Lattice Radiotherapy (LRT) is a technique in which heterogeneous doses are delivered to the target so large tumors can have optimal doses of radiation without compromising healthy tissue sparing. To date, case reports and case series documented its application for bulky tumors mainly in the pelvic region. LRT not only provides dosimetric advantages but also promotes tumor control by triggering some radiobiological and immunological pathways. We report two cases of giant liver metastases for whom other treatment options were not suitable. We treated both patients with Magnetic Resonance Image-Guided Radiotherapy (MRgRT) with online adaptive LRT (OALRT) technique. Adaptive plans were generated before each fraction. Tumors were observed to have regressed interfractionally so the location and number of spheres were adapted to tumor size and daily anatomy of the surrounding organs at risk (OAR). Both patients had good treatment compliance without any Grade 3+ side effects. They are both under follow-up and report improvement. By reporting the first application of OALRT by using MRgRT in liver metastases, we show that MRgRT is a promising modality for LRT technique with better target and OAR visualization as well as online adaptive planning before each fraction according to the daily anatomy of the patient. Treatment of voluminous tumors represents a challenge to radiation oncologists as delivery of high doses to the target lesions bring along the risk of not securing healthy tissue sparing. Spatially Fractionated Radiation Therapy (SFRT) modality was introduced in order the overcome this problem with the idea that the tumor could be irradiated with a heterogeneous dose distribution . SFTR noHerein we report the management of two cases with bulky liver metastases presenting to our clinic. Both patients were not amenable to surgery due to volumes of metastatic lesions. We decided to treat the patient with online adaptive LRT (OALRT) with the aim of preserving the surrounding tissue and benefiting from heterogeneous dose distribution. We have treated both patients by using MRgRT, which enabled better soft-tissue visibility, adaptive planning, and online continuous cine tracking of tumors during the treatment. To our knowledge, this report is the first on LRT using MRgRT in liver metastases.Case 1A 59-year-old female with a non-significant past medical history and family history was diagnosed with squamous cell cancer of the anal canal in 2018 and staged as T2N0M0. Positron emission tomography-CT (PET-CT) revealed a distal rectum tumor with pathological fludeoxyglucose (FDG) uptake in the lymph nodes at the level of the right presacral and lumbal 3 vertebrae. Chemoradiotherapy was offered to the patient, but she refused the treatment. In March 2020, she received 20 Gy in 5 fractions to the pelvic area as a palliative treatment. In the follow-ups, the tumor responded well to treatment but a metastatic lesion in the liver was detected. The patient declined to take chemotherapy and went with alternative medicine. In October 2020, liver metastases were found to have progressed. She did not receive any treatment. In May 2021, she received 36 Gy to pelvic lymph nodes and 50 Gy to the primary tumor bed in 20 fractions with a simultaneously integrated boost technique. Computed tomography was performed in July 2021, and it revealed giant metastatic liver lesions in segments 4B and 6. At that time, her aspartate aminotransferase (AST) was 33 IU/L, and alanine aminotransferase (ALT) was 13 IU/L. Her Gamma-glutamyltransferase was above normal limits and it was 147 IU/L. Chemotherapy was refused by the patient. She was referred to our clinic for stereotactic body radiotherapy (SBRT).Case 2A 70-year-old male patient underwent colonic resection and liver metastasectomy due to rectosigmoid adenocarcinoma in March 2016. Pathological staging was consistent with T3N1M1. He received adjuvant chemotherapy. Between 2019 and 2021, he received chemotherapies and underwent radiofrequency ablation and transarterial chemoembolization (TACE) due to the recurrence of his liver metastases. In 2021, contrasted abdominal CT revealed a large mass in the right lobe of the liver as well as progression in the mass to which TACE was performed. Due to the pandemic, the patient delayed the treatment and he presented to our clinic in October 2021. His last PET-CT report of August 2021 showed up to eight nodules with FDG uptake, and it was consistent with progression compared with his previous PET-CT results. The patient's ALT was 36 IU/L and AST was slightly above the reference range at 50 IU/L.We decided to go with SBRT with lattice treatment planning in MRIdian Linac .SBRT-Lattice TreatmentWe decided to perform SBRT using MRIdian Linac for both patients given the lesions were giant masses located in the liver. The patients were scanned by using a planning CT for electron density map, diagnostic MRI, and Truefisp sequence of MRIdian Linac. Sequences were taken under end-inspirium breath-hold. All the available images were fused for treatment planning.Gross Tumor Volume (GTV) was delineated as the primary tumor. Planning Target Volume (PTV) was extended 3 mm in all directions from GTV. OARs were determined as: esophagus, stomach, spinal cord, superior mesenteric artery, portal vein, pancreas, lungs, liver, bowels, right and left kidney, heart, duodenum, celiac artery, and aorta. A total dose of 30-50 Gy in 5 fractions (6-10 Gy/fraction) was prescribed to the metastatic lesion as valley and lattice structures as the peak. A step and shoot Intensity Modulated Radiotherapy (IMRT) treatment plan was generated with 18 and 23 fields in a total of 119 and 123 segments, respectively, with a single isocenter and 6 megavolt (MV) flattening filter free (FFF) photon energy.For case 1, the dose-volume histogram (DVH) revealed that the dose to 700 ccs of the liver was 10.7 Gy; V33 and V36 of the stomach were 0, and the mean dose to the right kidney was 11.04 Gy while the mean dose to left kidney was 11.4 Gy. The patient's duodenum could not be separately contoured because of the patient's anatomical variations and V36 of bowels was 0.04 cc and V33 of bowels was 1.29 cc. The GTVs were heterogeneous, so the homogeneity index (HI) was calculated for the whole PTV, and it was 0.65, and the conformity index (CI) was 1.03.For case 2, the dose to 700 ccs of the liver was 28.49 Gy; V33 and V36 of the stomach were 0, and the mean dose to the right kidney was 9.1 Gy while the mean dose to the left kidney was 3.37 Gy. V33 and V36 of the duodenum were 1.03 and 0.23 cc respectively and V33 and V36 of bowels were both 0. HI was calculated as previously mentioned and it was 0.58. CI was 1.33.An adaptive online plan was generated during all fractions (5 of 5 fractions) for both patients. The lattice spheres were relocated (or erased) if needed due to gross tumor volume shrinkage and proximity to OARs during each fraction. The plan of the original and reoptimized target volumes and spheres as well as the close proximity between GTV and OARs for case 1 are demonstrated in Figures Figures Both patients tolerated the treatment well without any acute Grade 3-4 toxicity according to CTCAE (Common Terminology Criteria for Adverse Events) v4. For Case 1, the mass was visibly regressed at the end of 5 fractions. PET-CT scan in the third month revealed that the mass showed near-complete regression. The patient accepted the chemotherapy and she will be evaluated for surgery. Case 2 showed a mild increase in liver function tests in the second fraction, but it did not persist. One month after the treatment, an abdominal CT revealed a regression in the dimension of GTV (pre-treatment GTV 494 cc to post-treatment GTV 230 cc).Spatially Fractionated Radiation Therapy differs from the standard radiotherapy approach by delivering in homogeneous doses to the target tissue. It has been decades since this concept was defined and the primary idea behind it was to palliate symptoms caused by bulky tumors by giving less dose to the areas close to OAR while still reaching a high biologically effective dose at the tumor . The hisIn GRID radiotherapy, the tumor is divided into valley and peak areas. Valleys, also called cold spots, are areas that are receiving the lower radiation dose, whereas peaks, also called hot spots, are the areas where the higher dose is prescribed . In the It was not until 2010 that we were introduced to LRT which can be defined as a three-dimensional extension of GRID radiotherapy . LRT proThe dosimetric advantage yielded by LRT not only demonstrates technical advantages but also helps disease control by triggering various radiobiological and immunogenic mechanisms . The rad. In their retrospective analysis of 10 non-small cell lung cancer patients, Amendola et al. report a mean tumor size reduction of 52% with no grade 2-3 radiation pneumonitis, hence they declared that LRT is a safe technique to apply cytotoxic radio-therapeutical doses [Indications of LRT are expected to widen in the future with its use not only limited to bulky tumors since we can also benefit from its radiobiological and immune-modulator effects . It is eal doses . They apal doses .Duraseti et al. reported a case series of 11 patients with >12 cm tumors. They prescribed a dose of 2000 cGy to the entire tumor and 6670 cGy to peak regions. The tumors were of different histologies from various sites. Dose constraints could be achieved in all patients except one in whom the tumor was in close proximity to skin and all plans achieved adequate coverage. They concluded that the planning can be successfully performed for bulky tumors . In theiHerein, to our knowledge, we present the first documented case of LRT application in an MR-Linac device with adaptive planning in each fraction. MRgRT allowed us to visualize the tumor with the advantages of better soft tissue discrimination in MRI with daily adaptive planning and modulation of sphere location and sizes by seeing the regression in the target volume. By means of this, the spheres that would be in proximity to surrounding tissue due to tumor shrinkage were deleted and high doses to vulnerable tissues were avoided. The patients tolerated the treatment well with visible tumor regression. LRT is a promising technique in bulky tumors, and it enables the delivery of higher doses without compromising on dose constraints. MRgRT can be safely and effectively used for LRT.


Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to predict enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify genomic regions with histone modifications consistent with enhancer activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high-confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for predicted transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Footprinting analyses provided further evidence that these potential TF binding sites are likely to be occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of TFs in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. We found that our predicted GRNs were independently supported by existing TF RNAi/RNA-seq datasets, providing further evidence that our work predicts active enhancers that regulate adult stem cells and regenerative mechanisms. The molecular and evolutionary mechanisms of regeneration remain underexplained compared to animal development. This can be attributed to the greater technical difficulty historically associated with studying the molecular mechanisms of adult biology compared with development. However, an ever-broadening repertoire of model organisms for regeneration, progress in understanding the variety of cellular and molecular mechanisms used across taxa, and advances in experimental tools are serving to close this gap. One area of slower progress has been our knowledge of the regulatory logic of regenerative mechanisms, with relatively few studies in highly regenerative models providing precise insight into the epigenetic regulation of regeneration. While transcription factors (TFs) have been assigned functions, and in some cases a list of likely targets through transcriptome analysis, regulatory logic has been studied in only a few cases . This isNothobranchius furzeri, and the fruit fly Drosophila melanogaster, and at the whole-body level in the acoel worm Hofstenia miamia and the cnidarian Hydra vulgaris  is known to mark active enhancers together with mono-methylation of lysine 4 on histone H3 (H3K4me1), whereas H3K4me1 alone marks predetermined or poised enhancers , while the new coding and non-coding transcripts were much shorter  . HoweverS. mediterranea (70 %), but 30% had a homolog in the Dugesia japonica genome . The naming of planarian TFs in the literature was mostly consistent, but some inconsistencies were found , many of which have not received much consideration yet in planarian regeneration research. While some of these unstudied ZNFs could be assigned to well-known ZNF families such as GATA, KLF, EGR, and PRDM, many could not be assigned to well-described families. The nomenclature of ZNFs was based on the naming of human proteins to which they have the highest identity, and hence many appear in the ZNF and ZNP categories . InteresNkx and Six, and a few TFs belonging to families not previously described in planarians were also discovered, such as Lmx and Shox .We proceeded to allocate proportional expression values to the TFs with respect to the X1, X2, and Xins cell compartments . The diszfp-7, pbx, prep, gli-3, and rfx7, while the least informative motifs were for hesl-2, pou2/3, irx, tead1, and lmx1  (zic-1) (zeb-1 and snail-1/2)    , and TFs (zic-1)  and cellail-1/2) . An enha foxA(P) . A distaed ets-1 . A dista foxA(P) , neural A(P)  smad1/9 , isl-1, and prep  and multiple bound motifs at enhancer-like regions linked to tbx genes    (We identified the same five planarian d tbx-20 . In mammem cells . Nonethe motifs)  and. tbx-1, sp5) , TFs inv-1, sp5) , and var-1, sp5) .Next, we studied enhancer-like regions and ppotential regulatory links associated with the well-known planarian PCGs. Planarians are a primary model system to understand how positional information guides and directs stem cell function during regeneration , and thesp5 and the Hox genes post-2c, post-2d, lox5a, and hox4b are rapidly downregulated  animals. Upon wnt1 knockdown, posterior Hox genes hox4b, post-2c, post-2b, lox5a, lox5b and wnt11-1, wnt11-2, fzd4, and sp5 were downregulated, replicating the results of β-catenin knockdown  and a few motifs were found to bind to the enhancer-like regions linked to prep, zic-1, isl-1, and foxD1  and nkx2-2/3/4  causes blastemal defects both at the anterior and posterior ends, while knockdown of prep leads to defects in the anterior, suggesting that nkx2-2 might work upstream of prep at the anterior end (anarians . Interesnd foxD1 . Prep hax2-2/3/4 . Knockdorior end .patched-1 (ptch-1) and found an intronic enhancer-like region containing multiple bound motifs, including the Hedgehog pathway TFs gli-1/2 as would be expected, for the Wnt effector sp5, the Hox genes lox5a/b, BMP signaling components , the anterior pole TFs prep and zic-1, the cell migration factors snail-1/2, and the muscle TFs myoD and twist  (foxD1(RNAi)  , foxD1(RD1(RNAi) , pax2/5/x2/5/8-1 , myoD of whole worms, we observed eight DETFs, and by collating these two datasets, we obtained pou4-1 as the only DETF containing a putative bound coe footprint . This puS. mediterranea by integrating available RNA-seq data and identified new coding and non-coding transcripts. We reviewed the literature on both computationally and experimentally derived planarian TFs and defined a high-confidence set of TFs. If possible, we also predicted binding motifs for the planarian TFs. We developed and performed ATAC-seq on the proliferating stem cell compartment and determined genomic regions of open chromatin. We analyzed genome-wide profiles of the histone modifications H3K27ac and H3K4me1, along with chromatin accessibility data, and used these epigenetic datasets to delineate putative active enhancers in planarian stem cells. Lastly, we identified TFs binding to the enhancers of potential target genes and constructed hypothetical GRNs active in the stem cells.In this study, we improved the current genome annotation of We found more than 50,000 new isoforms to known transcripts in our annotation. This information has already helped to improve the clustering of single-cell RNA-seq data . Our appAltogether, we annotated 551 TFs that were distributed evenly with respect to enrichment across all three FACS cell compartments in planarians . In planarians, most studied functionally have been involved in regulating differentiation. Here, we define many neoblast-enriched TFs that have not been formally studied, with potential roles in maintaining neoblast pluripotency and potentially early lineage commitment. A future systematic functional screen of these may help to uncover the GRN network that maintains pluripotency analogous to that in mammals and vertebrates . In partOverall, for the main goals of this study, our analysis and identification of TFs allowed us to confidently assign likely binding motifs to just under half of the annotated planarian TFs . In the Based on combining epigenomic experiments and data types, we could identify putative intergenic and intronic enhancers in the planarian the genome of proliferating stem cells. The combined use of ChIP-seq data, RNAi of a histone methyltransferase combined with ChIP-seq, ATAC-seq data, and footprinting analyses together provided strong evidence for the identification of bona fide planarians stem cell enhancers. In the future, genome-wide ChIP-seq against the transcriptional cofactor p300 could serve as a complementary high-throughput approach to further substantiate these enhancers , if avaiVerification of the function and targets of planarian enhancers is currently not possible using traditional approaches as no transgenic reporter technologies enabling enhancer-reporter constructs to be assayed are available. Therefore, we are bound to rely on less direct evidence from genome-wide sequencing technologies. Here, we assigned enhancers to target genes based on distance and the expression of the target gene, similar to established protocols used by others . We findTaken together, our definition of enhancers in stem cells genome wide creates a foundation for constructing detailed GRNs to help understand regenerative mechanisms driven by stem cells in planarians.The sexual genome SMESG.1 genome, the SMESG high-confidence annotation (SMESG-high release 1), and the SMESG annotation filtered for repeats (SMESG-repeat release 2) were downloaded from PlanMine . Availabhttps://github.com/jakke-neiro/Oxplatys/raw/gh-pages/Schmidtea_mediterranea_Oxford_v1.gtf.zip) was compared to the SMESG-high annotation with gffcompare . Hierarchical clustering of FACS proportions was performed using Euclidean distance and Ward’s method with the package hclust . The freThe motifs of the TFs  were pre2, 0.1% NP-40) was added, the cells were centrifuged , and the nuclei pellet was collected. Then, the transposase mix  was added, and the cells were resuspended and incubated . Finally, DNA was isolated using the Zymogen Clean & Concentrator Kit, and eluted in EB buffer.A standard protocol was used for preparing the ATAC-seq library . The X1 Subsequently, the eluted DNA was used for PCR amplification and library purification. DNA was amplified using a standard reaction . Finally, the libraries were cleaned with the AMPure bead purification kit. Samples were paired-end sequenced on the Illumina NextSeq. The sample replicates can be found on Sequence Read Archive (SRR18923214 and SRR18923213).The H3K27ac ChiP-seq library was prepared and sequenced using established protocols . A totallpt(RNAi) was reanalyzed . We certainly agree that each individual predicted enhancer and regulatory interaction of interest needs to be studied for its exact role in stem cells and/or during regeneration and will make sure the revision of the text reflects this.We have added extensive new analyses to address two of the reviewers’ comments that further functional evidence was required through new experiments using RNAi of TFs coupled to RNA-seq, to find independent evidence of for links between TFs and predicted targets. As this research project was affected by the pandemic, we were unable to currently afford this significant extension to the work in terms of lab time or resources, rather we see this as the work of many labs in the coming years. Nonetheless, we acted on the reviewers’ suggestions by using existing TF RNAi/RNA-seq data sets for TFs that we predict to be active in the stem cell regulatory networks predicted in our initial submission . Despite these experiments being performed in mixed tissues  we still observe clear evidence for differential expression of TFs predicted by our enhancer and footprint predictions to be downstream of the TF targeted by RNAi. This improves the confidence of our original findings significantly and we are grateful for the reviewers’ for suggesting this approach. Overall, we strongly agree with the reviewers’ comments that our work will be a valuable resource for those studying regeneration and animal stem cells.Reviewer #1 ):In this manuscript Neiro et al. aim to expand our knowledge on the regulation of gene expression in stem cells of the planarian model organism.As a first step the authors used published available data to expand the repertoire of the planaria transcriptome. By combining 183 RNAseq datasets the authors were able to identify thousands of new coding and non-coding transcripts. They then screened for TF motifs in the new annotations, identifying 551 putative TFs, of which 248 were already described in the planarian literature.The most substantial contribution of this work to the field of stem cells and planaria biology is the characterization of new putative enhancers that were identified by performing H3K27ac ChIP-seq and ATAC-seq and combining these data with previously published H3K4me1 ChIPseq dataset.We thank the reviewer for their careful assessment of our work, we agree that the identification of likely enhancers genome wide is a substantial contribution. Equally the improved annotation of all genes, including the collected set of transcription factors we choose to focus on here, is a substantial step forward for the planarian research community.By overlapping H3K27ac and H3K4me1the authors find 5,529 new enhancers, for which they report a higher chromatin accessibility than random points in the