Crop genetic diversity is essential for adaptation and productivity in agriculture. A previous study revealed that poor allele diversity in wheat commercial cultivars is a major barrier to its further improvement. Homologs within a variety, including paralogs and orthologs in polyploid, account for a large part of the total genes of a species. Homolog diversity, intra-varietal diversity (IVD), and their functions have not been elucidated. Common wheat, an important food crop, is a hexaploid species with three subgenomes. This study analyzed the sequence, expression, and functional diversity of homologous genes in common wheat based on high-quality reference genomes of two representative varieties, a modern commercial variety Aikang 58 (AK58) and a landrace Chinese Spring (CS). A total of 85,908 homologous genes, accounting for 71.9% of all wheat genes, including inparalogs (IPs), outparalogs (OPs), and single-copy orthologs (SORs), were identified, suggesting that homologs are an important part of the wheat genome. The levels of sequence, expression, and functional variation in OPs and SORs were higher than that of IPs, which indicates that polyploids have more homologous diversity than diploids. Expansion genes, a specific type of OPs, made a great contribution to crop evolution and adaptation and endowed crop with special characteristics. Almost all agronomically important genes were from OPs and SORs, demonstrating their essential functions for polyploid evolution, domestication, and improvement. Our results suggest that IVD analysis is a novel approach for evaluating intra-genomic variations, and exploitation of IVD might be a new road for plant breeding, especially for polyploid crops, such as wheat. Triticum aestivum L.) is a hexaploid crop species containing subgenomes AA, BB, and DD, formed by natural hybridizations of three diploid ancestors [Genetic diversity has an essential impact on the adaptation and productivity of crops in agriculture . Analysincestors . Previouncestors ,4,5.ZIP4 (Ph1) and CENH3, are essential for chromosome pairing, mitosis, and meiosis [Btr1 for spike brittleness is a key gene in wheat domestication. The dwarfing gene Rht1 and photoperiod response gene Ppd1 contribute significantly to the ‘Green Revolution’.Homologs within an individual include paralogs and orthologs in polyploids, which constitute a large component of the complete sets of genes in a species such as wheat. Paralogs are a set of duplicated genes derived from a common ancestral gene in the genome . Gene du meiosis . Btr1 foHomolog diversity or IVD refers to variation in sequence, expression, and function within a variety. Homologs coordinately confer plant growth, development, reproduction, and adaptation. Therefore, IVD analysis (IVDA) is expected to be an important and new area of diversity study.Here, we carried out an IVDA of Aikang 58 (AK58), a modern leading wheat variety in China with high yield and diverse environmental adaptability, and Chinese Spring (CS), a landrace, both with high-quality reference genomes available, to reveal the sequence, expression, and functional diversity of homologs in the common wheat genome. We also evaluated how homolog diversity has shaped wheat evolution, domestication, and improvement.A total of 119,448 high-confidence genes were annotated in the AK58 genome, including orthologs, paralogs, and singletons. We divided them into four types based on the distribution of homologous genes in the subgenomes: (1) IPs , duplicated genes in a single subgenome; (2) dyads, duplicated genes in two subgenomes; (3) triplets, duplicated genes in all the three subgenomes, of which OPs  are paralogous genes with collinearity between subgenomes in triplets; and (4) single-copy orthologs (SORs) with one copy in each subgenome. There were 12,762 gene families containing 44,568 paralogous genes and 13,780 gene families with 41,340 SOR genes, accounting for 71.9%  of the total genes in common wheat . The triTo test whether IVD was detected correctly and efficiently, we selected 16 gene ontology (GO) terms with the most homologs (from 312 to 1040 genes) to analyze IVD. Homologs conferring signal transduction, phosphorylation, and response to biotic and/or abiotic stress had a high diversity, while those conferring translation, abscisic acid-activated signaling pathway, and embryo development ending in seed dormancy had low diversity . These rWe analyzed the DNA nucleotide diversity of four regions with a gene—2 Kb upstream, 5′ UTR, coding sequence, and 3′ UTR of 8957 IPs and 13,131 OPs in the AK58 and CS genomes—for comparison of SORs in corresponding regions. The homologs had an average nucleotide diversity (π) of 0.2219, with the greatest diversity in OPs (0.3719), similar to SORs and IPs A. We comKs of 1.114 with dispersed distribution  were diverged in co-expression modules, followed by 32.7% of the SOR families and 20.6% of the IP families, indicating that homologous expression diversity of subgenomes significantly differs in co-expression . The higIPs, OPs, and SORs had tissue-specific expression patterns. IPs were expressed most actively in early seed development. The expression of OPs peaked in roots under salt stress and in anthers. SORs had the highest expression in the stem and anther B. DifferSequence and expression diversity often generate functional changes in homologs ,18. CandGenerally, duplicated genes are prone to be lost or subject to pseudogenization. However, over 40,000 duplicated genes were stable in the wheat genome, suggesting that duplicated genes have important functions. Therefore, we examined how IVD contributed to wheat evolution, domestication, and improvement.2+)-dependent protein kinases (CDPK) play an important function in the Ca(2+)-mediated signal transduction. Twenty-nine CDPK genes have been identified in the rice genome. OsCPK10 mediates drought tolerance and blast disease resistance, OsCPK12 is involved in salt stress tolerance and blast disease resistance, Oscpk7 confers tolerance to both salt and drought stresses, and OsCPK26 is involved in pollen development. These results suggest that the duplication genes in a family have divergence in their functions during the long period of evolution. However, the retention of common duplicated genes in different species demonstrates that the genes for plant development and tolerance to abiotic stresses are relatively conserved, and more genes are required to ensure crop development and adaptation to various environments. We detected 504 Gramineae WGDPGs with collinearity (Whole-genome duplication (WGD) and polyploidization are important in crop evolution . To analinearity B and othinearity A, as mosThe species-specific homologs in subgenomes A, B, and D were significantly enriched in the regulation of biosynthetic processes, nucleic acid binding, and negative regulation of coenzyme metabolic processes, respectively . The speGene expansion is a specific type of homolog that often contributes to specific species traits ,23. We dLeguminosae, such as soybean. Eight grass-specific or dominant expansion families, including 172 genes on average, were detected . These results suggest that the high protein contents of wheat and soybean are such as the result of the expansion of storage protein genes, and large and diverse expansion families contribute to wheat adaptation, making wheat a world crop.Wheat has the highest protein content over other major food crops, such as rice, maize, and millet. The exact number of gene-encoding proteins in wheat has been unclear due to the difficulty in assembling the high tandem duplication. However, using the high-quality AK58 genome, we detected eight expansion storage protein families, with approximately 80 copies in each subgenome C, comparCENH3 and Ph1 (ZIP4), play key roles in these processes in hexaploid wheat. Notably, both of them are OPs.Polyploidization and its subsequent diploidization are two important evolutionary processes in wheat evolution. Two genes, CENH3 encodes a centromere-related protein associated with plant height [CENH3 was found in rice and maize, but two copies (α-CENH3 and β-CENH3) were detected in diploid, tetraploid, and hexaploid wheat [α-CENH3 and β-CENH3) on wheat chromosomes 1A and 1D. Furthermore, we found three copies in tandem duplication, one α-CENH3 and two β-CENH3, on chromosome 1B of hexaploid and tetraploid wheat, including durum wheat (T. durum) [T. dicoccoides)   and wildccoides) A. We det β-CENH3 , suggestA and D. B. We alsA and D. C. All gend roots B. We mapZIP4 (Ph1) promotes homologous pairing during meiosis [ZIP4 copies, ZIP4-3A, ZIP4-3B, ZIP4-3D, and ZIP4-5B, were observed in common wheat. The first three are orthologs, and the ZIP4-5B sequence had a high similarity with ZIP4-3B , GW2 (Grain Weight2), and VRN1 . In addition, 41 transcription factors (TFs) were co-expressed with ZIP4  contributed greatly, combining with the other three copies to wheat post-polyploid diploidization, and may be a pleiotropic gene.The analysis of chromatin accessibility and histone modification revealed that r copies D. Althoupression B. In add ZIP4-5B , includiith ZIP4 , includiModern wheat experienced two major processes to shape its genome—domestication and improvement—with IVD significantly contributing to both processes. To reveal the contribution of IVD to wheat domestication and improvement, we collected the cloned 187 agronomic important genes and analyzed their IVD. These genes were associated with six agronomic traits and grouped into homologous types . Except Btr1. This gene confers brittle rachis brittleness, derived from its ancestral paralog Btr-like [Btr1-A (TtBtr1-A) and Btr1-B (TtBtr1-B) [Btr1-A and a 4 Kb insertion in Btr1-B. In the present study, we found that Btr1 and Btr2 and their associated-like genes were tandem duplications with three, five, and three copies located on chromosomes 3A, 3B, and 3D, respectively, for Btr1/Btr1-like, and three, four, and seven copies for Btr2/Btr2-like , and other copies had weak or non-expression . It is worth noting that both prolamin and Btr genes were tandem duplication. However, most of the prolamin genes, Gli- and Glu-, were actively expressed, possibly due to artificial selection.Next, we focused on analyzing the domestication gene Btr-like . It has tBtr1-B) . The domtr2-like E. Thus, Btr and Btr-like and their duplicate copies, may also be associated with epi-modification. The paralogs of Btr and Btr-like were DNA hypomethylated, with enriched levels of H3K27me3 in various tissues, including seedlings, leaves, stems, sheaths, flag leaves, and spikelets  was used to construct genomic synteny blocks with parameters -s 3 -e 1 × 10−5 [https://wheat-urgi.versailles.inrae.fr, accessed on 30 April 2023), and the AK58 genome data were obtained from a recent study at https://ngdc.cncb.ac.cn/gwh/Assembly/9670/show (accessed on 30 April 2023). The diploid T. uratu [Ae. tauschii [T. dicoccoides [We performed BLAST searches using NCBI BLAST v2.6.0+ to identify duplicate genes from all high-confidence genes in genomes of AK58 and other wheat varieties. We retained matches with identity ≥70% and e-values < 1 × 10−5 . OrthoMC−5 . We then1 × 10−5 . Circle 1 × 10−5 . The CS T. uratu , Ae. tautauschii , tetraploccoides , and hexoccoides  genomes Ka/Ks) sites of paralogs and SORs in AK58 using the MA method [−5, identity > 60%, and coverage > 60%) used for gene pairs in the Ka/Ks calculation. Only significant Ka/Ks values (p < 0.05) were used to ensure the reliability of the results. Other parameters were set to default values. These cutoffs were also used in the approach of CACTAs captured gene pairs. We removed the gene pairs with Ks ≥ 3 to be conservative. Such genes did not alter the Ka/Ks distribution even though they were excluded.The KaKs_Calculator v2.0 was used to estimate substitution rates at nonsynonymous/synonymous . The nucleotide diversity calculation of homologs was obtained with the index Pi [p < 0.05). All statistical analyses were performed using R version 3.6.3 .The homolog diversity analysis of gene families associated with important GO terms was carried out based on function annotations to identify homologous gene families. First, we searched for homologous gene families containing copies associated with 16 high-frequency annotated terms, retaining families with isolated copy numbers ≥ 2. Next, multiple sequence alignments were performed within each subfamily (copies with a target term) using MUSCLE v3.8.31 . Finallyindex Pi .