The role of the allantoin in promoting fracture healing in osteoclast-deficient zebrafish.

Fracture healing is a rigorous and orderly process with multiple steps that are mediated by multiple cells. During this process, osteoclast-mediated bone remodeling plays a critical role, and its abnormal activity leads not only to fracture susceptibility but also to impaired fracture healing. However, few studies have focused on impaired healing caused by osteoclast defects, and clinical drugs for this type of impaired fracture healing are still lacking. The cell types and regulatory pathways in the zebrafish skeletal system are highly similar to those of mammals, making the zebrafish skeletal system being widely used for skeletal-related studies. To study the process of fracture healing disorders caused by osteoclast defects and discover potential therapeutic drugs, we established an in vivo osteoclast-deficient fracture model using a previously generated fms gene mutant zebrafish (fmsj4e1). The results showed that reduced functional osteoclasts could affect fracture repair in the early stages of fracture. Then we applied an in vitro scale culture system to screen for osteoclast-activating drugs. We found the small molecule compound allantoin (ALL) being able to activate osteoclasts. Subsequently, we verified the activation role of ALL on osteoclasts and the promotion of fracture repair in an in vivo fmsj4e1 fracture defect model. Finally, by examining the osteoclastogenesis and maturation process, we found that ALL may promote osteoclast maturation by regulating RANKL/OPG, thus promoting fmsj4e1 fracture healing. Our study provides a potential new approach for the future improvement of fracture healing disorders caused by osteoclast defects.

Targeting the PTP1B-Bcr-Abl1 interaction for the degradation of T315I mutant Bcr-Abl1 in chronic myeloid leukemia.

Small-molecule-induced degradation of mutant Bcr-Abl1 provides a potential approach to overcome Bcr-Abl1 tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML). Our previous study reported that a synthetic steroidal glycoside SBF-1 showed remarkable anti-CML activity by inducing the degradation of native Bcr-Abl1 protein. Here, we observed the comparable growth inhibition for SBF-1 in CML cells harboring T315I mutant Bcr-Abl1 in vitro and in vivo. SBF-1 triggered its degradation through disrupting the interaction between protein-tyrosine phosphatase 1B (PTP1B) and Bcr-Abl1. Using SBF-1 as a tool, we found that Tyr46 in the PTP1B catalytic domain and Tyr852 in the Bcr-Abl1 pleckstrin-homology (PH) domain are critical for their interaction. Moreover, the phosphorylation of Tyr1086 within the Bcr-Abl1 SH2 domain recruited the E3 ubiquitin ligase c-Cbl to catalyze K27-linked ubiquitin chains, which serve as a recognition signal for p62-dependent autophagic degradation. PTP1B dephosphorylated Bcr-Abl1 at Tyr1086 and prevented the recruitment of c-Cbl, leading to the stability of Bcr-Abl1. This study unravels the action mechanism of PTP1B in stabilizing Bcr-Abl1 protein and indicates that the PTP1B-Bcr-Abl1 interaction might be one of druggable targets for TKI-resistant CML with point mutations.

Expanding the spectrum of "mesenchymal" tumors of the central nervous system.

In this review, we summarize the clinical, histopathological, and molecular features of central nervous system (CNS) tumors with BCOR internal tandem duplication, intracranial mesenchymal tumor with FET/CREB fusion, CNS CIC-rearranged sarcomas and primary intracranial sarcoma DICER1-mutant, now included in the 2021 WHO classification of CNS tumors. Possible relationships between tumors occurring in the CNS and their systemic counterparts are discussed.

Glyoxalase I activity affects Arabidopsis sensitivity to ammonium nutrition.

