AML is a heterogeneous and complex disease. RUNX1/RUNX1T1 is a fusion oncogene resulting from the chromosomal translocation t and plays a crucial role in AML. However, its impact on the transcriptomic profile of different age groups among AML patients is not completely understood. We aimed to investigate the deregulated gene expression profiles in RUNX1/RUNX1T1-positive AML patients, and compare their functions and regulatory networks between adult and pediatric patients. Our data corroborate that the RUNX1/RUNX1T1 fusion reprograms a large transcriptional network to establish and maintain leukemia via intricate protein–protein interactions and kinase-driven phosphorylation events.limma, GEO2Enrichr, and X2K. For validation purposes, we used the TCGA-LAML (adult) and TARGET-AML (pediatric) patient cohorts. We also analyzed the protein–protein interaction (PPI) networks, as well as those composed of transcription factors (TF), intermediate proteins, and kinases foreseen to regulate the top deregulated genes in each group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses were further performed for the DEGs in each dataset. We found that the top upregulated genes in (both adult and pediatric) RUNX1/RUNX1T1-positive AML patients are enriched in extracellular matrix organization, the cell projection membrane, filopodium membrane, and supramolecular fiber. Our data corroborate that RUNX1/RUNX1T1 reprograms a large transcriptional network to establish and maintain leukemia via intricate PPI interactions and kinase-driven phosphorylation events.Acute myeloid leukemia (AML) is a heterogeneous and complex disease concerning molecular aberrations and prognosis. RUNX1/RUNX1T1 is a fusion oncogene that results from the chromosomal translocation t and plays a crucial role in AML. However, its impact on the transcriptomic profile of different age groups of AML patients is not completely understood. Here, we investigated the deregulated gene expression (DEG) profiles in adult and pediatric RUNX1/RUNX1T1-positive AML patients, and compared their functions and regulatory networks. We retrospectively analyzed gene expression data from two independent Gene Expression Omnibus (GEO) datasets (GSE37642 and GSE75461) and computed their differentially expressed genes and upstream regulators, using  Acute myeloid leukemia (AML) is a heterogeneous disease in respect of molecular aberrations and prognosis. It is the second most common type of leukemia in adults and the most common type of acute leukemia. Nevertheless, AML is relatively rare and it accounts for approximately 1% of adult cancers in the United States, but nearly 2% of cancer-related deaths . AML is In AML, drug resistance toward standard chemotherapeutic compounds is due to different factors, such as enhanced DNA repair, inhibition of cancer cell apoptosis, alteration of signaling pathways, alterations in drug metabolism enzymes, drug sequestration in intracellular organelles, and overexpression of efflux drug transporters ,7. In adRunt-related transcription factor 1 (RUNX1) is crucial for the development of normal hematopoiesis , acting To gain a better understanding of the regulatory functions of the RUNX1/RUNX1T1 fusion oncogene in the transcriptome of AML patients of different age groups, we investigated the upstream regulators of the differentially expressed genes (DEG) in two independent GEO datasets composed of adult and pediatric AML patients harboring the RUNX1/RUNX1T1 fusion. We validated our findings using two independent datasets, and then we annotated and extracted gene expression signatures to acquire a better insight into the link between the DEGs and leukemic cells that harbor the RUNX1/RUNX1T1 fusion, as well as to decipher differences between the two age groups. We focused on the deregulated TFs and the protein kinases that seem to act as “hubs” in the gene networks formed in RUNX1/RUNX1T1-positive AML cases.We extracted gene expression data from two Gene Expression Omnibus (GEO) datasets. The first contained a total of 140 adult AML patients, 7 of which harbored the RUNX1/RUNX1T1 fusion  ,24,25,26limma with cut-off |log2FC > 2| for upregulation and |log2FC < 1| for downregulation, along with an adjusted p-value of <0.05. We utilized the B-statistic to sort DEGs and run Gene Ontology (GO) enrichment analysis to functionally annotate the 50 most prevalent DEGs in RUNX1/RUNX1T1 AML [p-values, with a threshold of an adjusted p-value (adj-p) ≤ 0.05. We clustered together gene sets with similar GO terms, using Uniform Manifold Approximation and Projection (UMAP) [p < 0.05) GO terms for biological processes (GO-BP), molecular function (GO-MF), and cellular component (GO-CC). We calculated clusters with the Leiden algorithm [Differential gene expression between RUNX1/RUNX1T1-positive and -negative AML patients was identified using X1T1 AML ,30. The n (UMAP)  and highlgorithm  and creahttps://ocg.cancer.gov/programs/target/projects/acute-myeloid-leukemia) [The top deregulated genes and their identified upstream regulators were validated using two independent AML patient cohorts: the TARGET-AML project (eukemia)  (accesseeukemia) . The mRNRUNX1, RUNX1T1, LINC00189, M1AP, ADCY7, TPPP3, ADARB1, TSPAN32, GPR114, PALM, SLCO5A1, HDX, SHANK1, SNORD9, EGLN1, HIPK2, MAPK1, CSNK2A1, MAPK3, MAPK14, SUZ12, PPARG, SRF, CBX3, SALL4, CF3, AR, HNF4A, NFE2L2, EZH2, TP63, ESR1, SMC3, STAT3, KLF4, GATA1, CTCF, PPARD, TP53, CDK1, GSK3B, CDK2, AKT1, CDK4, and MAPK8.From the TARGET-AML study, we collected the following clinicopathological data: type of gene fusion, presence of the t translocation, age at diagnosis (days), and treatment with gemtuzumab ozogamicin, as well as gene expression levels for RUNX1T1, POU4F1, CACNA2D2, FBLN5, CAV1, CLEC2L, SIPA1L2, GPM6B, CD19, IL5RA, PALM, ITGB4, FAM81A, ROBO1, FOXL1, TRH, IGBP1, RNF39, FRMD1, INPPL1, FSCN2, NUBPL, UQCRQ, C12orf10, LINC00115, LINC00189, M1AP, ADCY7, TPPP3, ADARB1, TSPAN32, GPR114, SLCO5A1, HDXS, HANK1, SNORD9, EGLN1, CSNK2A1, AKT1, MAPK3, STAT3, SMAD4, IRF1, TCF3, SOX2, EZH2, PPARD, FOSL2, ATF2, ELF1, E2F1, ERG, MYOD1, FOXP2, TCF7L2, GABPA, FOXA1, CDK1, MAPK14, MAPK8, MAPK1, and SUZ12.From the TCGA-LAML study, we collected the following clinicopathological data: cytogenetic abnormality, prior treatment diagnoses, age at diagnosis, as well as gene expression levels for The TARGET-AML study contains 284 AML patients with the RUNX1-RUNX1T1 fusion, 46 of whom received treatment with gentuzumab ozogamicin (the other 122 did not). On the other hand, the TCGA-AML cohort does not contain a t, but two patients with 21,9, six patients with t, six patients with t, eight patients with t, and thirty-eight AML patients with genetic abnormality in chr21.Expression2Kinases (X2K) to infer the upstream regulatory networks of the DEGs in RUNX1/RUNX1T1 AML. We further performed transcription factor (TF) enrichment analysis, expansion analysis of the protein–protein interaction (PPI) networks, kinase enrichment, and we inferred the gene networks governing the expression of the DEGs in RUNX1/RUNX1T1 AML patients [We used patients .p < 0.05 as a threshold for statistical significance. The expression levels of the genes that were significantly deregulated in both validation cohorts (TCGA-AML and TARGET-AML) were compared using the Mann–Whitney U test with Bonferroni correction .We assessed statistical differences in gene expression between RUNX1/RUNX1T1-positive and RUNX1/RUNX1T1-negative AML samples through the Mann–Whitney U test, with an adj. RUNX1T1, POU4F1, CACNA2D2, FBLN5, and CAV1 were among the top upregulated genes in RUNX1/RUNX1T1 AML patients, followed by CLEC2L, SIPA1L2, GPM6B, CD19, IL5RA, PALM, ITGB4, FAM81A, SLCO5A1, ROBO1, FOXL1, and TRH . They were also overrepresented in epidermal growth factor receptor binding, transmembrane receptor protein tyrosine kinase activity, neuregulin binding, neurotrophin TRK receptor binding, and neurotrophin TRKA receptor binding (GO-MF), as well as in collagen-containing extracellular matrix, cell projection membrane, sarcolemma, basement membrane, and membrane raft (We first detected the significantly deregulated genes in adult RUNX1/RUNX1T1 AML patients (GSE37642), and focused on the top (and bottom) 50 DEGs . As expe and TRH . The topane raft .In contrast, the bottom 50 DEGs included IGBP1, RNF39, FRMD1, INPPL1, FSCN2, NUBPL, UQCRQ, C12orf10, LINC00115, and LOC100129198, and were enriched in mitochondrial electron transport, ubiquinol to cytochrome c, aerobic electron transport chain, mitochondrial ATP synthesis coupled electron transport, mitochondrial respiratory chain complex I assembly, and NADH dehydrogenase complex assembly (GO-BP). They