In this study, we aimed to understand the characteristics of the gut microbial composition in a healthy Chinese population and to evaluate if they differed across different regions. In addition, we aimed to understand the changes in the gut microbial composition over time. We collected 239 fecal samples from healthy Chinese adults living in four regions and performed a 1-year time cohort study in a small population in Beijing. The Chinese gut microbiota share 34 core bacterial genera and 39 core bacterial species, which exist in all collected samples. Several disease-related microorganisms (DRMs), virulence factors, and antibiotic resistance genes were found in one or more healthy Chinese samples. Differences in gut microbiota were observed in samples from different regions, locations, individuals, and time points. Compared to other factors, time was associated with a lower degree of change in the gut microbiota. Our findings revealed spatial and temporal changes in the gut microbiota of healthy Chinese individuals. Compared to fecal microbiomes of 152 samples in the publicly released the Human Microbiome Project (HMP) project from the United States, samples in this study have higher variability in the fecal microbiome, with higher richness, Shannon diversity indices, and Pielou evenness indexes, at both the genus and species levels. The microbiota data obtained in this study will provide a detailed basis for further understanding the composition of the gut microbiota in the healthy Chinese population.IMPORTANCE China accounts for approximately 1/5th of the world’s total population. Differences in environment, ethnicity, and living habits could impart unique features to the structure of the gut microbiota of Chinese individuals. In 2016, we started to investigate healthy Chinese people and their gut microbiomes. Phase I results for 16S rRNA amplicons have been released. However, owing to the limitations of 16S rRNA amplicon sequencing, the gut microbiome of a healthy Chinese population could not be examined thoroughly at the species level, and the detailed changes in the gut microbiota over time need to be investigated. To address these knowledge gaps, we started a phase II study and investigated the basis for variations in the gut microbiome composition in a healthy Chinese population at the species level using shotgun metagenomics technology. In the phase II study, we also conducted a time scale analysis of fecal samples from healthy Chinese subjects, as a pioneered study, which quantitatively clarified the changes in the gut microbiota at both the spatial and temporal levels and elucidated the distribution pattern of DRMs in healthy Chinese individuals. Population-scale studies of the human microbiome, especially the gut microbiome, such as the Metagenomics of the Human Intestinal Tract (MetaHit) , the NIHth of the world’s total population. Differences in environment, ethnicity, and living habits could impart unique features to the structure of the gut microbiota of Chinese individuals. Since 2016, the Chinese Pathogen Identification Net, which is a laboratory-based surveillance and early warning network for bacterial infectious diseases, has investigated healthy Chinese people and their gut microbiomes (Chinese Microbiome Project [CMP]) through epidemiological surveys in multiple regions, fecal sampling from healthy people, and examination of the microbiome using sequencing and bioinformatics technology. Phase I CMP results for 16S rRNA amplicon sequencing have been released  in healthy Chinese individuals.From 2016 to 2019, we pretested and examined 850 human fecal samples from individuals living in four regions in China . DetaileThe volunteers were selected from four geographical regions . The average age of the volunteers was 28.6 years . The male:female ratio was 1:1.37. The mean body mass index (BMI) of the participants was 21.75. Detailed information about the CMP data set is listed in Table S1.(i) Taxonomy. Using the bioinformatics pipeline described in Materials and Methods, we identified 584 bacterial and viral genera in the CMP data set and found some common characteristics in this healthy Chinese population. First, Bacteroides organisms were present in all samples (100%), with a corresponding high stability . The high percentage of Bacteroides in the Chinese gut samples was supported by the reports of previous studies using 16S rRNA amplicon sequencing. Second, 34 bacterial genera were identified as the core genera because they are present in all samples .(ii) DRMs and virulence factors. In this study, we investigated the distribution of 155 disease-related microorganisms (DRMs) in healthy individuals at the species level based on the “List of Human Pathogenic Microorganisms”  (Table S2). As conditionally pathogenic bacteria, 45 DRMs (29%) were found in one or more healthy samples (Table S2). For example, Enterobacter hormaechei was positive in 16.2% samples from China, Klebsiella pneumoniae, in 46.0%, and Salmonella enterica, in 22.7%. A total of 110 DRMs  were not found in any of the samples; this includes Brucella ovis and Chlamydia trachomatis, which are clearly related to infectious diseases and are human health risk markers. This is in line with the results obtained using 16S rRNA amplicon sequencing  ARGs. We also investigated the resistome  in the gut samples of the CMP data set at the gene level. Among 3,109 antibiotic resistance genes (ARGs) in the ResFinder database (https://cge.food.dtu.dk/services/ResFinder/), 2620 ARGs (84.3%) were not found in any of the healthy samples; these included NDM-1 and its 23 subtypes, which could be used as human health risk markers. A total of 489 ARGs were found in the CMP data set. For example, the aminoglycoside resistance gene (aph2-If_2_AY701528) is common in samples from China ; this supports the hypothesis that several ARGs have reached normal community transmission in healthy populations and are stable in the gut microbiome. The distinct profiles and our interest in functions related to resistance resulted in the exploration of the resistome in greater detail at the drug class level. We manually curated these 489 genes into 17 drug groups. The tetracycline class was the most abundant ARG in the human gut microbiome . Several ARGs could be identified in bacteria of multiple phyla; for example, the aminoglycoside resistance gene (aph2-If_2_AY701528) could be found in bacteria of Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria. However, ARGs related to fosfomycin resistance (nine genes) and quinolone resistance (five genes) were only identified in contigs from the Proteobacteria phylum.Among the 489 ARGs identified in the CMP data set, 149 ARGs could be assigned to the taxa on the phylum level based on draft genome sequences (metagenome binning) . Most ARGS  were traced to three phyla ((iv) Functional profiles. Besides identifying virulence factors and ARGs, we also predicted metabolic pathways and their abundance using HUMAnN3  and clustered by region based on principal-coordinate analysis (PCoA) of the gut microbiota community . We found that the DRM patterns of the population from the four regions were also different . For 45 P < 0.001) supported the effect of spatial features on the gut microbiome on the region level. Samples from the Beijing region could be further divided into group samples from three different locations, labeled BJ1 (53 samples), BJ2 (40 samples), and BJ3 (9 samples). The Bray-Curtis dissimilarities between samples from different locations were also higher than those between samples from the same location, which supports the effect of spatial features—even at the location level—on the microbiota composition  was higher than that among the samples from the same region  (P < 0.001), which supports the higher level of variability in the fecal microbiome in the Chinese population.The effect of spatial features on the gut microbiome could also be supported by comparison with public data. Compared to fecal the microbiomes of 152 samples publicly released in the HMP project from United States , samplesTo further evaluate the shared proportion of the CMP data set on the sequence level, we generated a microbiome genetic similarity (GS) value to represent the overlap between samples, which is described Materials and Methods. We found that the average GS for human gut microbiota is 0.068. We also found that samples from the same region or the same location have a higher GS value than that from different cities , which represents the sequence variability of the fecal microbiome caused by the spatial feature.To compare the effects of the temporal features on the human gut microbiome, we conducted a 1-year cohort study in a small population from Beijing ; we collPrevotella, which was detected in all the samples, was the most variable core genus with high coefficient of variation value (0.61). The percentage of Prevotella fluctuated over time . PCoA supported this finding; samples from the same individuals were clustered. Permutational multivariate analysis of variance (PERMANOVA) indicated that the effect caused by the individual .The gut microbiome is not always stable over time; however, the level of change is low. The BC dissimilarity between samples originating from the same individual were significantly lower than that between samples originating from different individuals (P < 0.05) . Short-term represents a short time interval in which the samples were collected in two consecutive months. A long-term interval is an interval longer than 6 months. Based on the calculated Bray-Curtis dissimilarity for samples and comparison between groups, it was found that the differences in the gut microbiota between samples increased gradually with time. The Bray-Curtis dissimilarity in the short-term interval group was significantly lower than that in the long-term interval group  .P < 0.001). Although this GS value would slightly decrease with time, for a long-terms sample (>6 months), the average GS value was still 0.226, significantly higher than that between people , which also supported the finding that samples from the same person always had a higher similarity than samples from different people.In this 1-year cohort study, we also used sourmash  to estimIn this 1-year cohort study, we also identified 21 DRMs among 49 fecal samples on the species level. Of these 21, 12 DRMs (57.1%) were stable in one or more people. We did not find any stable existing bacterial virulence factors that were present in >60% of samples from the same individual. This finding supports the hypothesis that the existence of virulence factors is transient rather than stable in the gut microbiome.We also investigated the resistome stability on a time scale using these 49 samples from 7 individuals. Among the 49 samples, 182 ARGs were