genome as assessed by ATAC-seq. By using ATAC-footprints Neiro et al. refined the subset of TFs that have binding motifs in the predicted enhancer-like regions and present a list of 22,489 such factors.The manuscript is well written and organized and overall, the reported data will provide an important resource to study gene expression regulation in planaria's stem cells. However, this manuscript would greatly benefit from some functional validation to support the predicted gene regulatory networks. One option would be to use a CRISPR-dCas9-KRAB system to silence the putative enhancers identified in the manuscript and check by qPCR the expression of nearby genes.Currently mis-expression technologies, in order too directly test enhancer elements in driving expression, are still not available in planarians. This also preempts us using the suggested silencing system used in mammals and other animals with robust mis-expression tools.If this type of experiment is not feasible in planaria (I am not an expert in this model organism) another simple but key experiment would be to perform a knockdown of one (or more) putative enhancer-bound TFs identified in this study followed by RNA-seq. This would allow the authors to verify what are the target genes of the putative enhancer-bound TFs and if they correspond to the predicted gene networks they identified. Simultaneously, this experiment would allow the authors to verify if there are any changes in the expression of differentiation/pluripotency markers as a result of the knockdown of the putative enhancer-bound TF.These experiments are possible, but this would be the work of many labs in the future expert in studying those TFs, the organs and tissues they are expressed in, and their roles in planarian stem cells and regeneration. However, what we have done is analyze existing RNA-seq data collected after specific TF RNAi . There are several studies where TF have been studied and RNA-seq performed after RNAi. Although these studies are performed in specific experimental regenerative contexts, and not specifically in stem cells, it was nonetheless possible to look at expression changes of potentially downstream TFs with predicted enhancers for those TFs knockdown by RNAi. We analyzed these data and added it to the manuscript, rather than perform further TF RNAi experiments, as this was just about feasible within a 3-month revision time. We would add that currently there are no genes implicated in controlling pluripotency in the same way we might consider, for example, OSKM in mammals. Our identification of the TFs enriched in stem cell expression and implicated in binding predicted enhancers suggests future candidates.Our additional analyses  clearly show that TF predicted to be regulated by our enhancer predictions are significantly enriched amongst differentially expressed TFs after RNAi of the predicted regulating TF. We observed differences between the levels of unbound enhancers (10x enrichment) and bound enhancers with footprint evidence (21x enrichment) across all the TF RNAi/RNA-seq studies, and a correlation between the predictive power of our GRNs and the proportion of expression of the knockdown TF in the stem cell compartment. This suggests that our predicted GRNs are reflective of activity in stem cells, but not of other cells.Minor revision:The authors have mostly focused on the identification of enhancer-bound TFs. However, it would be interesting to look at differential enrichment of TFs in promoters versus enhancers and identify if there are specific factors that are enriched specifically at the planarian newly identified enhancer regions.We have not looked at potential TF binding sites near promoters/transcriptional start sites. We have added an analysis to look at this . This analysis does not reveal any TFs or TF families that show a pattern of having binding sites that are more proximal to promoters than others. We did not pursue this further as it was beyond the scope of our study.All tornado plots are missing a color bar We have fixed this error.There is a typo in the discussion: "the combined use of ChIP-seq data, RNAi of a histone methyltransferase combines with ChIP-seq" should be changed to "combined".We have fixed this and other typographical errors.Reviewer #1 (Significance (Required)):The manuscript is well written and organized and overall the reported data will provide an important resource to study gene expression regulation in planaria's stem cells.We thank the reviewer for their appreciation of our workReferees cross-commentingI agree with the other reviewers that additional functional data should be added to support the author's claims .See response above, with regard to adding further analyses , see Figure 8 and new Results section.In addition, as noticed by the third reviewer, all data should be made publicly available to the scientific community.We have made all data publicly available and have submitted all relevant data to public database repositories in advance of final publication.Reviewer #2 ):Summary:Schmidtea mediterranea. The authors start with the integration of transcriptome with genome sequencing data to more precisely annotate the genome of the planarian Schmidtea mediterranea. The second part of the manuscript actually then deals with the identification of potentially active enhancer elements in adult stem cells of this regenerating organism using genomic techniques like ATAC-seq and ChIP-seq of histone marks combined with motif searches and in silico footprint analysis. Using these data, the authors predict regulatory interactions potentially critical for pluripotency and regeneration in planarian adult stem cells.This manuscript aims at identifying enhancers in the planarian Major comments:- Are the key conclusions convincing?1) The authors claim  that their study identifies enhancers regulating adult stem cells and regenerative mechanisms. This is an over-statement found throughout the manuscript, as none of these enhancers are functionally tested nor is it shown that target gene expression changes when transcription factors predicted to interact with such enhancers are knocked down.We agree and it was not our intention to overstate our results, this is why we have tried to refer to putative enhancers, enhancer-like elements etc in manuscript from the title onwards. Only once we have demonstrated a set of elements with key conserved and widely supported characteristics do we suggest we have a set of higher confidence enhancers to study. However, we have adjusted the manuscript to reflect that our claims await direct testing as is the case for all enhancers implicated with the approaches used here.Another example is at the end of paragraph 1 of section 2.4. Here the authors claim that identifying many fate-specific transcription factor genes in the vicinity of potential enhancers is a further proof that the identified regions represent "real enhancers". It strongly supports this hypothesis, but no evidence for real enhancer activity.We agree the total body of evidence strongly supports that we have identified enhancer elements, but have adjusted the language to suggest further directed functional work will follow from many groups.Thus, although the authors state that the regulatory interactions and networks they predict from their data can be studied now in future, they should be more careful with their wording and correct these over-statements. Therefore, the key conclusion is that they identified by various techniques potential enhancers, which are close to genes controlling adult stem cells and potentially controlling these genes, which has to be shown by further analyses.We agree.Thus, also the title needs to be changed.We have changed ‘enhancer-like’ to “putative enhancer-like” and “defines” to “predicts” in the title as well as broadly adjusting the text to caveat that further work will clarify their functions and roles.The authors have no proof that the networks are active in planarian adult stem cells, as they do not show that the predicted networks are active in the presented way.We agree, see comments above. It was not our attention to claim we are showing pathways that were definitively active, but rather predicted by our experiments and analyses of the data from these experiments.2) Similarly, the identification of TF motifs within these potential motifs strongly suggests but not shows that these factors are binding, even when these sites were found to be bound by a protein using the ATAC-seq footprinting analysis. Thus, the authors need to be careful with their wording. One example is in the second paragraph of section 2.5, where the authors write that "We found that numerous FSTFs were binding to putative intronic enhancers.… ". The motif suggests that these factors bind, however, they have no experimental confirmation that these sequences are indeed bound by the planarian TFs.We agree and have been careful to emphasize “predicted” and “putative”Schmidtea mediterranea. The manuscript is informative yet descriptive, as tit presents no functional evidence for any of the predictions. If further toned down, the key conclusions are valid.In sum, this manuscript uses existing genomic tools to define potential enhancer regions in the planarian Future functional experiments to test the roles of all TFs and enhancers is now possible due to our work. The combination of data and analyses provides strong support of enhancer elements activity in stem cells across the genome. We have added analyses of previous RNAi work on TFs which provide strong cross validation for our predicted TF GRNs  and RNAi of the H3 K4 mono-methyl transferase provides genome wide functional validation of the predicted enhancer regions.– Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The experiments performed are well designed and in line with what is known in the field about enhancer architecture. However, as this model system is not very well characterized on that level and the authors do not provide real experimental evidence that any of the identified regions has really enhancer activity and that any of the identified motifs binds indeed the predicted TF, the authors need to be very careful with their statements. The authors should maybe emphasize even stronger that all the GRNs predicted under section 2.6 are really preliminary and need to be validated.Yes, we are happy to have been even clearer about this as the reviewer suggests– Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.One experiment that could provide more evidence for their predicted regulatory interactions is to knock-down one of the FSTFs for which motifs have been identified in potential enhancer regions and to study expression of associated genes (to confirm that the enhancers potentilla bound by these TFs control the expression of associated genes) or by analyzing the chromatin status of selected chromatin regions (by Q-PCR). These experiments would strongly support the claims of the authors. However, it also depends strongly on the journal whether I would consider these experiments essential or "nice to have".This suggestion of possible extra experiments is very similar to that of Reviewer 1. We are copying our earlier comment as this also addresses this point.These experiments are possible, but this would be the work of many labs in the future expert in studying those TFs and their roles in planarian stem cells and regeneration. However, what we have done is analyze existing RNA-seq data after specific TF RNAi, where this data is available. There are several studies where TF have been studied and RNA-seq performed after RNAi. Although these studies are performed in specific experimental regenerative contexts, and not specifically in stem cells, it was nonetheless possible to look at expression changes of potentially downstream TFs with predicted enhancers for those TFs knockdown by RNAi. We analyzed these data and added it to the manuscript, rather than perform further TF RNAi experiments, as this was just about feasible within a 3-month revision time. We would add that currently there are no genes implicated in controlling pluripotency in the same way we might consider, for example, OSKM in mammals. Our identification of the TFs enriched in stem cell expression and implicated in binding predicted enhancers suggests future candidates.Our additional analyses  clearly show that TF predicted to be regulated by our enhancer predictions are significantly enriched amongst differentially expressed TFs after RNAi of the predicted regulating TF. We observed differences between the levels of unbound enhancers (10x enrichment) and bound enhancers with footprint evidence (21x enrichment) across all the TF RNAi/RNA-seq studies, and a correlation between the predictive power of our GRNs and the proportion of expression of the knockdown TF in the stem cell compartment. This suggests that our predicted GRNs are reflective of activity in stem cells, but not of other cells.– Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.Schmidtea mediterranea, thus it is hard to judge how time consuming these experiments would be. Cost-wise they should be feasible, as it would include primarily Q-PCR experiments. And some functional back-up of their claims would be very helpful.This reviewer is not an expert in See previous comment regarding additional analyses. We have managed these just about within the 3 month revision window.– Are the data and the methods presented in such a way that they can be reproduced?For the parts I can judge, yes.– Are the experiments adequately replicated and statistical analysis adequate?It is not clear from the manuscript how many replicates of the ChIP-seq experiments were done.ChIP-seq replicate data (duplicates) has been explicitly added to the methodsMinor comments:– Specific experimental issues that are easily addressable.– Are prior studies referenced appropriately?For the literature I can judge, yes.– Are the text and figures clear and accurate? The figures are clear, the text (besides over-statements) is clear. However, the writing can be improved. A few examples: section 2.2 paragraph 1: "… we found 248 to be described in the planarian literature in some way." In which way described?; same paragraph: "… but significantly we could identify new homologs of.…" what does significantly mean? Which test etc? section 2.2, last paragraph: "Most TFs assigned to the X1 and Xins compartments and the least to the Χ2 compartment", "Very few TFs had expression in X1s and Xins to the exclusion of Χ2 expression as would be expected by overall lineage relationships"; what do these sentences mean?We thank the reviewer for paying careful attention to the language in our manuscript throughout. We will provide clearer explanation of the sentences indicated. We have better explained terms specific to the planarian model system that are obviously not intuitive and be clear in the figure legends, methods and included code notebook about statistical tests.– Do you have suggestions that would help the authors improve the presentation of their data and conclusions?No over-statements.See previous comments agreeing with the careful adjustment of our language to avoid this.Reviewer #2 (Significance (Required)):– Describe the nature and significance of the advance  for the field. This manuscript identifies genome-wide potential enhancers in adult planarian stem cells, and thus represents a very valuable resource for the community to study these enhancers and the gene regulatory networks they control in the future.– Place the work in the context of the existing literature .As I am not a planarian scientist, it is hard to judge this part.– State what audience might be interested in and influenced by the reported findings.In my opinion, this work will be primarily interesting for people working with planarian. When functional data exist, this might be also interesting for researchers working generally on regeneration.Given the nature of our data we also think all groups working on animal stem cells would be interested in our data and analyses.– Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.Schmidtea mediterranea.My field of expertise is transcriptional regulation using genomic techniques, however I am not familiar with the model Reviewer #3 ):Schmidtea mediterranea and new ChIP-seq and ATAC-seq data to use computational approaches to identify putative enhancers in the planarian genome. They integrate analysis of enhancers with transcription factor binding sites to generate testable hypotheses for the regulatory function of transcription factors active in stem cells or control of cell lineage trajectories. Their work creates an excellent resource for future work to resolve the regulatory logic underpinning stem cell biology and tissue regeneration in planarians.Neiro et al. capitalize on existing genomic data for the planarian We are glad the reviewer likes our research.Major: Overall, the work in this manuscript and methodology are well executed and presented. However, the authors should consider the following comments to improve the clarity and accessibility of the data and interpretations.1) The new transcriptome does not appear to be publically accessible. The links to Github resources are broken, and there is nothing on Neiro's Github page. Will the new transcriptome be integrated with Planmine?annotation has been available for over a year as we wished the community to have access to it ASAP . We tested the links in the paper before depositing our preprint and after review and they seemed to work for us both within and outside our institutional network. We can only apologize if they were broken or have not worked for the reviewer. We are unclear if this new annotation will be included in Planmine, but we have asked the colleagues maintaining this database to consider including it. Our recent analyses of Planmine suggest that our annotation is now all included as all the new genes have appeared, even if our work is not explicitly referenced.The new 2) Figure 1: Ternary plot in 1F. The legend is not clear or could be explained better. What is the metric? It could be my misunderstanding, but I didn't consider the ternary plots as insightful or unnecessary. Perhaps the authors can expand on what they are showing.These plots are important in demonstrating the distribution of mRNA expression of all genes across cell sorted compartments. Given the broad lineage relationship between sorted cell compartments This analysis allows us to identify genes expressed predominantly in one cell compartment or another, or across a specific transition. For example, genes enriched in Χ2 cells and Xins, but not X1 are likely to be enriched in post-mitotic differentiating progeny and differentiated cells. In contrast to single cell data where expression data can be sparse this analysis with bulk data allows identification and assignation of lowly expressed genes, like transcription factors. We will provide some further explanation of this in the revised text.1I is a map of exons, not alternative splicing. So, it isn't clear what the authors intend t show. Are the specific exons that are more likely to be spliced? Is the figure necessary?We wished to demonstrate the power of the annotation approach and the richness of the annotation for looking at alternate splicing. We have provided a more informative figure that indicates the variety of splice forms. We apologize for this oversight.3) Figure 2: 2A labels Xins as irradiation responsive. Is this the case (just making sure)?The reviewer is correct, this is wrong! This should read “insensitive” or “resistant” In Figure 1A. We thank the reviewer for spotting this error. We have fixed this.2F-G: Ternary plot in F seems redundant with G, but that could be my lack of understanding. In 2G, what is represented on the plots on the right of the hierarchical clusters?The ternary plot (2F) and heatmap of hierarchical clustering (2G) are complementary ways to visualize the proportional expression values of transcription factors. The ternary plot (2F) allows an overview of all the proportional expression values, while the heatmap (2G) shows how the proportional values may be grouped into clusters of similar expression profiles and displays the relative size of these clusters. For example, the heatmap shows that the clusters of X1 and Xins are more prominent than Χ2, suggesting that there are relatively a few Χ2-specific transcription factors. We have edited the text to better to explain this difference.4) Figure 3: The heat maps need a legend . In addition, labeling the figures could help the reader. For example, in G-J, a header about the different experiments above each map, such as "enhancers" and "random," etc., would make the figure more accessible.We agree the figures to be more easily interpretable and provided an independent scales and labels for the heatmaps.5) Figure 5: Although it is in the figure legend, the authors could label the 6th track as "RNA-seq in X1."We have added this to the figure.6) Section 2.6 second page last sentence of the first paragraph "GRN of asexual reproduction is not active in neoblasts" data in the supplement? Is it not shown?We apologize for this poorly written sentence. In line with Reviewer 2s comments this statement needs to be toned down and clarified. The raw information is included in the general table of enhancers (Supplementary File 2), but the genomic tracks visually highlighting the motifs at the promoters of lox5b and post2b are presented now Figure S4 A and B.7) Discussion: The discussion about pluripotency factors in planarians could be expanded. The authors could contrast the study's findings with Önal et al. 2012.We agree have expanded our discussion. But we very little is known with regard to TF roles in controlling pluripotency in adult stem cells.Minor: The manuscript has no page numbers or line numbers, so I'll provide a general location of the potential issues.1) Section 2 – newly identified isoforms are shorter (1656 vs. 1618). Is the order of the median length reversed?Yes, we have corrected this.2) No mention of Figure S1B in the text.We have added a correct reference to this figure in the presentation of transcript diversity.3) Figure 1H should be 1I in the text?Yes, we have corrected this4) The discussion contains some minor typos and grammatical errors.We have addressed with careful rereading.We thank the reviewer for spotting these errors and we will fix them in revision.Reviewer #3 (Significance (Required)):Neiro et al. provide an excellent resource for the planarian community. The paper is generally very well written and easy to read. The new transcriptome described, which improves the annotation of the planarian genome, should be made readily available. It would be excellent if the transcriptome could be incorporated in Planmine.The annotation  has been available since the pre-print for Garcia Castro, 2021, Genome Biology was deposited in BioRxiv.Schmidtea mediterranea. Their work provides insight into which factors are highly expressed in the stem cell compartment. Their computational identification of transcription factors and putative enhancers will be helpful to the growing community of researchers studying stem cell and regenerative biology using planarians. In addition, the large dataset generated in this study could inform studies in the evolution of regulatory sequences and transcription factor function.Referees cross-commentingFurthermore, the authors provide a comprehensive list of transcription factors in the planarian The data presented are well supported by previous studies. As noted by the authors, it is not possible to make transgenic planarians, and thus the field needs to rely on indirect methods. The authors focus on using the stem cell population, which can be isolated from the animals. Overall, I don't think additional experiments are necessary. Additional RNAi experiments combined with RNA-seq (using the stem cells) could take 6-12 months to complete. I believe this is a solid contribution that should be framed as a resource paper. The authors should pay close attention to Reviewer #2's suggestions and edit the paper accordingly.I have 20 years of experience in the field. It would be unreasonable to ask the authors to do more experiments, especially in this post-pandemic environment. I hope this helps.We thank the reviewer for their comments.


