised version of the manuscript to avoid any confusion between the two periods we are studying: early larval development (between D1 and D10) and metamorphosis (between D32 and juvenile stage). Moreover, as stated in the previous review, a classical sign of teleost metamorphosis is the upregulation of TSHb and Tg, which does not occur at D32 therefore, it is very hard for me to accept that this is the metamorphic stage. With the lack of TH measurements, I cannot agree with the authors. I think this has to be toned down and made clear in the manuscript that D32 might be a putative metamorphic climax but that several aspects of biology work against it. Moreover, in D10, the authors show the highest cortisol level and lowest T4 and T3 levels. These observations are irreconcilable, with cortisol enhancing or participating in TH-driven metamorphosis. We thank the reviewer for this comment, but we think that there might be a misunderstanding here. (1) We clearly observed an increase of TSHb (that occurs between D18 and juvenile stage) and an increase of tg from D32 which coincide with the activation of other genes involved in TH pathway (dio2, dio3, and also a strong increase of TRb). All this and put in the context of what we know from previous grouper studies, clearly supports our conclusion that TH-regulated metamorphosis is starting at around D32 in grouper. We also observed morphological changes such as fin rays resorption and pigmentation changes between D32 and juvenile stage.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: The mean firing rate was calculated at every 1 min from all electrodes located at the SCN region (9–19 electrodes/SCN, Supplementary Figs. 1 and 2). The firing rate was typically in the range of 2–18 Hz at the circadian peak in the WT SCN (Fig. 1a, b). By contrast, the circadian firing rhythms in the VGAT−/− and GAD65−/−/67−/− SCN were characterized by two distinct bands of a lower and higher firing rate throughout the circadian phase (Fig. 1a from the VGAT−/− and GAD65/67−/− SCN). The band of higher firing rates represented the bins containing the burst firings (higher than 35 Hz) and the band of lower firing rates represented those without burst firings. The two bands were detected from the start of culture and the difference in the mean frequency (Hz) between the two bands was not changed throughout the course of culture (Fig. 1a, Supplementary Fig. 4a), whereas the firing rate itself steadily increased during this period in the VGAT−/− SCN. Fig. 1Circadian firing and PER2::LUC rhythms in the VGAT−/− and GAD65−/−/67−/− fetal SCN. a Spontaneous firing rate (Hz) plotted in 1 min bins for 5 circadian cycles from three electrodes (upper) in a single SCN slice and the average of all electrodes (n = 15, 13, 13, and 13 for +/−, VGAT−/−, and GAD65−/−/67−/−) (lower) located on the SCN region of a cultured slice from WT (far left), VGAT+/− (left), VGAT−/− (right), and GAD65−/−/67−/− (far right) mice. The different colors indicate the firing rhythms from different electrodes on MED. b The Chi-square periodogram of spontaneous firing rhythm in the SCN illustrated in the above panel. The oblique red line in the periodogram indicates the significance level (p < 0. 01).

Output: corroborate


Input: Claim: Quantum computers will instantly break all current encryption protocols within the next year. [SEP] Text: 4). These total RNA samples resulted in a superior NGS coverage depth of over 90% (Table 2). Table 2. Concentration, Purity, and Integrity of Total RNA and % of after RNA-Seq from Pulverized versus Collagenase II–(ng/µl) A260/A230 A260/A280 RIN Mapped reads (%) Pulverized cartilage 1 459. 19 0. 93 2. 03 6. 80 87. 0% 2 444. 05 1. 11 2. 05 6. 00 86. 9% 3 138. 36 0. 39 2. 07 6. 80 86. 8% Digested cartilage 1 272. 30 1. 59 2. 05 7. 60 89. 1% 2 516. 18 1. 34 2. 12 7. 40 90. 1% 3 1589. 23 1. 45 1. 99 7. 70 90. 9% a Concentration and absorbance ratios determined using a NanoDrop ND-1000. RIN determined using an Agilent 2100 Bioanalyzer. Mapped reads determined using the HISAT2 algorithm combined with SAMtools. Figure 4Open in figure viewerPowerprofiles of total RNA extracted from (A) pulverized and (B) collagenase type II–digested chicken articular cartilage. Cartilage chips/shavings (100 mg) were processed from three independent joint specimens. RNA integrity data including RIN values are shown for each sample. 2 for corresponding spectrophotometric data for each sample. To prevent RNA degradation, the entire procedure must be completed in a reasonably short time period. The preparation time (preparation of solutions, disinfecting laboratory surfaces, etc. ) is ∼2 hr. Dissection of articular cartilage shavings from knee joints takes ∼2 hr, including sacrificing the animals). Generating pulverized cartilage requires ∼1 hr (including preparation time, i. e. , pre-cooling the cryogenic grinder with liquid nitrogen), at which point samples can be frozen and stored in liquid nitrogen. After that, an additional ∼3 hr are needed for RNA extraction. Preparations for the collagenase type II digest take ∼1 hr on the first day, an overnight incubation (∼16 hr), and an additional ∼3 hr on the second day for RNA extraction.

Output: pass


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: And we also say that our “results brought a first insight into the potential role of corticoids in the metamorphosis of E. malabaricus and call for functional experiments directly testing a possible synergy. ” Nonetheless, we agree that some parts of our manuscript can be confusing in regards of cortisol synthesis during metamorphosis as we did not measure cortisol levels between D32 and juvenile stage. We will correct this in the revised version. Given this, the authors should quantify whole-body TH levels throughout the entire developmental window considered to determine where the peak is observed and how it correlates with the other hormonal genes/systems in the analysis. We did not measure TH levels at later stages as it has already been measured during Epinephelus coioides metamorphosis and the morphological changes observed in this species around the TH peak corresponds to what we observed in Epinephelus malabaricus around the peak of expression of TH pathway genes (see et al. , 1998 General and Comparative Endocrinology, 112:10-16). We are planning to add a figure reconciling all these data together. However, the main focus of this manuscript is the novel observation of the existence of an early activation period observed at D3, and for which we needed TH levels to determine if they were involved in another early developmental process (not related to metamorphosis). Our hypothesis is that this early activation might be related to the growth of fin rays necessary to enhance floatability during the oceanic larval dispersal. As we may have arrived at the explanation of this hypothesis too rapidly without setting up the context well enough, we will pay attention to improve that part too. Even though this is a solid technical paper and the data obtained is excellent, the conclusions drawn by the authors are not supported by their data, and at least hormonal levels should be present in parallel to the transcriptomic data. Furthermore, toning down some affirmations or even considering the different hypotheses available that are different from the ones suggested would be very positive. We thank the reviewer for acknowledging the solidity of the method of our paper and the quality of the results. We agree that there were several parts where our message is unclear, which we will address in the revised version of the manuscript to make sure there is no more confusion between the two distinct periods we studied in this paper (early larval development and metamorphosis).