(1)Pi=2A phylogenetic tree was constructed in MEGA5 using the neighbor-joining and maximum-likelihood methods with 1000 replicates in the bootstrap test .p < 0.05). We restricted the co-expression analysis to gene families in which all copies had clear co-expression modules.We used the fragments per kilobase of the exon model per million mapped fragments (FPKM) of leaf organs at the one-leaf stage to estimate gene expression levels. For each gene family, the extreme deviation and coefficient of variation (CV) were obtained from all FPKM values; and expression patterns were considered significantly diverged if CV > 0.6 and absolute log2FC ≥ 1 .The tissue specificity expression index (τ) for each gene was determined by programming the following calculation in Python v3.5.6 :(2)τ=∑i=p < 0.05 were used for further downstream analysis steps.We used biological process, molecular function, and cellular component ontologies released by the GO Consortium . To deteThe genes were divided into three regions for analyzing TEs, GC content, and methylation: upstream 2 Kb, downstream 2 Kb, and gene body. The TE distribution on the above regions was programmed in Python code. The GC content was calculated in 200 bp windows. The bisulfite sequencing data were aligned to the AK58 genome using Bismark with bowtie2 v2.2.9 , adoptin−5. In the downstream analysis, genes with sequence identity and query coverage >70% were considered to construct gene families across different species, and the paired peptides cluster strategy used MCL v14-137 with the default settings [https://sourceforge.net/projects/mcscanx, accessed on 9 April 2023) with parameters -s 3, -e 1 × 10−5, respectively, to identify WGDPGs. Only paralogs in synteny blocks within and between genomes were considered common WGDPGs.We relied on a sequence homology approach to identify gene expansion, further identifying WGDPGs by synteny blocks generated in WGD events. We completed the first all-versus-all peptide alignments using NCBI BLAST+ v2.6.0+ for all proteins in AK58, rice, maize, millet, and soybean genomes with BLASTP parameters -max_target_seqs of 1000 and a maximum e-value of 1 × 10settings . Genomichttps://rapdb.dna.affrc.go.jp/download/irgsp1.html, accessed on 30 April 2023). The maize genome Zm-B73-REFERENCE-GRAMENE-4.0 and annotations were downloaded from the Maize Genomics Resource . The millet genome Setaria italica v2.0 and annotations were downloaded from Ensembl . The soybean genome Glycine max and annotations were downloaded from PlantGDB .The rice genome Os-Nipponbare-Reference-IRGSP-1.0 and annotations were downloaded from the Rice Annotation Project Database  and TFs within a genome, opening new frontiers in genomic analysis.The low level of allele diversity in common wheat, in particular commercial varieties, is found to be a main barrier to further wheat improvement. Intra-diversity analyses are a novel approach to examining genetic diversity within a genome. We carried out IDA to determine HD function in common wheat, representing the first intra-genome diversity analysis. Our results suggest that IDA is a powerful and efficient method for genomic research. Our IDA, based on the high-quality AK58 and CS genomes, showed that polyploids gain additional subgenome homolog diversity, i.e., OP and SOR, and thus have more genomic diversity than diploid species. Notably, subgenome homologs had a significantly higher proportion of agronomically important genes. Therefore, we proposed that subgenome homolog diversity is essential for polyploid heterosis. With the increasing availability of high-quality reference genomes and resequencing data, IDA could offer deep insights into genomic structure and function and become a routine tool in genomic research. Particularly, it could be used to evaluate crop germplasm diversity of individual accessions. The new capacity offered by IDA could consequently accelerate germplasm exploitation and utilization. This method could also be used to evaluate the diversity of gene families  than in previous reports by allelic diversity in wheat , which


The faithful formation and, consequently, function of a synapse requires continuous and tightly controlled delivery of synaptic material. At the presynapse, a variety of proteins with unequal molecular properties are indispensable to compose and control the molecular machinery concerting neurotransmitter release through synaptic vesicle fusion with the presynaptic membrane. As presynaptic proteins are produced mainly in the neuronal soma, they are obliged to traffic along microtubules through the axon to reach the consuming presynapse. This anterograde transport is performed by highly specialised and diverse presynaptic precursor vesicles, membranous organelles able to transport as different proteins such as synaptic vesicle membrane and membrane-associated proteins, cytosolic active zone proteins, ion-channels, and presynaptic membrane proteins, coordinating synaptic vesicle exo- and endocytosis. This review aims to summarise and categorise the diverse and numerous findings describing presynaptic precursor cargo, mode of trafficking, kinesin-based axonal transport and the molecular mechanisms of presynaptic precursor vesicles biogenesis in both vertebrate and invertebrate model systems. A neuron is a highly polarised cell with the cell body often distant from its synaptic terminals, where synapses are formed and maintained. Robust presynapse formation, maturation, and maintenance depend on the continuous delivery of presynaptic proteins from the neuronal soma, the predominant site of protein production. In the neuronal soma, newly synthetised presynaptic proteins are packaged into presynaptic transport vesicles or presynaptic precursor vesicles , exported from the soma to traffic anterogradely on polarised microtubules (MTs) along the axon to the consuming presynapse  1,2,3,4,6,7,8,9.Of note, although protein biosynthesis predominantly occurs in the neuronal soma, growing evidence is raised that pre- and postsynaptic local mRNA translation and protein synthesis significantly contribute to activity-dependent synapse plasticity mechanisms ,11,12,13The presynapse is composed of a variety of structurally and functionally highly diverse proteins, including synaptic vesicle (SV) proteins with integral membrane and membrane-associated proteins, cytosolic active zone (AZ) proteins and release factors, ion-channels, adhesion molecules, and presynaptic membrane proteins coordinating vesicle exo- and endocytosis  2,3,8,1,18,16. IDrosophila, the vertebrate specific proteins Piccolo and Bassoon, as well as Liprin-α/Syd-2 , Syd-1, and Spinophilin/Neurabin . I. I33]. IDrosophila leads to an accumulation of PV clusters in the neuronal soma . S. S34]. Snal soma . Electronal soma . Future 2+ channels, as shown by a study in hippocampal neurons, described epitope-tagged Neurexin co-trafficking with RIM1α and N-type Ca2+ channels, but not Bassoon ). I). IDrosoting PVs  [20,31,3trans-Golgi [Small GTPases including Rab2 recruit in their activated GTP-bound state effector proteins to dedicated membranes to concert vesicle fusion and/or fission events ,63, and ns-Golgi .trans-Golgi, including PV biogenesis but also the formation of neuronal DCVs. DCVs have a typical size of roughly 43 nm diameter with a dense core undergoing a complex maturation sequence to form mature neuropeptide transporting organelles [C. elegans system, Rab2/Unc108 is involved in neuronal DCV biogenesis at the trans-Golgi, as DCV morphology (size and shape) and PI(3)P-dependent cargo sorting during DCV maturation is aberrant in rab2−/− mutants [trans-Golgi [Drosophila neurons depends on Rab2 [Drosophila tissue [trans-Golgi on lysosomal membrane protein (LMP) carriers, a process mediated by Rab2 [trans-Golgi organise PV biogenesis using components of the other Golgi export pathways, or, as part of the PV maturation process, mediate fusion events with endo-lysosomal organelles.Early studies described Rab2 as a Golgi-resident to act in anterograde and retrograde ER-Golgi trafficking ,65,66. Hganelles ,68,69. I mutants ,56. Impons-Golgi ,57,58,59a tissue , and enda tissue . Interesa tissue ,70. As l by Rab2  and, Rab by Rab2 , Rab2 mitrans-Golgi export routes, possibly using components of the hitherto independently described molecular pathways to orchestrate PV biogenesis . This allows the fine-tuning of the kinesin activity, and motility to ensure accurate cargo load for cell- and synapse-specific cargo delivery. Kinesin-1 (KIF5A/B/C) and kinesin-3 (KIF1A) activation and termination have been extensively studied ,118. KinSpecificity of kinesin-dependent axonal PV transport requires a balanced regulatory machinery composed of kinesin-adaptor/regulatory protein complexes. During the last decades, a wealth of data was collected and numerous studies in different model systems using a wide set of molecular markers have expanded our understanding of the kinesin-based PV transport. Although it will not be possible to summarise all findings  is thought to act as major kinesin transporting PVs with an SV protein cargo [unc104 mutants [C. elegans, depleting Unc104 using the canonical unc-104D1497N allele with a mutation of the PH (pleckstrin homology) domain that diminishes cargo binding and causes protein degradation led to the accumulation of the SV protein Synaptogyrin in the neuronal soma, while its axonal transport was abolished [unc104 mutant, where Rab3 accumulated in the neuronal soma and proximal axon and, concomitantly, overexpression of Unc104 or gain-of-function mutations showed enhanced Rab3 transport with a decrease of the somatic and an increase of distal axonal and synaptic Rab3 [Similar to vertebrates, in in cargo ,118,125.in cargo . KIF1A P2 binding resulted in a failure of Synaptotagmin transport and a consistent accumulation in the neuronal soma [Other experiments