The reagent has the potential to act as a fluorescent labelling agent with azide-containing substrates, including biological molecules.We report the synthesis of a bipyridyl reagent containing a strained alkyne, which significantly restricts its flexibility. Upon strain-promoted alkyne-azide cycloaddition (SPAAC) with an azide, which does not require a Cu catalyst, the structure becomes significantly more flexible and an increase in fluorescence is observed. Upon addition of Zn( ii), the fluorescence is enhanced further.A bipyridyl reagent containing a strained alkyne 7, reacts with benzyl azide to give a significantly more flexible product 10 and an increase in fluorescence is observed. Upon addition of Zn( Indeed, misregulation of zinc in the brain is implicated in a range of neurological disorders such as epilepsy and Alzheimer's disease.4 Abnormal zinc levels are also intrinsically linked to cancer and are an important factor in apoptosis of malignant tumours.8 These factors have driven research into treatments or diagnostic tools for Zn(ii), through the design of complexation agents and sensors. Molecules which modulate their fluorescence upon undergoing a reaction have the potential to selectively indicate the location of specific functional groups or analyte, for example azide- or alkyne-containing proteins or other synthetic molecules.9 Azido coumarin 1 is an example of a reagent which increases significantly in fluorescence upon cycloaddition with an alkyne.9 In cases where the partner is a strained alkyne,10 the cycloaddition reaction can take place without the need for a copper catalyst,11 which is highly advantageous in a biological setting due to its bioorthogonality , which are known to be highly emissive.14 Related reagents based on the same dipyridyl scaffold have been reported,15 providing some precedent for our synthetic approach.Although strained alkynes such as 3–5 can be functionalised with a fluorescent group,16 from iodopyridine 9 using a new protocol, and then cyclising the reagent through reaction with but-2-yne-1,4-diyl bis(4-methylbenzenesulfonate), following our established protocol, to give 7 in 41% yield. The 1H NMR spectrum of 7 exhibited diastereotopic CH2 resonances indicating conversion between atropisomers is slow on the NMR timescale . Correspondingly, it was found that compound 7 was not an effective bidentate ligand, likely due to the large C–C biaryl torsional angle; our previous solid-state studies of biphenol strained alkynes evidence significantly non-planar C–C biaryl bond dihedral angles  accelerates the reaction however it is unlikely to chelate to the two pyridyl rings due to the likely wide torsion angle in the strained alkyne (vide supra).The cycloaddition of 7 with benzylazide was studied , using 12 are overlaid in 2 resonances are broad, indicating that the detected hydrogen atoms are alternating between different conformations due to restricted rotations on the NMR time scale, as previously observed for cycloadducts of this type.12 A variable temperature 1H NMR study of 10  revealed that the slow exchange of the atropisomers is reached at 243 K, contrasting 7 which is resolved at ambient temperature, indicating greater flexibility of 10, however coalescence was not reached at 323 K  was added to the uncomplexed NMR sample, resulting in sharp NMR peaks similar to the Zn(ii)-complexed product 10  added before the cycloaddition reaction has three singlet peaks that each integrate to two hydrogens, while the compound which had Zn(ii) added after the cycloaddition reaction has two singlet peaks, one of which integrates to four hydrogens and the other which integrates to two hydrogens. Since Zn(ii) is able to form complexes with up to three bipyridyl compounds, this could be due to a different ratio of Zn(ii) to bipyridyl in each sample.14 The observations indicate that the Zn(ii) complexation is likely to be responsible for the change in appearance of the NMR spectrum, possibly due to the flattening of the ring and reduction of the degrees of freedom (It is unclear why the compound which had Zn( freedom .ii) complexation with a bipyridyl strained alkyne cycloaddition adduct is novel, Zn(ii) complexation with bipyridyl compounds, to create highly fluorescent adducts, has been previously studied in detail.14 Zn(ii) complexation has been shown to increase fluorescence intensity, a property which could potentially be beneficial for fluorescence-based reagents. The effect of Zn(ii) complexation on fluorescence was also observed on both the cycloaddition bipyridyl product 10 and the precursor bipyridyl diol 8. All samples were made to the same concentration (10−5 M), and values were recorded at the same parameters on the same instrument (Whilst this Zn(strument . The str2 (ii) to the solution of 7 resulted in quenching of the fluorescence (ESIii) for cycloaddition bipyridyl product 10, it was complexed with several other metal salts to investigate the selectivity of the system. The 3D fluorescence spectra were recorded for each of the metal complexes -complexed bipyridyl 10 exhibited a significantly higher fluorescence intensity compared to the other metal complexes; none of the other metals enhanced the fluorescence of the bipyridyl 10; moreover, the other metal complexes had lower emission intensities than even the uncomplexed bipyridyl 7. 2-complexed and uncomplexed bipyridyl diol precursor 8 17 was also measured using 3D fluorescence spectroscopy (The Zn(troscopy .ii) complexation caused a dramatic increase in the fluorescent intensity of the compound, although the selection of excitation wavelength was crucial in establishing the sharpest difference between the complexed and uncomplexed emission. With excitation at 405 nm, sharply contrasting emission results were obtained compared to those at 360 nm excitation, where little difference was observed (ii) by diol 8.Once again, the Zn(observed . The higii) salts are added to it, which is selective across the ranges of metals studied.In conclusion, we have prepared a new strained alkyne based on a bipyridyl dioxy backbone and have applied this to strain-promoted cycloaddition reactions with benzyl azide. Metal complexation is precluded on the alkyne compound 7 due to the rigidity and non-coplanarity of the bipyridine. Upon triazole formation, product 10 increases in conformational flexibility, allowing coordination to a range of transition and alkaline earth metals, as evidence by photophysical studies. Importantly, the fluorescence intensity increases significantly when Zn supporting this publication can be accessed at The authors declare no conflicting interests.RA-009-C9RA06866J-s001


Different locations were also chosen to assess the influence of the position  and of the situation (shaded vs. sunny microclimate). The results show that the microorganism development rapidly responds to rainfall events but the response is more intense in winter as temperature is lower and relative humidity (RH) higher. Cyanobacteria are less sensitive to this seasonal effect as they are more resistant to desiccation than green algae. Based on all the data, different dose-response functions have been established to correlate RH, rain and temperature to the green algae concentration. The influence of the microclimate is considered via specific fitting parameters. This approach has to be extended to new campaign measurements but could be very useful to anticipate the effect of climate change.Buildings and monuments are often colonized by microorganisms that can result in colour change and aesthetical and physico-chemical damages. This bio-colonization is dependent of the material and on the environment. In order to better understand and correlate the microbial development at the surface of buildings with meteorological parameters, concentration of green algae and cyanobacteria have been measured using an  Building materials are affected by different biological and chemical processes that can induce their deterioration. Especially, different microorganisms are able to colonize building materials, like green algae and cyanobacteria in biofilm forms, bacteria, moss, lichen and fungi . CyanobaBiological colonization on façade is controlled by extrinsic (environment) and intrinsic factors  . The maiConcerning intrinsic factors, biogeochemical deterioration processes are controlled by the chemistry of the substrate and biogeophysical deterioration processes by substrate properties, such as porosity and roughness , 16. Howin situ concentration of microorganisms. The BenthoTorch (bbe Moldaenke ®) is one of this in situ instrument allowing a fast benthic microorganism  concentration measurement by chlorophyll a fluorescence. This instrument is generally used in river, freshwater or in caves, and recently on ceramic tiles [From a methodological point of view, most of the works dealing with microbial colonization used traditional techniques from cultivation in a rich medium until DNA extraction and amplified ribosomal DNA restriction analysis targeting the 16S rRNA genes. These techniques are effective in analyzing the presence of microorganisms at a given time, but unfortunately do not give an indication of their dynamics over time according to climatic factors. Especially, it is very important to know the key parameters controlling their presence and dynamics. Since several years, new non-destructive and fast techniques have been developed to measure the ic tiles .The objective of our study is to improve our knowledge about the climatic influence on microbial development and their dynamic over time in order to understand the impact of climate change on plaster and tiles deteriorations under temperate climate. For that, two different contrasted seasons were chosen: spring and fall winter.The studied site is a private habitation located in the Parisian suburb . Access was permitted by the homeowner. In the Parisian region, the climate is described as hot temperate (Cf. Koppen and Geiger classification) with the presence of rainfall in driest months. The annual average temperature is 11.6°C and annual average rainfall is 693.6 mm for 1981–2010 period (infoclimat.fr).Two campaigns of measurement were carried out: the first one between May 7th 2020 and June 18th 2020 (spring) and the second campaign between November 5th 2020 and December 30th 2020 . During both campaigns, climatic data (daily mean relative humidity (%), daily mean temperature (°C) and precipitation (mm)) were collected from the Roissy-en-France meteorological station (infoclimat.fr).Different zones of measurements were selected  to assesFor the vertical surface, two areas were selected: one close to a chestnut (sheltered situation), that limits the direct impact of rainfall  and not sheltered (HNA2) by the tree. Moreover, in order to understand biological colonization behavior, two tiles were cleaned with bleach: one on the soil HBS,  and one 3 on surface was determined by HCl test. The mineralogy was determined by X-Ray Diffraction (XRD) using a Panalytical Empyrean powder diffractometer equipped with a PIXcel detector fitted with a Cu anode tube (Kα1 = 1.5406 Å) operating at 45 kV and 40 Ma. The crystalline phases were identifying and quantifying with HighScorePlus using ISCD and COD database.The studied