were also enriched in syntaxin binding, fucose binding, NAADP-sensitive calcium-release channel activity, immunoglobulin receptor binding, and ubiquinol-cytochrome-c reductase activity (GO-MF), as well as in mitochondrial respiratory chain complex III, pseudopodium, and actin cytoskeleton (GO-CC) . Of noteLINC00189, RUNX1T1, M1AP, ADCY7, TPPP3, ADARB1, TSPAN32, GPR114, PALM, SLCO5A1, and HDX; whereas, among the top downregulated genes, we highlighted SHANK1, linc-RXFP2-1, SNORD9, and EGLN1.We similarly detected the significantly deregulated genes in pediatric RUNX1/RUNX1T1 AML patients . Among tThe first were primarily enriched in extracellular matrix (ECM) organization, protein polymerization, and cellular protein catabolic process (GO-BP). They were also enriched in choline transmembrane transporter activity, double-stranded RNA adenosine deaminase activity, and diphosphotransferase activity (GO-MF), as well as in an intrinsic component of the cytoplasmic side of the plasma membrane, the cell projection membrane, an integral component of the lysosomal membrane and microfibril (GO-CC) .ECM organization (GO-BP), cell projection membrane; filopodium membrane, and supramolecular fiber (GO-CC) were commonly found among the top 10 GO terms in both age groups of RUNX1/RUNX1T1-positive AML.Interestingly, the GO-terms In contrast, the bottom 50 DEGs were highly enriched in membrane depolarization during cardiac muscle cell action potential, regulation of ventricular cardiac muscle cell membrane repolarization, cardiac muscle cell contraction, and regulation of cardiac muscle cell membrane repolarization (GO-BP). Furthermore, they were enriched in phosphatidylinositol 3-kinase regulatory subunit binding, voltage-gated calcium (and sodium) channel activity involved in cardiac muscle cell action potential, ankyrin repeat binding, and phosphatidic acid transfer activity (GO-MF), as well as in sperm flagellum and 9 + 2 motile cilium (GO-CC) .In order to gain a better insight into the critical hubs that orchestrate the expression of the DEGs, we constructed the PPI networks. In the adult AML patient cohort (GSE37642), we found that SUZ12, SOX2, EZH2, SMAD4, and STAT3 (among other TFs), as well as MAPK14, CSNK2A1, MAPK1, CDK1, AKT1, and MAPK3 (among other kinases) regulate the co-upregulated genes upstream. Of these, we noticed that SUZ12, STAT3, SMAD4, IRF1, TCF3, SOX2, EZH2, and PPARD act as hubs in the PPI expansion network a.On the other hand, the co-downregulated genes seem to be orchestrated by FOSL2, ATF2, ELF1, E2F1, SUZ12, ERG, MYOD1, FOXP2, TCF7L2, GABPA, FOXA1, and other TFs, as well as by the kinases CDK1, MAPK14, MAPK8, JNK1, and MAPK1. Of these, E2F1, ATF2, MYID1, TCF7L2, ERG, and SUZ12 appear to act as hub proteins in the PPI network b.Similarly, we calculated the responsible upstream regulators for the co-deregulated genes among pediatric AML patients (GSE75461). We found RUNX1, RCONR1, NFE2L2, SUZ12, PPARG, SRF, CBX3, SALL4, TCF3, among other TFs, but also the kinases ERK1/2, HIPK2, CK2APLHA, MAPK1, CSNK2A1, and MAPK3/14, among others. Of these, SRF, PPARG, RUNX1, NFE2L2, TCF3, CBX3, IRF8, SUZ12, and RCOR1 were found to act as hub TFs in the PPI expansion network a.Additionally, among the co-downregulated genes, we found AR, HNF4A, NFE2L2, EZH2, TP63, ESR1, SMC3, STAT3, KLF4, SXO2, GATA1, CTCF, PPARD, and TP53, among other TFs, and the kinases CSNK2A1, MAPK14, CDK1, MAPK1/3, HIPK2, GSK3B, CDK2, ERK1/2, DNAPK, AKT1, CDK4, GSK3BETA, and MAPK8. Of these, we detected EZH2, NFE2L2, HNF4A, STAT3, TP63, and KLF4 as hub proteins in the PPI network b.TRH, POU4F1, TCF3, CAV1, PALM, ROBO1, SIPA1L2, SHANK1, IL5RA, CD19, CACNA2D2, ITGB4, SLCO5A1, M1AP, FBLN5, LINC00189, and EGL1 in RUNX1/RUNX1T1-positive patients; whereas, ADCY7, TSPAN32, ADAB1, PPARD, and FOXL1 were significantly downregulated in them compared to RUNX1/RUNX1T1 = negative patients ; whereas ELF1, FOSL2, IGBP1, STAT3, SRF, EZH2, NFE2L2, ATF2, SUZ12, IRF1, CTCF, UQCRQ, GSK3B, SNORD9, PPARD, CD19, and GPM6B, among others, were upregulated in the pediatric AML cohort (TARGET-AML)  c.Leukemia is characterized by somatic mutations and genetic rearrangements that impede signal transduction and expression programs, resulting in the disruption of chromatin modifiers and transcription factors. In AML, transcriptional and epigenetic reprogramming can be seen in the RUNX1 transcription factor and its oncogenic derivative RUNX1/RUNX1T1 . The RUNRUNX1 and intron 1 of RUNX1T1 [RUNX1 is a TF responsible for the differentiation of hematopoietic cells and the development of myeloid cells ,38. RUNX RUNX1T1 ,43. The  RUNX1T1 . The ove RUNX1T1 . In turn RUNX1T1 ,45. The  RUNX1T1 ,47,48. I RUNX1T1 ,50, but  RUNX1T1 , all of  RUNX1T1 ,53. It a RUNX1T1 ,55.RUNX1T1, which is known to be highly expressed in RUNX1/RUNX1T1 AML, POU4F1, CACNA2D2, FBLN5, and CAV1 are also upregulated in adult RUNX1/RUNX1T1 AML patients, followed by CLEC2L, SIPA1L2, GPM6B, CD19, IL5RA, PALM, ITGB4, FAM81A, SLCO5A1, ROBO1, FOXL1, and TRH. Here, we explored the upstream regulators of the DEGs in adult and pediatric AML patients with the RUNX1/RUNX1T1 fusion oncogene. We found that apart from SLCO5A1 is a member of the solute carrier organic anion transporters . OrganicLINC00189, RUNX1T1, M1AP, ADCY7, TPPP3, ADARB1, TSPAN32, GPR114, PALM, SLCO5A1, and HDX. Of these, the membrane protein ADCY7 has been shown to support leukemogenesis by decreasing apoptosis [In contrast, in pediatric RUNX1/RUNX1T1 AML, the top upregulated genes were poptosis . Adenosipoptosis ,65. Partpoptosis .FBLN5, CACNA2D2, M1AP, TPPP3, and TSPAN32 are mentioned to be upregulated in AML, indicating that perhaps other pathways are also likely implicated in the disease.Several G protein-coupled receptor members (GPCRs) are also overexpressed and enriched in adhesion receptor subfamilies ; whereaCACNA2D2, which encodes for a new subunit of the Ca2+-channel complex, has been postulated as a possible link in the pathogenesis of lung cancer [M1AP [LINC00189 and PALM are rarely reported in human diseases.Of these, FBLN5 is an anti-oncogene that belongs to the family of fibulins, and its aberrant expression has been reported in lung, breast, hepatocellular, and other types of cancer ,70,71,72g cancer , similarer [M1AP . Furtherer [M1AP . In addier [M1AP ,77. On tRUNX1T1, PALM, and SLCO5A1, and play a role in the regulation of the dopamine receptor signaling pathway, the negative regulation of adenylate cyclase activity, and the sodium-independent organic anion transport, among others.Interestingly, we found three genes being commonly upregulated between pediatric and adult RUNX1/RUNX1T1 AML patients. These are IGBP1, RNF39, FRMD1, INPPL1, FSCN2, NUBPL, UQCRQ, C12orf10, LINC00115, and LOC100129198; whereas, in pediatric RUNX1/RUNX1T1 AML patients, they included SHANK1, linc-RXFP2-1, SNORD9, and EGLN1.We also identified the top 50 downregulated genes in adult RUNX1/RUNX1T1 AML patients. These included Similar to the CBFB/MYH11 fusion, the RUNX1/RUNX1T1 fusion protein is associated with unique TF networks, which have some common nodes across all AML types and are post-translationally modified ,78. In tIn addition, we calculated the responsible upstream regulators of the co-deregulated genes between the two datasets. Our findings show that SRF, PPARG, RUNX1, NFE2L2, TCF3, CBX3, IRF8, SUZ12, and RCOR1 seem to act as hub TFs in the formed PPI network of the upregulated genes; whereas, EZH2, NFE2L2, HNF4A, STAT3, TP63, and KLF4 orchestrate the co-downregulated genes. The co-expression networks that our data highlight corroborate that the RUNX1/RUNX1T1 fusion protein reprograms a large transcriptional network.Interestingly, TCF3 was a common upstream regulator in both cohorts. Furthermore, STAT3 and EZH2 were found to be upregulated in the adult AML cohort, but downregulated in the pediatric one, suggesting that they act differently between pediatric adult AML patients.The expression pattern of TFs seems to have an important impact on the PPI network. The polycomb group of proteins (PcG) is a major class of epigenetic regulators that repress target genes involved in cell cycle regulation and differentiation ,80. PcGsA large number of studies have recently revealed that AML patients have repeated mutations in several important epigenetic mediators . MutatioThe effect of RUNX1 on target gene expression is highly context-dependent, determined by the composition of the transcriptional complexes in which RUNX1 functions at a particular gene. These complexes contain transcriptional cofactors as well as epigenetic modifiers, which are recruited to gene promoters as well as other regulatory regions, and