shed light on how Unc104 can bind membranes of SV transporting PVs. Unc104 contains a C-terminal PH domain that specifically binds phosphatidylinositol-4,5-bisphosphate P2) . A subsenal soma . The ArlSupporting the Unc104 loss of function analysis, overactivation of KIF1A/Unc104 by disruption of the intramolecular autoinhibition of KIF1A/Unc104 causes an increase of SVs in dendrites . HyperacC. elegans study, unc104−/− mutants showed no defect in axonal ELKS-1 distribution; however, RIM/Unc10 showed a mild accumulation in the proximal axon, similar to the SV protein gradient observed [Drosophila, Unc104 is required for both AZ and SV protein delivery to the synapse. In unc104−/− mutants, synaptic terminals (neuromuscular junctions = NMJs) were atrophic, boutons were not forming and devoid of SVs, and presynapses reduced in number in both embryonic and larval neurons [unc104 mutants provide evidence that the presynaptic AZ protein Liprin-α and the VGCC Cacophony (Cac) are also transported by Unc104 [In a recent observed , suggest neurons ,104. Upo neurons ,104,106.Notably, a recent study suggests an additional signalling role of Unc104 possibly causing the above summarised phenotypes independent of an Unc104 transport function. The “Wallenda (Wnd)/DLK MAP kinase axonal damage signalling pathway” is activated upon Unc104 depletion, and this activation was proposed to account for the observed reduction of presynaptic proteins at the synaptic terminal due to reduced expression levels of AZ and SV proteins .KIF5 is the primary kinesin motor for transport of mitochondria and lysosomes ,129, witDrosophila kinesin-1 (Kinesin heavy chain = KHC) is crucial for anterograde transport of mitochondria [khc mutants [khc mutants [mutAkhc). Interestingly, the axonal traffic jam was now removed and, at least, SV proteins (Synaptotagmin and CSP) did not accumulate in axons anymore, implying a restoration of SV protein trafficking [unc116−/− mutans both proteins still localise along the entire axon and are not accumulating in the proximal axon [As in vertebrates, chondria ,131. Addchondria . Hypomorchondria ,132. Thechondria ,98,132. chondria . The SV  mutants . However mutants  are caus mutants ,133,134.fficking . Notablymal axon . Even inmal axon , suggestDrosophila, alike to the findings in vertebrates, the kinesin adaptor protein FEZ/Unc76 was shown to bind the C-terminus of KHC, and loss of FEZ/Unc76 caused defects in axonal transport of SV proteins, e.g., Synaptotagmin 1 accumulated in axons [C. elegans, loss of FEZ/Unc76 function caused axonal transport defects of Syntaxin 1 as it accumulated in axons and neuronal cell bodies. [In in axons . Additioin axons . Coheren bodies. .Drosophila homologue APLIP-1 (APPL-interacting protein 1) phenocopies the khc mutant phenotype, including tail flipping, axonal swelling, and accumulation of the SV proteins Synaptotagmin and Synaptobrevin [jip1/jip2 knockout mice did not show a defect in Synapsin 1 distribution [liprin-α mutants, clear cored vesicles accumulate in axons as well as the SV proteins Synaptotagmin and Synaptobrevin, phenotcopying the khc mutant phenotype, possibly suggesting a direct role of Liprin-α during PV transport [KHC interacts with specific cargo binding adaptors, such as the JNK scaffolding adaptor JIP-1 (JNK-interacting protein 1), regulating c-Jun N-terminal kinase (JNK) activity, as shown in vertebrates ,136. Funtobrevin ,108 and tobrevin  in the aribution . Finallyransport . Interesransport , suggestunc104−/− mutants, and synaptic terminals are devoid of ANF-positive DCVs, which accumulate ectopically in the soma [Drosophila [Of note, transport of neuropeptide-filled DCVs marked by ANF–GFP, a fusion construct of the rat atrial natriuretic peptide and GFP behaving as an endogenous neuropeptide , is abolthe soma ,126. Alsthe soma  and KHC osophila  are requosophila ,118,124.Synapse growth and maintenance are dependent on the contingent of locally available presynaptic protein. Decreased or impeded axonal transport feeding presynaptic material into the synaptic terminal, e.g., in absence of the anterograde microtubule motor kinesin-3 KIF1A/Unc104) ,103,104, ,103,104Cargo specificity of a kinesin can be determined by adaptor or linker proteins associating a specific cargo to a specific kinesin. This paragraph highlights and attempts to summarise an intensely characterised adaptor complex implied in PV transport studied in different species: the Arl8/BORC complex.C. elegans and Drosophila [C. elegans to regulate SV-protein-carrying PV trafficking by activation of Kinesin-3 (KIF1A/Unc104) by unlocking the kinesin autoinhibition [C. elegans revealed that Rab3 transporting PVs can bind more Kinesin-3 (KIF1A/Unc104) motor proteins in presence of Arl8 than in its absence [arl8−/− mutant worms, the SV protein Rab3, the AZ proteins RIM/Unc10, Liprin-α/Syd-2, and a calcium channel subunit accumulated in proximal and were lost from distal axons, a typical transport defect phenotype [unc104−/− mutants, Liprin-α/Syd-2 and partially also RIM/Unc10 axonal distribution was not altered [arl8−/− mutant worms [Drosophila, Arl8 was detected by axonal life imaging on AZ- and SV-protein-transporting PVs, and in arl8−/− mutants, both SV and AZ proteins accumulated in the neuronal cell body and were absent from the synaptic terminal, while axonal trafficking was impeded [Arl8 is a conserved Arf-like small GTPase (ADP ribosylation factors = Arfs), and Arfs form a subfamily of small GTPases mainly implied in the regulation of membrane trafficking . Arl8 isosophila . Arl8 wahibition . Elegant absence . In arl8henotype ,70, alth altered . Of notent worms . In Dros impeded . In addi impeded .Drosophila, and in mouse hippocampal neurons, Lamp1 was transported together with Bassoon and VGlut (85% of VGlut-positive anterogradely trafficking vesicles are Lamp1-positive and 40% of Bassoon-positive vesicles) [Originally, Arl8 was described as a lysosomal resident and adaptor protein to control lysosome positioning and fusion events both in vertebrates and invertebrates ,154,155,esicles) . A lateresicles) . Future Drosophila were shown to interact with the adaptor protein SKIP (SifA and kinesin-interacting protein) to hook lysosomes onto KIF5 (kinesin-1 family), thus allowing for an MT plus-end-directed outward movement of lysosomes [Drosophila, the BORC complex subunits  were shown to be required for Arl8 localisation and activity to regulate lysosomal positioning, autophagosome–lysosome fusion, and endosome–lysosome fusion [In vertebrates, Arl8B is recruited to the lysosomal membrane by the BORC complex (biogenesis of the lysosome-related organelle complex 1 (BLOC-1)-related complex) . The BORysosomes ,160,161.ysosomes . Arl8A oysosomes ,161. In ysosomes . Amountsysosomes . While Kysosomes . Also ine fusion . As Arl8C. elegans, depletion of specific components of the BORC complex, e.g., BLOS-1/2 and Myrlysin, phenocopied the Kinesin-3 (KIF1A/Unc104) mutant phenotype, and SVs accumulated ectopically in the proximal axon [In mal axon . Howevermal axon . In vivomal axon . In contmal axon . ConsistPresynaptic precursor vesicles raised scientific interest already in the 1980s and a wealth of experimental data has been collected to date, fusing into a widely accepted model derived from several independent experimental approaches ranging from live imaging over ultrastructural electron microscopic to immunofluorescence labelling analysis across different species. It is likely that PVs traffic at least to some extent as multivesicular complexes or transport packets composed of unitary vesicles with different morphologies, including 40–60 nm sized clear or slightly dense cored vesicles and 80 nm sized dense cored vesicles C. Addititrans-Golgi. Small GTPases, including Rab2, likely control early steps of PV assembly, possibly recruiting effector proteins to the nascent PV membranes and thus initialising vesicle budding from the Golgi, and possibly also directing cargo load and sorting. Interestingly, mature PV membranes seem to share, to some extent, a lysosome-like identity and require the lysosomal kinesin adaptor Arl8 for PVs export from the neuronal soma. This raises the interesting possibility that additional fusion events with components of the endo-lysosomal system could contribute to the PV maturation. Finally, as Rab2 plays a pivotal role in dense core vesicle maturation at the trans-Golgi, molecular mechanisms and molecules of the DCV assembly pathway might contribute to PV biogenesis, a promising direction for future investigations (Regarding the mechanisms and molecular pathways governing PV assembly, early and recent findings synergise in a common model, suggesting that PV biogenesis occurs at the exit sites of the Golgi, the igations .Anterograde PV transport along the axon to the consuming synapse is performed by kinesins. Live imaging experiments using fluorescently tagged presynaptic proteins in combination with genetic mutations of the different kinesins raised a substantial amount of experimental data deciphering which kinesin and kinesin adaptors are responsible for PV transport . Clearly


Type 2 diabetes (T2D) is one of the major metabolic disorders in humans caused by hyperglycemia and insulin resistance syndrome. Although significant genetic effects on T2D pathogenesis are experimentally proved, the molecular mechanism of T2D in South Asian Populations (SAPs) is still limited. Hence, the current research analyzed two Gene Expression Omnibus (GEO) and 17 Genome-Wide Association Studies (GWAS) datasets associated with T2D in SAP to identify DEGs . The identified DEGs were further analyzed to explore the molecular mechanism of T2D pathogenesis following a series of bioinformatics approaches. Following PPI (Protein-Protein Interaction), 867 potential DEGs and nine hub genes were identified that might play significant roles in T2D pathogenesis. Interestingly, CTNNB1 and RUNX2 hub genes were found to be unique for T2D pathogenesis in SAPs. Then, the GO (Gene Ontology) showed the potential biological, molecular, and cellular functions of the DEGs. The target genes also interacted with different pathways of T2D pathogenesis. In fact, 118 genes (including HNF1A and TCF7L2 hub genes) were directly associated with T2D pathogenesis. Indeed, eight key miRNAs among 2582 significantly interacted with the target genes. Even 64 genes were downregulated by 367 FDA-approved drugs. Interestingly, 11 genes showed a wide range (9–43) of drug specificity. Hence, the identified DEGs may guide to elucidate the molecular mechanism of T2D pathogenesis in SAPs. Therefore, integrating the research findings of the potential roles of DEGs and candidate drug-mediated downregulation of marker genes, future drugs or treatments could be developed to treat T2D in SAPs. Type 2 diabetes (T2D) is a complex metabolic disorder that has sparked much research interest globally. In 2021, 536.6 million people had diabetes, and 783.2 million are expected to have T2D by 2045 [T2D is the cumulative effect of genetic and environmental factors. Multiple susceptible genetic signatures responsible for T2D have already been identified in various South Asian countries