Androgen receptor signaling is crucial for prostate cancer growth and is positively regulated in part by intratumoral CYP3A5. As African American (AA) men often carry the wild type CYP3A5 and express high levels of CYP3A5 protein, we blocked the wild type CYP3A5 in AA origin prostate cancer cells and tested its effect on androgen receptor signaling. q-PCR based profiler assay identified several AR regulated genes known to regulate AR nuclear translocation, cell cycle progression, and cell growth. CYP3A5 processes several commonly prescribed drugs and many of these are CYP3A5 inducers or inhibitors. In this study, we test the effect of these commonly prescribed CYP3A5 inducers/inhibitors on AR signaling. The results show that the CYP3A5 inducers promoted AR nuclear translocation, downstream signaling, and cell growth, whereas CYP3A5 inhibitors abrogated them. The observed changes in AR activity is specific to alterations in CYP3A5 activity as the effects are reduced in the CYP3A5 knockout background. Both the inducers tested demonstrated increased cell growth of prostate cancer cells, whereas the inhibitors showed reduced cell growth. Further, characterization and utilization of the observation that CYP3A5 inducers and inhibitors alter AR signaling may provide guidance to physicians prescribing CYP3A5 modulating drugs to treat comorbidities in elderly patients undergoing ADT, particularly AA. Androgen depletion therapy (ADT) is the standard first line treatment in advanced prostate cancer . ThroughOur previous work shows that CYP3A5 has a critical role in AR signaling as it promotes AR nuclear translocation and downstream signaling promoting growth . CYP3A5 Healthy prostate epithelia are shown to express high basal levels of CYP3A5, but CYP3A5 expression in prostate cancer tissues is less well-characterized ,18,19. DPreviously we demonstrated that CYP3A5 facilitates nuclear translocation of androgen receptor in prostate cancer cells . We haveWe have previously shown that CYP3A5 is expressed in androgen receptor positive prostate cancer cell lines  and promotes activation of AR and prostate cancer growth . CYP3A5 To test if wild type full length CYP3A5 regulates AR nuclear activation in a similar fashion, we used MDAPCa2b cells, which express wild type CYP3A5 (*1/*3). MDAPCa2b cells were treated with NT and CYP3A5 siRNA pool to specifically block CYP3A5 and then induced with DHT in charcoal stripped phenol red free media to monitor AR nuclear translocation and activation. The cytoskeletal fraction, which contains CYP3A5, shows reduced CYP3A5 protein after siRNA treatment as compared to non-target (NT) control A. Of not2 PCR pathway array deciphering changes in signaling targets downstream of Androgen receptor shows downregulation of several genes listed in To further evaluate the downstream signaling effect of CYP3A5 knockdown, cDNA prepared with RNA extracted from MDAPCA2b cells treated with NT and CYP3A5 siRNA pool was used for gene expression analysis. RTThe average age at detection is 66 for prostate cancer patients; hence, they often have comorbidities, and are prescribed other medications to treat these co-morbidities, while undergoing androgen deprivation treatment (ADT). CYP3A5 is known to process 33% of the commonly prescribed drugs and these co-prescribed drugs can be an inducer/inhibitor of CYP3A5. Since AR is central to prostate cancer progression and is a main therapeutic target in treating prostate cancer, any alteration in AR signaling can alter efficacy of these regimens. Based on our observation that CYP3A5 alters AR activity, we wanted to test the effect of CYP3A5 inducers/inhibitors drugs on AR signaling, as CYP3A5 can be modulated by these co-prescribed drugs. To evaluate the effect of known CYP3A inducers and inhibitors on AR nuclear translocation and downstream signaling, we used two CYP3A inhibitors, amiodarone (5 µM) and ritonavir (35 µM); and two inducers, phenytoin (50 µM) and rifampicin (30 µg/mL) . AmiodarWe tested the effect of these CYP3A5 inhibitors and inducers on total AR expression. Cell lysates prepared from LNCaP and MDAPca2b cells incubated with the selected drugs were analyzed by western blotting to verify if protein expression of AR is affected. None of the drugs tested affects total AR protein expression in LNCaP and MDAPCa2b cells, except ritonavir A. We observed reduced AR nuclear translocation in the cells treated with CYP3A inhibitors (amiodarone and ritonavir) and increased AR translocation in cells treated with CYP3A inducers (phenytoin and rifampicin) as compared to control cells that received no drugs (vehicle treated) B in bothTo test our hypothesis that AR nuclear localization is dependent on the changes in CYP3A5 expression caused by the CYP3A5 inducers/inhibitors, we performed AR nuclear localization assays after CYP3A5 and NT siRNA treatment. The MDAPCa2b cells treated with CYP3A5 siRNA and CYP3A5 inducer (phenytoin and rifampicin) do not show increased nuclear AR in contrast to NT control . This reProstate specific antigen (PSA) expression is regulated by androgen receptor and is an established marker to monitor AR downstream signaling. To evaluate downstream effects of AR nuclear translocation due to pharmacologic modulation of CYP3A5 expression, we analyzed the level of PSA protein expression in the phenytoin, rifampicin, and amiodarone treated cells. In both LNCaP and MDAPCa2b cell lines, CYP3A-inducing drugs phenytoin and rifampicin increased expression of PSA. The fold change in the MDAPCa2b cell line, which carries a wild type CYP3A5 A, was moWe used a luciferase-based reporter assay to determine the effect of commonly prescribed CYP3A5 inhibitors/inducers on their ability to modify AR downstream signaling. Both LNCaP and MDAPCa2b cell lines were transduced with a viral construct carrying androgen response elements (AREs) fused with luciferase; positive clones were selected after antibiotic selection. Negative controls were setup with constructs carrying only the TATA promoter without ARE. A pool of positive clones was used to monitor changes in luciferase activity after treatment with the CYP3A5 inducers/inhibitors. The reporter assay using MDAPCA2b cells show increased luciferase activity with CYP3A5 inducers  and decreased luciferase activity with inhibitors  (less AR activation) A. LNCaP Androgen signaling pathway is involved in cell growth; based on our observation that CYP3A inhibitors and inducers alter AR nuclear translocation, we hypothesized that they should also alter cancer cell growth. To test our hypothesis, we monitored the effect of these inhibitors and inducers on prostate cancer cell growth. Both LNCaP and MDAPCa2b cell lines were incubated with different dose range of inducers (phenytoin (0–60 µM), rifampicin (0–35 µM)] and CYP3A inhibitors [amiodarone (0–6 µM), ritonavir (0–40 µM)). Our results indicate that CYP3A inhibitors amiodarone and ritonavir decreased cell growth whereas CYP3A inducers phenytoin and rifampicin reduce cell growth of both cell lines increasing concentrations . The effOur previous work shows that CYP3A5 inhibition can lead to growth inhibition in LNCaP cells due to blocking of AR activation and downstream signaling. In keeping with previously published results for LNCaP, the MDAPCa2b, which carries one copy of wild type CYP3A5 (*1), also promotes AR nuclear localization. CYP3A5 is polymorphic with the wild type variant encoding full length translated protein being expressed in 73% of AAs, whereas only 5% of this variant is expressed in NHWA ,23. SincSince MDAPCa2b carries a wt CYP3A5, we used this cell line for the PCR based pathway array to study the effect of CYP3A5 inhibition on AR downstream signaling. The 11 genes that show maximum fold change (≥ 2.5) with CYP3A5 siRNA treatment are known to play an important role in prostate cancer growth and severity. SLC45A3, also known as prostein, is downregulated (−4.56 fold) with CYP3A5 siRNA treatment and belongs to solute carrier family 45. Protein expression is seen in both normal and malignant prostate tissue; its messenger RNA and protein are upregulated in response to androgen treatment in prostate cancer cells. ,27. FKBPCYP3A5 is an enzyme whose activity can be physiologically altered by many prescribed drugs that are activators or inhibitors of CYP3A5. Men with prostate cancer undergoing ADT are often elderly and have comorbidities requiring concomitant prescription medications, many of which are CYP3A5 inducers or inhibitors. The modulation of CYP3A5 by concomitant prescribed drugs may enhance or interfere with ADT, of great relevance to the AAs expressing wild type CYP3A5. Our data show that commonly prescribed CYP3A5 inducers promote AR nuclear migration, whereas CYP3A5 inhibitors block AR nuclear migration. The results of the study indicate that the CYP3A5 inhibitors show less nuclear AR and less PSA expression similar to CYP3A5 siRNA. Conversely, the inducers promoted nuclear AR translocation with and without DHT induction. Both the cell lines show similar effect since both they have different AR and CYP3A5 expression; we were not able to derive a quantitative difference between both the cell lines. Luciferase reporter assays showed a concordant response with respect to AR downstream signaling. Although CYP3A5 is not the intended target of any of these drugs, it was shown that a predictable effect on AR signaling is due to the changes in CYP3A5 and not due to the primary target of these drugs . The speBoth CYP3A inducers increase the proliferation of the cells (LNCaP and MDAPCa2b) and inhibitors reduce cell