by doing so, RUNX1 can influence both transcriptional activity as well as the epigenetic status of target genes .RUNX1 has been found to complex with an array of epigenetic modifiers, with the outcome for both RUNX1 function and target gene activity dependent on the balance of activating and repressive factors associated with RUNX1 at a particular time. These studies thus demonstrate an idea of RUNX1 itself as a target of epigenetic modifiers, which post-translationally modify the RUNX1 protein, altering its coactivator/corepressor interactions, thus influencing its transcriptional activity. In addition, RUNX1 recruits these coactivators and corepressors to target genes, resulting in the modification of the chromatin environment, thus affecting gene activity at this second level. An outstanding example of this multi-leveled interaction between RUNX1, epigenetic modifiers, and the epigenome is demonstrated in the recently described interaction of RUNX1 with protein arginine methyltransferase 6 (PRMT6) .EZH2 in AML agree with previous reports, suggesting that its overexpression (apart from mutation) promotes cell proliferation in hematologic and other malignancies. EZH2 controls the expression of genes involved in stem cell maintenance and differentiation and its down-regulation inhibits apoptosis, affects the expression of MAD2 and CDC20, and promotes chromosomal instability in AML cells [What is more, our findings regarding the deregulated expression of ML cells ,90,91,92ML cells ,94. The ML cells .RUNX1/RUNX1T1 fusion have been shown to be sensitive to the inbitition of the JAK-STAT pathway [Furthermore, we shed light on the Signal Transducer and Activator of Transcription 3 (STAT3), another TF that is crucial in leukemogenesis as it blocks the differentiation of myeloid cells ,96. STAT pathway . Of note pathway ,102,103.RUNX1T1,POU4F1,CACNA2D2,FBLN5, and CAV1, which play a role in axonogenesis, ECM organization, and negative regulation of cell differentiation and ECM assembly. The top downregulated genes in this group included IGBP1,RNF39,FRMD1,INPPL1,FSCN2,NUBPL, and UQCRQ, among others, playing a role in mitochondrial electron transport, ubiquinol to cytochrome c, aerobic electron transport chain, and mitochondrial ATP synthesis coupled electron transport. The most upregulated genes in pediatric RUNX1/RUNX1T1-positive AML included RUNX1T1,LINC00189,M1AP,ADCY7,TPPP3,ADARB1, and TSPAN32, playing key role in ECM organization, protein polymerization, and cellular protein catabolic processes. Finally, the top downregulated genes in this group involved SHANK1, linc-RXFP2-1, SNORD9, and EGLN1, being primarily enriched in membrane depolarization during cardiac muscle cell action potential.To conclude, our data corroborate that hematopoiesis is strictly regulated by different levels of intercellular signaling, and is affected by various intricate transcriptional regulations. The RUNX1/RUNX1T1 fusion aberrates many intracellular pathways that involve tumor suppressors and oncogenes. The regulatory networks governing the top deregulated genes in adult and pediatric RUNX1/RUNX1T1 AML patients include several transcription factors, intermediate proteins and kinases, which orchestrate the establishment and maintenance of leukemia. The most upregulated genes in adult RUNX1/RUNX1T1-positive AML were


Non-coding microRNAs (miRNAs) play critical roles in tumor progression and hold great promise as therapeutic agents for multiple cancers. MicroRNA 29a (miR-29a) is a tumor suppressor miRNA that inhibits cancer cell growth and tumor progression in non-small cell lung cancer. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), which plays an important role in lung cancer progression, has been identified as a target of miR-29a. Here, we evaluated the therapeutic efficacy of a peptide vector capable of delivering miR-29a intracellularly using the acidic tumor microenvironment in a lung adenocarcinoma xenograft mouse model.A miRNA delivery vector was constructed by tethering the peptide nucleic acid form of miR-29a to a peptide with a low pH-induced transmembrane structure (pHLIP) to enable transport of the miRNAs across the plasma membrane. Tumor suppressive effects of pHLIP-miR29a on lung adenocarcinoma development in vivo were assessed using a BALB/c xenograft model injected with A549 cells.Incubation of A549 cells with pHLIP-miR-29a at an acidic pH downregulated endogenous CEACAM6 expression and reduced cell viability. Intravenous injection of the mice with pHLIP-miR-29a inhibited tumor growth by up to 18.1%. Intraperitoneal injection of cisplatin reduced tumor volume by 29.9%. Combined pHLIP-miR-29a + cisplatin treatment had an additive effect, reducing tumor volume up to 39.7%.Delivery of miR-29a to lung adenocarcinoma cells using a pHLIP-mediated method has therapeutic potential as a unique cancer treatment approach.The online version contains supplementary material available at 10.1186/s12885-023-11352-w. Lung cancer is the major cause of cancer-related death worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 80% of lung cancer cases . DespiteCEACAM6, mir-29a also targets LASP1 [CDC42 [We demonstrated previously that miR-29a acts as a tumor suppressor in NSCLC by targeting carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) . Overexp1 [CDC42  and regu. [. used peptide nucleic acids (PNAs) as antisense oligomer analogs. PNAs consist of nucleobases that are connected by intramolecular amide bonds. The backbone of PNAs is composed of peptide-like structure of repeated N-(2-aminoethyl)-glycine units. The lack of anionic phosphodiester groups increases the PNAs binding affinity for complementary nucleic acids. In addition, PNAs have high resistance to nucleases and proteases, and exhibit exceptional stability over a broad pH range [Overexpression of tumor suppressor miRNAs and inhibition of oncogenic miRNAs have shown therapeutic potential in model systems . Moreove.  demonstrpH range .Recently, we demonstrated the therapeutic effect of delivery of siCEACAM6 into cells in the acidic tumor microenvironment using a PNA form of CEACAM6-specific siRNA (siCEACAM6) as a peptide vector . In the PNA-pHLIP was generated according to the protocols described previously . PNA ver−1 cm−1 (A), 6,600 M−1 cm−1 (C), 11,700 M−1 cm−1 (G), 8,800 M−1 cm−1 (T), 200 M−1 cm−1 , and 32,300 M−1 cm−1 (TAMRA). After quantification, the pHLIP-PNAs were freeze-dried.The pHLIP sequence AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG was synthesized to generate the pHLIP-PNA constructs. The C-terminus of each PNA oligomer was conjugated to pHLIP through a disulfide bond. pHLIP and PNA  were reacted overnight in the dark in a mixture of dimethylformamide (DMF)/Tris (pH 7.5). After conjugation, pHLIP-PNA was purified by RP-HPLC and characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. pHLIP-PNAs were quantified by UV/VIS spectrophotometry at 260 nm using the following extinction coefficients: 13,700 M2 atmosphere. For all pH-controlled cell culture experiments, cells were incubated in complete culture medium with 10% FBS and buffered at pH 7.4 with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or pH 6.2 with 2-(N-morpholino)-ethanesulfonic acid (MES).The human lung adenocarcinoma cell line A549 and human colon adenocarcinoma cell line HT-29 were purchased from the American Type Culture Collection . A549 and HT-29 cells were maintained in RPMI containing l-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum (FBS) and were cultured at 37°C in a 5% COWestern blot analysis was conducted according to the protocols described previously . Cells w4 /ml) were treated with pHLIP-miR-29a  in 96-well plates for 48 h. Assays were performed in parallel using pHLIP-scr as a control. All treatments were performed at the indicated pH. After 48 h, the extent of cell growth was assessed using the Cell Proliferation Kit II (XTT). Briefly, XTT solution (50 μl) was added to each well and the cells were incubated for 2 h at 37 °C. Absorbance at 450 nm was then determined using a Lambda Bio-20 multiplate reader . Cell proliferation was expressed as a percentage of that of control cells.The effects of pHLIP-miR-29a on the proliferation of A549 and HT-29 cells were measured using a Cell Proliferation Kit II (XTT) . The cells (5 × 107) were subcutaneously injected into the right flanks of 5-week-old male BALB/c athymic nude mice (n = 5 mice/group). Tumor growth (V) was determined by measuring the length (L), width (W), and height (H) with calipers and using the formula V = (L × W × H) × 0.5. Upon tumor formation (approximately 44.8 mm3), mice were intravenously injected with pHLIP-PNA constructs