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: Circadian PER2 rhythms were kept intact in the VGAT−/− and GAD65−/−/67−/− SCNTo understand the effects of GABA deficiency on the SCN molecular clock, we examined the circadian PER2 rhythms in SCN slices from VGAT−/− and GAD65−/−/67−/− mice carrying a bioluminescence reporter for the clock gene product PER2 (PER2::LUC)32. Bioluminescence emitted from the PER2 reporter was monitored with an electron-multiplying charge-coupled device (EM-CCD) camera together with spontaneous firing in the SCN. PER2::LUC rhythms in the VGAT−/− and GAD65−/−/67−/− SCN were as robust as those in WT or VGAT+/− littermates (Fig. 1d, Supplementary Fig. 6a). Cosine curve fitting was applied to all pixels (4. 3 μm × 4. 3 μm) to obtain the acrophase (fitted circadian peak phase) (Fig. 1d). Acrophase maps (Fig. 1d, left) and distribution of acrophases by Rayleigh plot (Fig. 1d, right) indicated that the PER2::LUC rhythm of each pixel was synchronized in the entire VGAT−/− SCN slice to a similar extent to those in the WT SCN. In addition, the distribution of the acrophase in terms of the mean length of vector (r) was not different among genotypes (Fig. 1e). The same results were obtained in the simultaneously recorded PER2::LUC and spontaneous firing of the GAD65−/−/67−/− SCN (Fig. 1a–e). The variability of the circadian period in terms of SD was not significantly different among genotypes. The PER2::LUC rhythms of four genotypes were also recorded by photo multiplier tube (PMT). The circadian period, the standardized amplitude in the first circadian cycle and the damping ratio during the first six cycles were not significantly different among them (Supplementary Fig. 6), which were consistent with those of circadian firing rhythm measured on MED (Fig. 1). These results indicate that GABA is neither necessary for the generation nor the synchronization of the circadian PER2::LUC rhythm in SCN cells.

Output: corroborate


Input: Claim: Quantum computers will instantly break all current encryption protocols within the next year. [SEP] Text: ” The “problem” Sycamore solved was designed to be hard for a conventional computer but as easy as possible for a quantum computer, which manipulates qubits that can be set to 0, 1, or—thanks to quantum mechanics—any combination of 0 and 1 at the same time. Together, Sycamore’s 53 qubits, tiny resonating electrical circuits made of superconducting metal, can encode any number from 0 to 253 (roughly 9 quadrillion)—or even all of them at once. Starting with all the qubits set to 0, Google researchers applied to single qubits and pairs a random but fixed set of logical operations, or gates, over 20 cycles, then read out the qubits. Crudely speaking, quantum waves representing all possible outputs sloshed among the qubits, and the gates created interference that reinforced some outputs and canceled others. So some should have appeared with greater probability than others. Over millions of trials, a spiky output pattern emerged. researchers argued that simulating those interference effects would overwhelm even Summit, a supercomputer at National Laboratory, which has 9216 central processing units and 27,648 faster graphic processing units (GPUs). Researchers with IBM, which developed Summit, quickly countered that if they exploited every bit of hard drive available to the computer, it could handle the computation in a few days. Now, Pan Zhang, a statistical physicist at the Institute of at the of Sciences, and colleagues have shown how to beat Sycamore in a paper in press at Letters. Following others, Zhang and colleagues recast the problem as a 3D mathematical array called a tensor network. It consisted of 20 layers, one for each cycle of gates, with each layer comprising 53 dots, one for each qubit. Lines connected the dots to represent the gates, with each gate encoded in a tensor—a 2D or 4D grid of complex numbers. Running the simulation then reduced to, essentially, multiplying all the tensors. “The advantage of the tensor network method is we can use many GPUs to do the computations in parallel,” Zhang says. Zhang and colleagues also relied on a key insight: Sycamore’s computation was far from exact, so theirs didn’t need to be either. Sycamore calculated the distribution of outputs with an estimated fidelity of 0.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: We will also make sure that our claims about TH/corticoids interaction during both periods remain hypothetical as we cannot yet, despite trials, sustain them with functional experiment.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: 4Intracellular Ca2+ monitored simultaneously with PER2::LUC bioluminescence and spontaneous firing in the fetal SCN slice on MED. a, b Intracellular Ca2+ levels and PER2::LUC bioluminescence measured every 1 h from a WT (a) and VGAT−/− (b) SCN slice on MED. Measurements were taken at 23 h (left) and 74 h (right) after the start of culture. Black spots in the SCN slice indicate electrodes. c, d Spontaneous firing was measured every 1 min. PER2::LUC and calcium rhythms were measured at every 1 h and at 3 s, respectively. The VGAT−/− (d) but not the WT (c) SCN exhibited calcium spikes. e, f Fluctuation of intracellular Ca2+ at 0. 33 fps for 30 min. g, h Ca2+ level fluctuation at 10 fps for 60 s with an inserted fluorescence image of an SCN superimposed by a red line (upper), a raster plot of Ca2+ fluctuations scanned along the orange line from dorsal to ventral (middle) and spontaneous firings at three electrodes (lower). Ca2+ levels are expressed in pseudocolor, the color key of which is shown at the right margin. Calcium spikes were synchronous throughout the entire SCN slice and coincided in time with burst firings in VGAT−/− SCNFull size imageVGAT deficiency produced calcium spikes in the fetal SCNGenetic deficiency of GABA in the SCN modulated circadian rhythms in neural firing, without affecting the circadian molecular oscillation (Fig. 1, Supplementary Fig. 5). Intracellular Ca2+ showed circadian rhythms33,34 and played an important role in the transmission of input and output signals to and from the cellular circadian oscillation in the SCN33,34,35,36. To investigate the effects of VGAT deficiency on circadian Ca2+ rhythms, hSyn-GCaMP6s, a fluorescent intracellular Ca2+ sensor was introduced into the cultured SCN slice carrying a PER2::LUC reporter using adeno-associated virus (AAV). The slice was set on an MED probe for simultaneous measurement of fluorescence (Ca2+), bioluminescence (PER2::LUC), and spontaneous firing.