growth. Interestingly the effect of inducers and inhibitors on growth are more pronounced in MDAPCa2b, which carries the wild type CYP3A5, as compared to LNCaP(*3/*3), with the exception of ritonavir. The observed difference can be because the other three tested inhibitors only effect the AR nuclear localization, whereas ritonavir also affects total AR levels. Although CYP3A5 inducers and inhibitors are well known to contribute to drug-drug interactions, a potential mechanism that may impact androgen receptor signaling has not been suggested or demonstrated previously. These data strongly suggest that concomitant CYP3A5 inhibitor/inducers prescribed to patients undergoing ADT may alter the efficacy of ADT. Although our data are highly suggestive and provocative, we recognize that they are preliminary proof of concept and may not be generalizable in the clinical setting. Prospective clinical studies are further needed to determine the clinical impact of concomitant prescription medications in men with prostate cancer undergoing ADT.LNCaP, MDAPCa2b, 22RV1, and E066AAhT cells were purchased from ATCC and maintained in RPMI , F-12K medium (ATCC® 30-2004), RPMI, and DMEM  media, respectively. Supplements were added as recommended by ATCC. C4-2 was a gift from Dr. David Nanus and maintained in RPMI media. RC77 T/E (tumor) and RC77 N/E  cell lines were obtained from Dr. Johng S Rhim and are maintained in Keratinocyte SFM media supplemented with epidermal growth factor and bovine pituitary extract .Antibodies against Androgen receptor (ab74272), were obtained from Abcam, . Anti-CYP3A5 (MA3033) was from Thermo Fisher  and anti-GAPDH (10R-G109A) was from Fitzgerald Industries . Anti-α tubulin (2125S) and anti Lamin A/C (4C11) were obtained from Cell Signaling Technologies . The secondary antibodies (IR dye 680 and IR dye 800) were from LI-COR .CYP3A inducers phenytoin (PHR1139) and rifampicin (R3501); and inhibitors ritonavir (SML0491), amiodarone hydrochloride (A8423), and azamulin (SML0485) were purchased from Sigma-Aldrich . The siRNA transfection reagents, oligofectamine, and OPTIMEM were from Invitrogen; the siRNAs were from Dharmacon . MTS cell titer reagent was from Promega .Cells were washed in phosphate buffer (PBS) and lysed in RIPA buffer  supplemented with protease and phosphatase inhibitors for total protein isolation. GAPDH was used as an internal control for total protein. Infrared fluorescent-labeled secondary antibodies were used for detection using Odyssey CLx. All the densitometry analysis and fold change calculations are shown in Nuclear and cytoplasmic cell fractionation was prepared using a NE-PER Nuclear and cytoplasmic extraction kit from Thermo Scientific (Cat No. 78833) and the manufacturer’s instructions were followed. The cells were treated with drugs in charcoal-stripped phenol red free media 48 hours after plating. Drugs and DHT were added at specified concentration and duration as indicated. Cells were washed in PBS once before the cells were suspended in CER buffer. Protease, phosphatase inhibitors, and EDTA was added prior to cell lysis. The pellet remaining after cytoplasmic isolation was washed twice with PBS. The pellet was suspended in NER buffer for nuclear fraction extraction according to guidelines and the samples were stored at −80°C until further processing. Cytoskeletal fraction was prepared using the Qproteome cell compartment kit from Qiagen LLC . The cytoskeletal fraction was dissolved in CER 4 buffer as instructed and used for western analysis of CYPA5. Tubulin was used as internal controls for cytoplasmic and cytoskeletal fractions and lamin was used as a control for nuclear fractions.The cells were plated in complete media without antibiotics on poly D-lysine-coated plates . After 48 h of growth, the cells were transfected using RNAimax according to the manufacturer’s instructions. The smart pool non-target (NT) siRNA  was used as a transfection control with the experimental target gene siRNAs. A pool of four siRNA  against the CYP3A5 were used to block the expression. The final concentration of the siRNA (NT and targets) used was 30 nM. 5-conjugated Donkey Anti-rabbit (711-175-152), and Alexa Fluor 488—conjugated Donkey Anti-Mouse (715-545-150) from Jackson Immuno Research, West Grove, PA, were diluted at 1:50 in a staining buffer and incubated for 30 minutes at room temperature. The cells were washed three times (10 minutes) in PBS and stained with 1 µg/mL DAPI  in PBS for 5 minutes at room temperature. The cells were stored in PBS at 4°C until imaging was completed.Cells were seeded into a 35-mm glass bottom dish . The cells were fixed in 4% paraformaldehyde for 20 minutes and permeabilized using a permeabilizing buffer (0.2% Tween 20 in PBS) for 5 minutes. The cells were blocked using 10% goat serum diluted in a permeabilizing buffer with 1% BSA for 15 minutes. Primary antibodies were diluted at 1:100 in staining buffer (1% BSA in PBS) and incubated for 60 minutes at room temperature. The cells were washed three times (10 minutes each) in PBS. Secondary antibodies, Cy5 405 nm, 488 nm, 561 nm, and 638 nm solid-state lasers were used, respectively. FITC and TRITC emissions were collected using GaAsP detectors on the A1R+ microscope. NIS-Elements software from Nikon was used for recording the data.The cells were imaged using confocal laser scanning microscopy on a Nikon A1R using a Galvano scanner and a 60× Apo-TIRF oil immersion objective. To excite DAPI, FITC, TRITC, and CY2 first strand kit (Cat No. 330404) Qiagen, according to the manufacturer’s instructions. The cDNA was diluted and used as template to analyze for gene expression pattern using individual gene assay or RT² Profiler PCR Arrays (Cat No. 330231) specifically designed to probe panel of Human Androgen Receptor Signaling Targets (PAHS-142Z). The real-time PCR reaction data was collected using ABI 7500 fast real-time PCR system. For CYP3A5 qRT-PCR, the primer was obtained from Qiagen (Cat No. PPH01219F-200); GAPDH was used as control. For the profiler array, a total of 96 genes were profiled and data analysis was done using the Geneglobe portal on the Qiagen website. Samples (triplicates) were grouped into control (Non-Target) and test (CYP3A5 siRNA), and normalized with Beta-2-microglobulin (B2M) and Ribosomal protein, large, P0 (RPLP). A set of genes were identified based on fold change cutoff value of 2.0 and p value of 0.005. RNA was isolated using the RNeasy Mini kit (Cat no.74104) from Qiagen  using the manufacturer’s instructions. cDNA synthesis was performed using RTCignal Lenti AR Reporter (luc) from Qiagen  was used to generate an AR pathway sensing LNCaP and MDAPCa2b cell lines for the study of the AR signal transduction pathway. These lentivirus particles have androgen response elements (ARE) fused to luciferase, which detect any changes in AR downstream signaling. The cells were transfected according to the manufacturer instructions. Negative Control (only TATA box in place of ARE) transfected cell lines were also generated to measure background luciferase activity. The cells were maintained under puromycin selection pressure to select for stable chromosomal integration of the lentiviral constructs. The selected cells were tested for AR signaling pathway activation in response to DHT treatment after drug treatment using Bright-Glo Luciferase Assay from Promega (Cat No. E264A). The cells were collected into an Eppendorf tube and divided into two equal aliquots. One aliquot was used for luciferase assay and lysed with a Glolysis buffer (Cat No. E266A); the manufacturer’s guidelines were followed. The second aliquot was lysed with RIPA buffer for protein quantification.DNA was isolated from cell lines using the QIAam DNA mini kit (Cat No. 51304) from Qiagen, according to the manufacturer’s instructions. TaqMan™ Drug Metabolism Genotyping Assay (Cat no. 4362691) from Applied Biosystems with 7500 Fast System was used to determine CYP3A5 *1 and *3 allelic status. The assay is a q-PCR based assay with two primer and two probe sets. One probe set identifies the wild type allele (*1) and is labelled with VIC dye (5’); the other identifies the *3 mutant and is labelled with the FAM dye (5’). Both the probes have a non-fluorescent quencher attached to the 3’ end, which increases the specificity of detection. The allelic differentiation probe/primer set has a minor groove binder (MGB); this modification increases the melting temperature (Tm) for a given probe length and results in greater differences in Tm values between matched and mismatched probes, which produces more robust allelic discrimination. After the end of the PCR reaction, the plate is read for the end point signal generated by both the reporter dyes (VIC and FAM), and allelic discrimination analysis is run to detect the specific alleles.Based on our data, we suggest that taking CYP3A5 inhibitors concomitantly may clinically benefit patients undergoing ADT (enhancing its effect), whereas taking CYP3A5 inducers may reduce the efficacy of the ADT treatment (countering its effect). These observations suggest that the effect of these inhibitors and inducers may be more relevant in AA patients, as they tend to carry the wild type CYP3A5 and may result in therapeutic resistance. This study also suggests that care be taken while prescribing CYP3A5 inducers when patients are undergoing ADT. In addition, it also suggests that genetic testing for CYP3A5 polymorphism in patients may provide significant information about the potential impact of these interactions, facilitating personalized treatment regimens.


