Dengue virus infection (DVI) is a mosquito-borne disease that can lead to serious morbidity and mortality. Dengue fever (DF) is a major public health concern that affects approximately 3.9 billion people each year globally. However, there is no vaccine or drug available to deal with DVI. Dengue virus consists of four distinct serotypes (DENV1-4), each raising a different immunological response. In the present study, we designed a tetravalent subunit multi-epitope vaccine, targeting proteins including the structural protein envelope domain III (EDIII), precursor membrane proteins (prM), and a non-structural protein (NS1) from each serotype by employing an immunoinformatic approach. Only conserved sequences obtained through a multiple sequence alignment were used for epitope mapping to ensure efficacy against all serotypes. The epitopes were shortlisted based on an IC50 value <50, antigenicity, allergenicity, and a toxicity analysis. In the final vaccine construct, overall, 11 B-cell epitopes, 10 HTL epitopes, and 10 CTL epitopes from EDIII, prM, and NS1 proteins targeting all serotypes were selected and joined via KK, AAY, and GGGS linkers, respectively. We incorporated a 45-amino-acid-long B-defensins adjuvant in the final vaccine construct for a better immunogenic response. The vaccine construct has an antigenic score of 0.79 via VaxiJen and is non-toxic and non-allergenic. Our refined vaccine structure has a Ramachandran score of 96.4%. The vaccine has shown stable interaction with TLR3, which has been validated by 50 ns of molecular dynamics (MD) simulation. Our findings propose that a designed multi-epitope vaccine has substantial potential to elicit a strong immune response against all dengue serotypes without causing any adverse effects. Furthermore, the proposed vaccine can be experimentally validated as a probable vaccine, suggesting it may serve as an effective preventative measure against dengue virus infection. In addition to Dengvaxia, many other dengue vaccines are under development and being evaluated in preclinical studies. The prM and E proteins are the primary focus of vaccine development because they are thought to elicit protective immune responses in humans . FurtherAll dengue virus serotypes –4 can in22.1The protein sequences of EDIII, prM, and NS1 of all serotypes (DENV1-4) were downloaded from NCBI GenBank , the Vir2.2The retrieved protein sequences of EDIII, prM, and NS1 were subjected to multiple sequence alignment (MSA) by ClustalW  integrat2.3http://tools.iedb.org/bcell/   /bcell/   with Bep/bcell/  . Similar2.4http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html), and the allergenic epitopes were predicted by AllerTOP v2.0 (https://www.ddg-pharmfac.net/AllerTOP/). To determine the toxicity of the peptides, ToxinPred2  energy was selected. For the visualization of interaction, the UCSF Chimera server  and BIOV2.6The multi-epitope subunit vaccine was constructed against all DENV1-4 serotypes by integrating prioritized B- and T-cell epitopes from prM, EDIII, and NS1 proteins. Using a special linker, EAAAK, AAY, and GGGS epitopes were joined together. B-cell, HTL, and CTL epitopes were joined by KK, AAY, and GGGS linkers, respectively. In addition, B-defensins (PDB ID: 1IJV) were incorporated into the vaccine as an adjuvant and positioned at the N termini of epitopes to enhance the immunogenicity of the vaccine. The first epitope was linked to the adjuvant through an EAAAK single linker, and a thrombin site and His tag were employed at the C terminus of the designed vaccine construct.http://protein-sol.manchester.ac.uk/).The vaccine was further evaluated for antigenic assesssment by the VaxiJen and AntigenPro tools. Additionally, the vaccine underwent testing on the AllerTOP v.2.0 server to make sure it would not cause any allergic reactions. Collectively, these studies offer insight into the vaccine’s efficacy and safety   2.8The PDB structure of TLR3 was extracted from the RCSB Protein Data Bank (PDB ID:1ZIW). Active and passive residues of the designed construct and TLR3 were achieved using C-PORT  before p2.9E. coli K12 strain (http://snapgene.com/) was used to cloned the improved construct into the pET21(+) vector.The protein sequence of the vaccine construct was reverse translated into nucleotide sequences by EMBOSS Backtranseq  followed2 strain . The GC 2.10http://tools.iedb.org/population/) with the prioritized MHC-I and MHC-II epitopes.Population coverage prediction is critical for vaccine design to ensure that the designed vaccine construct covers the highest possible global population. The IEDB population coverage tool was used to predict the population coverage of the vaccine construct  simulation was conducted with a vaccine-docked complex using GROMACS, a Linux-based program . The vac33.1The retrieved EDIII, prM, and NS1 protein sequences were subjected to BLASTp using default parameters to obtain the most identical sequences. The reference sequences with their accession numbers retrieved from different regions of the world, including Pakistan, are shown in Multiple sequence alignment of EDIII, prM, and NS1 proteins of all serotypes revealed the conservation among different serotypes. For downstream analysis, we selected only the conserved protein sequences of each serotype protein .3.2The epitopes were shortlisted based on different selection criteria, such as IC50, percentile score, and antigenic score. For an epitope to be considered high affinity, the IC50 value should be less than 50 nM. An IC50 value <500 nM implies intermediate affinity, while values <5,000 nM indicate low affinity . All epiThe cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes for EDIII for DENV1-4 were predicted using the Immune Epitope Database (IEDB) and were 110 and 117, respectively. Only 28 CTL and 22 HTL epitopes showed good antigenicity scores (>0.47), were non-allergenic and non-toxic, and had IC50 values <50, indicating significant binding potential with the anticipated HLA alleles. Likewise, for prM DENV1-4 serotypes, overall, 39 CTL epitopes and 30 HTL epitopes were predicted; out of these, 21 CTL and 22 HTL epitopes were screened. For NS1 protein DENV1-4 serotypes, overall, 85 CTL and 54 HTL epitopes were predicted, and out of these, 20 CTL epitopes and 28 HTL epitopes were predicted to be potential epitopes and had antigenic scores >0.47, IC50 values <50, and were non-allergenic and non-toxic.After considering their binding affinities with respect to IC50 values and the highest antigenic score, we shortlisted the best B- and T-cell epitopes from each serotype. To prioritize the epitopes, we chose one epitope of each serotype that had the highest antigenic score and an IC50 <50 nM. In total, 12 B-cell epitopes from EDIII, prM, and NS1 were identified as potential antigenic epitopes. Similarly, a total of 24 T-cell epitopes, including HTL and CTL epitopes, were selected from EDIII, prM, and NS1 from the DENV1-4 serotype to construct a vaccine, as shown in Additionally, during the selection process, we observed that the prioritized B-cell epitope of DENV2-EDIII contained KALKLSWFKKG and the smaller fragment of the DENV1-EDIII B-cell epitope, KALKLSW; therefore, the larger epitope was retained from these two epitopes in the final vaccine construct to ensure the best possible outcome . SimilarAdditionally, in our study, we also considered the DENV2 epitope RQEKGKSLLF associated with HLA-B*15:01 and DEV4 epitope EIRDVNKEKVVGRII associated with allele HLA-DRB1*07:01, despite having slightly higher IC50 values, i.e., 61.97 and 79.2, respectively. They were selected for inclusion in the final vaccine design because of favorable antigenicity scores and binding affinities. These epitopes are represented in bold in 3.3Molecular dockings were carried out between the modelled epitope and their putatively interacting HLA alleles. This was essential to perform structural analysis of the interaction between the epitope and the HLA alleles. Furthermore, the docking of epitopes with HLA molecules is the first stage in triggering specific immune responses. During the development of vaccines, determining the epitopes that have a statistically significant binding affinity for HLAs should be prioritized for inclusion in vaccine formulations. Epitopes are presented on the surface of antigen-presenting cells (APCs), such as dendritic cells; when they interact with HLA molecules, the resulting interaction triggers a series of immunological responses that ultimately leads to the generation of immune cells that are particular to the antigen  The struThe chosen T-cell epitopes had strong interactions with their corresponding HLA alleles and the PDB structure with the lowest ACE score (atomic contact energy) was selected. The best HLA protein-epitope docked complexes of some epitopes with their respective alleles are visualized in 3.4The vaccine construct was developed by incorporating the screened epitopes based on the criteria discussed above. In total, 11 B-cell epitopes, 10 helper T-cell (HTL).epitopes, and 10 cytotoxic T-cell (CTL) epitopes were chosen from DEN1-4  and joined using KK, AAY, and GGGS linkers, respectively. Following the completion of the merging process, it was determined that the total length of the construct was 500 amino acids. A 45-amino acid-long adjuvant (GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK) was added at the N terminus of the construct by the EAAAK linker. Subsequent to the incorporation of linkers in addition to the adjuvant, a thrombin cleavage site and 6×histidine tag were inserted at the C terminus. The final vaccine construct was determined to be 557 amino acids long. The sequence of the final vaccine construct formulated is shown in 3.5in vitro half-life of the vaccine was identified as 30 h in mammalian reticulocytes, whereas in vivo it was 20 h in yeast and 10h in Escherichia coli. The protein instability index was 29 (<40), which indicates a high probability of stability. The aliphatic index was 75.03; a higher aliphatic index value of proteins indicates thermostability, even at extreme temperatures. Moreover, a GRAVY (grand average of hydropathicity) score of -0.379 was obtained; a negative GRAVY value shows the hydrophilic characteristics of proteins  is necessary for the intracellular transport of an antigen molecule and the activation of the proper downstream immune pathways. In this regard, we conducted docking of a multi-epitope tetravalent vaccine construct with TLR3 to assess the intermolecular interactions and binding energy .The CPORT analysis before docking showed that the vaccine construct has active residues at positions 1, 2, 3, 4, 5, 6, 9, 10, 12, 13, 34, 262, 263, 266, 267, 270, 271, 273, 274, 281, 282, 284, 285, 288, 291, 292, 293, 294, and 324, and TLR-3 active residues are present at positions 322, 323, 353, 355, 356, 379, 380, 614, 616, 618, 634, 638, 639, 643, 644, 647, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664. Similarly, the HADDOCK server revealed a docking score of –108.02+/–6.4 for the docked complex. A docked complex of vaccine-TLR3 is visualized in 3.9In silico cloning was conducted to validate the expression of recombinant vaccine protein using an E. coli-K12 model  at multiple cloning sites. Thus, the complete clone was 6,991 base pairs in length , substantially increased after the first injection of the vaccine construct (antigen). The secondary immune reaction is characterized by elevated levels of IgM and IgG  and develops in response to repeated exposure to the same antigen. Aditionally, there was a notable rise in the concentration of IgM and IgG and a fall in antigen concentration. IgM, IgG1, and IgG2 levels also increased significantly, as shown in 3.11The molecular dynamic simulation was conducted on docked complexes using GROMACS 5.0 software to verify the stability of contacts between the multi-epitope tetravalent vaccine complex and the receptor TLR-3. Energy minimization was performed for 50,000 steps to remove clashes, and equilibration was conducted to bring the system to the desirable conditions in terms of temperature, pressure, density, etc. The energy minimization process was completed at 3,000 ps, as represented in 4Flaviviridae, consisting of four serotypes (DENV1-4) that cause DF, dengue shock syndrome, and dengue hemorrhagic fever (Dengue virus (DENV) is an arbovirus belonging to the family ic fever . It is eic fever . AlthougDengvaxia, developed by Sanofi-Pasteur, has been the only licensed vaccine against DENV infection. Dengvaxia varied in its protection among the DENV serotypes, demonstrating a protective efficiency of 50.3% against DENV1, 42.3% against DENV2, 74.0% against DENV3, and 77.74% against DENV4. Additionally, Dengvaxia