Here, we examined phenotypically and genotypically three commonly used allograftable mouse PDAC cell lines (C57BL6 genetic background): Panc02 , and two cell lines originated from PDACs developed in two different KPC  mouse models. Our study (i) characterized the ability of a widely used attenuated oncolytic vesicular stomatitis virus VSV-ΔM51-GFP to infect, replicate in, and kill mouse PDAC cells; (ii) examined their innate antiviral responses; (iii) compared their permissiveness to a non-attenuated VSV-Mwt-GFP and chemotherapeutic drugs; and (iv) analyzed their karyotype and exome. Mouse PDAC cell lines showed high divergence in their permissiveness to VSV-ΔM51-GFP, which negatively correlated with their abilities to mount innate antiviral responses, while all three cell lines were highly permissive to VSV-Mwt-GFP. No correlation was found between resistance to VSV-ΔM51-GFP and chemotherapy. Also, mouse PDAC cell lines showed high divergence in their karyotype and exome. The exome analysis demonstrated that more VSV-ΔM51-GFP-permissive mouse PDAC cell lines harbor mutations in multiple important antiviral genes, such as TYK2, JAK2, and JAK3.Oncolytic virus (OV) therapy is a promising virus-based approach against various malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies demonstrated that human PDAC cell lines are highly variable in their permissiveness to OVs. Mouse PDAC cell lines, which are widely used for in vivo examination of the adaptive immune responses during cancer therapies. Mouse PDAC cell lines showed high divergence in their permissiveness to oncolytic vesicular stomatitis virus (VSV), which negatively correlated with their abilities to mount innate antiviral responses. Also, we discovered that more VSV-permissive mouse PDAC cell lines harbor mutations in multiple important antiviral genes, such as TYK2, JAK2, and JAK3. Our study provides essential information about three model mouse PDAC cell lines and proposes a novel platform to study OV-based therapies against different PDACs in immunocompetent mice.Oncolytic virus (OV) therapy is a promising virus-based approach against various malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies using various human PDAC cell lines demonstrated that they are highly variable in their permissiveness to OVs. In this study, we examined phenotypically and genotypically three commonly used allograftable mouse PDAC cell lines, which are widely used for  Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that accounts for approximately 95% of pancreatic cancers and is the fourth cause of cancer-related deaths in the USA. The 5-year survival rate for PDAC patients has remained around 10%, while survival rates for other cancers have significantly improved .Mononegavirales, family Rhabdoviridae, genus Vesiculovirus) and a promising OV (Oncolytic virus (OV) therapy is a promising anticancer approach that utilizes replication-competent viruses that preferentially infect, replicate in, and kill cancer cells , 4. Vesiising OV –7. VSVising OV . The panising OV . The oncising OV –12, beising OV .in vivo PDAC mouse model systems fail to recapitulate all key characteristics of human PDAC disease   –16. Ime, etc.) . This ise, etc.) –20. MoIn this study, we examined three different allograftable mouse PDAC cell lines. Two of these cell lines originated from genetically engineered mouse models (GEMMs) of PDAC. GEMMs are created by introducing specific gene mutations in oncogenes and/or tumor suppressor genes that are central in human PDAC, effectively recapitulating PDAC in the mouse. The most robust and well-described PDAC GEMM is the KPC mouse, which is characterized by mutations in the Kras and Trp53, both of which are driven by a pancreas-specific Cre recombinase (via the Pdx1 promoter) which is expressed in all cells of the pancreas from early stages in development . ImportaIn this study, we examined phenotypically and genotypically three mouse PDAC cell lines (two KPCs and one non-KPC). Our study (i) characterized the ability of a widely used attenuated oncolytic virus VSV-ΔM51-GFP to infect, replicate in, and kill these mouse PDAC cells; (ii) examined their innate antiviral responses; (iii) compared their permissiveness to a non-attenuated VSV-Mwt-GFP and chemotherapeutic drugs; and (iv) analyzed their karyotype and exome. Mouse PDAC cell lines showed high divergence in their permissiveness to VSV-ΔM51-GFP, which negatively correlated with their abilities to mount innate antiviral responses, while all three cell lines were highly permissive to VSV-Mwt-GFP. No correlation was found between resistance to VSV-ΔM51-GFP and chemotherapy. Also, mouse PDAC cell lines showed high divergence in their karyotype and exome. Our study provides essential data about three allograftable model mouse PDAC cell lines and proposes a novel platform to study OV-based therapies against phenotypically different PDACs in immunocompetent mice.2 atmosphere at 37°C. For all experiments, cells were kept for no more than 15 passages. All described experiments were approved by the University of North Carolina at Charlotte Institutional Biosafety Committee (IBC).The recombinant virus VSV-ΔM51-GFP was previously described , in whic2, then read using a multiwell plate reader at 450 nm. This was repeated every 24 h for 7 days. Cell doubling time (TD) was calculated using the equation TD  =  (0.693t)/ln (Nt/N0) where t =  time difference in h during log phase, Nt =  absorbance value at time t, and N0 =  absorbance value at the initial time.1,000 cells per well in DMEM with 10% FBS were seeded into 96-well plates. Cells were given 24 h to adhere. After each 24-h period, WST-8  cell viability reagent was added to each well for 4 h at 37°C in 5% CO2 for the duration of the experiment. Virus-encoded GFP fluorescence was measured periodically over a 72-h time course using a fluorescence multiwell plate reader. GFP fluorescence was measured at 485/535 nm.Virus titers were calculated using standard plaque assays on BHK-21 cells in 12 or 24-well plates. For virus replication kinetics experiments, cells were seeded into 96-well plates and were given 24 h to adhere. Virus dilutions were prepared in DMEM with 0% FBS. Cells were washed with phosphate-buffered saline (PBS), followed by the addition of the virus for 1 h at 37°C. Virus-containing medium was then aspirated and fresh DMEM with 5% FBS was added back to cells and incubated at 37°C in 5% CO2, then read using a multiwell plate reader at 450 nm. Results are expressed as fold change compared with mock treatment.In a 96-well plate layout, cells were seeded at 90% confluence and were given 24 h to adhere. Cells were then washed once with PBS and mock-infected, or infected at either multiplicity of infection (MOI) 1, 0.1, 0.01, 0.001, or 0.0001. After 1-h incubation at 37°C, the virus was removed and a fresh medium containing 5% FBS was added to each well. At 70 h p.i., WST-8  was added to each well for 4 h at 37°C in 5% COTwelve-well plates were seeded at 90% confluence and were given 24 h to adhere. Cells were infected with VSV-ΔM51-GFP dilutions or mock-infected (control) for 24, 72, and 120 h. One hour after infection, the virus was aspirated and wells were overlaid with 2% agarose (VWR Agarose I-0710) in DMEM with 5% FBS. After 24, 72, or 120 h, formalin was added to fix cells for 4 h. After fixation, agarose was removed and cells were stained with crystal violet . For the plaque assay figures, each row represents one 12-well plate that was cut and stitched to view the highest amount of virus to the least amount of virus from left to right.Cells were seeded into 12-well plates at 90% confluence and were given 24 h to adhere. The medium was removed and cells were washed once with PBS. Cell lines were either mock-treated or infected with VSV-ΔM51-GFP at MOIs of 0.1 and 0.001 either based on virus titer on BHK-21 cells or based on virus titer on each cell line, in medium with 0% FBS and incubated for 1 h at 37°C. After 1-h incubation, the medium was removed and fresh medium with 0% FBS was added to each well. Cells were lysed and total protein was collected 24 h after infection using buffer as described previously . Total pCells were seeded into 12-well plates at 90% confluence and were given 24 h to adhere. The medium was removed and washed once with PBS. VSV-ΔM51-GFP was added at MOIs of 1 and 0.1 (based on each cell line) in a medium with 0% FBS and incubated for 1 h at 37°C. After 1-h incubation, fresh medium was added to each well containing 0% FBS. After 24 h, cell supernatants (for cytokine array) and lysates (for western blot) were collected and stored at −80°C. Supernatants were then sent to EVE Technologies Corp for Mouse Cytokine/Chemokine 44-Plex Discovery Assay Array (MD44).50 values were calculated using GraphPad Prism 9.3.1.Cells were seeded into 96-well plates at 90% confluence and were given 24 h to adhere. The medium was removed and cells were washed once with PBS. The virus was then added at MOI of 0.01 in medium with 0% FBS and incubated for 1 h at 37°C. After 1-h incubation, the medium was removed and fresh medium (0% FBS) containing mouse IFN alpha  was added at 2,500, 500, 100, 20, 4, or 0 units/mL. Virus-encoded GFP fluorescence was measured at 485/535 nm periodically over a 73-h time course using a fluorescence multiwell plate reader. ICSix-well plates were seeded at 90% confluence and were given 24 h to adhere. Cells were infected with sixfold serial dilutions of VSV-ΔM51-GFP or VSV-Mwt-GFP from 1E−4 to 7.7E−8 or mock-infected for 72 h. One hour after infection, the virus was aspirated and wells were overlaid with 2% agarose (VWR Agarose I- 0710) in DMEM with 5% FBS. After 72 h, formalin was added to fix cells for 3 h. After fixation, agarose was removed and cells were stained with crystal violet .Twelve-well plates were seeded at 90% confluence and were given 24 h to adhere. Cells were infected with threefold serial dilutions of VSV-ΔM51-GFP or VSV-Mwt-GFP from 1E−4 to 2E−9 or mock-infected for 72 h. One hour after infection, the virus was aspirated and wells were overlaid with 2% agarose (VWR Agarose I-0710) in DMEM with 5% FBS. After 24 h, virus-driven GFP focus forming units (FFUs) were counted using a fluorescent microscope. The results shown are relative to the titer on the KPC-Luc-4580 cell line.Cells were seeded into 12-well plates at 95% co0nfluence and were given 24 h to adhere. The medium was removed, and cells were washed with PBS. Cells were placed on ice approximately 5 min before virus infection to cool cells. Virus in DMEM with 0% FBS was added to cells on ice, and cells were incubated for 1 h at 4°C. After incubation, the virus-containing medium was aspirated, and cells were washed three times with cold PBS to remove any unbound virus. Samples were then either collected for cell lysates immediately (to examine attachment) or incubated for an additional 7 h at 37°C (to examine replication) and then cell lysates were collected. Total VSV protein was analyzed by western blot as described above. Membranes were blocked in 5% nonfat milk in TBS-T. Membranes were then incubated with a 1:5,000 dilution of rabbit