The ability of HCMV to reactivate from latency is exquisitely linked to changes in cell signaling, which result in HPC differentiation. The Wnt/β-catenin pathway is tightly linked to CD34+ HPC homeostasis and differentiation. The majority of the viral and cellular factors involved in the maintenance of HCMV latency and reactivation are still unknown. Our group previously discovered a viral hematopoietic cytokine (pUL7) that promotes cellular differentiation and viral reactivation. Here, we show that the UL7-related RL11 family member UL8 is also required for efficient viral reactivation in CD34+ HPCs by interacting with components of the (Wnt)/β-catenin pathway. Pull-down experiments demonstrate that UL8 and β-catenin interact with Dishevelled-2 (DVL2) through their PDZ-binding domains, and this interaction promotes β-catenin stabilization and transcriptional activation. Disrupting the interaction among UL8, β-catenin, and DVL2 blocks HCMV reactivation in vitro and in vivo, similar to deletion of UL8. This is the first observation that Wnt signaling components are essential for HCMV reactivation, unlike α- and γ-herpesvirus that require an active Wnt pathway to maintain latency. Detailed understanding of how the Wnt network is manipulated by herpesviruses will improve our ability to develop targeted therapeutics.Human cytomegalovirus (HCMV) is a species-specific virus that establishes a persistent/latent infection in CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/β-catenin pathway as an important component of viral reactivation. We further define that pUL8 and β-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with β-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.CD34 Latic cells . The mecDrosophila, are necessary for proper function and development of hematopoietic cells  impairs viral reactivation. We also determined that the formation of the UL8-β-catenin-DVL2 complex promotes β-catenin stabilization and transcriptional activation. These results indicate that UL8 contribution to β-catenin stabilization and transcriptional activation is essential to activate the Wnt/β-catenin pathway to promote viral reactivation.In the current study, we observed that deletion of UL8 impairs reactivation + HPCs were infected with HCMV WT or ΔUL8 for 48 hours, and viable CD34+ GFP+ cells were sorted by fluorescence-activated cell sorting (FACS), which were then seeded into long-term bone marrow culture (LTBMC) over stromal cell support (P < 0.0001). Analysis of viral DNA in both HCMV WT- and HCMVΔUL8-infected CD34+ HPCs at 14 days post-infection (dpi) indicated similar amounts of HCMV genomes in both cell populations (+ HPCs are engrafted into NOD-scid IL2Rγcnull mice (huNSG) followed by HCMV infection , similar to ΔUL8-infected cells .To determine whether, like UL8, signaling through the Wnt/β-catenin pathway is required for efficient viral reactivation, we treated HCMV-latently infected CD34ed cells . ReducedOverall, these results provide evidence that HCMV requires an active Wnt/β-catenin pathway to efficiently reactivate from latency and suggest a key role for UL8 in the modulation of the pathway through the interaction with the effector β-catenin.The pUL8 22-aa cytoplasmic tail has a PDZ-binding domain [DTEL (aa 321 to 324aa)] completely conserved among all strains of HCMV . β-CatenThese results indicate that UL8 and β-catenin form a complex with DVL2 through their PDZ-binding domains and that the Pen-N3 peptide can efficiently target and disrupt these interactions.Previous studies on HCMV and β-catenin have shown that during lytic replication, levels of β-catenin that are membrane-associated, in cytosolic pools, and in nuclear fractions, are reduced after infection . ConversP = 0.0103) and UL7 (P = 0.0114) controls. Moreover, interaction between UL8 and β-catenin is required to promote stabilization of the protein as demonstrated by the decrease levels of active β-catenin in cells transduced with the UL8-mutDTEL construct compared to full length UL8 (P = 0.0403).We next investigated if UL8 had an impact on active β-catenin levels outside of the context of HCMV infection. As shown in c-myc and survivin (Birc5). We transduced CD34+ HPCs with Ad-GFP, Ad-UL8, or Ad-UL8-mutDTEL and FACS isolated a pure population of viable CD34+ GFP+ cells to determine the effect of UL8 on c-myc and Birc5 mRNA levels. As shown in c-myc and Birc5 mRNA levels were increased compared to the control GFP . Mutagenesis of the PDZ-binding domain significantly decreased UL8-mediated transcriptional activation of c-myc (P = 0.0002) and Birc5 (P = 0.0077). No significant difference was found between β-catenin (Ctnnb1) mRNA levels in CD34+ HPCs transduced with Ad-UL8 or Ad-UL8-mutDTEL compared to Ad-GFP, indicating that regulation of β-catenin occurred at the protein, rather than at the transcriptional level. Importantly, a similar change in transcriptional levels of c-myc and Birc5 was observed in CD34+ HPCs stimulated with Wnt3A , suggesting that the stabilization of β-catenin by UL8 in CD34+ HPCs is functionally relevant.To test whether increased levels of active β-catenin had functional consequences, we examined expression of Wnt target genes + HPCs.Taken together, these data indicate that interaction between β-catenin and UL8 promotes stabilization and transcriptional activation of β-catenin-dependent genes in CD34+ HPCs with Pen-N3 peptide at the time of reactivation. As shown in P = 0.0131) decreased the ability of the virus to reactivate. The reduction in reactivation was not due to an inhibitory effect of the peptide on lytic replication or a cytotoxic effect as demonstrated by the single and multistep growth curves and cytotoxicity assay . We then generated a UL8 mutant virus in which the DTEL sequence was mutated into four alanine residues (DTEL>AAAA), and a V5-tag was added at the C-terminus (UL8-mutDTEL). We first performed UL8 pulldowns in fibroblasts infected with WT and then treated with Pen-N3 or DMSO or UL8-mutDTEL mutant virus. As shown in P = 0.02) and decreased levels of DVL2 (P = 0.01). Treatment with Pen-N3 peptide also reduced active β-catenin and DVL2 levels compared to WT . Lastly, we performed latency and reactivation assays in CD34+ HPCs infected with WT or UL8-mutDTEL virus in vitro and in vivo. As shown in P = 0.0047) similarly to the Pen-N3 peptide. Analysis of UL8-mutDTEL growth curve  and genomes  demonstrated that the lack in reactivation was not due to reduced growth capacity of the mutant compared to WT or inability to retain the genome during latency. Furthermore, infection of huNSG mice with UL8-mutDTEL virus resulted in reduced levels of viral DNA (P = 0.0025) in the spleen of the animals treated with G-CSF and AMD3100 compared to WT  approach, we discovered that UL8 is in proximity to several proteins in the Wnt signaling pathway, including β-catenin and DVL2. Immunoprecipitation analysis verified the interaction between UL8, β-catenin, and DVL2. In addition, we have shown that UL8 is required for β-catenin stabilization and transcriptional activation. Finally, we observed that the binding of UL8 and β-catenin to the DVL2 PDZ domain is required for efficient viral reactivation. Taken together, HCMV manipulates the Wnt/β-catenin pathway for efficient viral reactivation, and UL8 plays a critical role in the stabilization and activation of the effector of the pathway, β-catenin.In this study, we demonstrated for the first time that activation of Wnt/β-catenin signaling by UL8 is important for reactivation of HCMV in CD34+ T cells  E6 and E7 oncogenes appear to contribute to activation of β-catenin signaling in HPV16-positive oropharyngeal squamous carcinoma cells, the hepatitis C virus (HCV)-encoded core protein potentiates Wnt/β-catenin signaling in hepatocellular carcinoma cells , 33, and T cells . Using t T cells , and UL8 T cells . The for T cells  and trannd Birc5 .in vitro and in vivo  as previously described  WAe001-A, NIH approval no. NIHhESC-10-0043]. Cells were thawed and plated on Matrigel-coated six-well plates in complete mTeSR1 (Stem Cell Technologies). CD34escribed . NeonataUL7-8 from TB40E (EF999921) containing a HiBiT sequence (5′-gtg agc ggc tgg cgg ctg ttc aag aag att agc-3′) after the signal peptide, HiBiT-UL8-V5, HiBiT-UL8-mutDTEL-V5, where amino acids Asp-Thr-Glu-Leu were substituted to Ala-Ala-Ala-Ala, UL8-V5-TbID  inserts g for 10 min at 4°C. One milligram of protein lysates was incubated at 4°C overnight with 500 µL of streptavidin conjugated to magnetic beads . Beads were washed once in 1.5 mL of wash buffer 1 (2% SDS in H2O), once with wash buffer 2 , once with wash buffer 3 , and then twice with wash buffer 4 . To evaluate sample integrity, 10% of the total was retained for immunoblots. The remaining beads were spun at 2,000 × g and resuspended in 50 µL of 50 mM ammonium bicarbonate for mass spectrometry.HEK293 cells were transfected with pDNA3.1-UL8-V5 or pDNA3.1-UL8-V5-TbID for 48 hours with Lipofectamine 2000, and then, 50 µM of biotin was added to the media for 6 hours. Cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 8, 1% Nonidet P-40, 150 mM NaCl, and 1× protease inhibitor cocktail , 1× phosphatase inhibitor cocktail 1 , 1× phosphatase inhibitor cocktail 2 , and insoluble debris were pelleted at 10,000 × Galk/kan ORF was amplified from pYD-C255, a GalK-kan  dual-expression plasmid , using the primers shown in Table S1. To replace the Galk/Kan cassette in the second step, HiBiTUL8, UL8-V5, and HiBiTUL22A were amplified from pcDNA3-HiBiTUL7-8, pcDNA3-UL8-V5-TbID, and pMS-HiBitUL22A-FLAG plasmids using the primers shown in Table S1. TB40E-UL8-mutDTEL-V5 was generated by inserting the Galk/kan cassette at 3′ end of UL8 ORF, and in the second step, we used oligonucleotides (Table S1) with overlap to UL8-mutDTEL-V5 to substitute amino acids Asp-Thr-Asp-Leu to Ala-Ala-Ala-Ala. To construct a TB40/E based mutant deleted for UL8, we performed en passant recombination in E. coli strain GS1783  selection marker with primers shown in Table S1. Hence, 50 bp upstream of the UL7 termination codon was repeated in both primers, creating a 50-bp long repeated region flanking the recombination insert. The PCR product generated to replace exon 2 of ORF UL8 was inserted into the TB40 BAC backbone by homologous recombination through heat shock induction of the λ phage-derived Red recombination genes in E. coli strain GS1783. Recombinants were selected on kanamycin containing agar plates, and positive clones were screened by HindIII digest and Sanger sequences across the altered genomic locus to control from genome integrity and to select for clones that contained the desired genomic configuration. The KanR selection maker was removed seamlessly by homologous recombination through the arabinose-induced induction of I-SceI expression resulting in a DNA double-strand break at the introduced I-SceI recognition sequence in the BAC allowing the artificially created 50-bp-long repeat regions to overlap and recombine after heat shock induction of the λ phage-derived Red recombination genes in GS1783. Clones were