In this study, a total of 634 DEGs were identified. They were significantly enriched in 14 GO terms, such as integral component of membrane, plasma membrane, and G-protein-coupled receptor signaling pathway. Meanwhile, there were eight terms of KEGG pathways significantly enriched, such as Olfactory transduction, Cytokine–cytokine receptor interaction, and TNF signaling pathway. The top 10 hub genes  were identified by the PPI network. DEGs were classified into seven modules by WGCNA. According to qRT-PCR validation of the five genes of interest , the expression level of Rtp1 mRNA was significantly decreased in the AR group compared with the control group, while there are enhanced Rps27a, Penk, Cxcl2, and Anxa1 mRNA expressions in the AR mice group compared with the control group. Western blot was also performed to further explore the expression of Anxa1 in the protein level, and the results showed a similar expression trend.This study aimed to identify allergic rhinitis (AR)-related hub genes and functionally enriched pathways in a murine model. Dataset GSE52804  was downloaded from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and protein–protein interaction (PPI) analyses of DEGs were performed to identify the hub genes in AR. The DEGs were classified into different modules by using the weighted gene co-expression network analysis (WGCNA). Moreover, to verify the potential hub genes, nasal mucosa tissues were obtained from murine AR models ( Allergic rhinitis (AR) is a common chronic hyperresponsive upper respiratory disease that affects all age groups . It is aGene Expression Omnibus (GEO) integrated the independent published datasets in recent years, which enlarged the sample size and benefited further research . WeighteTo the best of our knowledge, this is the first study to integrate bioinformatics analyses and animal models to investigate the pathogenesis of AR.1 database to identify the hub genes related to AR. The platform of the GSE52804 dataset was GPL6246 (Affymetrix Mouse Gene 1.0 ST Array). This dataset included three AR mice models and three normal controls  (Series matrix files of GSE52804 were downloaded from the GEO8 weeks) . Mice in2. The “impute” package3 was carried out to fill in missing values. To identify the differentially expressed genes (DEGs) between the AR group and the control group, the “limma” package  package  in R sof4 to perform the GO and KEGG pathway analyses analyses . DAVID ianalyses . GO anal5. The cutoff point of correlation between proteins was set as greater than 0.9, and Cytoscape software  .1, UAS)  was used.1, UAS) .Weighted gene co-expression network analysis was used to identify the potential function and clusters highly correlated genes of DEGs in AR. The DEG co-expression modules were constructed by the “WGCNA” package in R software . The topn = 5 per group): the AR group and normal control group. AR murine models were induced according to the previously established protocol  and raised in a SPF (specific pathogen free) environment in the Department of Laboratory Animal Science of Shanghai Medical college of Fudan University. Mice experimental protocol was approved by the Animal Care and Use Committee of Fudan University. Mice were randomly divided into two groups , and each sample was analyzed in triplicate.TM  was used for qRT-PCR by ABI PRISM 7500 Sequence Detection System . Both target genes and GAPDH were amplified in separate wells in triplicate. The qRT-PCR primers were designed and synthesized by Sangon Biotech . BLAST 6 was applied to verify the primer design and to ensure the specificity of the primers. Primer sequences used are shown in Nasal mucosa tissues were harvested under a microscope. Total RNA was extracted from nasal mucosa tissues by TRIzol reagent . The purity and integrity of RNA were measured using a NanoDrop ND-2000 . PrimeScript RT Master mix  was applied to synthesize complementary DNA (cDNA), and SYBR Premix Ex TaqProtein samples (30 μg) obtained from nasal mucosa tissues were separated by using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes  according to the manufacturer’s guidelines for incubation with anti-ANXA1 antibody . Meanwhile, the expression of β-actin  was performed as an internal control. Image J software  was applied to analyze data, which was shown as densitometry units. The workflow of this study is shown in p-value < 0.05 was considered statistically significant.Differentially expressed genes identification and WGCNA were performed by R software . Results were presented as the mean ± standard deviation (SD). GraphPad Prism software  was applied to statistical analyses. p < 0.05, |logFC| > 0.5)  , and the| > 0.5) . There w| > 0.5) .DAVID online tool was used to perform GO and KEGG analyses and to identify the biological functions of the 634 DEGs in AR. GO analysis revealed that the DEGs were significantly enriched in 14 GO terms. They were mainly enriched in an integral component of membrane, plasma membrane, and G-protein-coupled receptor signaling pathway . MeanwhiThe PPI network of DEGs was constructed based on STRING database. The correlation between proteins was set as >0.9 score and then 602 nodes and 486 edges were produced . Further“WGCNA” package in R software was performed to merge the coherent expression genes into modules . Then, sp < 0.0001) (p < 0.0001).Sneezing was characterized by a sudden expiration, and nose rubbing was characterized by scratching of the nose with either one or both of the paw. Symptoms of sneezing and nose rubbing in the AR group and the normal control group were observed, and they were more severe in the AR mice model than in the control mice model ( 0.0001) . The ELIp < 0.05), while the Rps27a, Penk, Cxcl2, and Anxa1 mRNA expression levels were significantly increased in the AR group , the expression level of Rtp1 mRNA was significantly decreased in the AR group compared with the control group . A previous study showed that Rtp1 (Receptor Transporting Protein 1) strongly expressed in the peripheral olfactory organs (p < 0.0001) (p < 0.01)  . The exp < 0.01) . As far  < 0.01) .In general, this study was performed to identify the hub genes and pathways involved in AR by bioinformatics analysis. The hub gene was verified by murine models. It is reasonable to suppose that Anxa1 is important in the pathogenic progress of AR, and it deserves further research. Interestingly, genes associated with olfactory activity may also be relevant in AR, which indicates a new research direction of our laboratory.All datasets generated for this study are included in the article/The animal study was reviewed and approved by Animal Care and Use Committee of Fudan University.LC designed the study, participated in the experiments, analyzed the results, and drafted the manuscript. LS participated in model induction and analyzed the results. YM analyzed the results and participated in the experiments. CZ revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Blocking androgen receptor (AR) transcriptional activity by androgen deprivation therapy (ADT) improves the response to radiotherapy for intermediate and high risk prostate cancer. Unfortunately, ADT, antiandrogens, and abiraterone increase expression of constitutively active splice variants of AR (AR-Vs) which regulate DNA damage repair leading to resistance to radiotherapy. Here we investigate whether blocking the transcriptional activities of full-length AR and AR-Vs with ralaniten leads to enhanced sensitivity to radiotherapy. Combination therapies using ralaniten with ionizing radiation were evaluated for effects on proliferation, colony formation, cell cycle, DNA damage, and Western blot analyses in human prostate cancer cells that express both full-length AR and AR-Vs. Ralaniten and a potent next-generation analog (EPI-7170) decreased expression of DNA repair genes whereas enzalutamide had no effect. FACS analysis revealed a dose-dependent decrease of BrdU incorporation with increased accumulation of γH2AX with a combination of ionizing radiation with ralaniten. An additive inhibitory effect on proliferation of enzalutamide-resistant cells was achieved with a combination of ralaniten compounds with ionizing radiation. Ralaniten and EPI-7170 sensitized prostate cancer cells that express full-length AR and AR-Vs to radiotherapy whereas enzalutamide had no added benefit. Radiotherapy is one of the primary treatment options for localized prostate cancer and when combined with androgen deprivation therapy (ADT) there is significant improvement in disease-free and overall survival for intermediate and high-risk disease ,2,3,4. UAR is a ligand-activated transcription factor comprised of a C-terminal ligand-binding domain (LBD) linked to the DNA-binding domain by the hinge region. The N-terminal domain (NTD) contains all of the AR’s transcriptional activity within its activation function-1 (AF-1) region. AR regulates the expression of many genes in prostate cells including those controlling cellular proliferation and survival thereby providing the rationale for ADT to block AR signaling as a systemic therapy for prostate cancer. ADT reduces the levels of androgens and includes orchiectomy, LH-RH agonist/antagonists and also the CYP17 inhibitor abiraterone acetate. Nonsteroidal antiandrogens such as enzalutamide and the other “-lutamides” compete with androgen for binding in the ligand-binding pocket in AR-LBD to reduce its transcriptional activity. Both ADT and antiandrogens are AR-LBD inhibitors and provide their therapeutic response by blocking AR signaling, but these therapies are not curative. Resistance to ADT and antiandrogens results in lethal castration-resistant prostate cancer (CRPC) by mechanisms that include: Expression of constitutively active truncated AR splice variants (AR-Vs) that lack an LBD; de novo biosynthesis of androgens; and gain-of-function point mutations in the AR-LBD. Targeting the Achilles heel of the AR, the NTD, with the ralaniten (EPI-002) compounds blocks all of these mechanisms of resistance to inhibitors of AR-LBD  ,12,13,14Radiotherapy mediates a therapeutic response by inducing DNA double-strand breaks (DSB) that results in cell death when DSBs are not repaired and the cell continues to progress through the cell cycle. The most prominent arrest of most cancer cells in response to IR is in the G2 phase . Most DSIn prostate cancer, the AR regulates the expression of genes involved in DDR ,9,10. ThOne mechanism proposed to underlie poor responses to radiotherapy combined with ADT or AR-LBD inhibitors is the expression of AR-Vs. Prostate cancer cells that express AR-Vs either alone or together with full-length AR do not exhibit synergism between ADT and radiotherapy that is observed in cells that express solely full-length AR . The cliRalaniten and its analogs are the only known drugs proven to inhibit both full-length AR and AR-Vs by a mechanism of directly binding to AF-1 in the AR-NTD to block protein–protein interactions that are essential for its transcriptional activity ,31,32. HRalaniten-acetate (EPI-506) is the prodrug of ralaniten and was tested in first-in-human Phase 1 clinical trials (NCT02606123). Ralaniten-acetate is immediately converted to the active compound, ralaniten. Of the 500 ralaniten analogs that we have tested, here we describe EPI-7170 that has improved potency A. 3-luciferase reporter that contains three AR-V7 binding sites linked in tandem in front of a minimal promoter [PSA(6.1kb)-luciferase and probasin (PB)-luciferase reporters are highly induced by androgen via ligand-bound full-length AR binding to androgen-response elements (AREs) within the enhancer and promoter regions of these reporters. In LNCaP human prostate cancer cells that express functional full-length AR, an IC50 for EPI-7170 was