KEY MESSAGE: Elevated methylglyoxal levels contribute to ammonium-induced growth disorders in Arabidopsis thaliana. Methylglyoxal detoxification pathway limitation, mainly the glyoxalase I activity, leads to enhanced sensitivity of plants to ammonium nutrition. Ammonium applied to plants as the exclusive source of nitrogen often triggers multiple phenotypic effects, with severe growth inhibition being the most prominent symptom. Glycolytic flux increase, leading to overproduction of its toxic by-product methylglyoxal (MG), is one of the major metabolic consequences of long-term ammonium nutrition. This study aimed to evaluate the influence of MG metabolism on ammonium-dependent growth restriction in Arabidopsis thaliana plants. As the level of MG in plant cells is maintained by the glyoxalase (GLX) system, we analyzed MG-related metabolism in plants with a dysfunctional glyoxalase pathway. We report that MG detoxification, based on glutathione-dependent glyoxalases, is crucial for plants exposed to ammonium nutrition, and its essential role in ammonium sensitivity relays on glyoxalase I (GLXI) activity. Our results indicated that the accumulation of MG-derived advanced glycation end products significantly contributes to the incidence of ammonium toxicity symptoms. Using A. thaliana frostbite1 as a model plant that overcomes growth repression on ammonium, we have shown that its resistance to enhanced MG levels is based on increased GLXI activity and tolerance to elevated MG-derived advanced glycation end-product (MAGE) levels. Furthermore, our results show that glyoxalase pathway activity strongly affects cellular antioxidative systems. Under stress conditions, the disruption of the MG detoxification pathway limits the functioning of antioxidant defense. However, under optimal growth conditions, a defect in the MG detoxification route results in the activation of antioxidative systems.

Comprehensive Characterization of the Coding and Non-Coding Single Nucleotide Polymorphisms in the Tumor Protein p63 (TP63) Gene Using In Silico Tools.

Single nucleotide polymorphisms (SNPs) help to understand the phenotypic variations in humans. Genome-wide association studies (GWAS) have identified SNPs located in the tumor protein 63 (TP63) locus to be associated with the genetic susceptibility of cancers. However, there is a lack of in-depth characterization of the structural and functional impacts of the SNPs located at the TP63 gene. The current study was designed for the comprehensive characterization of the coding and non-coding SNPs in the human TP63 gene for their functional and structural significance. The functional and structural effects of the SNPs were investigated using a wide variety of computational tools and approaches, including molecular dynamics (MD) simulation. The deleterious impact of eight nonsynonymous SNPs (nsSNPs) affecting protein stability, structure, and functions was measured by using 13 bioinformatics tools. These eight nsSNPs are in highly conserved positions in protein and were predicted to decrease protein stability and have a deleterious impact on the TP63 protein function. Molecular docking analysis showed five nsSNPs to reduce the binding affinity of TP63 protein to DNA with significant results for three SNPs (R319H, G349E, and C347F). Further, MD simulations revealed the possible disruption of TP63 and DNA binding, hampering the essential protein function. PolymiRTS study found five non-coding SNPs in miRNA binding sites, and the GTEx portal recognized five eQTLs SNPs in single tissue of the lung, heart (LV), and cerebral hemisphere (brain). Characterized nsSNPs and non-coding SNPs will help researchers to focus on TP63 gene loci and ascertain their association with certain diseases.

Design, synthesis, and biological evaluation of novel Bcr-AblT315I inhibitors incorporating amino acids as flexible linker.

Despite the success of imatinib in CML therapy through Bcr-Abl inhibition, acquired drug resistance occurs over time in patients. In particular, the resistance caused by T315I mutation remains a challenge in clinic. Herein, we embarked on a structural optimization campaign aiming at discovery of novel Bcr-Abl inhibitors toward T315I mutant based on previously reported dibenzoylpiperazin derivatives. We proposed that incorporation of flexible linker could achieve potent inhibition of Bcr-AblT315I by avoiding steric clash with bulky sidechain of Ile315. A library of 28 compounds with amino acids as linker has been developed and evaluated. Among them, compound AA2 displayed the most potent activity against Bcr-AblWT and Bcr-AblT315I, as well as toward Bcr-Abl driven K562 and K562R cells. Further investigations indicated that AA2 could induce apoptosis of K562 cells and down regulate phosphorylation of Bcr-Abl. In summary, the compounds with amino acid as novel flexible linker exhibited certain antitumor activities, providing valuable hints for the discovery of novel Bcr-Abl inhibitors to overcome T315I mutant resistance, and AA2 could be considered as a candidate for further optimization.

Comparative transcriptomic analysis of the l-4i silkworm (Lepidoptera: Bombyx mori) mutants and its wild-type strain P33 by RNA-Seq.