screened for the absence of Galk or KanR, and negative clones were analyzed by HindIII restriction digest analysis and Sanger sequencing. The final BAC clones were furthermore analyzed by next-generation sequencing using an Illumina iSeq sequencer to exclude off-target mutations. Virus was reconstituted by transfecting the BAC genomes into NHDF. Plaque purification was performed, and viral stocks and titers were generated as previously described  was previously engineered to express green fluorescent protein (GFP) as a visual marker of infection . The TB4n GS1783 . Brieflyescribed .c-myc, Birc5, Ctnnb1, and GAPDH primer/probe sets were obtained from Thermo Fisher Scientific. Relative expression was determined using the ΔΔCt method using 18S as the standard control. For UL7 expression, we used the following set of primers and probe: UL7_F primer 5′-ACTACGTGTCGTCGCTGGATT-3′; UL7_R primer 5′-ACAACTTCCACCACCCCATAAT; UL7 probe 6FAM-CATGGCCTTGGTAGGTG-MGBNFQ. For UL8 expression, we used the following set of primers and probe: UL8_F primer 5′-TCACGGAAACCGCCAATAC-3′; UL8_R primer 5′-AACGATAATCAGCATCCAAGAG-3′; UL8 probe 6FAM-TTCGGCSSCGCAACTACGGTTATTCCAC-TAMRA.Total RNA was isolated from infected, transfected, or treated cells using the Trizol RNA isolation method following the manufacturer’s directions. cDNA was prepared using 10 to 1,000 ng of total RNA and random hexamer primers. Samples were incubated at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. Real-time PCR (Taqman) was used to analyze cDNA levels in transfected or infected samples. An ABI StepOnePlus Real Time PCR machine was used with the following program for 40 cycles: 95°C for 15 s and 60°C for 1 min. Pme I and subsequently recombined into E. coli BJ5183 cells containing the adenoviral backbone plasmid pAdEasy-1 (AdEasier-1 cells). Recombinants were selected for kanamycin resistance, and the recombination confirmed by restriction endonuclease analyses. Finally, the recombinant plasmids were linearized with PacI before transfection with Lipofectamine 2000 (Thermo Fisher) into the adenovirus packaging cell lines HEK293 cells. The control vector Ad-GFP, Ad-HiBiTUL7, Ad-HiBiTUL8-V5, and Ad-HiBiTUL8-mutDTEL-V5 were produced, purified, and titered in HEK293, as previously described (pAdTrack-CMV (Addgene plasmid #16405) and AdEasier-1 cells (Addgene #16399) were gifts from Bert Vogelstein . The pAdescribed .+ HPCs were resuspended at low volume in Iscove's modified Dulbecco's medium (IMDM) containing 10% BIT serum supplement, L-glutamine, low-density lipoproteins, B-ME, and stem cell cytokines as previously described  as previously described , and equal quantities of protein were used for immunoprecipitation. Immunoprecipitation was performed by incubating 300–500 μg of protein lysate with 1:100 of primary antibody for 3 h at 4°C, followed by capture of antigen-antibody complexes with 20 µL of protein A or G magnetic beads . The resin beads were then rapidly washed five times with Tris-buffered saline buffer with 0.1% Nonidet P-40. Immunoprecipitates were eluted from the affinity resin in 2× Laemmli Buffer for 10 min at 80°C and then subjected to SDS-PAGE and Western blotting as described below.Extracts were run on 8–12% SDS-PAGE gels, transferred to Immobilon-P Transfer Membranes (Millipore Corp.), and visualized with antibodies specific for β-catenin (E-5) , β-catenin , β-catenin , Non-Phospho (active) β-catenin , DVL2 , anti-HiBiT , FLAG M2 , anti-FLAG , V5 Tag , V5-tag , GAPDH Ab (6C5) , NanoGlo HiBiT Blotting System , and UL44 .+ HPCs were infected at an MOI of 2 for 48 h. Following infection, pure populations of viable, infected (GFP-positive) CD34+ HPCs were isolated by Wolf Cell Sorter  using 7AAD Viability Staining Solution (Invitrogen eBioscience) and PE-conjugated antibody specific to human CD34 . Long-term cultures were maintained in Myelocult supplemented with hydrocortisone in transwells above an irradiated M2-10B4 and S1/S1 stromal cell monolayer for 12 days  via IP injection. A control group of engrafted mice was mock infected using uninfected fibroblasts. The virus was reactivated as previously described  at Oregon Health and Science University. NOD-escribed . The aniescribed .+ HPC cultures was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s directions. Primers and a probe recognizing HCMV UL141 were used to quantify HCMV genomes . Viral genomes in humanized mice were normalized to 1-µg input DNA. Viral genomes synthesized during infection in CD34+ HPCs were normalized to total cell number determined using human-globin as a reference . The reaction was initiated using TaqMan Fast Advanced Master Mix (Applied Biosystems) activated at 95°C for 10 min followed by 40 cycles (15 s at 95°C and 1 min at 60°C) using a StepOnePlus TaqMan PCR machine. Results were analyzed using ABI StepOne software.Total DNA from mouse tissues was extracted using the DNAzol Reagent (Thermo Fisher Scientific) according to the manufacturer’s directions. Total DNA from primary CD34t-test or one-way ANOVA with Tukey or Bonferroni post-test as indicated. A P value of <0.05 or lower was considered significant, and exact P values are indicated.Data are expressed as the mean ± SEM. Statistical analysis was performed using GraphPad Prism (v9) for comparison between groups using Student’s


Tg(fabp10a:pt-β-catenin), we present that peroxisome proliferator-activated receptor-alpha (PPARα) activation augments LPC-to-hepatocyte differentiation. We found that treating Tg(fabp10a:pt-β-catenin) larvae with GW7647, a potent PPARα agonist, enhanced the expression of hepatocyte markers and simultaneously reduced the expression of biliary epithelial cell (BEC)/LPC markers in the regenerating livers, indicating enhanced LPC-to-hepatocyte differentiation. Mechanistically, PPARα activation augments the differentiation by suppressing YAP signaling. The differentiation phenotypes resulting from GW7647 treatment were rescued by expressing a constitutively active form of Yap1. Moreover, we found that suppression of YAP signaling was sufficient to promote LPC-to-hepatocyte differentiation. Treating Tg(fabp10a:pt-β-catenin) larvae with the TEAD inhibitor K-975, which suppresses YAP signaling, phenocopied the effect of GW7647 on LPC differentiation. Altogether, our findings provide insights into augmenting LPC-mediated liver regeneration as a regenerative therapy for chronic liver diseases.Despite the robust regenerative capacity of the liver, prolonged and severe liver damage impairs liver regeneration, leading to liver failure. Since the liver co-opts the differentiation of liver progenitor cells (LPCs) into hepatocytes to restore functional hepatocytes, augmenting LPC-mediated liver regeneration may be beneficial to patients with chronic liver diseases. However, the molecular mechanisms underlying LPC-to-hepatocyte differentiation have remained largely unknown. Using the zebrafish model of LPC-mediated liver regeneration,  Upon two-thirds partial hepatectomy (PHx) in rodents, the lost liver mass recovers within 8 days through hepatocyte proliferation2. However, despite this robust regenerative capacity, prolonged liver damage can impair the ability of hepatocytes to proliferate. In such situations, the liver co-opts cell fate conversion-mediated regeneration, thereby generating hepatocytes from biliary epithelial cells (BECs) or through liver progenitor cells (LPCs)1. When hepatocyte proliferation is severely impaired in rodents pharmacologically4, genetically8, or by long-term chronic injuries10, BECs convert to hepatocytes, thereby contributing to liver regeneration. This phenomenon also occurs in zebrafish when nearly all hepatocytes are ablated13 or when oncogenes are overexpressed in hepatocytes14. With these rodent and zebrafish models, some of the molecular mechanisms underlying the cell fate conversion-mediated liver regeneration have been identified17, but the mechanisms are still largely unknown.As a highly regenerative organ, the liver regenerates in two distinct ways: (1) hepatocyte proliferation and (2) cell fate conversion18 suggests that LPCs fail to differentiate efficiently into hepatocytes in the diseased liver. However, if the differentiation can be restored, it may promote cell fate conversion-mediated liver regeneration, contributing to liver recovery in patients. Thus, it is clinically important to understand the molecular mechanisms of LPC-to-hepatocyte differentiation and to identify means to promote the differentiation. Given this clinical significance, we sought to identify compounds that can promote LPC-to-hepatocyte differentiation and, herein, discovered the peroxisome proliferator-activated receptor alpha (PPARα) agonist GW7647 as such a compound.The correlation between the number of LPCs and the severity of liver diseases in patients20, PPARα also controls various biological processes by interacting with multiple signaling pathways. For example, following 2/3 PHx, PPARα facilitates the nuclear translocation of YAP in hepatocytes, thereby enhancing YAP signaling in the hepatocytes and promoting hepatocyte proliferation21. Moreover, PPARα promotes cardiomyocyte maturation by enhancing Yap1 expression22. In addition to this crosstalk with YAP signaling, PPARα interacts with Notch and WNT signaling. PPARα controls vasculoprotection and vascular remodeling by enhancing Notch signaling through fatty acid oxidation (FAO) in endothelial cells23. PPARα also protects diabetic kidneys from renal fibrosis by blocking WNT signaling in renal proximal tubules24.Although PPARα is well known to control lipid metabolism by regulating the expression of multiple genes important for the metabolism15. Given that FXR and PPARα play opposite roles in multiple processes in the liver, including FAO, gluconeogenesis, and autophagy20, we hypothesized that PPARα activation could promote LPC-to-hepatocyte differentiation. By testing this hypothesis, we here present that PPARα activation indeed promotes LPC-to-hepatocyte differentiation by suppressing YAP signaling.We recently reported that farnesoid X receptor (FXR) suppresses LPC-to-hepatocyte differentiation in zebrafishTg(fabp10a:pt-β-catenin)s704 line, which expresses an oncogenic, stable form of Xenopus β-catenin in hepatocytes under the hepatocyte-specific fabp10a promoter14. The hepatocyte-specific expression of the oncogene induces senescence and apoptosis in hepatocytes, which elicit LPC activation and inflammation in the liver. BECs and surviving hepatocytes dedifferentiate into LPCs, and the LPCs gradually and slowly differentiate into hepatocytes14. Since LPCs are predominant in the Tg(fabp10a:pt-β-catenin) liver between 12/13 dpf and 14/15 dpf, we treated Tg(fabp10a:pt-β-catenin) larvae with 1 μM GW7647, a potent PPARα agonist25, for 2 days from 12 or 13 dpf  livers compared with DMSO-treated Tg(fabp10a:pt-β-catenin) livers  was enhanced, and the expression of BEC marker genes (epcam and her9) was reduced in GW7647-treated Tg(fabp10a:pt-β-catenin) livers, suggesting enhanced LPC-to-hepatocyte differentiation  livers by in situ hybridization  larvae