measured at 1.08 ± 0.55 μM compared to 9.64 ± 3.72 μM for ralaniten to inhibit androgen-induced PSA-luciferase activity B. Antianpromoter . This repromoter . ActivitTo ensure that EPI-7170 blocked endogenous gene expression driven by full-length AR and AR-Vs, LNCaP95 human prostate cancer cells that express both functional full-length AR and AR-Vs were utilized ,28,36. ARalaniten and its stereoisomers have specificity for AR-dependent proliferation with an IC50 in the range of 10 μM to block androgen-induced proliferation of LNCaP cells A, left ,12,31. CEPI-7170 was compared to ralaniten-acetate  for efficacy against CRPC LNCaP xenografts that express full-length AR in castrated hosts. A daily oral dose of 23.3 mg/kg body weight of EPI-7170 resulted in significant reduction of LNCaP tumor burden compared to vehicle control B. A dailPOLA2, PCNA, FEN1, RAD51, and RPA2 to name a few in the top 15 enriched genes. To determine if ralaniten and its more potent analog EPI-7170 would block expression of DDR genes in a prostate cancer cell line that is driven by AR-Vs, a second approach employing a custom DDR qPCR array card was tested using LNCaP95 cells. Both ralaniten and EPI-7170 decreased the expression of DDR genes compared to vehicle  knocks down full-length AR and AR-Vs, whereas the second siRNA designed to exon 7 (encodes LBD) knocks down levels of full-length AR, but not AR-Vs ,42. DecrBlocking full-length AR transcriptional activity enhances DNA damage in response to radiotherapy ,8,9,22 bDDR is dependent on the cell cycle phase and checkpoints. Consistent with blocking AR transcriptional activity, ralaniten induces LNCaP cells to arrest in G1 similar to ADT and AR-LBD inhibitors ,34. A fuInterestingly, ralaniten caused a 3-fold reduction in the percentage of cells incorporating BrdU compared to the DMSO control after 24 h and this reduction was mainly in early S-phase cells C. This rγH2AX is a marker for DNA damage ,47,48,49Cell lines were obtained from the following: LNCaP cells from Dr. Leland Chung  in September 1993, LNCaP95 from Stephen R. Plymate , PC3, VCaP and HEK293 were from ATCC , DU145 from Victor Ling  in October 1998. Each cell line was maintained in RPMI-1640 , MEM (for HEK293), or DMEM  medium supplemented with 5% or 10% FBS or charcoal-stripped serum (CSS). Enzalutamide was purchased from Omega Chem . Bicalutamide from a gift from Dr. Marc Zarenda . Ralaniten and EPI-7170 were synthesized by us.3-luciferase and AR-V7 plasmids and transfection protocols for LNCaP cells have been described [PSA(6.1kb)-luciferase, PB-luciferase, V7BSescribed ,32,36. ILNCaP cells (5000 cells/well) and PC3 cells (2000 cells/well) were plated in 96-well plates in their respective media plus 1.5% CSS. The next day, PC3 cells were treated with compounds for 2 days. LNCaP cells were pretreated with compounds before incubating with 0.1 nM R1881 for 3 days. Proliferation and viability were measured using AlamarBlue cell viability assay  following the manufacturer’s protocol. LNCaP95 cells (6000 cells/well) were seeded in 96-well plates in RPMI media supplemented with 1.5% CSS for 48 h. Cells were treated with compounds for 48 h. BrdU incorporation was measured after 2 days using BrdU Elisa kit .n = 3 independent experiments with technical triplicates for each sample. Expression levels were normalized to levels of RPL13A transcript. Primers have been described [LNCaP95 cells  were plated in 6-well plates. The cells were treated with vehicle, ENZA, and EPI-7170 for one hour prior to treating with, or without 1 nM R1881 for 48 h. Total RNA was isolated using pure link RNA isolation kit (ThermoFisher Scientific) and reverse transcribed with high capacity RNA to cDNA kit (ThermoFisher Scientific). Quantitative real-time PCR was performed using escribed . www.cdrd.ca). Analysis of raw signal output was done using GeneSpring software (version 13.1). Hierarchical clustering was performed by conducting a 2-Way ANOVA on data with a significance threshold set at 0.05, and expression was normalized to DMSO/EtOH control samples. The Benjamini–Hochberg correction was applied to reduce the false discovery rate (FDR).Total RNA was extracted from LNCaP cells treated with ralaniten (35 µM), enzalutamide (5 µM), bicalutamide (10 μM), or vehicle (DMSO) and stimulated with either 1 nM R1881 or ethanol (EtOH) vehicle. RNA was reverse transcribed and cDNA was hybridized to the GeneChip Human Transcriptome Array 2.0 from Affymetrix. RT-PCR, cDNA hybridization and chip reading were carried out at CDRD’s Target Validation Division at the University of British Columbia  was used, and the difference in the expression levels between vehicle and drug treatment for each gene was analyzed based on the Molecular Signatures Database set H . The permutation number was set to 1,000. Those enrichment gene sets revealed by GSEA as exhibiting a nominal p < 0.05 and FDR < 0.05 were considered to indicate a statistically significant difference. The default parameters were used in GSEA software. Hierarchical clustering was performed on normalized enrichment scores (NES) generated for each sample using ClustVis software .Data generated from the microarray analysis was used to identify differences between AR antagonists. GSEA version 7.0 software (A custom Taq Man 96-well Fast Gene expression plate (Thermo Fisher Scientific) was used according to the manufacturer’s protocol using StepOne Plus Real-Time PCR System. Data were analyzed using ExpressionSuite Software Version 1.1 (Thermo Fisher). Quant Studio 6 Flex Real-Time PCR System  and Platinum SYBR Green qPCR SuperMix (Thermo Fisher Scientific) were used for conventional qPCR. Expression levels were normalized to levels of RPL13a transcript. siRNA targeting AR exon 1 and exon 7 were designed according to previous reports ,55 and pLNCaP95 cells were pretreated for two hours with compounds prior to IR with doses of 2 or 4 Gy using a Precision X-Ray XRAD320 . BrdU incorporation was measured 48 h after IR, using the Cell Proliferation ELISA BrdU kit .LNCaP95 cells were treated with DMSO vehicle, ralaniten, or ENZA in 6-well plates. Two hours later, cells were irradiated with doses of 0, 2, or 4 Gy and incubated for an additional 14 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 20 min and then photographed using ChemiDoc . Colonies containing more than 50 cells were counted using ImageJ software ver. 1.45s .Cells were seeded in 6-well plates and treated 24 or 48 h (LNCaP95) later with DMSO, ralaniten, EPI-7170 or ENZA in media supplemented with 1.5% CSS. Cells were harvested 24 h later and 20 µg of total protein was separated on Tris-Glycine gels and transferred to PVDF membrane (0.45 µm PVDF from Millipore and 0.2 µm PVDF from Bio-Rad). The membranes were immunoblotted with the following antibodies and dilutions (1:1000) AR N-20 (SC-816), Chk1  from Santa Cruz Biotechnology ; RAD51 , γH2AX  and PSA (#2475) from Cell Signalling Technology , and β-actin . After overnight incubation with primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Western blots were developed with Amersham ECL or ECL Prime Western Blotting Detection Reagent . Membranes were stripped in Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific) for re-probing. ImageJ was used for the densitometry analyses.LNCaP95 cells seeded into 10 cm dishes in 1.5% CSS were pretreated with DMSO, ralaniten, or ENZA. Two hours later, cells received IR doses of 0 or 4 Gy and then were incubated for an additional 48 h. Cells were labelled with BrdU for 2 h before being harvested and fixed in 70% ethanol. For cell cycle analysis, cells were incubated with BrdU-FITC antibody . DNA was stained with 7-aminoactinomycin D . For γH2AX analysis, cells were incubated first with γH2AX antibody  followed by AlexaFluor 488 anti-rabbit (1:200). DNA was stained with 7-AAD. Data were acquired using a FACS Calibur (BD Biosciences) and analyzed using FlowJo software ver.10.3 .6 cells/tumor in both flanks. When the tumor volume reached 50–100 mm3, mice were castrated and 7 days later they received daily oral doses of DMSO, EPI-506  or EPI-7170 . For the LNCaP95 xenograft model, NOD-SCID gamma mice were castrated two weeks before subcutaneous inoculation with 2 × 106 cells/tumor in both flanks. When the tumor volume reached 50–100 mm3, the animals received daily oral doses of DMSO, EPI-7170  or ENZA . Body weight was measured every day and tumor volume was measured twice a week using a caliper. Tumor volume was calculated by the formula: length × width × height × 0.5236. All animal experiments were approved by the University of British Columbia Animal Care Committee. For the LNCaP xenograft model, six- to eight-weeks old male NOD-SCID gamma mice were inoculated subcutaneously with 5 × 10p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.The data reported are shown as the mean ± SEM. Unless stated otherwise, statistical significance was determined by ANOVA using Dunnett’s or Tukey’s multiple comparisons test. An alpha level of 0.05 was used for all statistical tests, which is summarized in the figures as * In summary, these data reveal that blocking the transcriptional activities of both full-length AR and truncated AR-Vs with ralaniten inhibits the expression of DDR genes, cell proliferation, as well as cell survival, thereby leading to increased sensitivity of prostate cancer to radiotherapy. Clinical applications include localized prostate cancer  as well as for advanced disease including CRPC, oligoprogressive CRPC, and oligometastatic prostate cancer by combining ralaniten analogs with stereotactic radiation therapy, radium-223, or targeted alpha emitters such as 225Ac-labeled PSMA-617.


The rehabilitation process following cochlear implant (CI) surgery is carried out in a multimodal therapy according to German national guidelines and includes technical and medical aftercare. In times of the corona pandemic surgery and rehabilitation appointments were cancelled or delayed leading to a more difficult access to auditory rehabilitation. Newly implemented hygiene modalities due to the SARS-CoV‑2 pandemic have changed medical aftercare and the rehabilitation process. The aim of this study was to evaluate the quality of rehabilitation under corona conditions.An anonymous survey of adult cochlear implant patients was carried out by a non-standardized questionnaire. Demographics were analyzed and the quality of medical aftercare, speech therapy, technical aftercare, psychological support and the hygiene modalities were compared to previous rehabilitation stays.In total 109 patients completed the questionnaire. The quality of rehabilitation and individual therapy were rated as qualitatively similar or improved. The threat of the pandemic and fear of corona were rated unexpectedly high with 68% and 50%, respectively. The hygiene measures during the rehabilitation stay eased subjective fears at the same time. The majority of patients were annoyed by wearing face masks but visors, protection shields and social distancing were more tolerated.The implementation of the new hygiene modalities within the therapeutic rehabilitation setting was well-accepted by patients allowing access to auditory rehabilitation. A successful rehabilitation should ensure a fear-free environment by adhering to the necessary hygiene modalities.www.springermedizin.de. Please enter the title of the article in the search field, the supplementary material can be found under “Ergänzende Inhalte”.The online version of this article (10.1007/s00106-020-00923-z) includes the study questionnaire. Article and supplementary material are available at  The coronavirus pandemic has caused massive changes in working procedures in hospitals, general practices, and in rehabilitation clinics. With the start of the so-called lockdown, outpatient