polyclonal anti-VSV antibodies (raised against VSV virions) in TBS-T with 5% nonfat milk, followed by a 1:10,000 dilution of antirabbit secondary antibodies. To verify the total protein in each sample, loaded membranes were stained with Coomassie blue.50 values were calculated using GraphPad Prism 9.3.1.For gemcitabine and 5-FU determination, cells were seeded into 96-well plates for approximately 50% confluency in a medium supplemented with 10% FBS. The next day, cells were treated with serial dilutions of either gemcitabine  or 5-FU . The serial dilution ranges for gemcitabine and 5-FU were 1,000–0.008 µM and 300–0.006 µM, respectively. A WST-8  cell viability assay was performed 72 h later. ICEach cell line was grown to 70% confluence in a T-75 flask (Greiner #658170) and sent to the CytoGenomics Core Laboratory at the University of Minnesota. Adherent cells were harvested with colcemid arrest, treated with 0.75 M KCl hypotonic solution, and fixation with 3:1 methanol:acetic acid. The resulting cells were spread onto glass slides according to standard cytogenetic protocols. A spectral karyotyping (SKY) slide was processed according to the manufacturer’s protocol [Applied Spectral Imaging (ASI)]. SKY uses a unique combination of five fluorescent dyes to paint all 24 chromosomes. Seven metaphase cells per sample were examined by SKY using the Olympus BX61 microscope with DAPI and SKY fluorescence filter sets. G-band and SKY metaphase cells were imaged and karyotyped using ASI software. The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH Grant #P30 CA077598.6 cells), trypsinized, pelleted, frozen, and sent to Azenta US  for DNA extraction, library preparations, sequencing reactions, and bioinformatic analysis. DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Genomic DNA samples were quantified using Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Enrichment probes were designed against the region of interest and synthesized through Twist Biosciences—Twist Mouse Comprehensive Panel . Library preparation was performed according to the manufacturer’s guidelines. Briefly, the genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument. Fragmented DNA was cleaned up, end-repaired, and adenylated at the 3′ends. Adapters were ligated to the DNA fragments, and adapter-ligated DNA fragments were enriched with limited cycle PCR. Adapter-ligated DNA fragments were validated using Agilent TapeStation (Agilent Technologies) and quantified using Qubit 2.0 Fluorometer. Adapter-ligated DNA fragments were hybridized with biotinylated baits. The hybrid DNAs were captured by streptavidin-coated binding beads. After washing, the captured DNAs were amplified and indexed with Illumina indexing primers. Post-captured DNA libraries were validated using Agilent TapeStation (Agilent) and quantified using Qubit 2.0 Fluorometer and Real-Time PCR (KAPA Biosystems). Libraries were sequenced 2 × 150 on an Illumina HiSeq instrument. For data analysis, sequencing adapters and low-quality bases in raw reads were trimmed using Trimmomatic 0.39. The reads were then aligned to the GRCm37 reference genome using Sentieon 202112.01. Aligned sequences were then sorted and PCR/Optical duplicates were marked, producing BAM files. Somatic SNVs and small INDELs were called by using Sentieon 202112 . The VCF files generated by the pipeline were then normalized  using bcftools 1.13. Overlapped transcripts were identified for each variant and the effects of the variants on the transcripts were predicted by Ensembl Variant Effect Predictor v104. The most severe impact was selected for each variant and they are used for downstream cohort analysis. The impact of the variants was also classified based on MAF document specifications. The exome data were deposited to the NCBI GenBank Sequence Read Archive (SRA) database under BioProject PRJNA944630. Exome analysis of three murine pancreatic cancer cell lines was deposited under the following accessions: PANC02-Luc , KPC-Luc-4580 , and KPC-Luc-A .Each cell line was grown to 90% confluence  were performed using GraphPad Prism 9.3.1 software. Tests used are indicated in the legends of the figures.in vivo.In this study, we examined three clinically relevant allograftable mouse PDAC cell lines: PANC02-Luc, KPC-Luc-A, and KPC-Luc-4580. These cell lines originated from C57BL6 mice that had three different genetic backgrounds in the pancreas. The PANC02 cell line  is one of the most widely used mouse PDAC cell lines, and it was generated by chemically inducing PDAC in mice via the implantation of 3-methyl-cholantrene (3-MC) (a carcinogen) into the pancreas of genetically unaltered C57BL6 mice  Fig. 1AFig. 1A. TD) of these three cell lines  cell lines, which may contribute to the observed difference in growth kinetics. The KPC-Luc-4580 cells displayed the fasted growth with a doubling time of 21.23 days, and KPC-Luc-A cells were intermittent with a TD of 24.03 days. Although the KPC cell lines were generated similarly, differences in the overall history of each cell line  may contribute to the differences in growth kinetics.First, we compared growth kinetics and determined the doubling time . In addition, BHK-21, a baby hamster kidney cell line, was used as a reference cell line for comparison as it is highly permissive to VSV and widely used for VSV amplification and plaque assays. Also, our previous studies demonstrated that there is a wide range of permissiveness of human PDAC cells to OV therapy, from highly permissive to highly resistant, and the success of OV therapy greatly depends on permissiveness of human PDAC cell lines to OVs –20, 33To further examine the ability of VSV to replicate in these mouse PDAC cell lines, we analyzed VSV-driven GFP fluorescence using a panel of human and mouse PDAC cell lines and BHK-21 cells. Cells were seeded in 96-well plates and either mock-infected or infected with VSV-ΔM51-GFP at an MOI of 10, 1, 0.1, 0.01, 0.001, 0.0001, or 0.00001 (based virus titer on BHK-21 cells). As shown in To examine the effect of VSV infection on cell viability, cells were infected with VSV-ΔM51-GFP at different MOIs  for 70 h, followed by a cell viability assay . In geneIn general, our data indicate that VSV-ΔM51-GFP differs in its ability to replicate in, spread, and kill each of the three mouse PDAC cell lines. Our data show that PANC02-Luc cells are the overall most resistant to VSV-ΔM51-GFP, followed by KPC-Luc-A and KPC-Luc-4580.The differences in VSV-ΔM51-GFP replication, spread, and cell lysis observed between the mouse PDAC cell lines suggest potentially varied levels of antiviral signaling. Our previous studies have established that the capacities for type I IFN antiviral signaling between human PDAC cell lines vary dramatically and are strongly correlated with resistance to VSV and other OVs , 18–2050) for mIFNα, the amount of mIFNα needed to result in 50% inhibition of virus replication  (50 = 5.37 U/mL), followed by PANC02-Luc (IC50 = 33.1 U/mL), KPC-Luc-A (IC50 = 61.6 U/mL), and KPC 4580 (IC50 = 354.8 U/mL). These data support our hypothesis that resistance of mouse KPC cells to VSV is at least in part due to their abilities to mount antiviral responses. In agreement with this hypothesis, we see that PANC02-Luc cells have both the greatest sensitivity to mIFNα and the greatest overall resistance to VSV-ΔM51-GFP among tested mouse PDAC cell lines.To further study the role of antiviral signaling in mouse PDAC cell lines, we examined the antiviral effect of mouse IFNα (mIFNα) on VSV-ΔM51-GFP replication in each cell line. We also used the reference human PDAC cell line, SUIT-2, as this cell line is extensively used in our lab and exhibits an intermediate level of type I IFN antiviral signaling. Importantly, human cells are known to be also sensitive to mIFNα , 46. Oure cells) . The anaAs mentioned above, the functional innate immune responses require not only the ability of cells to respond to IFN but to produce and secrete IFNs and other antiviral cytokines. We hypothesized that cell lines would differ in their ability to produce and secrete such cytokines. To test this hypothesis, cells were either mock-treated or infected with VSV-ΔM51-GFP at MOI 1 and 0.1 (based on virus titer on each cell line). At 24 h p.i., cell supernatants were collected and analyzed using the Mouse Cytokine/Chemokine 44-Plex Discovery Assay Array (MD44), which includes critical antiviral and proinflammatory cytokines known to control viral infections. Out of the 44 different cytokines that were measured, we show in Our data suggest that the observed differences in permissiveness of mouse PDAC cell lines to VSV-ΔM51-GFP are likely due to the higher levels of antiviral restriction factors (constitutive and/or induced) present in more resistant cell lines. This hypothesis is consistent with our previous studies demonstrating that the ability of cells to mount functional type I IFN antiviral responses is a major determinant of resistance of human PDAC cell lines to VSV-ΔM51-GFP , 20. HowTo compare VSV-ΔM51-GFP and VSV-Mwt-GFP, we first tested the abilities of each virus to initiate infections  and spreWe previously showed that, while abnormal or residual type I IFN antiviral activities play a major role in resistance of some human PDAC cell lines to VSV-ΔM51-GFP, some human PDAC cell lines also show inefficient attachment of VSV . Our dat50 for each drug on each cell line that was used as a measure of drug resistance. We found no positive correlation between the level of resistance to VSV and resistance to either gemcitabine or 5-FU. In sharp contrast, PANC02-Luc cells, which are the most resistant mouse PDAC cell line among our three to VSV, demonstrated the lowest IC50 for both gemcitabine and 5-FU, at 28.8 and 2,880 nM, respectively. The IC50 values for gemcitabine and 5-FU in KPC-Luc-A cells were 131.8 and 12,020 nM. Interestingly, the mouse PDAC cell line most permissive to VSV among our three, KPC-Luc-4580, demonstrated the greatest resistance to gemcitabine and 5-FU with IC50 values of 199.5 and 48,900 nM. These data reveal that the level of chemoresistance in mouse PDAC cell lines does not correlate with the efficacy of VSV-based OV-therapy and, promisingly, suggest that chemoresistant tumors can be good targets for OV therapy.We have recently shown that some human PDAC cell lines, which acquire resistance to chemotherapeutical drugs, can also simultaneously develop resistance to OV therapy . In addiin vivo, to our knowledge, they have never been tested in detail for overall chromosomal abnormalities or all genomic mutations in protein-coding sequences. To examine the karyotype of these cells, each cell line was grown to 70% confluence and sent to the Cytogenomics Core Laboratory at the University of Minnesota for SKY. We found that each cell line contained numerous chromosomal aberrations , and two KPC cell lines originated from PDACs developed in different modified KPC mice encoding major driver mutations KrasG12D and Trp53R172H in the pancreas. We found that the three mouse PDAC cell lines were genotypically and phenotypically distinct and showed very different permissiveness to OV-based