has poor protection efficiency in children under the age of nine. Therefore, it is debatable whether Dengvaxia can be used to prevent DENV infection . The devin vitro. The findings revealed that when TLR3 is expressed in HEK293 cells, they induce the secretion of IL-8 in response to DENV recognition. In human monocytic cells (U937), the primary mechanism triggering IL-8 production after viral identification involves TLR3 following endosomal acidification. Silencing TLR3 in U937 cells results in a notable reduction in DENV-induced IL-8 production. One of the most critical requirements for a vaccine is that it establishes strong interactions with immune receptors to facilitate its optimal absorption and distribution across the host body. The results of molecular docking of the vaccine with TLR3 demonstrate that only a minimal amount of energy is needed to create a stable complex with strong interactions between the vaccine and TLR3. These findings suggest that our designed vaccine can potentially stably bind to immune receptors . We accomplished this by employing the PatchDock server, a powerful and efficient program for protein-peptide binding, which showed a robust correlation within epitopes and their associated human leukocyte allele(s). Accordingly, a tetravalent vaccine was constructed by joining the prioritized B-cell, HTL, and CTL epitopes via linkers along with the β-defensin adjuvant. The β-defensins as an adjuvant work as an antimicrobial and immunomodulatory agent. Defensin peptides help the host defend itself against microbial infection and play a role in adaptive immunity by attracting naive T cells and immature dendritic cells to the place of infection, thereby boosting antiviral immunity. Similarly, the linkers contribute significantly to extend conformation (flexibility), the folding of proteins, and the separation of functional domains. As a result, the protein structure is rendered more stable because of the contributions of the linkers . The HTLin silico cloning techniques assure enhanced expression of the designed vaccine in the E. coli expression system. Based on these findings, we conclude that the tetravalent subunit vaccine we anticipated has a high binding affinity with immune receptors and satisfactory computational evaluation.The antigenicity score of the multi-epitope construct was predicted to be 0.7968 by the online tool VaxiJen, with a molecular weight of 60.23 kDa. Furthermore, it is generally believed that proteins with a molecular weight of less than 110 kDa are considered more antigenic in nature. In a three-dimensional refined model of the vaccine, six discontinous B-cell epitopes were identified. Our proposed vaccine can generate substantial antibody formation. This is because discontinuous B-cell epitopes serve an important role in humoral immune responses by generating antibodies . However5Over the past few decades, dengue virus has spread rapidly across the world, becoming a major public health problem in tropical and subtropical regions, and millions of new cases are recorded. By employing an immunoinformatics approach, we designed a multi-epitope-based tetravalent vaccine targeting DENV1-4 serotypes using the conserved sequences of the proteins EDIII, prM, and NS1. The resulting vaccine construct was further investigated for allergenicity, antigenicity, and toxicity and underwent an immune simulation analysis. All of these analyses predicted that our designed vaccine is highly immunogenic, non-allergenic, and non-toxic. In addition, immune stimulation results demonstrated that both antibody and cell-mediated immune responses would be elicited against DENV by injecting the multi-epitope vaccine. Molecular docking and MD simulation were carried out to validate the interactions within the vaccine-TLR3 complex; the results showed that it exhibited a higher binding affinity and stable interaction. Our results propose that a designed multi-epitope vaccine has significant potential in provoking a balanced immune response against all dengue serotypes without causing any undesirable effects. Moreover, the designed vaccine can be further experimentally validated before human immunization to validate its efficacy and safety profile against dengue virus infections.The original contributions presented in the study are included in the article/AB: Data curation, Methodology, Software, Writing – original draft, Writing – review & editing. SJ: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. BA: Formal Analysis, Writing – review & editing. MF: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing.


ATXN1 gene and is characterized mostly by a profound loss of cerebellar Purkinje cells, leading to disturbances in coordination, balance, and gait. At present, no curative treatment is available for SCA1. However, increasing knowledge on the cellular and molecular mechanisms of SCA1 has led the way towards several therapeutic strategies that can potentially slow disease progression. SCA1 therapeutics can be classified as genetic, pharmacological, and cell replacement therapies. These different therapeutic strategies target either the (mutant) ATXN1 RNA or the ataxin-1 protein, pathways that play an important role in downstream SCA1 disease mechanisms or which help restore cells that are lost due to SCA1 pathology. In this review, we will provide a summary of the different therapeutic strategies that are currently being investigated for SCA1.Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder that affects one or two individuals per 100,000. The disease is caused by an extended CAG repeat in exon 8 of the  Spinocerebellar ataxia type 1 (SCA1) is a progressive, autosomal dominant neurodegenerative disorder characterized by ataxia, speech, and swallowing difficulties, spasticity, and abnormal control of eye movements. At later stages of the disease, patients show signs of muscular atrophy and cognitive defects ,3,4. EveATXN1 gene [ATXN1 gene is organized in nine exons of which the first seven fall in the 5′ untranslated region (UTR). The 5′UTR exons can undergo alternative splicing, suggesting that transcriptional and translational regulation of the SCA1 encoded protein, ataxin-1, may be complex [ATXN1 gene results in an expanded polyglutamine tract in the ataxin-1 protein, thereby inducing a polyglutamine-induced, toxic gain-of-function mechanism leading to SCA1 disease pathology [SCA1 is caused by an expanded CAG repeat in the coding region of the human XN1 gene . Normal XN1 gene ,12,13,14XN1 gene . The ATX complex ,17. The  complex . Expansiathology .Currently, there is no disease-modifying treatment available for SCA1 patients, but several therapies, including physiotherapy, occupational therapy, and speech therapy are offered which provide some symptomatic relief and help in improving the quality of life . MultiplSCA1 pathophysiology is characterized mainly by degeneration of the cerebellar cortex, deep cerebellar nuclei, and brainstem . Loss ofSca1 knock-out mice do not display any signs of ataxia, motor incoordination, or cerebellar degeneration [Sca1 knock-out mice do demonstrate deficits in several learning and memory behavioral tests and neurophysiological studies. These results show that SCA1 is not caused by a loss-of-function mechanism and indicate a role for ataxin-1 in learning and memory [Drosophila models leads to relevant disease phenotypes and pathology, suggesting that SCA1 is caused by a toxic gain-of-function mechanism [In the last decades, research has been conducted to study the molecular mechanisms behind SCA1 pathogenesis. However, the disease mechanisms are still not fully understood ,25. Geneneration . Howeverd memory . On the echanism ,28,29. Drosophila screen highlight the role of protein folding and clearance in SCA1 [The polyglutamine repeat expansion is thought to exert its toxicity by altering the interactions of several factors with key domains, including the C-terminus and AXH domain in ataxin-1 . ATXN1L, or Boat (brother-of-ataxin-1), shares 33% homology with ATXN1, including the conserved AXH domain, where both ATXN1 and ATXN1L interact with the transcriptional repressor CIC [ATXN1 binding to RNA decreases when the repeat expansion increases, possibly altering its role in RNA metabolism [Ataxin-1 additionally contains an AXH domain of 120 amino acids, which mediates many protein–protein interactions and RNA-binding activity ,25. The echanism ,40. By iechanism . Mutatioogenesis ,42. The ogenesis ,44. Addissor CIC ,25,45. Ttabolism . 154Q/2Q  promoter to ensure overexpression of human ifically . The humifically ,48. Miceifically ,48. Howeifically ,49.154Q/2Q knock-in mice expresses mutant Atxn1, with 154 CAG repeats under the control of endogenous regulators, thereby expressing mutant Atxn1 throughout the brain and spinal cord. SCA1154Q/2Q mice display motor incoordination and muscle wasting, as well as premature lethality, reduced adult hippocampal neurogenesis, and kyphosis  [The SCA1l bones) . Furtherl bones) ,51.Drosophila melanogaster fly model expresses human 82Q ATXN1 in the eye retina using the GAL4/UAS system. This leads to nuclear inclusion formation in the eye photoreceptor cells and neurons of the CNS, subsequently resulting in degeneration of the retina and neurodegeneration [In addition to mouse models to study SCA1, a fly model was developed ,47. The neration . HoweverATXN1 RNA act by either modulating RNA levels or by blocking translation. Here, we will provide an overview of the different ataxin-1 modulating therapies that are currently being explored for SCA1 , or post-transcriptional gene silencing, is a conserved biological process in which noncoding double-stranded RNA molecules are involved in suppression of gene expression through translational or transcriptional repression. ,53. The ATXN1 sequences improved motor coordination, restored cerebellar morphology, and resolved ataxin-1 inclusions in PCs of these mice  vectors expressing shRNAs targeting human  benefit . Importa benefit . To studas found . There was found . Altogetas found ,58,59,60as found . RNA-seqas found . By alteas found .A second RNA-based therapy approach to reduce gene or protein expression is the use of antisense oligonucleotides (ASOs). ASOs are chemically modified, single-stranded oligonucleotides of generally 12 to 30 bases, which bind to RNA through Watson–Crick–Franklin base pairing. Depending on the sequence and modifications, ASOs can alter RNA functions through several mechanisms. They can be used to reduce expression of a toxic protein, to modify mutant proteins, to reduce their toxicity, or to restore protein expression  or intracerebroventricular (ICV) injections ,56,67. AAtxn1 gapmer ASO353 in pre-symptomatic SCA1154Q/2Q mice, a reduction of Atxn1 levels at 6 and 24 weeks after ASO administration was shown. Furthermore, a rescue of disease-associated phenotypes was demonstrated, including motor performance, survival, analysis of neurochemicals, and transcriptional disease signatures 7 in all brain regions. Most importantly, ASO levels in the different brain regions and the spinal cord were similar, despite the differences in distance from the injection site [Steric or RNA blocking ASOs can inhibit (or activate) protein translation through binding to regulatory elements, including the upstream open reading frame  was shown to play a significant role in protein toxicity  on either the wildtype allele or the mutant allele, it was shown that targeting S776 phosphorylation could ameliorate SCA1 pathology [Since phosphorylation of ataxin-1 at the serine 776 residue , also contribute to the altered PC activity observed in SCA1  are potassium channel blockers with a high affinity for A-type potassium channels. Subcutaneous injection of APs has been shown to normalize the firing frequency of PCs and the motor dysfunction of early symptomatic B05 mice . However154Q/2Q mice with the Ro0711401 PAM led to a significant increase in motor performance after 30 min [154Q/2Q mice resulted in markedly reduced motor function [The observed reduction in mGluR1-mediated EPSC amplitude can be modulated by so-called positive allosteric modulators (PAMs), which amplify the mGluR1 receptor function ,94,95. Sr 30 min . Surprisr 30 min . Additiofunction . ContradFurthermore, the modulatory effects of baclofen on mGluR1 have been tested . Subarac154Q/2Q mice [ATXN1 [154Q/2Q mice ameliorated PC climbing fiber deficits, rescued dendritic spine loss, and improved behavioral outcomes [In addition to targeting mGluR1 directly, mGluR1-related signaling molecules can be targeted to ameliorate SCA1 pathology. For instance, a reduction in Homer-3, which is an adaptor protein of mGluR1, has been observed in early symptomatic