with CAY10599, a potent PPARγ agonist. Unlike GW7647 treatment, CAY10599 treatment did not enhance the expression of Bhmt, another hepatocyte marker27, in Tg(fabp10a:pt-β-catenin) livers  larvae promotes LPC-to-hepatocyte differentiation through Pparaa activation.To investigate the effect of PPARα activation on LPC-mediated liver regeneration, we used the ers Fig. A. Notablcyp7a1, gc, serpina1) and the reduced expression of the BEC marker epcam in GW7647-treated Tg(fabp10a:pt-β-catenin) livers  livers  livers by performing RNA-sequencing. Consistent with the above data  livers compared with DMSO-treated Tg(fabp10a:pt-β-catenin) livers  livers  livers  larvae  livers 34, we also found that YAP activity was highly enhanced in Tg(fabp10a:pt-β-catenin) livers and that the activity was present in most LPCs, which express Anxa4, an LPC/BEC marker  livers is simply the consequence of enhanced LPC-to-hepatocyte differentiation rather than the consequence of PPARα activation. To exclude this possibility, we examined the expression of ccn1 and ccn2 in Tg(fabp10a:pt-β-catenin) larvae treated with AG1478, an EGFR inhibitor, known to promote LPC-to-hepatocyte differentiation14. qRT-PCR analysis showed that AG1478 treatment enhanced the expression of hepatocyte marker genes  but not YAP target genes (ccn1 and ccn2)  livers. We also validated the reduced YAP signaling identified from RNA-sequencing data with qRT-PCR and Yap1 immunohistochemistry  livers.The pathway analysis also revealed that lipid metabolism-related pathways were enhanced in GW7647-treated ers Fig. C, as coners Fig. A. Given vae Fig. C. In addngs Fig. D. To furn2) Fig. A, B, sup37 and hepatocyte-to-BEC transdifferentiation39, we hypothesized that suppression of YAP signaling might promote LPC-to-hepatocyte differentiation. To test this hypothesis, we suppressed YAP signaling with K-975, the TEAD inhibitor40, which suppresses the transcription of YAP target genes by preventing the interaction of TEAD with YAP. In K975-treated Tg(fabp10a:pt-β-catenin) livers compared with DMSO-treated Tg(fabp10a:pt-β-catenin) livers, the expression of hepatocyte marker genes  was significantly enhanced and the expression of BEC marker genes (epcam and her9) was reduced considerably  livers. This promotion phenotype was confirmed by in situ hybridization  larvae.Given that YAP signaling positively controls the differentiation of hepatoblasts into BECsTg(fabp10a:pt-β-catenin) larvae treated with GW7647. To enhance YAP signaling, the Tg(hs:cayap1) line41, which expresses constitutive-active Yap1 (caYap1) under the heat-shock promoter, was used with a single heat-shock 6 h before GW7647 treatment  livers  fish that express nitroreductase (NTR) specifically in hepatocytes13. NTR converts metronidazole (Mtz) into a cytotoxic drug; thus, treating Tg(fabp10a:CFP-NTR) fish with Mtz ablates hepatocytes that express NTR. In this model, upon massive hepatocyte ablation, BECs first dedifferentiate into LPCs, and the LPCs subsequently differentiate into hepatocytes13. Tg(fabp10a:CFP-NTR) male adults were treated with Mtz for 5 h and subsequently with GW7647 from R1d to R3d for 48 h, and their livers were harvested at R3d for qRT-PCR analysis  model, GW7647 treatment increased the expression of two hepatocyte marker genes  and decreased the expression of the Yap target genes   Fig. B, sugges43, it is elusive whether PPARα also regulates cell fate conversion-mediated liver regeneration, particularly LPC-to-hepatocyte differentiation. Previous in vitro studies45 produced a discrepancy in the role of PPARα in LPC-to-hepatocyte differentiation. Here, using the zebrafish in vivo model, we demonstrate that PPARα activation promotes LPC-to-hepatocyte differentiation. Furthermore, we present the mechanism by which PPARα activation promotes the differentiation: suppressing YAP signaling in LPCs. Given the positive relationship between PPARα and YAP signaling in hepatocytes21 and cardiomyocytes22, our finding about their relationship in LPCs is rather surprising. However, our rescue experiments with enhanced YAP signaling  and Tg(fabp10a:CFP-NTR), we hereby present in vivo evidence.YAP signaling is one of the crucial signaling pathways in hepatobiliary plasticityTg(fabp10a:pt-β-catenin) model. We surmise that these variations could stem from different cellular origins of LPCs between the two models. Specifically, in the Tg(fabp10a:pt-β-catenin) model, ~ 70% and ~ 30% of LPCs originate from hepatocytes and BECs, respectively14, whereas in the adult hepatocyte ablation model, nearly all LPCs originate from BECs56. Given that hepatocyte-derived LPCs are distinct from BEC-derived LPCs in gene expression profiles57, we speculate that upon GW7647 treatment, hepatocyte-derived LPCs might differentiate into hepatocytes better than BEC-derived LPCs.In the adult hepatocyte ablation model, we observed rather subtle differences in the expression of hepatocyte markers and Yap1 target genes between DMSO- and GW7647-treated groups compared with those in the In summary, we provide the in vivo evidence that suppression of YAP signaling promotes LPC-to-hepatocyte differentiation. Furthermore, we provide a positive role for PPARα in LPC-to-hepatocyte differentiation. Our study suggests PPARα agonists as potential drugs that cannot only reduce a hepatic lipid level but also promote LPC-mediated liver regeneration.Danio rerio) were maintained at 28.5 °C on a 14 h light/10 h dark cycle. Embryos and adult fish were raised and maintained under standard laboratory conditions58. We used the ppargc1asa34243 mutant and the following transgenic lines: Tg(fabp10a:pt-β-catenin)s704, Tg(fabp10a:CFP-NTR)s931, Tg(hs:cayap1)zf622, and Tg(hCCN2:GFP)ia48. Their full names and references are listed in Table 59. For all experiments, we used embryos from multiple clutches; our breeding strategy is mass breeding to put 3–4 males and 5–6 females in the same breeding tank. We drew conclusions following at least three independent experiments.Zebrafish (Tg(fabp10a:pt-β-catenin) larvae were treated with 0.1% dimethyl sulfoxide (DMSO), 1 µM GW7647 , 2 µM K-975 , 15 µM Etomoxir , 2 µM AG1478 , or 7 μM CAY10599  for 48 h from 12 or 13 days post-fertilization (dpf). Compounds were refreshed every 24 h. The larvae were fed prior to the drug treatments but fasted during the treatments. They were harvested at 14 or 15 dpf for subsequent analyses.Tg(fabp10a:CFP-NTR) fish were treated with 5 mM Mtz in system water containing 0.5% DMSO for 5 h to ablate hepatocytes. 24 h after Mtz washout (R1d), the fish were treated with 1 mM GW7647 for 48 h and harvested at R3d.Six month old male ® III First-Strand Synthesis SuperMix  according to the manufacturer’s protocols. qRT-PCR was performed as previously described14 using the QuantStudio 12K Flex machine  with the iTaqTM Universal SYBR® Green Supermix . eef1a1l1 was used for normalization as previously described60. At least three independent experiments were performed. For each replicate with larval livers, the control value was first set to 1, and then the values of the experimental groups were calculated relative to the control value. The primers used for qRT-PCR are listed in Table Total RNA was extracted from 30 to 40 dissected livers for each condition using the RNeasy Micro Kit ; cDNA was synthesized from the RNA using the SuperScriptLivers were dissected from 30 to 40 DMSO- and GW7647-treated larvae. For adult experiments, the individual liver was dissected without pooling. Total RNA was extracted from the dissected livers using RNeasy Micro kit  and sent to the University of Pittsburgh Genomics Core for library preparation and sequencing. Library preparation was performed using the TruSEQ Stranded mRNA Sample Preparation Kit  according to the manufacturer’s instructions. Sequencing was performed on a NextSeq 500  for 2 × 75-bp paired-end reads. These data have been deposited in NCBI’s Gene Expression Omnibus (GSE226923).Raw sequencing data were imported into CLC Genomics Workbench, and reads were mapped to the zebrafish reference genome. Differentially expressed genes (DEGs) used in the pathway analysis were determined between DMSO- and GW7647-treated groups using filters to select genes with Expr Fold Change ≥ |1.5| and Expr False Discovery Rate ≤ 0.05. DEGs were imported into Ingenuity Pathway Analysis to identify signaling pathways and upstream regulators affected by PPARα activation.14. cDNA from 14- or 15-dpf larvae was used as a template for PCR to amplify genes of interest; PCR products were used for in situ probe synthesis. The primers used for the probe synthesis are listed in Table gc61 and epcam62, their probes were synthesized using plasmids containing the genes. Immunostaining was performed as previously described14, using the following antibodies: mouse anti-Bhmt , mouse anti-Anxa4 , rabbit anti-Abcb11 , and Alexa Fluor 488-, 568-, and 647-conjugated secondary antibodies . For section, larvae were fixed with 4% paraformaldehyde/PBS, embedded in Tissue Freezing Medium , and cryo-sectioned to 10-µm thickness.Section in situ hybridization was performed as previously described63. Samples were cut into 5-µm sections, and the sectioned samples were microwaved for 12 min in pH 6.0 sodium citrate buffer for antigen retrieval. After cooling, samples were placed in 3% H2O2 for 10 min to quench endogenous peroxide activity. After washing with PBS, slides were blocked with Super Block  for 10 min. Samples were incubated with rabbit anti-Yap1 antibodies  and then with biotinylated secondary antibodies  for 15 min at room temperature. After washing with PBS, samples proceeded with Vectastain ABC Elite kit , and the signal was developed with DAB Peroxidase Substrate Kit . Slides were counterstained with hematoxylin  and dehydrated to xylene, and coverslips were applied with Cytoseal XYL .Zebrafish larvae were fixed with Dietrich's fixative  at room temperature for 24 h and processed for paraffin embedding. Paraffin block was prepared as previously describedTg(hs:cayap1) larvae were heat-shocked at 12 or 13 dpf 6 h before compound treatments. The larvae were transferred into egg water pre-warmed to 38.5 °C and kept at 38.5 °C for 30 min, as previously described64.ppargc1a genotyping, genomic DNA was amplified with either a wild-type (5′-CTGTCTTCATGCTCTCCCTC-3′) or mutant (5′-CTGTCTTCATGCTCTCCCTA-3′) allele-specific forward primer and a common reverse primer (5′-TGGGCCCTTCCGAATAGAGC-3′). Both the wild-type and mutant alleles displayed 102-bp PCR products.For 65. All figures, labels, arrows, scale bars, and outlines were assembled or drawn using the Adobe Illustrator software . Statistical analyses were performed using the GraphPad Prism software . Differences among groups were tested by unpaired Student’s t-tests, Fisher’s exact test, or chi-square test. Differences were considered statistically significant when P < 0.05 .Zeiss LSM700 confocal , Leica M205 FA , and Nikon Eclipse Ti inverted  microscopes were used to obtain image data. Confocal images were analyzed using the ImageJ softwareSupplementary Information.Supplementary Table S4.