wall surface is made out of roughcast that is a mix between coarse sand (millimetric clasts) and concrete . Presencin situ measurement of benthic microorganisms concentration  using in vivo fluorescence of chlorophyll on various substrates such as stones and sediments. The BenthoTorch directly calculates the biomass based on the chlorophyll-a content and determines the distribution over the different categories of algae. All measurements were conducted in triplicate and realized each day evening (~9 pm).Microorganism concentrations  were daily measured on each zone using a BenthoTorch (bbe Moldaenke®). This portable field instrument consists of As significant differences have been observed between vertical spots for spring and winter seasons, the sequencing was focused on these vertical positions. Sampling with sterile swab stick has been realized on each point of BenthoTorch measurement on July 23, 2021. Total genomic DNA was extracted from sampling swab with Fast DNA Spinkit for Soil (MPBio®) according to manufacturer’s instructions. DNA extract was amplified and sequenced on Eurofins genomics platform for 16S on V3-V5 region for bacteria and ITS on ITS1 region for fungi. Green algae were not sequenced. Sequencing data analyses were performed on FROGS pipeline. FROGS allow to obtain abundance table of operational taxonomic unit (OTU) and their taxonomic affiliations. Databases used for taxonomic affiliation are Silva 138 for 16S and UNITE 8.0 for ITS. Alpha-diversity and Beta-diversity indices were calculated using Easy 16S, Migale platform to observe the richness of each sample and the phylogenic difference between the samples, respectively.Statistical analyses were carried out using Rstudio ® for the Pearson correlation matrices. Data have been fitted using Matlab®.Wall surfaces are covered by a plaster roughcast that is a mix between coarse sand (millimetric clasts) and concrete . XRD anaThe climatic data are presented in 2, 2 and the concentrations in cyanobacteria were systematically higher than green algae. Rainy events are followed by a strong increase in cyanobacteria concentration for VA1, contrary to green algae concentration that seems to be less sensitive. For VA3, cyanobacteria and green algae displayed the same concentrations and have the same behavior with a proliferation more important during a rainy event.For VA2, VNA4 and VNA5, the concentrations remained always lower than limit of detection of the apparatus , Vermiconia calcicola (VA1 18.7% and VNA6 16.8%), Solenospora sp. (VA1 35.2% and VNA6 1.5%). Other species were present in significant proportions according to their position such as Catillaria lenticularis, Pleosporales sp. for VA1 position, Lecanorales sp. and Cladosporium delicatulum for VA3 position or Verrucariaceae sp. and Stilbella sp. for VNA6 position.Fungi identification is presented in For bacteria , as a fuDifferent indices relative to the Alpha-diversity have been calculated . The thrFungal Bray Curtis dissimilarity  had highPearson correlation matrices  were calBased on this correlation matrix, a dose-response function (DRF) that correlates meteorological parameters and the biological colonization on vertical surfaces was developed. On horizontal surfaces, contamination can occur. It was also decided to focus on green algae concentration as cyanobacteria concentration is relatively similar. The dataset is given in Four sets of data were used separately: VA1 and VA2; VA3; VNA4 and VNA5; VNA6. Parameters of As green algae concentration is generally higher for rainy days, the rain parameter was added in the DRF:Different values of c were tested to fit the data. It was noted that c is around 20 in fall-winter and 40 for spring. This is consistent with previous observations that rain leads to higher biological development in winter. Thus, as there is approximately a factor 2 between average temperature (T) during the spring period (16.4°C) and during the fall-winter (7.9°C), c was estimated to be equal to 2.5 / T to take into account this seasonal effect:Therefore, 4 dose-response functions were established for the 4 situations: sheltered from tree but exposed to sun (VA1-VA2), sheltered from tree and few exposed to sun (VA3), not sheltered and exposed to sun (VNA4-VNA5), not sheltered and close to soil (VNA6), respectively:Then, the calculated values have been compared to measured data as a function of time . ResultsThe objective of this study was to improve the understanding about the climate influence on microbial development and their dynamic over time in order to understand the impact of climate (change) on plaster and tiles deteriorations under temperate climate during two contrasted seasons: spring and fall-winter.The cumulative concentration in microorganisms  vary accGlobally, for vertical samples, the microorganisms concentrations are systematically higher in winter than in spring. Regarding the environmental data registered for both periods (see §3.2.), the temperature is lower in winter than in spring, whereas precipitation  and RH  are higher. This indicates that humidity favors the microbial development. These results are consistent with the literature [For horizontal samples, after each rainfall event, a proliferation of microorganisms was observed whatever the season. However, it can result from the contamination of the paving stones close to the measured one. The comparison with a previously bleach-cleaned tile confirmed that this proliferation is caused by the remobilization of microorganisms present on the stones.For vertical samples, the results show also differences according to the sheltered from the tree or not situation. VA samples have generally higher values that VNA samples except for VNA6 that is close to the ground and can be affected by rising damp. This shows that the tree contributes to keep the surface humid. Beyond temperature, rain and humidity that are key parameters for the proliferation of microorganisms, local factors such as insolation must also be taken into account . As it iCyanobacteria concentrations are less sensitive to the season than green algae. Also, in spring, cyanobacteria concentrations were higher than green algae concentrations whatever the positions , whereas in winter, green algae concentrations were higher than those in cyanobacteria excepted for some vertical positions. The difference between cyanobacteria and green algae can be explained by the ability of cyanobacteria to survive and recover faster from desiccation than algae . Their pBased on the examination of data and on statistical analyses, dose-response functions have been established (Eqs  to 7)) ) 7)) forThese DRF have of course to be completed by new measurements campaigns and are at this stage relatively specific. However, they confirmed the influence of key parameters, they give a first estimation of the contribution of each of them and they could be used to assess the potential effects of climate change. For example, the Parisian climate during 1901–2000 period showed an increase of annual mean temperature of 1.6°C and precipitation of 13% with an acceleration on second part of the century (Parisian climate agency 2021). Short-duration extreme precipitations will be intensified for all scenarios . The VolVerrucariaceae sp. and Stibella sp. The first one is well known to be a lichenized-fungi, in symbiont often with green algae, and lives frequently on rock surface [Stibella sp. belongs to Sordariomycetes that are essentially parasites and are involved in decomposition and nutrient cycling [In this paper, we focused on the monitoring of green algae and cyanobacteria concentration. However, literature data have shown the influence of a huge kind of microorganisms for the deterioration of buildings. Our analysis of the bacterial and fungal diversities using next generation technique permits to support the literature data. At our knowledge, no studies have reported NGS analysis on plaster of modern building and on the influence of microclimate on microbial diversity. The calculation of the Beta-diversity for each position have highlighted an effect on the microclimate on the microbial diversity whatever the kind of microorganisms (fungi and bacteria). VA1 and VA3 positions have very similar microbial community with Beta-diversity index of 0.95 for fungi and 0.94 for bacteria. The comparison of these positions with VNA6 shows lower dissimilarity index, which highlights the microbial communities difference between VA1, VA3 and VNA6. For fungi, two species are added on VNA6 in comparison with VA1 and VA3:  surface . Stibell cycling .Moreover, bacteria present on building surface are common to microbial community observed on limestone around the world , 31. GenThis study is a first attempt to monitor the biological development on building surfaces at the daily scale as a function of meteorological parameters to assess the reactivity and the dynamics of this development. The results show that BenthoTorch is a suitable tool to perform this fine monitoring as peaks of green algae and cyanobacteria occur a few hours after a rain event. The data also highlights that the colonization by these photolithotrophic microorganisms is more important in winter than in spring and is very dependent of water availability . From the data, it was possible to implement first dose-response functions that correlate the microbial development to the environmental parameters. They need to be improved to account for the microclimate effect.S1 Table and concentrations of green algae  and cyanobacteria (‘cyano’) for each day of the 2 measurements campaigns.(DOCX)Click here for additional data file.


This paper links prescriptions data for the Northern Ireland population with data from the Northern Ireland Longitudinal Study and localized ambient air pollution data from 2002 onwards to estimate the association between long-term exposure to ambient air pollutant from fine particulates (PM2.5) and Parkinson’s Disease (PD).Cox Proportional Hazards models are used to examine the impact of air pollution on PD, first unconditionally, and then conditioning on a rich set of observable individual, family and contextual characteristics. Long-term exposure to PM2.5 is defined as exposure averaged over the previous 5 years. Onset of PD is proxied by first receipt of a prescription for PD medication. Estimates are presented in the form of hazard ratios for the effect of long-term PM2.5 exposure on the risk of PD onset.There is a non-trivial magnitude and statistically significant unconditional association between long-term exposure to ambient PM2.5 pollution and receiving a prescription for PD, with those exposed to higher levels of pollution more likely to receive a prescription for PD. This estimated association disappears (becomes insignificantly different from zero), however, when the model accounts for confounding variables at household, individual and geographical levels.This study contributes to an emerging literature examining the association between ambient PM2.5 pollution and onset of PD. Despite finding an unconditional association, we find no evidence for an association once individual, family and contextual characteristics are controlled for, at least in the relatively low-pollution context of Northern Ireland.