and/or intraperitoneally injected with cisplatin, as indicated. Mice were euthanized by carbon dioxide asphyxiation. Tumor volumes were measured every 2–3 days.Animal experiments was conducted according to the protocols described previously . All aniAfter euthanasia, tumors were harvested from mice, fixed in formalin, and paraffin embedded. The frozen tumor specimens were then cryosectioned into 2-μm sections. The sections were fixed with cold acetone, rinsed with phosphate-buffered saline (PBS), reacted with hydrogen peroxide to inactivate endogenous peroxidases, and then washed with PBS. The nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). All washes to remove surface-bound pHLIP were performed using PBS at pH 7.4. Images were acquired using a LSM 710 META confocal microscope  and processed using Zen software .Mice were euthanized and the tumors harvested, fixed in formalin, and paraffin embedded. The formalin-fixed, paraffin-embedded tumor specimens were sectioned (4 μm thickness) and then stained. Fully automated immunostaining was performed using a BenchMark XT autostainer . The following antibodies were used: monoclonal mouse anti-CC3  and anti-Ki-67 . The number of positive cells per tumor area was quantified.t-tests. Immunostaining data from each group were also compared using Student’s t-tests. Survival curves were estimated using the Kaplan–Meier method and compared using the log-rank test. P ≤ 0.05 was deemed statistically significant. All statistical tests were two-sided and performed using Prism 8 software .Two-way analysis of variance was performed to compare the cell viability data from the three groups. For animal experiments, mouse and tumor weights for each group were compared using Student’s To create the pHLIP-PNA miR-29a construct, the C-terminus of the PNA form of a miR-29a mimic was congugated to pHLIP. A control construct, pHLIP-scr, was also synthesized. A single isomer of 5-carboxytetramethylrhodamine (TAMRA) label was added to the PNA of the pHLIP-PNA oligomer. Verification of pHLIP-PNA oligomer production was conducted through RP-HPLC and mass spectrometry.  were injected twice a week with different doses of pHLIP-miR-29a (2 and 4 mg/kg) for 3 weeks. The mice were sacrificed after 3 weeks of treatment. Tumor volumes of mice treated with pHLIP-miR-29a were significantly smaller than those of control mice treated with pHLIP-scr . At 4 mg/kg, pHLIP-miR-29a treatment reduced tumor size by 40.9% compared with treatment with pHLIP-scr (P < 0.001). The ability of pHLIP-miR-29a to deliver miR-29a to lung adenocarcinomas in vivo was assessed by confocal imaging of the isolated tumor tissues. Strong fluorescent TAMRA signals were observed on the surfaces of tumor cells from the pHLIP-miR-29a-treated mice, indicating efficient in vivo intracellular delivery of miR-29a  As shown in Fig. P = 0.02; : (i) vehicle, (ii) pHLIP-miR-29a, (iii) cisplatin, and (iv) pHLIP-miR-29a + cisplatin. Based on the in vivo results, a dose of 2 mg/kg pHLIP-miR-29a was selected for the combination treatment. Two weeks after cell line grafting, pHLIP-miR-29a was tail-vein injected into the mice and a dose of 2 mg/kg of cisplatin was intraperitoneally injected twice per week for 3 weeks. As shown in Fig. P = 0.004). Cisplatin treatment reduced the tumor volume by 29.9% (P < 0.001), whereas pHLIP-miR-29a + cisplatin treatment reduced the tumor volume by 39.7% (P < 0.001). These results indicate that systemically delivered miR-29a and cytotoxic chemotherapy had additive anti-tumor effects in vivo. None of the mice in the therapeutic experiments experienced overt body weight loss  or cisplatin alone . However, tumors from mice treated with pHLIP-miR-29a + cisplatin showed significantly higher numbers of CC3-positive cells than those from mice treated with pHLIP-miR-29a alone (P = 0.009) or cisplatin alone (P = 0.02), as shown in Fig. To assess further the biologic effects of combined systemic miR-29a and cisplatin treatments, tumor samples were stained for markers of proliferation and apoptosis. As shown in Fig. We found that systemic administration of a pHLIP-miR-29a conjugate resulted in successful delivery of tumor suppressive miR-29a to lung adenocarcinoma cells in mice by targeting the acidic tumor microenvironment. At acidic pH, pHLIP forms an α-helix that inserts into the lipid bilayer, making it an attractive targeting moiety for selective labelling and tracing of acidic tissues in vivo . As acidThe pHLIP-mediated delivery of the miR-29a mimic inhibited CEACAM6 protein expression in A549 lung adenocarcinoma cells and reduced cell proliferation. The expression of LASP1 and CDC42 proteins were also downregulated by pHLIP-miR-29a. In vivo delivery of pHLIP-miR-29a inhibited the growth of lung adenocarcinoma tumors. These findings were consistent with the results of our previous study in which injection of mice with A549 cells expressing miR-29a inhibited the tumor growth in a lung adenocarcinoma mouse model [. [MiR-29a appears to exert its tumor suppressor effects by affecting the regulation of multiple biological processes , 31, 32.. , KDM5B [. , QKI-6 [. , MMP2 [3. , and TNF.  in varioIn the mouse lung adenocarcinoma xenograft model, the anti-tumor effects of the combination of miR-29a and the chemotherapeutic drug cisplatin were superior to those of either treatment alone. Previous studies have found that reduced levels of miR-29 lead to increased resistance of ovarian cancer cells to cisplatin  and miR-None of the mice in our experiments exhibited any overt signs of toxicity. The tumor-targeting property of pHLIP ensured the miR-29a delivery was limited to tumor tissues; however, additional studies are required to delineate the impact of miR-29a delivery on normal tissues and the tumor microenvironment.KRAS mRNA have been tested in phase I clinical trials for treatment of NSCLC and other advanced solid tumors [RNA-based therapies hold promise to expand the range of druggable targets from proteins to RNAs and the genome. Lipid nanoparticle-formulated siRNA delivery is safe and well-tolerated in first-in-human clinical trials and in some metastatic cancer cases elicited complete responses . In addid tumors . DevelopIn summary, the present study demonstrated the efficacy of small RNA-based therapy in a lung adenocarcinoma xenograft model. A pHLIP vector was used to improve tumor-targeting and mediate the successful delivery of a tumor suppressor miRNA. The effective delivery of therapeutic miRNAs to lung cancer cells using the pHLIP peptide increases the prospect of using small RNAs for cancer therapy.Additional file 1.Supplementary Figure 1. RP-HPLC elution profiles of a PNA (siCEACAM6) and pHLIP reaction mixture.Additional file 2.Supplementary Figure 2. Activity of pHLIP-miR-29a targeting CEACAM6 in HT-29 colon adenocarcinoma cells. (A) Western blotting analysis of CEACAM6 protein levels in HT-29 cells incubated with the indicated concentration of pHLIP-miR-29a at pH 6.2. (B) Effects of pHLIP-miR-29a on the viability of HT-29 cells at neutral and acidic pH. Data are presented as the mean ± s.d. ***P < 0.001.Additioanal file 3.Supplementary Figure 3. Activity of pHLIP-miR-29a targeting LASP1 and CDC42 in A549 cells. Western blotting analysis of LASP1 and CDC42 protein levels in A549 cells incubated with pHLIP-miR-29a at pH 6.2.Additional file 4.Supplementary Figure 4. Toxicity assessment of intravenously administered pHLIP-miR-29a. (A) Changes in the body weights of experimental mice during the treatment period (n = 5 mice/group). Data are presented as the mean ± s.d. (B) Representative histological analyses of kidney, liver, and heart specimens collected from mice treated with pHLIP-miR-29a or pHLIP-scr. All sections reveal an absence of microscopic changes associated with toxicity in pHLIP-miR-29a-treated mice. Original magnification ×400; scale bar = 50 µm.Additional file 5.Supplementary Figure 5. Toxicity assessment of combination treatment with pHLIP-miR-29a and cisplatin. (A) Changes in the body weights of experimental mice during the treatment period (n = 5 mice/group). Data are presented as the mean ± s.d. (B) Representative histological analysis of kidney, liver, and heart specimens collected from mice treated with pHLIP-miR-29a or pHLIP-miR-29a+cisplatin. All sections revealed an absence of microscopic changes associated with toxicity in pHLIP-miR-29a-treated and pHLIP-miR-29a+cisplatin-treated animals. Original magnification ×400; scale bar = 50 µm.Additional file 6.Supplementary Table 1. The tumor volume of each animal in Figure 2A.Additional file 7.Supplementary Table 2. The tumor volume of each animal in Figure 4A.Additional file 8. Raw images.