Spinal Muscular Atrophy (SMA) is a severe genetic neuromuscular disorder that occurs in childhood and is caused by misexpression of the survival motor neuron (SMN) protein. SMN reduction induces spinal cord motoneuron (MN) degeneration, which leads to progressive muscular atrophy and weakness. The link between SMN deficiency and the molecular mechanisms altered in SMA cells remains unclear. Autophagy, deregulation of intracellular survival pathways and ERK hyperphosphorylation may contribute to SMN-reduced MNs collapse, offering a useful strategy to develop new therapies to prevent neurodegeneration in SMA. Using SMA MN in vitro models, the effect of pharmacological inhibition of PI3K/Akt and ERK MAPK pathways on SMN and autophagy markers modulation was studied by western blot analysis and RT-qPCR. Experiments involved primary cultures of mouse SMA spinal cord MNs and differentiated SMA human MNs derived from induced pluripotent stem cells (iPSCs). Inhibition of the PI3K/Akt and the ERK MAPK pathways reduced SMN protein and mRNA levels. Importantly, mTOR phosphorylation, p62, and LC3-II autophagy markers protein level were decreased after ERK MAPK pharmacological inhibition. Furthermore, the intracellular calcium chelator BAPTA prevented ERK hyperphosphorylation in SMA cells. Our results propose a link between intracellular calcium, signaling pathways, and autophagy in SMA MNs, suggesting that ERK hyperphosphorylation may contribute to autophagy deregulation in SMN-reduced MNs. Survival motor neuron 1 (SMN1) gene, located at the telomeric region of the chromosome 5q3, resulting in deficient levels of SMN protein. It is well known that SMN deficiency provokes cellular degeneration, although the underlying molecular mechanisms are not fully understood [Spinal Muscular Atrophy (SMA) is a neuromuscular disease characterized by degeneration of alpha motoneurons (MNs) located in the ventral horn of the spinal cord , leadingderstood .MN development and physiology relies on a continuous and regulated trophic support induced by neurotrophic factors (NTFs) coming from innervated tissues, afferent neurons and glial cells , 8. NTFsWhile the canonical view of ERK emphasizes its anti-apoptotic properties, its activity has been linked to different modes of cell death . Among oOur objectives are to further analyze the contribution of PI3K/Akt and ERK MAPK pathways on SMN protein regulation in MNs, and to investigate whether these intracellular pathways participate in autophagy deregulation in SMA cells. Results provided evidence indicating that inhibition of ERK or Akt signaling pathways decreased SMN at both protein and mRNA levels in human differentiated MNs from induced pluripotent stem cells (IPSCs). We also found that increased ERK phosphorylation observed in SMN-reduced cells may contribute to the autophagy alterations in SMA MNs. Pharmacological inhibition of the ERK pathway reduced mTOR phosphorylation, p62, and LC3-II protein levels in SMA differentiated MNs. Importantly, treatment with the intracellular calcium chelator BAPTA prevented ERK overphosphorylation in SMA cells, suggesting a link between calcium, signaling pathways, and autophagy in SMA cells. Our data provide new insights on the molecular mechanisms leading to MN degeneration in SMA MNs, and identify new potential targets for SMA therapeutic strategies.To explore the role of PI3K/Akt pathway on SMN regulation, human iPSCs from an SMA patient and a non-affected control (Control)  were differentiated to MNs Fig. . After 7p < 0.0001; Cytoplasm: Control 45.91 ± 1.35, SMA 29.94 ± 0.77, p < 0.0001; Nucleus: Control 63.37 ± 1.34, SMA 59.07 ± 1.15, p = 0.015)  and Akt phosphorylation  were reduced in SMA cells compared to the control condition (data not shown). LY294002 addition significantly decreased the ratio of Akt phosphorylation versus total Akt in both Control  and SMA  cells, compared to the Control and SMA LY294002 untreated (-LY) conditions, respectively  nor in SMA  cells, compared to their respective -LY controls  and SMA  conditions compared to their respective non-treated (-LY) controls  by quantitative RT-PCR (qRT-PCR). Human Gapdh gene was used as a control. Seven-day differentiated human Control and SMA MNs were treated with or without 25 µm LY294002 during 24 h. To quantify SMN transcript level, total RNA was extracted and reverse-transcribed to cDNA. The results indicated a significant reduction in SMN mRNA expression in LY294002-treated conditions  compared to non-treated (-LY) controls, suggesting that PI3K inhibition regulates SMN at transcriptional level in human Control and SMA MNs  Fig. . Seven dely Fig. . LY29400ols Fig. . Analysiols Fig. . To evalMNs Fig. .−/−; SMN2+/+, mutSMA) MNs and 7-day differentiated MNs from human iPSCs cells were treated with 20 µM U0126. At 24 h, cell lysates were obtained and submitted to western blot analysis using anti-phospho-ERK, anti-ERK, and anti-SMN antibodies    , compared to their respective untreated (-U0) controls  Fig.  and huma01) Fig. , compareSMN gene expression in U0126 treated cells, we quantified Smn mRNA by qRT-PCR, using Gapdh gene as a control in CD1 mouse MNs. Six-day cultured CD1 MNs were incubated with or without 20 µM U0126. At 20 h, total RNA was extracted for reverse transcription and amplification of Smn transcript. U0126 addition was associated with a significant reduction in Smn mRNA expression  in CD1 MNs, compared to the non-treated controls  compared to control non-treated conditions  or mTOR  protein level in U0126-treated Control cultures, compared to their respective untreated controls  and mTOR  protein levels, compared with the non-treated condition (-U0)  Fig. . Inhibitp = 0.035; LC3-II 2.53 ± 0.66, p = 0.0099) MNs, compared to Control cells . However, Control cultures showed no differences in p62 and LC3-II protein levels in U0126-treated (+U0) cells compared with their respective non-treated controls (-U0)   Fig. . Togethep < 0.0001) and SMA  cultures, compared to the respective EV controls  cultures compared to the Control condition. Nevertheless, SMN overexpression did not induce significant differences in the p-ERK/pan-ERK ratio in Control (p = 0.5871) or SMA (p = 0.2822) ovSMN cultures, compared to EV controls  control or SMN overexpression (ovSMN) constructs. Twenty-four hours later, lentivirus medium was replaced with fresh culture medium and cells were maintained in these conditions for 7 days. Lentivirus transduction efficiency was microscopically monitored by observing green fluorescent protein (GFP) positive cells Fig. . Cell lyols Fig. . In agreol p = 0.871 or SMp = 0.0037) cells compared with the Control MNs  MNs but not in Control  MNs, compared with their untreated respective controls  and SMA (p = 0.999) MNs compared to their respective untreated controls.To elucidate the intracellular mechanisms involved in ERK phosphorylation increase observed in SMA MNs, we examined the effect of reducing intracellular calcium level using the calcium chelator BAPTA. To this end, 7-day Control and SMA human differentiated MNs were treated or not with 10 µM BAPTA . At 24 h, protein extracts were obtained and submitted to western blot using anti-SMN, anti-p-ERK, and anti-pan-ERK antibodies. In Fig. MNs Fig. . BAPTA aols Fig. . BAPTA tp = 0.0004) MNs, compared to the Control condition. BAPTA-AM treatment significantly reduced LC3-II protein level in SMA  cultures compared to SMA non-treated (-BAPTA-AM 2.122 ± 0.27) cells. In Control cultures, no differences on LC3-II protein level were observed between BAPTA-AM-treated and untreated cells (p > 0.99). These results together indicate that intracellular calcium reduction prevented ERK hyperphosphorylation and decreased LC3-II autophagy marker in SMA human differentiated MNs.Finally, we analyzed the effect of intracellular calcium reduction on the autophagy marker LC3-II. To this aim, 7-day differentiated Control and SMA human MNs were treated with 10 µM of BAPTA-AM during 24 h. Protein extracts were obtained and submitted to western blot analysis with anti-LC3, anti-p-ERK and anti-SMN antibodies Fig. . As expeSMN at transcriptional level. Both PI3K/Akt and ERK MAPK pathways have been linked to the positive regulation of SMN transcription through Akt/CREB and ERK/Elk-1, respectively [The precise intracellular mechanisms causing MN degeneration in SMA disease are not fully understood. Here, we report a link between ERK signaling pathway alteration and autophagy deregulation in SMA MNs. The activity of ERK MAPK and PI3K/Akt pathways is essential for the survival and maintenance of several neuronal types, including MNs . SMA celectively , 25. Accectively . Conversectively . Our obsectively . For insectively . Hence, Previous findings in SMA cells suggest increased autophagy markers and mTOR phosphorylation, correlating with the increase of autophagosome formation and reduced autophagic flux . Our curThe exact mechanism(s) by which ERK phosphorylation is increased in SMA cells remains unclear. Although ERK appears to be linked to SMN transcriptional regulation, our results suggested that SMN increase does not regulate ERK phosphorylation in human Control and SMA MNs Fig.  or in CDOverall, these results suggest a connection between intracellular calcium levels and the activity of ERK signaling pathway, which in turn may regulate autophagy and SMN in SMA MNs Fig. . Nonethe89AhmbSmn1tm1Msd/J  was used for experimental procedures. Heterozygous females and males were crossed to obtain Smn−/−; SMN2+/+ (hereafter referred to as mutSMA) and Smn+/+; SMN2+/+  mice.The severe SMA mouse model FVB·Cg-Tg (SMN2)A piece of the tail from neonatal offspring was collected for genotyping. The REDExtract-N-Amp Tissue PCR Kit  was used for genomic DNA extraction and PCR setup, with the following primers: WT forward 5′-CTCCGGGATATTGGGATTG-3′, SMA reverse 5′-GGTAACGCCAGGGTTTTCC-3′ and WT reverse 5′-TTTCTTCTGGCTGTGCCTTT-3′.All procedures were done in accordance with the Spanish Council on Animal Care guidelines and approved by the University of Lleida Advisory Committee on Animal Services (CEEA02–01/17).CD1 or SMA embryonic 13 (E13) mice were used for spinal cord MN primary cultures. Cells were isolated and plated in laminin-coated four-well dishes  as described , 36.v/v; Gibco), horse serum , l-glutamine  and 2-mercaptoethanol , and a cocktail of neurotrophic factors . At 24 h after plating, 2 μg/ml of aphidicolin (Sigma) was added to the culture medium and was maintained throughout the experiment.Culture medium was NBM complete (NBMc): neurobasal medium  supplemented with B27 . SMA patient was previously classified as SMA type I, but several data supported re-classification to SMA type II. Control and SMA cells were differentiated to MNs as described [The human iPSCs were acquired from Coriell Institute for Medical Research . The GM23411*B iPSC cell line  was used as a control (Control) and GM23240*B iPSC cell line (SMA) was from a patient with SMA type II  and for cell area analysis (blinded) the NIH ImageJ software was used.For experiments, dissociated neurospheres were plated in the following conditions: 80,000 cells/well in laminin-coated four-well tissue culture dishes  for western blot analysis, 500,000 cells/well in laminin-coated six-well tissue-culture dishes  for qRT-PCR experiments, or 15,000 cells/well on 1 cmSMN1 cDNA (NCBI accession number NM000344) was subcloned into FCIV plasmid and lentivirus containing overexpression constructs was obtained as described [For SMN overexpression, the open reading frame of the human escribed . For lenWestern blots were performed as previously described . Total cCultured cells were fixed with 4% paraformaldehyde (Sigma) for 10 min and with cold methanol (Sigma) for 10 additional min. Cells were permeabilized with 0.3% Triton X-100 and incubated for 30 min with 10% BSA in PBS. The primary antibody ; anti-Islet1/2 antibody, 1:50, Cat. No. 39.4D5, Developmental Studies Hybridoma Bank ; or anti-beta-III-tubulin, 1:400, Cat. No. 5568, Cell Signaling Technology) was diluted in 0.3% Triton-X-100 and incubated overnight with 10% BSA in PBS. After washing, the secondary antibody was added: anti-mouse ALEXA555 antibody, 1:400, Cat. No. A21422 or anti-rabbit ALEXA488 antibody, 1:400, Cat. No. A11008 ; or anti-sheep ALEXA649 