using genome-wide association studies (GWAS) . In SoutDue to considerable intergroup cultural differences, SAP possesses a significant genetic diversity . ResearcTCF7L2, FTO, PPARG2, IRS1, SLC30A8, CDKN2A, HHEX, CDKAL1, EXT2, ADIPOQ, IGF2BP2, WFS1, LOC387761, CAPN10, CDKN2B, MTHFR, KCNJ11, SGCG, ADAM30, THADA, GCK, LOC646279, TCF-2/ HNF1B, NOTCH2, VEGFA, and HOMA-β genes were found to be associated with T2D pathogenesis [Indeed, several genes have already been identified that are associated with T2D pathogenesis in SAP ,5,14. Inogenesis .Although different genes and loci have already been identified for T2D pathogenesis among SAP, how these genes interact at transcript and protein levels in disease association, different metabolic pathways, biological systems, and drug interaction are still unknown that needs to be elucidated. In addition, extensive genetic diversity among different SAP guides further research on population-specific disease associations . BioinfoTherefore, we have designed the research to identify and predict the functions of DEGs associated with T2D pathogenesis in SAP using GWAS catalog data and gene expression omnibus (GEO) data. These screened DEGs were utilized for further analyses following protein-protein interaction, gene ontology, pathway enrichment, miRNA target regulatory, disease association, and drug-gene interaction to elucidate the mechanisms of T2D pathogenesis among SAPs.https://www.ncbi.nlm.nih.gov/gds), and each GSE file was separated into control and disease states. Both datasets were from the South Asian populations (Singaporean and Indian). The dataset of the Singaporean population (GSE26168) is based on the GPL6883 platform, and we have used eight controls and nine T2D-affected samples within 60 samples of the dataset. While the dataset of the Indian population (GSE78721) is based on the GPL15207 platform, and we have used 16 controls and 19 T2D-affected samples within 130 samples of the dataset. The differentially expressed genes (DEGs) in T2D-affected populations were identified using the GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r) database. GEO2R is a web-based application that analyzes two or more GEO datasets to elucidate the DEGs under various experimental conditions. The modified Benjamin and Hochberg’s false discovery rate and the P-values were utilized to balance the identification of statistically significant DEGs with limitations of false positives. DEGs in T2D were identified using the fold change value, |log FC| > 1.5 and adj. P 0.05 [The GEO is a publicly available functional genomic resource that comprises information from chips, microarrays, and high-throughput gene expression investigations. Two microarray datasets (GSE26168 and GSE78721) were obtained from the NCBI database called the GEO database (. P 0.05 –21.https://www.ebi.ac.uk/gwas/) was used to explore the DEGs associated with T2D in SAP. The database is used to analyze SNP-trait correlations for observing DEGs and SNPs associated with different diseases. We have chosen 17 GWAS catalog datasets of T2D  among SAPs where, overlapping genes were omitted [The publicly available GWAS catalog database ( omitted .https://string-db.org/). After inputting the ID of the identified DEGs onto the STRING database, the species "Homo sapiens" was selected, and the high confidence (0.900) interaction score was set to create the PPI interaction network. Subsequently, Cytoscape software was used to visualize the PPI networks [http://apps.cytoscape.org/apps/mcode) and Cytohubba on Cytoscape (http://apps.cytoscape.org/apps/cytohubba) plugins to determine the most critical subnetwork modules. Cytohubba follows topological algorithms to visualize protein associations. MCODE gives clusters of sub-networks. We have set Node Score Cutoff = 0.2, Degree Cutoff = 2, and K-Score = 2 during analysis in MCODE [The PPI of the translated proteins of identified DEGs was constructed using the publicly available STRING database (networks –25. Thenin MCODE .https://toppgene.cchmc.org/enrichment.jsp) to conduct the functional enrichment of DEGs. We have used the default settings of the ToppGene suite portal with the p-value of 0.05 with corrected an FDR value. In the default setting, Correction value = FDR, p-Value cutoff score = 0.05, Gene Limits 1< = n< = 2000 were maintained. Then, the top ten significant roles in cellular components, biological processes, and molecular functions were presented [The GO analysis was done using the ToppFun tool of ToppGene  database was used to analyze the KEGG pathways enrichment, setting the FDR  value of 0.05 as the cutoff value [Homo sapiens was the reference genome during the KEGG pathway enrichment analysis, where Gene Symbol ID was used as the gene ID [The publicly available Web-Gestalt (WEB-based Gene SeT AnaLysis Toolkit)  database was used to predict the regulatory network of the identified DEGs-miRNAs associated with T2D. In the database, the genes option was selected and gene IDs were inputted to identify DEGs-miRNA association for T2D following the default setting of Homo sapiens species. The regulatory network of DEGs-miRNA was developed and visualized using Cytoscape. The top 30 degrees of the node was chosen to visualize the DEGs-miRNAs regulatory network [The publicly available miRNet  was used to analyze disease associations of the DEGs following the default setting of the Homo sapiens genome. Web-Gestalt database uses Benjamini and Hochberg approach and a hypergeometric statistical test to determining the false discovery rate. The top ten most significant disease associations were presented [The Web-Gestalt database (resented .https://www.dgidb.org/). The candidate drug-gene interaction pairs were obtained using known and FDA-approved drugs. Following that, Cytoscape software was used to analyze and visualize the drug-gene interaction network [The interaction networks of candidate drug-gene were predicted using the publicly available DGIdb database (Drug Gene Interaction Database)  were analyzed. Here, we have found 221 DEGs in the GSE26168 (GPL6883) dataset (The CDC42 gene showed both up and down-regulated), including 95 upregulated genes and 127 downregulated genes. In the GSE78721 (GPL15207) dataset, 28 DEGs were found, where 26 genes were upregulated and two were downregulated. In the Venn diagram, no overlapped gene was observed in these two datasets. Finally, 249 DEGs were detected in the selected GEO datasets .In 17 GWAS catalog datasets, 1378 DEGs were found to be associated with T2D in SAP. All the DEGs found in the 17 GWAS catalog datasets are as follows: 76 DEGs in GCST002352 datasets, 7 DEGs in GCST001213 datasets, 24 DEGs in GCST008833 datasets, 111 DEGs in GCST004894 datasets, 18 DEGs in GCST001033 datasets, 7 DEGs in GCST001759 datasets, 12 DEGs in GCST001809 datasets, 33 DEGs in GCST005414 datasets, 695 DEGs in GCST010557 datasets, 107 DEGs in GCST010553 datasets, 36 DEGs in GCST007515 datasets, 34 DEGs in GCST007516 datasets, 174 DEGs in GCST006867 datasets, 15 DEGs in GCST010272 datasets, 12 DEGs in GCST011337 datasets, 14 DEGs in GCST011329 datasets and 3 DEGs in GCST011321 datasets. After omitting overlapped genes, 843 unique genes were found to be associated with T2D pathogenesis in SAP .HNF1A, CTNNB1, PSMC2, PSMA3, RUNX2, TCF7L2, TLE1, PSMD6, and CTBP1) were identified as hub genes following MCODE analysis  is a framework to classify how gene plays a role in molecular functions, biological processes, and cellular components. GO analysis of all 867 potential genes was performed using the ToppGene database. The top 10 significant molecular activities, biological processes, and cellular components were selected . ChangesThe analysis of KEGG pathway enrichment based on the highest enrichment ratio and an FDR of < 0.05 revealed that DEGs regulate several metabolic pathways . In the The regulatory networks found that 825 DEGs were interrelated with 2582 miRNAs. Indeed, several miRNAs regulate the expression of a single gene . As obseThe disease association analysis was performed to identify the diseases associated with identified DEGs. The results revealed that gestational diabetes, T2D, obesity, hyperglycemia, endocrine system diseases, endocrine disorder NOS (Not Otherwise Specified), endocrine disturbance NOS, and nutritional and metabolic diseases are associated with identified DEGs . Among 8Research on drug-gene interaction networks is crucial for drug discovery and development. Here, the networks of candidate drug-gene were built based on the interactions and effects of the medications. The candidate drug-gene interactions were constructed using 867 DEGs obtained from the PPI network, that may guide to explore the mechanism for treating T2D . In drugComprehensive analysis of the microarray dataset guides expression patterns of DEGs and their integrative biological functions under different conditions in living organisms . To idenHere, we have screened 1085 DEGs from microarray (GEO) and GWAS catalog datasets after removing overlapped genes. Microarray dataset analysis revealed that 249 genes were associated with T2D pathogenesis, where 121 were upregulated and 128 downregulated . More spIn the PPI network, we have identified 867 candidate genes that are significantly associated with T2D pathogenesis. In addition, we have found nine hub genes among the 867 candidate genes for T2D pathogenesis. Among the nine hub genes, two genes  were only found in the South Asian population. However, the remaining seven hub genes  were observed in the South Asian population in addition to other major populations .Further, the integrated results of module selection  and diseThe results of GO analysis demonstrated that most biological processes were linked to regulating RNA and DNA-based metabolic and biosynthetic processes. Furthermore, most cellular components were found to be linked with granule and vesicle lumen activities. The result is consistent with our previous research except for differences in population group . By bindDEGs are observed in the secretory-granule lumen, vesicle lumen, and platelet alpha granule lumen tissues in T2D . InsulinMODY is a genetic heritable diabetes characterized by beta-cell dysfunction, non-insulin-dependent diabetes (NIDD), and autosomal dominant inheritance at a young age . It is amiRNA plays a regulatory role in disease progression through epigenetic modification, histone modification, and DNA methylation. miRNAs are also associated with the diagnosis and response for the treatment of diseases . miRNAs Furthermore, 367 FDA-approved drugs for T2D significantly downregulated the candidate genes in drug-gene association analysis . D-phenySince genetic signatures play vital roles in T2D pathogenesis, a series of bioinformatic systems were applied to analyze 2 GEO and 17 GWAS catalogue datasets from SAPs to explore the DEGs associated with the diseases. Following critical PPI analysis, 867 DEGs were found to be associated with T2D pathogenesis. Indeed, nine hub genes were also identified for the pathogenesis. Among these, CTNNB1, and RUNX2 could be the markers for T2D pathogenesis in SAPs as only found in that populations. In GO analysis, most of the identified DEGs showed significant contributions in molecular activities, biological processes, and cellular components of T2D. Following KEGG pathway analysis, MODY, EGFR tyrosine kinase inhibitor resistance, insulin secretion, HIF-1, and PI3K-Akt signaling pathways were found to be significantly enriched by the DEGs. Two genes (HNF1A and TCF7L2) among 118 identified genes that significantly contributed to T2D, were found in both hub genes and disease association. Even, 825 DEGs were also interrelated with 2582 miRNAs. Not surprisingly, several miRNAs regulate the expression of a single gene or vice versa . Among the 64 genes that interacted with 367 drugs of T2D, ABCC8, ACE, ACHE, ADRB1, ADRB3, BRAF, HTT, INSR, KCNJ11, PDE3A, and PPARG genes were downregulated by wide range of (9–43) FDA approved drugs for T2D. Indeed, different FDA-approved drugs significantly downregulated the expression of target genes. Therefore, the findings of the research might guide to explore the mechanism of how the DEGs progress T2D pathogenesis by interacting different biological functions, pathways, and miRNAs. Considering the above-mentioned findings, precise medication could be recommended after diagnosing the molecular mechanism of T2D pathogenesis and observing the expression levels of marker genes in T2D patients among SAPs.S1 FigThe pathway is based on the FDR value and enrichment ratio.