Output: corroborate


Input: Claim: Cryptography can be used to make digital information entirely unhackable forever. [SEP] Text: 2 Related WorkOne may feel some similarity of the results in this paper to a famous result by Canetti, Goldreich, and Halevi [6] showing that there is a scheme involving a (keyless) hash function that is provably secure when the hash function is modeled as a random oracle but becomes insecure for any concrete implementation of the hash function. In some sense, both of the present paper and theirs reveal gaps between cryptography based on idealized frameworks (ideal randomness/ROM) and that based on real objects (PRGs/hash functions). We emphasize, however, that there exists the following difference between the two results; the “real objects” in [6] (hash functions) themselves do not have provable security, while the present paper shows that even provably secure “real objects” (PRGs) can cause insecurity in implementation, which may have stronger impact. (On the other hand, a point of the present paper weaker than theirs is that our result here shows the existence of at least one “problematic” real object, while [6] shows that any such real object is “problematic”. )We also note another related result by Hirose [18] that for any (keyless) hash function under a certain model of construction that is secure when an ideal block cipher is used in the construction, there exists a block cipher that is provably secure but by which the resulting hash function becomes insecure. This result also focused on insecurity caused by provably secure building blocks, but our result in this paper covers wider situations, not just hash functions. One may also feel that the topic of the present paper seems to be related to some other topics concerning non-ideal randomness in cryptography, such as cryptography based on so-called “imperfect randomness” (e. g. , [10, 12]) and the security issues caused by “backdoored PRGs” (e. g. , [8, 9]). But actually, the former topic above mainly deals with randomness that is significantly far from being ideal; in contrast, the present paper focuses on the use of randomness that is significantly close to ideal.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: Skip to Content eLife home page your research Search by keyword or author Reset form my search to Chromosomes and Research 10. 7554/eLife. 94573. 3 Open access Copyright information Version of RecordSeptember 16, 2024 Read the peer reviews Version of RecordAugust 9, 2024 Reviewed preprintv2 July 26, 2024 Reviewed preprintv1 March 22, 2024 Open annotations (there are currently 0 annotations on this page). 563 views 69 downloads 2 4573. Copy to clipboard Download BibTeX Download. RIS Full text Figures and data Peer review Side by side Peer review process #1 (Public Review): Author response Peer review process Version of Record: This is the final version of the 10. 7554/eLife. 94573. 3. sa1 Author response The following is the authors’ response to the original reviews. Responses to recommendations Reviewer #1 (The Authors): Describe more precisely how gene expression graphs are built (tissues, reads counts). For example, how were read counts normalized? Were they from DESeq2 data, which only works by comparing two samples? If so, all samples should be independently compared to a reference and the normalized expression value of the reference will change from sample to sample. . . thus introducing a pure technical artifact. We have added additional information about the normalisation method to the Material and Methods section (Lines 597-598: “Lastly, expression levels shown in figures 2-5 are normalised gene counts produced by DESeq2. ”) and figure legends (lines 247, 286, 372, 404: “Gene expression data was generated from whole fish. Expression levels were derived from DESeq2 normalised gene counts. ”) to address this recommendation. DESeq2 provides a reference independent normalisation through a median of ratios method a good explanation can be found here: The normalised expression values are independent of any reference, and therefore will not change from sample and sample as suggested in this comment. In contrast, the pairwise comparisons are done when analysing significantly differentially expressed genes between two treatments using a Wald test, which is done against a reference and generates log2 fold change information and p-values. ; however, this is different to the normalisation we described above. Provide bioinformatics workflows and, if possible, the set of parameters used, the computing resources, etc.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: slice showed robust circadian PER2::LUC, intracellular Ca2+, and spontaneous firing rhythms (Fig. 4a, c, 1) as previously reported33,37. Interestingly, intracellular Ca2+ in the VGAT−/− SCN occasionally showed a spike-like increase which was superimposed on the circadian Ca2+ rhythm, while PER2::LUC exhibited robust circadian rhythms similar to those of the WT SCN (Fig. 4d, 2). The circadian firing rhythms showed two distinct bands. Time-lapse Ca2+ imaging at 3 s intervals for 30 min revealed the calcium spikes in the VGAT−/− but not in the WT SCN (Fig. 4e, f, 3). In order to examine the temporal relationship between the burst firing and the calcium spike, Ca2+ imaging was performed with a higher time resolution at 100 ms intervals for 1 min (Fig. 4g, h, 4). The calcium spikes occurred synchronously throughout the entire SCN slice for several seconds (Fig. 4d, f, h). The membrane potential also showed burst firings throughout the slice in synchrony with calcium spikes (Fig. 4h). These findings indicate that lack of GABA signaling induces a spike-like increase in intracellular Ca2+ throughout the entire SCN slice in synchrony with burst firings. VGAT deletion in the SCN attenuates circadian behavioral rhythmsTo understand the role of GABA in the SCN in the output signal to behavior, mice with an SCN-specific deletion of VGAT were generated by injecting AAV9-hSyn-GFP-Cre into the SCN of VGATflox/flox mice (SCN-VGAT depleted mice). As control, AAV9-hSyn-hrGFP was injected into the SCN of VGATflox/flox mice. Three weeks later, mice were transferred from a light–dark (LD) cycle to constant darkness (DD). Circadian behavioral rhythms were persisted in SCN-VGAT depleted mice but the activity was disrupted under DD (Fig. 5a–e). The amount of behavioral activity in the dark period of the LD cycle was reduced in the SCN-VGAT depleted mice (Fig. 5d) and the subjective day–night difference in the activity was also reduced under DD (Fig. 5f).

Output: corroborate


Input: Claim: Once encrypted, data is impossible to recover, even by those who encrypted it. [SEP] Text: -19 PreprintsProtocolsVideosJournalsTools & a 10. 21203/rs. 3. rs-2722738/v1This work is licensed under a CC BY 4. 0 LicenseStatus:PostedVersion 1posted You are reading this latest preprint versionAbstractBackground: Accurate models are crucial to estimate the phenotypes from high throughput genomic data. While the genetic and phenotypic data are sensitive, secure models are essential to protect the private information. Therefore, construct an accurate and secure model is significant in secure inference of phenotypes. Methods: We propose a secure inference protocol on homomorphically encrypted genotype data with encrypted linear models. Firstly, scale the genotype data by feature importance with Xgboost or Adaboost then train linear models to predict the phenotypes in plaintext. Secondly, encrypt the model parameters and test data with CKKS scheme for secure inference. Thirdly, predict the phenotypes under CKKS homomorphically encryption computation. Finally, decrypt the encrypted predictions by client to compute the 1-NRMSE/AUC for model evaluation. Results: 5 phenotypes of 3000 samples with 20390 variants are used to validate the performance of the secure inference protocol. The protocol achieves 0. 9548, 0. 9639, 0. 9673 (1-NRMSE) for 3 continuous phenotypes and 0. 9943, 0. 99290 (AUC) for 2 category phenotypes in test data. Moreover, the protocol shows robust in 100 times of random sampling. Furthermore, the protocol achieves 0. 9725 (the average accuracy) in an encrypted test set with 198 samples, and it only takes 4. 32s for the overall inference. These help the protocol rank top one in the iDASH-2022 track2 challenge. Conclusion: We propose an accurate and secure protocol to predict the phenotype from genotype and it takes seconds to obtain hundreds of predictions for all phenotypes. Homomorphically EncryptionGenotype to -2022FiguresFigure 1Figure 2Figure 3IntroductionThe research on genotype-to-phenotype are crucial to uncover the gene functions and the mechanisms in distinct phenotypic outcomes [1]. The high throughput genomic data makes it may be possible to predict the phenotype from genotype. While the inference of genotype to phenotype is a complex problem due to the intricate factors such as genotypes, epigenetic variants and their interactions [2].