antibody, 1:400, Cat. No. 713-496-147 . To identify nuclear localization in cells Hoechst  staining was used. Samples were mounted using Mowiol  medium. Microscopy observations were performed in an Olympus FV10i confocal microscope.Smn-specific primers: Smn exon 1-forward (5′-GATGATTCTGACATTTGGGATG-3′) and Smn Exon 2-reverse (5′-TGGCTTATCTGGAGTTTCAGAA-3′) and specific primers of mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh): Forward (5′-TGCACCACCAACTGCTTAG-3′) and reverse (5′-GGATGCAGGGATGATGTTC-3′) as internal control. For human differentiated MNs, Real-Time was performed using human SMN-specific primers: SMN exon 6-forward (5′-CCGCCACCCCCTCCCATCTCT-3′) and SMN Exon 8-reverse (5′- CATCTCCTGAGACAGAGCTGA-3′) and specific primers of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward (5′- TGCACCACCAACTGCTTAG-3′) and reverse (5′-AGAGGCAGGGATGATGTTG-3′) as internal control. Quantification was completed using Bio-Rad CFX Manager real-time detection system software . Each sample was measured in triplicate; relative fold change gene expression levels were calculated with the formula 2^−(ΔΔCq).CD1 or human differentiated MNs were plated  in laminin-coated six-well tissue-culture dishes . Once the cells were attached, MNs were treated with or without 25 μM LY294002 (LY)  or 20 μM U0126 (U0)  for 24 h. Total RNA was extracted using the RNeasy® Mini Kit . Eighty nanograms of total RNA from each condition were used for each individual qRT-PCR reaction. The assays were performed in a CFX96 Real-Time System  using iTaq™ Universal SYBR® Green One-Step Kit from Bio-Rad. For CD1 experiments, Real-Time was performed using mouse t-test, and differences between more than two groups one-way ANOVA with Tukey’s multiple comparisons test was used. If the data was not following a normal distribution, the non-parametric two-tailed Mann-Whitney test was applied to compare differences between two groups, and for more than two groups the non-parametric Kruskal-Wallis with Dunn’s multiple comparisons test was performed. Values were considered significant when p < 0.05. Statistical significance is shown with “*” when a parametric test was applied and with “#” when a non-parametric test was applied.All experiments were performed at least three independent times (from three separate mouse offspring for primary MN cultures or three separate iPSCs differentiation cycles). Values were expressed as mean ± estimated standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism, version 9.4.1 . Normality of the data was tested with Shapiro-Wilk test. If the data distribution was normal, differences between two groups were assessed by two-tailed Student Original western blot


Skin interstitial fluid (ISF) has emerged as a fungible biofluid sample for blood serum and plasma for disease diagnosis and therapy. The sampling of skin ISF is highly desirable considering its easy accessibility, no damage to blood vessels, and reduced risk of infection. Particularly, skin ISF can be sampled using microneedle (MN)-based platforms in the skin tissues, which exhibit multiple advantages including minimal invasion of the skin tissues, less pain, ease of carrying, capacity for continuous monitoring, etc. In this review, we focus on the current development of microneedle-integrated transdermal sensors for collecting ISF and detecting specific disease biomarkers. Firstly, we discussed and classified microneedles according to their structural design, including solid MNs, hollow MNs, porous MNs, and coated MNs. Subsequently, we elaborate on the construction of MN-integrated sensors for metabolic analysis with highlights on the electrochemical, fluorescent, chemical chromogenic, immunodiagnostic, and molecular diagnostic MN-integrated sensors. Finally, we discuss the current challenges and future direction for developing MN-based platforms for ISF extraction and sensing applications. Identification and detection of biomarkers in body fluids have become increasingly important in advanced medical diagnosis and disease therapy ,2,3,4. BMicroneedles (MNs), well-known as micron-scale needles, can penetrate the outer skin barrier  and collect cutaneous ISF in a minimally invasive way ,33,34. TIn this review, we will systematically describe the construction and related operating principles of MN’s integrated sensors for ISF extraction and metabolic analysis . We begiIn recent years, MNs have attracted more and more researchers’ interest due to their potential in minimally invasive diagnosis and have gradually been applied to extracting ISF from skin tissues . MNs conSolid MNs represent a class of MNs without gaps inside their tips, and most of them are made from macromolecular hydrogels and widely utilized for transdermal drug delivery . HydrogeNormally, solid MNs are used to collect and extract ISF for in vitro detection of cholesterol, glucose, nucleic acid, protein, drugs (such as vancomycin), and so on . In one In fact, most solid MNs are made up of photopolymerized hydrogels, which are easily prepared by mold methods. As shown in Hollow MNs are a kind of MN with a hollow channel inside the needle tips. Biomarkers in ISF can be collected by flowing through a network of microcapillaries in a single capillary using hollow MNs  Figure a. UnlikeIn early studies, hollow MNs were fabricated by a series of procedures, including standard lithography, buffered hydrofluoric acid (HF) etching, and potassium hydroxide (KOH) etching b 64]. T. T64]. T3 and HF solution, and the hollow channel was retained at the edge. Similarly, Wu et al. made hollow MNs with an oblique channel such as a hypodermic syringe  enzyme . The catCompared with other types of MNs, the thinner coating of coated MNs can collect ISF rapidly in a several seconds. Moreover, the coating MNs can not only be modified on the coating, but also maintain the own characteristics of the inner MNs, such as the strong mechanical property of the solid MNs and the capillary action of the porous MNs.In addition to the single MN-based extraction platforms, more and more efforts are being made to explore MN-integrated sensors with multiple modalities of multi-period extraction, multi-substance monitoring, and their combinations ,87. MNs Electrochemical MN-integrated sensors are a kind of sensor according to the electrochemical performance of the analyte, in which the chemical quantity is converted into electric properties for sensing and monitoring ,94,95,96Nowadays, electrochemical sensors have been integrated with MNs to detect small molecular physiological indexes in ISF. As an example, Yanxiang Cheng et al. exploited a touch-driven biosensor for collecting and monitoring glucose in ISF based on “MN transdermal-reverse iontophoresis (RI) extraction-electrochemical detection” . This ty+/K+ from cutaneous ISF . T. T92]. TImmunodiagnostic MN-integrated sensors are committed to detecting the humoral or cellular immune response based on the extraction action of MNs and the analytic functions of immunological techniques ,108,109.Today, a series of MNs based on Ag-Ab-specific binding have been developed for distinguishing specific protein markers from cutaneous ISF. These MNs are stripped of their skin and incubated with enzyme-labeled antibodies for a few minutes. The targeted protein markers are subsequently observed and quantified by fluorescent microscopy or enzyme-substrate coloration. For example, Zhang et al. proposed novel immunodiagnostic MNs integrated with photonic crystal (PhC) barcodes that enriched specific Ab onto their probe-modified PhC balls . The capCell populations in local tissues cannot often be monitored by traditional blood draws. The concentration of antigen-specific T cell populations in the systemic circulation (blood) is extremely low and of strong clinical significance, especially at the time of vaccination or infection. Therefore, in situ T-cell detection by immunodiagnostic MNs is of great significance for monitoring the local immune response. Anasuya Mandal et al. designed a microneedle array loaded with adjuvant and specific Ag to draw in antigen-presenting cells (APC) in skin for subsequent cell phenotype and functional analysis without changing the immune status of local tissues . This meMolecular diagnostic MN-integrated sensors provide a novel method for in situ detection, analysis, and diagnosis of genetic materials in tissues by integrating MNs with molecular biology technologies ,33,111. In situ detection of microRNA (miRNA) in tissues has become a new approach for clinical health monitoring, or in situ or local molecular diagnosis . In one MN-integrated sensors have been recognized as powerful platforms for skin ISF extraction and detection applications ,114,115.The integration of advanced MNs and multiple sensors presents a promising ISF-detection platform for clinical transformations ,92. Howe


In addition, TOPFlash/FOPFlash reporter assays showed that overexpression or downregulation of SFN obviously increased or decreased, respectively, the activity of the Wnt/β-catenin pathway. Our results indicated that SFN plays an important role in HCC, possibly providing a prognostic factor and therapeutic target for HCC.Abnormal stratifin (SFN) expression is closely related to the progression of several human cancers, but the potential roles of SFN in hepatocellular carcinoma (HCC) remain largely unknown. In this study, we found that SFN was upregulated in HCC cell lines and tissues and was positively associated with tumor size, poor differentiation, Tumor Node Metastasis (TNM) stage, and vascular invasion. In addition, high expression levels of SFN were associated with poor overall survival and disease-free survival. Biologically, downregulation of SFN suppressed tumor cell proliferation, epithelial–mesenchymal transition (EMT), invasion, and migration in vitro and tumor growth in vivo. However, overexpression of SFN promoted cell proliferation, EMT, invasion, and migration in vitro and tumor growth in vivo. Mechanistically, overexpression of SFN activated the Wnt/ Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related death worldwide , 2. HepaStratifin (SFN) is a checkpoint protein of the cell cycle and a regulatory factor of nuclear mitosis . SFN is β-catenin signaling pathway. Our results indicate the role and potential mechanism of SFN in HCC, possibly providing a therapeutic target and prognostic marker for HCC. A preprint of this manuscript has previously been published on the Research Square online platform [In this study, we explored the role of SFN in HCC and found that SFN was upregulated in HCC tissues and cell lines. The expression of SFN was positively associated with large tumor size, poor differentiation, advanced Tumor Node Metastasis (TNM) stage, vascular invasion, and poor prognosis in HCC. We also found that SFN promotes the proliferation, migration, invasion, EMT, and tumorigenesis of HCC cells. Mechanistically, SFN activates the Wnt/platform .A total of 34 pairs of HCC tissues and adjacent nontumor tissues were obtained from HCC patients in the Department of General Surgery of the First Affiliated Hospital of Nanchang University. All of the patients underwent radical resection between October 2017 and January 2018. The fresh specimens were stored in liquid nitrogen. The clinical data were collected from electronic medical records, and the survival information was obtained by follow-up. None of the patients received any adjuvant therapy before surgery and signed a written informed consent form. Our study was conducted in compliance with the Declaration of Helsinki and was approved by the institutional review of The First Affiliated Hospital of Nanchang University.