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Orally administered small-molecule drugs including tyrosine kinase 2 (TYK2) inhibitors and phosphodiesterase 4 (PDE4) inhibitors are new candidates for systemic therapy in plaque psoriasis. However, no previous articles evaluated the benefit and risk profile of TYK2 and PDE4 inhibitors in psoriasis.The objective of this study was to compare the efficacy and safety of oral small-molecule drugs, including TYK2 and PDE4 inhibitors, in treating moderate-to-severe plaque psoriasis.PubMed, Embase, and Cochrane library were searched for eligible randomized clinical trials (RCTs). Response rates for a 75% reduction from baseline in Psoriasis Area and Severity Index (PASI-75) and Physician’s Global Assessment score of 0 or 1 (PGA 0/1) were used for efficacy assessment. Safety was evaluated with the incidence of adverse events (AEs). A Bayesian multiple treatment network meta-analysis (NMA) was performed.In total, 13 RCTs (five for TYK2 inhibitors and eight for PDE4 inhibitors) involving 5274 patients were included. The study found that deucravacitinib at any dose (except for 3 mg QOD), ropsacitinib (200 and 400 mg QD), and apremilast (20 and 30 mg BID) had higher PASI and PGA response rates than placebo. In addition, deucravacitinib , and ropsacitinib (400 mg QD) showed superior efficacy than apremilast (30 mg BID). In terms of safety, deucravacitinib or ropsacitinib at any dose did not lead to a higher incidence of AEs than apremilast (30 mg BID). The ranking analysis of efficacy revealed that deucravacitinib 12 mg QD and deucravacitinib 3 mg BID had the highest chance of being the most effective oral treatment, followed by deucravacitinib 6 mg BID and ropsacitinib 400 mg QD.Oral TYK2 inhibitors demonstrated satisfactory performance in treating psoriasis, surpassing apremilast at certain doses. More large-scale, long-term studies focusing on novel TYK2 inhibitors are needed.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022384859, identifier CRD42022384859.PROSPERO (ID: CRD42022384859), available from:  Psoriasis is a chronic, systemic, immune-mediated, inflammatory disorder of skin affecting 2-3% of the global population . CharactVarious inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-12, interleukin (IL)-17, IL-22, and IL-23 and interferon (IFN)-γ, contribute to the immunopathogenesis of psoriasis , 6. TreaCurrently, oral small molecules available for psoriasis treatment include phosphodiesterase 4 (PDE4) inhibitors and Janus kinase (JAK) inhibitors. PDE4 and JAK are two important classes of molecules involved in the inflammatory process of psoriasis, and are viewed as viable targets for psoriasis therapy –17. ApreSystemic therapy is an important treatment for psoriasis. Though the molecular mechanisms underlying treatment efficacy are distinguished, oral small-molecule PDE4 and TYK2 inhibitors provide similar advantages in terms of patient convenience, reduced healthcare costs, and improved quality of life . No prevThis systematic review and network meta-analysis was reported following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) for RCTs. Additionally, the study protocol was registered at PROSPERO (ID: CRD42022384859).A systematic search was conducted by two independent investigators (Y.X. and Z.L.) across the PubMed, Embase, and Cochrane library databases until May 10, 2023. The detailed search strategies used by the investigators across different databases are displayed in Studies were considered eligible for inclusion if they met the following criteria: (1) studies that enrolled patients with moderate-to-severe plaque psoriasis; (2) studies that aimed to compare the efficacy or safety profile of any type of oral TYK2 inhibitors or PDE4 inhibitors to placebo; (3) studies that recorded efficacy outcomes including Psoriasis Area and Severity Index (PASI) scores or Physician’s Global Assessment (PGA) scores or safety outcomes including the incidence of adverse events (AEs), with corresponding time points; (4) RCTs. Exclusion criteria were: (1) studies that did not report efficacy outcomes of PASI scores or safety outcomes; (2) studies of observational studies or other types of clinical trials, and studies without complete original data, such as editorials, comments, reviews, protocols, and conference presentations; (3) studies of repetitive publications from the same study group.Two investigators (Y.X. and Z.L.) independently conducted the data extraction process and cross-checked the extracted data. The information extracted as baseline characteristics included: study name, study design, type of intervention, the number of patients, sex, age, BMI, disease duration, treatment duration, and outcome measure. In this study, the primary endpoint for efficacy was 75% improvement in the Psoriasis Area and Severity Index (PASI-75) from baseline. The Physician’s Global Assessment achieving “clear” or “almost clear” (PGA 0/1) was also analyzed for efficacy assessment. The incidence of AEs was assessed for safety. Therefore, the number of patients who achieved PASI-75 and PGA 0/1 at the end of the trial and the number of AEs were extracted. Any discrepancies between the investigators during the process of data extraction were resolved through discussions with a senior investigator (L.G.).All statistical analyses were conducted using the GeMTC package in R software (version 4.0.3). We performed a Bayesian multiple treatment network meta-analysis (NMA) with fixed effects and non-informative priors. An initial burn-in of 20,000 simulations was executed, followed by an additional 50,000 simulations to generate the outputs in each analysis. The convergence of simulations was confirmed with the potential scale reduction factor (PSRF) and Gelman-Rubin-Brooks plots . Fixed-eThe systematic search across multiple databases yielded 890 records, of which 775 articles remained after removing duplicates. Screening of titles and abstracts led to the exclusion of 750 articles, leaving 25 records for full-text review. Of these, 13 were considered eligible for the final analysis, comprising eight articles on oral PDE4 inhibitors –33, and The Cochrane Collaboration’s tool for assessing the risk of bias in randomized trials was used to evaluate the quality of the studies. As a result, all of the trials included were deemed to have an acceptable risk of bias and were qualified for further analysis. More details on the quality assessment can be found in Except for one study , the othThe result of the NMA revealed that, in comparison to placebo, the low-dose groups of oral TYK2 and PDE4 inhibitors did not exhibit superior efficacy. However, when administered at sufficient dosages, the oral TYK2 and PDE4 inhibitors displayed significantly greater PASI-75 response rates than placebo. Notably, apremilast 20 or 30 mg BID, ropsacitinib 200 or 400 mg QD, and deucravacitinib at all dosages except for 3 mg QOD exhibited significantly greater efficacy than placebo. Moreover, when compared to apremilast 30 mg BID, the NMA demonstrated that oral TYK2 inhibitors, specifically deucravacitinib 3 mg BID, deucravacitinib 6 mg QD, deucravacitinib 6 mg BID, deucravacitinib 12 mg QD, and ropsacitinib 400 mg QD, displayed significantly higher efficacy, as shown in the forest plot . This was followed by deucravacitinib 3 mg BID , deucravacitinib 6 mg BID , and ropsacitinib 400 mg QD   and deucravacitinib 3 mg BID  had the highest chance of being the most effective oral treatment options, which were followed by deucravacitinib 6 mg BID  and ropsacitinib 400 mg QD  , followed by deucravacitinib 3 mg QD  and ropsacitinib 50 mg QD   and PGA 0/1 (53.6% vs. 32.1%) response rate at week 16 , 36. OurOral small-molecule PDE4 and TYK2 inhibitors appear effective and safe in treating psoriasis, but they work through different mechanisms of action. PDE4 is a major class of enzyme capable of hydrolyzing cyclic adenosine monophosphate (cAMP), a key “secondary messenger” that can modulate the cellular immune response by regulating the network of pro-inflammatory and anti-inflammatory cytokines . PDE4 inOn the other hand, JAKs, comprising JAK1, JAK2, JAK3, and TYK2, function as important signal transducers in intracellular signaling pathways stimulated by cytokines . The binThis study has several limitations. First, a relatively small number of patients using ropsacitinib were available for our analysis, leading to wider confidence intervals for the corresponding results. In addition, a clinical trial on brepocitinib was excluded from the analysis due to differences in study design and a small number of participants, resulting in an inability to compare the efficacy and safety profile of brepocitinib with other TYK2 and PDE4 inhibitors . TherefoIn conclusion, this NMA has validated the potential of orally administered small-molecule drugs, including PDE4 and TYK2 inhibitors, in the treatment of moderate-to-severe plaque psoriasis. Oral TYK2 inhibitors have displayed promising efficacy and safety profiles, surpassing oral PDE4 inhibitors at certain doses. Specifically, deucravacitinib at various doses  and ropsacitinib (400 mg QD) were more effective than apremilast (30 mg BID) in treating plaque psoriasis, with no increased risk of AEs reported at any dosage. Therefore, TYK2 inhibitors have demonstrated satisfactory performance in treating plaque psoriasis, warranting further development and more long-term, large-scale clinical trials for novel TYK2 inhibitors.The original contributions presented in the study are included in the article and All authors contributed to the article and approved the submitted version. YX and ZL searched the literature and extracted the data. YX and SW performed the statistical analyses. YX wrote the article. LG and XJ reviewed and revised this article.