therapy and, therefore, can serve as promising cell lines to use and address intertumoral heterogeneity 50 values for each cell line represent the relative ability of each cell line to respond to IFN. Between the three tested mouse cell lines, PANC02-Luc displayed the lowest mIFNα IC50 (33.1 U/mL), versus the highest IC50 in KPC-Luc-4580 cells (354.8 U/mL), and intermediate IC50 of 61.6 U/mL for KPC-Luc-A. This means that it takes almost 11 times the amount of mIFNα to bring KPC-Luc-4580 cells to the same level of type I IFN antiviral signaling as PANC02-Luc cells. We further highlight the differential levels of type I (and III) IFN antiviral signaling between these cell lines in We found that each of the three mouse PDAC cell lines exhibited varying levels of permissiveness to VSV infection and VSV-mediated cell killing . We also2/M cell cycle phase are transcriptionally repressed, leading to a decreased ability of cells to mount antiviral responses  to VSV could be also explained by the presence of KrasG12D driver mutation in these 2 KPC cell lines, while it is absent in PANC02-Luc . Severalther OVs –63. Fother OVs . Additiother OVs . Therefother OVs , which mInterestingly, we found that KPC-Luc-A and KPC-Luc-4580 cell lines, even though isolated from PDAC tumors generated from cells that had the same driver mutations, exhibited differential responses to VSV infection. Multiple possible reasons for the differences in phenotypes and genotypes between KPC-Lu-A and KPC-Luc-4580 include: (i) different environmental factors and selective pressures during tumor development in mice; (ii) different selective pressures throughout cell culture adaptation, and (iii) random genetic changes. To our knowledge, the only difference in the original engineering design between KPC-Luc-A and KPC-Luc-4580 cell lines was the method by which each cell line was engineered to express luciferase . In KPC-Whatever the reason for the phenotypic and genotypic differences between the tested KPC cell lines, these differences help contribute to their clinical translatability, as they likely better represent characteristic heterogeneity among and within PDAC patients.50 for both gemcitabine and 5-FU, and the least resistant cell line to VSV (KPC-Luc-4580) had the highest IC50 for gemcitabine and 5-FU. These data suggest that even if PDAC in patients is inherently chemoresistant, VSV-based OV therapy remains a viable treatment option.As VSV-based OV therapy becomes a more commonly tested therapeutic option in clinical trials , it is necessary to better understand how the effectiveness of VSV-based OV therapy is affected by the chemoresistance status of cancer cells. It has been observed in our previous studies and others , 57, 66 The ideal PDAC mouse model system should address both the intertumoral heterogeneity and the ability to detect and evaluate innate and adaptive immune responses against both tumor and OV. In In conclusion, our study provides essential data about three allograftable model mouse PDAC cell lines and proposes a novel platform to study OV-based therapies against phenotypically and genotypically distinct PDACs in immunocompetent mice. This study will be useful for ongoing and future studies in the field of PDAC therapeutics. Although this study focused on VSV as the oncolytic agent, alternative OVs can be tested using the model cell lines characterized in this study.


Pnpla7 expression, underscoring the possibility that insulin regulates PNPLA7 in skeletal muscle. The first aim of this study was to establish whether PNPLA7 is functionally expressed in cultured human skeletal muscle cells. The second aim was to determine whether PNPLA7 is regulated by insulin, glucocorticoids, cAMP/protein kinase A pathway, and/or glucose. Cultured human skeletal muscle cells expressed PNPLA7 mRNA and protein. Gene silencing of PNPLA7 in myoblasts reduced the phosphorylation of 70 kDa ribosomal protein S6 kinase and ribosomal protein S6 as well as the abundance of α1-subunit of Na+,K+-ATPase and acetyl-CoA carboxylase, indirectly suggesting that PNPLA7 is functionally important. In myotubes, insulin suppressed PNPLA7 mRNA at 1 g/L glucose, but not at low (0.5 g/L) or high (4.5 g/L) concentrations. Treatment with synthetic glucocorticoid dexamethasone and activator of adenylyl cyclase forskolin had no effect on PNPLA7 regardless of glucose concentration, while dibutyryl-cAMP, a cell-permeable cAMP analogue, suppressed PNPLA7 mRNA at 4.5 g/L glucose. The abundance of PNPLA7 protein correlated inversely with the glucose concentrations. Collectively, our results highlight that PNPLA7 in human myotubes is regulated by metabolic signals, implicating a role for PNPLA7 in skeletal muscle energy metabolism.Expression of patatin-like phospholipase domain containing protein 7 (PNPLA7), also known as neuropathy target esterase-related esterase (NRE), a lysophospholipase, increases with fasting and decreases with feeding in mouse skeletal muscle, indicating it is regulated by insulin, counterregulatory hormones, such as glucocorticoids and catecholamines, and/or nutrients. In cultured mouse adipocytes insulin reduces  PNPLAs via destabilization of a direct PNPLA7 ligand apolipoprotein-E  or TPP . Advanced MEM, DMEM, OptiMEM, GlutaMAX, MEM vitamin solution, fetal bovine serum (FBS), Fungizone (250 µg/mL of amphotericin B), gentamicin (10 mg/mL), trypsin-EDTA, Pierce Enhanced Chemiluminescence (ECL) Western Blotting substrate, High Capacity cDNA Reverse Transcription Kit, TaqMan Universal Master Mix with UNG and TaqMan gene expression assays were purchased from Thermo Fisher Scientific . 4-12% Criterion Bis-Tris polyacrylamide gels, XT MES electrophoresis buffer, goat anti-mouse IgG-horseradish peroxidase conjugate, anti-rabbit IgG-horseradish peroxidase conjugate, PCR plates and PCR plate sealing films were purchased from Bio-Rad . Amersham ECL Full-Range Rainbow Molecular Weight Marker was purchased from . Polyvinylidene fluoride (PVDF) membrane, forskolin, dibutyryl-cAMP (db-cAMP), bovine serum albumin (BSA), and all other reagents were purchased from Merck/Sigma-Aldrich . Insulin (Actrapid) was obtained from NovoNordisk (Denmark). E.Z.N.A HP Total RNA Kit was purchased from Omega Bio-Tek . Dexamethasone was purchased from KRKA (Slovenia).2.2semitendinosus muscle obtained during routine orthopaedic surgery as surgical waste, with informed written consent signed by the participants as described  FBS, 1% (v/v) MEM vitamin solution, 0.3% (v/v) Fungizone and 0.15% (v/v) gentamicin). Experiments were performed on myotubes after 7-10 days of differentiation.Human skeletal muscle cells were prepared from samples of the escribed –32. One 2HPO4, 1.8 mM KH2PO4, pH=7.4) and switched to serum- and insulin-free DMEM (without antibiotics and antimycotics), which contained 0.5 g/L, 1 g/L, or 4.5 g/L glucose. Treatments with insulin (0.1 µg/mL and/or 10 µg/mL), dexamethasone (1 µM), forskolin (5 µM in DMSO), db-cAMP (200 µM) and/or vehicle lasted for 16 h. At the end of experiment myotube samples were lyzed and collected for the subsequent mRNA and protein analysis.Experiments were performed in serum-free DMEM. To remove FBS and insulin, which is present in Advanced MEM, myotubes were washed with warm PBS  with the following sequence: CEVGYQHGRTVFDIWGRSGVLEKMLRDQQGPSKKPASAVLTCPNASFTDLAEIVSRIEPAKPAMVDDESDYQTEYEEELLDVPRDAYADFQSTSAQQGSDLEDESSLRHRHPSLAFPKLSE. Briefly, primary antibody against PNPLA7 was incubated with the excess of PNPLA7 peptide antigen used for immunization. Incubation mixture contained 10 μL of PNPLA7 antibody (0.4 μg/mL), 1.1 μL of PNPLA7 PrEST antigen (0.8 μg/mL) and 29 μL of primary antibody buffer with 1 M urea. The mixture was incubated for 3.5 h at 22°C and constant shaking (300 rpm). After incubation, the mixture was diluted with the primary antibody buffer (containing 1 M urea) to the final volume of 5000 μL, the final antibody dilution thus reaching 1:500. The solution containing the neutralized PNPLA7 antibody solution was subsequently used for immunoblotting. To detect PNPLA7, homogenates of human semitendinosus muscle or human myotubes were used. Tissue homogenates, prepared in Laemmli buffer, were collected from our previous study  Antigen  were seeded into 6-well plates and cultured overnight in Advanced MEM supplemented with 10% (v/v) FBS, 1% (v/v) GlutaMAX and 1% (v/v) MEM vitamin solution, without antibiotics. The transfection was performed using Lipofectamine 2000 reagent (Thermo Fisher Scientific) and siRNA according to the manufacturer’s protocol. As a negative control, non-targeting scrambled RNA (siSCR) was used. Lipofectamine and siRNA were separately diluted in Opti-MEM and combined just before the addition to the cell culture medium. Final concentration of siRNA against PNPLA7  and scrambled siRNA  in the medium was 10 nM. Final concentration of Lipofectamine was 2 ‰(v/v). After 24 h of incubation in the presence of the siRNA, the cell medium was replaced with the complete growth medium. Analyses of mRNA and protein levels were performed 48 and 72 h after the beginning of siRNA treatment, respectively.2.6® Universal Master Mix II with UNG and TaqMan gene expression assays (PNPLA7 (Hs00295012_m1), IL-6 (Hs00174131_m1), acetyl-CoA carboxylase 1 , acetyl-CoA carboxylase 2 , β-actin , 18S rRNA (Hs99999901_s1)) in a 96-well plate. β-actin (ACTB) and 18S rRNA were used as reference genes (endogenous controls). Results were calculated as gene expression ratios: (1+Ereference)Ct,reference/(1+Etarget gene)Ct,target gene where E is the PCR efficiency and Ct is the threshold cycle. Since two reference genes were used for normalization, geometric mean of both expression ratios was calculated at the last step, while the efficiency of PCR was analyzed with the LinRegPCR software  on Epoch™ spectrophotometer . RNA extracted from cell lysates was reverse transcribed to cDNA with High Capacity cDNA Reverse Transcription Kit and was performed in PTC-100 Programmable Thermal Controler (MJ Research Inc). qPCR was performed on Quant Studio 3  using TaqMansoftware , 35, as  Hs001741_m1, acet2.7Immunoblotting was performed as described –32. Brie2.8post hoc test. The difference between the groups was considered statistically significant when p ≤ 0.05.Data are presented as means with the standard error of the mean (± SEM). In all experiments, n refers to the number of donors (primary cultures obtained from different donors). Statistical analysis was performed with Microsoft Excel (Microsoft Office 2016) and GraphPad Prism 6 (GraphPad Software). Statistical significance was determined using t-test or a two-way ANOVA with Tukey 33.1Pnpla7 mRNA in mouse skeletal muscle was determined using Northern blot . Taken together, these results indicated that primary human skeletal muscle cells express PNPLA7 protein and that the ~150 kDa band, obtained with HPA009130 could be used for its quantification.As an additional control, the antigen sequence of HPA009130 was tested for possible alignments with other proteins using BLAST , which r3.2+,K+-ATPase (NKA aka sodium pump), whose function is known to be affected by lysophosphatidylcholine and membrane lipid environment , which by producing malonyl-CoA catalyzes the first step of de novo synthesis of fatty acids, and the ACC2 isoform , which by producing malonyl-CoA inhibits carnitine palmitoyltransferase and β-oxidation, were similar in control and knock-down cells  AMPK (at Thr172) was significantly reduced (The AMPK pathway was assessed by measuring the expression and phosphorylation of AMPK substrate acetyl-CoA carboxylase (ACC), which plays a major role in fatty acid metabolism by catalyzing the conversion of acetyl-CoA to malonyl-CoA. The abundance of the total ACC was reduced in PNPLA7 knock-down cells   of STAT3. The expression of IL6 mRNA (6 (IL-6) , and lowpression . To dete effects , 39, 40,Collectively, these results suggested that deficiency of PNPLA7 selectively affects various aspects of myoblast function, thereby indirectly indicating a role for PNPLA7 in human skeletal muscle.3.3Pnpla7 mRNA levels in mouse skeletal muscle , a well-characterized down-stream target of PKA , a cell-permeable analogue of cAMP, at low (0.5 g/L ≈ 2.78 mM), physiological (1 g/L), or high (4.5 g/L) glucose concentrations. The 4PNPLA7 mRNA in skeletal muscle is dependent on hormonal or metabolic status as well as that PNPLA7 deficiency leads to myopathy, highlighting the functional significance of PNPLA7 in skeletal muscle physiology. However, little is known about the expression and regulation of PNPLA7 in human skeletal muscle. Our preliminary results suggested that human myotubes might express PNPLA7 protein  by mTORC1, p70S6K phosphorylates ribosomal protein S6, thus promoting protein translation , 42. WhiPnpla7 mRNA levels in skeletal muscle  was not sufficient to observe a decrease in PNPLA7 protein levels. Interestingly, insulin did not alter PNPLA7 mRNA levels at low (0.5 g/L) or high (4.5 g/L) glucose, showing that glucose modulated its expression. Consistent with this view, the protein abundance of PNPLA7 in cultured myotubes inversely correlated with glucose concentrations. Taken together with previously published work  modulates expression of La cells . Further pathway . It woul pathway , 45, 46,r PNPLA7 , there ir PNPLA7 , 10.PNPLA7 did not affect expression level of IL6 mRNA or phosphorylation of STAT3, indicating PNPLA7 is not important for IL-6 signalling in human skeletal muscle cells. Conversely, in Pnpla7 knock-out mice the concentrations of IL-6 in plasma were increased   . While tPNPLA7 depends on the interpretation of the immunoblotting data, which might be affected by non-specific binding of the primary antibody HPA009130. To avoid technical artefacts, we performed antibody validation experiments, which included antibody neutralization and gene silencing of PNPLA7. Antibody neutralization experiments indicated that both the ~150 kDa and ~225 kDa likely reflect specific binding of HPA009130. Taken together with the predicted molecular weight of PNPLA7 (~145.7 kDa) and gene silencing experiments, which resulted in marked suppression of the ~150 kDa band, it is highly likely that the ~150 kDa band represents PNPLA7. Conversely, the ~225 kDa band, which was not detectable during incubation with PrEST-treated HPA009130, was not affected by gene silencing, suggesting it does not represent a protein encoded by the PNPLA7 gene. The discrepancy could be explained if a dimer or another PNPLA7-containing complex has a longer half-life than a PNPLA7 monomer, meaning that incubation was too short to observe a significant decrease in the ~225 kDa immunoreactivity. However, while patatin, isolated from Solanum tuberosum, is expressed as a dimer . Full statement: The primary human skeletal muscle cell cultures were prepared from the samples of semitendinosus muscle obtained during routine orthopaedic surgery as surgical waste, with informed written consent signed by the participants as described –31. PrepKM and AM-L performed experiments. MK, AM-L, and KM analyzed the data and prepared the first draft of the manuscript. TM planned experiments and analyzed the data. SP analyzed the data, edited, redrafted, and finalized the manuscript. SP and MK provided funds, planned and supervised the experiments. SP, KM, and MK conceptualized the study. All authors contributed to the article and approved the submitted version.


As effective therapies for relapse and refractory B-cell acute lymphoblastic leukemia (B-ALL) remain problematic, novel therapeutic strategies are needed. Artemis is a key endonuclease in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Inhibition of Artemis would cause chromosome breaks during maturation of RAG-expressing T- and B-cells. Though this would block generation of new B- and T-cells temporarily, it could be oncologically beneficial for reducing the proliferation of B-ALL and T-ALL cells by causing chromosome breaks in these RAG-expressing tumor cells. Currently, pharmacological inhibition is not available for Artemis. According to gene expression analyses from 207 children with high-risk pre-B acute lymphoblastic leukemias high Artemis expression is correlated with poor outcome. Therefore, we evaluated four compounds , previously generated from a large Artemis targeted drug screen. A biochemical assay using a purified Artemis:DNA-PKcs complex shows that the Artemis inhibitors 827171, 827032, 826941, 825226 have nanomolar IC50 values for Artemis inhibition. We compared these 4 compounds to a DNA-PK inhibitor (AZD7648) in three patient-derived B-ALL cell lines  and in two mature B-cell lines (3301015 and 5680001) as controls. We found that pharmacological Artemis inhibition substantially decreases proliferation of B-ALL cell lines while normal mature B-cell lines are not markedly affected. Inhibition of DNA-PKcs (which regulates Artemis) using the DNA-PK inhibitor AZD7648 had minor effects on these same primary patient-derived ALL lines, indicating that inhibition of V(D)J hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis. Our data provides a basis for further evaluation of pharmacological Artemis inhibition of proliferation of B- and T-ALL. The proArtemis, a DNA nuclease in the metallo-β-lactamase family, is an enzyme that is specifically responsible for opening these DNA hairpins . TherefoOne strategy that we have been pursuing for targeting ALL cells takes advantage of the fact that over 90% of ALL patient cell lines express the RAG complex . TherefoThis rationale was supported here by examining the expression level data for Artemis in primary and relapse human ALL patients. Based on this rationale, a high throughput screen was done using a proprietary library to evaluate inhibition of the Artemis catalytic domain and its nuclease activity (to be reported elsewhere). Following hit confirmation and medicinal chemistry optimization to improve potency and selectivity, four compounds were assessed for inhibition of primary and relapse ALL cells.Bone marrow (BM) and peripheral blood (PB) samples from ALL patients were acquired in compliance with the Institutional Review Board regulations of each institution. Informed consent for cell banking was obtained from all human subjects. Leukemia cells were processed and cultured as previously described . Human sNormal mature human B cell lines, immortalized lymphoblastoid cell lines 3301015 and 5680001, were generated using EBV-transformation of peripheral blood from patients with no hematopoietic abnormalities.BCR-ABL1 or hypodiploidy) were excluded from the study (http://www.R-project.org/).Clinical and gene expression microarray data from 207 high-risk B-precursor ALL patients from the COG Clinical Trial P9906 were obtained from the GEO database (GSE11877) . The pathe study . CryopreD binding affinity to Artemis from 20 to 150 nM. The commercially available DNA-PK inhibitor (AZD7648)  was evaluated in parallel as a positive control. Proliferation was determined by measuring the green fluorescence intensity per image (9 images were captured per well) by Incucyte every 8 h for 3 days. Raw values were normalized to the zero-time DMSO control. On day 3, viability of the treated cells was determined by annexin V/DAPI staining via flow cytometry.Lentivirally green fluorescence protein (GFP) labeled patient-derived B-ALL cells  or mature B cell lines 3301015 and 5680001 were plated on irradiated murine stromal OP9 cells and treated with four compounds optimized for Artemis inhibition  . These compounds ranged in Kλex = 485 nm, λem = 520 nm) with 1-min reads for 40 min. Compounds were tested in 10-point concentration (10 different concentrations with 3-fold dilutions) responses using the slopes of the enzyme readouts to determine percent inhibition which was fit to a 4-parameter logistic curve to calculate the IC50 of each compound. We have previously published the purity of the protein preparations  on the 3’ end. The assay buffer used was 25 mM MOPS, pH 7.5, 10 mM MgCl2, 10 mM KCl, 10 μM ATP, 1 mM DTT, 0.05% (w/v) CHAPS with 5 nM FL-Artemis, 5 nM DNA-PKcs, and 200 nM DNA FAM hairpin substrate. The fluorescence signal was monitored kinetically on a Perkin Elmer Envision microplate reader with FITC filters  and Artemislow expressing cases . Artemishigh leukemias were associated with inferior outcome with lower overall survival and relapse-free survival. This supported the rationale for targeting Artemis.To determine the role of Artemis in ALL, expression of Artemis mRNA (DCLRE1C) in 207 ALL patients uniformly treated with the COG P9906 clinical trial  was corrA high throughput screen of Artemis active site inhibitors using a proprietary library to assess for inhibition of the Artemis catalytic domain and its nuclease activity identified four small molecule inhibitors . The med2+ conditions is entirely inactive without DNA-PKcs  were transduced with GFP and 100,000 cells were plated in 24-well plates seeded with irradiated OP9 cells. Increasing green fluorescence intensity is indicative of proliferation as measured by Incucyte every 8 h. Although the GFP expression varied between cell lines , the norOur studies indicate that the 827171 compound substantially inhibits the proliferation of primary patient B-ALL lines, and it is comparable to the AZD7648 compound . The advThe effects of 827171 on viability, in contrast to proliferation, are among the mildest of the compounds evaluated. This likely reflects that DSBs in nondividing cells may not cause death immediately or at all. In contrast, an unrepaired DSB in proliferating cells represents more of a challenge. This increased effect of DSBs on proliferating cells may be because of a much higher probability of losing large chromosome fragments. Such a loss is because only the portion of the chromosome with the centromere is retained at the metaphase plate during cell division, and the chromosomal fragment lacking a centromere portion is easily lost. In addition, inhibition of Artemis may interfere with the ability of cells to exit from S phase .The large majority of acute lymphoblastic leukemias (ALL) produce the RAG enzyme complex encoded by the RAG genes . The RAGHere, we evaluated four of the Artemis inhibitors generated from a high-throughput screen and medicinal chemistry effort. These were assessed in three primary human ALL samples, with human mature B cell lines used for comparison. For comparison with the Artemis inhibitors, we used a known DNA-PKcs inhibitor, AZD7648, because DNA-PKcs is a kinase which regulates Artemis activity, and this compound has been evaluated in clinical trials for utility in a variety of non-lymphoid human malignancies .We found that the 827171-compound reduced proliferation of the three B-ALL lines at 1, 5, and 10 µM without affecting the mature B-cell control lines. In contrast, the DNA-PKcs inhibitor, AZD7648, showed little inhibition of the B-ALL lines, and instead suppressed the growth of the mature B cell control lines. These findings are encouraging for future work to make derivatives of the 827171 compounds. Importantly, the DNA-PKcs inhibitor AZD7648 decreased the proliferation less than 827171 in these same lines. This suggests that inhibition of hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis, namely, DNA-PKcs. This may be because Artemis can be activated by DNA ligase IV, even when DNA-PKcs is not present . TherefoWhile the 827171 compound shows promise for inhibition of the active site of Artemis, the brief study here also raises the interest in parallel strategies for inhibiting Artemis. Future work may take advantage of the recently described cryo-EM structure for the basal state of the Artemis:DNA-PKcs complex to develop inhibitors that prevent Artemis from being activated . Combina