services were reduced, operations cancelled or postponed, and rehabilitation programs were no longer conducted or only to a very limited extent after March 17, 2020. Curtailing of elective programs set intensive care capacities free to enable treatment of COVID-19 patients under special hygiene measures. It is estimated that up to 38.9% of surgical interventions in malignant diseases and up to 81.5% in non-malignant diseases were cancelled and/or postponed in the ear–nose–throat (ENT) area [Two months later, with the step-wise relaxing of hygiene measures, surgical capacities were reactivated and the rehabilitation clinics recommenced their work, although to a limited extent. In principle, special hygiene measures, social distancing, and obligatory masks were enforced, along with limited visiting rules and the resultant numerical reduction in capacities.This situation was found to be a special challenge in rehabilitation after CI. According to the guidelines, rehabilitation is an integral component in CI care . The intThe Q‑Reha-certified CI rehabilitation is conducted for adults at the Implant Centre Freiburg (ICF) on 20 rehabilitation days as an interval rehabilitation. The 5‑day basic therapy is followed by 2‑ to 3‑day rehabilitation stays over the course of 24 months. Due to the coronavirus pandemic, rehabilitation was re-started in early May 2020 with a reduced number of patients. The patients and the persons accompanying them underwent medical examination on admission, including temperature measurement, and were given information on hygiene measures. The usual scheduling plan was modified according to hygiene rules, since group therapies or discussions were not possible. Technical fittings, logopedic diagnostics and therapy, psychological sessions, music therapy, and consultations required the use of mouth–nose masks, face shields, spit barriers, and maintaining the required distance. Meals were served in the cafeteria to reduced numbers and in shifts. Owing to the reduced number of patients to approximately 50% compared with pre-coronavirus times, the patients had a lower chance of contact for personal interaction.This present study is intended to examine the extent to which the current changes resulting from the application of the required hygiene measures, social distancing rules, and obligatory masks influence the course and the subjective success of CI rehabilitation in comparison with the pre-coronavirus situation. Moreover, the question arises of how CI patients rate the pandemic. To date, there are no studies on rehabilitation after CI under COVID-19 conditions.We conducted an anonymous patient survey in the period from May 13, 2020 to June 25, 2020. Adult patients who were attending rehabilitation after CI for the second or multiple times at the ICF were included.All patients planned for in-patient rehabilitation were contacted by telephone 3–5 days before admission and asked about the state of their health and possible contact with SARS-COV-2-infected persons. An anonymous evaluation was made of the number of patients who wanted to cancel or postpone their appointment.The hygiene measures were determined in close cooperation and consultation with the Institute for Infection Prevention and Hospital Epidemiology. Social distancing and mouth–nose masks (MNM) had priority. Face shields and spit barriers were used in situations in which distancing or MNM were not possible for medical or therapeutic reasons. Attention was paid to the disinfection of hands according to hospital-wide rules.The patients who came for rehabilitation were informed about the study during the admission examination and were given written patient information and the questionnaire . The latter was handed in anonymously at the end of the 2–3-day stay.The non-standardized questionnaire comprised 44 questions in the medical, psychological, therapeutic, and technical areas. The survey addressed the qualitative and quantitative comparison of rehabilitation before and under coronavirus conditions. Anxiety regarding the coronavirus pandemic, the quality of the rehabilitation, and the attainment of goals under pandemic conditions were queried. The use of the various hygiene measures—MNM, face shields, spit barriers, and distancing—was evaluated. The importance of exchange among rehabilitation patients was evaluated, as was the reduction in contacts resulting from the hygiene measures. The age group , the subjective estimate of belonging to a risk group, rehabilitation experience, inclusion of an accompanying person, and gender were recorded.The assessment was made descriptively. This study was registered under No. 00021680 with the German Registry of Clinical Studies (Deutsches Register klinischer Studien). With application No. 10020/20 of the Ethics Commission of the University of Freiburg, exemption from obligatory consultation was granted, since only anonymized data were recorded and evaluated.n = 109).A total of 129 questionnaires were issued to rehabilitation patients between May 13, 2020 and June 26, 2020. The response rate was 84.5% (n = 120 of the planned 249 Reha patients) cancelled the appointment during the pre-evaluation by telephone. The distribution of cancellations remained constant over calendar weeks 17–26.Triage results: 48.1% of the adult patients  is shown in Fig. The age distribution of the patients (The majority of the patients rated the admissions examination as positive: The admitting doctor was sympathetic (86.1%), information on hygiene measures was given (89.8%), and MNM were provided (95.3%).n = 19) of the patients wished for and received an additional session. The majority did not want to broach the issue of coronavirus (15 of 19 patients). Overall, suggestions for everyday problems could be made (13 of 15 patients), the patients felt relieved (11 of 18 patients), and connections could be clarified (14 of 18 patients).An initial psychological session is regularly conducted only during basic therapy. During the rehabilitation stay, 18.3%  compared with pre-coronavirus times. Music therapy was rated as unchanged in quality, but only 44.3% of the patients received this therapy due to the limitation of group therapies.While 86.9% of the rehabilitation patients rated the intensity of the therapies compared with the pre-coronavirus period as just right, 11.2% found the intensity too low. Overall, 89.6% of the patients could achieve or nearly achieve their therapy goals, while 10.4% could not do so, or only to a limited degree.The evaluation of whether the ICF offers the proper therapy for each problem was rated positive by 82.4% of the respondents; only three patients (2.5%) were of the opinion that the ICF does not offer the proper therapies.The use of MNM was rated as annoying by the majority (65.1%), and 48% of the patients considered treatment more difficult due to the masks. Nearly half of the patients 43.6%) considered their own personal mask (MNM) and 25.5% the masks of the therapists annoying; 30.9% of the patients found both irritating (their own mask and the mask of the therapist). The rating of the individual protective measures showed that social distancing was considered the least annoying compared with the face shields, spit barriers, and MNM. The MNM was rated as the most detrimental to therapy  considered conversations with other rehabilitation patients important or very important. The required protective measures negatively affected these conversations to a moderate or high degree for 50% of the patients.For a large majority of the rehabilitation patients . This creates a further patient backlog. Moreover, shifts within an interval rehabilitation, whether as in- or outpatient, always bring a risk that the interval between the individual therapies and adjustments will be too long, so that the full potential will not be achieved or there will be programming that needs improvement, resulting in limited speech comprehension. For deaf and hearing-impaired patients, it also applies that chronic disease does not take a “Corona break” .The use of hygiene measures made the accustomed rehabilitation more difficult. The MNM was considered to be the most annoying for patients as well as the most disruptive for treatment. Studies by Saile and Gregori in 2020  showed aIt is interesting that the rehabilitation patients definitely noticed the limitations due to hygiene measures, but rated the quality of rehabilitation as unchanged or even improved. This applies to all areas of rehabilitation, such as logopedics, technical aspects, and music therapy. The altered setting with MNM, face shields, spit barriers etc. had no negative effect on the rehabilitation success and achieving of goals. This can be interpreted, on the one hand, as an expression of acceptance of the required measures by the patients, but also as the successful and empathetic implementation of the measures by the doctors, therapists, and technicians.Fear of the coronavirus pandemic in the general public has been cited at ca. 30% since early May 2020 ). It is We only surveyed patients who were at the ICF for therapy and who did not cancel or postpone their stay because of concerns about the pandemic, which is a limitation of this study. This limitation also means that hearing-impaired or CI patients may possibly experience limitations and anxiety due to coronavirus to a much higher extent than the normal population. This is particularly important as it must be postulated that especially anxious patients are more likely to cancel the rehabilitation appointments. For further studies, structured interviews with this patient group are important so as to optimize dealing with the anxieties of hearing-impaired patients in times of pandemics.Another limitation of this study is the use of a nonstandardized questionnaire. This was applied due to the completely new situation of a worldwide pandemic and the actual relevance of the rehabilitation subject and because of non-existing validated questionnaires.The results of our study are a confirmation of the hygiene measures implemented at the ICF. The hygiene concept does not only affect the patients but needs to demonstrate the care of management for the therapists and ensure safety for the caregivers, creating a cool-headed and quiet working atmosphere. The team at the ICF put every effort into creating such a workspace. Weekly telephone conferences of management including all department heads and colleagues in charge of quality management were very helpful. The topics discussed are made transparent by distributing the minutes to all staff members. Indirectly, all this was evaluated positively by the patients since the risk posed by the coronavirus was generally perceived as high, but at the ICF as low.The current pandemic situation leads to limitations for hearing-impaired patients. Access to auditory rehabilitation appears more difficult. Despite all relaxation to date, a high percentage of these patients cancel their rehabilitation appointments. This may mean that patients use their speech processors with inadequate settings over longer periods and are thus not able to achieve their full hearing potential; this is contrary to the goal of inclusion.The use of electronic media for rehabilitation is common in Germany in individual cases to support rehabilitation (learning apps etc.), but it could provide another form of therapy in the future or supplement existing concepts. There are reports now on remote mapping , but thiThe “Krankenhausentlastungsgesetz”  supports hospitals and rehabilitation clinics financially during the coronavirus pandemic. This support will not be sufficient to balance the financial losses, according to our own experience. The currently necessary reduction in patient numbers and the changes within the rehabilitation process according to the newly implemented hygiene measures  will increase the length of waiting lists further. This will make the rehabilitation process more expensive and should be considered in future negotiations with health insurance providers.A successful, relatively fear-free rehabilitation under COVID-19 conditions is possible.The patients can effectively adapt to the situation, even if hygiene measures, especially the mouth–nose mask (MNM), are considered annoying by the majority.The use of face shields or transparent MNM should be self-evident for facilitated communication.The task for all those involved in rehabilitation is to create a safe environment for the optimal auditory rehabilitation under the best-possible protection for patients and coworkers, even under pandemic conditions.Study questionnaire