Lung cancer is the most prevalent type of cancer worldwide, with 2.21 million cases and 1.80 million fatalities in 2020. The mainfactor influencing lung cancer is smoking, and the most common form of lung cancer, non-small cell lung cancer (NSCLC), accounts foraround 80% of instances compared to small cell carcinoma, and about 75% of patients are already in an advanced stage when they aredetected. Despite significant early detection and therapy improvements, the five-year survival rate for NSCLC is not encouraging.Therefore, it is essential to look into the molecular origins of non-small cell lung cancer to develop more effective therapeuticstrategies-the binding affinities and energy landscape with the proteins. Cyclin Dependent kinase 2 (CDK2) and Insulin-like GrowthFactor 1 Receptor (IGF1) were more substantial and sustained in lung cancer that was chosen as the two primary target proteins in this.We screened the entire Drug Bank-prepared library of 1,55,888 compounds and found -7-(Methylsulfonyl)-3--1,2,3,4-tetrahydropyrido benzimidazol-2-aminium (Mefluhybenamine) to be a significant inhibitor. Mefluhybenamine showed stronghydrogen bonding and other bonding topologies, such as van der Waals force, in its high docking scores of -6.168 Kcal/mol and -5.26Kcal/mol, and ADMET results showed excellent bioavailability, remarkable solubility, no side effects, and toxicity. The moleculardynamicsimulation confirmed the compound's stability and interaction pattern for 100 ns in an SPC water medium with the slightestdeviation and fluctuation. Data shows that Mefluhybenamine is a potential candidate. However, validation of the compound is essential. One of the deadliest cancer types is lung cancer, partly because the disease often manifests at an advanced stage and becausea significant portion of its growth can occur without any symptoms ,2. Lung Non-small cell lung cancer- More than 87 percent of lung cancer occurrences are non-small-cell lung cancer, making it the mostprevalent type. The three possible forms are squamous cell carcinoma, adenocarcinoma, and large-cell carcinoma, 6.Small cell lung cancer- A less frequent type of lung cancer called small-cell lung cancer typically spreads more quickly thannon-small-cell lung cancer , 8.Lung cancer indicators are not commonly available in the advanced stages. Early symptoms include expected back discomfort andwarning indications like shortness of breath. Back discomfort might develop when tumours impinge on the patient's lungs, spread tothe spinal cord, or affect the ribs. Lung cancer's early signs can be challenging to detect, but the earlier it shows some diagnosisfeatures. However, a better chance of treatment options is available if it is identified in its earlier stage,11,12. We considered two proteins, Cyclin-Dependent kinase 2 (CDK2) with PDBID: 1AQ1 and Insulin-like Growth Factor 1 Receptor (IGF1) withPDBID: 1K3A, which show primary indicators and play a role in the pathogenesis of lung cancer ,26.ProtDrug Bank is a vast online database with free access to information on drug candidates. We downloaded the complete Drug Banklibrary and used the LigPrep tool in maestro to prepare the complete library ,33. TheThe prepared proteins were used for the grid generation using the Glid Grid generator tool, which was generated around the completeprotein to perform the blind docking to find the optimal position for the ligand. The Virtual Screening Workflow (VSW) tool was usedto do the screening. The prepared library was browsed and verified in VSW before QikProp was used to generate the ADMET attributesthat had been Lipinski's Rule prefiltered , 35. TheBiologists frequently utilise the approach known as molecular dynamics (MD) to simulate the physical stability and flexibility ofatoms and molecules in order to assess the complexity and stability of any protein or protein-ligand complex in various solute mediumsand temperatures. The Desmond package, considered the MD algorithm's quickest and most accurate calculation, was used to run the MDsimulation . The 3D Computerised criteria are defined for each candidate that is analysed and categorised in the event of a docking score, and throughthe docking parameters, we have identified a compound name Mefluhybenamine against Lung Cancer. We have also demonstrated extensiveanalyse of the interactions between ligands and proteins. Mefluhybenamine with Cyclin-Dependent Protein Kinase 2 (CDK2) also PDBID:1AQ1 has been shown to have a docking score of -6.168 Kcal/mol and MM\GBSA score of -37.7 Kcal/mol (Table 1-see PDF), and it was fitto inside of the pocket  while interacting through the hydrogen bond with LYS129 residue with O atoms. Anotherhydrogen bond also formed among ASP86 residue with NH3+ atom, and one salt bridge also formed among the interaction of ASP86 withNH3+ atom of the ligand . While in the case of Insulin-like Growth Factor 1 Receptor (IGF1) in complex with theligand Mefluhybenamine (PDBID: 1K3A), has induced a docking score of -5.26 Kcal/mol and MM\GBSA score of -38.65 Kcal/mol(Table 1-see PDF) when fitted inside the binding pocket  that interacts through the hydrogen bonding formed amongthe THR1053 residue and NH3+ atom of the ligand . Other than the docking and MM\GBSA scores, more interaction scoressuch as Prime Hydrogen bonds, Prime Vander wall's forces, ligand efficiency sa and ligand efficiency are providedin Table 1(see PDF).The standard values against the descriptor are given while comparing the computed values of the ligand Mefluhybenamine with theQikProp tool for fully comprehending the ADMET. All the calculated values of the ligand satisfy all criteria and indicate that theMefluhybenamine compound can be used against Lung Cancer (Table 2-see PDF). Although it also shows no significant side effects andtoxicity, experimental validation in the in-vitro condition is essential before moving to the in-vivo or human use.Molecular Dynamics (MD) simulation is a computation tool for analysing the molecular and atomic motions and physical propertiesof ligand-protein complex interactions and stability. In the present study, we considered MD simulations of complexes kept at 100nstime duration with 1000 recordings of each complex individually in the SPC water model to analyse the Root Mean Square Deviation(RMSD), Root Mean Square Fluctuation (RMSF) and Simulative Interaction Diagram (SID). Further, the detailed analysis is as follows:Root Mean Square Deviation (RMSD) is commonly used to calculate the statistical information that reveals the strength of the bondsbetween proteins and ligands. RMSD provides the molecular level deviation against time duration in a nanosecond with Angstrom valueÅ. The Cyclin-Dependent Protein Kinase 2 (1AQ1) protein with Mefluhybenamine has initially deviated to 1.13 Å, and theligand shows deviation to 0.48 Å at 0.10 ns, while at 100 ns, the protein CDK2 shown deviation at 2.28 Å andMefluhybenamine showed deviation at 26.27 Å. If the initial deviation is ignored, the complete RMSD of protein and ligand wastrustable and acceptable . For the complete 100ns, protein has shown deviations below 2 Å. However, theligand has shown deviation to some extent. The most deviation was during 40ns-60ns, and after the 60ns ligand also stabilised andshowed a deviation of less than 2 Å. From the above points, it can be stated that the complex is stable, and it is suggestedto take the analysis in in-vitro conditions. The Insulin-like Growth Factor 1 Receptor (1K3A) protein has an initial deviation of1.25 Å, and ligand Mefluhybenamine provides a deviation of 1.27 Å at 0.10 ns. At 100 ns, protein deviated at 2.93 Åwhile ligand deviated at 7. Å. The whole complex was initially and then stabilised. However, after ignoring the initial deviationstage, the complex was satisfied . The protein and ligand both deviated till 10ns and then showed stable performance. Thecomparative understanding suggested that the complex of Insulin-like Growth Factor-1 (1K3A) with Mefluhybenamine has shown more stableperformance during simulative analysis, while the Cyclin-Dependent Protein Kinase-2 (1AQ1) with ligand Mefluhybenamine has showndeviation till some extent.Root Mean Square Fluctuation (RMSF) plot shows the protein  and ligand fluctuation at atomic and residue index. In theplot, the protein  is shown in blue, whereas the ligand is green against the residue index. The protein Cyclin-DependentProtein Kinase-2 (1AQ1) with ligand Mefluhybenamine has achieved very steadily, not much fluctuation, and only a few residues showfluctuation that gone beyond 2 Å are LEU25, THR26, PHE152, GLY153, VAL154, PRO155, VAL156, THR158, TYR159, THR160, HIS161,GLU162, VAL164, VAL289, LEU296, ARG287, LEU298, NMA298 . The Insulin-like Growth Factor-1 Receptor (1K3A) with ligandMefluhybenamine was noticed that a few fluctuations residues are VAL958, PRO959, ASP960, GLU961, GLY976, GLN977, GLY978, SER979,PHE980, GLY981, GLY990, VAL991, VAL992, LYS993, ASP994, GLU995, PRO996, GLU997, GLU1007, ALA1008, ALA1009, SER1010, MET1011, ARG1012,GLU1013, ILE1015, GLU1016, GLY1042, PRO1044, GLU1067, MET1068, GLU1069, ASN1070, ASN1071, PRO1072, VAL1073, LEU1074, ALA1075, PRO1076,GLY1139, GLY1140, LYS1141, GLY1142, LEU1144, GLY1185, SER1187, ASN1188, LYS1256, NMA1256 . When analysing the whole RMSFplot, the protein was steady, and it gives many interactions with the ligand Mefluhybenamine, accepted that ligand Mefluhybenamine hasfit and stable that entering into the pocket to stop the action and activity of the protein. The figure's green colour shows the ligandcontacts during the simulative period. While comparing both conditions, Mefluhybenamine has shown many interactions with 1AQ1,dispersed sufficiently from initial to end residues.The SID or the Simulative Interaction Diagram proves that the interaction between ligand and protein during the simulation periodswas much better with web-like formations. At the same time, to understand the bonding types and dynamic angles of residues, we haveextracted the interaction count for both conditions and plotted it in figure 4. The complex of protein Cyclin-Dependent ProteinKinase-2 (1AQ1) with ligand Mefluhybenamine gives enormous interaction bonding by Pi-Pi stacking with benzene ring among PHE4 residue,and hydrogen bonds interact with ASP145, ASN23 residues with N atom, hydrogen bonds also interact with ASP86, GLN131 residues withNH3+ atom of the ligand Mefluhybenamine . The Insulin-like Growth Factor 1 Receptor (1K3A) with ligandMefluhybenamine interacts with hydrogen bonding among ASP1056 with NH3+ atom; hydrogen bond also interacts with MET1052 with O atomof the ligand Mefluhybenamine .Lung diseases are widespread nowadays, and lung cancer is among the most dangerous. While compared to other cancers, lung cancerremains at the top and is on the world health organisation's top priority list to find a better cure. In this study, we havescreened a huge library and identified a drug compound name -7-(Methylsulfonyl)-3--1,2,3,4-tetrahydropyrido benzimidazol-2-aminium (Mefluhybenamine) that is showing an excellent binding score with two main proteins participatingin the case of lung cancers are Insulin-like Growth Factor-1 and Cyclin-Dependent Protein Kinase-2. Also, the complexes have shown goodMM\GBSA scores and ligand efficiency during the ADMET analysis. Further, the simulation extended the analysis for validation, and theresults exceeded expectations. From the above analysis, it can be concluded that the identified drug can work for lung cancer again.However, validation is needed before any prescription.