The silkworm (Bombyx mori) is a domesticated holometabolous insect, and more than 400 Mendelian mutations have been identified. Investigating the mechanism behind these silkworm mutants is essential for understanding the development of silkworms and other lepidopterans, and lethal genes could be used for pest control. The lethal silkworm mutant in the fourth instar (l-4i) has been recently found; however, the underlying mechanism is not yet clear. Herein, we studied the l-4i mutant and its wild-type strain P33 using RNA sequencing (RNA-seq). Our results revealed that 2013 genes were significantly downregulated, and 20 biological processes, including spliceosomal snRNP assembly, protein folding and protein catabolic process, were significantly enriched in these downregulated genes. Moreover, 2405 genes were significantly upregulated in the l-4i mutant, and 20 biological processes, including purine nucleobase metabolic process, nucleoside metabolic process and de novo IMP biosynthetic process, were significantly enriched in these upregulated genes. The study suggests that the imbalance of multiple biological processes and pathways and abnormal protein generation from RNA alternative splicing may cause the death of the l-4i mutant.

Repurposing cabozantinib to GISTs: Overcoming multiple imatinib-resistant cKIT mutations including gatekeeper and activation loop mutants in GISTs preclinical models.

Despite of the great success of imatinib as the first-line treatment for GISTs, the majority of patients will develop drug-acquired resistance due to secondary mutations in the cKIT kinase. Sunitinib and regorafenib have been approved as the second and third line therapies to overcome some of these drug-resistance mutations; however, their limited clinical response, toxicity and resistance of the activation loop mutants still makes new therapies bearing different cKIT mutants activity spectrum profile highly demanded. Through a drug repositioning approach, we found that cabozantinib exhibited higher potency than imatinib against primary gain-of-function mutations of cKIT. Moreover, cabozantinib was able to overcome cKIT gatekeeper T670I mutation and the activation loop mutations that are resistant to imatinib or sunitinib. Cabozantinib demonstrated good efficacy in vitro and in vivo in the cKIT mutant-driven preclinical models of GISTs while displaying a long-lasting effect after treatment withdrawal. Furthermore, it also exhibited dose-dependent anti-proliferative efficacy in the GIST patient derived primary cells. Considering clinical safety and PK profile of cabozantinib, this report provides the basis for the future clinical applications of cabozantinib as an alternative anti-GISTs therapy in precision medicine.

(-)-Epigallocatechin-3-gallate induces cell apoptosis in chronic myeloid leukaemia by regulating Bcr/Abl-mediated p38-MAPK/JNK and JAK2/STAT3/AKT signalling pathways.

Epigallocatechin-3-gallate (EGCG), a major polyphenolic constituent of green tea, possesses remarkable chemopreventive and therapeutic potential against various types of cancer, including leukaemia. However, the molecular mechanism involved in chronic myeloid leukaemia (CML), especially imatinib-resistant CML cells, is not completely understood. In the present study, we investigated the effect of EGCG on the growth of Bcr/Abl+ CML cell lines, including imatinib-resistant cell lines and primary CML cells. The results revealed that EGCG could inhibit cell growth and induce apoptosis in CML cells. The mechanisms involved inhibition of the Bcr/Abl oncoprotein and regulation of its downstream p38-MAPK/JNK and JAK2/STAT3/AKT pathways. In conclusion, we documented the anti-CML effects of EGCG in imatinib-sensitive and imatinib-resistant Bcr/Abl+ cells, especially T315I-mutated cells.

Exploring the Influence of Mutation on Transthyretin Aggregation in Heart Disease.

BACKGROUND: Transthyretin (TTR) is the transporter protein (55 kDa) that carries retinolbinding protein and Thyroxin (T4) in its functional tetramer form. Presence of the mutation in this protein (TTR) may lead to the dissociation of tetramers to monomer which unfolds and self-associates to form amyloid aggregates. Aggregation of this protein has been found to be associated with various lifethreatening disorders such as Coronary Artery Disease (CAD) which is the major cause of mortality and morbidity worldwide. METHODS: In the present communication, we have predicted mutation prone residues of TTR with the help of suspect server. Substitution (T139R with 95 score) occurring at the thyroid hormone binding site was selected for studying the mutational consequences on TTR. The effect of mutation on stability, functionality, aggregation and folding rate was analyzed by MuPro, DUET, SDM, SNAP2, Polyphen2, PASTA2.0, Aggrescan and Folding RaCe servers. The presence of TTR monomer in CAD plasma has been observed through Western blot analysis. RESULTS: T139R mutation may expose the buried regions of TTR protein which help in the self association and the increase in the stability may help in the TTR deposition. Structural analysis indicated that F and H strands of TTR are more prone to aggregation. Thus, T139R mutation might cause these residues to be aggregation prone and change in folding rate and validated TTR monomer in diseased cases by Western blot analysis. CONCLUSION: The observed results clearly indicated that the occurrence of this mutation is causing the impact on the structural and functional significance of TTR by interfering in the formation of tetramer. Thus, hindrance created to thyroxin transportation resulted in higher lipid levels in the blood that ultimately might promote the progression of the CAD.