identified; 13 ARGs (7.1%) were stable in all 49 samples from the 7 individuals; 60 ARGs (33.0%) were identified in all samples from a single person. These 73 ARGs were called time-stable ARGs. The other ARGs (59.9%) were only transiently positive in one or more time point samples; this supports the partially stable nature of the ARGs in the human gut microbiome.The time-stable ARGs exhibited differences in their occurrence in the samples from different people. The beta-lactam ARG (blaOXA-85_1_JANA01000064) was positive in all five samples from P7 but was not found in any other samples. The aminoglycoside ARG [aph(3″)-Ib_5_AF321551] is a time-stable ARG in P4 and P5 but was transient in P6.Bacteroidetes at the phylum level at the T4 (September 2018) sample point but to the Firmicutes at the T11 point (May 2019), which supported the idea that some ARGs may also come from different bacterial strains.For 182 ARGs identified in 49 samples from 7 individuals, 105 ARGs could be assigned to the taxa on the phylum level. We found that the tetracycline ARG (tet32_2_EF626943) is a time-stable ARG in P5, but it was from a bacterial strain assigned to the We used PERMANOVA to explore the associations between gut microbiota and host characteristics. We found that individuals exerted the strongest effect  and 4A, Bifidobacterium spp. than those in the Yogurt– group .In this study, volunteers who consumed yogurt every day or often (more than 3 times in the past month) were grouped as “yogurt+,” while those who never ate yogurt were classified as “yogurt–.” Yogurt+ people had a higher percentage of Bifidobacterium spp. than the male individuals .In this study, we did not find any significant difference between the male and female participants with respect to the gut microbiome richness; however, the female individuals had a higher percentage of P < 0.05) Shannon index than those who did not. We used linear discriminant analysis effect size (LEFSE) to distinguish the marker genera between the drink+ and drink– groups. In the drink+ group, the sample had a higher percentage of Enterobacter spp., Klebsiella spp., and Desulfovibrio spp.Alcohol consumption is associated with health risks. In this study, people who consumed alcohol had a significantly higher . Sk1 virus and T4 virus are common in most HMP samples, but not in CMP samples. However, a similar divergence between countries was not observed for archaea.In CMP phases I and II, we determined the effects of spatial factors and lifestyle on the gut microbiota composition. The impact of spatial features on the fecal microbiome at the levels of country, region, and location were supported by the results from this study; samples from different countries/regions/locations exhibit higher divergence than those from the same places. Compared to the HMP data set, samples in this study have higher variability in the fecal microbiome, with higher richness, Shannon diversity indices, and Pielou evenness index, as well as having more ARGs in several classes . BesidesP < 0.05).In CMP phase II, we investigated the changes in the gut microbiota in a healthy Chinese population over time. Analyzing the gut samples from the same individuals for 1 year, we found that the gut microbiota community from the same people always has a higher similarity than that from different people, which supports community stability over time. However, in samples from P7, the enterotype changed with time. The Bray-Curtis dissimilarity for the short-term interval groups was significantly lower than that for the long-term interval groups .“Healthy” is not equivalent to the complete absence of disease-causing microorganisms (DRMs). In this study, we found that even for people without any disorder symptoms, 45 DRMs, which are conditionally pathogenic bacteria, were detected in healthy volunteers, and 12 DRMs were found to be stable, existing in gut samples from the same people sampled at different time points. Based on phase II findings for healthy Chinese individuals, we released a program  for users to quickly obtain information about their DRMs and virulence factors based on the metagenomic results of their gut samples (P > 0.1); however, these factors influenced the occurrence of specific species, such as Bifidobacterium spp.“Healthy” gut microbiota is not equivalent to the “same.” One important objective for this field is to make the health status measurable and comparable. Based on the information available for the volunteers in this study and the Mantel test, we compared the influence of ten factors on gut microbiota . The most influential factor was the individual (the host of the samples), followed by the country (the sample project HMP or CMP) and region (sample location). Physical activities, BMI, yogurt consumption, and sex were not found to have a significant impact on gut microbiome richness ; P > 0.1Individuals are the main factor influencing the gut microbiota; the samples from the same individual have higher similarity than those from different individuals. We also found differences in the gut microbiota composition with time; the relatively small change in the human gut microbiota over a short period (<1 month) enhanced its potential for use in host source traceability in the future. It is noteworthy that the conclusion about the host of the samples being the most influential factor is only based on samples from seven individuals, whereas the rest of the factors are based on a big data set. Thus, we still need more work to support this conclusion, as well as more data to make it clearer for the temporal changes in the gut microbiota. Our study also had the limitations of having only four sample regions and employing only DNA sequencing data. In our future studies, we will process a larger number of samples and also evaluate RNA sequencing data. These limitations notwithstanding, our results have helped establish that the characteristics of gut microbiota change with time and space.Conclusions. In summary, our findings revealed changes in the Chinese gut microbiota over time and spatial levels and examined the distribution patterns of disease-related microorganisms (DRMs) at the species level. The microbiota data obtained in this study on the species level will provide a detailed basis for the healthy gut microbiome composition in the Chinese population.From 2016 to 2019, 239 fecal samples were sampled from people living in four cities in China . These four regions are located in the northern, southern, central inland, and western regions of China, respectively. All volunteers in the groups were given a pretest and a detailed questionnaire about their age, career, drug/medical history of both themselves and their immediate families, and their living habits  (Table S1) to determine the health level of the volunteers. We also measured the height, weight, blood pressure, and blood glucose levels on-site and constructed detailed inclusion/exclusion criteria to filter out candidate factors affecting the microbiome. Detailed information about the questionnaire process and inclusion/exclusion criteria is provided in the supplemental material.Briefly, only adult (age 18~69) volunteers were included in this project . People All fecal samples were collected within 24 h after finishing the pretest and questionnaire. The feces were collected from a disposable bedpan given to the volunteers and were not obtained from a flush toilet, to avoid the complication of collecting toilet water. Samples were stored in ice boxes and transported to the laboratory within 4 h. The study was approved by China CDC; all experiments were performed in accordance with relevant guidelines and regulations, and all people provided informed written consent.Each month from June 2018 to June 2019, we collected fecal samples from 7 healthy Chinese individuals residing in Beijing, China. The eligibility criteria for participation in this study and the sampling process were same as described in the previous section. Details regarding the characteristics of the subjects are provided in the supplemental material. In total, 49 fecal samples from 7 individuals were collected, and DNA was extracted and successfully sequenced. The number of samples collected from each subject, corresponding to a total of 12 time points, varied in the range 5 to 11 .The daily habits  of the subjects were also evaluated each month to determine their health status. Additionally, their heights, weights, blood pressure values, and blood glucose levels were recorded on-site every month. Thereafter, we constructed a detailed report on candidate-specific factors that could affect their gut microbiome. Detailed information in this regard is provided in the supplemental material, personal information questionnaire.DNA was extracted from the samples within 24 h using the QIAamp Fast DNA stool minikit , as per the manufacturer’s instructions.7, and the percentage of bases with values higher than a quality value of Q30 was above 80%. After quality control of raw reads, we removed the human sequences using Bowtie 2 (The shotgun metagenomics library was built using the TruePrep DNA library prep kit for Illumina and sequenced using an Illumina HiSeq platform with paired-end sequencing generating reads of 150 bp. Using FastQC  and FastBowtie 2  (human dhttp://www.hmpdacc.org/). After filtering out samples without metadata, only samples from healthy individuals and those released with shotgun metagenomics data of >100,000 reads were kept for further analysis. Finally, we got raw sequencing reads from 152 healthy individuals in the HMP project.The public fecal microbiome data used in this study were downloaded from the HMP database .The analysis pipeline used in this study was released in GitHub .The microbiome of human fecal samples was analyzed using HUMAnN3 (v3.1.1)  with theThe DRMs identified in this study represent the bacterial species that cause or might potentially cause disease in humans or in other bacterial species in the same genus as those that have been proven to cause disease. Based on the “List of Human Pathogenic Microorganisms” , we listed 155 DRMs with genomes which covered Chinese national legal infectious diseases and several pathogens detected in recent outbreaks as well as several novel bacterial species proven to cause disease; all listed DRMs are listed in Table S2. To detect the existence of DRMs, all shotgun metagenomics sequences were classified using the Kraken 2 method  based onVirulence factors were identified using the Bowtie 2 method  based onhttps://cge.food.dtu.dk/services/ResFinder/). The analysis script was released in GitHub .The same pipeline was used for detection of ARGs. The ResFinder database was used .All sequencing data in the project, as well as metadata (Table S1), were deposited in the NCBI SRA database (BioProject no.


Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using  Prior to cell division, DNA must be entirely and accurately duplicated to be transmitted to the daughter cells . In metaXenopus egg extracts  in S-phase egg extracts (see data availability). Among the proteins enriched more than 3-fold in Yap-co-IP versus Mock-co-IP conditions, we mostly identified factors functionally associated with mRNA metabolic process, ribonucleoprotein complex assembly and translation . This isications . Of noteications . InteresWe confirmed this Yap/Rif1 interaction in egg extracts by reciprocal co-IP assays . We furtXenopus egg extracts , cells divide very rapidly, rather synchronously for a series of 12 divisions and present a cell cycle structure without gap phases. As a result, variations of the number of cells at a given time during this developmental period reflect alteration of the time spent in the S and M phases. We thus decided to deplete Yap from embryos and assess the outcomes on the rate of embryonic cell division. Since Yap protein is maternally expressed (Xenopus for another target using Trim21 mRNAs (yap translation blocking morpholino oligonucleotides (MO) to prevent de novo protein synthesis (yap mRNA (gain of function) have larger and less numerous cells than in controls  of the retina  and in retinal stem cells (i.e. increase in early-like replication foci patterns). It is thus tempting to speculate that Rif1 and Yap act in concert to regulate replication dynamics.The molecular control of the RT program remains elusive. Regarding key factors, Rif1 was the first mammalian factor shown to temporally control DNA replication, acting negatively on origin activation . This fuyap (rif1 knockdown (this study) rather suggests an impact on the partition between early and late replication-firing. In Rif1-depleted Hela cells, the overall replication foci were similarly found to be extensively rearranged, with cells displaying predominantly early S-phase-like patterns . How cell cycle kinetics is differentially regulated in both cases remains to be investigated. It is well known that Yap also transcriptionally regulates cell cycle genes, which may contribute to such different outcomes. Interestingly, among direct targets genes regulated by Yap, there are also essential factors involved in replication licensing, DNA synthesis and repair , and used as an antigen to raise antibodies in rabbits at a commercial facility . A cDNA encoding recombinant His-tagged Xenopus Yap (from the L subgenome) was cloned in pFastBac1vector, expressed in the baculovirus Bac-to-Bac expression system (Invitrogen), purified by Nickel-Sepharose chromatography as described by the supplier (Amersham Bioscience) and then dialyzed overnight against 25 mM Hepes pH 7.8, 250 mM NaCl, 5 mM imidazole, 5% glycerol, 7.5 mM MgCl2, 1 mM DTT, 1 mM EDTA. Purified His-Yap was then used as an antigen to raise antibodies in rabbits at a commercial facility .A detailed list of the antibodies used in this study for immunohistochemistry (IHC), immunodepletion and western blot (WB) is provided in the Key Resources Table. HLTV-hTRIM21 was a gift from Leo James. Recombinant His-geminin, and His-hTRIM21 were prepared as described respectively . C-termiYap-Morpholinos  or 1 pmol of rif1-MO or 2 pmol of standard control MO together were microinjected into one-cell stage embryos. The Trim-Away experiments were conducted in a similar way using a mixture of recombinant hTRIM21, anti-Rif1 or anti-Yap antibodies together with 1 or 2 pmol of rif1-, Yap-, or control-MO . MO sequences used in this study can be found in Key Resources Table.Embryos were injected at the one-cell stage with different components  along with a fluorescent tracer  to ascertain the correctness of the injections. A total of 200 pg of mRNA  were injected, corresponding to the coding region of Yap (FJ979828), Rif1(NM_001280649.1) or GFP as a control. For in vivo depletion experiments, 2 pmol of Xenopus eggs and sperm nuclei from testis of male frogs were prepared as described (2). Loading of the MCM complex (pre-RC assembly) was prevented by the addition of 100 nM of recombinant geminin to the extracts.Replication competent extracts from unfertilized escribed . Sperm n2). For Yap depletions, coupled beads were then incubated 1 hr at 4 °C in egg extracts (volume ratio 1:3). For Rif1 depletions, coupled beads were incubated 30 min at 4 °C in egg extracts (ratio 1:1) and egg extracts after a first round were re-incubated another 30 min at 4 °C with coupled beads.Rabbit anti-Yap antibody  or rabbit anti-Rif1 antibody , pre-immune serum or rabbit IgG  were coupled overnight at 4 °C to protein A Sepharose beads . Coupled beads were washed three times in EB buffer  were added to replication reactions in the presence of 20 µM rhodamine-dUTP (Roche). At each time point, 20 µl aliquots were diluted in 500 µl PBS and fixed by the addition of 500 µl paraformaldehyde 8%. Nuclei were spun onto coverslips through 1 ml of 20% sucrose cushion in PBS, and counterstained with Hoechst 33,258 as previously described . Coversl32P]dCTP (3000 Ci/mmol) and reactions were stopped at indicated time points. DNA was recovered after DNAzol treatment (Invitrogen protocol) followed by ethanol precipitation and specific incorporation was measured in cpm in a liquid scintillation analyzer . Total DNA synthesis (ng/µl) was calculated as described (Sperm nuclei (2000 nuclei/µl) were incubated in Mock or Yap depleted extracts and one-fiftieth volume of  with 0.1% formic acid) and separated using a C18-PepMap column (Thermo Fisher Scientific) with a solvent gradient of 2–100% Buffer B (0.1% formic acid and 98% acetonitrile) in Buffer A at a flow rate of 0.3 µl/min. The peptides were electrosprayed using a nanoelectrospray ionization source at an ion spray voltage of 2300 eV and analysed by a NanoLC-ESI-Triple TOF 5600 system (AB Sciex). Protein identification was based on a threshold protein score of >1.0. For quantitation, at least two unique peptides with 95% confidence and a p-value <0.05 were required.Rabbit anti-Yap antibody  or rabbit IgG  were coupled 2 hr at RT to protein A Sepharose beads . Coupled beads were covalently crosslinked using dimethyl pimelimidate according to standard procedures, washed with PBS and kept in PBS, 0.02% sodium azide at 4 °C. For IP experiments, crosslinked beads with rabbit anti-Yap antibody or rabbit IgG were washed three times in EB buffer  were performed using a Mann-Whitney test or Wilcoxon signed ranked test as mentioned in the figure legends. Xenopus egg extracts and embryos, before the initiation of transcription, this manuscript now elegantly identifies a new role of YAP in DNA replication dynamics, thus slowing down cell division rate.The YAP protein is well-known as a major regulator of tissue growth and repair, acting as a co-factor of transcription to promote cell proliferation and de-differentiation. In  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "A non-transcriptional function of Yap orchestrates the DNA replication program" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) The quantification of the amount of YAP in Figure 1B is confusing. The legend of the chart states "Control in light grey and presence of geminin in black", but the bar colors are of different shades of grey. It is not clear how to evaluate them. The graph should be improved by properly labeling the legend of the figure or, simply, by showing the relative amount of YAP intensity values relative to a loading control like H3, normalized to the 90 min control condition.The higher rate of DNA synthesis observed in the absence of Yap in Figure 1D is not very evident from the gels in Figure 1- supplement 3B. The timing of the experiments is continuously changing throughout the figures. It is therefore difficult to compare them. Also, comparisons across different gels are difficult to interpret.Most importantly, relative quantification on gel images cannot support the claim of increased DNA synthesis in the absence of YAP. To accurately quantify the replication of DNA added to the extract, the total amount of DNA synthesized must be quantified. This can be easily done by counting labeled nucleotide incorporation on precipitated DNA . Without these data, the central claim of the paper is meaningless.It is necessary to analyze the dynamics and the abundance of chromatin-bound replication proteins associated with the active replication fork after Yap depletion using chromatin binding assays. This would further confirm the increase in the fork density observed by DNA combing experiments.2) Why are only 2 replicates instead of 3 provided in Figure 2? This is especially true for the conclusion that eye length is unaltered -it appears that there is a subset of eye length that is increased in 2F, which might reach significance if triplicates were performed.3) The authors suggest that YAP and RIF1 work together, – it would be interesting to co-deplete YAP and RIF1. If YAP works solely through Rif1, it should not be more effective than loss of YAP alone.Does loss of RIF1 affect YAP loading onto chromatin?The efficiency of depletion for both RIF1 and YAP is different in Figure 4B and Figure 4A, supplement 1. Moreover, the combined use of the TRIM-away approach with injections of MO led to a stronger and prolonged YAP depletion but also triggered toxicity in the tadpoles, which display severe abnormalities. Therefore, the outcomes of these procedures are unlikely to be specific, unless the authors can provide a rescue experiment. Quantitation of the Western blots in panel A of Figure 4 supplement 1 is needed to be convincing that trim away and morpholino combined are more effective.4) It is difficult to see the points the authors wish to communicate in Figure 6. If the authors add yellow and red arrows in B to C and D, this might make it clearer. There is almost no EdU in the YAP-MO -does this affect the ability to recognize the different patterns in this region