Furthermore, the multivariate logistic analyses showed that bivalirudin (vs heparin) was independently correlated with lower risk of NACEs , BARC 2-5 bleeding events , and BARC 3-5 bleeding events ; other independent risk factors for NACEs, MACCEs, or BARC bleeding events included history of diabetes mellitus, emergency operation, multiple lesional vessels, stent length >33.0 mm, and higher CRUSADE score . Thus, bivalirudin presented a better safety profile than heparin among Chinese STEMI patients undergoing PCI.Although bivalirudin has been recently made available for purchase in China, large-scale analyses on the safety profile of bivalirudin among Chinese patients is lacking. Thus, this study aimed to compare the safety profile of bivalirudin and heparin as anticoagulants in Chinese ST-segment elevation myocardial infarction (STEMI) patients undergoing percutaneous coronary intervention (PCI). A total of 1063 STEMI patients undergoing PCI and receiving bivalirudin  or heparin  as anticoagulants were retrospectively enrolled. The net adverse clinical events (NACEs) within 30 days after PCI were recorded, including major adverse cardiac and cerebral events (MACCEs) and bleeding events (bleeding academic research consortium (BARC) grades 2-5 (BARC 2-5)). The incidences of NACEs (10.1  Cardiovascular diseases are the leading cause of death globally, taking the lives of an estimated 17.9 million people ,3. So faBivalirudin is an oligopeptide anticoagulant with several advantages, including direct inhibition of thrombin, rapid onset, short half-life, good safety profile, etc. ,13. SeveThe current study aimed to explore the incidence of and risk factors for total NACEs, major adverse cardiac and cerebral events (MACCEs), and bleeding events of bivalirudin and heparin as anticoagulants in 1063 Chinese STEMI patients receiving PCI.This retrospective cohort study included 1063 STEMI patients who were treated with PCI and received bivalirudin or heparin as anticoagulants in HanDan Central Hospital (China) between December 2017 and February 2022. The screening criteria were: a) diagnosed with STEMI according to the European Society of Cardiology Guidelines ; b) overPatients received bivalirudin or heparin (unfractionated heparin or low molecular weight heparin) as anticoagulants based on the current disease status, physician advice, and patient willingness. GPIs were administrated if needed. Patients who received bivalirudin were considered as the bivalirudin group (n=424) and patients who received heparin were considered as the heparin group (n=639). The regimens of bivalirudin and heparin were in accordance with a previous study . In the Data of STEMI patients were obtained, which included demographic characteristics, medical history, disease characteristics, and treatment information. In addition, NACEs within 30 days after PCI were recorded, which included MACCEs and bleeding events (bleeding academic research consortium (BARC) grades 2-5 (BARC 2-5)) ,21. MACCt-test, chi-squared test, or Wilcoxon rank sum test. Independent factors for NACEs, MACCEs, BARC 2-5 bleeding events, or BARC 3-5 bleeding events were assessed by forward stepwise multivariate logistic regression analysis with all parameters included. P<0.05 was considered significant.Statistics were performed using SPSS v 22.0 , and figures were designed by GraphPad Prism v 6.1 . Differences between the bivalirudin group and the heparin group were analyzed by Student's In the heparin group, the mean age of patients was 64.4±12.0 years, and there were 179 (28%) females and 460 (72%) males. In the bivalirudin group, the mean age of patients was 63.3±11.3 years, and there were 108 (25.5%) and 316 (74.5%) females and males, respectively. Moreover, no differences were found between groups in demographic characteristics, medical history, disease characteristics, and treatment information (except GPIs); the number of patients in the heparin group receiving GPIs (431 (67.4%)) was higher than those in the bivalirudin group  .vs 15.6%) (P=0.010) (vs 6.4%) (P=0.131) (vs 10.3%) (P=0.003) (vs 5.5%) (P=0.007)  . HoweverP=0.131) . FurtherP=0.003) . A lowerP=0.007) . To furtP=0.007) .vs heparin) as treatment had a lower risk of NACEs (odds ratio (OR)=0.508, P=0.002), while history of diabetes mellitus (vs no) , emergency operation (vs elective operation) , multiple (vs single) lesional vessels , and stent length >33.0 mm (vs ≤33.0 mm)  were associated with a higher risk of NACEs  was the only factor independently correlated with a higher number of MACCEs .A history of diabetes mellitus (vs heparin) as treatment  was related to a lower risk of BARC 2-5 bleeding events, while history of diabetes mellitus (vs no) , emergency operation (vs elective operation) , multiple (vs single) lesional vessels , and stent length >33.0 mm (vs ≤33.0 mm)  were associated with an increased risk of BARC 2-5 bleeding events  as treatment  was associated with a lower risk of BARC 3-5 bleeding events, while a higher CRUSADE score  and stent length >33.0 mm (vs ≤33.0 mm)  were associated with a higher risk of BARC 3-5 bleeding events  was independently associated with lower risk of NACE, BARC 2-5 bleeding events, and BARC 3-5 bleeding events. This study indicated that bivalirudin presented a better safety profile than heparin among Chinese STEMI patients undergoing PCI.The current study found that: 1) the incidences of NACE, BARC 2-5 bleeding events, and BARC 3-5 bleeding events were lower in the bivalirudin group compared to the heparin group; 2) bivalirudin , followed by heparin (7.6%) and heparin plus GPI (9.8%) among STEMI patients undergoing PCI . MoreoveThe findings of the current study might be due to the fact that heparin indirectly suppresses the activity of thrombin while bivalirudin is a direct thrombin inhibitor. In addition, bivalirudin reduced platelet activity compared to heparin ,13,24, svs heparin) was associated with a lower risk of NACEs, BARC 2-5 bleeding events, and BARC 3-5 bleeding events; other factors associated with NACEs, MACCEs, or BRAC bleeding events included history of diabetes mellitus, emergency operation, multiple lesional vessels, stent length >33.0 mm, and higher CRUSADE score, which was partly in line with the previous studies  bivalirudin as an anticoagulant during PCI might reduce the risk of severe bleeding complications in STEMI patients, which is especially crucial during high-risk procedures like PCI; 2) bivalirudin was associated with fewer NACEs compared to heparin. The lower rate of NACEs indicated that using bivalirudin might result in better overall patient outcomes; 3) for STEMI patients undergoing PCI who were at a higher risk of complications, such as those with a history of bleeding or other medical conditions that increase the risk of adverse events, bivalirudin might be a suitable anticoagulant option. Evaluating clinical events within a specific time frame such as 30 days ensured safety, compliance with regulations, proper maintenance, cost-effectiveness, environmental care, quality control, and adherence to insurance requirements. BARC 2-5 bleeding events included any overt sign of hemorrhage, while BARC 3-5 bleeding events included overt bleeding plus a hemoglobin drop of 3 to 5 g/dL or intracranial hemorrhage (severe bleeding) .Several limitations in the current study should not be ignored: 1) although this was a large-scale research, the low incidence of NACEs, MACCEs, and bleeding events led to low statistical power; 2) the retrospective and single-center nature of the study; 3) the short follow-up; 4) the different number of patients in the bivalirudin and heparin groups.In conclusion, bivalirudin exhibited a better tolerance compared to heparin among Chinese STEMI patients undergoing PCI.


FGFR2) fusion, in addition to less frequently observed targets such as BRAF V600E, deficient mismatch repair/high microsatellite instability (dMMR/MSI-H), and high tumor mutation burden (TMB-H). These targets are observed in varying frequency among patients with intrahepatic CCA and extrahepatic CCA. Multiple novel therapies have been developed to exploit each of these targets, with some having received United States Food and Drug Administration approval for use in the second-line setting. In the current review, we discuss targets of interest in CCA and summarize current evidence evaluating available therapies directed at these targets.Primary tumor resection and liver transplantation are the only curative treatment options for the management of cholangiocarcinoma (CCA). However, for patients with advanced or metastatic disease, palliative systemic therapy remains the only treatment option. The development of targeted therapeutics has begun to shift the treatment paradigm in CCA. Targets of interest in CCA include mutated isocitrate dehydrogenase-1 (mIDH-1), human epidermal growth factor receptor 2 (HER2) overexpression/amplification, and fibroblast growth factor receptor 2 ( Cholangiocarcinoma (CCA) is a malignancy of the biliary epithelium that accounts for approximately 3% of gastrointestinal malignancies globally . CCA is p = 0.001] and median overall survival (mOS)  [Previously, standard first-line systemic therapy for advanced CCA consisted of gemcitabine plus cisplatin based on the ABC-02 trial that demonstrated survival benefit with the combination regimen compared to gemcitabine monotherapy, and this remained the standard of care for over a decade . More rep = 0.0034). This regimen has not yet received U.S. Food and Drug Administration (FDA) approval but represents an alternative first-line option for advanced CCA. More recently, KEYNOTE-966, which was a randomized, double-blind, placebo-controlled, phase 3 trial, evaluated patients with advanced, previously untreated biliary tract cancers who were randomized to receive gemcitabine plus cisplatin with pembrolizumab, an anti-programmed cell death-1 (anti-PD-1) antibody, or placebo . Patientp = 0.031) [p = 0.004) [2 in NIFTY but was 80 mg/m2 in NALIRICC-AIO-HEP-0116. At this time, fluoropyrimidine plus irinotecan may be considered a second- or third-line option in CCA, but the efficacy of irinotecan-based regimens is not well established. Select trials establishing first- and second-line standards of care are listed in Following progression on first-line systemic therapy, second-line therapy may consist of folinic acid, fluorouracil, and oxaliplatin (FOLFOX), after the ABC-06 trial, which was an open-label, randomized, phase 3 trial consisting of patients with advanced biliary tract cancer who had progression on first-line gemcitabine plus cisplatin and were randomized to active symptom control with or without FOLFOX, found that patients in the FOLFOX arm exhibited longer mOS  . Alterna= 0.031) . The iri= 0.004) . However= 0.004) .The diffGiven the marginal difference in response rate and overall survival with different combinations of cytotoxic chemotherapy and immunotherapy in advanced CCA and the detection of actionable targets in nearly 50% of CCA cases, greater interest has been generated in targeted therapy for the management of CCA . This haIDH-1 mutation (mIDH-1) may result in a gain of function, facilitating the conversion of α-ketoglutarate to D-2-hydroxyglutarate, which is an oncometabolite [IDH-1 has been implicated in glioma and acute myeloid leukemia, as well as iCCA, where it may be detected in 13–25% of cases [Isocitrate dehydrogenase-1 (IDH-1) is a metabolic enzyme that functions to convert isocitrate to α-ketoglutarate, and mutations of this enzyme have been implicated in oncogenesis . Specifitabolite ,22. Accutabolite . Mutatioof cases ,24,25. Dof cases .Ivosidenib is a targeted inhibitor of mIDH-1 and was first used in the management of mIDH-1 acute myeloid leukemia . It was p < 0.0001). Final survival analysis failed to demonstrate a statistically significant improvement in survival, with an mOS of 10.3 months in the ivosidenib arm versus 7.5 months in the placebo arm  [p = 0.0008). In addition to the above-mentioned benefits, those receiving ivosidenib did not experience a decline in quality of life when compared to placebo. Based on the results of ClarIDHy, the U.S. FDA granted approval of ivosidenib for patients with advanced mIDH-1 CCA who had progressed on first-line therapy.This trial was subsequently followed by ClarIDHy, which was a multicenter, randomized, double-blind, placebo-controlled, phase 3 trial evaluating ivosidenib in 230 patients with mIDH-1 CCA who had progressed on at least one prior line of therapy . PatientHuman epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that, when activated, results in activation of the PI3K-AKT-mTOR pathway ,32. OverTrastuzumab is a monoclonal antibody that binds domain IV of HER2, resulting in antibody-dependent cell-mediated cytotoxicity, while pertuzumab, which has shown synergism when used with trastuzumab, is a monoclonal antibody that binds subdomain II of