Saccharomyces cerevisiae made this organism a suitable model organism for elucidating molecular mechanisms of copper transport and homeostasis. In this study, the dynamic transcriptional response of both the reference strain and homozygous deletion mutant strain of CCC2, which encodes a Cu2+-transporting P-type ATPase, were investigated following the introduction of copper impulse to reach a copper concentration which was shown to improve the respiration capacity of CCC2 deletion mutants. The analysis of data by using different clustering algorithms revealed significantly affected processes and pathways in response to a switch from copper deficient environment to elevated copper levels. Sulfur compound, methionine and cysteine biosynthetic processes were identified as significantly affected processes for the first time in this study. Stress response, cellular response to DNA damage, iron ion homeostasis, ubiquitin dependent proteolysis, autophagy and regulation of macroautophagy, DNA repair and replication, as well as organization of mitochondrial respiratory chain complex IV, mitochondrial organization and translation were identified as significantly affected processes in only CCC2 deleted strain. The integration of the transcriptomic data with regulome revealed the differences in the extensive re-wiring of dynamic transcriptional organization and regulation in these strains.Copper is a crucial trace element for all living systems and any deficiency in copper homeostasis leads to the development of severe diseases in humans. The observation of extensive evolutionary conservation in copper homeostatic systems between human and  Besides being a vital element, copper may also be highly toxic in case of accumulation due to its high redox potential2. Copper toxicity occurs due to the formation of reactive oxygen species (ROS) via the Fenton reactions. ROS accumulation results in the damage of the cellular elements including proteins, lipids, and nucleic acids, and eventually in the cell death. Since copper is both essential and toxic, a tight control of its uptake, transport, utilization, and export is required. Deficiency in the copper uptake results in an increase in oxidative stress, abnormal iron metabolism, crosslinking in the extracellular matrix, and altered signaling in tissues and organs. On the other hand, the presence of free ions in excess amounts leads to the reaction with hydrogen peroxide and formation of hydroxyl radicals deleterious for the cells3.Copper is an essential trace element involved in a variety of biological processes including iron metabolism, respiration, DNA damage response, lipid metabolism, and oxidative stress response3. Copper trafficking is carried out through a basic set of steps in all organisms. Following the transfer of copper ions across the cell membrane by copper transporters, copper is bound to the chaperones for the delivery to target proteins within the cytosol, nucleus, mitochondria and secretory intracellular compartments2. The requirement of copper as well as the distribution of copper transporters differs between the cell types in complex organisms; however, the key players of this homeostatic system remain almost the same4. Since most of the members of copper homeostasis in yeasts have counterparts in mammalian cells, S. cerevisiae is considered as a suitable model organism to elucidate the general molecular mechanisms of copper sensing, intra- and extra- cellular transportation and copper homeostasis5.Complex homeostatic systems were developed in both prokaryotic and eukaryotic organisms in order to maintain intracellular copper levelsCTR1 and CTR3 are activated under low copper conditions by Mac1, which is downregulated by copper import through Fet4 in yeast6. Low affinity copper transport is carried out by Fet4 and Smf1, dominantly by Fet46. In the presence of high extracellular copper, Ace1 is activated upon conformational changes and induce CUP1 and CRS5, which encode copper binding metallothioneins (MTs) and SOD1, which encodes copper, zinc superoxide dismutase8. The localization of both copper-sensing regulators of copper homeostasis in the nucleus provides evidence for the presence of changing copper concentrations in nucleus; however, there is still less known about copper transport into the nucleus4.High affinity copper transporter genes 4. Copper is transferred to the Zn, Cu superoxide dismutase Sod1 by Ccs1 in the cytosol and the intermembrane space of mitochondria10; however, based on the fact that Ccs1 is not expressed in several species an independent route of copper transport to Sod1 was also suggested10. Copper is transported to the cytochrome c complex in the inner mitochondrial membrane via Cox17, Sco1 and Cox1113. Pic2 and Mrs3 were identified as potential mediators of copper import to the mitochondrial matrix15. Several important human diseases, such as red blood disorders, cardiomyopathy, skeletal myopathy and neurodegenerative diseases, were found to be strictly associated with defect in mitochondrial copper and iron homeostasis16. Despite the increasing knowledge, the molecular mechanism of copper transport into mitochondria remains to be elucidated.Upon uptake, copper is reduced by the reductases, and this reduced form may react with GSH, and form Cu-GSH complex, which may be involved in the mediation of copper delivery to the copper binding proteins within different cellular compartments17. Copper transporting P-type ATPase Ccc2 is homologous to human Atp7a and Atp7b, which are associated with Menkes and Wilson diseases, respectively. Lack of CCC2 was reported to cause defective iron uptake and respiratory deficiency which can be overcome by copper or iron supplementation in S. cerevisiae18. An endocytosis mediated alternative path of copper transport to Ccc2 independent of Atx1 has been also reported, and this may be also associated with the endocytic internalization of Ctr119. Furthermore, analysis of the transcriptional response of yeast cells to the deletion of ATX1 and to different concentrations of copper indicated the presence of an Atx1-independent path for the transfer of copper to Ccc220.Copper may be transferred to Ccc2 via the cytosolic chaperone Atx1 and delivered into the trans-Golgi network in yeast. Copper is then incorporated into the human ceruloplasmin ortholog Fet3 subunit of the iron responsive Fet3-Ftr1 complex. This copper insertion into Fet3, which takes part in the high affinity iron transport, is required for proper iron uptakeS. cerevisiae cells which were grown in batch fermenters grown until steady state in the presence of three different copper concentrations revealed the effects of the CCC2 deletion or the presence of different bioavailable extracellular copper concentration as well as their interactive effects on these cells21. In the present study, the dynamic transcriptional response of the reference and CCC2 deleted S. cerevisiae strains to a copper impulse was investigated in order to elucidate further the mechanism(s) involved in copper homeostasis. The collected dynamic transcriptional data was comparatively analyzed by using different clustering techniques based either on time dependent gene expression or co-expression profiles in order to identify the significantly affected biological processes and pathways in response to a switch from copper deficient environment to elevated copper levels. The integration of this data with regulome revealed the dynamic transcriptional and regulatory organization of the sets of genes in these two strains of S. cerevisiae in response to copper.Yeast cells are affected by the availability of copper in the medium and/or from the mutations in the genes encoding proteins involved in the copper homeostasis. An integrative comparative analysis of the transcriptional and metabolic response of hoΔ/hoΔ) and CCC2 deleted (ccc2Δ/ccc2Δ) strains of S. cerevisiae to a switch from copper deficient medium to elevated copper containing environment was investigated following the introduction of copper as an impulse at the steady-state. The cells were grown in continuous cultures using a copper deficient defined medium in fully controlled bioreactors. Samples were collected within the first two hours following the copper addition to reach a copper concentration of 0.5 mM which was shown to improve the respiration capacity of respiratory deficient CCC2 deletion mutants21, in addition to the steady-state sampling.Dynamic transcriptional response of both the reference  were identified  genes in the reference and CCC2 deleted strain were significantly associated with protein acetylation, oxidoreduction coenzyme metabolic process, and regulation of biological quality. On the other hand, the genes which were upregulated throughout the experiment in the reference strain were significantly associated with methionine biosynthetic process, sulfur compound metabolism, sulfate assimilation, and NADH oxidation. The genes which were significantly associated with the methionine, cysteine and homoserine biosynthetic processes, response to nutrient levels, to chemical and to heat, as well as the sulfur metabolism pathway displayed an upregulation within the first hour, and downregulated within the second hour in CCC2 deleted strain.The genes which were downregulated throughout the experiment, were significantly enriched with biological regulation and ribosome pathway in the reference strain whereas the genes which were similarly downregulated over the course of experiment in CCC2 deleted strain, for a duration of approximately 15 min were significantly associated with the biological regulation, positive and negative regulation of transcription, histone exchange, endocytosis, Golgi to endosome transport, autophagy, Ras protein signal transduction, response to stress, heat, nutrient levels, and starvation, and carbon catabolite regulation of transcription.The genes, which were significantly associated with the processes such as biological regulation, transcription, ATP-dependent chromatin remodeling, endocytosis, carboxylic acid metabolic process, and cellular response to stimulus, displayed an initial upregulation for a duration of 5–10 min in the reference strain. These genes displayed a slight repression, and then, returned to their initial steady levels. The genes which were significantly associated with DNA replication, mitochondrial fission, and negative regulation of transcription, displayed also a similar behavior in this strain. The genes which display an initial upregulation in the CCC2, were significantly associated with ribosomal processes, cytoplasmic translation, and mitochondrial membrane organization. On the other hand, several genes which were also significantly enriched with cytoplasmic translation and ribosomal GO terms, as well as mitochondrial respiratory chain complex assembly, displayed a downregulation within the first hour and started to increase in the second hour in this strain.The