After this article  was publDisregarding the erroneous bioinformatic analysis results about HNF-4α expression in the GSE84402 dataset, the article’s main conclusion relies on data obtained in cell line experiments. There are concerns about the validity of the control cell line used in comparisons of HCC cell line vs. control cell line expression: as is noted in the Discussion, the control cell line used is of embryonic hepatic origin and as such may not serve as an appropriate control for experiments using adult HCC cell lines. Furthermore, the study did not include loss of function studies or studies in native tissue samples to support claims about a role of HNF-4α in HCC proliferation.PLOS ONE Editors concluded that the article’s conclusions are not adequately supported when disregarding the erroneous bioinformatic analysis results and considering the methodological concerns discussed above. Therefore, the PLOS ONE Editors retract this article.The All authors did not comment on the retraction decision.


Human pluripotent stem cells (hPSCs) can be used as a renewable source of endothelial cells for treating cardiovascular disease and other ischemic conditions. Here, we present the derivation and characterization of a panel of distinct clonal embryonic endothelial progenitor cells (eEPCs) lines that were differentiated from human embryonic stem cells (hESCs). The hESC line, ESI-017, was first partially differentiated to produce candidate cultures from which eEPCs were cloned. Endothelial cell identity was assessed by transcriptomic analysis, cell surface marker expression, immunocytochemical marker analysis, and functional analysis of cells and exosomes using vascular network forming assays. The transcriptome of the eEPC lines was compared to various adult endothelial lines as well as various non-endothelial cells including both adult and embryonic origins. This resulted in a variety of distinct cell lines with functional properties of endothelial cells and strong transcriptomic similarity to adult endothelial primary cell lines. The eEPC lines, however, were distinguished from adult endothelium by their novel pattern of embryonic gene expression. We demonstrated eEPC line scalability of up to 80 population doublings (pd) and stable long-term expansion of over 50 pd with stable angiogenic properties at late passage. Taken together, these data support the finding that hESC-derived clonal eEPC lines are a potential source of scalable therapeutic cells and cell products for treating cardiovascular disease. These eEPC lines offer a highly promising resource for the development of further preclinical studies aimed at therapeutic interventions. Cardiovascular diseases including atherosclerosis, ischemic stroke, myocardial infarction, and ischemic cardiomyopathy currently represent a large economic and societal burden due to their high mortality and disability rates . ImpairmPCDHB2, and their lack of fetal/adult gene markers such as COX7A1 that are preferentially expressed in cells that have traversed the embryonic-fetal transition [We have previously developed a method of generating purified and embryonic site-specific somatic cell types through the propagation of hPSC-derived clonal human embryonic progenitor cell lines to overcome current issues of purity and scale . We desiansition . These hansition . Due to ansition ,23, seveansition ,25, and ansition  from ouransition ,22,23.In the present study, we derived novel hEP cell lines with endothelial properties and characterized a panel of 14 embryonic endothelial progenitor cell (eEPC) lines to explore their potential as a scalable source of human endothelial cells and cell products for further therapeutic development. We compared the eEPC lines to a diverse panel of adult endothelial cell (AEC) lines using comprehensive transcriptomic analysis. We found that 13 out of 14 eEPC lines expressed a broad array of endothelial specific genes shared across various endothelial subtypes. We further assessed these eEPC lines for endothelial identity using cell surface markers and well-established vascular tube forming assays. Interestingly, the eEPCs were clearly distinguished from AECs based on embryonic specific gene expression and molecular network analysis. In addition, we were able to achieve high scalability typical of previously identified hEP cell lines and stable secretion of angiogenic exosomes, suggesting that eEPCs may be advantageous for developmental research and for treating cardiovascular disease in a large patient population.Research grade hESCs (ESI-017) were differentiated using a two-step progenitor derivation protocol. In the first step, human embryonic stem cells were exposed to a 7-day procedure on Matrigel coated plates involving specific differentiation factors, followed by exposure to two different growth media to generate two cultures of heterogeneous cells designated as Candidate Cultures (CC). Afterwards, each CC subpopulation was placed at clonal densities in their same growth medium for two weeks. Then, individual clonal colonies were selected with cloning cylinders and expanded from a 24-well plate to multiple T225 flasks A.As described above and in 2 and 5% O2.A total of 14 eEPC lines and 15 human adult endothelial cell (AEC) lines were cultured in endothelial growth medium  on gelatin-coated plates. All the information on cell lines including cell origins and sources were described in t-test and p (Corr) cut-off = 0.05 while applying Pearson metric. The dendrogram was created using hierarchical cluster analysis using the average agglomeration method. For each cell culture sample, the differential expression values were calculated against a selection of 14 eEPC clones versus 15 adult endothelial cell lines versus 17 non-endothelial cell lines including embryonic and adult origin. All the information on samples is described in RNA was prepared upon lysis with RLT with 1% β-mercaptoethanol, using Qiagen RNeasy mini kits (Cat# 74104) following manufacturer’s directions. The extracted RNA was then quantitated using a NanoDrop (ND-1000) spectrophotometer. Library Construction was performed using the Illumina Truseq mRNA library prep kit following manufacturer’s directions. Library QC and library pooling were accomplished using an Agilent Technologies 2100 Bioanalyzer to assay the library fragments. qPCR was used to quantify the libraries. Libraries were pooled, which had different barcodes/indexing and sequencing, in one lane. The paired-end sequencing was performed using the Illumina HiSeq4000 sequencing instrument, yielding 100-bp paired-end reads. The sequencing was performed by BGI Americas Corporation. Data analysis of the transcription levels  was performed using Gene Spring GX Suite . Hierarchical and correlation analysis were performed with a moderate Total DNA was extracted from cells using Qiagen DNeasy mini kits according to instructions supplied by the manufacturer. DNA concentrations were measured using a Nanodrop spectrophotometer. Whole-genome expression was obtained using Illumina Human HT-12 v4 BeadArrays. In preparation for Illumina BeadArrays, total DNA was linearly amplified and biotin-labeled using Illumina TotalPrep kits . The sample quality was measured using an Agilent 2100 Bioanalyzer before being hybridized to Illumina BeadChips, processed, and read by an iScan microarray scanner according to the manufacturer’s instructions . Values under 130 relative fluorescence units (RFUs) were considered as nonspecific background signal. Analysis of microarray data was performed using the GeneSpring suite. Raw microarray data were normalized with the R BeadArray library . Mergingt-test. Independent experiments of samples over time were analyzed by repeated measures of ANOVA with the Holm adjustment. Differences were considered significant at probability values of p < 0.05.All data are expressed as mean ± standard deviation. Statistical analysis between groups at each time point was performed by the unpaired student’s 6 cells per 1 mL for 10 min. The cells were stained by incubating them with monoclonal antibodies  for 30 min. Primary antibodies were diluted according to the manufacturer’s instructions. Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), Mouse IgG1 Isotype-FITC were used as controls. The cells were scanned with an LSRII flow cytometer  and analyzed with the FlowJo software version 10.6 .Cells were dissociated into a single-cell suspension by using TrypLE  and fixed in BD Cytofix buffer  for 20 min at room temperature. The cells were permeabilized by washing and incubating them with BD Permeabilization/Wash  buffer at 1 × 10For detection of CXCR4 and CD31, cells were washed once with PBS and fixed in 4% paraformaldehyde for 30–60 min at room temperature (RT). Fixed cells were washed three times with PBS, permeabilized, and blocked by incubation in blocking buffer  for 1 h at RT. The cells were then incubated overnight at 4 °C with CD31 monoclonal antibody with Alexa Fluor 488  and primary rabbit anti-human CXCR4 polyclonal antibody  at a dilution of 1:500 in 5% normal donkey serum, 0.5% BSA, and 0.05% Triton X-100 in PBS. Then, the cells were washed four times with PBS plus 0.05% Triton X-100 (PBS-Triton) and incubated for 1 h at RT with Alexa Fluor 568 donkey anti-rabbit IgG antibody  at a 1:500 dilution in PBS-Triton. Isotype controls were stained under identical conditions except that total rabbit IgG  was used as primary antibody. Cells were counterstained with DAPI at 0.1 ng/mL for 10 min at RT and imaged on a Nikon Eclipse TE2000-U inverted microscope.2). Exosomes for tube formation assay were isolated from eEPC and BM-MSC  culture medium using the precipitation method following the manufacturer’s instructions . Total exosome particles were counted using nanoparticle tracking analysis (NTA).Tube formation angiogenesis assay was performed using the Cell Player Angiogenesis PrimeKit  to assess tube network growth in eEPC lines. eEPCs were labeled using TagRFP control vector, which consists of the lentiviral backbone vector, pLKO.1-puro, containing a gene encoding red fluorescence protein (RFP), driven by the CMV promoter . RFP labeled eEPCs were plated with monolayer, and 10ng/mL of recombinant human VEGF protein  was treated to enhance tube formation. Images were taken every 6 h for 10–14 days using an IncuCyte imager. Tube formation was quantified using the IncuCtye Angiogenesis Analysis Module by dividing the lengths of all tube networks by the image area at each time point  to assess tube network growth effect of eEPCs-secreted exosomes. HUVECs with 104 cells/well were seeded in µ-Slide 15 well 3D  coated with Matrigel and followed by the treatment of eEPC-exosomes in PBS, compared with control conditions . Tube formation networks were analyzed using the ImageJ version 1.44 Angiogenesis Analyzer.Tube formation angiogenesis assay was also performed using human umbilical vein cells (HUVECs) that were seeded onto growth factor reduced MatrigelWe have previously demonstrated that a large variety of distinct, highly scalable, and clonally pure progenitors can be isolated from human pluripotent stem cells using a two-step process of differentiation under diverse conditions followed by clonal selection of scalable cell lines . In our To understand the molecular characteristics of the clonally selected hEP lines, we analyzed whole transcriptomic gene expression. We compared three groups: (1) 14 human embryonic endothelial progenitor cell (eEPC) lines, 30-MV2-6, 30-MV2-3, 30-MV2-4, 30-MV2-10, 30-MV2-17, 30-MV2-19, 30-MV2-2, 30-MV2-7, 30-MV2-9, 30-MV2-14, 30-MV2-24, 30-MV2-8, 30-SM2-1, and 30-SM2-3; (2) 15 primary adult endothelial cell (AEC) lines including HBMEC, HCPEC, HDMEC, HDLEC, HLEC, HIMEC, HRGEC, HHSEC, HCMEC, HAEC, HOMEC, HUVEC, HUAEC, HMVEC, and HAEC; and (3) a panel of 17 non-endothelial cell lines . The description of cell origin, cell sources, and ages of RNAseq samples are described in Hierarchical clustering analysis with a representative heatmap in KDR and vWF) (PDGF-B), inhibitor of differentiation 1 (ID1), and C-X-C Motif Chemokine Receptor 4 (CXCR4), with comparable level to AEC lines  31, VE-Cadherin (CD144), vascular endothelial growth factor receptor (VEGFR-2)/KDR, and Von Willebrand factor (vWF), which are all widely recognized as endothelial-specific markers ,29 and Cand vWF) . InteresEC lines . It has culation , and regculation , respectin vitro. To assess tube network growth in eEPC lines, eEPCs were labeled using TagRFP and co-cultured with normal human dermal fibroblasts (NHDF). This model allows us to demonstrate all phases of the angiogenesis process, including proliferation, migration, and, eventually, differentiation and angiogenic networks [We next investigated the angiogenic functionality of eEPC lines for vascular tube formation networks . ImagingAPLNR), differentiation marker of hematopoietic stem and progenitor cells [IGF2) promoting EPC homing [MAFB, controlling endothelial sprouting [IGFBP5), promoting angiogenesis [H19, a long non-coding RNA increasing EC tube formation [CPT1C) promoting human mesenchymal stem cells survival [ACP5), promoting cell differentiation and proliferation [EGLN3), a regulator associated with resistance to stress) [To better understand the molecular signature of eEPC lines compared to AEC lines, we performed a differential gene expression analysis of the transcriptomic RNAseq data. Although the overall global gene expression patterns of the 13 eEPC lines were similar to 15 AEC lines from various origins, we found that there was a significant subset of genes that were differentially expressed in the eEPC lines . The RNAor cells , insulinC homing , MAFB, cprouting , insulinogenesis , H19, a ormation , Carnitisurvival , Tartratferation , and enh stress)   that is almost exclusively expressed in fetal and adult cells [COX7A1 expression level in eEPC lines was observed to be undetectable (Log2 FC = 0.1), compared to AEC lines (Log2 FC = 4.3), suggesting that eEPC lines retained an embryonic progenitor stage (CCL2), which is associated with diabetes [(BACE2), which is linked to increased risk and earlier disease onset such as Alzheimer’s disease [CAT), which plays an important role in endothelial cell shear stress response [CXCL1), which regulates TNF-stimulated endothelial cells [IFI27), which is a stimulator of VEGF-A mediated angiogenesis [MGP), which regulates differentiation of endothelial cells [MEG3) [We previously identified lt cells . COX7A1 or stage B. In adddiabetes , β-site  disease , an antiresponse , chemokial cells , interfeogenesis , matrix al cells , and a ls [MEG3) , were alWe examined cell growth characteristics by measuring scalability and functional stability of a representative eEPC line, 30-MV2-6. It has been suggested that adult EPCs have therapeutic potential and clinical implications in ischemic diseases ,50,51. T6 exosomes/well  of cells was compared to primary MSCs. We found that MSCs begin to senesce and lose proliferative and differentiation capacity after 12 pd, consistent with previous reports ,53. In cmes/well . The res6 exosomes/well of BM-MSC exosomes showed equivalent angiogenic activity as 30MV2-6 exosomes at 200 × 106 exosomes/well, indicating a 6-fold difference in potency  versus late passages (50 pd). The results show that the early passage eEPC exosome angiogenic activity was stable at later passage C. To invPrevious studies have suggested that mesenchymal stem cells (MSCs) ,5, endotin vitro. Interestingly, our study showed that eEPC lines retained embryonic progenitor characteristics. We found a subset of genes that were upregulated in eEPC lines compared to AEC lines. For example, eEPC overexpressed apelin receptor (APLNR), insulin-like growth factor 2 (IGF2), the MAF BZIP transcription factor B (MAFB), insulin-like growth factor binding protein 2 (IGFBP2), non-coding RNA H19, carnitine palmitoyltransferase 1 C (CPT1C), tartrate-resistant acid phosphatase 5 (ACP5) and enhanced prolyl hydroxylase domain (PHD)-3 (EGLN3) relative to AEC. APLNR signaling is required for the generation of cells that give rise to HSCs [IGF2/IGF2R system in promoting endothelial progenitor cells homing [MAFB gene encodes a transcription factor that controls endothelial sprouting in vitro and in vivo [IGFBP2 is known as a developmentally regulated gene that is highly expressed in embryonic and fetal tissues and markedly decreases after birth [H19 is expressed during EC differentiation and has functional effects on EC tube formation [CPT1C promotes human mesenchymal stem cells survival under glucose deprivation through the modulation of autophagy and is strongly associated with the epithelial-mesenchymal program [ACP5 is upregulated by transforming growth factor-β1 (TGF-β1) and subsequently enhances β-catenin signaling in the nucleus, which promotes the differentiation, proliferation, and migration of fibroblasts [EGLN3, a member of the EGLN family of prolyl hydroxylases, increases during skeletal myoblast differentiation [In the present study, we provided comprehensive transcriptomic profiling analysis demonstrating that eEPCs differentiated from hEPs have endothelial phenotypes expressing endothelial markers such as CD31, CXCR4, KDR, and vWR, and EPC marker, CD34, as well as generating tube formation s homing . The MAF in vivo  and play in vivo ,63. IGFBer birth . It promer birth . H19 is ormation . CPT1C p program . ACP5 isroblasts . EGLN3, ntiation  and is antiation . UpregulBACE2), catalase (CAT), CXCL1, Interferon α-inducible protein 27 (IFI27), matrix Gla protein (MGP), and long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) compared to AEC lines. The BACE2 gene is linked to increased risk and earlier disease onset of Alzheimer’s disease. CAT encodes an antioxidant enzyme that plays an important role in endothelial cell pathophysiology, in shear stress response, and ultimately, in arterial aging [CXCL1 was only expressed by old osteoblasts but not young cells [IFI27 is known as an oncogene, a strong stimulator of angiogenesis, by increasing secretion of vascular endothelial growth factor (VEGF-A) [MGP stimulates differentiation of endothelial cells [MEG3 negatively regulates angiogenesis and proliferation in vascular endothelial cells. MEG3 overexpression was significantly suppressed by inhibition of miR-9 resulting in proliferation and in vitro angiogenesis in vascular endothelial cells [We also found a subset of genes that were downregulated in eEPCs. For example, eEPC lines expressed little to no β-site APP cleavage enzyme 2  . MGP stial cells , but alsal cells . MEG3 neal cells . DownregIFITM1, NNAT, H19, PLVAP, and IGF2, which were highly upregulated and CYTL1, KRT7, CXCL1, and CXCL8, which were downregulated in HUVECs and eEPC lines, but not the AEC lines  have been a robust source of ECs with proven capability of forming stable microvessels, which are capable of capillary morphogenesis compared to hPSC-ECs . From ouEC lines . Our finDerivation of eEPC lines that are homogeneous clonal cells with batch-to-batch consistency in production provides a unique competitive advantage for unparalleled industrial scale production that is needed for therapeutic application in various ischemic diseases. The overall advantages of hEP lines are (1) identity and homogeneity, (2) scalability and stability, and (3) diversity  stem cells such as endothelial progenitor cells (EPCs), MSCs, and CDCs for clinical application in the treatment of ischemic disease. However, the source of these primary cell lines results in cellular heterogeneity both within a donor line and between donor lines. Thus, there is a manufacturing bottleneck for the consistent production of therapeutic ECs on an industrial scale needed to treat a large cardiovascular disease patient population. We addressed this issue by developing a bank of embryonic progenitor cell lines derived by early-stage clonal isolation from partially differentiated hPS cells . This meHere, we demonstrate the ability to derive clonally pure endothelial cells (eEPCs) from hPS cells using our two-step process. We derived 13 eEPC lines and characterized them as endothelial by transcriptomic analysis, biochemical markers, and vascular function. Further, we have shown early evidence of the angiogenic potential of exosomes secreted from eEPC lines. We are currently developing eEPC exosomes manufacturing methods and investigating the potential therapeutic uses of eEPC exosomes for treating ischemic conditions like stroke by improving vascular and neurological recovery. Our findings suggest that eEPC lines will be a valuable source of both cells and exosomes for use in therapeutic vascular regeneration.Exosomes from Clonal Progenitor Cells, 2022 (US 11274281); Exosomes from Clonal Progenitor Cells, 2019 ; Methods and compositions for targeting progenitor cell lines, 2019 .