of the eye?5) The title of the manuscript is "A non-transcriptional function of YAP orchestrates the DNA replication program". It is not clear that YAP "orchestrates" DNA replication – for this to be true, it would have to be signal responsive. Since the authors did not reveal any links to YAP activity (such as YAP phosphorylation or nuclear/cytoplasmic distribution) it is not "orchestrating" DNA replication. Please adjust the title accordingly and also provide a clear indication of the biological system under investigation.6) StatisticsPlease indicate the statistical test in the legend of Figures 1B, 2E-F, 5C. Essential revisions:1) The quantification of the amount of YAP in Figure 1B is confusing. The legend of the chart states "Control in light grey and presence of geminin in black", but the bar colors are of different shades of grey. It is not clear how to evaluate them. The graph should be improved by properly labeling the legend of the figure or, simply, by showing the relative amount of YAP intensity values relative to a loading control like H3, normalized to the 90 min control condition.We apologize for this confusion. This has been corrected and the Figure 1B is now properly labelled.The higher rate of DNA synthesis observed in the absence of Yap in Figure 1D is not very evident from the gels in Figure 1- supplement 3B.Since Figure 1- supplement 3B was not clear enough, we replaced this image of the gel electrophoresis of nascent strands by another one, now displayed in Figure 1—figure supplement 2. We think that this new figure better illustrates both the quantitative aspect of replication differences in terms of raw intensities measured of whole lanes on gels but also the qualitative view of replication. We think that it is important for the readers to visualize and to be able to compare the length of nascent strains between Mock- and Yap-depleted extracts, which is also a proof of experimental quality of the DNA synthesis in vitro, which is not visible in absolute quantification after scintillation counting.The timing of the experiments is continuously changing throughout the figures. It is therefore difficult to compare them. Also, comparisons across different gels are difficult to interpret.The referee is correct but as explained in the MM section page 14 of the original manuscript, the replication extent (percent of replication) differs for a specific time point from one extract to another, because each egg extract prepared from one batch of eggs from one frog replicates nuclei with its own replication kinetics. To overcome this problem and to compare different independent experiments performed using different egg extracts, the data points of each sample were normalized to maximum incorporation value.Most importantly, relative quantification on gel images cannot support the claim of increased DNA synthesis in the absence of YAP. To accurately quantify the replication of DNA added to the extract, the total amount of DNA synthesized must be quantified. This can be easily done by counting labeled nucleotide incorporation on precipitated DNA . Without these data, the central claim of the paper is meaningless.Although we do not agree that relative quantification on gel images cannot support the claim of increased DNA synthesis in the absence of Yap, we thank the reviewer for his suggestion since we now provide additional data clearly strengthening our conclusion.Xenopus replication laboratories have quantified DNA synthesised after 32P-dCTP incorporation followed by separation by agarose gel electrophoresis using percentage of maximal incorporation or arbitrary units . Nevertheless, as the referee suggested, we quantified the total amount of DNA synthesized in three new independent experiments. These new results, presented page 5, lines 34-39 and shown in Figure 1G, support our conclusion, as they also show that Yap depletion increases total DNA synthesis. Please note that the DNA combing results presented in Figure 2 already showed that replication is increased after Yap depletion when quantifying total replication percentages of all combed DNA. Finally, we also added another set of experiments to Figure 1 to further confirm these findings. We used the incorporation of rhodamine-dUTP followed by the quantification of the fluorescence intensity within nuclei. This nuclei-fluorescence based method is frequently used in proliferation assays to assess nucleotide incorporation resulting from the DNA replication process in other organisms. Our new results demonstrate that DNA synthesis is increased 1.5-fold in six biological replicates and represent a third independent method, in addition to DNA combing and 32P-dCTP incorporation, showing that DNA synthesis is increased upon YAP depletion. These new results are now presented page 5, lines 2734 and shown in Figure 1D-F.Many studies, published in high standards journals and coming from different Considering all these additional data, we had to move and replace the alkaline gel from the original Figure 1D to Figure 1—figure supplement 2.It is necessary to analyze the dynamics and the abundance of chromatin-bound replication proteins associated with the active replication fork after Yap depletion using chromatin binding assays. This would further confirm the increase in the fork density observed by DNA combing experiments.We thank the referee for this suggestion and we added a western blot of chromatin bound proteins after Yap depletion. This shows that two replication proteins associated with the active replication fork, namely Cdc45 and PCNA, are enriched after Yap depletion compared to the control at the beginning of S-phase. This observation further supports the DNA combing results showing that more forks are active after YAP depletion. This new data is now presented page 6, lines 25-32 and displayed in Figure 2H.We would like to stress here that with these additional methods added to the revised version, five different methods in total  show that DNA synthesis and origin activation is increased after Yap depletion.2) Why are only 2 replicates instead of 3 provided in Figure 2?.Answer: We apologize for this confusing presentation. In Figure 2A, three fibers were shown as visual examples illustrating the results of this experiment (and not one fiber per replicate). We have two replicates with two different time points, as stated in the text each, and shown in the tables. We modified the legend of the figure to make this clearerWe also changed the fibers images  to better illustrate the data set (see point below).This is especially true for the conclusion that eye length is unaltered -it appears that there is a subset of eye length that is increased in 2F, which might reach significance if triplicates were performed.The scatter plot in Figure 2F makes it look like that there are more eyes with larger sizes after Yap depletion, but please note that there are also more EL measured as stated in the legend (ΔMock n=182 versus ΔYap n=311). To highlight this important parameter, we added these numbers below the scatter plot in the revised Figure 2F, as we have done consistently for all of the experiments presented in the revised Figures. The means of these two EL distributions are indeed numerically different but since both distributions are not Gaussian (d'Agostino and Pearson test), only non-parametric tests can apply (Mann-Whitney or Kolmogorov Smirnow test). The results of the two non-parametric tests showed that the distributions are not significantly different as mentioned in the legend. However, we cannot rule out that after Yap depletion, some larger eyes may arise from fusions of forks or from a higher fork speed, but again, the tests applied to a high number of measurements show no significant statistical differences. For later time points, this issue is discussed in the original manuscript p9 and still now in the revised manuscript page 6, line 22-25.3) The authors suggest that YAP and RIF1 work together, – it would be interesting to co-deplete YAP and RIF1. If YAP works solely through Rif1, it should not be more effective than loss of YAP alone.Xenopus in vitro system which clearly shows that Rif1 depletion leads to a strong increase of origin activation during early S-phase. We therefore found that Rif1 depletion and Yap depletion qualitatively show the same main effects: an increase of DNA synthesis and fork density, that are more pronounced in early S-phase. We also noticed quantitative differences in the direct fluorescence after rhodamine incorporation of whole nuclei and fork density, with stronger effects after Rif1 depletion compared to Yap depletion. This suggests that there might be an additional mechanism for Rif1 in regulating origin activation. These new data, presented page 7, lines 4-29, have been added in the form of an entire new Figure 4.Double immunodepletions are technically very challenging. We did not manage to efficiently co-deplete Yap and Rif1 from egg extracts while maintaining a sufficient replication efficiency. We did however directly compare the effects of YAP depletion to those of Rif1 depletion alone. As for Yap depletion, we first quantified rhodamine-dUTP incorporation after Rif1 depletion by direct fluorescence microscopy that demonstrated a clear increase of DNA synthesis, consistent with Alver et al., 2017. Second, we performed 2 independent DNA combing experiments after Rif1 depletion with a large number of fibers analyzed  in egg extracts that show a marked increase in DNA replication and fork density like those seen after Yap depletion, spanning from very early to mid Sphase. Please note that this is also the first replication kinetics analysis by DNA combing after Rif1 depletion in the Does loss of RIF1 affect YAP loading onto chromatin?We thank the referee for this interesting question, which we addressed already after the preprint deposition of our manuscript on BioRxiv upon a request of a reader. We could not observe any major changes in Yap