Pennisetum purpupoides) in complete feed on the productivity of male Ongole steer calves.This study aimed to assess the nutritional values of corn stover and king grass (thweek by collecting feces. Dry matter (DM), organic matter (OM), crude protein (CP) digestibility, neutral detergent fiber (NDF), acid detergent fiber (ADF), body weight gain, and feed conversion data were analyzed.This study was conducted in two steps: Cattle adaptation and data collection. Cattle adaptation was carried out for 2 weeks, and the cattle were fed the experimental feed. The experimental feed was formulated into five combinations: R1 (50% king grass); R2(12.5% corn stover +7.5% king grass 50% concentrate); R3(25% corn stover+25% king grass); R4(37.5% corn stover+12.5% king grass); and R5(50% corn stover). All experimental feeds were added with 50% concentrate. Data were collected in five intervals, and each interval was of 4weeks. In every interval, weight gain and digestibility were measured every day, beginning from the 4R4 and R5 treatment significantly increased (p<0.05) the feed nutritional value and weight gain of male Ongole steer calves. Interestingly, treatment with R4 had the maximum increase (p<0.05) on the digestibility value of each variable: DM, 68.85%; OM, 71.89%; CP, 73.90%; NDF, 59.10%; ADF, 55.35%; and weight gain, 0.61/gr/day.R4 treatment found to be the best nutritional value for improving the productivity of male Ongole steer calves. Corn stantation . Furtherantation . SupplemPennisetum purpupoides). It possesses high protein content and palatability [Another valuable forage for ruminants is the king grass . Feed adaptation was carried out for 14days. Feeds were given The pre-collection stage was carried out 7days before data collection. Afeeding limit of 85% established in the preliminary stage. Feed restriction was performed to maximize feed intake.The collection phase was carried out for 14days for every period (for five periods).The resting period was carried out for 14days. Livestock were given treatment feed to adapt them to the treatment feed and to nullify the effects of the previous period.thweek, by collecting the feces. This study aimed to determine feed formulation from a combination of two feed sources (corn stover and king grass) in complete feed. Thus, the treatment (R1-R5) is a comparison/combination of two feed sources, and it does not include controls. In this study, various treatments (combinations) were compared to determine the best formulation.Weight gain measurements were taken in every stage, whereas digestibility was measured every day, beginning from the 4Twenty-five Ongole male calves aged 26months and weighing 220-245kg were included. Livestock were placed individually in a metallic cage. The effect of corn stover and king grass feed supplemented with concentrate was evaluated by comparing it with complete feed contained isoprotein (13-14%). Proximate analysis of the nutrient content of the feed ingredients used in this study is presented in et al. [The chemical composition of corn stover and king grass was analyzed using a standard procedure for DM according to the AOAC procedure . NDF, ADet al. . Feed coDigestibility of corn stover and king grass was measured according to Tilley and Terry :et al. [Body weight gain (BWG) was measured, according to Tillman et al. . BWG wasData were analyzed using one-way analysis of variance, which performed with the SPSS for Windows . Significance of difference between all treatment groups was indicated by p<0.05.The evaluation of the digestibility value of DM, OM, CF, cellulose, and hemicellulose of corn stover and king grass in complete feed on organochlorine (OC) cattle is presented in The results showed that the DMD of feed comprising of corn stover and king grass in this study ranges from 65.95% to 68.85%. Analysis of variance showed a significant difference (p≤0.05). The highest digestibility was found with R4 treatment (68.85%), which included a combination of feed containing 37.5% corn stover+12.5% king grass+50% concentrate. R5 treatment also showed similar results (DMD– 68.70%), with complete feed containing 50% corn stover, 0% king grass, and 50% concentrate. The DMD values were higher than those obtained with R1, R2, and R3 treatments, which included complete feed containing 0.25-25% corn stover, 25-50% king grass, and 50% concentrate, respectively. The increase in digestibility of DM feed seems proportional to the increasing amounts of corn stover in the complete feed. Data from the proximate analysis  showed tThe average OMD of complete feed was observed to be between 67.98% and 71.89%. Analysis of variance showed a significant difference (p≤0.05). The highest average OMD was observed with R4 treatment (71.89%), which was not significantly different from that with R5 treatment (71.72%) but was significantly higher than that with R1, R2, and R3 treatments. The lowest OMD was significant (p=0.05) with R1(67.78%) followed by that with R2 and R3.The average CPD in complete feed was observed to be between 70.90% and 73.90%. Analysis of variance showed a significant difference (p≤0.05). R4 and R5 showed no significant difference (p>0.05), but R1, R2, and R3 were significantly different (p<0.05) from R4 and R5. The highest digestibility was observed with R4(73.90%), which included complete feed containing 37.5% corn stover, 12.5% king grass, and 50% concentrate, and the lowest digestibility was observed with R1(70.90%), which included complete feed with 0% corn stover, 50% king grass, and 50% concentrate.The CPD followed the pattern of DM and OMD. The digestibility of CP in the treatment feed significantly increased with the proportion of corn stover in the complete feed. The increase in DMD was due to the higher metabolic energy content of corn stover compared to that of the king grass. Corn stover has kernels rich in RAC.The average NDF digestibility in this study ranged from 57.75% to 59.10%. Analysis of variance showed that the treatment had a significant effect (p<0.05) on the NDF digestibility in complete feed that included corn stover and king grass. The highest NDF digestibility was found with R4(59.10%), with complete feed containing 37.5% corn stover, 12.5% king grass, and 50% concentrate and was significantly different (p<0.05) as compared to that with R1, R2, and R3, but not with R5. The lowest NDF digestibility was observed with R1(57.74%). The difference in NDF, CP, CF, and energy content between corn stover and king grass in complete feed caused the variability in NDF digestibility. ADF digestibility is determined by the microbial population and cellulolytic microbial activity in the rumen. ADF has the same digestibility pattern as NDF because both have almost the same fiber component. ADF consists of cellulose, lignin, and silicates without hemicellulose and only dissolves in acidic solvents. On the other hand, NDF dissolves in neutral solvents and contains hemicellulose, which can be easily digested. In this study, the average ADF digestibility of complete feed that comprised of corn stover and king grass ranged from 51.70% to 55.35% and was lower than NDF digestibility (57.74%-59.10%).The BWG of complete feed comprising of corn stover and king grass for each calf ranged from 0.49 to 0.61/gr/day. Analysis of variance showed a significant difference (p≤0.05). The highest increase was observed with R4(0.61/gr/day). R5 led to a non-significant decrease (0.59/gr/day) and a significant increase was observed with R1, R2, and R3 treatments. The lowest BWG was observed with R1(0.49/gr/day) with a complete feed formulation of 50% king grass and 50% concentrate without the administration of corn stover followed by R2(0.53/gr/day) and R3(0.55/gr/day).The study showed that the average proportion of young kernels in corn stover was 17%. This readily digested carbohydrate can serve as an energy source for optimal rumen microbial development. The availability of RAC spurs rumen microbial growth so that it has a positive impact on increasing the DMD. In the rumen, the microbial process of degradation of proteins obtained from the feed is faster than the process of providing energy , so the presence of corn stover-based RAC increases the efficiency of rumen microbial protein synthesis.et al. [The study conducted by Nasriya et al.  reportedThe effect of the treatment on the digestibility of feed OM increased in line with the increasing proportion of corn stover in complete feed. The data revealed that the protein and energy content of corn stover were higher than those observed for the king grass. Feed protein is degraded into ammonia by rumen microbes as a nitrogen source, and they also require energy  The syntProtein from feed undergoes degradation into ammonia by rumen microbes as a source of nitrogen for rumen microbes. The degradation rate of protein from feed is determined by the availability of metabolic energy from the feed consumed. The higher the energy obtained from the easily digested carbohydrates, the higher the protein digestibility. CP from feed is degraded by rumen microbes into amino acids and then undergoes deaminase into ammonia as a source of nitrogen and carbon skeleton (α-keto) for rumen microbial growth. The higher CP in the feed consumed, the higher the protein to be degraded to ammonia in the rumen.Rumen microbes digest NDF more easily than ADF because NDF consists of ADF and hemicellulose, which are easier to be digested by rumen microbes while ADF has no hemicellulose . The levThe degradation rate of NDF in the rumen was higher than the degradation rate of ADF . The resThe higher average BWG after the treatment of complete feed based on corn stover compared with one being treated with complete feed based on king grass was due to the high digestibility of DM, OM, CP, NDF, and ADF on corn stover-based complete feed. This trThe increase in BWG was affected by the increase in the proportion of corn stover, which had better nutrient quality than king grass. Acomplementary effect was observed when king grass was added, which then tended to decrease with R5, although not significantly; hence, there was no complementary effect. Data from the proximate analysis  show thaad libitum and concentrate of 3kg/day, was 0.51-0.62 kg/day, which was lower than the weight gain of local OC cattle (Javanese and Sumba cattle) crossed with imported cows , which was 0.71kg.The availability of this RAC spurs rumen microbial growth; therefore, it has a positive impact on increasing the digestibility of feed nutrients . The higBased on the results of this study, we conclude that the corn waste-and king grass-based feed  provided best nutritional value for local Ongole crossbred calves.RAVT designed and conducted the experiment, acquisition of data, and drafting of the manuscript. SSM conducted the experiment and collected the data. SAEM analyzed the data and drafting the manuscript. All authors read and approved the final manuscript.