μg/ml streptomycin (Gibco). All cells were placed in a humidified incubator with 5% CO2 and at 37°C.The HCC cell lines MHCC-97H and MHCC-97L were obtained from the Liver Cancer Institute of Fudan University . The HCC cell lines Huh7, SMMC-7721, HepG2, and the normal liver cell line L02 were obtained from the Chinese Academy of Sciences . All cells were cultured in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum , 100 U/ml penicillin, and 100 Premix Ex Taq™ II (Tli RnaseH Plus) Kit . β-Actin was used as an internal control. All gene expression levels were calculated by the 2−△△CT method. The primers used in the current study were as follows: SFN: forward primer: 5′-TGACGACAAGAAGCGCATCAT-3′, reverse primer: 5′-GTAGTGGAAGACGGAAAAGTTCA-3′.Total RNA was obtained from the cultured cells or fresh-frozen tissues by using TRIzol Reagent  and was reverse transcribed into cDNA by using the PrimeScript RT Reagent Kit with gDNA Eraser  by the manufacturer's instructions. Quantitative real-time polymerase chain reaction (RT–qPCR) was performed in a StepOnePlus™ Real-Time PCR System  by using the TB Green® 5 MHCC-97H or SMMC-7721 cells were seeded in 6-well plates in DMEM with 10% FBS. The cells were transfected when the cell confluence was approximately 60%. The medium was replaced with 1 ml of fresh medium with 20 μl of lentivirus solution. After 4 hours, another 1 ml of fresh medium was added to each well. The medium was replaced after 24 hours. The stably transfected cell lines were selected by puromycin at a final concentration of 2 μg/ml. The efficiency of transfection was verified by RT–qPCR and western blotting (WB).The knockdown and ectopic expression lentiviruses for SFN and the corresponding control lentivirus were synthesized by HanBio . The full-length human SFN cDNA sequence was inserted into the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO retroviral vector to generate the pHBLV-SFN vector (named SFN), and the empty vector was considered the negative control vector (named Vector). Two shRNA oligonucleotide sequences against SFN were inserted separately into the retroviral vector to generate the pHBLV-shSFN vectors (names shSFN#1 and shSFN#2). The shRNA sequences were as follows: shSFN#1, 5′-GACGACAAGAAGCGCATCATT-3′; and shSFN#2, 5′-GCTCTCAGTAGCCTATAAGAA-3′. The scramble shRNA sequence was inserted into the vector, and the resulting construct was considered the shRNA negative control vector (named shNC). Transfection of cell lines was performed according to the manufacturer's protocol. In brief, 1 × 103 MHCC-97H or SMMC-7721 cells were seeded into 96-well plates after stable transduction with lentiviral vectors for 48 hours, with 5 wells established per group. Then, at 0, 24, 48, 72, and 96 hours, 10 μL CCK-8 reagent mixed with 100 μL culture medium was added to each well, and the plates were incubated in an incubator for 2 hours. Finally, a multimode plate reader  was used to measure the absorbance at 450 nm.Cell proliferation assays were conducted using a Cell Counting Kit-8  according to the manufacturer's protocol. Briefly, 5 × 10Transduced cells (1000 cells/well in the SFN overexpressing group and 500 cells/well in the SFN knockdown group) were plated in 6-well plates, and then the medium was replaced every five days. After two weeks, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Then, the 6-well plates were washed with running water. The cell colonies were counted in the images.μL pipette tip by drawing a line on the surface of the cells in each well of 6-well plates. Images of wound healing at 0 and 24 hours were photographed using a microscope (10×) in each well.After transduction for 48 hours, MHCC-97H or SMMC-7721 cells were seeded into 6-well plates. When the cells were 95% confluence, a scratch wound was made with a 200 μm, and 24-well plates were used to perform the cell migration and invasion assays. For the migration experiments, 5 × 104 cells were seeded into the upper chambers in 200 μL of DMEM without FBS, whereas 700 μL of DMEM containing 20% FBS was added to the bottom plates. For the invasion experiments, 50 μL Matrigel/DMEM  was added into the upper transwell chambers and then incubated overnight in a 4°C refrigerator for the next experiment. The cells were seeded and maintained in the same culture medium and conditions used in the migration experiments, although the number of cells was different (1 × 105 cells). After incubation for 24 hours, the cells in the lower chamber or on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Then, the cells or Matrigel on the upper surface of the membrane were removed with a cotton swab. HCC cells were counted in five random fields by using a microscope (10×).Transwell chambers  containing a membrane with a pore size of 8 6 lentivirally transduced HCC cells in 100 μl DMEM containing 50% Matrigel were subcutaneously injected into the right upper flank regions of the mice. The tumor length (L) and width (W) were measured every three days. The formula 1/2 × (L × W2) was used to calculate the tumor volume. After 34 days, the mice were placed into the euthanasia chamber, and carbon dioxide was injected into the chamber at a rate of 30% of the volume of the euthanasia chamber per minute. After the animals stopped breathing and their pupils dilated, the next experiments were carried out. The tumors were excised for further study. All animal studies were approved by the Medical Experimental Animal Care Commission of the First Affiliated Hospital of Nanchang University.Six-week-old male BALB/c nude mice purchased from Hunan SJA Laboratory Animal Co., Ltd., were used to investigate the role of SFN in tumor growth (six mice for each group). A total of 5 × 10The reporter plasmids encoding TOPFlash or FOPFlash with TCF/LEF DNA-binding sites and the control plasmid pTK-RL were purchased from Beyotime Biotechnology (China). Infected cells were plated in 24-well plates, and the pTK-RL and Flash plasmids were cotransfected into the cells. After 48 hours, firefly luciferase activity was measured and normalized to Renilla luciferase activity.μg of protein from cells or tissues was separated by 8–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride  membranes. The PVDF membranes were blocked with 5% skim milk for 1 hour at 37°C and incubated with primary antibodies against SFN , β-actin , E-cadherin , N-cadherin , vimentin , Matrix metalloproteinase 2 (MMP-2) , Matrix metalloproteinase 9 (MMP-9) , β-catenin , non-phospho (active)-β-catenin , Glycogen synthase kinase-3 beta (GSK-3β) , phospho-GSK-3β (Ser9) , c-Myc , and Histone H3  overnight at 4°C. After washing with Tris-buffered saline in Tween buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit  or HRP-conjugated anti-mouse  secondary antibodies at 37°C for 1 hour. Both the primary and secondary antibodies were diluted with Primary & Secondary Antibody Diluent Buffer . The band intensities were quantified by ImageJ , and the specific details were described previously [Total protein from cells and tissues was extracted with Radio Immuno Precipitation Assay (RIPA) buffer containing protease inhibitors. After centrifugation, the supernatants were collected for subsequent experiments. NE-PER™ Nuclear and Cytoplasmic Extraction Reagents  were used to extract nuclear proteins. The BCA Protein Quantitation Kit  was used to measure the protein concentration. A total of 30 https://portal.gdc.cancer.gov/repository) and consisted of data for 374 HCC tissues and 50 adjacent nontumor tissues. The limma package (R) was used to analyze the expression of SFN in tumor and nontumor tissues [The gene expression profiles of liver HCC (LIHC) obtained from the RNA-Seq HTSeq-FPKM platform were downloaded from The Cancer Genome Atlas (TCGA) via the GDC Data Portal ( tissues .https://www.ncbi.nlm.nih.gov/geo/), we downloaded ten microarray expression profiles  containing data for HCC tissues and nontumor tissues. Except for the difference in SFN expression in GSE77314, which was directly calculated from the XLSX data downloaded from GEO datasets, the difference in SFN expression between tumor and nontumor tissues in the other GEO series was determined by the Limma package (R) [Regarding Gene Expression Omnibus (GEO) datasets (https://www.oncomine.org/), four studies were comparing SFN expression between HCC and normal samples. We comprehensively analyzed the data in these four studies with the following thresholds: P value ≤1 × 10−4, fold change ≥2, and gene rank in the top 10%.In the Oncomine database (https://www.proteinatlas.org/).Immunohistochemical images were used to assess the difference in SFN protein expression between HCC tumors and normal liver tissues in The Human Protein Atlas (HPA) database (https://cran.r-project.org/web/packages/) based on the R language. Data downloaded from the Kaplan–Meier plotter database  were used for survival analysis [To investigate the prognostic value of SFN gene expression in patients with HCC, we conducted survival analysis and univariate and multivariate analyses. Univariate and multivariate Cox regression analyses and overall survival (OS) analyses were performed by the “survival” and “survminer” packages .Furthermore, the clinicopathological data of 374 patients with HCC obtained from TCGA were used to evaluate the prognostic value of SFN in HCC by univariate and multivariate analyses. Due to the incompleteness of the clinicopathological data for many patients, we selected some common features  for further analysis and excluded unavailable data, including unknown, missing, Tx, Nx, and Mx data. Finally, 212 HCC patients were enrolled in this study. The univariate analysis indicated that TNM stage, T stage, M stage, and SFN expression level were significantly associated with OS  cells, with the lowest SFN expression level, and we downregulated SFN expression by lentivirus-mediated knockdown in MHCC-97H (named MHCC-97H-Lv-shSFN) cells, with the highest SFN expression level. RT–qPCR and WB were used to estimate the knockdown efficiency Figures . To idenTaken together, these data demonstrated that SFN promoted HCC cell proliferation, migration, and invasion in vitro and tumor growth in vivo.EMT is an important process in tumor progression and is closely related to the processes of invasion and migration . To furtβ-catenin signaling is a crucial pathway for HCC progression [β-catenin signaling to promote HCC progression. To verify this hypothesis, we explored the role of SFN in the expression of key markers of the Wnt/β-catenin signaling pathway, such as β-catenin and GSK-3β [β-Catenin is a key member of the Wnt/β-catenin signaling pathway and plays an important role in tumor progression [β-catenin was downregulated  protein, but overexpression of SFN showed the opposite effect. Translocation of β-catenin into the nucleus is the best biomarker for activation of Wnt/β-catenin signaling [β-catenin in the nucleus and found that SFN overexpression promoted β-catenin accumulation in the nucleus and that SFN knockdown exerted the opposite effect  with the Wnt signaling pathway-specific antagonist XAV-939. As shown in Supplemental Figure 2A, XAV-939 significantly inhibited the activity of the Wnt signaling pathway. The results indicated that the change trends in the levels of EMT-related markers were reversed by XAV-939 treatment in SFN-overexpressing cells -β-catenin, and phospho-Gsk-3β (Ser9) in SFN-overexpressing cells were increased. Cells with SFN knockdown showed decreases in these levels. To further confirm the effect of SFN on the Wnt/β-catenin pathway, we examined the protein level of β-catenin in the nucleus and found that SFN overexpression promoted the accumulation of β-catenin in the nucleus and that SFN knockdown exhibited the opposite effect. c-Myc is a downstream molecule of the Wnt/β-catenin signaling pathway, and its expression increases when β-catenin is translocated into the nucleus [β-catenin (Figures β-catenin signaling pathway [β-catenin signaling pathway. The results indicated that overexpression of SFN activates the Wnt/β-catenin signaling pathway, whereas knockdown of SFN inhibits the Wnt/β-catenin signaling pathway. In addition, we provided further support for the hypothesis that Wnt/β-catenin signaling is related to the mechanism, by which SFN promotes HCC progression. XAV-939, an inhibitor of Wnt/β-catenin signaling, was adopted to inhibit Wnt/β-catenin signaling in SFN-overexpressing cells [β-catenin signaling in SFN-overexpressing cells (Supplement Figure β-catenin signaling activity, treatment with XAV-939 led to the obvious abolishment of the process of EMT and a decreased ability of migration and invasion in SFN-overexpressing cells. Taken together, these results suggest that SFN can promote the progression of HCC by activating the Wnt/β-catenin signaling pathway. A previous study indicated that SFN can interact with Gsk-3β and inhibit Gsk-3β activity [β catalytic activity in SFN-transfected cells was significantly lower than that in nontransfected cells. In line with the inhibition of catalytic activity, the phospho-Gsk-3β level was increased in SFN-overexpressing cells. Gsk-3β inactivation is controlled by its phosphorylation by the destruction complex [β is phosphorylated and dissociated from the multiprotein destruction complex, β-catenin is released [β and then activates the Wnt/β-catenin signaling pathway to promote the progression of HCC, which is the possible main mechanism revealed by the current research. However, this is just a preliminary exploration of SFN in HCC, and a more in-depth mechanism needs to be further investigated, especially its impact on the inflammatory process.In the current study, we further showed that SFN facilitated HCC cell proliferation, migration, invasion, tumor growth, and EMT by activating Wnt/n of HCC . Gsk-3β  pathway , 27. Stag output , 26. Our nucleus . Our res Figures , which a pathway . Our resng cells . Treatmeactivity . Our res complex . Once Gsreleased . Thus, Sβ-catenin signaling pathway. The results of the current analysis may identify a prognostic factor and therapeutic target for HCC in the future.In conclusion, SFN is overexpressed in HCC tissues and cell lines, and SFN expression is correlated with tumor size, differentiation, vascular invasion, and TNM stage. High expression of SFN was associated with worse survival outcomes. SFN promotes cell proliferation, migration, invasion, and tumor growth and may be an oncogene in HCC. Moreover, SFN induces EMT and activates the Wnt/