SCA1/2Q mice ,93. Thise [ATXN1 ,93. To ie [ATXN1 . AAV-Homoutcomes . N-methyl-D-aspartate receptor (NMDAR) is involved in SCA1, administration of memantine, which is a NMDAR antagonist that preferentially inhibits the extra-synaptic NMDAR, had beneficial effects , BDNF, and lithium carbonate have been tested in mouse models , Table 3154Q/2Q mice as well as human patients, possibly through a repressive effect of the mutant ataxin-1 [154Q/2Q mice models [154Q/2Q mice [VEGF is another trophic factor, which additionally functions as an angiogenic factor. VEGF is widely expressed in PCs in the cerebellum and is downregulated in SCA1ataxin-1 ,102. Redataxin-1 . To incre models ,102. ICV/2Q mice ,102. How154Q/2Q mice additionally showed amelioration of motor and cognitive deficits as well as improved cerebellar and hippocampal pathology [Brain-derived neurotrophic factor (BDNF) is an important trophic factor for cognitive and motor function and plays key roles in both survival signaling and neuroplasticity. BDNF levels are reduced in the cerebellum and medulla of SCA1 patients . Furtherathology .154Q/2Q mice [154Q/2Q mice resulted in a rescue of motor function as well as spatial learning and memory impairment [Lithium carbonate has an effect on several cellular functions, and it has shown to exert neuroprotective effects, possibly by affecting gene transcription or by reducing oxidative stress , thereby inhibiting damage repair [154Q/2Q mice with an AAV-based method (AAV1-HMGB1) showed an increase in lifespan and improvement in motor activity [In SCA1, the expression of mutant e repair . HMGB1 ie repair ,108. To Drosophila as well as in silico models have shown that RpA1 might be involved in DNA damage repair in SCA1 pathology [154Q/2Q mice. Treatment with AAV-RpA1 showed a recovery of motor function, DNA damage, dendritic shrinkage, and spine abnormalities [RpA1 is another molecule that is involved in multiple DNA damage repair pathways, including homologous recombination and non-homologous end joining . Geneticathology ,117. Themalities . It is smalities . Howevermalities .ATXN1 [ATXN1 clearance through interfering with the proteasome-mediated degradation, possibly by inhibiting a ubiquitin E3 ligase or other factors in the proteasome pathway that normally promote ubiquitination and degradation of ATXN1. Indeed, pharmacological inhibition of all PAKs resulted in a reduction in Atxn1 levels in SCA1154Q/2Q mice, suggesting that PAK1 regulates ATXN1 stability and toxicity [As SCA1 is a protein aggregation disease, therapeutics that target the proteostatic machinery to promote protein breakdown and prevent aggregation have also been tested. P21-activated kinases (PAKs) are a family of serine/threonine kinases 154Q/2Q mouse model [MSCs are multipotent and reside in several tissues but mainly in the bone marrow and fat, where they support hematopoiesis and produce cells of the mesodermal lineage. Additionally, they also have immunomodulatory and neurotrophic properties . The undse model . Resultsse model ,121,122.se model . Howeverse model . 154Q/2Q mice to assess the effects of these therapies on the PNS, as motor neuron degeneration in this area is observed in SCA1 patients. IT administration of GFP-labeled MSCs into the subarachnoid space in pre-symptomatic SCA1154Q/2Q mice showed distribution of the MSCs into the ventral root of the spinal cord, which resulted in larger axons and bigger myelin sheaths compared with non-treated SCA1154Q/2Q mice [154Q/2Q mice [In addition to transgenic B05 mice, MSC therapies were also tested in SCA1/2Q mice . In this/2Q mice . These r/2Q mice . In anot/2Q mice . Mice ei/2Q mice . Both ad/2Q mice .Next to MSCs, NPCs can also be used as a cell source for stem cell therapy ,125. NPCPre-clinical studies have shown that replacement therapies using MSCs and NPCs can improve neuropathology and motor coordination in SCA1 mice ,124,125.As outlined in this review, several therapeutics for SCA1 have been tested in pre-clinical animal models. Therapies focus either on modulating the levels of ataxin-1, restoring the normal function of several targets involved in SCA1 pathophysiology, or replacing the cells that have been lost due to neurodegeneration. Pre-clinical studies have shown promising results for these strategies, and some therapies were additionally tested in clinical trials ,128. Most clinical trials in SCA1 have focused on restoring the PC activity by targeting the potassium channels and the glutamatergic channels. A clinical, open label trial with the slow-release form of 4-AP  was performed in 16 patients with chronic cerebellar ataxia, including 3 patients with SCA1 ,130. TreOther clinical studies have been performed to improve the electrical function of PCs by using Riluzole, Troriluzole, or CAD-1883, which all act by promoting the opening of small-conductance calcium-activated potassium channels and by enhancing glutamate transporters . A rando7 ASO (VO659) has recently entered a phase I/II open-label clinical trial, including 95 SCA1, SCA3, and Huntington’s disease patients to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of ascending doses of intrathecally administered V0659 .Additionally, oral lithium has been investigated in an open-label, non-randomized phase I trial (NCT00683943), but results have never been published . MoreoveIn one open label clinical study, 14 SCA patients were IT injected with umbilical cord MSCs for 4 weeks . TreatmeAlthough results from these clinical studies on MSC transplants seem efficient and safe, a meta-analysis of these studies showed no significant differences in the ICARS scores before and after treatment, low certainty in estimates of quality, and a high risk of bias, as both studies were uncontrolled clinical trials . FurtherAlthough several human clinical trials have been performed, no therapies for SCA1 have reached market authorization yet. For future SCA1 therapeutics to reach the clinic, several challenges, mainly in the translation of preclinical findings into clinical trials and in clinical trial design, need to be overcome ,139. MouBecause of the BBB, delivery of drugs to the brain is a major challenge. Finding the right administration route and dose of a drug in order to reach a therapeutic dose in the brain without inducing toxicity, remains challenging and should be assessed for each therapeutic strategy . AnotherDrug development is a long and expensive process, and as SCA1 is a relatively rare disease, it is less attractive for industry to invest in SCA1 drug development . TherefoIn SCA1, disease progression is slow, and clinical rating scales might not be sensitive to measure effects in trials that run for one year ,146. ImpSCA1 is a devastating disease, and to date only symptomatic treatment is available. With the advancements made in understanding SCA1 pathology and the progression of different intervention strategies, there is great promise that a therapy to treat SCA1 patients will become available in the coming years. There are some pharmacological treatments targeting dysregulated pathways in SCA1 as well as promising strategies modulating ataxin-1 levels, with several RNA targeting therapies for SCA1 in advanced stages of preclinical development. Another promising strategy for SCA1 is gene editing using CRISPR/Cas9, which has already successfully been tested in a pre-clinical study in hiPSCs ,152. How


Interferons (IFN) are an assemblage of signaling proteins made and released by various host cells in response to stimuli, including viruses. Respiratory syncytial virus (RSV), influenza virus, and SARS-CoV-2 are major causes of respiratory disease that induce or antagonize IFN responses depending on various factors. In this review, the role and function of type I, II, and III IFN responses to respiratory virus infections are considered. In addition, the role of the viral proteins in modifying anti-viral immunity is noted, as are the specific IFN responses that underly the correlates of immunity and protection from disease. Interferons (IFNs) were first discovered during a study on viral interference in egg chorioallantoic membranes. The early study found that a factor was released which had the ability to induce viral interference, hence “interferon” . This soRIG-1) receptors, and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are the principal IFN-stimulating PRRs [The development of innate and adaptive antiviral immunity is influenced by pattern recognition receptors (PRRs). The activation of innate immunity by respiratory viruses can occur either on the cell surface through host cell receptors or by the interaction of intracellular and cytosolic PRRs. These receptors detect viral components such as RNA or DNA, resulting in the generation of downstream pathways that lead to antiviral immunity . When PRing PRRs ,15,16. Ting PRRs . RIG-liking PRRs  and signing PRRs . The bining PRRs . These ping PRRs ,21. Binding PRRs . The JAKing PRRs .IFIT family of ISGs is commonly induced by type I and type III IFNs. IFIT genes suppress viral infection primarily by limiting viral RNA and DNA replication and impairing the entry of enveloped viruses [The primary function of ISGs is to detect viral RNA, thus inhibiting viral replication . The act viruses . Oligoad viruses . ISG15 i viruses . ISG20 i viruses . Viperin viruses . As mult viruses .IFNγ is different from types I and III and has a unique structure and function. It is primarily secreted by activated CD4 T helper and CD8 T cells, natural killer (NK) cells, NK T cells, and professional antigen-presenting cells (APCs) . The proInfants initially have a Th17/Th2-biased immune response with lower levels of IFNs ,41,42. AAt birth, the expression of IFNγ and other Th1-associated responses is reduced but increases as age progresses ,51,52. TMice are often used in translational research due to their easy housing and relatively low cost, as well as the availability of a range of immunology reagents. However, IFN responses vary between strains of mice and between mice and humans. In mice, there is a notable difference in the expression of type III IFN compared to humans. Humans possess up to four functional subtypes of IFNλ, while mice only have functional IFNλ2/3 and a pseudogene for IFNλ1 ,62. StudMx1 gene, which is a dominant antiviral resistance gene known as Mx for ‘myxovirus resistance’ [Mx1 gene are able to survive infection with mouse-adapted influenza A virus at doses that would be lethal for standard inbred strains [Mx1 gene remain just as susceptible to influenza challenge as their Mx1-negative counterparts [Mx1-competent mice become resistant, similarly to their adult counterparts, whereas neonates without a functioning Mx gene do not respond to IFN treatment [Mx genes [MxA gene has an important role in inhibiting viral replication by interfering with the assembly of viral ribonucleoprotein, although the exact mechanism is yet to be determined [Certain inbred strains  lack a functional istance’ ,69. Mice strains . Interesterparts . Howeverreatment ,73. Thesreatment ,73. OtheMx genes ,75,76. Itermined ,78.RSV is a virus that can cause serious respiratory tract diseases and death in certain groups of people, including young children (up to 60 months old), the elderly, and those with weakened immune systems . A 2019 RIG-1 activation by binding to the N-terminal caspase activation and recruitment domain (CARD) of RIG-1, thus preventing downstream interaction with MAVS [RIG-1 [Research has shown that the RSV NS1 and NS2 proteins are effective in suppressing the IFN response. Removing these proteins results in a weakened response making NS1/NS2 deletion mutants’ potential candidates for a vaccine ,90,91. Nith MAVS . In contS [RIG-1 ,95. In mS [RIG-1 . OverallRIG-1-dependent manner [The RSV G protein has been found to affect the immune response through a CX3C motif that is present in all RSV strains and is similar to the natural chemokine fractalkine . The G pt manner ,102. Thit manner .It has also been shown that the administration of IFNα to mice before RSV challenge can decrease IL-4Rα and Th2 polarization . In a clRIG-1 activation [The function of type III IFNs in RSV infection is an area that requires further investigation. Research has shown that in an in vitro model of primary human nasal epithelial cells, RSV infection induced IFNλ1 through tivation . Pretreativation . Surpristivation . These ctivation . Anothertivation . Higher tivation . Insteadtivation . These sWhen infants are infected with RSV, a Th2 polarization is often linked to RSV immunopathology, and the lack of a strong Th1 response in infants and young children can lead to more severe illness ,118,119.Type I IFNs can promote IFNγ expression by