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: Were some assembly finishing steps carried out (by long-range PCR? ) and experimental validations (especially for allelespecific transcripts, by conventional RT-PCR based on diagnostic mutations)? We have added additional information on the bioinformatics workflows where required, including parameters used (Lines 530, 536, 549-551, and 574-583. ). No finishing steps other than HiC scaffolding were performed. No allele-specific analysis was done as part of this manuscript. To further improve transparency, we have also uploaded all the scripts used for this study to and the gene models and functional annotation to annotation/199909. This information has been added to the manuscript in lines 600601 and 609-611. Reviewer #3 (The Authors): General author response: All the recommendations of this reviewer are very relevant and would certainly provide a lot of information, but they are constituting a full project in themselves as they would imply establishing this grouper species as an experimental model in our lab. Currently we only have access to the larval and juvenile stages via a collaboration with the Center, which is an hour drive from our lab, and is limited to the grouper spawning season. If we want to do all what is suggested, we need to have a regular and easy access to the fishes. This would require establishing this model in our marine station, which is not possible due to space and time issues. These groupers grow to a very large size (1-2 m in length, and up to 150 kg in weight) and only mature into males after > 6 years. First and foremost, I would advise the authors to extend their TH and cortisol levels measurements to the entire developmental time considered in their analysis. For the reasons stated above we could not perform these experiments. We must emphasize that the data regarding TH are available for a closely related species (e. g. , Epinephelus coioides, de Jesus et al. 1998) and there is no reason to think that the situation will be drastically different in E. malabaricus. In addition, given that we have now studied several coral reef fish species in the same context (clownfish, surgeonfish, damselfish, gobies) we observed that the transcriptomic data are more robust, more sensitive, and more precise than hormone measurements.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: Although the resulting phase response curves are different, a dark pulse type in behavioral rhythms versus a light pulse type in dispersed SCN neurons, GABA is possibly involved in the neural system of rhythm entrainment52. The present finding was not contradictory to the above hypothesis, because the burst firing due to GABA deficiency covered all circadian phases, which could cancel out the phase-shifting effects of GABA, if any. The present findings support the idea of differential neural networks for the regulation of circadian oscillation in the SCN21. GABA is a major inhibitory transmitter in the central nervous system but also an excitatory transmitter depending on the developmental stage53 and on the areas of brain. According to a current agreement54, the activation of GABAA receptor opens chloride (Cl−) channels on the cell membrane to allow inward flow of Cl− resulting in hyperpolarization (inhibitory action), when the Cl− equilibrium potential level is lower than the resting membrane potential. The opposite (excitatory) is the case, when the Cl− equilibrium potential level is higher than the resting potential. In the SCN, the switch from inhibitory action to excitatory and vice versa was reported to occur depending on the circadian phase and the area in the SCN18,20. We demonstrated previously that GABA action was changed alternatively in some synchronous SCN neurons in which GABA was excitatory at the circadian peak and inhibitory at the circadian trough12. In the present study, GABA application to the fetal SCN slice of VGAT−/− mice exerted tonic inhibition and GABA receptor antagonists to the WT SCN exerted tonic excitation, both of which did not interfere with the circadian molecular rhythm. It is not known whether the burst firing occurs in the adult SCN lacking GABA signaling. The perinatal and adult circadian pacemakers differ in several aspects. The SCN circadian system is immature in the perinatal period with respect to light entrainment and oscillatory coupling11,55. Before neural pruning which occurs around the postnatal day 2–556, the number of neurons is more abundant but the synaptic connections are less abundant in the perinatal SCN than in the adult56. The perinatal SCN entrains not to light cycles but to maternal circadian rhythms57.