Mentoring plays a pivotal yet poorly understood role in shaping a physician’s professional identity formation (PIF) or how they see, feel and act as professionals. New theories posit that mentoring nurtures PIF by functioning as a community of practice through its structured approach and its support of a socialisation process made possible by its assessment-directed personalized support. To test this theory and reshape the design, employ and support of mentoring programs, we evaluate peer-mentor experiences within the Palliative Medicine Initiative’s structured research mentoring program.Semi-structured interviews with peer mentors under the Palliative Medicine Initiative (PMI) at National Cancer Centre Singapore were conducted and triangulated against mentoring diaries to capture longitudinal data of their PMI experiences. The Systematic Evidence-Based Approach (SEBA) was adopted to enhance the trustworthiness of the data. SEBA employed concurrent content and thematic analysis of the data to ensure a comprehensive review. The Jigsaw Perspective merged complementary themes and categories identified to create themes/categories. The themes/categories were compared with prevailing studies on mentoring in the Funnelling Process to reaffirm their accuracy.Twelve peer-mentors participated in the interviews and eight peer-mentors completed the mentoring diaries. The domains identified were community of practice and identity work.The PMI’s structured mentoring program functions as a community of practice supporting the socialisation process which shapes the peer-mentor’s belief system. Guided by a structured mentoring approach, stage-based assessments, and longitudinal mentoring and peer support, peer-mentors enhance their detection and evaluation of threats to their regnant belief system and adapt their self-concepts of identity and personhood to suit their context. These insights will help structure and support mentoring programs as they nurture PIF beyond Palliative Medicine. Medical education is tasked with shaping the Professional Identity Formation (PIF) of medical students and physicians (henceforth clinicians), or how they think, feel and act as professionals as they navigate their medical practice , 2. ExtaNew insights into mentoring practice at the Palliative Medicine Initiative (PMI), a structured research mentoring program at the National Cancer Centre Singapore (NCCS), may offer a better perspective into the development of professional identities in this aspect of medical education. Indeed, mentoring is a recognised means of developing PIF in medical education. However, before we evaluate PMI experiences, its role in nurturing PIF and its links with CoP and the Socialisation Process, we must foreground our approach with explanations of the terms and theories we will use.desired characteristics); learning objectives, goals, timelines, professional standards, codes of conduct, roles, responsibilities, expectations, implicit norms, culture, artifacts, sociocultural norms and expectations and legal requirements (henceforth codes of conduct). They are also reintroduced to the current education approaches; the program’s value, support and assessment systems; the settings and stages of training, as well as the formal curriculum (henceforth host organization related facets).To begin, the PMI employs a combined novice-, peer- and e-mentoring approach (henceforth CNEP) , 13 thatFollowing their regular bi-weekly online meetings vis-à-vis ad-hoc and informal meetings, peer-mentors make an entry into their mentoring diaries to document their experiences. These entries are privy to only specific members of the administrative staff to safeguard the anonymity and privacy of the peer-mentors whilst ensuring that timely and appropriate support can be provided if any mentoring, mental health, and physical problems are detected.The PMI also establishes a spiral curriculum for peer-mentors that sees them regularly revisit key training skills and competencies along the training trajectory Fig. . This spThese roles are represented by three concentric rings. The centre ring represents the role of a senior mentor whilst the middle and outermost ring denote the roles of the peer-mentor and mentee respectively in the CoP. Progress from one role to the next reflects achievement of the requisite skills and competencies, alongside concurrent changes in perspectives, belief systems, decision making and actions. Shepherding this developmental process is personalised, timely, appropriate, longitudinal, and often holistic mentoring support drawn from the mentoring umbrella, guided by mentoring assessments and provided by the two trained mentors. It is this combination of personalised mentoring support framed around the PMI’s structured approach and the mentoring course mapped out by the mentoring framework that facilitates the shift away from individual self-interests amongst mentees to furthering the interests of the PMI program that practices ‘paying it forward’. Facilitating this spiral course between the circles is a combination of experiential learning , graduatThe clearly defined trajectory and expectations set out provide a unique opportunity to study the longitudinal influence of mentoring on the PIF of peer-mentors –10, 13. codes of conduct, desired characteristics and host organization related facets [In particular, there are two features that are imperative to our study. The first is the mentoring umbrella , 10, 24 d facets –30; mentd facets  that nurd facets ; stage-sd facets ; longitud facets ; mentor d facets . Collectthe characteristics, values, and norms of the medical profession… resulting in an individual thinking, acting and feeling like a physician” [The mentoring umbrella plays a key role in supporting the Socialisation Process, or the internalisation of “ysician” . Here, tysician” , 32, 33,a persistent, sustaining social network of individuals who share and develop an overlapping knowledge base, set of beliefs, values and history and experiences focused on a common practice and/or enterprise” [The Socialisation Process is, in turn, dependent on the presence of an organized, supportive sense of community provided by the PMI through its curated mentoring environment and mentoring structure. This likens the PMI to a Community of Practice (CoP). Indeed, the PMI’s mentoring structure does meet the key features of a CoP as “erprise” .With its shared sense of identity, structure, culture and mentoring support, the PMI provides peer-mentors with a holistic and personalised means of inculcating new beliefs, values, principles, insights, and experiences into regnant belief systems within the confines set by the mentoring structure , 35, 36.The RToP builds on the notion that shifts in a clinician’s belief systems change how they see themselves as persons and as professionals. This allows the RToP to map changes in individual belief systems in the clinician’s Innate, Individual, Relational and Societal identities and capture corresponding changes in the Innate, Individual, Relational and Societal rings of personhood Fig. .Fig. 2ThThe Innate Identity is derived from the peer-mentor’s regnant spiritual, religious, theist, moral and ethical belief systems within the Innate Ring. The Individual Ring’s belief system revolves around the peer-mentor’s beliefs, values and principles surrounding conscious function and informs the peer-mentor’s thoughts, conduct, biases, narratives, personality, and decision-making processes, thus shaping the peer-mentor’s Individual Identity. The Relational Identity is born of a belief system governing those relationships that the peer-mentor determines to be personal and important. The Societal Identity is shaped by regnant societal, cultural, religious, professional, and legal roles and expectations, and belief systems which inform their interactions with colleagues and acquaintances.The Individual Identity also manifests in the peer-mentor’s overall identity, balancing the influences of the Innate, Relational and Social Identities. This balancing process shapes and is shaped by the beliefs systems within each domain of personhood. When there is consistency between the beliefs, values, and principles being introduced and the current belief system within the ring or rings of the RToP, there is ‘resonance’. ‘Synchrony’ occurs when current values, beliefs and principles within the ring or rings of the RToP are reprioritised to better fit with the practice beliefs, values, and principles. In contrast, inconsistencies between new and prevailing belief systems lead to ‘dissonance’. Dissonance within one ring of the RToP precipitates ‘disharmony’ whilst ‘dyssynchrony’ manifests from dissonance between rings.The KPM explores the effects of resonance, synchrony, dyssynchrony and disharmony (henceforth event) on the belief system and PIF Fig. . The KPMIt is within these conditions where the PMI functions as a CoP supporting the Socialisation Process and that changes in the peer-mentor’s belief system may be captured by the KPM and RToP that we pose the primary research question, “How does peer-mentoring in a structured mentoring program impact PIF?”. In providing a comprehensive perspective of peer-mentoring experiences in the PMI, we proffer the secondary research questions, “What features of the CoPs and the socialisation process are present in the PMI peer-mentoring experience?” and “What impact does mentoring have on Professional Identity Formation?”.Acknowledging peer-mentoring as a sociocultural construct shaped by individual, psycho-social, academic, professional, clinical, research and environmental considerations, we adopt a Constructivist perspective and a Relativist lens. Accordingly, we adopt a qualitative approach to study the lived experiences of peer-mentors in the PMI. To capture a longitudinal and holistic perspective of peer-mentoring experiences, we employ a semi-structured interview questionnaire and peer-mentoring diaries. We analyzed the data from the semi-structured interviews and diaries using Krishna’s Systematic Evidence Based Approach (SEBA).In advancing a balanced, accountable and reproducible study and analysis, an expert team consisting of a librarian from the National University of Singapore’s (NUS) Yong Loo Lin School of Medicine (YLLSoM) and local educational experts and clinicians at YLLSoM, National Cancer Centre Singapore, Palliative Care Institute Liverpool, and Duke-NUS Medical School navigated the stages of SEBA Fig. .Fig. 4ThEligible participants comprised of peer-mentors in the PMI who had completed mentoring programs as mentees and had been subsequently selected, trained and completed PMI mentoring programs as peer-mentors. Purposive sampling was conducted and email invitations containing a participant information sheet and consent form were sent out. The invitations stressed the participant’s rights to privacy and anonymity, as well as their right to withdraw from the study at any point without prejudice. All participants provided written and verbal informed consent.Individual semi-structured interviews were arranged with each peer-mentor upon return of the endorsed consent forms. These 30–45-min audio-recorded interviews conducted over the Zoom video conferencing platform took place in quiet offices that ensured privacy to facilitate in-depth exploration of personal belief, experiences and practices. The interviews were carried out between February and May 2021 by experienced and trained interviewers, AP and CQWL. As non-clinicians with neither previous interactions nor dependent relationships with the participants, the trained interviewers sent out the email invitations and arranged the meetings. This enhanced the participant anonymity from the research and expert teams. Audio recordings were transcribed verbatim using the NVivo 12 Software, anonymized and their integrity verified.Ethics approval (reference number: 202010–00084 and 202103–00057) was obtained from the Singhealth Combined