genes which display a repression within the first minutes and started to increase around the tenth minute in the reference strain following the copper impulse were significantly associated with the amino acid transport and protein processing in ER pathway. Furthermore, several genes which were significantly associated with ribosomal GO terms were observed to be downregulated within the first ten minutes in this strain. The expression patterns of these genes were diversified after 30 min. Genes which were downregulated within the first 15 min, and then, upregulated to return back to the steady-state levels in the absence of CCC2 deleted strain were significantly associated with the chromatin organization, biological regulation, transcription, gene silencing, response to stress, carbohydrate catabolic process, iron ion homeostasis, and ubiquitin mediated proteolysis pathway.The genes which were downregulated as of the fifteenth minute in the 24, used to identify the co-expressed modules consisting of highly correlated significantly expressed genes in response to copper impulse, revealed seven co-expressed modules in both strains and significantly associated biological processes associated with these clusters were identified. Largest modules consisted of 688 and 847 genes in the reference and CCC2 deleted strains, respectively ely Fig.  online.FCCC2 deleted strains. A differential co-expression network, which displays the differences in the co-expressions in response to copper impulse, was created by Diffany25. The resulting differential co-expression network contained five connected components with five or more members. The largest connected component of the differential network contained 2307 nodes and 10,977 interactions  enriched with ribosome biogenesis, intracellular transport, regulation of translation, and carboxylic acid biosynthetic process. 4810 edges specific to the reference strain were found to connect 1406 nodes. These nodes were significantly associated with  regulation of transcription, regulation of glycolysis, ribosome biogenesis, and osmosensory signaling pathway via Sho1 osmosensor.The co-expressions that are detected in both strains were included in the consensus network. The consensus network contained three connected components with five or more members, and the largest one contained 42 nodes and 79 interactions. The largest component  in the reference but not in the CCC2 deleted strain were determined. On the other hand, 43 DC clusters which were significantly co-expressed in the CCC2 deleted strain but not in the reference were identified. However, significantly (FDR < 0.05) enriched biological process GO terms were found for three DC clusters of each strain  in the reference or CCC2  which are possibly responsible for the diverging transcriptional activities of these sets of genes at the bifurcation/split points were identified based on the enrichment of their target genes in the splits its Fig. .Figure 6CCC2 deleted strains, respectively  associated with carboxylic acid biosynthetic process, alpha-amino acid metabolic process, and endocytosis. This split of genes based on the divergence of gene expression patterns was found to be regulated by Swi6, Stb4, Gcn4, and Sko1.The middle branch after the first bifurcation contained 2911 genes which were significantly associated with cytoplasmic translation, and Fhl1 and Rap1 were identified to be involved in the control of this split of genes. The lowest branch of this bifurcation point which consisted of 886 genes were significantly enriched with ribosome biogenesis and sulfur compound transport , Cad1, and Nrg1. These genes were significantly associated with sulfur compound biosynthetic process, methionine biosynthetic process, energy derivation by oxidation of organic compounds, and oxidative stress response , which is involved in stress response and iron metabolism30, were responsible for the upregulation of the genes in both strains within the first five minutes. However, stress response , cellular response to DNA damage, DNA repair and replication were identified as significantly affected biological processes in only CCC2 deleted strain in response to copper impulse. The genes involved in these processes were either induced for a duration of 15 min following the impulse or remained at their steady levels before downregulation. The repair of the DNA damage was reported to be associated to chromatin modifications by histone modifications and ATP-dependent chromatin remodeling31. The observed DNA damage may be possibly caused by ROS generated from copper exposure. The observation of the high level expression of metal reductase activities in mutants defective in DNA repair, induction of copper import genes and repression of MT genes in response to DNA damaging agents indicated a regulatory relationship between DNA damage and copper homeostasis. Although the molecular basis of this relationship remains elusive, this relationship was supported by the observation of the changes in the redox state of Mac1 in response to copper or MMS32. Signaling and signal transduction  were observed to be significantly affected in the mutant strain.Copper has a redox activity and elevated levels of copper may induce oxidative stress and formation of reactive oxygen species. Alterations in intracellular redox and any defect in maintaining redox homeostasis may affect a number of signaling pathways which regulate cellular division and stress response systems+/NADPH and GSSG/2GSH in reduced state and detoxifying systems are also very important. The maintenance of a high NADPH/NADP+ ratio is critical to keep a reduced environment33. Although NADPH is mainly produced by the pentose phosphate pathway, this pathway did not appear as a significant term in any clusters of both strains. Manual investigation of the variations in the expression levels of the genes involved in cellular detoxifying system and NADPH generation indicated that these genes were clustered into different clusters in both strains.Redox homeostasis and maintenance of redox environment is very important to maintain several important cellular processes. Although GSH homeostasis is of vital importance, the maintenance of redox couples such as NADPCCC2 deleted strain in response to copper impulse. Analysis of dynamic transcriptional re-organization of the genes in response to copper impulse revealed that Met32 was involved in this re-organization after the first minute in reference strain whereas the regulator of amino acid biosynthesis Gcn4 was involved following the copper impulse. The transcriptional re-organization of the genes associated with these processes was identified to be under the control of Met31 and Met32 following the impulse in the CCC2 deleted strain. Cbf1, which may act in concert with Met434, was also involved in this control at the first minute in this strain.Sulfur compound metabolism, methionine and cysteine biosynthetic processes were found to be significantly affected in response to high copper levels in both strains. The genes associated with these processes in the reference were upregulated throughout the experiment whereas they were upregulated within the first hour, and then, downregulated within the second hour in 35. GSH is a small antioxidant molecule which protects the cells from damage causing effects of ROS and is the main redox buffer in S. cerevisiae. GSH, together with glutaredoxin, catalases, and peroxidases, plays also an important role in redox homeostasis of the cells, and it is involved in a number of important biological processes such as protein and DNA synthesis, amino acid transport36. Intracellular concentration of GSH is strictly controlled and degradation of this molecule is considered to be an important element of GSH homeostasis37. Furthermore, a key role of cellular GSH in copper uptake was proposed in mammalian and human cells39. Although GSH metabolic process was not identified as a significantly affected process in neither strain, manual investigation of the transcriptomic data indicated that several genes associated with GSH metabolic process were present among the differentially expressed genes which were clustered into different clusters.The biological pathways and chemical reactions, which involve sulfur or sulfur containing compounds including methionine, cysteine or GSH, constitute the sulfur compound metabolism, which is of vital importance. Methionine and de novo recycling of homocysteine is the main precursor of the GSH21. Ccc2 in yeast mediates the transport of copper from the cytosol to Golgi to incorporate into Fet3 which is the yeast ortholog of ceruloplasmin. Moreover, these metal ion metabolisms may be also related to each other based on the ROS balancing4. The iron and copper ion transport processes were not identified among the significantly affected biological processes in any cluster of both strains following the excessive copper addition as pulse. Three transcription factors (TFs) which regulates iron metabolism  in the reference and only one TF (Cad1) in CCC2 deleted strain were identified to be involved in the transcriptional re-organization in response to copper within the first minute in both strains. The identification of Aft1 and Aft2 in the first minute of the dynamic organization of transcription indicates the involvement of iron regulon in the reference strain. Iron homeostasis was found to be a significantly affected process in CCC2 deleted strain in response to elevated copper levels and the genes significantly associated with this process displayed a downregulation after 15 min following the impulse. It might be suggested that the deletion of CCC2 may cause a decrease in the utilization of iron in the mutant cells, and this defect may be restored by the addition of excess copper into the medium. This findings are in good agreement with our previous results, and this amount of copper may compensate the respiratory deficiency of CCC2 deleted strain21. The observation of similar response of the genes involved in iron homeostasis in ATX1 deleted and reference cells to high copper levels indicated that an Atx1 independent pathway may exist to transfer copper to Fet320.Copper and iron are essential for the organisms owing to their redox properties and activities as cofactors. Copper and iron metabolisms are intertwined and dependent on each other such that iron uptake through Fet3-Ftr1 complex is dependent on the copper load, and cells repress iron transport genes under copper starving conditionsCCC2 deleted strain. These genes displayed a downregulation during the first hour of the experiment, and then, an upregulation in the second hour. Furthermore, the comparative analysis of differentially co-expressed genes indicated the co-expression of a number of genes significantly associated with respiratory complex IV biogenesis, regulation of mitochondrial organization and mitochondrial translation in the reference strain, and this