TP53, RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I (Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel therapeutic approach in OS. The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years. Mutations in the genes  Osteosarcoma (OS) is the most frequently occurring primary malignant bone tumour in children and young adults, with the highest peak incidence during the second decade of life . Currentc-Myc and mTOR) and tumour suppressors  promote Pol I transcription activity [Dysregulated cell growth is one of the representative hallmarks of cancer  and accoactivity ,14,15. ICX-5461, the first-in-class small molecule inhibitor of Pol I-mediated transcription, suppresses rRNA transcription by preventing the binding of the Pol I-specific transcription initiation factor, SL-1, to the rDNA promoter region ,17. CX-5TP53 and RB1 are highly mutated [50) values for CX-5461 in two OS cell lines, and a high concentration of CX-5461 was used in cell cycle and autophagy studies [TP53 mutant human OS xenograft, (ii) a TP53 wild-type human OS xenograft and (iii) a TP53 wild-type murine allograft.Recent studies in OS indicate that Pol I transcription activity is very likely to be dysregulated in OS. c-Myc protein, a key upstream regulator of ribosome biogenesis , is freq mutated ,28,29,30 mutated . A previ studies . These r2PO4 (pH4.5) and diluted in growth media immediately prior to use in vitro. For the in vivo studies, 30 mg/kg of CX-5451 was prepared in 50 mM NaH2PO4 (pH4.5).CX-5461 was purchased from SYNkinase , and 10 mM stocks of CX-5461 were prepared in 50 mM NaH®, Manassas, VA, USA), and a further five patient-derived osteosarcoma cell lines, KOS-1, KOS-2, KOS-3, KOS-6 and KOS-7, were kindly provided by HS KIM from Seoul National University Hospital [2.Five commercially available human osteosarcoma cell lines, HOS, 143B, MNNG/HOS, U-2-OS and SJSA-1, were purchased from American Tissue Culture Collection . The assay was conducted following the manufacturer’s instructions (Single-nucleotide variants (SNVs) and copy number variants (CNVs) in ten OS cell lines were analysed using The Ampliseqructions .2; HOS: 6.0 × 102; MNNG/HOS: 6.0 × 102; U-2-OS: 1.0 × 103; SJSA-1: 1.0 × 103; KOS-1: 1.0 × 103; KOS-2: 1.0 × 103; KOS-3: 1.0 × 103; KOS-6: 1.0 × 103; and KOS-7: 1.0 × 103) and murine OS cells (2.0 × 103) following exposure to fresh medium containing vehicle or various concentrations of CX-5461 for 72 h was undertaken as previously described [50) were calculated using Graph-Pad Prism (version 9.1.0).The proliferation of human OS cells (143B: 4.0 × 10escribed . The dos6) and human fibroblast cells (1.0 × 106) as previously described [The protein levels of p53, MDM2, c-Myc and RB1 were assessed in the ten OS cell lines  was estimated by quantitative real-time PCR (qRT-PCR) as previously described [TM Thermocycler , and then cDNA synthesis was performed with SuperScriptTM IV Reverse Transcriptase , Hexameric Random Primers  and dNTPs  in a T100TM Thermocycler. All synthesised cDNA samples were loaded into 96-well PCR plates with SYBRTM Green master mix, primers that targeted the external transcribed spacer-2 (ETS-2) of the pre-47S ribosomal RNA and the housekeeping gene, beta-2-microglobulin (β2M) . All settings for cDNA synthesis and RT-PCR are described in 50) were calculated using Graph-Pad Prism (version 9.1.0).rDNA transcription in 1.0 × 10escribed ,34. Briein (β2M) , and rea50 CX-5461 concentrations (determined from the cell proliferation assay), then labelled with 10 µM BrdU , harvested and stained with PI. Flow cytometry (Becton Dickinson LSR II) was used to assess the level of staining, and FlowJo software (version 10.0) was used for data analysis.All OS cell lines were analysed for cell cycle progression using Bromodeoxyuridine (BrdU) and Propidium iodide (PI) staining as previously described . Briefly6) in 50 µL of Hank’s Balanced Salt Solution (HBSS)  or SJSA-1-Luc cells (5 × 106) in 50 µL of HBSS containing 50% (v/v) Matrigel  were injected subcutaneously into the right flanks of mice. For the allograft mouse model, K7M2 cells (1 × 106) in 50 µL of HBSS were injected subcutaneously into BALB/c wild-type mice on the right flank. Tumour-bearing mice were monitored daily. Once the average tumour volume reached 30~50 mm3, mice were randomised to receive either CX-5461 or vehicle. Thrice (3 times) weekly for 3 weeks, 30 mg/kg of CX-5461 in 50 mM NaH2PO4 (pH4.5) or the corresponding vehicle (50:50 mixture of 50 mM NaH2PO4 and 50 mM Citric Acid) was administered via oral gavage. The experiments were terminated 15 days following the start of drug administration or prior if mice reached the ethical endpoint.All in vivo experiments were conducted following approval from the Australian National University Animal Experimentation Ethics Committee (A2017/16 and A2020/36). Rag 2 knockout (KO) mice were obtained at 6 to 8 weeks of age from the Animal Resource Centre . 143B-Luc cells  was assessed using two-way ANOVA. Statistical analyses were performed using GraphPad Prism software, version 9.1.0. The dose–response curve fit and 50% inhibitory values were obtained using non-linear regression. Comparisons of GICTP53 are reported to confer chemo-resistance and a poor prognosis in OS patients [TP53 gene and normal p53 protein expression) (TP53 gene or dysregulated p53 protein expression)   A and abnression) B OS cell 139 nM) C. There 50) range in normal p53  and abnormal p53 OS cell lines   B. In the50 of CX-5461 for 72 h and analysed by flow cytometry. CX-5461 treatment was found to induce G2-phase cell cycle arrest in all OS cell lines analysed, both p53 normal and abnormal cell lines  cell lines, and U-2-OS, SJSA-1 and KOS-3 were identified as normal p53 (p53 wild-type) cell lines . Previoull lines . p < 0.05) after the first week (Day 8) of drug dosing (The anti-tumour activity of CX-5461 was investigated in two human OS xenograft mouse models in Rag 2 knockout (KO) mice using 143B (p53 mutant-type) and SJSA-1 (p53 wild-type) cell lines . Each exg dosing B. Similag dosing C. CX-546g dosing D,E. To investigate whether CX-5461 had the same in vivo therapeutic efficacy in the presence of a functional immune system, the anti-tumour effect of CX-5461 was evaluated in an allograft mouse model using the murine OS cell line K7M2 . As seen50 and tIC50 values for each OS cell line, (ii) demonstrated the on-target effect of CX-5461 on Pol I activity, (iii) analysed the effect of CX-5461 on cell division in a range of cell lines and (iv) demonstrated the therapeutic effect of CX-5461 in vivo using three different mouse models. The advancement of diagnostic and therapeutic strategies for OS has not improved outcomes over the last 30 years, and chemo-resistance is a major problem. There is, therefore, an urgent need for new therapeutic strategies against this disease ,7,8,37. TP53, RB1, TSC1, TSC2 and ATRX and amplifications of c-Myc, MDM2 and CDK4. Additionally, the dysregulated expression of p53, RB1, c-Myc and MDM2 proteins was demonstrated, all of which have the potential to impact Pol I activity. These molecular results are consistent with other genetic studies of OS and highlight the significant diversity in gene and protein alterations that may lead to OS and enhanced Pol I activity, and they emphasise the potential for Pol I inhibitors to be effective against the majority of OS cases.Alterations in tumour suppressors and oncogenes that occur in many cancers influence Pol I transcription activity ,14. The Multiple studies have revealed the mechanism of the therapeutic effect of CX-5461 on human cancers ,36,42,43TP53 gene mutation status [TP53 mutations had a significantly higher risk of death within 2 years than patients with wild-type TP53 tumours [In clinical studies, the response to chemotherapy and the survival rate of OS patients varied according to the n status ,46. A me tumours . Our in  tumours . The dru50 value in the cancer cell lines of 147 nM (range 3–5500 nM), whereas the 5 normal cell lines all had a GIC50 value of approximately 5000 nM [50 values obtained for the 10 OS cell lines in this study (33–188 nM), therefore, closely align with the median values previously reported for other cancer cell lines. More importantly, these values are well below those required to impact the proliferation of normal cell lines; thus, these doses could be considered selective for OS cells. The lack of obvious side effects in our mouse studies, with a maximum weight loss of 10% measured, reflects the results of early phase trials in humans in which CX-5461 was well tolerated [Previous studies of the anti-proliferative effect of CX-5461 in 50 cancer cell lines and 5 normal cell lines demonstrated a median GIC 5000 nM . The GIColerated . InteresAs with most chemotherapy agents, resistance to RNA Pol I inhibition may develop as a result of new mutations or compensatory mechanisms. Combining therapies that target different pathways increases the treatment effect and decreases the likelihood of resistance occurring. CX-5461 was successfully combined with the mTORC1 inhibitor everolimus to demonstrate a synergistic anti-tumour effect in lymphoma-bearing mice . Future In conclusion, this study has identified mutations in genes associated with Pol I activity in 10 OS cell lines and has confirmed the alteration of proteins well recognised to be involved in the regulation of Pol I transcription. Furthermore, this study has demonstrated that targeting elevated Pol I transcription in OS shows significant promise as an effective novel therapeutic strategy. Given the pre-clinical results provided and the safety profile of CX-5461 already obtained in human clinical studies, CX-5461 should be considered a potential therapeutic option for OS.