We found that quinacrine enhanced TRAIL sensitivity or reversed TRAIL resistance in all the ovarian cancer cell lines tested. Mice treated with quinacrine and TRAIL remained disease-free for up to 20 weeks, however, mice treated with TRAIL or quinacrine alone and in control group died within ~8 weeks after treatment. Intraperitoneal delivery of quinacrine and TRAIL is rational and practical with extraordinary synergistic anti-cancer effects in preclinical models of ovarian cancer. Clinical investigation of combining quinacrine with TRAIL for ovarian cancer treatment is warranted.Quinacrine has been identified as a potent DR5-inducing agent that sensitizes cancer cells to TRAIL-induced apoptosis. In the current study, we found that quinacrine increased DR5 mRNA levels significantly in ovarian cancer cell lines regardless of p53 status. Further study showed the half-life of DR5 in quinacrine-treated cells was significantly prolonged, indicating that DR5 protein degradation was inhibited by quinacrine. We tested if the combination of TRAIL and quinacrine could be effective in ovarian cancer treatment  The extrinsic apoptotic pathway has been well-investigated and characterized; however, targeting this pathway for cancer treatment has not been successful , 2. The TRAIL induces apoptosis in cancer cells by engaging death receptors, DR4 and DR5. Subsequently pro-caspase 8 is cleaved to become active caspase 8 by recruiting FADD (fas-associated death domain) to form DISC . Cleaved caspase 8 then induces a series of caspase reactions and eventually activates caspase 3/7 to execute extrinsic apoptosis pathway. In most circumstances, cleaved caspase 8 also activates bid, which sends death signals to mitochondrion and simultaneously promotes intrinsic pathways to induce apoptosis. Death signaling in normal cells is well-controlled and regulated, either by limiting production of death receptors or facilitating degradation to keep a low level of death receptors. It has been reported that negative regulators such as FLIP (FLICE [Fas-associated death-domain-like IL-1beta-converting enzyme]-inhibitory proteins) could prevent DISC activation . SubcellDespite attempts to reverse TRAIL resistance using DNA-damaging agents , kinase In this work, we demonstrated that quinacrine induced high level of DR5 and reversed TRAIL resistance in human ovarian cancer cell lines. Quinacrine not only significantly induces high mRNA level of DR5, but also reducesDR5 protein degradation, most likely in lysosomes. Further experiments showed that quinacrine induces permeabilization of lysosomal membranes, as manifested by aggregation of galectin-3 in lysosome, which may also contribute to the synergistic effect of quinacrine and TRAIL in executing cancer cell death. Surprisingly, interperitoneally delivery of TRAIL and quinacrine showed remarkable synergistic effect in treating mice bearing A2780 ovarian cancer xenografts. Our data provides conclusive evidence for rational combination of quinacrine and TRAIL for ovarian cancer treatment.2) at 37°C. Quinacrine, z-VAD-fmk, and cycloheximide (CHX) were obtained from Millipore Sigma . D-Luciferin, potassium salt, was purchase from BioVision .Ovarian cell lines SK-OV-3, OVCAR-4, OVCAR-8, and A2780 were cultured in RPMI-1640 (Gibco) containing 10% Fetal Bovine Serum , L-glutamine and 1% penicillin-streptomycin in a humidified incubator (5% COThe Cell Counting Kit-8 (CCK-8) for cell viability assay was purchased from Dojindo Molecular Technologies . Cells were plated in 96-well plates in culture overnight and treated with TRAIL, quinacrine, or in combination at indicated concentrations for 16 h. CCK-8 solution was added to each well; 10 μl/well. Absorbance at 450 nm was measured by the Tecan Spark 10M multimode microplate reader. Drug synergy analysis was conducted using CompuSyn software . AlternaThe Caspase-Glo® 3/7 Assay kit was purchased from Promega . Ovarian cancer cells were plated in a black 96-well plate with clear bottom. After overnight incubation, cells were treated for 16 h with TRAIL, quinacrine, pan-caspase inhibitor z-VAD-fmk or in combination. Luminescence intensity was recorded by a Tecan Spark 10M multimode microplate reader and caspase 3/7 activity was calculated per the manufacturer's instructions.The human TRAIL cDNA fragment (coding amino acids 94–281) was codon optimized, synthesized by IDT  and inserted into a bacterial expression vector, pQE80L (Qiagen) to generate a His-tagged human recombinant TRAIL, which was purified per a protocol published previously . The humAntibodies against human caspase-3, caspase-8, caspase-9, and DR5 were purchased from Cell Signaling Technology . Antibodies against p53, DO1, was purchased from Santa Cruz Biotechnology . The Ran antibody was purchased from BD Biosciences . Cell lysates were prepared using RIPA lysis buffer. Protein samples were subjected to SDS-PAGE on 4–20% mini-protein TGX gels  and transferred onto Immobilon™ membranes . The membrane was blocked using blocking buffer for 1 h and probed with primary antibody for 1 h at room temperature or overnight at 4°C, washed, and then subjected to secondary antibodies labeled with IRDye . Blots were scanned using the Odyssey CLx Imaging System (LI-COR).Total RNA was extracted from cells using the RNeasy Mini Kit . Complementary DNA was synthesized using the Super-script III first-strand cDNA synthesis Kit . For quantitative real-time PCR, cDNA and specific primer pairs were mixed with Power SYBR Green PCR Master Mix , and run on the ABI Prism 7900HT Sequence Detection System . The following primers were used for amplification of DR5: DR5 (sense) 5′-ATGGAACAACGGGGACAGAAC-3′ and (antisense) 5′-CTGCTGGGGAGCTAGGTCT-3′. The threshold cycle number (Ct) for gene expression was calculated, and GAPDH was used as a reference. Delta-delta Ct values for gene expression were presented as relative fold induction.Cells were treated with the test compounds for 16 h and cells were collected upon trypsinization and centrifugation. The pellets were washed with PBS and incubated with Annexin V and DAPI  in binding buffer for 15 min. Then, 400 μl of cells in binding buffer was loaded and cells were analyzed by flow cytometry.A2780 cells in a chamber slide treated with quinacrine  were fixed with 4% formaldehyde solution in PBS buffer (pH 7.4) for 10 min in room temperature, then blocked in 3% bovine serum albumin in PBS for 30 min. Anti-Lamp2  was added and incubated for 1 h. Goat anti-rabbit IgG AlexaFluor-568  was used as secondary antibody. Nucleus was counterstained with DAPI . Images were captured with a ZEISS LSM 880 confocal laser scanning microscope .6) in 100 μL of PBS were injected intraperitoneally into NSG mice. Five days later tumor cells in the mice were imaged after intraperitoneal injection of 2 mg/mouse D-luciferin by SPECTRAL Lago X Imaging System . Bioluminescent images were captured and the intensity was quantified using the Aura Imaging Software developed by Spectral Instruments Imaging as a baseline on day 0. Mice were then randomly divided into four groups and treated with quinacrine, TRAIL, quinacrine plus TRAIL, or control with vehicle as follows: quinacrine was delivered at a dose of 50 mg/kg on day 1. TRAIL was injected intraperitoneally at 10 mg/kg on day 1, 2, 3, and 4. On day 5, mice were reimaged using the Lago X Imaging System. A repeated cycle was started on day 7. Mice were monitored closely and euthanized when cancer-related symptoms developed such as weight loss, dehydration, abdomen distention, and impaired ambulation per the IACUC protocol.All animal experiments were carried out in accordance with animal care guidelines approved by the Institution Animal Care and Use Committee (IACUC) of City of Hope. Female NSG mice (6–8 weeks old with body weight range from 19 to 23 grams) were purchased from The Jackson Laboratory. The animals were acclimated for 1 week prior to the study. A2780-luc cells . Combination index was calculated using CompuSyn software  and two-way variance analysis. Differences between groups were considered statistically significant when the software  developeWe chose four commonly used ovarian cancer cell lines and tested their sensitivity to TRAIL—A2780, an ovarian adenocarcinoma, endometrioid type cell line; OVCAR-4, a grade 2 ovarian adenocarcinoma cell line; OVCAR-8, a grade 3 ovarian adenocarcinoma cell line; and SK-OV-3, a high grade serous ovarian carcinoma cell line. OVCAR-4 and SK-OV-3 were relatively sensitive to TRAIL, while A2780 and OVCAR-8 were resistant to TRAIL . After tWe further confirmed that quinacrine induces apoptosis in combination with TRAIL by flow cytometry using Annexin V and DAPI stain . ApoptosWe measured caspase 3/7 activity in quinacrine and TRAIL treated cells. Combination of TRAIL and quinacrine induced significant caspase 3/7 activity, while quinacrine or TRAIL alone failed to induce significant caspase 3/7 activity at the concentration and the time point illustrated . Pre-treWe tested TRAIL receptor DR5 protein and mRNA levels. As shown in We further investigated whether the elevated DR5 protein level is due to increased production or reduced degradation. We treated OVCAR-8 and A2780 cells with quinacrine at indicated concentrations for 8 h. DR5 mRNA levels were significantly induced in OVCAR-8 and A2780 cells by quinacrine . In the To track DR5 in the cells, we constructed a retroviral vector, pBabe/Puro/DR5-EGFP, expressing a fragment from amino acid 1–334 with the death domain deleted, fused with EGFP, and codon optimized for better expression in mammalian cells. Cancer cells infected with the retrovirus carrying DR5-EGFP and selected by puromycin showed weak expression of DR5 on the cell surface with rare intracellular deposits visualized under a laser microscope. After treatment with quinacrine for as little as 1 h, intracellular deposits of DR5-EGFP manifested as bright dots or vesicular structures increased significantly . We counAs fluoresce of EGFP is usually quenched in an acidic environment such as in lysosomes, the bright green signal of DR5-EGFP accumulation indicates elevated pH in lysosomes. One of the major causes of pH elevation in lysosome is lysosomal membrane permeabilization (LMP). To assess whether quinacrine treatment led to LMP, we constructed a lentivirus vector expressing galectin-3 fused with mCherry at the N-terminus, codon optimized for optimized expression in mammalian cells, and overexpressed in A2780 cells. We treated the cells with quinacrine and then visualized the expression pattern of galectin-3, which is normally diffused in the cytoplasm and nucleus but translocates to leaky lysosomes rapidly to form visible puncta when LMP takes place . We founin vivo study because of its unique feature of local spreading within abdomen and pelvis and practical intraperitoneal delivery of chemotherapeutics that has been proved more effective than systemic delivery under certain circumstances. After treatment, we detected minimum bioluminescence in the group of mice treated with quinacrine and TRAIL, while mice treated with quinacrine or TRAIL alone showed no difference compared to the vehicle control group   is the fQuinacrine, introduced in the 1920's, was originally used for malaria treatment and prophylaxis , 30, treMost recently, quinacrine emerged as a potential anti-cancer agent. Quinacrine is actively involved in multiple signaling pathways including apoptosis, cell cycle arrest, DNA repair, autophagy, and arachidonic acid metabolism , 30 by tBased on our previous findings that quinacrine can significantly induce DR5 protein levels and synergize with TRAIL to induce apoptosis , we combThe anti-cancer effects of quinacrine in combination with TRAIL in ovarian cancer xenograft models we observed are extraordinary. Two cycles of one dose of quinacrine (50 mg/kg) followed by four doses of TRAIL (10 mg/kg) resulted in mice that were cancer free for 20 weeks when euthanized while all other mice  died ~8 weeks after cancer cell inoculation. No therapeutic effect of TRAIL or quinacrine alone was observed at the doses delivered in our experiments. The remarkable synergistic effect we observed may be partly explained by the route of administration—intraperitoneal injection of quinacrine and TRAIL, instead of oral delivery and systemic injection, respectively, which we hypothesize may lead to longer exposure of cancer cells to therapeutic agents in higher concentrations within the peritoneum.Intraperitoneal delivery of therapeutics in the adjuvant setting is a practical approach and has been exclusively practiced as an effective way to treat ovarian cancer allowing close contact of tumor cells with therapeutic agents at higher concentrations in the peritoneal cavity, which is the principle site of spread and