CD8 T cells via STAT4 in conjunction with TCR activation  and in NCOVID-19 is caused SARS-CoV-2. This virus belongs to the family Coronaviradae and has a single-stranded, positive-sense RNA enclosed in an envelope . Its genSimilarly to other respiratory viruses, SARS-CoV-2 triggers an IFN response through the recognition of various viral PAMPs by PRRs ,131. TheRIG-1 [Several other SARS-CoV-2 viral proteins have been shown to inhibit the IFN response, specifically NSP1, NSP3, NSP5, NSP12, NSP13, NSP14, NSP15, ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, N, and M reported to inhibit IFNß expression by suppression of RLR-mediated signaling ,142,144.RIG-1 . NSP 6 hRIG-1 .While SARS-CoV-2 primarily replicates in the nasopharyngeal and type II alveolar epithelial cells, recent evidence has shown that ACE2 expression (the receptor for SARS-CoV-2) and TMPRSS2  are also present in other cells . A mouseIn contrast to other respiratory viruses, children and neonates are not more likely than adults to develop severe COVID-19, possibly due to differences in IFN signaling ,155,156.The findings suggest that the neonatal immune system changes rapidly in early years of life affecting the IFN response. Neonates are less likely to suffer severe outcomes from SARS-CoV-2 infection, but pre-term infants are at higher risk of COVID-19 and pre-term birth due to medically induced birth.Influenza virus is an enveloped ssRNA virus belonging to the Orthomyxoviridae family . It has RIG-1/MAVs, inducing the production of type I and III IFNs [RIG-1-mediated IFN production by preventing TRIM25 multimerization and CARD domain ubiquitination [Influenza virus activates TLRs 3, 7, and 8, as well as III IFNs ,172. TheIII IFNs ,174 inclIII IFNs ,176. NS1III IFNs . Additiotination . Influentination ,179,180.tination ,182,183.tination , but typtination . IFNα antination .ISGs and lower viral loads on days 2 and 7, which was attributed to asymptomatic respiratory virus infection prior to the administration of LAIV [Neonatal mice are more susceptible to influenza virus due to poor IFN responses . Reduced of LAIV . Childre of LAIV ,193,194.NS1, NS2, and G genes are key effectors in this response [NSP1, ORF6, and NSP13 genes have a particularly potent effect on IFN. Influenza virus has the NS1 gene as the canonical IFN antagonist [PA, PB1, PB1-F2, and PB2 genes [IFNs have an early and crucial role in the body’s antiviral response. Three major respiratory viruses affecting humans, i.e., RSV, influenza virus, and SARS-CoV-2, have IFN antagonistic features (summarized in response , while ftagonist , but antB2 genes .In infants and young children, type I, II, and III IFN responses are lower compared to adults ,42. The A beneficial IFN response is associated with lower viral loads, faster viral clearance, and reduced disease severity . IFN tre


KIT/PDGFRA-activating mutations are major driver alterations in the development of GISTs, leading to ligand-independent activation of KIT/PDGFRA receptors. The activated KIT/PDGFRA receptor is sensitive to the TKI imatinib. However, not all GIST patients respond to imatinib. Precise characterization of the driver mutations in GISTs—in particular, in the KIT and PDGFRA genes—and an understanding of the molecular mechanisms underlying resistance to imatinib and other TKIs should allow clinicians to select the most effective targeted drug as part of regular clinical practice. The correct choice of the TKI in the sequence of targeted agents should lead to improved survival for metastatic patients.Gastrointestinal stromal tumors (GISTs) are rare malignancies of the gastrointestinal tract recognized by their clinical presentation and using specific immunohistochemical staining for CD117 and DOG1. In recent years, prognoses of GISTs patients have significantly improved due to the introduction of tyrosine kinase inhibitors (TKIs) in clinical practice. KIT or PDGFRA genes represent the driving mutations in more than 90% of all GISTs. These patients exhibit good responses to targeted therapy with tyrosine kinase inhibitors (TKIs). Gastrointestinal stromal tumors lacking the KIT/PDGFRA mutations, however, represent distinct clinico-pathological entities with diverse molecular mechanisms of oncogenesis. In these patients, therapy with TKIs is hardly ever as effective as for KIT/PDGFRA-mutated GISTs. This review provides an outline of current diagnostics aimed at identifying clinically relevant driver alterations and a comprehensive summary of current treatments with targeted therapies for patients with GISTs in both adjuvant and metastatic settings. The role of molecular testing and the selection of the optimal targeted therapy according to the identified oncogenic driver are reviewed and some future directions are proposed.Gastrointestinal stromal tumors (GISTs) are soft tissue sarcomas that mostly derive from Cajal cell precursors. They are by far the most common soft tissue sarcomas. Clinically, they present as gastrointestinal malignancies, most often with bleeding, pain, or intestinal obstruction. They are identified using characteristic immunohistochemical staining for CD117 and DOG1. Improved understanding of the molecular biology of these tumors and identification of oncogenic drivers have altered the systemic treatment of primarily disseminated disease, which is becoming increasingly complex. Gain-of-function mutations in  Incidence varies worldwide from 4.3/1 × 10per year ,6,7. Theper year . Most GIper year ,9. Gastrper year ,12. They(PDGFRA) ,14. Arouyndrome) ,19,20,21Surgery is the only curative treatment for GISTs ,23. ImprThis review provides an outline of current knowledge on the molecular mechanisms of GISTs and the selection of the optimal systemic treatment according to the identified molecular mechanisms, both in limited and metastatic disease, and proposes future directions for research.KIT encodes the KIT receptor, which is a type III receptor tyrosine kinase (RTK); it belongs to a family of RTKs that also includes PDGFRA, platelet-derived growth factor receptor beta (PDGFRB), colony-stimulating factor 1 receptor (CSF1R), and Fms-like RTK 3 (FLT3). The receptor tyrosine kinase KIT (CD 117) is normally expressed in Cajal cells in the gastrointestinal tract. It plays an important role in the development of a normal pacemaker system in the gut [KIT mutations are the major mechanism of GIST oncogenesis [KIT lead to the formation of a permanently active protein that is a target for imatinib binding. Of equal importance—and the second most common driver mutations in GISTs—are mutations in PDGFRA, which encodes the PDGFRA receptor tyrosine kinase. The PDGFRA RTK itself is homologous to the KIT RTK and, again, an activating mutation in PDGFRA results in the formation of a permanently active RTK that is also a target for imatinib. KIT/PDGFRA-induced oncogenesis mediates the rapidly accelerated fibrosarcoma (RAF)–mitogen-activated protein kinase (MEK)–mitogen-activated protein kinases (MAPK) (RAF-MEK-MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) (PI3K-AKT) signaling pathways [KIT/PDGFRA-activating mutations are mutually exclusive [KIT/PDGFRA, while in the remaining 10–15%, the molecular mechanism of oncogenesis has not been determined  GISTs). With advances in molecular diagnostic techniques—in particular, with the use of next-generation sequencing (NGS) technology—the mechanisms of oncogenesis can be more precisely defined, even in historical WT GISTs. With the increased sensitivity of the NGS method, it has been discovered that driver mutations in KIT/PDGFRA are also the most frequent in historical WT GISTs. Taking this fact into account, KIT/PDGFRA mutations represent the driving mutations in as many as 92–93% of all GISTs [KIT/PDGFRA mutations are detected and the SDH complex is competent, various very rare driver alterations have been identified: alterations in the Rat sarcoma virus (RAS) gene family, the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) gene, the NF1 gene, the neurotropic tyrosine receptor kinase 1–3 (NTRK1–3) genes, and the fibroblast growth factor receptor 1–4 (FGFR1–4) genes [The proto-oncogene  the gut . In 1998ogenesis . Activatpathways ,31,32. Axclusive . In recell GISTs ,36,37,38ll GISTs ,44,45,464) genes ,49,50,514) genes ,35,36,37Different molecular mechanisms in GISTs lead to different clinical courses of the disease and, in particular, different responses to systemic imatinib treatment. Therefore, it is necessary to identify the driving alterations before initiating the first systemic therapy, both in (neo)adjuvant settings and as the first systemic therapy for metastatic disease ,23.KIT/PDGFRA/BRAF is demonstrated, they advise immunohistochemical (IHC) staining to determine SDH B protein (SDHB) expression. In GISTs where no driver alteration in KIT/PDGFRA/BRAF is established and SDHB is expressed, they further recommend excluding driver alterations in NF1 [KIT/PDGFRA and who have a competent SDH complex, as confirmed by IHC staining, should be tested with NGS to identify any targetable driver alterations in other genes  [Molecular analysis is an important factor influencing decision about systemic treatment for both limited and metastatic disease. Molecular markers have both prognostic and predictive value ,53,54,55s in NF1 . The NatK, FGFR) .The human KIT proto-oncogene is located on chromosome 4 (q12). This gene was discovered in 1987 . A nonmuActivated KIT triggers different signaling pathways: RAS/MAP/MAPK, PI3K/AKT, phospholipase C gamma (PLC-gamma), Janus kinase (JAK)/signal transducer and activator of transcription (STAT) (JAK/STAT), and Scr kinase pathways. Which pathway will be activated depends on which tyrosine residue of the IC domain is phosphorylated .In humans, the proto-oncogene PDGFRA is also located on chromosome 4 (q12), in the same region as KIT, and encodes a class III RTK that is structurally homologous to KIT RTK. The activation of downstream pathways is also similar to KIT; PDGFRA activation predominantly activates the RAS/RAF/MAPK and PI3K/AKT signaling pathways ,31,32. IKIT/PDGFRA-mutated GISTs with high risk for disease recurrence prolongs recurrence-free survival (RFS) (ten-year RFS: 52.5% vs. 41.8%) and OS (ten-year OS: 79.0% vs. 65.3%) compared to 1 year of treatment alone [KIT/PDGFRA mutation. Patients with PDGFRA mutations, KIT exon 11 duplications, insertions, and substitutions have longer RFS than patients with KIT exon 11 deletions or indel mutations. Patients with KIT exon 9 mutations (most commonly an AY duplication) have the shortest RFS. Patients with KIT exon 11 deletions or indels  have a significantly longer RFS if treated with 3 years of imatinib compared to 1 year. This difference in RFS with regard to the duration of adjuvant imatinib treatment has not been observed in other groups (KIT exon 11 substitutions and KIT exon 9 mutations) [KIT exon 9-mutated metastatic patients with the 800 mg dose compared to the 400 mg/day dose, the question of an escalated dose of imatinib for KIT exon 9 mutation also arises in the context of adjuvant therapy. Results from a multi-institutional European retrospective study of patients with KIT exon 9-mutated GISTs treated with adjuvant imatinib revealed that a higher daily dose of 800 mg versus 400 mg did not improve survival outcomes (RFS: hazard ratio (HR), 1.24; mRFS: HR, 1.69; imatinib failure-free survival (IFFS): HR, 1.35; 95% CI, 0.79–2.28) [Three years of adjuvant treatment with 400 mg/day imatinib in nt alone ,60,61. Ant alone ,63. The tations) ) and bet79–2.28) ,66,67.Taking into consideration that a longer duration of adjuvant imatinib therapy leads to longer survival, another dilemma appears regarding further prolongation of adjuvant imatinib therapy beyond 3 years. In a phase 2 clinical trial involving imatinib-sensitive GISTs with 5 years of adjuvant imatinib therapy following resection in patients with a high risk of recurrence, the five-year RFS estimate was 90% and the five-year OS estimate was 95% . Two morKIT/PDGFRA mutation is not the only factor influencing adjuvant treatment efficacy. Other factors, such as the duration of adjuvant treatment, primary site ), tumor size (larger tumor size being associated with a poorer DFS), mitotic index (high mitotic index being associated with poorer DFS), and female sex (an independent prognostic factor for a higher PFS and OS), have also been shown to contribute [The type of ntribute ,69.KIT/PDGFRA-mutated GISTs with imatinib is to prolong survival with good quality of life. The initial dose of imatinib is 400 mg/day, except for patients where KIT exon 9 mutation is known to be the oncogenic driver; in these patients, treatment is started upfront with an escalated dose of 