Output: corroborate


Input: Claim: Once encrypted, data is impossible to recover, even by those who encrypted it. [SEP] Text: In [1] the problem of filtering encrypted data upon traffic monitoring which is outbound from a computer or a network, so-called egress filtering, is considered. Many aspects and properties of the problem are brought to attention and an extensive account for various evaluation techniques is mentioned. Different measures for discriminating a given distribution from the normal distribution are the topic in [2]. For discrimination between encrypted and non-encrypted data other distributions than the normal are more relevant but some of the measures considered in this paper can also be used for the more general case. In [3] machine learning methods are deployed to a fully automated method to the distinction between encrypted and compressed data, claimed to be the first of its kind. A dataset for further evaluation and benchmarking is also provided. A method for discriminating between compressed and encrypted data in the case when the data is voice over IP with constant and variable bit rate codecs is proposed in [4]. The method is evaluated utilizing the NIST [5] and the ENT [6] test suits. Reference [7] does not suggest a solution to the discrimination problem but rather a method for fast and distribution true simulation of data reflecting the properties of encrypted and compressed data respectively. In Ref. [8] a classification procedure, based on Gaussian mixtures and hidden Markov models, is detailed. An elaborate comparative evaluation is carried out taking 9 other attempts to solve the same problem into account. The study concludes that the proposed method renders a better classification at a lower computation complexity. A lucid convolution neural network method to classify data into being encrypted or non-encrypted is presented by [9]. The proposed method is thoroughly evaluated for various kinds of network traffic and some different performance measures in the field of machine learning. 1. 2 Methods for discrimination in case of static dataA different need for discrimination between encrypted and unencrypted data is the following. In police investigations concerning heavy criminality and organized crime where evidence of criminal activity is residing as data on a computer hard disk drive (HDD) the capability to distinguish encrypted files from non-encrypted ones may be primordial [10, 11, 12]. When the police seize an HDD containing data belonging to a suspected criminal, that data can be material of evidence in a subsequent trial. But criminals often try to make sure that the police will be able to use that data. Possible actions to obstruct data access are then to encrypt and/or to quick delete that data.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: Consider carrying out in situ hybridisation of TSH with putative CRH receptors to determine if thyrotrophin could be competent to respond to HPA axis signals. We agree studying the interplay between corticoids and thyroid hormones at the neuroendocrine level would be desirable and we fully agree with the experiment suggested by the reviewer, but this is impossible in our current situation. We are not working with an establish animal model like zebrafish or Xenopus, but with a large, long-lived marine fish that reproduces in spawning aggregations and whose husbandry is notoriously difficult. Consider conducting cortisol treatment experiments to functionally determine if indeed cortisol is involved in grouper metamorphosis. We tried to do TH and cortisol treatments specifically on the early larval stages corresponding to the early TH peak to see how this would impact the development of the fin spines, but our trials were unsuccessful. The larvae at that stage are extremely fragile and even putting them into small volumes of treatment drugs induced massive mortalities. Again, this would mean establishing this grouper species as a model organism and would require a massive effort to improve larval rearing as discussed above. We feel that our data stands on its own in the meantime and adds valuable information to the existing literature by studying a rarely investigated species. Responses to comments Reviewer #1 (Public Review): Weaknesses: The manuscript needs proper editing and is not complete. Some wordings lack precision and make it difficult to follow e. g. line 98 "we assembled a chromosome-scale genome of. . . " should read instead "we assembled a chromsome-scla genome sequence of. . . ". Also, panel Figure 2E is missing. We made the suggested change of adding “sequence” in lines 32 and 121. Concerning additional changes, we have carefully edited our manuscript and looked for any incomplete sections. Unfortunately, it is difficult to see what other issues are being raised here without any further information. As for panel E of figure 2, it is not missing. The panel is located to the right, just below “Target Cells”. The shortcomings of the manuscripts are not limited to the writing style, and important technical and technological information is missing or not clear enough, thereby preventing a proper evaluation of the resolution of the genomic resources provided: Several RNASeq libraries from different tissues have been built to help annotate the genome and identify transcribed regions. This is fine.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: VIP, an important neuropeptide for coherent circadian rhythms in the SCN, shows an endogenous circadian rhythm in the neonatal SCN but not in the adult, indicating that VIP is regulated by the circadian pacemaker more strongly in the perinatal period than in the adulthood11. Recently, the neural network for the coupling of cellular circadian oscillators is suggested to be different among the embryonic, neonatal, and adult SCN11,55,58,59,60,61. However, the developmental change in the GABA system remains to be studied. The mechanism of burst firing generation has not been elucidated62. Burst firings are general phenomena in the central nervous system outside the SCN under physiological conditions63. In contrast, the WT SCN slice lacks burst firings. The intact SCN neurons in a dispersed cell culture showed more noisy firing rhythms superimposed by fast oscillation at 1–2 min intervals40 than in a slice culture with sinusoidal firing rhythm3,6,23. These features have similarities to the burst firings in the SCN slice lacking GABA signaling. Neural communication becomes less potent as the distances among the SCN neurons get larger in dispersed cell cultures12. Similar to dispersed neurons, the SCN neural network may attenuate its potency to communicate with other neurons with GABA signaling and becomes noisy. Burst firings could be an intrinsic nature of neurons or neural networks, and GABA in the SCN, at least in the perinatal SCN where the neural networks are immature, erases noises from the circadian signals. The firing rates in the cultured SCN increased during culture, suggesting an increase in neural connections (Fig. 1a, Supplementary Fig. 4a). The burst firing was detected from the very beginning of SCN culture and the difference in burst and non-burst firings (difference between two bands) did not change throughout the culture period, indicating that changes in neural networks during culture do not contribute to the appearance of burst firings. The burst firing was associated with an abrupt increase in intracellular Ca2+ (Fig. 4h). In presynaptic neurons, GABA release is induced by an influx of Ca2+ through voltage-gated ion channels. The firing rate determines the amount of Ca2+ influx, altering the intracellular Ca2+.

Output: corroborate


Input: Claim: Once encrypted, data is impossible to recover, even by those who encrypted it. [SEP] Text: In case of quick delete of data, the pointers to the files are destroyed but the content of the files is still left. The other action is to encrypt files. Using state of the art tools for encryption of files (such as VeraCrypt, Bitlocker, Ciphershed and thereby provided ciphers) there is no general procedure of breaking the encryption other than brute force attack by guessing the cipher algorithm and systematically guessing the encryption key [13]. If some of the files are encrypted and others not, it is then a delicate matter to distinguish between the two if the code has been quick deleted. Nevertheless, the importance of discriminating between encrypted code and plaintext is also stressed by the fact that brute force attacking all clusters of data on the hard drive would be an impossible task while being able to separate the encrypted code from the non-encrypted would make a substantial improvement of the chances for successful cryptanalysis. The police authorities usually have software to brute force those encrypted data but this procedure may be very time consuming if the amount of data is so large that different parts of it have been encrypted with different keys. In juridical cases, time is typically of the essence since the chances of success in prosecution and proceedings against a criminal is depending on deadlines (such as time of arrest, time of trial etc) any time savings in the procedure of extracting evidence from the HDDs is essential. Thus time has to be spent on the appropriate tasks: code-breaking only on the encrypted data rather than trying to decypher non-encrypted data. Given a single file, the task of determining whether it is encrypted or not is usually easy; but given a whole HDD without knowing where the encrypted files are, this is trickier. There are several software solutions for indicating whether a file is encrypted or not, mostly checking the header of the file and looking for some known pattern like the EFS (System on Windows), BestCrypt, or other softwares’ headers. Such alternatives cannot be used in the case when the user has performed a (quick) delete of the HDD because then the pointers to all files are lost. Nevertheless, the files remain on the HDD: upon deleting a file on an HDD, the pointers to the beginning and end of the physical space on the HDD containing the information of the file are removed. Still, the physical space on the HDD containing the information of the file is not overwritten because that operation would be slow, i. e.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: But all along the manuscript, gene expression changes are summarized into a single panel where it is not clear at all which tissue this comes from (whole embryo or a specific tissue), or whether it is a cumulative expression level computed across several tissues (and how it was computed) etc. This is essential information needed for data interpretation. No fertilised eggs or embryos have been sequenced. The individual tissues derived from juvenile fish were used for the genome annotation only, using ISOseq. The whole larval fish were used for the developmental analysis using RNAseq, as well as the genome annotation. We have added additional information in the figures and text that the results shown are from whole larvae, and added more detail to the material and methods section about which type of sample was analysed in which way. Specifically, we have added “Lastly, expression levels shown in figures 2-5 are normalised gene counts produced by DESeq2. ” to lines 597-598 in the Material and Methods section, “Gene expression data was generated from whole larvae. ” to line 191, and “Gene expression data was generated from whole fish. Expression levels were derived from DESeq2 normalised gene counts. ” to the figure legends in lines 247, 286, 372, 404. Additionally, we have added clarifications in lines 489, 497, 530, and 536. The bioinformatic processing, especially of the assemble and annotation, is very poorly described. This is also a sensitive topic, as illustrated by the numerous "assemblathon" and "annotathon" initiatives to evaluate tools and workflows. Importantly, providing configuration files and in-depth description of workflows and parameter settings is highly recommended. This can be made available through data store services and documents even benefit from DOIs. This provides others with more information to evaluate the resolution of this work. No doubt that it is well done,but especially in the field of genome assembly and annotation, high resolution is VERY cost and time-intensive. Not surprisingly, most projects are conditioned by trade-offs between cost, time, and labor. The authors should provide others with the information needed to evaluate this. We have added additional information on parameters used in the genome assembly, annotation and transcriptome analysis in lines 549-551, 577, 579, 580, and 582.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: overt expression of circadian rhythms is a manifestation of the suprachiasmatic nucleus (SCN). This integrated complex function based on the transcriptional/translational feedback loops (TFFLs), neurotransmitters, genes, networking, and synchronization is essential for this molecular mechanism to operate effectively. Neurotransmitters by participating in the entrainment to the environmental light conditions and synchronization contribute to the robustness of the rhythm. Neurotransmitter signaling is the hallmark of circadian rhythm expression. Even during development, neuropeptides contribute to the dramatic cellular, genetic, and network circuit changes. Participating neurotransmitters are seen in afferent inputs, efferent output, and the SCN. There are numerous neurotransmitters involved in SCN function. Astrocytes co-exist with neurons in the SCN. Autonomous clocks seen in astrocytes can drive circadian behavior like neurons. Astrocytes and neurons are acting as two arms of the clock. Coupling through glutamate released from astrocytes gives additional evidence for the role of astrocytes. Glutaminergic signaling from astrocytes may also be responsible for timekeeping. The neurotransmitters can independently and in combination execute the functions making SCN a unique pacemaker for the overt expression of circadian rhythms. This reassessment also highlights its role in underlying molecular mechanisms, genetic linkage, and the recently known role of astrocytes. suprachiasmatic nucleus, neurotransmitters, astrocyte, neurotransmitter signaling, genetic linkage IntroductionNeurotransmitters, neuropeptides, and neuromodulators in a broader sense synthesized by the suprachiasmatic nucleus (SCN) neurons participate in the coordination of endogenous oscillators and entrainment of the SCN. Neuronal diversity along with diverse neurotransmitters and their abundance in the adult SCN makes the study of neurotransmitter function complex. Our timekeeping system focused on the SCN requires a multitude of peptide hormones and neurotransmitters. Hundreds of neuropeptides and small molecules like gamma-aminobutyric acid (GABA), nitric oxide (NO), and others are present in the SCN [1]. An estimate shows that among the heterogeneous population of neurons of about 20,000, they secrete more than 100 neurotransmitters, neuropeptides, cytokines, and growth factors [2].