Synthesis and identification of GZD856 as an orally bioavailable Bcr-AblT315I inhibitor overcoming acquired imatinib resistance.

Bcr-AblT315I induced drug resistance remains a major challenge to chronic myelogenous leukemia (CML) treatment. Herein, we reported GZD856 as a novel orally bioavailable Bcr-AblT315I inhibitor, which strongly suppressed the kinase activities of both native Bcr-Abl and the T315I mutant with IC50 values of 19.9 and 15.4 nM, and potently inhibited proliferation of corresponding K562, Ba/F3WT and Ba/F3T315I cells with IC50 values of 2.2, 0.64 and 10.8 nM. Furthermore, GZD856 potently suppressed tumor growth in mouse bearing xenograft K562 and Ba/F3 cells expressing Bcr-AblT315I. Thus, GZD856 may serve as a promising lead for the development of Bcr-Abl inhibitors overcoming acquired imatinib resistance.

Prediction of Phenotypic Effects of Variants Observed in LOC_Os04g36720 of FRO1 Gene in Rice (Oryza sativa L.).

In rice, ferric-chelate reductase-1 (FRO1) (LOC_Os04g36720) gene was present on chromosome number 4 and its beginning and ending coordinates where coding sequence lies are 22182599 and 22186943, respectively. It plays a vital role in metal homeostasis and iron transportation in plants. Based on the alignment results, location of single-nucleotide variants is located in open reading frame and their effects of variants were predicted using SIFT sequence tool. The non-synonymous variants at position 342 and 436 lies in helical and coil parts of the protein, respectively, as predicted by Psi-pred server. PSI-Blast which resulted in significant hits and the most similar protein sequence (Accession ID: NP_001052896) with available sequence features displayed 100 % identity with query cover of 99 %. Results suggest the non-synonymous variant at position 436 (Accession ID: TBGI204002) lies in FAD-binding domain and nsSNV at position 342 (Accession ID: TBGI203998) lies in periphery of NADP. The SNPs were also analyzed for the deleterious effect by PANTHER subPSEC scores and I-mutant score, and it was postulated that SNPs would be hampering on biological as well as molecular function of FRO 1 gene of rice. A cutoff of -3 corresponds to a 50 % probability that a score is deleterious. From this, the probability that a given variant will cause a deleterious effect on protein function is estimated by P deleterious, such that a subPSEC score of -4 corresponds to a P deleterious of 0.79. Hence, to study the phenotypic consequences of variant TBGI204002, we performed comparative molecular docking studies of native modeled protein and protein with induced mutation as receptors and FAD as ligand to be utilized for binding. The docking process was performed by AutoDock 4.2 software with Lamarckian Genetic algorithm as computational algorithm. Results suggest binding energies are higher in case of mutation-induced protein which suggests presence of variant TBGI204002 enhances binding of FAD ligand at FAD-binding domain site. In case of TBGI203998, similar comparative docking procedure was performed with FAD as binding ligand, which suggests presence of variant does not impact FAD binding at the domain site. We revealed impact of SNPs on the protein structure and its function using sequence-based tools.

Metabolic characterization of imatinib-resistant BCR-ABL T315I chronic myeloid leukemia cells indicates down-regulation of glycolytic pathway and low ROS production.

Long-term imatinib treatment induces drug-resistant chronic myeloid leukemia (CML) cells harboring T315I gate keeper mutation of breakpoint cluster region (BCR)-ABL oncogenic kinase. However, although cell proliferation is coupled with cellular energy status in CML carcinogenesis, the metabolic characteristics of T315I-mutant CML cells have never been investigated. Here, we analyzed cell proliferation activities and metabolic phenotypes, including cell proliferation, oxygen consumption, lactate production, and redox state in the KBM5 (imatinib-sensitive) and KBM5-T315I (imatinib-resistant) CML cell lines. Interestingly, KBM5-T315I cells showed decreased cell proliferation, lactate production, fatty acid synthesis, ROS production, and down regulation of mRNA expression related to ROS scavengers, such as SOD2, catalase, GCLm, and GPx1. Taken together, our data demonstrate that the lower growth ability of KBM5-T315I CML cells might be related to the decreased expression of glycolysis-related genes and ROS levels, and this will be used to identify therapeutic targets for imatinib resistance in CML.