chromatin recruitment after Rif1 depletion nor the reverse in our conditions . However, since after Rif1 depletion we can still detect some residual Rif1 left on chromatin, we cannot completely exclude that this residual Rif1 is sufficient to recruit Yap. We were unable to further improve Rif1 depletion without severely compromising the replication capacity of the Rif1depleted egg extract. This difficulty could be explained by the abundance of Rif1 and/or its binding to insoluble nuclear structure. Since the result is not 100 % conclusive for us to be published, we prefer not to include it into the manuscript and we show the blots for information to the referees, see The efficiency of depletion for both RIF1 and YAP is different in Figure 4B and Figure 4A, supplement 1.We agree with the referee that the efficiency of depletion is different in both figures. This is explained by the fact that the extent of the depletion varies from experiment to experiment. We work with different batches of in vitro fertilized embryos and extracts, so these differences simply reflect the technical/biological variability.Moreover, the combined use of the TRIM-away approach with injections of MO led to a stronger and prolonged YAP depletion but also triggered toxicity in the tadpoles, which display severe abnormalities. Therefore, the outcomes of these procedures are unlikely to be specific, unless the authors can provide a rescue experiment.It is important to point out that abnormal development is not always attributable to a toxic effect. Many losses of gene function result in malformations without being ascribed to toxicity or unspecific effects. However, we agree with the reviewers on the need to present a rescue experiment, which is now shown in new Figure 5C and new Figure 5—figure supplement 1B. In addition, we also provide gain-of-function (GOF), data for YAP in early embryos. In brief, we find that the Yap GOF leads to opposite outcomes than those of its depletion with embryos at the same stage of development, having fewer and larger cells than the control. Furthermore, we show that the effects of Yap depletion, i.e. embryos with more and smaller cells than the control at the same developmental stage, are rescued by the addition of mRNA encoding Yap to restore the protein level. This is true for both embryonic divisions  and development, as we obtained normal-looking neurula after Yap rescue . Overall, these data now clearly show that Yap is both sufficient and necessary to maintain the rate of embryonic divisions and that this phenotype is specific since it can be rescued by expressing Yap alone. These new data are presented pages 8, lines 2-10.Quantitation of the Western blots in panel A of Figure 4 supplement 1 is needed to be convincing that trim away and morpholino combined are more effective.The quantification is now presented in new Figure 5—figure supplement 1A. At the 2 -cell stage, we observe some fluctuations in the amounts of Yap between samples, the origin of which we do not fully understand. At the 4-cell stage, a reduction in Yap is observed regardless of the depletion strategy used. It is from the 8-cell stage onwards that differential effects between the depletion methods can be appreciated. From this point, the quantifications confirm that the TRIM-away and Morpholino combined are more effective than taken separately with regard to Yap .4) It is difficult to see the points the authors wish to communicate in Figure 6. If the authors add yellow and red arrows in B to C and D, this might make it clearer.We thank the reviewer for this suggestion. We have added different coloured stars according to the replication patterns directly on the nuclei observed on the CMZ enlargements . This representation makes it possible to immediately identify the different patterns without obscuring the image.There is almost no EdU in the YAP-MO -does this affect the ability to recognize the different patterns in this region of the eye?Our observations show that there are fewer EdU positive cells in the Yap-MO but not “no EdU”. The fluorescence intensity in the green-labelled nuclei in Figure 7 C after Yap MO does not appear different from that in the control-MO. Under these conditions, there is no reason to think that one pattern is more difficult to recognise than the other one.5) The title of the manuscript is "A non-transcriptional function of YAP orchestrates the DNA replication program". It is not clear that YAP "orchestrates" DNA replication – for this to be true, it would have to be signal responsive. Since the authors did not reveal any links to YAP activity (such as YAP phosphorylation or nuclear/cytoplasmic distribution) it is not "orchestrating" DNA replication. Please adjust the title accordingly and also provide a clear indication of the biological system under investigation.By “orchestrate”, we had in mind an actor able to modulate the speed of execution of the replication process. Nonetheless, we acknowledge that this may be open to interpretation. We thus propose to replace “orchestrates” by “regulates”.6) StatisticsPlease indicate the statistical test in the legend of Figures1B, 2E-F, 5C.We have now added the statistical tests in the legends for these Figures.


Functional near-infrared spectroscopy (fNIRS) studies have demonstrated associations between hearing outcomes after cochlear implantation and plastic brain changes. However, inconsistent results make it difficult to draw conclusions. A major problem is that many variables need to be controlled. To gain further understanding, a careful preparation and planning of such a functional neuroimaging task is key.Using fNIRS, our main objective is to develop a well-controlled audiovisual speech comprehension task to study brain activation in individuals with normal hearing and hearing impairment (including cochlear implant users). The task should be deductible from clinically established tests, induce maximal cortical activation, use optimal coverage of relevant brain regions, and be reproducible by other research groups.The protocol will consist of a 5-minute resting state and 2 stimulation periods that are 12 minutes each. During the stimulation periods, 13-second video recordings of the clinically established Oldenburg Sentence Test (OLSA) will be presented. Stimuli will be presented in 4 different modalities: (1) speech in quiet, (2) speech in noise, (3) visual only , and (4) audiovisual speech. Each stimulus type will be repeated 10 times in a counterbalanced block design. Interactive question windows will monitor speech comprehension during the task. After the measurement, we will perform a 3D scan to digitize optode positions and verify the covered anatomical locations.This paper reports the study protocol. Enrollment for the study started in August 2021. We expect to publish our first results by the end of 2022.The proposed audiovisual speech comprehension task will help elucidate neural correlates to speech understanding. The comprehensive study will have the potential to provide additional information beyond the conventional clinical standards about the underlying plastic brain changes of a hearing-impaired person. It will facilitate more precise indication criteria for cochlear implantation and better planning of rehabilitation.DERR1-10.2196/38407 Disabling hearing loss is a major communication and health problem that affects over 6% of the overall population and over 50% of adults above the age of 65. For adults, deafness leads to social isolation, unemployment, and reliance on social services. This problem will increase with demographic change. It is estimated that by 2050, 10% of the global population will be living with disabling hearing loss ,4, improIn the absence of auditory input, the sensory deprivation induces reallocation of cortical areas . This leads to functional reorganization within the auditory and auditory-related brain cortex, with new functions being assigned to these brain regions . As an eWe use functional imaging to study these described plastic brain changes. However, in CI recipients, there are important considerations to make. Despite the efforts of CI manufacturers to allow structural magnetic resonance imaging (MRI) with a surgically implanted device, the technique has limitations. The outer speech processor cannot be worn during MRI scanning and thus cannot be used to assess evoked auditory responses associated with functional MRI. Furthermore, the implanted magnet and electrode array of the CI cause imaging artifacts in MRI and stimulation artifacts in electroencephalography (EEG) -20.Functional near-infrared spectroscopy (fNIRS), on the other hand, is ideal for this patient population . The tecPrevious fNIRS studies with implanted adults showed evidence of cortical reorganization. However, when comparing study findings, there are contradictory results. For example, some studies suggest that strong activation of the auditory cortex during lipreading tasks is a negative predictor of speech understanding with the implant ,23. OtheAccording to a recent review on fNIRS measurements in CI patients, at the current stage, it is difficult to draw a general conclusion about the potential positive or negative effects of cortical reorganization. Instead, methodological aspects must first be clarified . The effThe aim of our study protocol is to develop a well-controlled and reproducible fNIRS task to evaluate brain activation in response to speech comprehension in individuals with normal hearing, those with hearing impairments, and CI users. Our hypothesis is that through such a task, we can identify cortical networks that are clearly correlated to hearing performance with the implant. Identified brain activation patterns may later be used preoperatively as biomarkers of speech understanding with the implant.Using fNIRS, our main objective is to develop an audiovisual speech comprehension task to measure functional brain activity regarding speech understanding. The