Output: corroborate


Input: Claim: Once encrypted, data is impossible to recover, even by those who encrypted it. [SEP] Text: as slow as writing the same file again. The operating system’s designers rather leave the file in the HDD intact but indicate that this location is free to host some other data. If nothing has been overwritten, this means that the file is still stored in the HDD for some time which allows recovery software to recover those files. Recovery software might help to simply recover the files but might also overwrite the data contained in HDD which could result in loss of evidence in case of an investigation. In this case, the police would have to locate the encrypted files without using recovery software or “header-detector” software. Here methods to locate quick deleted encrypted data are presented and detailed. First, a description of how encrypted data is different from other data is presented. This is followed by the introduction to statistical change-point methods for discrimination between encrypted and non-encrypted data. Finally, the results of these procedures are presented along with some experimental values to evaluate the methods. However, such a method will only work on mechanical HDDs and not with flash memory devices: in flash memory (like USB memory sticks or Drive (SSD)), as soon as a file is removed it is erased from the memory because data cannot be overwritten. Therefore as soon as data is deleted, the operating system will choose to delete the pointers and the data to gain time for the time when the user will decide to save data at this location. But erasing in flash memory also takes longer since the pointer have to be deleted and all the files removed which is as long as copying new files on the device. This application differs from the one mentioned in Subsection 1. 1. In the previous case when data is commonly considered being transmitted in packets in a network while here, there is static access to the data. In the previous situation, data consists of sometimes small files where statistical methods for making a foundation indicating if the data is encrypted or not becomes weaker [14] as opposed to the situation here where there is a large amount of data possibly of both kinds, encrypted and non-encrypted. In the previous case with dynamic data in all senses—variable in size, access, time, kind—methods of machine learning which could pick up on the current circumstances were preferable while here, in the case of fixed data, fast and efficient methods of change-point detection becomes a far more advantageous choice.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: Additionally, we have uploaded all scripts to github as outlined in the Code and section (lines 599-614). The genome assembly did not use a specific workflow (e. g. , nextflow), but was done with a simple command and standard parameters in IPA. Scaffolding was carried out by using their standardised proprietary workflow, of which a detailed description provided by can be found in the supplementary material. Quantifications of T3 and T4 levels look fairly low and not so convincing. The work would clearly benefit from a discussion about why the signal is so low and what are the current technological limitations of these quantifications. This would really help (general) readers. The T3/T4 levels are consistent with other published work in fish. In the present manuscript for grouper we have a peak level of 1. 2 ng/g (1,200 pg/g) of T4 and 0. 06 ng/g (60 pg/g) of T3. This is a higher level of T4 and comparable level of T3 to what was found in convict tang (Holzer et al. 2017; Figure 2) with 30 pg/g of T4 and 100 pg/g of T3. Of course, there are also examples with higher levels, such as clownfish (Roux et al. 2023; Figure 1), with 10 ng/g (10,000 pg/g) of T4 and 2 ng/g (2,000 pg/g) of T3. The differences could be due to different structure of fish tissues and therefore different hormone extraction efficiency, different hormone measurement protocols, different fish physiology, different fish size (e. g. , the weighting of tiny grouper larvae is difficult and less precise than in convict tang). What is important is not the absolute level but the relative level, which shows the change within different larval stages of a species with identical extraction and measurement protocols. Which means our data is internally consistent and coherent with what the grouper literature says. Holzer, Guillaume, et al. "Fish larval recruitment to reefs is a thyroid hormonemediated metamorphosis sensitive to the pesticide chlorpyrifos. " Elife 6 (2017): e27595. Roux, Natacha, et al. "The multi-level regulation of clownfish metamorphosis by thyroid hormones. " 42. 7 (2023).