800 mg/day [The goal of systemic treatment of metastatic or unresectable 0 mg/day . In most0 mg/day ,74,75,76KIT exon 11 mutations who, at the same time, bear polymorphisms in genes involved in imatinib metabolism (reducing metabolic capacity), patients with KIT exon 9 mutations, patients without KIT/PDGFRA mutations (WT GISTs), and patients with primary resistance to imatinib, especially those carrying PDGFRA D842V and KIT D816V mutations [KIT exon 9 mutation achieve a PFS of only 12.6–16.7 months with 400 mg/day imatinib [p = 0.017) but without improving the OS [PDGFRA D842V or KIT D816V mutations [PDGFRA D842V mutation have been described [However, there is a group of patients who respond worse to imatinib initiation; namely, some patients with utations ,77,78. Pimatinib ,66,67. Hg the OS . Patientg the OS ,66,67. Putations ,80,81. Nescribed ,37,82.Disease progression is frequently due to the occurrence of secondary mutations resulting from evolutionary selection pressure during imatinib treatment . They ocIn everyday clinical practice, we do not strive to confirm the exact mechanism of resistance due to the heterogeneity of mutations occurring at progression. If the progression is focal (a “nodule within a mass” up to one or a few nodule(s)/mass(es) while the rest of the disease is still responding), surgery or nonsurgical procedures  may be selected ,23. WhenKIT or PDGFRA. However, the response to second-line TKI treatment is not always the same and depends on the mechanism of resistance and the efficacy of the TKI applied. Secondary mutations, typically missense mutations, most commonly affect the coding region of the ATP binding site (exons 13 and 14 of KIT and exon 14 of PDGFRA), as well as the coding region of the TKD activation loop (exon 17 of KIT and exon 18 of PDGFRA) [Secondary mutations occur mainly at specific sites in  PDGFRA) . KIT/PDG PDGFRA) ,89,90,91 PDGFRA) ,93,94,95Following disease progression with imatinib, the current guidelines recommend the use of three multitargeted TKIs—sunitinib, regorafenib, and ripretinib. There are also data in the literature on the efficacy of other multitargeted TKIs in this setting , but none of them exceeded a 6 month barrier for mPFS ,23,96. TPDGFRA are poorly understood, with well-known primary resistance to imatinib only in PDGFRA D482V (and its homologue KIT exon 17 mutation D816V) [PDGFRA D842V. In a phase 3 clinical study, it exhibited good efficacy and an objective response of 84% [The mechanisms of secondary mutations in n D816V) . Avaprite of 84% .Other mechanisms of GIST resistance to imatinib are infrequent and poorly understood. Resistance could potentially be induced by activation of other oncogenic pathways—e.g., dysregulation of cyclin-dependent kinase inhibitor 2A (CDKN2A); loss of tumor protein 53 (TP53) function; inactivation of dystrophin; genomic alterations in chromosomes 1p, 14q, and 22q; and overexpression of KIT ,99,100.p < 0.0001) [KIT exon 9, sunitinib elicits improved PFS in OS compared to those with the primary mutation in KIT exon 11 [p < 0.0001) [p < 0.0001). The efficacy of ripretinib is also not universal, and it depends on the type of secondary mutation [PDGFRA D842V mutations, regardless of prior systemic therapy, achieved an objective response in 88% of patients  [KIT/PDGFRA-mutated, imatinib-pretreated (PDGFRA D842V excluded) patients is only modest, with an ORR of 17%, median duration of response (mDOR) of 10.2 months, and mPFS of 3.7 months [ 0.0001) . The eff exon 11 ,102. Fur exon 11 ,86,102.  0.0001) . The eff 0.0001) ,64. In amutation . In a phesponse) . The eff7 months .SDHA tumor suppressor gene (TSG) is located on chromosome 5 (p15.33), the SDHB TSG on chromosome 1 (p36.13), the SDHC TSG on chromosome 1 (q23.3), and the SDHD TSG on chromosome 11 (q23.1). They encode the four subunits  of heterotetrametric enzyme SDH, the key enzyme in the Krebs cycle and respiratory chain in mitochondria.The SDHA, SDHB, SDHC, or SDHD Miettinen et Lasotation of SDHB [IGF1R) and VEGFR. Furthermore, a lack of dioxygenase activity triggers hypermethylation of DNA; i.e., epigenetic silencing. Consequently, activation of IGF1R and VEGFR and/or an increase in DNA methylation lead to malignant transformation of normal interstitial Cajal cells into GISTs [The SDH enzyme complex catalyzes the oxidation of succinate to fumarate. Loss of function of mitochondrial SDH  in combination with a somatic mutation  [SDHC (post-zygotic hypermethylation of the promoter region). The specific hypermethylation pattern of the SDHC gene is associated with a rare non-hereditary Carney triad syndrome  [Deficiency in the SDH complex is a rare event in GISTs, present in up to 5–7.5% of GIST patients ,44,45,46tations) ,46. Germtations) ,17. In tcomplex) ,46,108.Gastrointestinal stromal tumors with a deficient SDH complex do not respond to imatinib treatment but, in line with the mechanism of oncogenesis, they respond to multitargeted TKIs, which are potent antiangiogenetic agents, such as sunitinib, regorafenib, and pazopanib ,111,112.NF1 gene is a huge TSG located on chromosome 17 (q11.2). It codes for the protein neurofibromin, which plays a role in the RAS/MEK/MAPK and mammalian target of rapamycin (mTOR) oncogenic pathways. Neurofibromin acts as a guanine triphosphate (GTP) hydrolase (GTPase), converting RAS-GTP to the inactive RAS guanosine diphosphate (GDP). Inactivating mutations in NF1 result in the accumulation of RAS-GTP, followed by increased RAS signaling in the RAS/MEK/MAPK signaling pathway [NF1 inactivation causes neurofibromatosis type 1 (NF1), a relatively common autosomal dominant genetic disorder characterized by predisposition to cancer development. Clinical studies have demonstrated that different inactivating mutations of NF1 exhibit different phenotypic variants and clinical presentations [NF1 gene mutation [NF1 in GIST patients have been described as “missense” mutations, “nonsense” mutations, “frameshift-induced” protein truncation, “splice site” mutations, and larger deletions [KIT mutations and/or mutations in the Notch signaling pathway [NF1 gene result in increased signaling in this pathway independent of RTK KIT activation [NF1 mutations and the current lack of any effective systemic treatment for NF1 GISTs.The  pathway . Germlinntations . Due to mutation . Inactiveletions ,120,121.eletions ,118,120. pathway ,123. Lostivation . This exBRAF proto-oncogene is located on chromosome 7 (7q34). It encodes the BRAF protein, which is a serine threonine kinase with the function of activating the MAPK signaling pathway. Mutations in the BRAF gene are divided into three classes according to the impacts they have on the function of BRAF protein [BRAF are mutations in exon 15, the most common of which is a substitution of valine with aspartate at codon 600 (BRAF V600E). This mutation leads to phosphorylation of the activation domain of the BRAF kinase and, consequently, its constitutive activation [BRAF V600E mutation is a driver mutation in various solid cancers, but it is a rare event in GIST tumorigenesis [The human  protein . Class otivation . The BRAigenesis . Accordiigenesis ,130,131.BRAF V600E do not respond to treatment with imatinib and sunitinib, but a successful case of treatment with regorafenib has been described [BRAF V600E mutation and is approved by the US Food and Drug Administration (FDA), but not by the EMA, as tumor agnostic therapy [BRAF V600E GIST with the BRAF inhibitor dabrafenib alone (without the MEK inhibitor trametinib) has been described [BRAF rearrangements (PRKAR1B-BRAF), the clinical significance of which is so far unknown, have been recently recognized [Gastrointestinal stromal tumors with escribed . Treatme therapy . To dateescribed . There aescribed ,23. Addicognized ,136.KRAS proto-oncogene is located on chromosome 12 (12p11.1–12p12.1). It codes for KRAS protein, a hydrolase that converts the nucleotide GTP to GDP and is part of the RAS super-family of GTPases. Active KRAS has the GTP molecule bound and, under physiological conditions, allows transduction of signals downstream to the RAS/MEK/MAPK and PI3K/AKT signaling pathways. Mutations in KRAS, predominantly point mutations, can be either primary or secondary in GIST evolution. Both primary and secondary mutations result in a permanently active KRAS protein and, thus, incessantly active signaling pathways [KRAS are very rare in GISTs (less than 0.5% of cases). Secondary mutations in KRAS occur as an after-effect of imatinib treatment in KIT/PDGFRA mutant GISTs. The incidence is unknown [The human pathways . Primary unknown ,140,141.KRAS mutation do not respond to imatinib treatment. Sotorasib is a specific KRAS G12C inhibitor, the efficacy of which has been determined in a phase 1/2 clinical trial; however, it included no patients with metastatic, KRAS G12C-mutated GISTs [Gastrointestinal stromal tumors with a ed GISTs .NTRK1 proto-oncogene is located on chromosome 1 (q23.1), the NTRK2 proto-oncogene on chromosome 9 (q21.33), and the NTRK3 proto-oncogene on chromosome 15 (q25.3) [NTRK1, NTRK2, and NTRK3 encode a family of receptor tropomyosin kinases—TRKA, TRKB, and TRKC—involved in neuronal development. Rearrangements of the NTRK1, NTRK2, and NTRK3 genes with different partner genes result in the formation of a constitutively active (ligand-independent) TK, which thereby leads to the development of several solid cancers. Rearrangements are rare in common cancers and common in very rare cancers [NTRK rearrangements are agnostic driver alterations, and NTRK inhibitors are effective in tumors with NTRK fusions irrespective of the site of origin of metastatic disease [The  (q25.3) ,144. The disease ,146.ETV6-NTRK3 and LMNA-NTRK1 rearrangements in GISTs have already been described [NTRK rearrangements do not respond to treatment with imatinib or sunitinib but do respond to treatment with NTRK inhibitors (larotrectinib and entrectinib). A pooled analysis of three phase 1 and 2 clinical trials on the efficacy and safety of larotrectinib enrolled four patients with metastatic GISTs with NTRK rearrangements. All four patients treated with larotrectinib had an objective response [ETV6-NTRK3 rearrangement treated with larotrectinib have been reported, all of whom responded to treatment, one with a complete response [The escribed ,149,150.response . Furtherresponse . In anotFGFR1) proto-oncogene is located on chromosome 8 (p11.23), FGFR2 on chromosome 10 (q26.13), FGFR3 on chromosome 4 (p16.3), and FGFR4 on chromosome 5 (q35.2). They encode the FGFR family of proteins, which are transmembrane receptors with an IC tyrosine kinase domain [FGFR1–4 [FGFR driver alterations are very rare events in GIST evolution but have been described as a mechanism of resistance to imatinib in addition to secondary KIT/PDGFRA mutations [Fibroblast growth factor receptor 1 , while other multitargeted TKIs are currently in clinical trials in this setting: dovitinib, masitinib, ponatinib, lenvatinib, pazopanib, and nintedanib [Multitargeted TKIs that have been found to be active in cases of ntedanib ,92. Selentedanib .PIK3CA, MAX, MEN1, ARID1A, ARID1B, ATR CBL, LTK, MEN1, PARK2, SUFU, and ZNF217) [Since the introduction of NGS into routine clinical practice, several other rare alterations in various genes have been described in relation to GISTs ( ZNF217) ,99,157. Currently, there are several interventional prospective clinical trials active for GISTs, either in (neo)adjuvant or metastatic settings . Novel dKIT primary and secondary mutations, particularly after progression with imatinib, and in assessing tumor dynamics with serial monitoring [KIT-mutated GISTs [On the other hand, growing research interest has been focused on so-called liquid biopsies. In patients with advanced malignancy, circulating tumor DNA (ctDNA) assays could be utilized to identify clinically relevant mutations for directing targeted therapy. However, the limitations of these assays should be considered carefully . In metanitoring ,161. Added GISTs .Gastrointestinal stromal tumors are rare diseases that vary in their clinical and molecular characteristics. Decision regarding their systemic treatment should be governed by molecular predictive markers . Due to the rarity of the disease and the complexity of treatment necessitated by the variety of molecular characteristics, these patients should be managed in centers that offer the possibility of genuinely comprehensive treatment. The introduction of wide-ranging genomic profiling has improved knowledge about the molecular features of GISTs and opened new perspectives for systemic treatment. Last but not least, increased enrollment of patients in clinical trials is essential, as this leads to improved survival.