co-expression was dysregulated in the absence of CCC2. ATP synthesis coupled electron transport was found to be affected in response to high levels of copper in our previous studies21. The dysregulation of mitochondrial organization and translation in the CCC2 deleted strain was reported for the first time in the present study. Mitochondria are important sites of utilization of both copper and iron. The biogenesis of CcO which is the final enzyme of the respiratory chain is one of the copper toxicity targets40.The genes involved in the organization of mitochondrial respiratory chain complex IV  assembly were found to be significantly affected in response to elevated copper levels in 41. Mitochondrial Fe-S cluster (ISC) pathway, components of ISC export machinery  and cytosolic iron sulfur protein assembly (CIA) are very important for the maturation of cytosolic Fe-S proteins43. Failure in the assembly of these clusters result in an increased acquisition of iron and mitochondrial iron overload through regulation by iron-responsive TFs controlling cellular iron uptake and distribution42. Manual investigation indicated that gene expression profiles of 12 and 11 significantly expressed genes associated with Fe-S cluster assembly were found to be clustered into different clusters in the reference and CCC2 deleted strains, respectively. The role of CCC2 in the mitochondrial organization and in the assembly of Fe-S clusters requires further investigation.Mitochondria are essential players in the regulation of iron metabolism, in the assembly of Fe-S clusters, and heme biosynthesis. The assembly of Fe-S clusters in mitochondria is an essential process and any defect in this process leads to mitochondrial dysfunction+ is an electron carrier and an essential cofactor required for many biochemical reactions in the cell. Mitochondrial function is critical for the maintenance of NAD+/NADH homeostasis44. NADH oxidation was found to be significantly affected process in the reference strain. The genes involved in this process were co-regulated and displayed an upregulation throughout the experiment following the copper impulse in this strain. NADH oxidation was not identified as a significant process in the CCC2 deleted strain. However, oxidoreduction coenzyme metabolic process including NAD+ metabolism was identified as a significantly affected process and the genes involved in this process displayed a downregulation throughout the experiment in this strain. NAD+ metabolism was found to be affected from both the deletion of the CCC2 gene and the level of extracellular copper level, and supplementation of nicotinic acid was found to improve the respiratory deficiency of the CCC2 deleted strain21.NADCCC2 deleted strain in response to a switch from copper deficiency to high copper conditions. Golgi to endosome transport which regulates a number of cellular and developmental events by recycling of membrane proteins was also observed to be a significantly affected biological process in a cluster where the gene expression profiles display a similar upregulation during the first 15 min following the copper impulse in only CCC2 deleted strain. This observation is in good correlation with the previous findings indicating the induction of CTR1 gene increasing the amount of Ctr1 in the plasma membrane of yeast cells grown in copper deficient environment46 and the internalization of Ctr1 by endocytosis and degradation under high copper conditions48.Endocytosis, which is an important process in the internalization of extracellular fluid, particles, and plasma membrane transporters, was found to be a significantly associated GO biological process term in both strains. The genes involved in endocytosis showed an upregulation in the first 10 min in the reference strain and in the first 15 min in the 49. Autophagy which maintains intracellular homeostasis involved in important cellular functions under various stress conditions50 was found as one of the significantly affected processes in CCC2 deleted cells and the genes associated with this important process displayed an upregulation within the first 15 min following the impulse. Furthermore, a group of genes involved in the regulation of macroautophagy were significantly and differentially co-regulated in the CCC2 deleted cells, but not in the reference strain. Autophagy was not found to be a significantly affected process in the reference cells. On the other hand, protein processing in ER was identified as a significantly affected biological pathway and the genes involved in this process were upregulated after a brief period of downregulation in the reference strain. Although this process was not identified as a significant process, several genes involved in protein processing in ER and ERAD pathways were observed to be repressed after a period of 15 min following the impulse in CCC2 deleted cells. Furthermore, ubiquitin-mediated proteolysis was identified as a significantly affected pathway in CCC2 deleted strain and the genes associated with this process displayed a downregulation 15 min later following the switch. This process was not identified as a significant process in any of clusters of the reference strain. The genes which are involved in proteosome assembly were found to be upregulated in the CCC2 deleted strain.ER quality control (ERQC) is involved in the folding and modification of secretory and membrane proteins and misfolded proteins are eliminated by ER-associated degradation (ERAD) or by autophagy of which the recognition mechanisms of ERAD substrate is not knownCCC2 deleted S. cerevisiae cells in response to a switch from copper deficient to high copper containing medium revealed that large number of general processes such as replication, chromatin modeling/silencing, regulation of gene expression, transcription, RNA processing, ribosome biogenesis and metabolic processes were significantly affected in both strains. The importance of sulfur compound metabolism, methionine and cysteine biosynthetic processes in response to copper were also revealed for the first time in the present study. Integration of genome wide transcriptomic data with regulome indicated an extensive and different time-dependent re-wiring of regulatory events in both reference and CCC2 deleted cells in response to increasing levels of copper. Additionally, transcriptional factors responsible in the regulation of different biological processes were identified.The analysis of time-dependent transcriptomic response of the reference and CCC2 deleted strain in response to copper impulse indicated the higher sensitivity of this strain in comparison to reference strain to increased copper levels. Signaling and signal transduction were also observed to be significantly affected only in the mutant strain. The organization of mitochondrial respiratory chain complex IV, mitochondrial organization and translation was found to be significantly affected only in this strain. The role of Ccc2 in these processes requires further investigation.The differences and similarities between these strains in response to a copper impulse could be revealed throughout the experiment. The identification of stress response, cellular response to DNA damage, iron ion homeostasis, ubiquitin dependent proteolysis, autophagy and regulation of macroautophagy, DNA repair and replication as significantly affected biological processes in only Time-series experiments carried out in the present study could reveal several above mentioned significantly affected biological processes and pathways which could not be detected before by static perturbation experiments carried out in our laboratory. The dynamic transcriptional response of the strains carrying deletions of these genes or other genes known to be involved in the copper homeostasis and the integration of this data with other omic studies such as metabolome, proteome and phosphoproteome will possibly help to elucidate the complete mechanism of copper sensing, signaling, and copper transport to different compartments of the cell.hoΔ/hoΔ and ccc2Δ/ccc2Δ, of Saccharomyces cerevisiae BY4743 (MATa/MATΔ his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/ + met15Δ0/ + ura3Δ0/ura3Δ0) were used in the experiments. hoΔ/hoΔ strain was used as the reference strain. The precultures were incubated in YPD medium  at 30 °C and 180 rpm in an orbital shaker.Homozygous deletion mutant strains, 51 without CuSO4. The cells were grown in Biostat B fermenters with a working volume of 1.5 L at 30 °C with an agitation speed at 800 rpm. The dilution rate and pH were kept at 0.1 h-1 and 5.5, respectively. The experiments were carried out under aerobic conditions with an air feed of 1.5 L/min. A solution of CuSO4 was injected into the copper deficient media as an impulse to reach a copper concentration of 0.5 mM following five-residence times at the steady-state. This concentration of copper was shown to improve the respiration capacity of respiratory deficient CCC2 deletion mutants21. Samples were collected at the 1st, 5th, 10th, 15th, 20th, 25th, 30th, 60th and 120th min, in addition to the steady-state sampling. The collected samples were kept at − 80 °C for further transcriptome analyses.The overnight grown precultures were inoculated into the synthetic defined F1 mediumRNA isolation was carried out with the Qiagen RNeasy Mini Kit in the robotic workstation, QIAcube  following the manufacturer’s protocol for the applications in yeast (RNeasy protocol for extracting yeast via enzymatic lysis). The quantity and quality assessments of the isolated RNA were carried out by using the NanoDrop UV–vis spectrophotometer  and Bioanalyzer 2100 , respectively.21. cDNA was synthesized and converted into a double stranded form by using GeneChip 3′ IVT Express Kit . Biotin-labelled aRNA was loaded onto GeneChip Yeast Genome 2.0 arrays  for hybridization. GeneChip Hybridization, Wash, and Stain Kit  was used in the hybridization, washing, and staining steps. Washing and staining were carried out in the fluidics station using the Affymetrix Command Console Software (AGCC) version 3.0.1  Fluidics Control Module with Mini_euk2v3. The chips were loaded onto the Affymetrix GeneChip Scanner 3000.Transcriptome experiments were carried out as described previously52 version 1.0 (https://sites.google.com/site/dchipsoft/). Quantile normalization and log-transformation steps were carried out with the RMA Express software53 version 1.2.0 (https://rmaexpress.bmbolstad.com/).The raw transcriptome data obtained by the microarray analysis was assessed for the presence-absence calls by using dChip software54 version 2.6.0 in R version 3.2.5, which was developed specifically for the testing of differential gene expression in time-course experiments, using the optimal discovery procedure55. Significance of gene expression was determined based on false discovery rate, which was estimated by data resampling. The genes which were differentially expressed