task should comply with the following criteria: it should (1) be deducible from clinically established hearing tests; (2) induce maximal cortical activation ; (3) align with the international 10-10 system of electrode placement, using optimally spaced optode positions with maximal coverage over the responsible brain regions. Short separation channels should allow noise reduction; (4) be time-efficient  be suitable for normal hearing, hearing impaired, and cochlear implant users; and (6) be reproducible by other research groups.We will correlate the fNIRS recordings with (1) data from patients’ history, (2) clinically validated questionnaires, and (3) performance during the fNIRS measurements .This research project is a prospective single-center study and will be conducted at the Department of Otolaryngology, Head and Neck Surgery at the Bern University Hospital, Inselspital, Bern, Switzerland.The protocol was designed in accordance with the ethical principles of the Declaration of Helsinki. The study setup was approved by the local ethical committee (reference number 2020-02978) and fulfils all the patient data regulations of Switzerland.All study participants must (1) be at least 18 years old, (2) be native German speakers, and (3) have preferably light and thin hair ,30. PartThe ear through which the acoustic stimulation will be presented needs to be implanted for at least 1 year. This will ensure that hearing rehabilitation after implantation is completed.Participants will be allocated to one of 3 groups: (1) normal hearing “control” cohort, (2) CI users with good speech understanding (“overperformer”), or (3) CI users with poor speech understanding (“underperformer”). CI users with moderate speech perception  will not be recruited because we want to investigate the functional mechanisms specifically for good and poor outcomes. Pilot measurements were performed on 10 participants to estimate an appropriate sample size. We compared the median relative change in the concentration of oxygenated hemoglobin in the auditory cortex. After acoustic stimulation (speech in quiet), an increase of 1.315 (SD 1.275) µMolar*cm was measured, while during the resting state, the value fluctuated close to 0. A power analysis to test a 2-sided hypothesis at 95% significance and 80% power showed that we need at least 15 participants with normal hearing to detect auditory activations. In addition, we considered previous findings from auditory fNIRS studies ,28,31,32Recruitment will be done through the CI center of our department. Potential study candidates will be screened based on their medical records and will be subsequently informed verbally or in writing about the study procedure. Candidates who are willing to participate and able to complete all tests required for the study will be asked to sign an informed consent form.Potential study candidates will be invited to an enrollment session. First, we will hand out the information sheet and answer any questions the candidates may have. To assess full eligibility, the candidates will have to fill in questionnaires and perform additional hearing tests before data collection. Bilateral CI users will be asked to turn off and remove the audio processor of the worse hearing ear to limit acoustic stimulation exclusively to the better ear. The worse hearing ear will be covered using an ear plug. The full enrollment session will take a maximum of 30 minutes.Questions on medical history will target the candidates’ handedness (Edinburgh Handedness Inventory) and the presence of diseases, which are among the exclusion criteria -35. AddiThe audiometric measurements and the fNIRS recordings will take place in an acoustic chamber (6 m × 4 m × 2 m) with a separate ventilation system and electromagnetic shielding. The broadband reverberation time is ~200 ms.In normal hearing participants, we will assess pure tone air-conduction hearing thresholds with a clinical audiometer . The findings must confirm that participants have no hidden or undetected hearing loss . For CI In all participants, we will measure the word recognition score for Freiburg monosyllabic word lists at a sound pressure level (SPL) of 65 dB . AdditioDuring fNIRS recording, each study participant will sit in a comfortable chair with an armrest, headrest, and lumbar support . A desk The stimulation protocol will be controlled by a custom-written script (Python 3.8.8) using Tkinter and python-vlc libraries. The script will send triggers via the serial interface to a trigger-box , which converts the signals to transistor-transistor Logic (TTL) levels. The TTL-encoded signals will then be received by the fNIRS machine .Participants will interact with the control laptop using the buttons of a mouse . The pointing function of the mouse will be disabled to ensure that participants control the experiment only by clicking and rolling. During the fNIRS measurement, the participants will be able to press an alarm button  positioned in a reachable distance on the table.We will select the regions of interest (ROIs) for the placement of the optodes considering previous studies. We expect responses related to audiovisual speech comprehension in the auditory and visual cortex, more specifically in the following ROIs: superior temporal gyrus (STG), primary visual cortex (V1), and visual association cortex (V2) ,31,46-50The montage will consist of 16 sources and 16 detectors placed on the surface of the skull according to the international 10-10 system of electrode placement A 54]. T. T54]. TThe optode holder cap will be assembled using the manufacturer's components  and custom 3D printed parts (colored optode markers and stabilizers for different head sizes). The parts will be designed in a solid modelling software  and printed using a 3D printer .At the end of the experiment, we will digitize the position of all optodes with a depth sensing camera  connected to an iPad . The depth sensing camera will be set up for optimized scanning of dark objects with low ambient infrared light. The infrared exposure time, gain, and depth resolution will be set to the highest available settings so that the colored optode markers can be easily identified on the 3D scan.During fNIRS recordings, we will instruct the participants to concentrate on the screen, follow the instructions, and reduce head movements. If the participants feel uncomfortable, an emergency button in front of them will be made available to stop the experiment. We will give all instructions both verbally and in writing. Before the recordings, the participants will conduct a short familiarization session with 4 example stimulations. Once the participant confirms that the task is understood, we will start the definitive recording. The functional recordings will begin with a 5-minute resting state period A. We wilAs stimulus material, we will use a video version of the female OLSA test ,58. A siA single stimulation block will contain 4 different stimuli, presented in one of the following modalities in a counterbalanced order B: (1) spAt random points, participants will be asked to answer questions to ensure attention and monitor speech comprehension during the test. The questions will be displayed in the nonstimulus epoch, for which the nonstimulus interval will be shortened to 10 seconds. The questions will ask to repeat the correct name or number of the last sentence from 4 possible answers. For example, if the sentence is “Nina gives 12 red flowers,” the question is either ”How many red flowers?“ or ”Who gives 12 red flowers?“ To answer the question, the participant will have to select 1 of 4 choices: 2 randomly selected numbers/names from the OLSA sentence matrix (wrong answers), an option if the respondent is not sure of the answer (skipped answer), and the correct answer. For the previous question, a possible combination could be (1) ”Britta,” (2) “Nina,” (3) “Peter,” and (4) “I cannot decide.” The participant will select an option with the roller on the computer mouse and confirm the answer with a double click. In the previous example, the participant must select the second option (”Nina“). The questions and the answers will be randomly generated, and the position of the question within the blocks will also be randomly chosen.The shortened nonstimulus interval of 10 seconds prior to the question window will allow us to evaluate the fNIRS responses. Therefore, the interleaved questions will not harm the overall effectiveness of the measurement. After the question is answered, the regular 20-25 second relaxation time will be applied to ensure that the brain responses return to baseline. Overall, 2 questions per modality will be asked, resulting in 8 questions throughout the entire fNIRS measurement.Following the breaks, before the first stimulation, there will be a stimulus-free interval of 20 seconds. This will ensure homogeneity of responses, meaning that all stimuli are perceived under similar circumstances. Overall, 10 blocks will be presented, resulting in 10 responses per stimulation modality, and the total fNIRS measurement time will be around 45 minutes. At the beginning of every event , a trigger will be sent from the control computer to the fNIRS machine through the trigger-box and stored as an extra channel in the fNIRS raw data.Following 5 stimulation blocks, we will ask every participant to rate their listening effort to the different stimuli, their rating of fatigue and their level of mind-wandering A 60-64]-6460-64]All written source documents will be completed in a neat, legible manner to ensure accurate interpretation of data. For each participant, a case report form (CRF) will be maintained, including the participant number. In CRFs and other project-specific documents, participants are only identified by a unique participant number. fNIRS measurements will be stored in a closed research environment . The secure web application is running on a local server maintained and backed up by the University of