Lilium is affected by many physiological and biochemical phenomena, including flower bud differentiation, starch and sucrose accumulation, photoperiod, carbon fixation, plant hormone transduction, etc. The transcriptome analysis of flower buds of Lilium hybrid ‘Siberia’ at different maturity stages showed that floral bud formation is associated with the accumulation of anthocyanins. The results of HPLC-MS showed that cyanidin is the major anthocyanin found in Lilium ‘Siberia’. Transcriptome KEGG enrichment analysis and qRT-PCR validation showed that two genes related to flavonoid biosynthesis (LhANS-rr1 and LhDFR) were significantly up-regulated. The functional analysis of differential genes revealed that LhMYB114 was directly related to anthocyanin accumulation among 19 MYB transcription factors. Furthermore, the qRT-PCR results suggested that their expression patterns were very similar at different developmental stages of the lily bulbs. Virus-induced gene silencing (VIGS) revealed that down-regulation of LhANS-rr1, LhDFR, and LhMYB114 could directly lead to a decrease in anthocyanin accumulation, turning the purple phenotype into a white color. Moreover, this is the first report to reveal that LhMYB114 can regulate anthocyanin accumulation at the mature stage of lily bulbs. The accumulation of anthocyanins is an important sign of lily maturity. Therefore, these findings have laid a solid theoretical foundation for further discussion on lily bulb development in the future.The bulb formation of  Lilium, spp.), a group of monocotyledonous ornamental plants, are widely grown for commercial purposes. They significantly contribute to the global ornamental industry and are utilized for commercial bulb and flower production, including outdoor and indoor fresh-cut flowers and potted plants, and for landscaping in private gardens [Lilies , and one WD40 repeat TF [R2R3-MYB type TFs in Arabidopsis, including MYB114, MYB113, MYB90, and MYB75, enhanced anthocyanin accumulation [PyMYB114 could inhibit anthocyanin biosynthesis, which was similar to the function in tobacco and strawberry [PyMYB114 to regulate anthocyanin biosynthesis in red-skinned pears [Hydrangea macrophylla revealed that MYB114 acted as a negative regulation transcription factor in anthocyanin synthesis [WD40 repeat gene (OsTTG1) regulated anthocyanin biosynthesis in rice [ZmC1 (MYB-type C1) and ZmR (bHLH R) are two TFs; their up-regulation controlled anthocyanin biosynthesis in maize [MYB TFs positively regulated anthocyanin biosynthesis, except for MdMYB16 [MdMYB114’s transcription level was increased with the deepening of apple color, and overexpression promoted the production and accumulation of anthocyanin in callus [With the cloning of anthocyanin-related transcription factors, anthocyanin biosynthesis in plants has been formally studied . In planepeat TF , and theepeat TF . Anthocyepeat TF . The ovemulation . Furtherrawberry . Furthered pears . The traynthesis . A WD40  in rice . ZmC1  and 70% methanol buffer to a volume of 50 mL, and this process was repeated 3 times. Then, the samples were shook for 1 min and extracted for 30 min by ultrasonication. It was hydrolyzed in a boiling water bath for 1 h, and then cooled in constant volume. Standing and taking the supernatant, it was filtered with 0.45 aqueous phase filter membrane, and stored at 4 degrees for test. The injection volume of 20 μL was passed through the chromatographic column C18. The mobile phase A is an aqueous solution containing 1% formic acid, and the mobile phase B is a 100% acetonitrile solution at 0.25 mL/min flow rate. It was detected by a wavelength of 530 nm at LC-QTOF . Delphinidin, cyanidin, petunidin, pelargonin, and mallow pigment were used as an internal standard for quantitation. Samples were more than 5 g. All samples were homogenized in a homogenizer and stored at −18 °C. The samples were mixed with the HCl (0.5% ® II 1st Strand cDNA Synthesis Kit (+gDNA wiper)  was used as reverse transcription kit. The quantity and quality of the cDNA were assessed by using a NanoDrop ND2000 spectrophotometer . cDNA library construction and transcriptome sequencing were performed by Novogene Technologies Co., Ltd. . The quality of the sample libraries was determined by an Agilent 2100 Bioanalyzer and Qubit2.0. Finally, the well-constructed library was sequenced using an Illumina NovaSeq 6000 platform . The R package DESeq2 (version 1.10.1) was used to identify differential expression analysis of two groups (E&F), and the gene expression values were calculated as reads aligned to the fold change of the normalized (RPKM) expression values by Ballgown (version 2.14.1). FDR was used to judge the threshold of p value. DEGs mainly depended on multiples hypotheses, such as the p-value threshold, false discovery rate (FDR) (≤0.05) and (log 2 FoldChange ≥ 1), which were considered as the significance of each gene expression difference.Total RNA was extracted from the samples of two developmental stages E,F usingLilium × formolongi EF-1a was selected as a reference gene and the relative expression was analyzed with the 2−∆∆Ct method [The real-time quantitative PCR (qRT-PCR) was performed on an ABI Q1 Real-Time PCR System with 10 µL mixture according to the following procedures: 95 °C for 1 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. The primers were designed using the software Primer Premier 6.0 and are listed in t method . Three iLhMYB114, LhANS-rr1, or LhDFR) by VIGS was performed as described earlier [Agrobacterium GV3101 strain. Then, monoclonal bacteria were picked up and inoculated in a 100 mL LB medium containing antibiotics, then grown overnight at 28 °C in a shaker. Agrobacterium cells were collected and resuspended in infiltration buffer  ethane sulfonic acid (pH 5.6) to a final OD600 of 1.8). Equal volumes of TRV1 and TRV2 (the control), as well as the TRV1 and TRV2 with target genes, were mixed together and kept in the dark for 3–6 h at room temperature before infiltration. The buds were infiltrated using a needleless 1 mL syringe and grew at room temperature for seven days. Then, the flower buds continued vernalization in a refrigerator at 4 °C under dark for 15 days.Silencing of the targeted gene  at 6 developmental stages. The cutting sections were soon fixed in 2.5% glutaraldehyde, and then dehydrated in a series of graded ethanol, as previously described [To evaluate the features of apical meristem structure, the apical meristem from escribed . The samp-values were <0.05 and <0.01, respectively.All data are presented as the mean ± standard deviation (SD) of at least three independent replicates. All statistical analyses in this paper were performed with SPSS  software. The significant and extreme differences were confirmed if Lilium flower buds, five types of anthocyanins were used as standard, including delphinidin, cyanidin, petunia, pelargonin, and mallow , including R2-R3 repeat domain and the bHLH binding domain , but there were no bands in the negative control (buffer) (Lilium and replicated and transferred in vivo. The qRT-PCR results suggested that the expression of three key genes was evidently reduced in gene-silenced buds compared with control buds (empty vector) at 15 dpi  B. The pht 15 dpi A–F. Othet 15 dpi G. These LhANS-rr1, LhDFR, and LhMYB114 are closely related to anthocyanin biosynthesis in lily buds. Our results (according to VIGS) suggested that three key genes might be closely related to anthocyanin accumulation in floral buds of lilies.The color is an important attribute that determines the quality of flowers ,41. The MYB-bHLH-WD40 (MBW) transcription complex [C4H, 4CL, CHS, CHI, UFGT, F3H, DFR, and ANS genes. Among the unigenes encoding six CCoAOMT, UDPG, HCT, C4H, 4CL, CHS, CHI, F3H, LhDFR, LhANS-rr1, and UFGT, the results suggested that the FPKM values of the three CCoAOMT, LhANS-rr1, and LhDFR unigenes were higher in the purple buds of bulbs than in the white buds, whereas the FPKM values of three CCoAOMT, UDPG, HCT, C4H, 4CL, CHS, CHI, F3H, and UFGT were not , which proved that they were related to anthocyanin biosynthesis [During the process of bulb development, the flower bud of a lily becomes larger with the expansion of bulb size, and anthocyanin accumulation simultaneously occurs. The coordinated expression of genes, which encode the anthocyanin biosynthetic pathway enzymes, usually controls the anthocyanin accumulation through a ternary  complex . Anthocy complex ,46. The were not A. Meanwhegulated D. Furtheynthesis .Lilium bulbs (There are three main downstream metabolic pathways of anthocyanins, i.e., cyanidin, pelargonium, and delphinidin, in plants. Though ANS and DFR genes do not specifically regulate anthocyanin biosynthesis, they are the key genes in the cyanidin biosynthesis pathway . Previouum bulbs B,C. FurtMYB TFs regulate the phenylpropanoid pathway, including lignin, flavonoids, and other metabolites, in plants [MYB TFs [LhMYB114, was involved in anthocyanin synthesis (LhMYB114 sequencing blast results suggest that it is the allele gene of Lilium regale LrMYB15 for transcription factor R2R3-MYB [LhMYB114 is homologous with anthocyanin-related genes in Arabidopsis thaliana, including AtMYB123, AtMYB5, AtMYB114, AtMYB113, AtMYB90, and AtMYB75. Most R2R3-MYB TFs can promote anthocyanin biosynthesis by up-regulating the expression level in plants [LhMYB114 expression level increases significantly in the mature stage of lily bulb development (n plants . The MBW[MYB TFs . It is iynthesis . The LhMR2R3-MYB . The Clun plants . Our reselopment A.MYB transcription factor or the MBW complex [Agrobacterium tumefaciens is a powerful tool to reveal gene functions in plants, which are difficult to transform, such as lily, potato, etc. [LhMYB114, 35S:LhANS-rr1, 35S:LhDFR, 35S:LhMYB114+35S:LhANS-rr1, 35S:LhMYB114+35S:LhDFR, and 35S:LhMYB114+35S:LhANS-rr1+LhDFR (LhMYB114 and LhANS-rr1 was more typical than that of LhDFR after VIGS experiments (C4H, 4CL, CHS, CHI, and F3H (The structural genes may be regulated by a single  complex . VIGS meto, etc. . Thus, tr1+LhDFR A. The faeriments A. At theeriments A–F. This and F3H G. The quLhMYB114, LhANS-rr1, and LhDFR were the key genes for regulating the accumulation of cyanidin in floral lily buds. Among them, LhMYB114 is an important regulatory transcription factor during lily bulb maturation. In a word, the data in our article contribute to the understanding for the molecular mechanism of anthocyanin biosynthesis and the judging of bulb maturity in Lilium flower bud development.In this study, it was revealed that the maturation of lily bulbs is closely associated with the amount of anthocyanin accumulation. Therefore, anthocyanin accumulation in flower buds is a very important marker in the development of lily bulbs, and the concentration of anthocyanins directly decides on the ripeness of bulbs. In addition, the maturity of lily bulbs will also directly decide on the time of bulb quality and lily vernalization. The transcriptomics and VIGS analysis firstly revealed that