over time in each strain were identified by comparing the gene expression profiles with the average profiles through within-class analysis method or steady-state profiles through between-class analysis method. The set of genes identified by any of these methods with a false-discovery level of 1% were determined as significantly and differentially expressed.The significance analysis was carried out via EDGE package56 by using Pearson correlation as the distance metric. Significantly expressed genes were clustered by using the web-based tool SwitchFinder22, in which the genes were grouped based on the change-points within the expression patterns. The number of clusters was chosen considering the confidence intervals within the clusters, as well as the similarity between the clusters.The experimental conditions were hierarchically clustered via MultiExperiment Viewer (MeV) 4.9.024 version 1.51 in R version 3.2.5. The network was reconstructed as a signed network, and soft-thresholding power was chosen as 9 in accordance with the use of filtered data and also the number of samples. Pearson correlation was used as the correlation type. Co-expression modules were identified setting the minimum connectivity value, module merging criterion, and minimum module size as 0.5, 0.25, and 30, respectively. Co-expression network of the differentially expressed genes in each strain was reconstructed with the Pearson correlation coefficient threshold of 0.75 and were visualized by using Cytoscape57 (https://cytoscape.org/) version 3.5.2. The differential co-expression network was constructed by using Diffany plugin25 version 1.0.0 through pairwise comparison. Enriched biological process gene ontology (GO) terms within specific network members were identified by using BINGO (version 3.0.3) plugin58 of Cytoscape with the Benjamini&Hochberg (FDR) corrected p-value threshold of 0.05.Co-expression network analysis was carried out via Weighted Gene Co-expression Network Analysis (WGCNA) package26 version 1.4 in R version 2.15.2, in which a resampling method was applied for the calculation of the significance of differential co-expression across conditions. Pearson correlation coefficient was used as the distance metric. The cut-off value for the tree cut height, the iteration number for resampling, and module significance threshold were determined as 0.4, 10,000, and 0.001, respectively. Significant gene subsets which have five or more members were further investigated for the enrichment of GO biological processes.Differential co-expression analysis was performed via CoXpress packageSaccharomyces Genome Database (SGD)59. The significantly enriched biological process GO terms and KEGG23 pathways for specific subsets were determined using DAVID60 version 6.8 with an FDR threshold of 0.05 removing the genes with only unknown biological process GO term in SGD59 .Gene descriptions were obtained from 27 (https://sb.cs.cmu.edu/drem/) version 2.0.3. The regulatory code of MacIsaac et al.61 was chosen as the source interactome data for the TFs and the target genes with a binding p-value threshold of 0.001 and motif presence requirement but without any conservation requirement. The threshold for the scores of key TFs, which are associated with the divergence of the gene expression patterns, was set as 0.001. The enrichment analysis of the biological process GO terms within the gene sets in the splits was also performed. The randomization test62 was chosen as the correction method in the calculation of the p-values for the GO terms, and the corrected p-value threshold was set as 0.05.Transcriptional regulation dynamics was examined by using The Dynamic Regulatory Events Miner (DREM) softwareSupplementary Information


In silico screening of connectivity maps, which link gene expression profiles to libraries of perturbagens, may facilitate the identification of such emerging therapies. The L1000 Connectivity Map is built from samples of transcripts taken from gene expression profiles of cells in various experimental conditions followed by computational inferences of the remainder of the transcriptome. Searching the L1000 Connectivity Map for modulators of a protease that facilitates coronavirus infection identifies plausible candidate drugs for repurposing as antiviral agents against SARS-CoV-2 following further investigation.Adaptive clinical trials are underway to determine the efficacy of potential therapies for COVID-19, with flexibility to include emerging therapies if there is sufficient preclinical evidence for their potential utility.  The design of the RECOVERY trial in particular allows for its Trial Steering Committee to amend its protocol to include or exclude treatment arms in response to new evidence.The pace of propagation of the COVID-19 pandemic has necessitated a search for effective therapeutics for the disease. Adaptive clinical trials such as the Randomised Evaluation of COVID-19 Therapy (RECOVERY) trial , which infects a target cell by engaging its spike (S) protein with the host cell receptor ACE2 and subsequently fusing its membrane with that of the cell. In order for the virion to enter the target cell, the S protein must be primed by the cellular serine protease TMPRSS2, the inhibition of which has been demonstrated to limit SARS-CoV-2 infection of a human lung cancer cell line meta-analysis http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE70138. The significance of differentially expressed genes in data was tested in the software using the Linear Models for Microarray data (Limma) algorithm to perform a moderated t-test on the normalised data 2 ratio change > 0.58 (roughly 1.5-fold change) to p < 0.05. This is an arbitrary threshold selected for screening of the connectivity map due to precedent for 1.5-fold change being considered differentially expressed in other published microarray experiments, and also because differential expression thresholds at that level are likely to represent a good trade-off between allowing the rejection of background noise and identifying biologically meaningful changes I searched the L1000 database using Genevestigator v8.0.1 . Genevestigator is a computer software platform that incorporates a suite of data analysis tools and a search engine for public high throughput functional genomics data that has been curated, quality controlled, annotated and normalised by the Nebion AG team to facilitate TMPRSS2 expression in A549 human lung epithelial adenocarcinoma cells in at least one of the tested drug concentrations. Of these, 9 are drugs with prior approvals for use in humans for alternative indications: alpelisib/BYL719, crizotinib, fedratinib/TG-101348, neratinib, nilotinib, nintedanib, ruxolitinib, selumetinib, and vemurafenib (TMPRSS2 expression changes.) None of these compounds significantly affected ACE2 expression. On the basis of literature searches, ruxolitinib and selumetinib are drugs of particular interest.Of the compounds in the curated dataset that were tested in 24-hour perturbation assays, 40 drugs and tool compounds were identified that significantly downregulated urafenib . (It shoRuxolitinib is an inhibitor of Janus kinase (JAK) enzymes that has been approved by the United States Food and Drug Administration (FDA) for the treatment of myelofibrosis, a condition characterised by abnormal clonal proliferation of haematopoietic stem cells. JAKs operate as transducers downstream of several receptors for pro-inflammatory cytokines, which makes them attractive candidates for treatment of conditions in which inflammation is dysregulated, as can be the case in COVID-19 in vitro data suggest it may have some antiviral activity. Selumetinib (as well as some other MAPK pathway modulators) was found to inhibit the infection of a human hepatoma cell line with Middle East respiratory syndrome coronavirus (MERS-CoV) in vitroin silico screen that suggests reduced mRNA expression of TMPRSS2 in cells exposed to selumetinib, identifies the MEK inhibitor as a plausible candidate for further evaluation in experimental models relevant to COVID-19.Selumetinib is a mitogen-activated protein kinase (MAPK/ERK) kinase (MAPKK/MEK) inhibitor that has FDA approval for the treatment of neurofibromatosis type 1, a multisystem disorder that is most often characterised by a propensity for neurofibroma formation. Although selumetinib has not been trialled in COVID-19 patients like ruxolitinib has, TMPRSS2 is under the transcriptional control of the androgen receptor (AR) AR itself is regulated by the transcription factor CREB1, which is activated by phosphorylated MAPK. MEK inhibitors such as selumetinib (because they are inhibitors of MAPK kinase) are therefore able to reduce the expression of AR in vivoTMPRSS2). It is plausible that reduced TMPRSS2 availability as a result of MEK inhibition would limit SARS-CoV-2 cell entry, but this hypothesised mechanism would need to be validated experimentally. Androgen regulation of TMPRSS2 expression may also provide a mechanistic explanation for the observation that male sex is associated with a higher COVID-19 case fatality rate than female sex in silico screening is an aid and a precursor to further in vitro and in vivo evaluation, not a replacement for it.Adaptive clinical trials like the RECOVERY trial will be pivotal in gaining control of the COVID-19 pandemic and candidate compounds for inclusion in such trials will need to be identified with sound scientific rationale. Connectivity maps such as L1000 are likely to prove essential in that regard, enabling researchers to screen for drugs that may be repurposed for new indications. Screening of the L1000 Connectivity Map in this case identifies selumetinib as a plausible antiviral candidate to treat COVID-19, but it is important to note that such Wezi Sendama: Conceptualisation, investigation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