Bern. All documents related to the study, including the CRFs will be considered as source data, and these will be stored at the measurement site in accordance with relevant standards.Data preprocessing will be performed in MATLAB (MathWorks) using the Homer2 (v2.3)  and NIRSA further correction step will be performed to reduce noise based on the principle that the concentration changes of oxygenated and deoxygenated hemoglobin should be negatively correlated .We will perform the postprocessing of the scans with a 3D mesh processing tool (MeshLab) and custom-written scripts (MathWorks) .We will manually select the coordinates of the optodes and anatomical landmarks with MeshLab on the obtained 3D scans. The list of coordinates will then be exported and projected into Montreal Neurological Institute (MNI) space. The MNI coordinates will be displayed on the preoperative MRI scan of every CI-user participant, and the exact source of measured hemodynamic activation will be determined. Additionally, the mean and the standard deviation of optode coordinates will be calculated and reported as quality measure for optode fittings ,76.t=24 seconds relative to the stimulus onset. The epochs will be baseline-corrected by subtracting the mean of the signal between t =−5 seconds and t=0 seconds. Using the Glover canonical hemodynamic response function [Data analysis will be performed in Python using the MNE-Toolbox  and MNE-function  a designfunction ,81. Aftefunction . The datThe answers from the questionnaires will be digitized, and correlation analysis will be performed to reveal relations between the measured brain activation patterns and the evaluated questionnaires. Additionally, further behavioral data will be obtained from the triggers, such as reaction time to questions across the measurement as a measure of fatigue or response accuracy for each stimulation type as a measure of speech understanding.The enrollment for the study described in this protocol started in August 2021. The first results are expected at the end of 2022.The postoperative adaptive or maladaptive effect of existing cross-modal reorganization in CI candidates is a complex question. The available studies show contradictory findings. A recent review states that it is important to discuss the methodological aspects of such functional neuroimaging examinations ,46,47,50First, the test should be deducible from clinically established hearing tests. We used the video version of a widely used clinical test (Oldenburg Sentence Test) . FunctioSecond, the task should induce maximal cortical activation . We use an optimized counterbalanced block design. The duration of 1 stimulus will be 13 seconds, and the interstimulus break will be between 20 and 25 seconds, comparable to hemodynamic responses ,59. Our Third, it should be in alignment with the international 10-10 system of electrode placement, using optimally spaced optode positions with maximal coverage over the responsible brain regions. Short separation channels should allow noise reduction. Our optode placement covers the following brain regions: superior temporal gyrus (STG), primary visual cortex (V1), visual association cortex (V2), left inferior frontal gyrus (LIFG), middle temporal gyrus (MTG), and middle frontal gyrus (MFG). This allows us to study not only audiovisual activations but also speech perception in noise, the effects of fatigue, and activity related to higher-order cortical processing. Many other studies have not had the opportunity to cover such a wide range of cortical regions, mostly due to hardware limitations -25,50. WAdditionally, we use a multidistance channel setup. The optodes of the regular channels are ~30 mm apart from each other. Additional short channels with a 15-mm interoptode distance over the auditory and visual cortex provide extracerebral information to remove confounding systemic signals. It is recommended to use a systemic physiology controlled fNIRS approach, although this has rarely been applied in previous studies .Fourth, it should be time efficient to avoid fatigue due to experiment duration. The longest task the participants will be required to complete will last 12 minutes, and the total measurement time will be around 45 minutes. Regular breaks will be provided, and the total duration of the experiment is expected to be around 120-150 minutes.Fifth, it should be suitable for participants with normal hearing, hearing impairments, and those using CIs. The audio material is presented through a loudspeaker, so the task is suitable for people with normal hearing as well as for hearing aid and CI users. Alternatively, an insert earphone or a direct CI audio input simulation would be feasible. However, these 2 approaches have the disadvantage that the 3 aforementioned groups cannot not be stimulated identically. Our optode placement was chosen to allow for easy attachment of the implant coil.Sixth, it should be reproducible by other research groups. The audiovisual version of the OLSA was published in 2021 and is now accessible . MoreoveIn summary, the proposed audiovisual speech comprehension task will help us understand neural correlates to speech understanding. In the first stage, we will perform these measurements postoperatively to better understand the corresponding neuronal networks with an activated implant. In the subsequent stage, we will perform the measurements pre- and postoperatively to make prognostic calculations. The comprehensive study will have the potential to provide additional prognostic information beyond the conventional clinical standards regarding the underlying plastic brain changes of a person with hearing impairment. Our study will facilitate more precise indication criteria for cochlear implantation and a better planning of rehabilitation.


Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypoplasia and respiratory morbidity. To assess whether respiratory morbidity during the first 2 years of life in infants with left-sided CDH is associated with fetal lung volume (FLV) evaluated by the observed-to-expected FLV ratio (o/e FLV) on antenatal magnetic resonance imaging (MRI). In this retrospective study, o/e FLV measures were collected. Respiratory morbidity in the first 2 years of life was studied according to two endpoints: treatment with inhaled corticosteroids for >3 consecutive months and hospitalization for any acute respiratory disease. The primary outcome was a favorable progression defined by the absence of either endpoint. Forty-seven patients were included. The median o/e FLV was 39% . Sixteen (34%) infants were treated with inhaled corticosteroids and 13 (28%) were hospitalized. The most efficient threshold for a favorable outcome was an o/e FLV ≥ 44% with a sensitivity of 57%, specificity of 79%, negative predictive value of 56%, and positive predictive value of 80%. An o/e FLV ≥ 44% was associated with a favorable outcome in 80% of cases. These data suggest that lung volume measurement on fetal MRI may help to identify children at lower respiratory risk and improve information during pregnancy, patient characterization, decisions about treatment strategy and research, and personalized follow-up. Congenital diaphragmatic hernia (CDH) is a rare birth malformation involving a diaphragmatic defect that allows the intraabdominal organs to migrate upward into the thorax and which compromises normal lung development. In 2017, the prevalence rates per 10,000 live births in Europe were 2.47, and this rate increased to 3.45 when including termination of pregnancy for fetal anomalies or death [The respiratory consequences of CDH in children are heterogeneous and can include very early death, need for oxygen therapy on day 28, which is defined as chronic lung disease (CLD), persistent need for oxygen therapy, no neonatal complications, and even delayed diagnosis. Neonatal care is also heterogeneous and can be affected by the caseload, the team’s experience, and neonatal facilities; this had led to the centralization of the treatment of patients with this rare disease in some countries.Identifying prognostic factors in the prenatal period remains an important issue. The use of magnetic resonance imaging (MRI), as proposed by the French “Protocole National de Diagnostic et de Soins” has led to significant progress in the evaluation of fetal pulmonary hypoplasia . A fetalTo our knowledge, no studies have evaluated the prenatal prognostic factors for respiratory outcomes during the first years of life. The main objective of our study was to identify a threshold value of o/e FLV that is associated with a better prognosis for respiratory morbidity during the first 2 years of life in a cohort of children with left-sided CDH born between 2009 and 2015 at the French Reference Centre for CDH, Lille University Hospital. The secondary objective was to examine the relationships between the o/e FLV measured on MRI and acute or chronic respiratory morbidity.Our study was carried out at Lille University Hospital in the French reference center for CDH.All patients born between 1 January 2009 and 31 December 2015 with a prenatal diagnosis of left-sided CDH, with at least one fetal MRI performed, and who remained alive at 2 years of age were included. We conducted a retrospective analysis of the data collected prospectively during the follow-up of these children between birth and 2 years of age using a standardized questionnaire. The data were an integral part of the issues routinely addressed during follow-up visits by neonatologists and recorded in medical reports; they were collected from September 2017 to March 2018. The study was approved by the French Data Protection Authority (CNIL) (No. DEC19-328).Data on sex, gestational age expressed in weeks, premature birth, and type of CDH  were collected from the local database. The type of ventilation support , or noninvasive ventilation), the need for ECMO, the total duration (days) and need for supplemental oxygen at day 28 (defined