The application of orthodontic forces causes root resorption of variable severity with potentially severe clinical ramifications.in vitro, experimental, and in vivo studies.To systematically review reports on the pathophysiological mechanisms of orthodontically induced inflammatory root resorption (OIIRR) and the associated risk factors based on We undertook an electronic search of four databases and a separate hand-search.in-vitro studies, the incidence root resorption in (2) animal studies, and (3) human studies.Studies reporting on the effect of orthodontic forces with/without the addition of potential risk factors on OIIRR, including (1) gene expression in Potential hits underwent a two-step selection, data extraction, quality assessment, and systematic appraisal performed by duplicate examiners.One hundred and eighteen articles met the eligibility criteria. Studies varied considerably in methodology, reporting of results, and variable risk of bias judgements.In summary, the variable evidence identified supports the notion that the application of orthodontic forces leads to (1) characteristic alterations of molecular expression profiles in vitro, (2) an increased rate of OIIRR in animal models, as well as (3) in human studies. Importantly, the additional presence of risk factors such as malocclusion, previous trauma, and medications like corticosteroids increased the severity of OIIRR, whilst other factors decreased its severity, including oral contraceptives, baicalin, and high caffeine.Based on the systematically reviewed evidence, OIIRR seems to be an inevitable consequence of the application of orthodontic forces—with different risk factors modifying its severity. Our review has identified several molecular mechanisms that can help explain this link between orthodontic forces and OIIRR. Nevertheless, it must be noted that the available eligible literature was in part significantly confounded by bias and was characterized by substantial methodological heterogeneity, suggesting that the results of this systematic review should be interpreted with caution.PROSPERO (CRD42021243431). Orthodontic tooth movement (OTM) is achieved by combining mechanical strain application and subsequent physiological adaptation of the periodontium . CrucialPeriodontal ligament cells (PDLs) are necessary mechanoreceptors and primary shock absorbers that respond to constant and intermittent loading . In addiOrthodontically induced inflammatory root resorption (OIIRR) is a clinically challenging, well-documented side effect of OTM . It is uTherefore, it is of critical importance for clinicians to understand why OIIRR occurs and which patients are, based on these mechanisms, most at-risk of severe OIIRR. A plethora of research focuses either on the underlying mechanisms of OIIRR or the risk factors that can predispose to severe resorption, but typically, the biological mechanisms and the clinical factors are seldom combined ,12.Generally, factors can be divided into patient-related risk factors or treatment-related factors. The patient-associated factors can be the race, the age that involves both chronological and dental age ,14,15, iThe local factors are type and severity of the malocclusion ,48, habiThe treatment-related or intervention-related are, for example, duration of treatment , directiin-vitro, in-vivo animal, and human studies.Therefore, we sought to systematically review reports on the pathophysiological mechanisms of OIIRR and the linked systemic and local clinical risk factors for OIIRR based on Before undertaking this review, we created a specific protocol in line with the PRISMA guidelines and checklist ,62. The (i) The research questions were developed according to the PI/ECOT format; that is, the Population Intervention/Exposure, Control, Outcome, and Time .Molecular responses(ii) PICOT 1A: Are molecular expression profiles [O] different in human periodontal ligaments cell culture models of OTM [P] with different active mechanical forces [I] compared to cell culture models without force application?PE/ICOTS 1B: Are molecular expression profiles [O] different in human periodontal ligaments cell culture models of OTM with active mechanical forces [P] with possible risk factors [E] compared to cell culture models without risk factors?Pre-clinical responses(i) PICOT 2A: Is the incidence of root resorption [O] different in teeth exposed to different active mechanical orthodontic forces [I] compared to teeth not exposed to mechanical force [C] after a minimum follow-up of 2 weeks [T] investigated in animal models [P]?PE/ICOT 2B: Is the incidence of root resorption [O] on teeth exposed to orthodontic forces [P] higher in animals/situations with potential systemic or local risk factors [E] compared to animals/situations without such risk factors [C] after a minimum follow-up of 2 weeks [T]?Clinical responses(ii) PICOT 3A: Is the incidence of root resorption [O] in systemically healthy patients [P] different in teeth exposed to orthodontic treatment with different active mechanical forces [I] compared to teeth not exposed to mechanical force [C] after a minimum follow-up of 4 weeks [T]?(iii) PE/ICOT 3B: Is the incidence of root resorption [O] in a population of patients undergoing orthodontic treatment [P] higher in patients/situations with potential systemic or local risk factors [E] compared to patients/conditions without such risk factors [C] after a minimum follow-up of 4 weeks [T]?- Comparative studies , controlled clinical trials (CCT), case-control studies, comparative cohort studies)- Reporting on high-throughput sequencing methodology- Written in English languageIn vitro human PDL cell culture modelsAnimal studies for PE/ICOT 2Group size ≥ 10 participants at the time of measurement of the outcomeMinimum follows up of two weeks for human studies and 4 weeks for animal studies- Non-English literature- Non-comparative studies - Systematic reviews- Not reporting on high-throughput sequencing methodology- Non-human, non-PDL cells, non-force culture in vitro models- Group size less than ten at the time of measurement.- Internal root resorption.- Root damage due to placement of a miniscrew or other surgical procedure.- Root resorption due to ectopic or unerupted teeth.- Abnormal tooth morphology.- Deciduous teeth.- Clefts of lip and palate and other craniofacial disorders- Auto-transplantation.- Abnormal tooth morphology.- Deciduous teeth.- Clefts of lip and palate and other craniofacial disorders (in human studies).- Auto-transplantation.The following electronic databases were searched electronically for peer-reviewed publications :MEDLINE .CENTRAL .Embase.SCOPUS.A manual search for eligible publications:The last 20 years of the following journals: European Journal of Orthodontics (volumes 3–43), Journal of Dental Research (volumes 59–100), The Angle Orthodontist, and American Journal of Orthodontics and Dentofacial Orthopedics (77–159).Reference lists of included publications.Controlled Trials in ClinicalTrials.gov.Experts in the topic.For search terms, please refer to First, titles and abstracts were screened by two independent reviewers . Abstracts that—according to the two reviewers—met all inclusion and exclusion criteria were considered. An initial calibration exercise was undertaken. The inter-rater reliability was calculated (percentage of agreement). Any disagreement was referred to a third reviewer (AK) and discussed to reach an agreement. For the next step—the full-text search was conducted independently by HD and VC. Any difference of opinion was consulted with a third reviewer (AK) for the final decision.Two reviewers  independently performed duplicate data extraction utilizing a pre-established and trialed spreadsheet as recommended by the Cochrane Handbook . In addiThe main outcome isThe genomic changes of PDL cells after mechanical force application.The additional outcomes are: relationship between the measured molecular and genetic expression and root resorption mechanism.The main outcome was:Incidence of OIIRRThe additional outcomes were:Gingival indexPeriodontal disease incidenceMolecular biomarkersHistological findingsThe main outcome was:Incidence of OIIRR.The additional outcomes were:Gingival indexPeriodontal disease incidenceMobilityTooth lossMolecular biomarkersHistological findingsPatient-related outcomes: pain and quality of lifeTwo reviewers  independently assessed the risk of bias using multiple tools. Any disagreements were resolved by a third reviewer (AK).The Cochrane Collaboration Risk of Bias (RoB 2.0 tool) was used for randomized trials .Risk Of Bias In Nonrandomised Studies of Interventions (ROBINS-I tool) .Newcastle-Ottawa-Scale was used for observational studies .The Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) was used for the animal studies .A modified Animal Research: Reporting of In Vivo Experiments (ARRIVE)  was usedet al.  approach ; Higginset al. .According to the GRADE methodology , the cerA total of 7824 studies were identified through electronic searches. However, 6027 hits remained after the removal of duplicates. After title and abstract screening, a total of 253 full texts were assessed for eligibility using full-text assessment. Overall, 113 individual studies, some applicable for more than one PICO, were included (The inter-rater raw agreement rate during the screening stage of the titles and the abstracts was 95.8%.k = 0.817) for the risk of bias analyses  tool was used for PICO 2A and PI/ECO 2B. However, most authors rarely mentioned if allocation was randomized or if the animals were kept in random houses or commented on the blindness of the assessors, resulting in unclear certainty in many domains .All results showed that force-tested groups have a significantly higher risk of OIIRR than the no-force control groups ,111,112.Is the incidence of root resorption [O] on teeth exposed to orthodontic forces [P] higher in animals/situations with potential systemic or local risk factors [E] compared to animals/situations without such risk factors [C] after a minimum follow-up of 2 weeks [T]?Twenty-eight articles were included for data extraction, 1 on mice  and 27 oPlease refer to section PICO 2A RoB, as the same tool was used for PE/ICO 2B.Higher risks of OIIRR were found in groups with high bone turnover, acute corticosteroid intake than chronic corticosteroid intake or control groups, 4-hexylresorcinol medication intake, hypofunctional periodontium, nicotine, and strontium ranelate ,108,109.Significantly less OIIRR was reported in animals with a chronic combined oral contraceptive, baicalin intake, sites with previous bone ceramic or bio osseous graft, systemic sodium fluoride, immature roots, osteopontin (OPN) deficiency, angiogenic cytokines, high caffeine, ovariectomy, and orchiectomy rats ,125,127.Studies investigating the effects of a diet with high casein phosphopeptides (CPP), high zinc in the diet, carboxymethyl cellulose and atorvastatin medication, lacunae size in amitriptyline medication, cetirizine intake, zoledronic acid, etoricoxib medicine, fluoxetine, budesonide, ovalbumin and in systemic fluoride did not report significant differences between test and control groups ,122,106.In terms of secondary outcomes, 19 studies reported histological and/or biological findings ,126,127.High quality of evidence assessed via GRADE Methodology indicates that orthodontic forces in PICO 2A and/or intervention or exposure in PI/ECO 2B are associated with the incidence or increased severity of OIIRR in animal studies .Is the incidence of root resorption [O] in systemically healthy patients [P] different in teeth exposed to orthodontic treatment with different