HER2, blocking dimerization and therefore activation of HER2 . HER2-diHERIZON-BTC-01 was a global, phase 2b trial evaluating zanidatimab, a monoclonal antibody binding HER2 at two domains, resulting in the formation of HER2-antibody clusters that are internalized, reducing HER2 expression, in patients with advanced, HER2-amplified biliary tract cancers with progression on gemcitabine-based therapy . Among 8DESTINY-PanTumor02 was an open-label, phase 2 trial evaluating trastuzumab deruxtecan, a topoisomerase inhibitor conjugated to trastuzumab, in patients with HER2-positive, previously treated solid malignancies, excluding breast, gastric, colorectal, and non-small-cell lung cancer . Among tFGFR2 fusion seen in CCA is most commonly due to chromosomal rearrangement causing a C-terminal substitution of FGFR2 to the region of a binding partner, allowing auto-dimerization of FGFR2 and constitutive activation of downstream signaling pathways [FGFR2 fusion is primarily seen in iCCA and rarely other cancers, including gallbladder carcinoma, hepatocellular carcinoma, pancreatic cancer, and sarcoma. In iCCA, frequency was initially reported in approximately 15% of cases, but in advanced iCCA, the frequency may be lower in 7–10% [FGFR2 alterations have been associated with improved survival in iCCA, even without the use of FGFR inhibitors [Fibroblast growth factor receptor 2 (FGFR2) is a member of a family of four receptor tyrosine kinases that, when activated by fibroblast growth factors, dimerize, leading to autophosphorylation of the intracellular kinase domain and activation of downstream signaling pathways ,43,44,45pathways ,49. FGFRin 7–10% ,51. FGFRhibitors ,53.FGFR2 is one of the best studied targets with regard to therapeutic options in CCA, and multiple FGFR inhibitors have received FDA approval for the indication of CCA, including pemigatinib, futibatinib, and infigratinib [gratinib .Pemigatinib is a selective inhibitor of FGFR1-3 and was the first FGFR inhibitor to receive FDA approval for treatment of CCA ,55. It wFGFR2 fusion or rearrangement, those with FGFR2 mutation, and those with no alteration of FGFR2 [FGFR2 fusion or rearrangement, 35.5% achieved an objective response, demonstrating clinical promise. Patients with FGFR2 fusion or rearrangement demonstrated an mPFS of 6.9 months (95% CI 6.2–9.6) and an mOS of 21.1 months (95% CI 14.8–not estimable). An additional phase 2 study evaluating pemigatinib in Chinese patients with advanced CCA who had progressed on prior therapy found an ORR of 50.0% [FGR2 fusion or rearrangement following progression on first-line therapy.This was followed by FIGHT-202, which was a multicenter, open-label, phase 2 trial enrolling 3 cohorts of patients with CCA who had progressed on prior treatment: those with of FGFR2 . Among pof 50.0% . Based oFGFR2 rearrangement [FGFR2 fusion or rearrangement.FIGHT-302 is currently ongoing, which is an open-label, randomized, active-control, phase 3 study evaluating first-line pemigatinib versus gemcitabine plus cisplatin in the treatment of CCA with angement . This stFGFR abnormality. The most frequently observed TEAEs included hyperphosphatemia (59%), diarrhea (37%), and constipation (34%), with 48% of patients experiencing grade 3 toxicity. Futibatinib was also evaluated in an additional phase 1 trial including 197 patients with advanced solid tumors, with an ORR of 25.4% in patients with FGFR2 fusion/rearrangement-positive iCCA [Futibatinib is an irreversible, pan-FGFR inhibitor . It was ive iCCA .FGFR2 fusion/rearrangement who had progressed on prior therapy [FGFR2 fusion/rearrangement-positive iCCA with progression on prior therapy. FOENIX-CCA3 was a planned phase 3 trial to evaluate futibatinib versus gemcitabine plus cisplatin in the first-line setting; however, this trial has been halted due to slow patient accrual [These were followed by FOENIX-CCA2, which was a multinational, open-label, phase 2 study including 103 patients with advanced iCCA with  therapy . An ORR  therapy . Based o accrual .FGFR alterations [Infigratinib is a pan-FGFR inhibitor that was first evaluated in a phase 1 dose escalation/expansion study that included 132 patients with solid malignancies with erations ,67. ThisFGFR2 fusion or alteration who had progressed on prior therapy [FGFR2 fusion, an ORR of 18.8% and a DCR of 83.3% were observed. The most commonly observed TEAEs included hyperphosphatemia (72.1%), fatigue (36.1%), stomatitis (29.5%), and alopecia (26.2%), with grade 3 or 4 TEAEs in 41%, including hyperphosphatemia (16.4%), stomatitis (6.6%), and palmar-plantar erythrodysesthesia (4.9%). An additional open-label, phase 2 study enrolling 122 patients with advanced CCA with FGFR2 fusion or rearrangement with progression on prior gemcitabine-based chemotherapy found an ORR of 23.1% (95% CI 15.6–32.2) [FGFR2 fusion or rearrangement. Despite this, its manufacturer will no longer pursue this clinical indication for infigratinib, and further trials have been discontinued.Infigratinib was subsequently evaluated in an open-label, phase 2 trial that enrolled patients with advanced CCA harboring an  therapy . An ORR .6–32.2) . Based oBRAF is a proto-oncogene that functions within the mitogen-activated protein kinase/extracellular signal-related kinase (MAPK/ERK) pathway and plays a role in cell proliferation [BRAF V600E is the most common mutation of BRAF, resulting in constitutive activation of this signaling pathway. BRAF V600E has been implicated in multiple malignancies, including melanoma, colorectal cancer, hairy cell leukemia, and multiple myeloma [BRAF V600E has been detected in 1–5% of cases of CCA and is primarily found in iCCA [BRAF V600E has prognostic implications in iCCA and is associated with shorter overall survival and disease-free survival [BRAF V600E to patients with non-V600E BRAF mutations, patients with BRAF V600E are more likely to have larger tumors, more synchronous tumors, and increased invasion of vessels or bile ducts [feration . BRAF V6 myeloma . BRAF V6 in iCCA ,74,75,76survival . Additiole ducts .BRAF V600E mutated solid malignancy, excluding patients with melanoma, thyroid, colorectal, and non-small-cell lung cancer [Dabrafenib, a selective BRAF inhibitor, and trametinib, a MEK1/2 inhibitor, were evaluated via subprotocol H (EAY131-H) of the NCI-MATCH platform trial, which was an open-label, single-arm study enrolling 35 patients with a g cancer . This stBRAF V600E-mutated, advanced biliary tract cancer with progression on prior therapy [BRAF V600E-mutated solid malignancies with progression on prior therapy.Dabrafenib plus trametinib was subsequently evaluated in ROAR, which was an open-label, single-arm, phase 2 basket study that included a biliary tract cancer cohort consisting of 43 patients with  therapy . The ORRMismatch repair proteins, including MSH2, MSH6, MLH1, and PMS2, function to correct errors that occur during DNA replication ,80. LossPembrolizumab was evaluated for use in non-colorectal, MSI-H/dMMR, advanced solid malignancies with progression on prior therapy in the phase 2 KEYNOTE-158 study . This stSimilarly, dostarlimab, an anti-PD1 antibody, has been evaluated in the phase 1 GARNET trial, which included 144 patients with dMMR or POLE mutated, non-endometrial solid tumors with progression on prior therapy . This stTumor mutation burden is the quantification of genetic mutations within a cell. This may be evaluated using next-generation sequencing of tumor DNA, and the presence of ≥10 mutations/megabase (mut/Mb) is frequently referred to as high tumor mutation burden (TMB-H) . A high A high tumor mutation burden is also a potential target in CCA. The previously mentioned KEYNOTE-158 trial was subsequently evaluated to determine the efficacy of pembrolizumab in patients with TMB-H advanced solid malignancies . An ORR CheckMate 848 was a phase 2 trial that enrolled 212 patients with advanced TMB-H (by tissue biopsy or blood-based assay) solid malignancies who were immunotherapy naïve and had progressed on prior therapy . PatientNTRK) is a family of three genes  that encode tropomyosin receptor kinases  that play a role in neuronal development [NTRK fusion, similar in mechanism to FGFR2 fusion, stems from chromosomal rearrangement, resulting in the replacement of the 3′ region of NTRK with a binding partner that allows for dimerization of TRK and activation of signaling in the absence of neurotrophins [NTRK fusion may be observed in <1% of solid malignancies, and has been reported in <1% of cases of CCA [Neurotrophic tyrosine receptor kinase , and all three manifested a response, suggesting promise in using entrectinib in the management of solid malignancies with NTRK1/2/3 fusion. Among all 119 phase 1 patients, the most common TRAEs included fatigue (46%), dysgeusia (42%), paresthesia (29%), nausea (28%), and myalgia (23%). Grade 3 TRAEs included fatigue (4%), weight increase (2%), diarrhea (1%), and arthralgia (1%). A subsequent analysis was conducted, including data from three phase 1/2 trials , and included patients with NTRK fusion-positive, advanced solid malignancies who were tyrosine kinase inhibitor naïve and received entrectinib 600 mg daily [Entrectinib is an inhibitor of TRKA, TRKB, TRKC, ROS1, and ALK . One stunhibitor . Thirty mg daily . Fifty-fLarotrectinib is a highly selective, small-molecule TRK inhibitor that was evaluated in a combined analysis of a phase 1, a phase 1–2, and a phase 2 study, including children and adults with advanced solid malignancies harboring a TRK fusion . This anNTRK fusion-targeted agents may have benefit in this small subpopulation of patients with CCA.Based on the results of these studies, RET proto-oncogene (RET) encodes a receptor tyrosine kinase, and fusion of the 3′ terminal of RET to the 5′ domain of RET finger protein has been implicated in oncogenesis [ogenesis . RET fusogenesis . RET fusion [RET-altered solid malignancies, including 3 patients with CCA [Pralsetinib is an inhibitor of RET kinases, including fusion proteins stemming from T fusion . ARROW wwith CCA . This stRET-fusion-positive advanced solid malignancies [Selpercatinib is a selective RET kinase inhibitor that was evaluated in LIBRETTO-001, which is an ongoing phase 1/2 basket trial evaluating selpercatinib in patients with gnancies . The intThese studies suggest a role for RET inhibition in patients with CCA with RET fusion, although the rarity of this mutation limits evaluation in this population.IDH-1 mutation, HER2 amplification/overexpression, or FGFR2 fusion/rearrangement, are more common, while others, including BRAF V600E, MSI-H, or TMB-H, are less common. These discoveries have paved the way for the development and clinical testing of targeted therapies aimed at these specific genetic anomalies. Multiple agents have been developed and have received FDA approval for use in the second-line setting, with ongoing phase 3 trials evaluating these agents in the first-line setting, although low enrollment has been a barrier in some instances.As our understanding of oncogenesis in CCA continues to evolve, multiple targets for intervention have been identified, allowing for the development of targeted, novel therapeutics. Some of these, such as IDH mutations, isoform switching, mitochondrial metabolism, and clonal selection [Despitethis evolving treatment paradigm, limitations regarding the use of targeted therapies in CCA remain. Reviewing current trial data, most targeted therapies result in, at best, a few months of PFS improvement, with modest improvement in OS. This suggests a need to better understand the resistance mechanisms that develop. For example, in the case of FGFR2 inhibitors, multiple resistance mechanisms have been identified, including mutation of the receptor or activation of feedback survival loops, impairing the duration of response ,109. In election . Future Additionally, future trials should aim to assess targeted agents in combination with immunotherapy and/or chemotherapy. For example, in the case of HER2 overexpressing biliary tract cancer, a planned trial in South Korea will evaluate the combination of trastuzumab with gemcitabine, cisplatin, and nivolumab (NCT05749900). However, historically, trials evaluating targeted therapies in advanced biliary tract cancer have struggled with patient accrual. In addition to new combinations, new novel therapies are also in development, and continued improvement in the way we inhibit these mechanisms will be important going forward .In summary, targeted therapies offer a new avenue of hope for patients with advanced CCA, though continued research is required to optimize these strategies, overcome resistance mechanisms, and identify the patient populations most likely to benefit. The aim of this article is to outline the current landscape and trial data evaluating targeted therapies in CCA and provide a foundational basis from which future drug optimization and development may grow.