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: Studies have also shown five different classes of neurons in the SCN based on the co-expression of various neuropeptides [3]. Another report [4] has identified eight major cell types with each group having a pattern of circadian gene expression. Based on connections, chemo architecture, anatomically and functionally distinct regions of the core that lie adjacent to optic chiasma and shell surrounding the core, are demarcated in the SCN, although the demarcation of core and shell is hardly clear-cut [5]. Studies have demonstrated [6] that core synthesizes GABA, calbindin (CalB), vasoactive intestinal peptide (VIP), calretinin (CALR), gastrin releasing peptide (GRP), neurotensin (NT) while arginine vasopressin (AVP) expressing neurons are seen in the shell. Others in the shell include angiotensin II (ANGII), met-enkephalin (m ENK), and galanin (Gal). The core receives dense visual and midbrain raphe afferents, and the shell has more non-visual cortical and subcortical inputs. It is seen that mouse SCN and its connections are observed in hamsters, rats, and humans [5, 6]. An estimate in rats has put 37% AVP, 24% and GRP each 14%, while others NT, ENK, somatostatin (SSA), and substance P (SP) constitute less than 5% each [7]. It is remarkable that a relatively small area of the brain, SCN, with a small number of neurons and one-third as many astroglia can synchronize circadian rhythms in the body and guide so many rhythms. A probable answer to this lies in the fact that genes within cells and cells in circuits work together coherently to carry out complex functions to enable normal body function. The intimate linkage of genes and neurotransmitters observed in the molecular basis of the working of the clock also multiplies the relevance of neurotransmitters in SCN function. The very fact that the in Medicine and Physiology for the year 2017 was awarded to three pioneers working in the molecular mechanisms in the circadian field speaks volumes about the research going on in the field and the fertile nature of this ongoing research area. The complex heterogenous structure with histological diversity underlies the functional specificity of the SCN.

Output: corroborate


Input: Claim: Once encrypted, data is impossible to recover, even by those who encrypted it. [SEP] Text: The machine learning methods need training data while the change-point methods can start from scratch with pre-defined parameter values optimized for the situation or with very little run-in data for calibrating the levels. The performance of the machine learning methods is still a matter of research since these are very highly structured procedures, sometimes black box techniques impossible to fully evaluate all properties of, while the change-point detection methods are long since optimized and very thoroughly evaluated from all kinds of aspects of performance through a much longer time. Advertisement 2. Method2. 1 DescriptionIf encrypted data is not uniformly distributed, the cryptosystem used to cipher those data has a bias and can therefore be attacked. For this reason, characters of the cyphertext produced by any modern high-quality cryptosystem are uniformly distributed [15, 16] i. e. the values of the bytes of the cyphertext are uniformly distributed on some character interval. Indeed, a cryptosystem that does not have this property would be weak since it would be possible to attack it on this bias (distinguishing attacks such as on the RC4 encryption algorithm). The other types of files do not possess this feature although the contents of some types of files are close to being uniformly distributed. The files coming closest without being encrypted are compressed/zipped files: those files are indeed very close to cipher files in terms of the distribution of their character’s byte numbers. Albeit small there is a difference in distribution making it possible to tell compressed files and encrypted files apart. A technique to quantify this distribution difference is by using chi-square statistic and more or less performing a chi-square test (see [17]) to tell whether the data is suspiciously uniform or not. Another classical method is to calculate the Kolmogorov-Smirnov distance, see e. g. [1, 2] for a more extensive account of ways to indicate whether data are encrypted or not. Procedures building on such preprocessors can then be defined. One attempt to exploit this distribution difference utilizing the chi-square statistic is [18] where a method to automate the discrimination between encrypted and non-encrypted (i. e. most critically compressed) data into a method with impressing performance. Another approach could be to use means of anomaly detection in the theory of machine learning to develop an adaptive method. The proposed method, however, stems from using statistical change-point detection.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: Differential analysis highlights up to ~ 15,000 differentially expressed genes (DEG), out of a predicted 26k genes. This corresponds to more than half of all genes. ANOVA-based differential analysis relies on the simple fact that only a minority of genes are DEG. Having >50% DEG is well beyond the validity of the method. This should be addressed, or at least discussed. The large number of differentially expressed genes is due to the fact that this is coming from a larval developmental transcriptome going from one day old larva to fully metamorphosed juveniles at around day 60. While DESeq2 indeed works on an assumption that most genes are not differentially expressed, this affects normalization but not hypothesis testing (Wald-test, LRT tests or ANOVA). However, normalisation in DESeq2 is fairly robust to this assumption. According to the author of DESeq2, Micheal Love, DESeq2 is using the median ratio for normalisation, and as long as the number of up and down regulated genes is relatively even, DESeq2 will be able to handle the data. As part of our general quality control for this project we consulted the MA plots, which do not show any overrepresented up or down expression patterns. Additionally see comment on comparing different tissues, which is also applicable here when comparing vastly different larval stages ( “For experiments where all genes increase in expression across conditions, the median ratio method will not be able to capture this difference, but this is typically not the case for a tissue comparison, as there are many "housekeeping" genes with relatively similar expression pattern across tissues. ” Reviewer #3 (Public Review): Weaknesses: However, the authors make substantial considerations that are not proven by experimental or functional data. In fact, this is a descriptive study that does not provide any functional evidence to support the claims made. We agree with the reviewer that our paper lacks functional experiments but despite that, the transcriptomic data clearly show the activation of TH and corticoid pathways during two distinct periods: an early activation between D1 and D10, and a second one between D32 and juvenile stage. These data are interesting as they call for further examination of (1) the existence of an early larval developmental step also involving TH and corticosteroids and (2) the possible interaction of corticoids and TH during metamorphosis.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: Electrically, chemically, and peptidergically, the heterogenous nature of the neurons is a fundamental feature of the SCN and these neurons individually generate circadian oscillations [8, 9]. An internal molecular oscillating clock based on transcriptional/translational feedback loops (TTFLs) of clock genes [10, 11] drives circadian rhythms in electrical activity and is more active during the day than at night [12]. This landscape nature regarding excitability within the SCN is being reinforced by external signals from the retina [13]. Genetic studies indicate autoregulatory feedback loops form the basis of circadian rhythms not only in the SCN but also in extra SCN oscillators [14]. TTFL generates noisy oscillations of gene expression and firing rates [15] of nearly all neurons because of the orchestrated coupled network of these neurons. Synchronization among the cells [16] with high precision [17] produced by the coupling mechanism is attributed to a variety of neuropeptides mainly VIP and AVP [18–21]. SCN generates rhythms of firing rate by adjusting light inputs through the retinohypothalamic tract (RHT) [22]. The connectome of the SCN along with afferent inputs and neural and humoral outputs are considered important aspects of the synchronization process of the SCN to signal time to the rest of the body [23]. Neurotransmitters are classified based on various parameters but no rigid classification is possible as parameters can be overlapping. An immunocytochemical analysis to identify and locate 25 neurotransmitters in the SCN and its projections has been reported. Many reviews have been cited [24–29] in the literature highlighting various neurotransmitters and their function. The purpose of this review is to revisit the neurotransmitters since there are new additions to the list of neurotransmitters, neurotransmitter-based changes in the molecular mechanism of the clock working, and advancement in the knowledge regarding genetically linked neurotransmitter working. Not only neurons but astrocytes are also reported to have circadian clocks and molecular mechanisms like the one seen in neurons. Neurotransmitters are secreted by the glia as well and are important for the integrated clock function. Hence a discussion on the role of astrocytes is also in order. The review is divided into two parts: part 1 is devoted to SCN, connections, input and output, receptors, and neurotransmitters present.