Institutional Review Board. Informed written and oral consent was obtained from all participants.Peer-mentoring diaries were hosted on Google Forms and were completed by all mentees and peer-mentors in the PMI on an ad-hoc basis between March to December 2021. Mentoring diaries were anonymized by independent research team members not involved in the PMI or the semi-structured interviews for analysis.Three independent teams, each guided by a senior trained PMI mentor, carried out the analysis of the anonymized data. The first and second teams employed Braun and Clarke ’s approaAt independent and regular online discussions, Sandelowski and Barroso ’s approaThis process combined overlapping themes and categories to create themes/categories.The themes/categories from the mentoring diaries and interviews were combined and funneled to create domains that frame the discussion in Stage 8 .Twelve peer-mentors participated in the study interviews, and a further eight peer-mentors completed the peer-mentoring diaries. Table Independent analysis of the interviews with 12 peer-mentors and 8 peer-mentor diaries revealed two domains—the PMI as a Community of Practice, and Identity Formation.The PMI displays features of a CoP described by existing literature –46. FirsThe PMI’s strong sense of community, welcoming environment, culture of ‘paying it forward’, and shared belief systems create the CoP’s culture whilst the mentoring umbrella-based support strengthens a peer mentor’s core values of responsibility, teamwork, empathy, respect, integrity and commitment, alongside their shared belief system and identity.Progress along the PMI’s mentoring trajectory guided by the mentoring umbrella-based support within the clear boundaries of the PMI’s structure and nurturing mentoring environment supports the Socialisation Process. This helps peer-mentors integrate the PMI’s belief system and indeed apply them in the program. This bolsters personal and professional development as peer mentors are encouraged and challenged to adopt new roles and responsibilities.In toto, the PMI’s sense of community, structured approach, support mechanisms and fostering of personal and professional development solidify its position as a CoP. Table Peer mentors exhibit features of PIF in their efforts to attend to resonance, synchrony, dyssynchrony and disharmony. Resonance, for instance, is apparent when peer mentors find that the values, beliefs, principles, and goals introduced in the PMI echo that of their prevailing belief systems. Resonance motivates continued engagement in the PMI and sees peer mentors reshape and reprioritise their regnant values, beliefs and principles to reflect synchrony and inspire action. Here, peer mentors show personal growth in taking on new endeavours not just for their individual self-development, but to find meaning in serving the wider community. However, peer-mentors also encounter obstacles in their PMI journey, alerted by their ‘sensitivity’ to instances of disharmony and dyssynchrony. Disharmony manifests when peer mentors reveal a mismatch between their prevailing skillsets and new responsibilities whilst managing ‘difficult’ mentees who miss deadlines or produce inadequate work heightens dyssynchrony.Dyssynchrony, disharmony, resonance, and synchrony may arise as an acute ‘event’, such as the rejection of a white-listed journal submission (P10) or dealing with ‘difficult’ mentees (P11). The precipitating ‘event’ may also be a slow process simmering or persistent issues such as dealing with a lack of ‘confidence’ (P5) and self-belief (P2), contending with changing perspectives of oneself and roles (P2), balancing competing demands on time and maintaining a work-life balance (P3).How and if dyssynchrony, disharmony, resonance, and synchrony are to be addressed is determined by the peer-mentor’s ‘judgement’ that accords attention, significance and urgency to the issue. Often, ‘judgement’ is perceived as a matter of goal setting, prioritising tasks, time and roles and managing expectations. Upon determining the magnitude of the situation, the peer mentor’s motivation to make adaptations to their self-concepts of identity pivots on their ‘willingness’. This is depicted as part of the peer-mentor’s responsibility (P6), accountability (P11), goals (P7) and aspirations (P4).Peer mentors also exhibit ‘balance’ in determining the changes to be carried out and prioritised in making adjustments to their self-concepts of identity. The process of balancing is also impacted by the speed in which events occur, the peer-mentor’s ‘sensitivity’, ‘judgment’ and ‘willingness’ and if there are further changes in the peer-mentor’s circumstances. This then culminates in peer mentors practicing ‘identity work’ that sees the integration of new work practices, values, beliefs and principles, ‘sensitivity’, ‘judgment’, ‘willingness’ and ‘balance’ to create context-specific self-concept of identity.Table This study reveals that a structured program like the PMI does act as a CoP supporting the socialisation process capable of nurturing PIF amongst peer-mentors. Through the lens of the KPM and RToP, it is possible to appreciate the impact of resonance, synchrony, dyssynchrony, and/or disharmony on belief systems and its effects upon the conduct and practice of peer-mentors as they progress along the mentoring trajectory set out Fig. .Fig. 5ThDependent on the peer-mentor’s individual experience, capabilities, goals, abilities, and availabilities, it is common that the course taken by new peer-mentors deviates from the ideal (blue line). Here, the role of the mentoring umbrella helps them stay on course and as close to the ideal trajectory. However, as the data suggests, changes in the peer-mentor’s personal, psycho-social, financial, clinical, research, academic, practice and/or psycho-emotional situation may lead to a significant ‘event’. Here, the presence of regular stage-based assessments help direct further personalised and appropriate support to peer-mentors. If effective, these interventions bring the peer mentoring trajectory back towards the ideal trajectory. It is posited that with these interventions, peer-mentors will eventually achieve their goals.Our data also reveals a further reason for deviations from the ideal mentoring trajectory. Peer-mentors appear to be simultaneously involved to varying degrees in different roles in concurrent projects. Whilst concurrent participation in multiple mentored programs within the PMI may impact progress in their primary project, evidence of multiple frequently overlapping programs within the PMI casts the PMI as a collection of projects, each functioning as a CoP. This implications of a “complex landscape of different communities of practice” invites the idea of the PMI being a Landscape of Practice (LoP) , 46–56 aFor peer-mentors involved in several concurrent projects (multi-membership), often in different capacities in a LoP, ‘events’ have wider connotations and are subject to wider influences. Here, subtle differences even amongst projects built on a common PMI belief system may be problematic due to possible differences in the support and advice provided by various mentors. This underscores the need for longitudinal and holistic evaluation of peer-mentor’s progress.On the surface, being a LoP underlines the importance of the PMI, ensuring consistency in the culture, goals, belief systems and structure within projects/CoPs. It also stresses the importance of ensuring that PMI mentors are effectively trained and supported, and that there is alignment of expectations; timely, stage-based and appropriate assessments; oversight; and seamless support across the PMI. On a deeper level, however, it does shift the manner in which progress is viewed within the PMI. Indeed, it may be suggested that progress within multiple PMI projects may be viewed as the peer-mentor’s overall progress towards refining their PIF Fig. .Fig. 6CoWith the KPM increasingly proposed as an evidence-based approach to better understand PIF in mentoring and potentially in medical education, ensuring that the KPM is clinically relevant is critical. The notion of the PMI being a LoP thus inspires a change in the manner that we view Krishna-Pisupati’s theoretical model Fig. .Fig. 7ThSituated within the LoP which also captures the wider contextual and environmental considerations, the advanced KPM is still focused upon the PIF of an individual. As a result, there remains due acknowledgment of the influence of the individual’s narratives. ‘Sensitivity’, ‘judgment’, ‘willingness’, and ‘balance’ leading up to the creation of working hypothesis for a context-specific identity that can straddle all the PMI projects now also considers the peer-mentor’s ability and responsiveness, as well as the notion that events need not be singular nor from one source, but from multiple sources. Events may also exhibit differing levels of importance and exigency. This underscores the importance of continued guidance and support of peer-mentors as they rank ‘events’ in terms of importance and significance.Experimenting with the working hypothesis or ‘adapted’ professional identity and belief systems is guided by available support, culture and structure of the CoPs and the LoP, as well as regnant codes of practice, boundaries, expectations, roles and responsibilities set out by the LoP and CoPs.Although insightful, this qualitative approach built upon the RToP has not been validated and is subject to bias interpretations by the authors. Moreover, although supplemented by mentoring diaries, the use of interviews as the primary source of data in this study remains as ‘snapshots’ or retrospective accounts that are susceptible to recall bias. There are also limitations due to the small sample size and the limit of the depth of the data collected.Whilst the categories drawn from directed content analysis emphasised the features of CoPs in the PMI and complemented the themes identified in Identity Formation, the Split Approach and Funnelling Processes are time and resource-heavy and could threaten the sustainability of the study. Similarly, use of independent team analysis and the Split Approach may not have fully attenuated the risk of bias.This study suggests that a structured mentoring program can shape professional identity in a consistent manner as long as there is due consideration for the needs of the peer-mentor and the influence of environmental factors. The impact of environmental factors is multiplied when multi-membership is present and underlines the need for portfolio use that will not only assess their competency, but their PIF and promote the use of reflective practice and mentoring diaries. This then must be an area for future study.Similarly, whilst this study will be of particular interest to program designers, host organizations and senior mentors, it does reiterate the need for host organizations to ensure careful stakeholder selection, training, assessment, and support; effective alignment of expectations; longitudinal support and assessment of the mentoring, communication, assessment, and oversight mechanisms within the program; and careful curation of the mentoring culture and structure. This underlines the need for effective program evaluations. These gaps in appraisals ought to be the focus of future studies as we plan to extend the PMI beyond Palliative Medicine.