Peripheral hIAPP plays a major role in the pathogenesis of AD, and high circulating hIAPP level increase AD risk in T2DM patients. However, there is no hard evidence for the role of brain-derived hIAPP in the pathogenesis of AD. Nevertheless, oxidative stress, mitochondrial dysfunction, chaperon-mediated autophagy, heparan sulfate proteoglycan (HSPG), immune response, and zinc homeostasis in T2DM could be the possible mechanisms for the induction of the aggregation of hIAPP which increase AD risk. In conclusion, increasing hIAPP circulating levels in T2DM patients predispose them to the development and progression of AD. Dipeptidyl peptidase 4 (DPP4) inhibitors and glucagon-like peptide-1 (GLP-1) agonists attenuate AD in T2DM by inhibiting expression and deposition of hIAP.Human Islet amyloid polypeptide (hIAPP) from pancreatic β cells in the islet of Langerhans has different physiological functions including inhibiting the release of insulin and glucagon. Type 2 diabetes mellitus (T2DM) is an endocrine disorder due to relative insulin insufficiency and insulin resistance (IR) is associated with increased circulating hIAPP. Remarkably, hIAPP has structural similarity with amyloid beta (Aβ) and can engage in the pathogenesis of T2DM and Alzheimer’s disease (AD). Therefore, the present review aimed to elucidate how hIAPP acts as a link between T2DM and AD. IR, aging and low β cell mass increase expression of hIAPP which binds cell membrane leading to the aberrant release of Ca Human Islet amyloid polypeptide (hIAPP) also called amylin is one of the main secretory products of pancreatic β cells in the islet of Langerhans . hIAPP hhIAPP is co-secreted with insulin from pancreatic β cells, and due to its ability to aggregate into pancreatic islet cells in type 2 diabetes mellitus (T2DM) so-called hIAPP .Notably, hIAPP was initially described in 1901 by weichselbam et al. and Opie et al. as an islet amyloid called islet hyalinization in patients with T2DM . HoweverExpression of hIAPP is not limited to the pancreatic β cells but is also expressed on α cells of the islet . In addiT2DM is an endocrine disorder due to relative insulin insufficiency and IR, characterized by hyperglycemia and cardiometabolic complications . T2DM reDeficiency of IDE which is involved in the degradation of hIAPP induces the development of hyperamylinemia which triggers the development of IR . In vitrNotably, hIAPP unlike rat IAPP is highly subjected to amyloidogenesis by forming amyloid fibril. The amyloidogenic propensity of hIAPP is related to the presence of 20–29 and 8–18 residues which have no proline as in rat IAPP as proline prevents aggregation of hIAPP . Higher In addition, a monoclonal antibody to hIAPP could be an early diagnostic biomarker for T2DM diagnosis . These fTherefore, the oligomerization of hIAPP is controlled by the balance between processing and degradation pathways . GeneticIt has been shown that the incidence and prevalence of T2DM are increased with age due to increase aggregation of hIAPP and the development of pancreatic β cells . Of inteThe underlying causative factors that convert soluble hIAPP oligomers to toxic non-soluble fibrils are largely unknown . Neverth2+ and activation of proteolytic enzyme calpain leading to a series of events causing loss of β cells [.Monomeric and oligomeric hIAPP disrupt cell membrane fluidity by increasing the production of ROS causing membrane leakage and cytotoxicity . Monomer β cells  Fig. 3)2+ and acIn response to oxidative stress and ER stress, chaperon-mediated autophagy is upregulated to prevent cell injury . ChaperoFurthermore, heparan sulfate proteoglycan (HSPG) can increase hIAPP aggregation . PerlecaMoreover, the immune response to the formed hIAPP fibrils triggers the release of pro-inflammatory cytokines mainly IL-1β which increases hIAPP fibrils-induced β cytotoxicity and development of IR and T2DM . In vitrIndeed, zinc prevents protein misfolding and aggregation of hIAPP . Zinc deIn sum, hIAPP-induced dysfunction of pancreatic β cells through induction of oxidative stress and ER stress. Zinc deficiency and exaggerated immune response against hIAPP aggregation trigger more injury of pancreatic β cells.AD is the most common type of dementia characterized by progressive neurodegeneration leading to memory and cognitive deficits . The patT2DM patients have been reported to hold a higher incidence of cognitive decline and AD; T2DM has been intensely associated with an increased risk of developing all types of dementia, including AD . A systeDisturbances in brain insulin signaling mechanisms represent early and progressive abnormalities and could account for the majority of molecular, biochemical, and histopathological lesions in AD . Increasvia a combination of four β-sheet-to-β-sheet interfaces and two packing orientations, focusing on a comparison of different matches of β-sheet layers [Amyloid formation is a hallmark in both AD and T2DM due to the deposition of hIAPP in both pancreatic β islets and the brain . Pancreat layers . Two crot layers . Both Aβt layers , 85. Inft layers . The inft layers . In contt layers .However, there was no correlation between peripheral hIAPP and AD risk in animal and human studies . In the Peripheral hIAPP serum level is not different in AD and controls . TherefoDeposition of hIAPP in both T2DM and AD is due to metabolic dysfunction, microvascular injury, and failure of Aβ clearance . Besides42 level [42 level even in non-diabetic status, hIAPP disrupts neurovascular injury, interacts with Aβ42 and acts as a seed to increase deposition of Aβ plaques [42 by increasing the production of ROS. Likewise, Aβ42 interacts, cross seed and increases the toxic effects of hIAPP [42 increases tau protein hyperphosphorylation through induction of oxidative stress and inflammatory changes causing progressive synaptic loss and synaptic dysfunction [In the normal state, unmodified (soluble) hIAPP crosses BBB and enhances clearance of Aβ4 to prevent the development and progression of AD neuropathology . When un42 level . Both hI42 level . The cor42 level . Interes42 level . In cont42 level , 91. In  plaques . In addiof hIAPP . Co-aggrfunction . IAPP cofunction , 95. Synfunction . Given tfunction , 96. How42 is prevented by hIAPP and hIAPP analogue pramlintide which attenuate the development of AD and other neurodegenerative diseases [A recent study involved postmortem brain tissues of AD patients together with in vitro and experimental studies illustrated that hIAPP promotes tau protein deposition . In addidiseases . Taken tInterestingly, hIAPP undergoes tertiary structural changes and can form aggregates that deposit in the pancreatic islet causing β cells dysfunction even in the prediabetic period . Both ADThe mode of toxicity by hIAPP and Aβ despite is mediated in a receptor-dependent manner. Receptor activity modifying protein 3 (RAMP3) and calcitonin receptors which are highly expressed in the brain act as a receptor for hIAPP to increase Aβ toxicity . In addiInterestingly, HSPG and perlecan which increase hIAPP aggregation  are dysrFurthermore, abnormal immune response to the aggregated hIAPP triggers releases of pro-inflammatory cytokines and expression of inflammatory signaling pathways that augment hIAPP-induced cytotoxicity in both T2DM  and AD . Of noteIn addition, zinc has a protective effect against protein misfolding and aggregation of hIAPP . Zinc deTaken together, oxidative stress, mitochondrial dysfunction, chaperon-mediated autophagy, HSPG, immune response, and zinc homeostasis in T2DM could be the possible mechanisms for induction of the aggregation of hIAPP which increase AD risk.It has been detected that IR is linked with mild cognitive impairment in AD patients compared to controls due to dysfunction of brain insulin signaling . AdminisFurthermore, the thiazolidinediones (TZDs), a family of peroxisome proliferator–activated receptor γ (PPARγ) activators like rosiglitazone and pioglitazone are antidiabetic agents that primarily act by improving peripheral insulin sensitivity . TZDs imAPOE-Ε4-negative or other analysis populations [It has been demonstrated that hIAP formation in vivo is markedly reduced by the effects of rosiglitazone and metformin in hIAPP transgenic male mice fed a moderate-fat diet for 1 year . Rosigliulations . These fDipeptidyl peptidase 4 (DPP4) inhibitors like sitagliptin are widely used and tolerated in the management of T2DM with minimal or no hypoglycemia as an adverse effect , 149. Pr2+ and activation of the proteolytic enzyme leading to series of events causing loss of β cells. hIAPP-induced dysfunction of pancreatic β cells through induction of oxidative stress and ER stress. Zinc deficiency and exaggerated immune response against hIAPP aggregation trigger more injury of pancreatic β cells. Peripheral hIAPP plays a major role in the pathogenesis of AD, and high circulating hIAPP level increase AD risk in T2DM patients. However, there is no hard evidence for the role of brain-derived hIAPP in the pathogenesis of AD. In sum, oxidative stress, mitochondrial dysfunction, chaperon-mediated autophagy, HSPG, immune response and zinc homeostasis in T2DM could be the possible mechanisms for induction the aggregation of hIAPP which increase AD risk. In addition, DPP4 inhibitors and GLP-1 agonists attenuate AD in T2DM by inhibiting the expression and deposition of hIAP. Despite these evidences the underlying mechanism linking pancreatic islet hIAPP in the development of AD need to be elucidated. Therefore, retrospective and prospective studies are recommended in this state.hIAPP is one of the main secretory products of pancreatic β cells in the islet of Langerhans. hIAPP has different physiological functions including inhibiting gastric empty, regulation of satiety and inhibiting the release of insulin and glucagon. T2DM is an endocrine disorder due to relative insulin insufficiency and IR, characterized by hyperglycemia and cardiometabolic complications. Interestingly, hIAPP has structural similarity with Aβ and can involve in the pathogenesis of T2DM and AD. The underlying causative factors that convert soluble hIAPP oligomers to toxic non-soluble fibrils are largely unknown. IR, aging and low β cell mass increases the expression of hIAPP which binds the cell membrane leading to the aberrant release of Ca