Output: corroborate


Input: Claim: Once encrypted, data is impossible to recover, even by those who encrypted it. [SEP] Text: But the larger the number of bytes that are in each cluster, the worse the precision to detect encrypted file values: indeed, if too many bytes were detected as being unencrypted (but were actually encrypted) a large amount of encrypted data might not be detected in the procedure. Here, the alphabet used was the numbers 0,1,…,255 representing the possible size in bytes of the characters in the data. These numbers were divided into K=8 classes (class 1: values of bytes in 0,1,2…31 to class 8: values of bytes in 224,225,226…255) and the clusters of size N=64 bytes making the expected values in each class Ekt=8. Assuming that encrypted data are uniformly distributed, the Q scores based on counts of characters in encrypted data are χ2 distributed, see Figure 1 and since 8 classes were chosen, the number of degrees of freedom is 8−1=7. Figure 1. Distribution of the Q scores of encrypted files (obtained by using more than 5000 files) with the distribution function of Q∈χ27. 2. 3 Distribution of non-encrypted dataFor non-encrypted data, the distribution is more complicated. Each type of file has its distribution. Consequently, the standardized squared deviances from expected counts under the assumption about uniform distribution are larger and so are the Q scores of the χ2 statistic. However, two problems emerge. Firstly, the size of these increased deviances depends on the type of data—i. e. is the data a text file, an image, a compiled program, a compressed file or some other kind of file? —and how should this information be properly taken into account? Secondly, what is the distribution of the Q score in the case when the data are not encrypted? To develop a method for distinguishing between encrypted and non-encrypted data it is sufficient to focus on the non-encrypted that is most similar to the encrypted and this turns out to be compressed data. Other types of files such as images, compiled programs etc. commonly render higher Q scores and are therefore indirectly distinguished from encrypted data by a method that is calibrated for discriminating between encrypted and compressed data. About the second question, this is not readily answered. Rather, we just suggest modeling the Q score as being scaled χ2 distributed, i. e.

Output: contrast


Input: Claim: Some insects can undergo complete metamorphosis, transitioning through larval stages. [SEP] Text: This is a question that is certainly not settled yet in teleost fishes and which is of great interest. Especially (1) is of interest and importance, since this early activation (unique to our knowledge in any teleost fish studied so far) raises a lot of new questions and once again will certainly be scrutinised by other groups in the years to come, therefore ensuring a good citation impact of this study. We hope that the reviewer, while disagreeing with some our statements, will recognize that our study will be stimulating at that level and that this is what scientific studies should do. We acknowledge the descriptive nature of the data and the lack of functional experiments in the Discussion in lines 443 to 445: “This may suggest that in some aspect, cortisol synthesis could work in concert with TH, as has been shown in several different contexts in amphibians, but functional experiments need to be conducted to confirm this hypothesis. ” As stated above doing such functional experiment would require establishing the grouper as an experimental model in our husbandry, which currently is not possible due to the large size of the adult fish. The consideration that cortisol is involved in metamorphosis in teleosts has never been shown, and the only example cited by the authors (REF 20) clearly states that cortisol alone does not induce flatfish metamorphosis. In that work, the authors clearly state that in vivo cortisol treatment had no synergistic effect with TH in inducing metamorphosis. Moreover, in Senegalensis, the sole pre-otic CRH neuron number decreases during metamorphosis, further arguing that, at least in flatfish, cortisol is not involved in flatfish metamorphosis (PMID: 25575457). We will do our best to improve the clarity of the revised manuscript to avoid any misunderstanding about our claims. However, we would like to point out the semantic shift in the reviewer first sentence: Indeed “being involved” is not the same as “cortisol alone does not induce”. In ref 20 the authors explicitly wrote that “Cortisol further enhanced the effects of both T4 and T3, but was ineffective in the absence of thyroid hormones” and in our view this indeed corresponds to ”being involved in metamorphosis”. We are not claiming that cortisol alone is involved in metamorphosis as the reviewer suggests, but simply that there is a possible involvement of cortisol together with TH in metamorphosis.

Output: pass


Input: Claim: The suprachiasmatic nucleus is the master regulator of circadian rhythms. [SEP] Text: Neurotransmitters and their probable locationNeurotransmittersAfferent inputSCNEfferent outputArginine vasopressin (AVP)++Vasoactive intestinal peptide (VIP)+++gamma-aminobutyric acid (GABA)+++Gastrin releasing peptide (GRP)+Pituitary adenylate cyclase-activating peptide (PACAP)+Dopamine+Glycine++Glutamate++Serotonin (5HT)+Neuropeptide Y+Acetylcholine+Cholecystokinin+Melatonin+Melanopsin++Prokineticin 2++Orexin+Pigment dispersing factor (PDF)+Nitric oxide (NO)+Histamine+Neurotensin (NT)+Met-enkephalin (m ENK)+Substance P++(NMS)+Somatostatin (SSA)++Calbindin (CalB)++Calretinin (CALR)++Angiotensin II (ANGII)++Galanin (Gal)++Oxytocin (OX)+Display full sizeSCN: suprachiasmatic nucleus; +: presence of the neurotransmitterIt is reported [26] that a wide range of neuropeptides and neuropeptide receptors are present in the neurons of the SCN for the execution of its function. These neuropeptide receptors mediate input from extra SCN afferent pathways as well as intra-SCN networks. Various signaling pathways are identified within SCN. As regards the action of neurotransmitters and neuropeptides, it is known that they activate receptors and ion channels on the plasma membrane to produce intracellular signaling events. This includes the activation of guanine nucleotide (G) protein-coupled receptors (GPCR), G protein-gated ion channels, ligand-gated ion channels, voltage-gated ion channels, ionotropic receptors, and tyrosine kinase receptors (Trk) [32]. Trk receptor belongs to a family of tyrosine kinases that regulate synaptic strength and plasticity in the mammalian nervous system. Neurotransmitters in afferent and efferent pathwaysNeurotransmitters are seen in the afferent inputs to the SCN, efferent output, and in the clock itself.

Output: corroborate