as CLD), were recorded for each patient.2O and a positive end-expiratory pressure (PEEP) between 2 and 5 cmH2O. The fraction of inspired oxygen (FiO2) and ventilator parameters are adjusted to target pre-ductal SpO2 85–95% and PaCO2 45–60 mmHg. The use of high-frequency oscillatory ventilation (HFOV) can be considered a means of reducing baro-volotrauma. It is indicated in situations of persistent hypercapnia (PaCO2 > 60 mmHg) and/or hypoxemia . ECMO is used in CDH infants in the following situations: preductal SpO2 < 85% while inspiratory pressure is > 28 cmH2O (or mean pressure > 16 cmH2O in HFOV) and signs of circulatory failure; or oxygenation index > 40 for more than 6 h; or occurrence of refractory intercurrent septic shock.Management of the CDH infants is based on the EURO Consortium guidelines and the French Reference Center guidelines ,12. NewbTo study the respiratory outcomes during the first 2 years of life, respiratory morbidity was defined according to two criteria: (1) daily treatment with inhaled corticosteroids (ICS) for >3 consecutive months and (2) occurrence of at least one unscheduled hospitalization for lower respiratory tract illness. These two objective criteria allowed a global approach to quantifying respiratory morbidity by using data about the daily treatment, which reflects chronic respiratory morbidity, and hospital admission, which reflects severe acute morbidity. These two criteria have been studied separately in the literature. In 2001, Muratore et al.  reportedAs part of the follow-up performed at the CDH reference center, one or two fetal MRI imaging scans were performed after the prenatal diagnosis of CDH: the first between 26 and 28 weeks and the second between 30 and 32 weeks of gestation. In cases of two MRIs, we used the first images. The fetal MRIs were performed using a Philips Ingenia 3 TESLA MRI imaging scanner. No premedication was given, and the mother was placed in supine position. The examination time was about 30 min. The protocol included three T2 planes (TE 70) and T1-weighted sequences in the axial and sagittal planes. The T2-weighted images were obtained using a single-shot half-Fourier TSE sequence in the orthogonal transverse coronal and sagittal planes according to the fetal orientation. The sequences affected by fetal movements were repeated. The cutting thickness was 4 mm. Measurements were performed by two radiologists qualified for this evaluation using PACS software  using a contour (freehand region of interest) cut. The contouring was performed on the sequence that allowed the best visualization, either coronal or sagittal . The o/eU test. The optimal threshold value of the o/e FLV for predicting a favorable respiratory outcome was determined by maximizing the Youden index. Sensitivity, specificity, and positive and negative predicted values for the optimal threshold value were calculated. All statistical tests were performed at the two-tailed α level of 0.05 using SAS software .Qualitative variables are reported as a number (percentage) and continuous variables as a median (interquartile range (IQR)). The normality of the distributions was assessed graphically and using the Shapiro–Wilk test. The o/e FLV was compared between two groups of patients using Mann–Whitney’s p = 0.047). The median durations of mechanical ventilation and oxygen therapy  were 5 days  and 14 days , respectively. Nine (19%) patients required HFO and two (4%) ECMO. A total of 13 (28%) patients were diagnosed as having CLD. During the hospitalization period, six patients (13%) were diagnosed as having ventilator-associated pneumonia. Four (8%) had a pleural effusion and four had a pneumothorax. Only one patient had chylothorax during his hospitalization.From 2009 to 2015, 62 prenatal diagnoses of left-sided CDH were made. Fifteen patients (24%) were excluded, including two who had no fetal MRI, three who were lost during the systematic follow-up, and ten who died before two years of age. The characteristics of these 10 children and the causes and times of their deaths are listed in p = 0.027). The o/e FLV did not differ between patients who were hospitalized at least once for respiratory reasons and those who were not  versus 42% ; p = 0.082).Sixteen patients (34%) were treated for >3 months with ICS. Thirteen (28%) patients were hospitalized at least once for an acute respiratory tract illness during the first 2 years of life: eleven (23%) in the first year, four (9%) in the second year, and two (4%) at least once a year. The median o/e FLV according to the two criteria are shown in p = 0.024).In total, 28 patients (60%) had a favorable outcome, and 19 (40%) had an unfavorable progression . The medThe optimal threshold value of o/e FLV to predict a favorable respiratory outcome was 44% with a sensitivity of 57%, specificity of 79%, negative predictive value of 56%, and positive predictive value of 80%. In other words, an o/e FLV ≥ 44% was associated with a favorable outcome in 80% of cases.Our study reports on the relationship between lung volume measured by MRI and respiratory morbidity during the first 2 years of life in infants with left-sided CDH. We found that o/e FLV may be useful for identifying patients with a lower risk of respiratory morbidity.Patients with CDH are at risk of pulmonary impairment, although the respiratory risk is heterogeneous. Therefore, it is crucial to identify factors that can help predict the respiratory outcome of these children. Several studies have searched for associations with the mortality or morbidity of different combinations of prognostic factors by focusing on the possible associations between antenatal markers, for example, the associations between o/e FLV and the development of CLD or between postnatal markers such as the dosage of N-terminal pro-brain natriuretic peptide and the need for ECMO support ,9,15.We chose to study o/e FLV, an antenatal marker, using MRI because this technique has many advantages over ultrasound. MRI allows pulmonary visualization in all three planes and a complete view of all thoracic structures, particularly the position of the liver. The study by Büsing et al.  demonstrp = 0.047). In another study by Cordier et al., o/e FLV > 45% was associated with a survival rate better than 75% [Many studies have pointed out that o/e FLV is a prognostic factor for mortality ,22,23,24Recent studies have focused on FLV as a predictor of respiratory morbidity beyond the neonatal period, including the development of CLD or the need for oxygen therapy at 1 year of age ,25. In aTo our knowledge, no study has examined the link between o/e FLV and long-term respiratory outcomes. It is crucial to be able to identify patients at risk for adverse outcomes as early as the antenatal period, as well as those most likely to have a positive outcome, by determining a threshold associated with lower chronic and acute respiratory morbidity; this was our aim in the present study. Identifying a threshold is important for improving information given to parents and for making decisions about treatment and outpatient follow-up care. In our study, an o/e FLV ≥ 44% of PV was associated with a favorable outcome in 80% of cases. By contrast, Debus et al.  reportedWe found a significant relationship between o/e FLV and chronic respiratory morbidity, as reflected in the long-term prescription of ICS. Chronic morbidity and ICS prescription are frequently observed in patients with CDH ,26. MuraWe found that the association link between o/e FLV and severe acute respiratory exacerbations leading to hospitalization was not significant. Respiratory complications leading to hospitalization occur most frequently in the first 3 years of life ,28. BenoThis study was conducted in only one reference center that provided homogeneous and expert management of patients with CDH. This study also included children born between 2009 and 2015, i.e., over a period of 6 years marked by continuity in the management strategy and without major changes. We included only children with a left-sided hernia because right-sided CDH occurs less frequently but has a more severe course and a higher risk of respiratory morbidity ,30,31. HOur study has some limitations. The number of patients was small because of the low prevalence of this rare disease, and this contributed to the low power of the statistical analysis. The small number of patients in this study is probably responsible for the lack of sensitivity of the o/e FLV to predict the risk of respiratory morbidity later in life. The performance of our threshold was moderate, especially in terms of sensitivity and negative predictive value. It will be crucial to confirm the association between o/e FLV and respiratory morbidity in a larger population. Nevertheless, our results can be helpful to the clinician to orient the parental antenatal information. A larger study would allow the prognostic value of this marker to be determined more accurately. The association between CDH and morbidity should be explored over a longer term, including childhood and adolescence. As CDH is associated with impaired lung development, an assessment of the association between lung volume and long-term respiratory morbidity, including lung function, is needed to identify high-risk populations who may benefit from multidisciplinary and personalized management.CDH is a complex disease that has significant heterogeneity in its morbidity. Our findings support the use of FLV as an easily measured component during pregnancy to predict respiratory morbidity during the first year of life. We suggest using a threshold value to identify infants likely to progress along a more favorable course. These data must be confirmed and validated in a larger population. Longitudinal follow-up studies are needed to assess the morbidity during childhood and adolescence. These data are important for improving parents’ access to information during pregnancy and for distinguishing low- from high-risk patients.