This study was designed to evaluate the anti-inflammatory effects of a curcumin treatment on the knee of rats with induced osteoarthritis. Fifteen adult rats were used and divided in three groups: the osteoarthritis group (OAG), control group (CG–without induction of osteoarthritis), and curcumin-treated osteoarthritis group (COAG). Osteoarthritis was induced in the right knee of rats in the OAG and COAG by administering an intra-articular injection of 1 mg of zymosan. Fourteen days after induction, 50 mg/kg curcumin was administered by gavage daily for 60 days to the COAG. After the treatment period, rats from all groups were euthanized. Medial femoral condyles were collected for light microscopy and immunohistochemical staining. The expression of SOX-5, IHH, MMP-8, MMP-13, and collagen 2 (Col2) was analyzed. The COAG exhibited an increase in the number of chondrocytes in the surface and middle layers compared with that of the OAG and CG, respectively. The COAG also showed a decrease in the thicknesses of the middle and deep layers compared with those of the OAG, and an increase in Col2 expression was observed in all articular layers  in the COAG compared with that in the OAG. SOX-5 expression was increased in the surface and deep layers of the COAG compared with those in the OAG and CG. Based on the results of this study, the curcumin treatment appeared to exert a protective effect on cartilage, as it did not result in an increase in cartilage thickness or in MMP-8 and MMP-13 expression but led to increased IHH, Col2, and SOX-5 expression and the number of chondrocytes. Osteoarthritis (OA) is one of the most burdensome diseases worldwide, and it is caused by a metabolic imbalance due to extrinsic factors, such as trauma, or intrinsic factors, such as various types of molecular dysfunction. The imbalance induces the production of a cascade of cytokines and inflammatory mediators that results in the release of nitric oxide (NO), leading to chondrocyte apoptosis and extracellular matrix (ECM) degeneration .During the articular inflammatory process, macrophages release some cytokines, such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α, as well as many growth factors. These cytokines induce the expression of matrix metalloproteinases (MMPs)  that digThe collagen degeneration in OA cartilage gradually spreads around the chondrocytes according to the cartilage layers, beginning in the surface layer and extending to the deep layer . The MMPSome transcription factors, such as the SOX trio , are crucial for the development of primordial cartilage, formation of the growth plate and maintenance of the chondrogenesis phenotypes .In addition, during limb development, Indian hedgehog (IHH) is initially detected in the precartilaginous condensation mesenchyme when Sonic hedgehog (Shh) signaling becomes weaker . Then, IConsidering the human diet and available treatments for inflammatory diseases, turmeric is used in Ayurveda and Chinese medicine due to its antioxidant, antimicrobial and anti-inflammatory properties –14. CurcBased on this information, the purpose of the present study is to confirm the efficacy of a curcumin treatment in an animal model of induced osteoarthritis using a histomorphometric method and immunohistochemical staining for proteins related to the articular structure and osteoarthritis development.The animal procedures were approved by Institutional Care and Use Committee (IACUC) of Federal University of São Paulo .Rattus norvegicus) that were bred and housed until they were aged 12 weeks in CEDEME  of Federal University of São Paulo on a natural dark and light (12:12 h) cycle and water and standard food available ad libitum were used in the present study. The animals were randomly distributed into three groups : the control group (CG): without induction of OA and treated with a saline solution; the osteoarthritis group (OAG): without treatment; and the curcumin-treated osteoarthritis group (COAG): animals with osteoarthritis that were treated with curcumin.Fifteen male Wistar rats , osteoarthritis was induced in the right knees of rats in the OAG and COAG by administering an intra-articular injection of a single dose of 1 mg of zymosan (Sigma-Aldrich CAS number 58856-93-2) dissolved in 50 μL of sterile saline . After t® CURCUMIN bcm-95, Scottsdale, Arizon, USA) were administered by gavage to the COAG daily for 60 days. The CG and OAG were administered sterile saline by gavage for 60 days. The curcumin dose was based on a previous study Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: “Evaluation of rat knee articular cartilage with induced arthritis and treated with Curcumin” by Tiago Nicoliche et al. attempts to evaluate the anti-inflammatory effects of curcumin in the induced osteoarthritis treatment in rats’ knee. I recognize the effort made or the authors to develop this histologic descriptive study about some keys elements of OA development. Conceptually this manuscript is very similar to other manuscripts published ; Osteoarthritis Cartilage. 2015 Jan;23(1):94-102; Oxid Med Cell Longev. 2019 May 16;2019; Biosci Rep. 2018 Jul 2;38(4); Mol Med Rep. 2017 Aug;16(29). So, what is the novelty of this study? I do not think that this manuscript adds significantly to what is already know and is not novel enough to warrant a new full length publication. I have the following suggestions for improvement.Major comments:1) The style of the manuscript should be improved. The discussion reads like a review at certain places. It should be more focused, discussing the experimental findings in light of the available data in the literature as they relate to the development of osteoarthritis. “In studies carried out by Fernandes et al. … Burrage et al. (2006) believed that … Tetlow et al. (2001) (40) found …” This paragraph reads like a list without concluding what is unknown, for example.2) As it is suggested that curcumin could be administered as part of a dietary supplementation aimed at preventing inflammation and reducing OA severity, it is important to fully analyse the effects of the tested dose on control animals in addition to those subjected to the OA model. Does orally administered curcumin also improved Col2, IHH, SOX-5 expressions and the number of chondrocytes in unchallenged joints or in naturally ageing animals?3) Does the curcumin lead to any reduction in synovial tissue inflammation? At least the presentation of wider field images of the joint including synovial and cartilage would help in understanding the therapeutic impact of the treatment.4) Please provide more specific reasons why intraarticular injection of Zymosan is a proper model of OA, since the mechanism of OA is not fully understood5) Is there clinical report or field study showing that people regularly consuming diet containing curcumin have lower rate of OA compared to those not consuming such diet?Minor comments:1) The Schedule of OA induction and treatment it is not clear. Animals were euthanized 8 weeks after induction of OA? or 8 weeks after curcumin was daily administered for 60 days?2) The results should be described in the same order than results are presented in figures.3) A figure describing possible model for how curcumin modulate inflammation could be good.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  27 Jan 2020It was sent a doc file in the previous steps responding all the questions.AttachmentResponse to the reviewers.docxSubmitted filename: Click here for additional data file. 26 Feb 2020Evaluation of the articular cartilage in the knees of rats with induced arthritis treated with curcuminPONE-D-19-17705R1Dear Dr. Nicoliche,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Chi ZhangAcademic EditorPLOS ONE 2 Mar 2020PONE-D-19-17705R1 Evaluation of the articular cartilage in the knees of rats with induced arthritis treated with curcumin Dear Dr. Nicoliche:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Chi Zhang Academic EditorPLOS ONE


The way strength recovers after reduction of pediatric fractures of the upper extremity has not previously been the specific scope of research. This is remarkable, since strength measurements are often used as an outcome measure in studies on trauma of the upper extremity. The aim of this study was to evaluate how strength recovers after sustainment of fractures of the forearm, wrist or hand treated by closed or open reduction in children and adolescents in the first 6 months after trauma. How much strength is lost at 6 weeks, 3 months and 6 months after trauma, and is this loss significant? Are there differences in the pattern of recovery between children who underwent a different treatment? And finally, which of the following factors are associated with an increase in the ratio between affected grip strength and expected strength: type of fracture, cast immobilization, occurrence of complications, and degree of pain?Prospective observational study.Children and adolescents aged 4–18 years with a reduced fracture of the forearm, wrist or hand.Grip strength, key grip and three-jaw chuck grip were measured twice in each hand 6 weeks, 3 months and 6 months after trauma. Details on fracture type and location, treatment received, cast immobilization and complications were obtained. Hand-dominance and pain were verbally confirmed.Loss of strength was more prominent and prolonged the more invasive the treatment, hence most extensive in the group receiving open reduction with internal fixation (ORIF), intermediate in the group receiving closed reduction with percutaneous pinning (CRIF), and least extensive in the group undergoing closed reduction without internal fixation (CR). Besides time passed, gender and age were of significant influence on strength, although there was no difference in pattern of recovery over time between children who received a different treatment. In the period of 6 weeks to 3 months after trauma, female gender, type of fracture sustained and occurrence of an unwanted event were associated with an increased ratio between affected and expected grip strength. For the later phase of recovery, between 3 and 6 months, this was only true for the occurrence of an unwanted event. Within the extensive arsenal of existing functional tests, strength measurements are conducted almost routinely in the follow-up after trauma of the upper extremity in adults because of their well-established role in the assessment of hand function. Strength measurements are quick to assess and have excellent intra- and interrater reliability.