Odontogenic cutaneous sinus tract (OCST) is defined as pulp necrosis caused by dental caries or trauma that forms a fistula on the body surface as a drainage channel for the infected pulp. OCST can be difficult to diagnose because subjective symptoms, such as pain in the affected tooth, may be minimal. In addition, lesions in the cervical region are extremely rare. In this report, we discuss the case of a 10-year-old girl who presented with inflammation, edema, and purulent exudation on the right neck. Her symptoms resembled those of lateral cervical cysts and fistulas. However, upon evaluation, she was diagnosed with OCST. Although OCST is an important differential diagnosis for head and neck lesions, it is often overlooked. OCST should be considered in the differential diagnosis of neck masses and fistulas. Odontogenic cutaneous sinus tract (OCST) is defined as pulp necrosis caused by dental caries or trauma that forms a fistula on the body surface as a drainage channel for the infected pulp . The diaA 10-year-old girl presented to our hospital with inflammation, edema, and purulent exudation on the right neck. One year earlier, she had been treated with antibiotics by her primary care physician for a mass on the right neck. She was initially diagnosed with lymphadenitis. However, the mass recurred periodically, requiring incision and drainage. A lateral cervical cyst or fistula was suspected, and the patient was referred to our hospital for workup. On examination, inflammation, edema, and purulent exudation were observed from the right neck, with mild tenderness on palpation (Figure Ultrasonography revealed a fistula extending from the body surface to the mandible Figure , and theHer dental history revealed that a carious tooth in the same area had been restored with a crown several years earlier. Suspecting odontogenic disease, she was referred to a dentist, who found recurrent caries in the crown restoration of the mandibular right first molar, accompanied by apical periodontitis Figure .On the basis of these findings, a diagnosis of OCST due to apical periodontitis of the mandibular right first molar was made, and the tooth was extracted and the granulation tissue was removed. Symptoms improved rapidly after surgery, with no recurrence of symptoms 10 months after surgery Figure .The main cause of OCST is apical periodontitis, a necrosis of the dental pulp caused by dental caries or trauma ,4. OCST The primary site of OCST is the mandibular molar, which has a high incidence of dental caries. Mandibular canines are the second most common cause of pyogenic pulpitis because their roots are long and do not exfoliate until old age ,11. OCSTPanoramic radiographs are an effective diagnostic tool for identifying OCST by demonstrating the radiolucency of the affected teeth . HoweverOnce OCST is confirmed, the first step is to treat the causative tooth. This may include endodontic therapy or surgical root canal therapy and apicoectomy for restorable teeth, or extraction of nonvital teeth to eradicate infection. While the standard protocol involves resection of the fistula in conjunction with the treatment of the causative tooth, even when the causative tooth is treated alone, the fistula often resolves within two weeks, resulting in a good outcome ,15. OCSTThis case resembles a lateral cervical cyst and fistula both in appearance and history . LateralThis case report highlights the importance of considering OCST as a possible differential diagnosis for cervical lesions in pediatric patients. Awareness among clinicians treating pediatric patients is necessary for the early diagnosis and appropriate management of OCST.


We characterized 212Pb-TCMC-YS5 in vitro and established a safe dose in vivo. We next studied therapeutic efficacy of a single dose of 212Pb-TCMC-YS5 using three prostate cancer small animal models: a subcutaneous mCRPC cell line-derived xenograft (CDX) model (subcu-CDX), an orthotopically grafted mCRPC CDX model (ortho-CDX), and a prostate cancer patient-derived xenograft model (PDX). In all three models, a single dose of 0.74 MBq (20 µCi) 212Pb-TCMC-YS5 was well tolerated and caused potent and sustained inhibition of established tumors, with significant increases of survival in treated animals. A lower dose (0.37 MBq or 10 µCi 212Pb-TCMC-YS5) was also studied on the PDX model, which also showed a significant effect on tumor growth inhibition and prolongation of animal survival. These results demonstrate that 212Pb-TCMC-YS5 has an excellent therapeutic window in preclinical models including PDXs, opening a direct path for clinical translation of this novel CD46-targeted alpha radioimmunotherapy for mCRPC treatment.We recently identified CD46 as a novel prostate cancer cell surface antigen that shows lineage independent expression in both adenocarcinoma and small cell neuroendocrine subtypes of metastatic castration resistant prostate cancer (mCRPC), discovered an internalizing human monoclonal antibody YS5 that binds to a tumor selective CD46 epitope, and developed a microtubule inhibitor-based antibody drug conjugate that is in a multi-center phase I trial for mCRPC (NCT03575819). Here we report the development of a novel CD46-targeted alpha therapy based on YS5. We conjugated The online version contains supplementary material available at 10.1186/s13046-023-02636-x. Given that YS5 is a clinical stage human antibody, this YS5-based 212Pb alpha particle therapy has potential of translation to the clinic for treatment of mCRPC patients.This study reports a novel CD46 targeted 212Pb has a half-life (t1/2) of 10.6 h that acts as an in vivo generator for the high LET α-emitting daughter isotopes 212Bi with t1/2 = 60.6 min and 212Po with t1/2 = 0.3 microseconds. RIT with 212Pb-TCMC-trastuzumab has been clinically tested in ovarian cancer , which showed an antitumor activity and was well tolerated [212Pb also showed promising therapeutic efficacy in different cancers [Targeted radiotherapy with α-particle emitting radionuclides is a promising approach to eliminate microscopic clusters of malignant cells due to the short path length 50–80 μm), high linear energy transfer LET; 100 keV/μm), and high relative biological effectiveness of α-particles [00 keV/μm cancers –8.0 μm, hig177Lu-J591 and 225Ac-J591), or radio-labeled small molecule PSMA-617  for the treatment of adult patients with PSMA-positive mCRPC who have been treated with androgen receptor pathway inhibition and taxane-based chemotherapy [Current efforts on targeted radio-ligand delivery in prostate cancer are centered on prostate-specific membrane antigen (PSMA) for the adenocarcinoma subtype using anti-PSMA antibodies . Inotherapy . Despiteotherapy ; (2) PSMotherapy . Not surotherapy . In addiotherapy , 17. Thu89Zr radiolabeled YS5 (89Zr-DFO-YS5) can detect prostate cancer in vivo in both PSMA positive and PSMA negative mCRPC cell line xenograft (CDX) models and patient derived xenograft (PDX) models of both the adenocarcinoma and neuroendocrine subtypes [We have previously developed a non-gene expression-based approach to identify tumor cell surface epitopes including those formed by conformational change and post-translational modifications , 19. We subtypes , and a f212Pb-radiolabeled human antibody YS5 (212Pb-TCMC-YS5) in multiple prostate cancer small animal models, including subcutaneous CDX, intraprostate orthotopic CDX, and prostate cancer PDX models.In this study, we report the synthesis and evaluation of tolerability and therapeutic efficacy of the 89Zr oxalate was purchased from the Cyclotron Laboratory at University of Wisconsin, Madison . The 224Ra/212Pb generators used for these studies were purchased through the Isotopes Program, US Department of Energy, Oak Ridge National Laboratory . All other chemical reagents and materials were the highest grade available from Sigma-Aldrich and used as received without further purification (unless otherwise described). Radioactivity measurements of samples from the in vitro and in vivo experiments were performed on a calibrated Capintec CRC® -55tR Dose Calibrator  with dial setting at 101 or 158 at equilibrium, or the calibrated Hidex Automatic Gamma Counters using an energy window centered on the main gamma peak from 212Pb at 238.6 keV. Radioactivity analyses were corrected for radioactive decay.  was purchased from Fisher Chemical. Pb-resin™ 100–150 μm) was purchased from Eichrom Technologies LLC . The fully human CD46-targeted monoclonal antibody, YS5, was produced and purified as described previously [0 μm was 89Zr labeling of DFO-YS5 were performed as described [Conjugation of YS5 with the chelator DFO and subsequent escribed .212Pb radiolabeling of YS5-TCMC and purification of the 212Pb-TCMC-YS5 were performed as previously described [212Pb was eluted with 2 M HCl from the 224Ra-212Pb generator and pre-concentrated by passing a column loaded with the Pb-resin. Following the pre-concentration step, about 7.4 × 107 Bq (2 mCi) to 1.85 × 108 Bq (5 mCi) 212Pb2+ was eluted from the Pb-resin column into a reaction vessel with pH = 6 NaOAc buffer as the radiolabeling reaction. The reaction vessel was pre-loaded with 200 μg to 500 μg YS5-TCMC in 500 μL pH = 5.4 NaOAc buffer and 50 μL human serum albumin (10 mg/ml) and the reaction vessel was heated at 37 °C for 1 h. 212Pb-TCMC-YS5 was purified by using a PD-10 column with 0.1 M phosphate buffer with human serum albumin (10 mg/ml) and used right after for in vitro and in vivo animal studies. Radiolabeling efficiency (the percentage of incorporated radionuclide from radiolabeling reaction) and radiochemical purity of 212Pb-YS5 was analyzed using size-exclusion HPLC using a Zenix-C-SEC-300 column  with 0.1 M phosphate buffer (pH 7.2) at the eluting rate of 1 ml/min. Fractions were collected and 212Pb activity was determined by a Capintec CRC®-55tR Dose Calibrator and the radioactivity recovery yield of all combined eluted fractions against injected radioactivity was calculated [YS5 was first conjugated with the bi-functional chelator 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane  at the molar ratio of TCMC/YS5 at 10:1. The average TCMC chelator/mAb ratios were determined by a spectrophotometric assay . 