active mechanical forces [I] compared to teeth not exposed to mechanical force [C] after a minimum follow-up of 4 weeks [T]?Nine articles were selected for data extraction ,132,133.Six studies were RCTs ,131,133,The experimental group in all studies with force application showed an increased incidence of resorption and/or severity of OIIRR. The reported OIIRR was the resorption crater volume ,133, theTwo studies reported relevant secondary outcomes ,132: no Is the incidence of root resorption [O] in a population of patients undergoing orthodontic treatment [P] higher in patients/situations with potential systemic or local risk factors [E] compared to patients/conditions without such risk factors [C] after a minimum follow-up of 4 weeks [T]?Forty-eight articles were selected for data extraction ,48,47. ATwo studies were RCTs, one with low RoB  and the Analysis for reporting bias across the studies revealed that nine reports were on the same cohort of the population ,33,24,24The main finding of these studies was an immediate significant increase in the risk of OIIRR with:Asthma ,Atopy  ,23,146Cortical thickness ,Incisor proclination Increase in age ,41,23,15Initial degree of RR ,15,Initial root length ,23,15,Initial size of the root ,Males ,27,148,Occlusion; overbite ,23,Overjet  and ClasPresence of periapical pathology ,Previous trauma ,23.RFT with increased age ,Root shape and width ,Small or peg shape tooth ,23Concerning genetic studies, a significantly increased risk of OIIRR was found in:Specific genotypes as in purinergic-receptor-P2X, ligand-gated ion channel 7 SNP rs208294 .IL1RN*12, *22 genotypes, the short two alleles  in the subgroup of girls and VNTR variants in girls .IL-1A variation with genotype 2-2 .GG genotype of rs1718119 from P2RX7 gene .Specific haplotype of P2RX7 (rs208294) and IL1RN (rs419598) with SNPs .RANK polymorphisms, in rs12455775, rs3102724, rs2875845, rs1032128, and rs3102728, osteoprotegerin polymorphisms, .The microsatellite marker D18S64 (tightly linked to TNFRSF11A) .Association of IL-1B polymorphism with Max CI EARR, IL-1B , which have a 5.6 fold increased risk of EARR greater than 2 mm, compared with subjects who were not homozygous for the IL-1B allele 1 .Homozygous [2/2(TT)] for the IL1B gene in root-filled teeth .IL1B gene in the comparative analysis of homozygous subjects (2/2[TT]) and (1/1[CC]), which led to an increased risk of experiencing post orthodontic EARR in root-filled teeth .In root-filled teeth in subjects with homozygous [1/1(TT)] for the IL1RN gene .IL-1B[1/1(CC)] and IL-1RN [1/1[(TT)] gene .The rs1143634 in allelic model, the A allele of IL-1β SNP , and X cSome studies also identified possible protective factors in reducing OIIRR severity, such asRoot-filled teeth compared to vital, immature teeth, and systemic fluoridation ,150,148.Homozygosity/hemizygosity for variant C from the IRAK1 gene and the heterozygous and homozygous osteopontin gene allele at position 89261521 ,27.In contrast, some studies found no significant differences between experimental and control groups regarding age, gender, tooth length, root shape, tooth type, asthma, root-filled teeth, habits, nail-biting, gingivitis, medications, and overbite ,50,147. High quality of evidence according to applied GRADE methodology indicates that orthodontic forces in PICO 3A and/or intervention or exposure in PI/ECO 3B are associated with the incidence or increased severity of OIIRR in human studies .in vivo studies. Further, we aimed to elucidate the underlying pathophysiological process of OIIRR by assessing cellular ‘-omics’ changes in vitro studies.This work aimed to systematically review the existing literature on the effect of mechanical force (± patient-related risk factors) on the incidence of root resorption from The systematic review of current literature demonstrated that OIIRR occurs under mechanical stress in humans and animals. Further, it can be concluded that different forms of pressure application are associated with differing severities of OIIRR.in vitro studies reported a stimulating effect of mechanical force on human PDL cells in an artificial environment. However, these investigations primarily focused on the interaction at the cellular level whilst not considering the interaction with the environment. These effects are expressed either as increased or decreased expression of genes and pathways, which differed between studies due to the magnitude of applied force, application method, extraction devices, and sequencing methodology. The results of the expression profiles were reposted using fold change compared to controls. Only the significant expression data were collected.All It was found that the CF elevated interleukin 6(IL-6), interleukin-1b, ADAMTS1, cyclooxygenase (COX)-2, and osteocalcin ,78,6. Boet al. identified 53 differentially expressed miRNAs from PDL cells under pressure compared to PDL cells without mechanical force; 26 were upregulated; and 27 were downregulated  may lead to more stress on the root and subsequently to an increase in odontoclastic effects, with high-quality human studies ,54,24,15Biological sex seems to have a controversial effect on the OIIRR among studies; Four studies with strong evidence reported increased OIIRR in male compared to female participants ,27,148. Higher age was associated with increased OIIRR in human observational studies, 4 of which are of high quality ,41,23,15Data from low risks observational studies conclude that the association between asthma and OIIRR is inconclusive; one shows a link to increase OIIRR risk , and theThe initial size of the root, initial root length, shape, and width are significant factors in some observational variable qualities studies ,23,15. IGenetic considerations may be an important perspective for the incidence and severity of OIIRR. These genetic variations might be associated with increasing or decreasing the incidence of OIIRR. It is important to note that all of the included genetic studies were only observational.Polymorphisms in some with substantial evidence studies were reported as an indicator for a higher chance of more severe OIIRR; one of the most common is the IL polymorphism, where several studies reported its effect on the severity of the OIIRR, such as IL1RN polymorphism, IL-1A variations, IL-1B, IL-6, and IL1RN ,33,26,41Conversely, according to other low risks studies, some genes may have a protective effect, such as variations in the OPN gene and the P2RX7 gene, osteopontin (OPN) deficient subjects, polymorphisms of osteoprotegerin and IRAK1 ,33,28,27Whilst other studies found there was no relationship between the severity of OIIRR and genetic variations in IL1b (rs1143634), P2RX7 (rs1718119), CASP1/ICE rs530537), P2RX7 rs208294), IL-1RN SNP (rs419598), polymorphisms in the RANKL gene, polymorphisms in TNFa and TNSALP, the haplotype of P2RX7 (rs208294 and rs1718119), SPP1 or TNFRSF11B ,25,29,304, IL-1RN537, P2RXMany articles discussing the genetic effect, both included and not included in our work, supported the belief of a possible relationship between genetic variations and OIIRR, which explains the difference in the amount of root resorption between individuals with other factors held constant.Multiple aspects of this systematic review accentuate the limitations of the currently available literature dealing with the question posed.in vitro studies are generally questionable regarding their generalizability and applicability to the natural environment. For example, in some studies with the force application model device, direct contact between the glass and cells may generate an electrical charge and gas exchange. In addition, using different devices and software may also lead to variability in outcomes. Furthermore, using a two-dimensional or three-dimensional medium is questionable; some studies used both, showing different cellular and molecular responses  but may also exhibit itself as the reduction in thickness, cervical resorption, creation of coves, lacunae, and mini cracks. These increase with biomechanics application and cannot be detected by 2D radiographs , panoramic, or cephalometric). Another drawback of the none standardized radiograph includes different magnification levels, angulation and superimposition of other structures, limiting the usefulness of this data  and environmental and lifestyle risk factors  may play a role .et al., genetic-association studies have four main problems First, careful case selection is essential to minimise phenotypic heterogeneity. Also, sufficient participants should contribute to the study’s statistical power. Further, replication, which can be considered the gold standard, and the complete capturing of the genetic information is essential. If all these factors were applied, this should result in a significant finding, but unfortunately, many OIIRR genetic studies miss one or more .Supplementary Table 3. Quality assessment of Supplementary Table 4. PICO 1A, studies’ summarySupplementary Table 5. PICO 1, detailed study descriptions and outcomes.Supplementary Table 6. PICO1 most upregulated genetic expression profile.Supplementary Table 7. PICO1 most downregulated genetic expression profile.Supplementary Table 8. Risk of bias in animal studies .Supplementary Table 9. PICO 2A, studies’ summarySupplementary Table 10. PICO 2A, detailed studies description and outcomes.Supplementary Table 11. PIECO 2B studies’ summary.Supplementary Table 12. PICO 2B, detailed studies description and outcomes.Supplementary Table 13. GRADE summary of findings for OIIRR outcome for animal studies PICO 2A and 2B.Supplementary Table 14. Risk of bias human studies (PICO 3A and 3B).Supplementary Table 15. PICO 3A, studies’ summarySupplementary Table 16. PICO 3A, detailed study descriptions and outcomes.Supplementary Table 17. PIECO 3B studies’ summary.Supplementary Table 18. PICO 3B, detailed study descriptions and outcomes.Supplementary Table 19. GRADE summary of findings for OIIRR outcome for human studies PICO 3A and 3B.Supplementary Table 20. List of abbreviations.cjad011_suppl_Supplementary_Table_S1Click here for additional data file.cjad011_suppl_Supplementary_Table_S2Click here for additional data file.cjad011_suppl_Supplementary_Table_S3Click here for additional data file.cjad011_suppl_Supplementary_Table_S4Click here for additional data file.cjad011_suppl_Supplementary_Table_S5Click here for additional data file.cjad011_suppl_Supplementary_Table_S6Click here for additional data file.cjad011_suppl_Supplementary_Table_S7Click here for additional data file.cjad011_suppl_Supplementary_Table_S8Click here for additional data file.cjad011_suppl_Supplementary_Table_S9Click here for additional data file.cjad011_suppl_Supplementary_Table_S10Click here for additional data file.cjad011_suppl_Supplementary_Table_S11Click here for additional data file.cjad011_suppl_Supplementary_Table_S12Click here for additional data file.cjad011_suppl_Supplementary_Table_S13Click here for additional data file.cjad011_suppl_Supplementary_Table_S14Click here for additional data file.cjad011_suppl_Supplementary_Table_S15Click here for additional data file.cjad011_suppl_Supplementary_Table_S16Click here for additional data file.cjad011_suppl_Supplementary_Table_S17Click here for additional data file.cjad011_suppl_Supplementary_Table_S18Click here for additional data file.