–3Scores Illustrative for the importance of strength measurements in the recovery of pediatric forearm fractures is the prospective study by Pershad et al. 2000. Results The aim of this prospective study is to evaluate how strength recovers in children and adolescents after having sustained fractures of the forearm, wrist or hand treated by closed or open reduction. The research questions were as follows. How much strength is lost at 6 weeks, 3 months and 6 months after trauma, and is this difference significant in comparison to the unaffected hand? Are there differences in pattern of strength recovery between children treated by means of closed reduction (CR), closed reduction with percutaneous pinning (CRIF), and open reduction using either percutaneous pinning, intramedullary pinning or plate fixation (ORIF)? And finally, which of the following factors are associated with an increase in the ratio between affected grip strength and expected strength: type of fracture, cast immobilization, occurrence of complications, and degree of pain?Children and their parents were informed about the study by one of the researchers (AMH/BB) and received additional written information about the study goals and procedures. Written consent was obtained from parents or the legal guardian. Children were only included if they themselves were willing to participate, and the researcher made sure parents as well as children knew that participation was neither mandatory nor would affect their treatment. The study received a waiver from the Medical Ethical Board of University Medical Center Groningen (M.14.150324).All children and adolescents aged 4–18 years with a reduced fracture distal from the olecranon treated at University Medical Center Groningen in a one year period were invited to participate. Exclusion criteria comprised neuromuscular and bone diseases, any condition interfering with normal growth, and fractures proven or suspected to be the result of child abuse. Also excluded were children who could not be properly instructed, for example due to a language barrier, or who received follow-up at a different hospital. Participating children had 3 appointments: at 6 weeks (T1), 3 months (T2) and 6 months (T3) after sustainment of the fracture. Participants were not measured in the week following cast or osteosynthesis removal. In those cases measurements were postponed. When appointments at the hospital could not be planned concurrently with measurement sessions, a home visit by the researcher was offered. Patients were assigned to each treatment regimen by the treating physician as part of the standard-of-care.General characteristics of the participants such as age, gender and hand dominance were registered. Details obtained on the fracture comprised location, type, (post) treatment, cast duration and potential complications. Grip strength was measured with the Jamar® hydraulic hand dynamometer . Participants were positioned according to the standardized testing position of the American Society of Hand Therapists (ASTH): seated subject, shoulders adducted and neutrally rotated, elbow flexed at 90°, wrist at 0–30° extension and 0–15° ulnar variation. The handDescriptive statistics were used to describe the main characteristics of the study population. For strength measurements the mean of the two attempts  of each hand was used in the analyses. To correct grip strength for the influence of hand dominance, the score of the affected hand was also compared to a calculated expected value of that hand (as if it were unaffected). This calculated value was derived from the adjusted scores of the unaffected hand according to findings from an earlier study by the current research group. Scores bTo examine in more detail if there were differences on pattern of recovery between children who underwent a different treatment, a mixed-model repeated measurements analysis was performed for possible confounders . Variables noteworthy of altering the -2 restricted log likelihood of grip strength were ultimately taken into the final model.Finally, multivariate linear regression analyses were performed to establish if the variables treatment type, fracture type, cast immobilization, occurrence of unwanted events (re-displacement or complication) and degree of pain were associated with an increase in the ratio between affected grip strength and expected strength. To this end, a ratio variable was created by dividing the affected value by the previously mentioned calculated expected value at all three measurement points. Extent of strength increase was used as the dependent variable and was defined as the difference in this ratio variable between measurement sessions (T2 minus T1 and T3 minus T2). In these analyses, pain was defined as occurrence of pain at 6 weeks or 3 months after trauma respectively. Results were considered to be significant when the associated p-value was <0.05. Statistical procedures were conducted using SPSS 23.0 for Windows (IBM SPSS Inc.).During the study period 97 children underwent an open or closed reduction of their fracture. Twenty children could not participate, 6 failed to meet criteria for inclusion, and another 14 could not be included due to other reasons . Bilateral fractures occurred in 7.8% (N = 6) of children, all right-dominant. In 3 cases both fractures required repositioning and thus met criteria for inclusion. Since analyzing these participants twice could induce dependency within the data, they were excluded. The final study population therefore comprised 74 participants. An enrollment flow diagram is shown in In 16 participants an unwanted event occurred, either secondary dislocation or the endurance of a complication. In 10 participants angulation or rotation either did not improve or worsened, for which a secondary repositioning was performed. Complications were related to problems with Kirschner wires, imminent malunion or child factors (e.g. second trauma during treatment). Slightly more than half of the study population (53%) was pain-free within 6 weeks of trauma versus 76% at 3 months and 6 months after trauma. None of the participants experienced continuous pain–only in specific situations–and more importantly, none experienced pain while performing the measurements in this study.For all participants with a unilateral fracture, grip strength of the affected hand was compared to that of the unaffected hand at 3 measurement sessions. Overall, loss of strength amounted to 32.3% at 6 weeks, 12.8% at 3 months and 4.7% 6 months after trauma. This was analyzed further by type of treatment. The average loss of strength amounted to 24.1%, 6.8%, and -0.2% for fractures that were treated by CR, versus 42.3%, 15.9%, and 4.9% respectively for fractures treated by CRIF. Finally, loss of strength for fractures treated by ORIF was more prominent, amounting to 37.3%, 20.0% and 10.2%. Results showed a significant difference between the strength of the affected and unaffected hand for all types of treatments at 6 weeks and 3 months after trauma. However, after 6 months only the ORIF group still showed a significant strength difference between the hands. An overview of these results can be found in To correct for the influence of hand dominance, grip strength of the affected hand was further compared to that of the calculated expected value, which was derived from the scores of the unaffected hand as described in the Methods section. This analysis did not lead to any changes in significance compared to the results as shown in Overall loss of strength was 22.0% at 6 weeks, 6.9% at 3 months and 1.8% at 6 months after trauma. For fractures treated by CR a significant loss of strength in key grip could only be observed at T1 (12.5%). Loss of strength after sustainment of fractures treated by CRIF and ORIF at 6 weeks was more prominent, 30.6% and 32.0% respectively, decreasing to 14.4% and 13.8% at 3 months. In both groups this difference was significant. Six months after sustainment of the fracture, loss of strength for the ORIF group was still 13.5%. An overview of these results can be found in Overall loss of strength amounted to 22.1% at 6 weeks, 4.7% at 3 months and 3.2% at 6 months after trauma. For both the CR and CRIF group a significant difference was limited to the 6-week measurement (17.7% and 33.1% respectively). By contrast, the ORIF group still showed a significant difference in strength at 3 months amounting to 14.5%. Six months after trauma no significant difference in strength could be observed in any of the groups. An overview of these results can be found in A mixed-model repeated measurements analysis was performed to examine for differences in the pattern of strength recovery of the affected hand over time between participants who underwent different type of treatments (treatment x time). Time, age and gender were found to be of significant influence on all 3 grasps, and were therefore incorporated in the overall model. The dominant hand being affected and location of fracture were not of significant influence on strength recovery of the affected hand, hence removed from the model. Final results showed no difference in the pattern of recovery of the affected hand for any of the grasps over time between participants who received a different treatment. An overview of the p-values of this analysis of can be found in Multivariate linear regression analyses were performed to establish which variables were associated with an increase in the ratio between affected grip strength and expected strength between the different measurement sessions. A larger ratio difference implies a larger strength increase towards ones expected (unaffected) strength during this timeframe, however not necessarily a better recovery as children with a larger delta could simply be worse off at the start of the timeframe. In the period of 6 weeks to 3 months female gender, type of fracture sustained (both-bone) and occurrence of an unwanted event showed to be significantly associated with a larger ratio difference. In the 3-6-month period the occurrence of an unwanted event still was associated with the increase in this ratio difference, whereas the same did no longer hold true for gender and type of fracture sustained. An overview of the p-values of these results can be found in To our knowledge, this is the first study to prospectively focus on how strength recovers after reduced fractures of the forearm, wrist or hand in children. Results showed that loss of strength as compared to the value of the unaffected hand was more prominent and prolonged the more invasive the course of treatment, i.e. most extensive in the group receiving ORIF and least extensive in the group receiving CR only. In participants treated by CR, grip strength was significantly impaired up to 3 months after trauma whereas key grip and three-jaw chuck grip recovered within this period. Grip strength was similarly impaired in children treated by CRIF. Key grip was also still impaired in this group 3 months after trauma. In participants treated by ORIF, both grip strength and key grip were still significantly impaired 6 months after sustaining the fracture. Also, the three-jaw-chuck grip was impaired prolongedly compared to the other groups–up to 3 months. There was however no difference in pattern of recovery between the groups, all following a similar trend. Time passed since sustainment of the fracture, age and gender were of significant influence on the strength of the affected hand over time. The increase in ratio between the affected grip strength and expected strength between 6 weeks and 3 months was associated with female gender, type of fracture sustained (both-bone) and occurrence of an unwanted event. The difference is due to this ratio being lower at the beginning of this timeframe for participants who sustained a both-bone fracture or endured an unwanted event (they were more affected at the start). Between 3 and 6 months after trauma only the occurrence of an unwanted event was still significantly associated with an increase in this ratio. Although around 25% of participants still experienced pain both 3 months and 6 months after trauma, no association between pain score and ratio between affected and expected strength was found. This is most likely because none of the participants experienced pain performing the strength measurements. The presence of pain has thus not influenced the outcome of the strength measurements, but should nonetheless not be