ABL kinase inhibitory and antiproliferative activity of novel picolinamide based benzothiazoles.

A series of novel picolinamide based benzothiazoles (17 final compounds), targeting both wild-type and the most resistant T315I mutant of Bcr-Abl kinase, has been designed and synthesized. Moreover, a selected array (8 compounds) was evaluated for its antiproliferative activity over a panel of 60 cancer cell lines. Compound 5l was the most potent derivative against both native and T315I mutant ABL with IC50 values of 18.2 and 39.9nM, respectively, and showed highly selective inhibitory activity (89.8%) towards the Bcr-Abl dependent leukemia cell (K-562) at 10µM concentration. Significance of C6-oxypicolinamide moiety and SAR study for the C2 aliphatic side chain of benzothiazole are discussed in detail.

CoagVDb: a comprehensive database for coagulation factors and their associated SAPs

The current state of the art in medical genetics is to identify and classify the functional (deleterious) or non-functional (neutral) single amino acid substitutions (SAPs), also known as non-synonymous SNPs (nsSNPs). The primary goal is to elucidate the mechanisms through which functional SAPs exert their effects, and ultimately interrogating this information for association with complex phenotypes. This work focuses on coagulation factors involved in the coagulation cascade pathway which plays a vital role in the maintenance of homeostasis in the human system. We developed an integrated coagulation variation database, CoagVDb, which makes use of the biological information from various public databases such as NCBI, OMIM, UniProt, PDB and SAPs (rsIDs/variant). CoagVDb enriched with computational prediction scores classify SAPs as either deleterious or tolerated. Also, various other properties are incorporated such as amino acid composition, secondary structure elements, solvent accessibility, ordered/disordered regions, conservation, and the presence of disulfide bonds. This specialized database provides integration of various prediction scores from different computational methods along with gene, protein, and disease information. We hope our database will act as a useful reference resource for hematologists to reveal protein structure-function relationship and disease genotype-phenotype correlation.

Computational pipeline to identify and characterize functional mutations in ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) (E.C. 2.1.3.3) is one of the enzymes in the urea cycle, which involves in a sequence of reactions in the liver cells. During protein assimilation in our body surplus nitrogen is made, this open nitrogen is altered into urea and expelled out of the body by kidneys, in this cycle OTC helps in the conversion of free toxic nitrogen into urea. Ornithine transcarbamylase deficiency (OTCD: OMIM#311250) is triggered by mutation in this OTC gene. To date more than 200 mutations have been noted. Mutation in OTC gene indicates alteration in enzyme production, which upsets the ability to carry out the chemical reaction. The computational analysis was initiated to identify the deleterious nsSNPs in OTC gene in causing OTCD using five different computational tools such as SIFT, PolyPhen 2, I-Mutant 3, SNPs&Go, and PhD-SNP. Studies on the molecular basis of OTC gene and OTCD have been done partially till date. Hence, in silico categorization of functional SNPs in OTC gene can provide valuable insight in near future in the diagnosis and treatment of OTCD.

Exploration of structural stability in deleterious nsSNPs of the XPA gene: A molecular dynamics approach.

BACKGROUND: Distinguishing the deleterious from the massive number of non-functional nsSNPs that occur within a single genome is a considerable challenge in mutation research. In this approach, we have used the existing in silico methods to explore the mutation-structure-function relationship in the XPAgene. MATERIALS AND METHODS: We used the Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping (PolyPhen), I-Mutant 2.0, and the Protein Analysis THrough Evolutionary Relationships methods to predict the effects of deleterious nsSNPs on protein function and evaluated the impact of mutation on protein stability by Molecular Dynamics simulations. RESULTS: By comparing the scores of all the four in silico methods, nsSNP with an ID rs104894131 at position C108F was predicted to be highly deleterious. We extended our Molecular dynamics approach to gain insight into the impact of this non-synonymous polymorphism on structural changes that may affect the activity of the XPAgene. CONCLUSION: Based on the in silico methods score, potential energy, root-mean-square deviation, and root-mean-square fluctuation, we predict that deleterious nsSNP at position C108F would play a significant role in causing disease by the XPA gene. Our approach would present the application of in silicotools in understanding the functional variation from the perspective of structure, evolution, and phenotype.