212Pb rescribed  with modD value of 212Pb-TCMC-YS5 binding to the CD46-expressing mCRPC cell line PC3 was determined by a saturation binding assay with concentrations at 5, 10, 25, 50, and 100 nM as previously described [212Pb at 238.6 keV.The apparent Kescribed . Radioac−/−) . Animal studies were approved by the University of Virginia Institutional Animal Care and Use Committee and performed in compliance with guidelines from the Public Health Service Policy and Animal Welfare Act of the United States.CDX studies were performed using 5–7 week old male athymic nude mice . PDX studies were performed using 6–8 weeks old male NSG mice  of 89Zr-DFO-YS5 in saline was administered through the tail vein, and animals were imaged at 24, 72, and 120 h post injection, with a 20-min acquisition time by using microPET/CT Albira Trimodal PET/SPECT/CT Scanner (Bruker Corporation). PET imaging data were acquired in list mode and reconstructed using Albira Software Suite provided by the manufacturer. The resulting image data were then normalized to the administered activity to parameterize images in terms of %IA/g. Imaging data were viewed and processed using an open source Amide software. CT images were acquired following PET, and the CT data were used for attenuation correction for PET reconstruction, and anatomic reference.Approximately 3–5 weeks after tumor implantation, animals with PC3 xenografts reaching 100 mmn = 5–7/group) were given doses of 212Pb-TCMC-YS5 at 0, 0.185 MBq, 0.37 MBq, 0.74 MBq, and 1.85 MBq  per mouse. Mice were monitored for health status, signs of stress and body weight twice weekly. Mice were sacrificed when loss of body weight was more than 20%, or if the mice showed visible signs of distress, including lack of appetite, excessive lethargy, or formation of sores and rashes.To determine the safe dose for therapy study, a dose escalation study was performed in nude mice without tumor to determine toxicity. Groups of nude mice . For blood chemistry, plasma was separated from blood by centrifugation  and was either studied immediately or stored at -80 °C for later analysis. Biomarkers for organ function such as albumin, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using enzyme-linked immunosorbent assay (ELISA) kits .To evaluate the potential toxicity of 3 in volume were given a single i.v. dose of 212Pb-TCMC-YS5 at 20 µCi (740 kBq) (n = 5) or the unlabeled YS5 antibody (n = 7) or no treatment (n = 4). Animal number per group was based on past experience with prostate cancer models, not power calculation. Graft failures  at the time of randomization were excluded from the study. Tumor dimensions were measured and recorded 3 times weekly with a digital caliper, and tumor volumes were calculated using the following modified ellipsoid equation: volume = 0.5 × length x width x width [For the therapeutic study, mice bearing PC3 tumors about 100 mm5) were injected into the murine prostate in 30 µL of PBS, as previously described [PC3-luc cells were derived from PC3 cells transfected with the firefly luciferase expressing plasmid from Addgene  . PC3-lucescribed –28. Tumo212Pb-TCMC-YS5 at 0.74 MBq (20 µCi) (n = 4) or the unlabeled YS5 antibody (n = 5). Tumor growth was monitored by in vivo BLI. Animal survival data was analyzed by Kaplan–Meier analysis.For the therapeutic study, 30 days after PC3-luc cells were inoculated and tumor engraftment were confirmed by in vivo BLI, the mice were given a single i.v. dose of 3, mice were given a single i.v. dose of 212Pb-TCMC-YS5 at 0.74 MBq (20 µCi) (n = 6), 0.37 MBq (10 µCi) (n = 8) or the unlabeled YS5 antibody (n = 4), each along with non-binding human IgG (0.5 mg per mouse) for blocking Fc receptors. The PDX is carried in NSG mice that are known to have a high background antibody binding compared with nude mice used in the CDX study, which can be reduced by injection of Fc blocking agents [89Zr-DFO-YS5 PET imaging was performed using microPET/CT Albira Trimodal PET/SPECT/CT Scanner (Bruker Corporation) 3 days post i.v. injection of 3.70 – 5.55 MBq  of 89Zr-DFO-YS5 in saline along with non-binding human IgG (0.5 mg per mouse) for blocking Fc receptors. In the blocking control group, the cold YS5 antibody was administered 2 days prior to 89Zr-DFO-YS5 injection. Tumor dimensions were measured twice weekly with a digital caliper. Tumor growth was calculated as mean percent change in volume relative to the initial volume at the time of dosing. Animal survival data was analyzed by Kaplan–Meier analysis. Body weight and signs of distress were assessed twice weekly.PDX mice with subcutaneously grafted tumor (Cat#: TM00298 PR1996) were purchased from The Jackson Laboratory . Animal number per group was based on past experience, not power calculation. When tumors size reaches about 100 mmP < 0.05 was considered significant. Survival was analyzed by Kaplan–Meier analysis. The significance of median survival  was determined by a Log-rank (Mantel-Cox) test.Two-tailed Student’s t-test and one-way analysis of variance (ANOVA) were used for analysis of two and multiple (more than two) groups, respectively. 212Pb radiolabeling of the YS5 antibody was performed as shown in Fig. 212Pb radiolabeling process was adopted from the Pb-resin pre-concentration and column extraction method described previously, with modifications [212Pb-TCMC-YS5 was > 98% by size-exclusion HPLC  212Pb-TCMC-YS5 to PC3 cells was determined by an in vitro cell binding assay and found to be 21.7 ± 7.3 nM  of 0.185 MBq (5 μCi), 0.37 MBq (10 μCi), 0.74 MBq (20 μCi), 1.85 MBq (50 μCi) and one control group receiving “cold” YS5 . As shown in Fig. We performed a dose escalation study to identify the safe dose range using non-tumor bearing nude mice. The study included 4 dose groups  and long-term (day-90 post treatment) toxicity in mice receiving 0.74 MBq 20 μCi) of 0 μCi of 89Zr-DFO-YS5 in the PC3 subcu-CDX model. We showed, by PET imaging : tumor reduction began within the first week with a volume decrease up to 70%, and the inhibitory effect lasted over 40 days post administration of 212Pb-TCMC-YS5 .We first studied anti-tumor efficacy of ing Fig. A and bioing Fig. B, that 8us study . We perf212Pb-TCMC-YS5 in an intraprostate orthotopic mCRPC CDX  .We further studied the therapeutic effect of a single dose CDX Fig. A. The PCCDX Fig. , was signt) Fig. C. Surviv89Zr-DFO-YS5 to confirm tumor targeting by YS5 in vivo. The result showed a clear evidence of tumor targeting by YS5 , and the lower dose of 0.37 MBq (10 μCi). As shown in Fig. , both doses inhibited tumor growth, with the higher dose of 0.74 MBq (20 μCi) showing a longer duration of inhibition. Body weight remained steady for the two treatment groups (0.37 MBq (10 μCi) and 0.74 MBq (20 μCi)) for the duration of the study .To broaden clinical applicability, we performed therapy study using a prostate cancer PDX model. We first used PET imaging with YS5 Fig. A with tuYS5 Fig. B. Uptake212Pb radiotherapy and studied efficacy in three prostate cancer animal models. We optimized the 212Pb labeling process that produced 212Pb-YS5 with high yield that retained high affinity binding to tumor cells. For therapeutic evaluation, we found that a single i.v. dose of 20 μCi of 212Pb-TCMC-YS5 was well tolerated without causing either acute or long-term toxicity as assessed by body weight change, general health condition, blood cell count and blood chemistry analysis. We next studied efficacy of this dose (a single injection) in three mouse models , and found potent tumor inhibition with an increase in animal survival in all three models.CD46 is a new prostate cancer cell surface target that we identified and validated, which shows lineage independent homogeneous expression in both adenocarcinoma and small cell neuroendocrine mCRPC , differe212Pb (10.6 h), the tumor inhibition effect from a single dose treatment lasted over 40 days post treatment, suggesting that 212Pb-TCMC-YS5 could be a promising candidate for prostate cancer therapy development. Several other studies also showed that 212Pb labeled full-length monoclonal antibodies were effective in tumor inhibition in animal models [212Pb. Thus, 212Pb radio-ligand therapy may be a promising approach not only for small molecule ligands [224Ra-212Pb and 203Pb as the imaging theranostic pair, the pace of 212Pb-based radio-ligand therapy development is expected to accelerate [Despite the relatively short half-life of l models –33 and i ligands  but alsocelerate –37.212Pb radiolabeling process employed in this study can be readily incorporated into an automatic synthesizer for clinical production. 212Pb eluted by strong acids from the 224Ra-212Pb generator requires further process before labeling [212Pb also puts a time constraint on the preparation protocol. As such, a more facile and rapid process may facilitate clinical translation. Pre-concentration of 212Pb via extraction chromatography resin was reported recently [212Pb-TCMC-YS5 within 2 h, suggesting that this method of 212Pb-TCMC-YS5 preparation could be further scaled for clinical production.It should be noted that the labeling , 33. The212Pb-TCMC-YS5 in two mCRPC CDXs and one PDX – in all three models efficacy is evident for a single dose of 0.74 MBq (20 μCi) and a single treatment cycle, with excellent tolerability and no clear toxicity detected by blood analysis 90 days post treatment. A single treatment of a lower dose (0.37 MBq or 10 μCi) of 212Pb-TCMC-YS5 also showed efficacy in the PDX model, suggesting opportunities for further optimization. Additional dosing schemes and multiple treatment cycles in future studies could further improve efficacy over toxicity and increase the therapeutic window.We studied the therapeutic effect of 212Pb-TCMC-YS5 is applicable to this subtype.We have shown previously that CD46 is expressed in both adenocarcinoma and small cell neuroendocrine prostate cancer . Nearly 225Ac and found potent anti-tumor activity in other prostate cancer models (BioRxiv 2022.10.13.512165). Further studies will compare efficacy and toxicity of those YS5 alpha particle therapies to select a candidate for clinical testing in human patients.The clinical stage human antibody YS5 is compatible with the development of radioimmunotherapy based on other alpha particles. For example, we have also conjugated YS5 with Finally, the YS5 human antibody binds to cynomolgus monkey CD46 but not mouse CD46 , prevent212Pb radioimmunotherapy study demonstrates that 212Pb-TCMC-YS5 is well tolerated and efficacious against prostate cancer in both CDX and PDX models. Given that CD46 is becoming a credentialed target in mCRPC, an YS5-based antibody–drug conjugate is in multiple clinical trials (NCT03575819 and NCT05011188) with an established safety profile, and an YS5-PET imaging agent is in a first-in-human study (NCT05245006), our YS5-conjugated radioimmunotherapy has potential for rapid translation into the clinic for prostate cancer treatment.The result from this first of its kind CD46-targeted Additional file 1: Table S1. Hematology parameters of acute (day-14) and long-term (day-90) toxicity studies. RBC: Red blood cell; WBC: White blood cell; PLT: Platelet; HCT: Hematocrit test (proportion of RBC in blood); HGB: Hemoglobin; Neu: Neutrophil; Lym: Lymphocyte; Mon: Monocyte; and Eos: Eosinophil. Mean ± SD, n = 5. Table S2. Blood clinical chemistry tests for liver and kidney function for acute and long-term toxicity studies. Figure S1. The calibration curve of Pb-Arsenazo (Pb-AAIII) spectrophotometric assay for determining the TCMC chelator number per antibody. The standard curve of UV (upper panel) is constructed by plotting the absorbance at 656 nm against Pb2+ concentration at various dilutions from a stock solution of Pb(II)-AA(III). The stock solution was prepared in a 0.15 M NH4OAc buffer at pH 7.0, containing 10 mM AAIII and 4.83 mM Pb(II).. The titration curve (bottom panel) is constructed by plotting the change of UV absorbance at 656 nm (%) against the mass of TCMC added to 100 µl of the Pb(II)-AA(III) complex solution. The solution had a Pb2+ concentration of 192 µM. Figure S2. Therapeutic effects of a single dose 212Pb-TCMC-YS5 in the PC3 subcutaneous CDX model. Tumor growth of individual mice within 55 days post treatment of 212Pb-TCMC-YS5 . Red: Study group receiving 212Pb-TCMC-YS5. Blue: Control group receiving unlabeled YS5 antibody only. Figure S3. BLI images of the PC3-Luc orthotropic CDX model treated by a single dose 212Pb-TCMC-YS5 (top), and the cold YS5 control (bottom). Two animals died in the control group on day-19 post treatment and were not imaged. Figure S4: Change (days post injection) of actual tumor volume of individual PDX mouse treated with a single dose of 0.37MBq (10 μCi) and 0.74 MBq (20 μCi) of 212Pb-TCMC-YS5.