In parallel, the goats learned a 4-choice visual initial discrimination task (ID) and three subsequent reversal learning (RL) tasks. The number of animals that reached the learning criterion and the number of trials needed (TTC) in each task were calculated. Our results show that goats with the lowest learning performance in ID needed more TTC in RL1 and reached the learning criterion less frequently in RL2 and RL3 compared to animals with better learning performance in ID. This suggests a close relationship between initial learning and flexibility in learning behavior. To study the link between personality and learning, we conducted two analyses, one using only data from the first OF- and NO-test , while the other included both tests integrating only animals that were stable for their specific trait . No relationship between personality and learning was found using data from only the first OF- and NO-test. However, stability in the trait boldness was found to have an effect on learning. Unbold goats outperformed bold goats in RL1. This finding supports the general hypothesis that bold animals tend to develop routines and show less flexibility in the context of learning than unbold individuals. Understanding how individual personality traits can affect cognitive abilities will help us gain insight into mechanisms that can constrain cognitive processing and adaptive behavioral responses.A wide range of species exhibit time- and context-consistent interindividual variation in a number of specific behaviors related to an individual's personality. Several studies have shown that individual differences in personality-associated behavioral traits have an impact on cognitive abilities. The aim of this study was to investigate the relationship between personality traits and learning abilities in dwarf goats. The behavior of 95 goats during a repeated open field (OF) and novel object test (NO) was analyzed, and two main components were identified using principal component analysis:  A wide range of species exhibit time- and context-consistent interindividual variation in behaviors, such as activity, exploration, boldness, aggressiveness and sociability, which are all traits related to an individual's personality –5. More Parus mayor) showed more flexible learning abilities than slow-exploring males, female slow explorers outperformed fast explorers, showing a sex dependency in the relationship between personality types and learning (Danio rerio) strains classified as proactive and reactive were able to learn and recall the fearful association of an associative fear conditioning task. While both coping style strains showed no differences in memory, reactive zebrafish acquired fear memory at a significantly faster rate than proactive zebrafish (Mungotictis decemlineata) tested the relationship between learning performance, social information and individual differences in boldness: learning performance of seven wild female groups  was tested with an artificial feeding box using a demonstrator-observer paradigm. Bold individuals as well as individuals in groups with demonstrators were faster in learning the task, while individuals without a demonstrator learned the task more slowly, indicating an interaction between personality traits, use of social cues and learning performance (Rhabdomys pumilio), proactive individuals  performed better than reactive males in two learning tests: a string-pulling task to obtain food and a door-opening task to reach the nest (Poecilia reticulata) exploration, sociability and boldness were not intercorrelated with learning performance  represent behavioral mechanisms that have a major impact on individual learning and cognitive performance . Currentlearning . Two difebrafish . Anotherformance . In malethe nest . On the formance , 31, agavia a screen and found that goats have clear categorization capacities . There is mounting evidence that these differences play an important role in various contexts in pigs –34, cowspacities . They wepacities  and werepacities , 52. In pacities , 54. Trapacities . In summpacities , 8, 45. In the current study, we investigated the potential relationship between specific personality traits and learning performance in female dwarf goats. A 4-choice visual discrimination task and three subsequent reversal learning tasks were applied to characterize the learning abilities of the goats. Reversal learning paradigms are commonly used to test behavioral flexibility, a type of phenotypic plasticity that can influence how animals cope with environmental changes. To investigate the relationship between the consistency of certain personality traits and learning, first only data from the first personality test and then the combined data from both personality tests were used separately to test the respective effect on learning performance.First of all, we expected a relationship between learning and reversal learning in that good learners in the visual discrimination task would need more trials to reach the learning criterion in the reversal learning task and vice versa. This is based on the findings of other studies showing that fast learners exhibited lower behavioral flexibility and, therefore, performed worse in a reversal learning paradigm compared to slow learners .Second, we expected no interrelation between the personality traits deriving only from the first personality test and discrimination learning or reversal learning. On the other hand, taking consistent personality traits into account , we expected the more active and bold individuals to disproportionately achieve the learning criterion in the discrimination task faster while inactive and unbold individuals would perform better in the reversal learning tasks, in line with studies regarding behavioral flexibility in the context of coping style , 57.Capra aegagrus hircus) bred from a line at the Leibniz Institute for Farm Animal Biology . Due to our breeding program, two experimental runs were conducted per year. From birth to weaning (2) were littered with wheat straw as bedding material and contained a hayrack and a round feeder to deliver concentrate . Hay and water were provided ad libitum. After weaning (seven weeks of age), female goat kids were kept in groups of up to 16 animals per group in pens as described above, with an additional climbing rack as an enrichment activity. All animals were ear tagged and wore a collar with a responder  for individual recognition at the free accessible automated learning device. The operating principle of the learning device has been described in detail elsewhere , which was only accessible at the learning device. For detailed information regarding the shaping procedure, see Langbein et al. . In shor2). To allow the goats to choose one of the symbols, four press buttons were mounted on the acrylic panel, one button beside each sector. After completing shaping, goats were regrouped to up to eight animals per group into identically equipped pens, including the learning device. The learning device consisted of a 15-inch LCD screen (resolution of 640 x 480 pixels) behind a transparent acrylic panel. The screen was divided into four virtual sectors. In each sector, we displayed a different symbol .During the first week, the goats were presented with a white screen, and all four buttons were rewarded. Next, the goats were presented with the first two training sets . The symTo investigate the impact of personality on the learning performance of the goats, a new 4-choice discrimination task was presented . This inAll animal care and experimental procedures were performed in accordance with the German welfare requirements for farm animals and the “Guidelines for the treatment of animals in behavioral research and teaching” . All proFrom 108 tested goats, we excluded those for whom technical issues concerning the learning device occurred . Finally, a total of 95 goats were included in all further analyses.p < 0.05) of correct responding was determined according to the Binomial test when n = 20 and p = 0.25, to be 46% of correct choices in at least two consecutive blocks of 20 trials (learning criterion). This reflects the same learning success compared to the learning criterion of 75% in similar studies applying the common two-choice design . Given the four-symbol choice task where chance responding to a given symbol is 25%, the criterion for a statistically significant level  for the initial OF- and NO-tests and their respective repetitions (please see 44 for further details). We selected 11 behavioral measures , we found two similar main principal components, with the first describing the interaction with the novel object  and the second describing active-like behavior , explaining 34% and 12% of the variation in the data, respectively. Finally, we calculated PC scores (from −3 to +3) for each personality trait for each animal in each of the two test periods, where each individual score was calculated from the standardized original data and the respective loadings of each PC separately for PCA1 and PCA2. These scores were used for any further statistical analysis.The personality traits ures see  out of 8ures see , 63. Usimomentary and stable personality traits using either the first or both OF- and NO-tests as the basis for the calculations. Using the personality scores resulting from the PCA1 (activity and boldness scores from −3 to +3), the respective quartiles were calculated to test the impact of a momentary personality trait on cognitive performance (see activity and boldness quartiles). Conversely, to investigate whether consistency in these personality traits had an effect on learning performance, we selected animals that showed stable activity and boldness scores in the repeated OF- and NO-tests . Stability was met if the scores from the first and second OF- and NO-tests were similar (both either positive or negative) for each personality trait (including a protection zone of ± 0.5 from the zero line).We tested the impact of the personality traits on learning performance and characterized All statistical analyses were conducted in SAS® 9.4 . The data were evaluated by various analyses of variance (ANOVAs) using the GLIMMIX procedure.To measure the impact of learning performance in ID, allocated among the four quartiles, on the overall achievement of the learning criterion during RL1-3, we used the variance function for the binomial distribution (binary data) and the logit link function in the statistical model. For more detailed analyses of learning, we ran a model with TTC in RL1-3 as our response variable and evaluated the effect of learning performance in ID , task  and their interaction, which were fitted as fixed effects. Both models contained motherID, pens and season nested in replicates as random effects.activity  and boldness  in PCA1 as fixed factors was tested on the number of animals reaching the learning criterion and on the TTC in RL1-3. In each model, motherID, pen and season nested in replicates were included as random effects.The impact of the quartiles of activity and boldness scores in the repeated OF- and NO-tests. Out of 95 animals, 32 animals showed a stable value in activity , and 42 animals showed a stable value in boldness  across the repeated OF- and NO-tests. The selected animals were then assigned to the groups stable in activity (inactive or active) or stable in boldness (unbold or bold), which were used as fixed effects on the TTC in ID and RL1-3 in the statistical model. In these models, motherID, replicates and season were included as random effects.To investigate whether consistency in specific personality traits had an effect on learning performance, we selected animals that showed stable p < 0.05 were considered significantly different.Least squared means (LSMs) and their standard errors (SEs) were computed for the fixed effects in all models, and pairwise differences in the LSM were tested using the Tukey–Kramer correction. All analyses included the animal as a repeated factor in the random statement, and mean differences with 3, 41 = 4.56, p < 0.01) and RL3 , while a tendency was found concerning RL1 . In other words, 46% of the low performers in ID (Q4) were not able to learn the first reversal task (n = 11). This effect was significant for the second  and third reversal tasks . Goats with the lowest learning performance in ID (Q4) reached the learning criterion in RL2 less frequently compared to goats with only poor learning performance in ID (Q3) (p < 0.01) and also tend to reach the learning criterion less frequently compared to goats with good  and very good  learning performance in ID. Additionally, low performers in ID (Q4) reached the learning criterion in RL3 less frequently compared to all other groups of learning performance in ID (Q1-Q3), as shown in p < 0.05, Q2: 0.01). Regarding the animals that successfully reached the learning criterion in RL1, RL2 or RL3, we found an effect of learning performance in ID on TTC in the reversal tasks . Pairwise comparisons indicate that low performers (Q4) needed significantly more TTC than the other learning groups (Q1-Q3) in RL1 . Furthermore, we found a significant effect of task on TTC  in that the animals significantly reduced their TTC in RL3 compared to RL1 and