Introduction: Acute myeloid leukemia (AML) is a heterogeneous myeloid malignancy with abnormal molecular diversity. Tissue kallikrein 2 (KLK2) is a kind of serine protease, and has a close relationship with the occurrence and development of malignant tumors. Single nucleotide polymorphism (SNP) of various genes are associated with susceptibility, treatment and survival of AML.Methods: We investigated the association of KLK2 SNPs rs198977 and rs2664155 with AML. We recruited 284 AML patients and 280 healthy controls from the Han population and genotyping KLK2 SNPs rs198977 and rs2664155 by MassARRAY system.Results: Using clinical data from AML patients and controls, including AML susceptibility, blood count, risk stratification, response to induced chemotherapy and survival, our results showed an increased risk of AML susceptibility with KLK2 rs198977 TT genotype in the recessive model. Regarding white blood cell counts in AML patients, the results showed an increased risk of hyperleukocytosis with the TT genotype of KLK2 rs198977 in a codominant model. Moreover, in the recessive model, AML with KLK2 SNPs rs198977 TT genotype had an increased risk of hyperleukocytosis. No significant correlation was found between KLK2 rs2664155 and AML.Discussion: This study suggests that KLK2 rs198977 may be an important genetic factor in the occurrence of AML and hyperleukocytosis in AML, providing a new perspective for disease progression and new therapeutic targets. Acute myeloid leukemia (AML) is a heterogeneous malignant tumor originating from hematopoietic stem cells, characterized by clonal proliferation, arrest of apoptosis and failure to differentiate of tumor cells . The divTissue kallikrein 2 (KLK2) is a kind of serine protease, which exists in various tissues and biological fluids. KLK2 has a variety of physiological functions, involved in physiological adaptation, physiological metabolism and regulation of brain development, and has a close relationship with the occurrence of a variety of malignant tumors . Recent To further explore the potential unique etiology of AML, we investigated the association of two important SNPs sites of KLK2 with AML, in 284 AML cases and 280 healthy controls. We analyzed the association between SNPs and disease susceptibility, blood routine, risk stratification, response to induction chemotherapy and survival of AML. Our study seeks to provide new insights into the molecular biological heterogeneity of AML patients and new potential therapeutic targets.9/L in AML patients. We used the 2017 European Leukemia Network (ELN) criteria to assess the assessment of post-chemotherapy response in AML  with a median age of 49 years (13–87 years) were recruited from the Department of Hematology, Qilu Hospital of Shandong University from May 2011 to December 2021. The diagnosis of AML patients was confirmed by the National Comprehensive Cancer Network (NCCN) guidelines (version 3.2017) . Hyperlee in AML . And 280The genomic DNA in the study was isolated from bone marrow mononuclear cells (BMMCs) by the TIANamp Blood DNA Kit, following the manufacturer’s instructions during the procedure . We measured the extracted DNA concentration with a spectrophotometer . SNP genotyping was analyzed using Sequenom iPLEX and ffight (MALDI-TOF) MassARRAY platform based on matrix-assisted laser desorption/ionization time . The platform is based on multiplex polymerase chain reaction (PCR) reactions, site-specific single base extension reactions, and MALDI-TOF spectroscopy. The primers of HK2 rs198977 and rs2664155 were designed as follows:KLK2 rs198977 forward: ACGTTGGATGAAAAGCCTGCTGTGTACACC.KLK2 rs198977 reverse: ACGTTGGATGTGCGATGGTGTCCTTGATCC.KLK2 rs2664155 forward: ACGTTGGATGTGGAGAGAGGTCCTTGAAAG.KLK2 rs2664155 reverse: ACGTTGGATGCCAGCTGCTTTACTAAAGAG.p values <0.05 were defined as statistically significant.SPSS 20.0 software  was used for statistical analyses. Hardy-Weinberg equilibrium (HWE) of KLK2 rs198977 and rs2664155 genotype in control group was determined using Pearson’s goodness-of-fit chi-square test. The 4 models  were used to analyze genotyping data. We compared the distribution of KLK2 SNPs with AML sensitivity, white blood cell count, hemoglobin, platelet count, risk stratification, response to induction chemotherapy, and prognosis using chi-square test or Fisher exact test. The univariate binary logistic regression analysis was used to analyze odds ratios (ORs) with corresponding 95% confidence intervals (95%CI), adjusted for age and sex. The overall survival (OS) was estimated by Kaplan-Meier curves. Two-tailed 9/L, with 77.0 (21.0–148.0) g/L of the median hemoglobin and 38.5 (2.0–235.0) × 109/L of the median platelet. Of all AML patients enrolled in this study, 60 cases were low-risk of cytogenetics and molecular stratification, 147 cases were intermediate-risk, and 77 cases were high-risk. Among AML patients who received induction chemotherapy, 108 achieved complete response (CR) after the initial course of induction chemotherapy. Two SNPs of KLK2 (rs198977 and rs2664155) were consistent with HWE of healthy controls (p > 0.05).The clinical and demographic details of AML patients and controls are presented in p = 0.039, p = 0.594, p = 1.000, p = 0.499, p = 0.807, p = 0.031, We used 4 gene models to analyze the relationship between KLK2 SNPs (rs198977 and rs2664155) and AML susceptibility. First, we applied the chi-square test or Fisher exact test for screening, and the results show that recessive genotypic of KLK2 rs198977 was significantly associated with AML susceptibility  and disseminated intravascular coagulation (DIC). Patients with Hyperleukocytosis of AML often have poor prognosis. We analyzed the relationship between KLK2 SNPs and hyperleukocytosis. Among the recruited AML cases, 55 cases had hyperleukocytosis at diagnosis and 229 cases did not have hyperleukocytosis. After initial screening by chi-square test or Fisher exact test, we found that codominant and recessive of KLK2 rs198977 were related to hyperleukocytosis  between the high and low HGB groups  (p = 0.418)  . The med= 0.418) .In this study, 284 AML patients (non-M3) and 280 healthy controls were recruited from Han Chinese population to investigate the relationship between two SNPS of KLK2 (rs198977 and rs2664155) and the pathogenesis, treatment, and prognosis of AML. Interestingly, our results revealed that KLK2 rs198977 polymorphism was obviously related to AML susceptibility under the TT genetic models. Moreover, AML patients with rs198977 genotype TT have an increased risk of developing hyperleukemia. However, the results of this study showed that KLK2 SNPs (rs198977 and rs2664155) were not associated with risk stratification, response to induction chemotherapy, and survival of AML. Our results suggest that KLK2 may be involved in the pathogenesis of AML and its complication hyperleukocytosis.KLK2 is one of the members of the human kallikreins family, which consists of 15 family members (KLK1 - KLK15) . KLK2 is9/L are called hyperleukocytosis of AML or hyperleukocytic AML (HAML). AML patients with hyperleukocytosis account for about 18% of AML. AML patients with hyperleukocytosis have a serious condition and a poor prognosis, and often die of various serious complications, such as leukostasis, tumor lysis syndrome (TLS) and disseminated intravascular coagulation (DIC) (AML patients with WBC count greater than 100 × 10on (DIC) . Althougon (DIC) . HoweverIn this study, we analyzed the relationship between two important SNPs of KLK2 (rs198977 and rs2664155) and blood count in AML patients. The TT genotype of KLK2 rs198977 was associated with an increased incidence of hyperleukocytosis in AML. Therefore, KLK2 gene may be related to the occurrence of hyperleukocytosis in AML. The specific mechanism leading to the proliferation of leukemia cells remains unclear. However, the hyperleukocytosis of AML must be related to the extreme proliferation and blocked apoptosis of leukemia cells. It was found that KLK2 combined with androgen receptor cofactor (ARA)70 plays an important role in promoting tumor cell proliferation and inhibiting apoptosis . The comOverall, this study identifies a new risk SNP that may serve as a risk biomarker for AML and hyperleukaemia, and it may contribute to new personalized targeted drug therapy regimens for AML. However, it is important to validate these results in a larger sample size of AML and healthy controls.In this study we found that the TT genotype of KLK2 SNP rs198977 was associated with increased risk of AML susceptibility. Moreover, the TT genotype of KLK2 SNP rs198977 was associated with a higher incidence of AML hyperleukocytosis. For the first time, our results show that the genetic variants of KLK2 are associated with susceptibility and hyperleukocytosis in AML. This study provides a new perspective on the pathogenesis of AML and hyperleukocytosis, and provides a new direction for exploring its targeted therapy. Due to the heterogeneity of AML patients and the limited number of patients in this study, the results need to be further confirmed in larger studies.


Due to its high efficacy agonist properties, [18F]F13640 binds preferentially to functional 5-HT1A receptors, which are coupled to intracellular G-proteins. Here, we characterize brain labeling of 5-HT1A receptors by [18F]F13640 in humans and describe a simplified model for its quantification.F13640  is a highly selective 5-HTn = 9) and test–retest protocol (n = 8). Several kinetic models were compared ; two models with reference region were also evaluated: simplified reference tissue model (SRTM) and the logan reference model (LREF).PET/CT and PET-MRI scans were conducted in a total of 13 healthy male volunteers (29 ± 9 years old), with arterial input functions (AIF) (18F]F13640 showed high uptake values in raphe nuclei and cortical regions. SRTM and LREF models showed a very high correlation with kinetic models using AIF. As concerns test–retest parameters and the prolonged binding kinetics of [18F]F13640, better reproducibility, and reliability were found with the LREF method. Cerebellum white matter and frontal lobe white matter stand out as suitable reference regions.[18F]F13640, its high receptor specificity and its high efficacy agonist properties open new perspectives for studying functionally active 5-HT1A receptors, unlike previous radiopharmaceuticals that act as antagonists. [18F]F13640’s kinetic properties allow injection outside of the PET scanner with delayed acquisitions, facilitating the design of innovative longitudinal protocols in neurology and psychiatry.The favorable brain labeling and kinetic profile of [Trial Registration EudraCT 2017–002,722-21.The online version contains supplementary material available at 10.1007/s00259-022-06103-1. The objectives of the present study were, therefore, to perform a full PET kinetic modeling of [18F]F13640 using arterial input function (AIF), to identify a reference region suitable for a simplified modeling method, and to assess reproducibility with test–retest scans. Ultimately, the present data characterizing [18F]F13640 as a selective agonist radiopharmaceutical will, for the first time, enable the investigation of changes in both 5-HT1A receptor expression and functionality in patients suffering from disorders arising from dysfunctional serotonergic neurotransmission.Very few 5-HTtagonist , and it oceptors . These soceptors , 19. In l models , 21. AltF]F13640 , it rema18F]F13640 (3-chloro-4-fluorophenyl)-[4-18fluoro-4-[[(5-methylpyridin-2-yl)methylamino]methyl]piperidin-1-yl]methanone; a.k.a. [18F]NLX-112 or [18F]befiradol) was synthesized as previously described F13640 activity curves slowly continued to vary even at later time points, suggesting that there may be further changes beyond 90 min. A PET-MRI acquisition of almost 4 h was tested to observe kinetics for a longer period. For the comfort of the subjects, the acquisition was carried out in two parts. In part 1 (PET1), list-mode PET data were acquired for 90 min directly after the injection of [18F]F13640 (150 MBq + 1 MBq/kg ± 10%) . Subjects were then taken out of the camera for a break. One hour later, for part 2 (PET2), subjects had a second PET-MRI scan for 75 min . Participants performed this PET-MRI protocol twice (test and retest sessions) 1 to 9 weeks apart.Participants underwent a PET-MRI acquisition on the Siemens mMR Biograph system. Because the pilot study group scans showed that F13640 were separated, and fractions were collected and counted in the gamma counter. The activity of the [18F]F13640 fraction was