ignored as it concerns a substantial amount of children and could very well affect other (more prolonged or intensive) activities that fall beyond the scope of the current study.Comparison to previous literature is difficult because studies taking strength measurements into account are scarce. Roth et al. 2014) evaluated functional outcome after manipulation of previously reduced re-displaced forearm fractures versus conservative treatment (no secondary manipulation) 1–8 years post-injury. The stud evaluateSinikumpu et al. 2013 also evaluated grip strength as an end result 9 to 14 years post-trauma. This was the only study using a control group to compare strength after sustainment of forearm shaft fractures in childhood.No signifA strong point of the current study was that besides grip strength, other standardized strength measurements often used by hand therapists–namely key grip and three-jaw chuck–were evaluated. All measurements were obtained at set moments in time corresponding to usual follow-up appointments. The follow-up rate was very high, with only one child withdrawing from further follow-up after the first measurement session. The lowest percentage of children completing a grip measurement session during the entire study period was 91.0%, for key grip and three-jaw grip at 6 months. A limitation of the current study was the heterogeneity of the study population itself, namely a large variance in age, type of fracture and type of treatment. This is why even though the study population was rather substantial to offer a first insight into the recovery of strength, subgroup analyses nonetheless quickly led to small groups. Future research should concentrate on a larger or less heterogenic study population. Also, pinch strength was unfortunately not evaluated even though it was initially intended. Researchers established that this specific measurement was difficult to perform on the smaller children and moreover that the set of measurements became too extensive to maintain the child’s interest. Pinch strength was therefore eliminated from the study protocol after the first measurement sessions.The current study had a descriptive nature, so no treatment alterations were made. The fact that the ORIF group scored worse than the CRIF (and the CRIF worse than the CR) might simply be a reflection of the severity of the fracture sustained. Therefore, the current study will not have consequences for the management of pediatric forearm fractures. However, the relation between treatment invasiveness and the duration and severity of strength loss has to our knowledge not been described previously. This combined with the trend from conservative treatment toward surgical intervention for displaced fractures of the forearm calls for further research into this topic.–20 IdealIn conclusion, since the extent and duration of muscle strength loss for all strength measurements tend to be more prominent the more invasive the treatment chosen, as well as the fact that a large percentage of children still experience pain 6 months after trauma, referral to a hand therapist for additional guidance should be easily accessible to all children with a reduced fracture. In particular, referral should be considered when ORIF is chosen as the course of treatment.S1 Checklist(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S2 Protocol(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Stickler syndrome is known to cause visual handicap due to the high incidence of retinal detachment. We aim to present an unusual case of a child with Stickler syndrome who had progressive visual loss secondary to atrophy of the outer retinal layers not associated with retinal detachment. This is a descriptive case report of a 9-year-old child with ocular history of high myopia who presented to our institution with suboptimal visual acuity in both eyes. After 2 years of follow up, he developed unilateral progressive visual loss with marked atrophy of the outer retinal layers and peripheral vascular leakage. Such a presentation has not been previously described in the literature to the best of our knowledge. It is the most common inherited vitreoretinopathy, estimated to affect 1 in 7,500 to 9,000 newborns.COL2A1 and accounts for 80-90% of cases; Type II, which is caused by mutation in COL11A1 and accounts for 10-20% of cases; and Type III, which occurs due to a mutation in COL11A2 and is characterized by non-ocular manifestations. The autosomal recessive types include Stickler type IV and V with mutations in the COL9A1 and COL9A2 genes, respectively.Mutations in several genes cause the different types of Stickler Syndrome. The autosomal dominant types are Stickler type I, which is due to a mutation in 2 Vitreous veils attached to the retina, radial perivascular atrophy, and retinal lattice degeneration are also common. Retinal detachments secondary to anterior giant retinal tears or posterior breaks are common, as well as pre-senile cataract.3The most common ocular manifestations are high myopia and vitreous syneresis (100% of patients). Stickler type I is characterized by membranous vitreous and type II by beaded, fibrillar vitreous.We present the case of a boy with high myopia and progressive visual loss not related to retinal detachment. After an exhaustive investigation including whole exome sequencing (WES), Stickler syndrome type I was diagnosed, with unusual ophthalmological findings not previously described in the literature.A 9-year-old patient was referred to our clinic due to suboptimal visual acuity. He had ocular history of high myopia, as did his father and grandfather.At presentation, logMAR best corrected visual acuity (BCVA) was 0.48 in the right eye (RE) and 0.18 in the left eye (LE). The refraction was RE -7.50 -1.00 x180 and LE -7.50 -0.75 x180. On eye examination, the anterior segments were normal and the vitreous was quiet, with a vitreous strand overlying the superotemporal retina in the LE. The retina was flat with fine macular and perivascular pigmentary changes (RE more than LE).Two years later, BCVA had decreased considerably to 1.0 logMAR in his RE and remained stable in his LE. During this examination there was evidence of bilateral marked vitreous syneresis with membranous formations. There were no vitreous cells and retinal findings remained unchanged. Repeated FA showed leakage from the peripheral vessels in the RE and focal areas of capillary nonperfusion. Fundus autofluorescence showed areas of hypoautofluorescence in the posterior pole. SD-OCT demonstrated total loss of the ellipsoid zone and marked atrophy of the outer retinal layers in the RE. The LE remained stable. Swept source OCT-angiography showed no abnormal vascularization . RepeateCOL2A1 gene, which suggested the diagnosis of Stickler syndrome type I. His parents were tested by Sanger sequencing for the genetic variant and were not found to carry the variant, indicating that it occurred as a de novo mutation in the child. WES was repeated by a laboratory specialized in inherited retinal diseases in order to rule out additional mutations that can explain a retinal dystrophy in this child, but no other mutations were identified.The child was referred for genetic testing. WES revealed a frame-shift pathogenic variant  in exon 42 of the The original anamnesis reported that the child was born with bifid uvula, and also described some mild orthopedic problems. Physical examination by a clinical geneticist following genetic tests results showed very subtle signs of malar hypoplasia with retromicrognatia and crowded teeth, bifid uvula and high arched palate, and camptodactyly of the fifth finger. These findings supported the diagnosis of Stickler syndrome.4 Non-ocular findings can include incomplete palate, which ranges from open cleft, submucous cleft, to bifid uvula like in our case. Hearing loss, joint hypermobility, and other skeletal manifestations are also seen.5Stickler Syndrome is a rare hereditary connective tissue disease. Clinical manifestations and targeted genetic testing are generally sufficient to reach a diagnosis. Most cases are inherited via autosomal dominant inheritance, while a minority of cases result from de novo mutations, as in our case.COL2A1 gene that can produce a phenotype with predominantly ocular manifestations.6 The majority of patients presenting to an ophthalmologist will have either type 1 or type 2 Stickler syndrome and are frequently high myopes.7When systemic signs are not evident, ophthalmologists play a major role in the diagnosis. This occurs especially in cases of mutations in exon 2 of the 8Our patient presented with progressive disruption of the outer retinal layers leading to visual loss in one eye. Peripheral vascular leakage  and possible thick choroid were also detected. These changes have not been previously described in Stickler syndrome and may be the result of mild vascular changes. Retinal capillaritis has been previously described in the setting of CRB1-associated retinal dystrophy; the authors suggested that capillaritis may be due to the active phase of the disease in young patients, although the influence of modifier genes could not be excluded.6 Abnormal ERG with progressive abnormalities of cone-rod function was seen in our patient and has already been described in Stickler Syndrome.1Our patient also presented with radial perivascular pigmentary degeneration which is known to be a characteristic manifestation of Stickler that develops in childhood and progresses with time.10 In 2018, Matsushita et al. studied the degree of foveal hypoplasia in patients diagnosed with Stickler syndrome type I and found that 82% of the subjects had mild foveal hypoplasia with persistence of the inner retinal layers in the fovea in OCT images.9Our patient also presented with bilateral foveal hypoplasia, with good vision in the LE. Recently, foveal hypoplasia has been associated with Stickler syndrome.11 Recent advancements and accessibility of high-resolution OCT imaging have shown that a lack of foveal pit does not always indicate poor visual acuity.Foveal hypoplasia had not been commonly reported in patients with Stickler syndrome probably because these patients have fairly good visual acuity.11 In our case, there was progressive visual loss secondary to total loss of the ellipsoid zone and outer retinal layer atrophy without retinal detachment, not previously described in Stickler cases. Other possible additional diagnoses such as posterior uveitis, infection, and retinal dystrophy were ruled out, raising the suspicion that this retinal atrophy was not a coincidental finding but a potential Stickler-related ocular manifestation not previously reported.Visual loss and blindness in children with Stickler syndrome has classically been related to the presence of retinal detachment.WES is a useful tool that assists ophthalmologists in reaching the correct clinical diagnosis and ruling out additional genetic pathology in complex cases.


Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype. The extensive application of biotechnology, genetic engineering, genome editing and genome breeding in modern agriculture and forestry leads to creation of a wide variety of unique genotypes  ,2,3. TheIn vitro culture is most acceptable for conservation of modified lines ,16. It iThe PVS2-based vitrification developed by Sakai et al.  is the mPopulus are the main model woody plants for various genetic manipulations. Though, this model plant is rather difficult to preserve ex vitro and in vitro collections due to a number of biological features. The article by Tsai and Hubscher [Populus functional genomics future research. Nowadays there is a lot of research works in creating transgenes based on the Populus genus plants. [Plants of the genus Hubscher  draws at plants. . Earlier plants. , it beco plants.  and wild plants. .Populus genus plants lasts for many years, and significant success is achieved [That is why, work on creating cryopreservation protocols of the achieved ,14,15,23achieved . It is pachieved  and therachieved ,30,31, bPopulus genus plants  long-term storage without changing their genetic status is an urgent and timely task which can help to reveal new opportunities in woody plants genomic analysis. Regarding to this, we considered in our article possible ways to increase the regenerate survival rate using the example of aspen transformed plants and wild types after the freezing—thawing procedure and storage in liquid nitrogen by changing the explant preparation conditions, and taking into account preservation of recovered plants genetic stability.Therefore, elaboration of new approaches for the To determine possible influence of various factors on the explant survival after the freezing - thawing procedure, experiments with different pretreatments were laid down. In vitro cryopreservation of microshoot axillary buds by rapid freezing in the presence of PVS2 was successful with all pretreatment options. After the thawing process, explant differentiated tissues of all experimental options died off, and only the axillary bud remained viable ensuring restoration A. MicrogRegenerants survival varied depending on the pre-treatment selected. The most reliable differences affecting survival were registered in the experiment using the OF pretreatment a. The avFurther analysis of microshoots obtained from regenerants after 8 weeks of incubation on the recovery medium showed that visually distinguishable morphological alterations after freezing-thawing were not found in all types of experiments C. Figuresp-Xeg gene was lower than for other transgenic and wild plant genotypes by an average of 7%. No significant relationship was found between the storage time in LN and the regenerants survival rate after the freezing - thawing procedure.Most favorable conditions were selected for transgenic aspen lines cryopreservation, which showed an increase in the regenerate survival on non-transformed Pt, f2 and PtV22 genotypes. Non-transformed plants were also included in the experiment. Microplants cultivated on growth medium with 1% agar-agar were used, four-day-old axillary buds were extracted after removal of apical buds, and then the explants were treated with OF. Further, explants were also transferred to PVS2 and stored in LN. After 3 and 6 months of cryostorage, explants were thawed and transferred to recovery medium. Complex action approach in the explant pretreatment made it possible to significantly increase the regenerant survival after the freezing-thawing procedure. Survival rate was 83% on average, which was significantly higher than all previously obtained survival rates in all types of experiments . StatistTo determine integrity of genotypes restored after the plant cryostorage, analysis was initially carried out on the presence of recombinant insertion of the plant transgenic genotypes: PtXIBar9a, PtXIBar14a, f2XIBar5a, PtV22XBar2a, Xeg-1-1a, Xeg-1-1c, Xeg-2-1b and Xeg-2-3b. PCR analysis showed presence of the recombinant gene introduction with required size in all transgenic lines .Genetic marking was performed using SSR markers at 6 loci for all experimental lines. Fragmentary analysis demonstrated that genetic profiles of plants that were exposed to the freezing-thawing procedure and of plants that were not exposed to these manipulations were identical .Analysis of plants under greenhouse conditions showed that the data on height of cryopreserved plants and plants without cryopreservation were similar . MorpholPopulus plants in the forest biotechnology are the main model plants, all variations of the obtained and wild genotypes preservation became an important problem. It was proven that aspen subcultivation in vitro leads to alterations in genetic material without visible external signs of somaclonal variability [Populus genus plants remains unresolved. Studies show that this genus regenerated plants obtained from in vitro culture survival rate varies greatly from 3% to 93% [Due to the fact that iability . Based o% to 93% . This is% to 93% ,29,30,31sp-Xeg gene, could be significantly lower in comparison with the others. This may be due to the peculiarity of the recombinant gene action, which leads to an increase in the cell size [Studies conducted on tops of the white poplar shoots  showed tell size , as a reFor the selected transgenic and non-transgenic lines, it was decided not to increase the number of passages, to reduce somaclonal variability. This is why cultivation on medium with a high sugar content to reduce the moisture level of the culture ,36 was nMeristem structure is another important parameter in increasing the survival rate, as it is shown by Thinh et al. . Fully cThe osmotic fluid (OF) pretreatment was used for additional dehydration of plant material. It showed an increase in the survival rate of regenerates by 20%. Osmotic fluid influences this way because of its hypertonic features. It allows to change the cell water content and prepares the cell to PVS2 solution action. The main feature of this mechanism is the following: it maximizes concentration of the cell solute molecules. It prevents formation of crystals from the remaining water molecules . The freTo recovery the culture we used rapid heating to thaw the explants; this improves the survival rate, as very small ice crystals could turn into larger and destructive ones, if the frozen system is being heated too slowly . In addiUsing pre-cultivation on medium with higher gelling agent content; removal of apical buds and introduction of osmotic fluid in pretreatment of explants allowed to increase survival rate from 55% to 83%. So, we can state that only the combined effect of different factors is able to significantly increase the survival rate of regenerants. This is why this technology is recommended for in vitro cryopreservation of aspen plants. Our research also shows that storage duration in the developed technology does not affect the degree of regeneration recovery. We calculated that according to recommendations on creating a cryoreservoir we should take at least 50 explants per genotype for successful and safe storage .sp-Xeg gene showed saving of changes in the interclonal variation, which was noted earlier [We analyzed the safety of genetic characteristics after cryopreservation. The results showed that there were no alterations in the genotype. The recombinant genetic construct in transgenic plants so as genetic status during genotyping using the SSR markers remain undamaged. The selected marker loci have a high degree of variability. They are used to determine genetic changes within one specie and are also used to identify somaclonal variability ,48,49. T earlier . This coPopulus tremula L.), and PtV22 (Populus tremula x Populus tremuloides). Plants with the Pt genotype are characterized by rapid growth and resistance to heartwood rot [bar [sp-Xeg [In the experiment, 11 genotypes of wild types of aspen and transformed lines obtained on their basis were used. Wild types of aspen: Pt and f2  were divided into three groups for different experiments of explants’ pretreatment , which included different types of explants, cultivation on medium with different densities and osmotic treatments. Axillary buds of microshoots and axillary buds obtained after preliminary removal of the apical bud and cultivation for 4 days on the WPM growth medium, agar-agar 0.8%, were used as explants in experiment A. In experiment B, explants of axillary buds of microshoots were used obtained after three times subcultivation on the WPM medium with agar-agar content in concentration of 0.8%, 1%, and 1.2%. Experiment C included treatment of the isolated explants with osmotic liquid (OF) (Murashige and Skoog medium (MS)  with 2 MCryotubes of all types in the experiments were transferred to a water bath with 45 °C for 1 min. After thawing, PVS2 was drained from cryotubes and replaced with liquid MS medium containing 1.2 M sucrose , where t®Plant Mini kit  for molecular analysis. PCR was performed using the Encyclo Plus PCR kit . Previously selected primers and conditions were used to determine the presence of bar and sp-Xeg construct [PTR2, PTR3, PTR4, PTR5, PTR6, PTR14 loci and selected for genotyping P. tremuloides were used in microsatellite marking [The approaches including variability molecular analysis and biometric analysis were used to determine the genotype integrity. The molecular analysis included determination of the recombinant construct presence in the transformed lines, as well as molecular microsatellite marking (SSR) of all lines after storing for 6 months in LN. Plants of all genotypes not exposed to cryostorage were used as control plants to analyze variability. DNA was isolated from all lines using DNeasyonstruct ,50. Prim marking ,49. For After assessing the survival rate after cryopreservation, part of the microshoots 4–5 weeks old 1.5–2 cm in size of the Pt, Xeg-1-1a, Xeg-1-1c, Xeg-2-1b and Xeg-2-3b genotypes were excised and rooted on the WPM medium at 22–24 °C and at photoperiod of 16 h to analyze the interclonal variation. The same genotypes, but not cryopreserved, were used as control species. Rooted plants were transplanted into protected ground conditions . Height of the plants was measured after 6 months. Plants of Pt genotypes not exposed to cryostorage were used as control plants to analyze genetic variability under greenhouse conditions.Each experiment with each cryopreservation line was repeated twice and consisted of at least three repetitions per treatment, 18–20 samples each. Plants were transferred to greenhouse conditions with an amount of 20 plants per genotype. Statistical analysis of percentages was carried out by the test for homogeneity of proportions, and significant treatment differences selected by a non-parametric statistical test, the Post Hoc Multiple Comparisons Test .Thus, we developed a technology that makes it possible to successfully preserve the wild aspen genotypes and the transformed lines with no changes in genetic status of culture. Average recovery rate in this technology is high reaching 83%. Such high percentage of survival rate is achieved due to the following technology features: preliminary cultivation on a denser growth medium; selection of axillary bud’s explants, obtained after removal of the apical bud; explants pretreatment with osmotic fluid; using the one-step freezing method and PVS2; the fast thawing rate. Usage of complex pretreatment made it possible to increase the regenerant survival rate from 55% to 83%. Genetic and morphological analysis showed that cryopreservation technology developed by us is the most suitable in long-term cryopreservation of aspen plants and does not lead to variation in the plant genotype. Our data demonstrated that survival rate of the explants could directly depend on specific genetic transformation that was carried out for a given genotype. It is important to take this into consideration in the process of cryopreservation. The results obtained allow us to conclude that the technology developed by us can be acceptable for long-term storage of valuable aspen genotypes. However, further genetic, histological and morphological studies are needed for a deeper understanding. We also plan to test this technology on other types of woody deciduous plants, in particular valuable birch genotypes.In this regard, in order to standardize and possibly increase the survival rate of regenerates, we plan to develop a cryostorage protocol using artificial intelligence models and optimization algorithms in plant cell and tissue culture. This approach has shown itself to be promising for various plants . Thus, f