RL2  that was mainly apparent in Q4 regarding the TTC in RL3 compared to RL1 (p < 0.05). However, the interaction between learning performance in the ID and RL tasks revealed no significant effect.A significant effect of learning performance in ID (Q1–Q4) was found on the number of goats that reached the learning criterion in RL2 (Factivity (A1-A4) or boldness (B1-B4), based on only the first conducted OF- and NO-tests, on the number of goats that reached the learning criterion during RL1-3. Furthermore, activity and boldness did not show any significant effects on the TTC either during ID nor during RL1-3.The statistical analysis indicated no significant effects of stability in activity or boldness on the number of goats that reached the learning criterion in RL1-3. Regarding TTC, stability in activity (inactive vs. active) was not found to have a significant effect on TTC in ID or RL1-3 (stability in boldness (unbold vs. bold) significantly affected TTC in RL1 . As shown in We found no significant effects of or RL1-3 . In contactivity and/or boldness and learning performance, either in the discrimination task or in the reversal tasks. However, including only the animals that were stable for personality trait activity and/or boldness (measured twice), no evidence for any link between activity and learning performance was found, while boldness revealed a significant link to learning performance in the first reversal task in that unbold individuals outperformed bold individuals in RL1.Our study investigated the potential relationship between specific personality traits and individual learning performance by using a visual discrimination paradigm. Goats learned to associate a visual stimulus with a reward in the initial 4-choice visual discrimination task (ID) presented 24/7 using an automated learning device and were able to reverse their associations in a serial reversal task (RL1-3). Their performance in the three consecutive RL's depended on their learning success in the ID in that low performers in the ID also performed low in the RL's. Taking specific personality traits (measured once) into account, we did not find any evidence of a relationship between The learning performance of the goats in the three consecutive reversal tasks depended on their learning success in the initial learning task. A higher proportion of goats performing low in the initial discrimination task did not achieve the learning criteria in the reversal tasks compared to the goats that learned the initial discrimination task better. This effect is not as obvious in the first reversal task but reaches significance in the second and third reversal tasks. Additionally, the low-performing goats that were able to reverse the initially learned association needed more trials to solve the reversal task, which was significant at least in RL1. This indicates a relationship between visual discrimination learning and reversal learning in goats in the sense that the learning speed in the visual discrimination task would predict the learning performance in a reversal task regarding low-performing learners. Unexpectedly, goats who performed low in the initial discrimination task also performed low in the reversal task. This finding contrasts other learning studies showing that good learners (high learning speed) seem to be poor reversal learners (low learning speed), which has been demonstrated in guppies  and parrmomentary personality traits activity and boldness (measured once) and learning performance either in the discrimination task or in the reversal tasks. We might have expected, based on previous studies concerning personality measures and learning/cognition, that bold and/or active individuals learn to associate a cue with a reward faster than unbold and/or inactive individuals  personality trait boldness on learning performance in RL1  but not in ID. This partly reflects the general assumption that bold or proactive individuals are better at learning a discrimination task, but unbold or reactive individuals are better at adapting to changes in an already learned task reflecting a higher behavioral flexibility  can be found below: The animal study was reviewed and approved by Committee for Animal Use and Care of the Ministry of Agriculture, Environment and Consumer Protection of the federal state of Mecklenburg-Vorpommern, Germany (Ref. 7221.3-2-005/14).JL conceptualized the study. M-AF and AK collected and analyzed the data and did the statistical analysis together with AT. M-AF, AK, JL, and BP contributed to the conception of the manuscript. M-AF, AK, and JL wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Deutsche Forschungsgemeinschaft .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: The scaphoid is the most commonly fractured bone among the carpal bones seen in orthopedic practice. The majority have good favorable prognosis, but some develop nonunion of fracture despite optimal treatment, which can lead to further complications if left untreated. It is recommended that displaced scaphoid nonunions (SNUs) should be reduced and fixed to prevent degenerative changes from occurring, even if they are asymptomatic. Many treatments have been described, from a percutaneous fixation with a k-wire or screw to open reduction and internal fixation with or without bone grafting, but none of them is the gold standard.Aim: To evaluate the outcome of an SNU treated with an Ilizarov fixator using two olive wires without bone graft.Methods: Eleven cases of non-union scaphoid fractures were considered in the study which was presented to the Department of Orthopedics of Kalinga Institute of Medical Sciences during the period of March 2015 to March 2018. This study has been approved by the scientific and ethical committees. The anatomical location of the fracture was graded according to the MAYO classification. An Ilizarov frame was applied with two cross olive wires for achieving compression at the fracture area and maintained for six weeks. A final outcome was assessed using the scaphoid outcome score.Results: One out of 11 patients operated on during the study period was lost in follow-up, so 10 patients were considered for analysis of the results. There were nine male patients and one female patient. The majority were right-sided and dominant-handed, with varied occupations. The average duration of nonunion, when presented, was 10.7 months (a range of 6-20 months). The average follow-up was 43.6 months (range 27-60 months). Union was achieved in an average of 12.9 weeks (range 10-18 weeks). All the patients returned to their pre-injury activity level in a mean of 17.1 weeks (range 13-23 weeks). Grip strength improved from a mean of 29.5 kg preoperatively to 39.4 kg postoperatively. At the final follow-up, the mean scaphoid outcome score was 9.1. An excellent outcome was obtained in five cases (50%), a good outcome in three cases (30%), a fair outcome in one case (10%), and in one case (10%), a poor outcome.Conclusion: With our technique of Ilizarov fixation and compression with two cross olive wires, SNU can be treated safely even without opening the non-union site and even without bone grafting. Since we excluded SNU patients with humpback deformity, carpal instability, carpal collapse, or avascular necrosis (AVN), our results might not be directly comparable to those of other SNU series in the literature. These would have predisposed to a poor outcome. Since we did not assign the patients at random, it is challenging to compare the Ilizarov technique to other widely used SNU treatments and determine whether it is more effective. However, the study's results are encouraging and show that the Ilizarov method using two olive wires for compression. Scaphoid fractures mostly result from a fall on an outstretched hand with forced dorsiflexion, and it is the most commonly fractured carpal bone -2. PrecaScaphoid nonunion (SNU) can be asymptomatic initially, but the natural history of untreated SNU progresses to degenerative and arthritic changes in adjacent intercarpal joints, leading to impairment of wrist joint function and affecting activities of daily living . Non-disMany treatments have been suggested, from a percutaneous fixation with a k-wire or screw to open reduction and internal fixation with or without bone grafting, but none of them is the gold standard , 7.The Ilizarov technique has been used to close gaps in long bones that do not join together and fix deformities by using mechanical forces to make new bone and improve blood flow -9. TreatThis study will look at what happens when a nonunion of a broken scaphoid is treated with an Ilizarov fixator and olive wire compression at the broken site instead of a bone graft.Some 11 cases of SNU fractures were considered in the study that was presented to the Department of Orthopaedics of Kalinga Institute of Medical Sciences during the period of March 2015 to March 2018. This study has been approved by the scientific and ethical committees with IRB approval number KIITS/012/OR2022. After getting their consent to take part in the study, subjects who met the inclusion and exclusion criteria were added to the study  and ulna  in a safe anatomical zone after tensioning the wire to 90-100 kg load. Similarly, the distal ring is applied to the mid metacarpal level by attaching to two similar wires of 1.5 mm, one passing from the radial border of the second metacarpal exiting from the third metacarpal and another passing from the ulnar border of the fifth metacarpal exiting from the fourth metacarpal at an angle of 30-40 degrees. One 1.5-mm olive wire is passed through the nonunion site from the proximal to the distal pole of the scaphoid bone, with the olive stopping at the proximal pole, and the sharp end of the wire is attached to the distal ring with a male post. Another similar olive wire is passed through the scaphoid from its distal pole, exiting the proximal pole obliquely through nonunion, with the olive stopping at the distal pole of the scaphoid bone. The sharp end of this wire is attached to the proximal ring with a wire fixation bolt. Before fixing, these two cross wires are gradually tensioned until the SNU site is compressed under fluoroscopy without opening the nonunion site at all. Four connecting rods are also fixed between the two rings Figure -D.Postoperative evaluationAfter the immediate post-operative radiograph, weekly serial radiographs were taken until the frame was taken off at a duration of six weeks to look for nonunion compression, wire detensioning, and pin tract osteolysis, and these problems were dealt with as needed. Every week, the fixator's hardware was checked for stability, and loose nuts were tightened on a regular basis. The finger small joints, elbow, and shoulder joints were all encouraged to move. The frame was removed at six weeks. Patients were encouraged to do an active range of motion at the wrist to overcome post-immobilization stiffness. Patients were evaluated clinically and radiologically at two-weekly intervals following surgery until the bony union was demonstrated on radiographs. They were also evaluated clinically at 6, 12, and 24 months after frame removal to look for any collapse, avascular necrosis with the fragmentation of the proximal pole, and arthritic changes. Grip strength at the final follow-up was measured with a Jamar analog hand dynamometer and compared with preoperative values. With the help of the scaphoid outcome score, the functional outcome was measured by comparing pain, function, range of motion, and overall satisfaction before and after surgery.One out of eleven patients operated on during the study period was lost in follow-up, so 10 patients were considered for analysis of the results. There were nine male patients and one female patient. The majority were right-sided and dominant-handed, with varied occupations. The average duration of nonunion, when presented, was 10.7 months range 6-20 months). Eight patients were initially treated with cast immobilization; the remaining two patients were fully neglected and did not receive any form of treatment (Table 0 months.The average follow-up was 43.6 months (range 27-60 months). Union was achieved in an average of 12.9 weeks (range 10-18 weeks). All the patients returned to their pre-injury activity level in a mean of 17.1 weeks (range 13-23 weeks). Grip strength improved from a mean of 29.5 kg preoperatively to 39.4 kg postoperatively. At the final follow-up, the mean scaphoid outcome score was 9.1. An excellent outcome was seen in five cases (50%), a good outcome in three cases (30%), a fair outcome in one case (10%), and in one case (10%), a poor outcome . These would have predisposed to a poor outcome. It is challenging to determine whether the Ilizarov approach is better because we did not randomly assign the patients and compare it to other well-established treatments for SNU. However, the study's results are encouraging and show that the Ilizarov method using two olive wires compression without bone grafting is an effective procedure. They also suggest that, in some circumstances, it may be a successful means of treating SNU. Further studies are required to establish this as the preferred treatment method in specific situations without associated complications.