divided by the total activity recovered from the gamma counter to give the plasma parent fraction of unmetabolized [18F]F13640 (PPf). For plasma free fraction (fp), 1 mL of plasma was centrifugated  for 20 min, 2000 g at 20–25 °C, and 100 µL of ultrafiltrate plasma was counted in a gamma counter. After counting, all samples were weighed, and counts were corrected. The fp was calculated from the ratio of concentrations in the ultrafiltrate and whole plasma. The AIF was derived from Cp (t) according to: AIF (t) = PPf (t).Cp (t).fp (t).Four participants underwent AIF measures during their test or retest session. AIF, free fraction, and metabolites were measured based on 29 blood samples. Samples were collected manually after arterial catheterization with local anesthesia (lidocaine patch 5%). Sample time points were as follows: every 5 s in the first minute, every 15 s in the second minute, every 30 s in the third minute, and at time 5, 10, 20, 30, 40, 60, 75, 90, 160, and 205 min. Whole blood radioactivity was counted on every sample using a gamma counter (Perkin-Elmer) to measure the whole blood curve (Cwb). Plasma was collected and counted after centrifugation  on 18 out of 29 samples (PET (t)) were modeled with three different AIF models for participants who performed AIF (n = 4): one-tissue compartment (1TC), two-tissue compartment (2TC), and the Logan graphical method (LOGAN) F13640 for a retest session that was slightly lower than the recommended dosages described in the study. Details are presented in Table Mean age of the participants was 29 ± 9 years in the test–retest study. No significant differences were found in the activity of the 8FF13640 A0 = 0.046 and T = 9.06 minMean plasma parent fractions were 99.40% ± 0.00%, 97.40% ± 0.01%, 96.20% ± 0.01%, 95.40% ± 0.01%, and 95.30% ± 0.01% at 2, 10, 30, 90, and 205 min respectively after injection. One value was discarded for participant 2 at 90 min due to non-interpretable value. Mean plasma parent fraction was modeled with a one-exponential function:Free plasmatic fraction was not constant over time and mean values were 0.48% ± 0.09%, 0.82% ± 0.08%, 1.14% ± 0.26%, 1.90% ± 0.71% and 1.61% ± 0.29% at 2, 10, 30, 90, and 205 min. Ratio plasma to whole blood was stable over time and mean value was 1.79 ± 0.03. Table 18F]F13640 when comparing the AIC but results in nonsensical numerical kinetics parameters (k3 and k4). Thus, the 1TC model was considered as the reference model for the results presented below. Fitted curves for 1TC and 2TC modeling are presented in online resource 1  was 320.88 ± 13.66 and 291.92 ± 25.46 for the two-tissue compartment model (2TC). Two-tissue compartment model better fits with the pharmacokinetic of F13640 which, thanks to its agonist properties, permits in vivo imaging of functional human 5-HT1A receptors. Several analyses were carried out: (i) full kinetic modeling of the radiopharmaceutical using dynamic scans and arterial blood sampling; (ii) comparison of four different reference regions and evaluation of two simplified modeling methods to measure binding potential values and obtain parametric images; and (iii) assessment of test–retest reliability for simplified models. The main finding is that [18F]F13640 constitutes a 5-HT1A receptor radiopharmaceutical with favorable brain binding properties, and the present study describes a PET acquisition protocol and a quantification method suitable for the clinical study of functional 5-HT1A receptors in neurology and psychiatry.In this study, we describe the first-in-human trial of a new PET radiopharmaceutical [18F]fallypride [18F]F13640 has a high affinity for the target sites, the low tissue clearance k2 observed on kinetics could be interpreted as a very low dissociation rate on 5-HT1A receptors. On the basis of these preliminary observations, we concluded that kinetic modeling of the tracer and parameters identification would require a longer acquisition protocol, which was then implemented for the main study. A 165-min long PET acquisition was implemented with a first scan lasting 90 min and a second one lasting 75 min, separated by a 1-h break outside of the camera. This longer protocol with two acquisition periods was facilitated using a hybrid PET-MR scanner, which is capable of brain anatomical realignment and motion correction between PET acquisitions.Initial tests used a protocol with 90 min PET/CT scans and AIF for study participants. However, preliminary analyses of the 5 first subjects revealed a slow washout from cerebral tissues of the radiopharmaceutical, a characteristic that has also been observed for some other radiopharmaceuticals, such as [llypride . This obllypride . Since [18F]F13640 were similar to those found with F13640  at pharmacological doses. Thus, the fraction of [18F]F13640 parent compound remained high (95%) in blood at 30 to 205 min after injection, consistent with observations on unlabeled NLX-112 in healthy volunteers . The ratio of [18F]F13640 binding in plasma to whole blood showed favorable results with a higher distribution in plasma (1.79 ± 0.03). Also, the fp of about 2% was similar between NLX-112 and [18F]F13640. However, we observed unreasonably high K1 values since they were higher than cerebral blood flow investigated with [15O]H2O PET F13640 since CERWM is known to have very low 5-HT1A receptors expression F13640 binding patterns, as discussed below.Overall, these results lead us to favor the LREF model with CERWM as reference region for future studies comparing patients to healthy volunteers or patients at different times. The low variability and high reliability (ICC > 0.7) of the LREF model with CERWM as reference region guarantee the power of future studies and the capacity to detect differences between groups due to pathological changes rather than inter-participant or protocol variability. However, the SRTM modeling method and FLWM as an alternative reference region are not to be totally discarded. Indeed, the choice of the modeling method and of the reference region will be driven by the pathology in the study. As an example, cerebellar atrophy could be a confounding factor when using CERWM as a reference region and FLWM would be a suitable alternative. In the present study, the LREF method with CERWM as reference region was used to evaluate F13640 with moderate BP values. Median and dorsal raphe nuclei were regions with the highest BP values but also with the most substantial variability. This can be explained by the fact that the raphe nucleus is the only region that is not defined on the basis of anatomical data but by functional data F13640 in the cerebellum could thus question its target specificity, but an interaction by the radiotracer with a possible off-target seems unlikely based on well-documented in vitro studies on F13640 [18F]F13640 binding in the cerebellum in different species [1A receptor agonist (8-OH-DPAT) or of an antagonist (WAY-100635) inhibited [18F]F13640 binding, indicating that its binding in cerebellum grey matter specifically reflects 5-HT1A receptor expression in this region. In addition to the present data with [18F]F13640, the assumption of a total lack of 5-HT1A receptors in the cerebellum was already challenged by previous PET studies revealing [carbonyl-11C]WAY-100635 binding in human cerebellum CUMI-101 which showed a significant reduction of labeling following 8-OH-DPAT or WAY-100635 pre-injection F13640.As described above, cerebellum grey matter and vermis both showed high BP values. Whereas a high expression of 5-HTn vermis , 37, it  studies , 39, 5-Hn adults . The pren F13640 . Moreove species , 22 and, WAY-1006 inhibiterebellum –43. Othenjection . Taken t1A radiopharmaceuticals, [18F]F13640 showed particularly high uptake in raphe nuclei, cortical regions, and vermis in limbic and paralimbic regions. Although [18F]MPPF also showed high uptake in raphe nuclei, it preferentially binds to limbic areas such as the hippocampus and amygdala whereas cortical regions showed less uptake [carbonyl-11C]WAY-100635, also showed high binding in the raphe and frontal cortex (like [18F]F13640) but it also showed marked binding to the hippocampus (like [18F]MPPF) [carbonyl-11C]WAY-100635, [18F]MPPF and [18F]F13640 are likely to be due to pharmacological differences between these 5-HT1A receptor radiopharmaceuticals [18F]F13640, express a high proportion of G-protein uncoupled 5-HT1A receptors. In contrast, regions with higher [18F]F13640 binding values, such as the cingulate cortex, frontal cortex, or vermis, may express a relatively higher proportion of functionally active, G-protein-coupled receptors. Finally, regions such as raphe which showed high binding with both antagonist and agonist radiopharmaceuticals may express both states of 5-HT1A receptors. Thus, differences in receptor binding patterns can be found between agonists and antagonists radiopharmaceuticals targeting the same receptor. An in vitro study using [18F]F13640 and [18F]MPPF already explored this hypothesis and showed differences in binding between agonists and antagonists radiotracer with 5-HT1A in Alzheimer’s disease NPA, showed a greater extent of than the antagonist [11C]raclopride binding in a rat Parkinson’s disease model F13640 has now the status of a radiopharmaceutical whose novel pharmacokinetic and radiopharmacological characteristics offer new perspectives in neurology and psychiatry.Based on the above observations, it can be considered that [18F]F13640 will allow studies to be performed with multiple acquisitions at different late time points following the same injection. This experimental paradigm could, for example, allow exploration of changes in receptor coupling states associated with circadian rhythms. As an illustration, a previous preclinical study demonstrated that [18F]F13640 is sensitive to fluctuations in serotonin levels F13640 binding using the LREF method showed excellent test–retest parameters, a profile which makes it attractive for studies involving repeated measurements on the same subject, including drug occupancy and intervention studies, or for exploring pathological states at different time points—situations where reproducibility and reliability of the PET measurements is crucial.As demonstrated in the present study, [18F]F13640 opens the way to informative imaging studies in neurology and psychiatry. For example, [18F]F13640 shows high binding values in the raphe, consistent with the therapeutic-like activity of 5-HT1A receptor agonists in rodent and non-human primate models of L-DOPA-induced dyskinesia (LID) in Parkinson’s disease [18F]F13640 also shows high binding values in cortical regions associated with antidepressant activity or control of negative symptoms of schizophrenia [1A receptors in the brainstem are also promising targets to alleviate respiratory dysfunction in disorders such as Rett syndrome [18F]F13640 could therefore be a promising tool to assess the role of 5-HT1A receptors in a variety of different disorders involving serotonergic mechanisms F1chanisms .18F]F13640 as the first 5-HT1A receptor agonist usable as a PET radiopharmaceutical. [18F]F13640 shows many favorable radiopharmacological and radiopharmaceutical characteristics: radiolabeling with fluorine-18, high selectivity over cross-reacting sites, high reproducibility, and long-term binding which facilitates the experimental protocols. [18F]F13640 shows pronounced binding in raphe nuclei and cortical regions, with notable differences in comparison with classical antagonist PET radiopharmaceuticals. All these characteristics confirm the interest of developing an agonist radiotracer able to specifically target functionally active 5-HT1A receptors in studies with long-term scans and test–retest protocols in order to investigate disease states in neurology and psychiatry.The present study reports the first-in-human validation and full kinetic modeling of [Supplementary file1 (PDF 453 KB)Below is the link to the electronic supplementary material.