recurrence of ovarian cancer , 54. It In summary, we show that intraperitoneal delivery of quinacrine and TRAIL is rational and practical with remarkable synergistic anti-cancer effects in preclinical models of ovarian cancer. Clinical investigation of combining quinacrine TRAIL for ovarian cancer treatment is warranted.All datasets generated for this study are included in the article/This animal study was reviewed and approved by City of Hope IACUC.RL: design and carry out most of the laboratory work and drafted the manuscript. YY: LMP assay and animal work. GW: TRAIL purification and animal work. EY: revise work of figures and manuscript. GY: animal work. XQ and LZ: tissue culture and cytotoxicity assay. YW: TRAIL expression vector construction and purification. TZ: design of the work and critical input in interpretation of the work. YZ: critical input and analysis and interpretation of data for the work. EW: substantial contributions to the conception and design of the work, analysis and interpretation of data, draft, revise, and finalize the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Limited studies are available regarding the pathophysiological mechanism of acquired atrioventricular block (AVB). Matrix metalloproteinases (MMPs) and angiotensin-converting enzyme (ACE) have been implicated in the pathogenesis of arrhythmia. However, the relationship between these molecules and acquired AVB is still unclear. One hundred and two patients with documented acquired AVB and 100 controls were studied. Gene polymorphisms of the MMP1 and ACE encoding genes were screened by the gene sequencing method or polymerase chain reaction-fragment length polymorphism assay, followed by an association study. The frequencies of the MMP1 −1607 2G2G genotype and MMP1 −1607 2 G allele were significantly higher in the AVB group than that in the controls . Consistently, the level of serum MMP1 was significantly greater in acquired AVB patients than that in controls . In addition, the MMP1 2G2G genotype showed a higher MMP-1 serum level than the other genotypes (1G1G/1G2G) . MMP1 1 G/2 G gene polymorphism may contribute to determining the disease susceptibility of acquired AVB by linking the MMP serum protein level. AVB is one of the major disorders for patients requiring pacemaker implantation2. In an AV block condition, the wavelets cannot reach the ventricles or are delayed through the AV node, slowing down the heart beat and causing a serious condition3. The main causes of acquired AVB include idiopathic fibrosis, aging process in cardiomyocytes, degeneration, ischemia, infarction, infiltration disease or drugs4. Progressive idiopathic fibrosis related to an aging process of the cardiac skeleton is the most common cause of chronic acquired AVB8. It is generally accepted that familial and inherited AVB are caused by gene mutations and dysfunction of the conduction system during heart development12. In contrast, the pathophysiological mechanism of acquired AVB is currently unclear.Atrioventricular block (AVB) is a group of abnormal AV conduction disorders caused by a delay or interruption in the impulse connection from the atria to the ventricles due to structural or functional impairment15. MMPs and extracellular matrix (ECM) have been shown to control cardiac conduction and have been linked to the modification of gap junction proteins for atrioventricular conduction16. Although the aging process and progressive fibrosis have been thought to be the key events for age-related conduction disturbances, ECM alteration was shown to cause impulse-conduction disorders18. Studies have shown that MMPs and RAS acting agents are involved in the pathogenesis of arrhythmia and cardiac conduction23. Studies in humans and animals demonstrated that modulation of MMP and RAS gene expression could lead to atrial fibrillation20, non-familial sick sinus syndrome21, and affect cardiac conduction after myocardial infarction17, apoptosis of the conductive system22 and occurrence of ventricular arrhythmia23. Here, we examined whether the polymorphisms of the genes encoding the MMPs and ACE could alter the disease susceptibility for acquired AVB by investigating the relationship between MMPs and ACE gene polymorphisms and disease susceptibility of acquired AVB. The results obtained indicated that MMP1 1 G/2 G gene polymorphism is associated with AVB via linking a higher serum protein level.Matrix metalloproteinases (MMPs) and renin-angiotensin system (RAS) acting agents, including the angiotensin-converting enzyme (ACE) and angiotensinogen, have been reported to promote tissue fibrosis and the aging process24. The criteria for inclusion were symptomatic second and third degree AVB, which met the indications for permanent pacemaker implantation. Patients with a history of familial AVB, severe systemic disease, acute coronary syndrome, drug-induced AVB, or AVB with reversible cause were excluded from this study. The control subjects free of AVB were randomly selected from the cardiovascular outpatient department and ward, and they were frequency-matched to AVB patients by gender and age at the China Medical University Hospital in Taiwan. The protocol of present study was approved by the Institutional Review Board of China Medical University Hospital in Taiwan (CMUH108-REC3-031). Written informed consent was obtained from each participant after a full explanation for the present study. The investigation conformed to the principles outlined in the Declaration of Helsinki. Here, we presented the data obtained according to the standard guideline, Strengthening the Reporting of Observational Studies in Epidemiology (STROBE), to improve the quality of this study25.During the period from Sep 1, 2018 to Aug 31, 2019, a total of 102 patients with documented acquired AVB were enrolled as the patient group, and another 100 age and sex frequency-matched healthy individuals were enrolled as a control group . Polymerase chain reaction (PCR) was performed with 100 ng genomic DNA, 2–6 pmol of selected primers, 1X TAG polymerase buffer, and 0.25 units of AMPLITAG GOLD polymerase (THERMO FISHER) in a final reaction volume of 50 μL using a programmable thermal cycler . The polymorphisms for MMP genes selected for screening included MMP-1–320 T/C (rs494379), −340 T/C (rs514921), −422 T/A (rs475007), −519 A/G (rs1144393) and −1607 1 G/2 G (rs1799750), while the polymorphisms for ACE genes selected included ACE insertion/deletion (I/D) polymorphism.The genotype analysis of the subjects was studied by electrophoresis, polymerase chain reaction-fragment length polymorphism assay or direct gene sequencing if appropriate. Genotyping for the ACE I/D polymorphisms was studied by polymerase chain reaction-fragment length polymorphism assay. The primers for the ACE I/D gene polymorphisms were 5-TGGAGACCACTCCCATCCTTTCT-3 (forward) and 5-CAGGTCTTCATATTTCCGATGTGG-3 (reverse). Two fragments with different PCR product lengths were presented for the different genotypes (ACE I or ACE D genotypes). Electrophoresis was performed for genotyping. Thereafter, 10 µL of the products were loaded into a 3% agarose gel with ethidium bromide staining and separated by electrophoresis. Electrophoresis of the amplified products allowed to identify a 495-base pair fragment (insertion) and a 208-base pair fragment (deletion), respectively, on the gel for genotyping the ACE I/D polymorphisms. The gene polymorphisms were categorized as homozygotes , homozygotes , and heterozygotes  as shown in Fig. An association study between gene polymorphisms and AVB was performed to measure and compare the genotype distribution and allele frequency of the candidate genes in AV block and control groups.Serum samples were randomly obtained from 75 acquired AV block patients and 74 control individuals. The samples were stored at −20 °C until the assays were performed. The serum MMP levels were detected using the enzyme-linked immunosorbent assay kit (ELISA) . The optical density (OD) was measured by an ELISA microplate reader. The concentration of MMP was determined from a standard curve according to the manufacturer’s instructions.26. Owing to the short distances between each polymorphism location on the MMP1 gene, gene polymorphisms may not separate by recombination and were in linkage disequilibrium (LD)27. Therefore, pairwise measurement of LD was performed to test the LD between the MMP1 gene polymorphisms. LD was estimated by D’. We also selected the r2 value to confirm LD because the magnitude of D′ significantly depends on the sample size28. Expectation-maximization (EM)-based haplotype frequency estimation was performed to determine whether any specific haplotypes were associated with acquired AVB on the basis of previous reports29. The statistical significance of LD was defined as r2 > 1/3 and D′ > 0.7, as suggested by previous reports29. A P value < 0.05 was considered statistically significant.Student’s t test was used when continuous data were collected. Categorical data were compared by the conventional chi-square test if the observation numbers in all categories were larger than 5; otherwise, the Fisher exact test was used. The genotype proportions for Hardy–Weinberg equilibrium (HWE) were assessed for each gene polymorphism by using the conventional goodness-of-fit test. Haplotype analysis for the gene polymorphisms was estimated by using HAPLOVIEW softwareThe clinical features of AVB patients and controls are summarized in Table 2. The D’ values of the loci pairs for −320/−340, −320/−422, −320/−519, −320/−1607, −340/−422, −340/−519, −340/−1607, −422/−519, −422/−1607 and −519/−1607 were 0.129, 0.137, 0.536, 0.265, 0.054, 0.758, 0.116, 1.0, 0.215, and 0.743, respectively. The corresponding values for r2 were 0.004, 0.012, 0.036, 0.031, 0.0, 0.009, 0.004, 0.045, 0.013, 0.085, respectively  indicate an incomplete linkage among these 2 loci pairs, which explains the wide distribution of haplotypes for the MMP-1 gene.Five polymorphic sites at positions −320, −340, −422, −519 and −1607 were found within the promoter region of the MMP-1 gene. The HWE for genotype distributions was assessed for each MMP1 promoter polymorphism at the −320, −340, − 422, −519 and −1607 positions for the AVB and control groups by the conventional chi-square goodness-of-fit test. The MMP1 genotype distribution in the AVB patients and controls did not significantly deviate from the HWE for the AVB patients  and control groups . The pairwise linkage among these five polymorphic sites on the MMP1 promoter gene was evaluated by the LD test using D’ and rACE I/D gene polymorphisms were found for the ACE gene. The ACE I/D genotype distribution in AVB patients and the control group did not significantly deviate from HWE .Five MMP1 gene polymorphisms  and one ACE gene polymorphism (I/D) were identified in our study. In addition, an association study showed that the ACE I/D polymorphisms were not associated with acquired AVB. A significant difference was noted in the distribution of the genotypes of MMP1 −1607 1 G/2 G polymorphisms between AVB and control subjects (P = 0.027). The 2G-dominant genotype (2G2G/1 G2G) frequency of the −1607 1 G/2 G polymorphism was significantly higher in acquired AVB patients than that in controls . The 2G2G genotype frequency of the −1607 1 G/2 G2G polymorphism was significantly higher in the AVB group than that in the control group . The 2G allele frequency of −1607 1 G/2 G was significantly higher in the AVB group than that in the control group  . In addition, the 2G2G genotype showed a greater MMP-1 serum level than the 1G2G and 1G1G genotypes   genotype frequency of the MMP-1 −1607 1G/2 G gene polymorphism was also significantly higher in the AV block group than in the controls. In the haplotype analysis, we found 7 major haplotypes of the MMP1 promoter polymorphism in the studied population Table . The 2G-Consistently, the MMP1 serum protein level was significantly higher in the 2G2G genotypes than that in the other genotypes in the study population. The serum MMP1 protein levels were also higher in AVB patients than in the controls. These data suggested that MMP1 1 G/2 G gene polymorphism is associated with disease susceptibility for acquired AVB via linking a higher serum MMP1 protein level. Taken together, the results implicated that gene polymorphisms of MMP molecules are associated with the disease susceptibility of acquired AVB via linking the MMP1 serum protein levels.6. Tissue fibrosis is associated with MMP molecule expression, the renin-angiotensin system and TGF beta15. Studies using transgenic mice demonstrated that the overexpression of the angiotensin II type I (AT1) receptor in the myocardium is associated with AVB30. In a post MI rat model, downregulation of MMP has been demonstrated to protect disturbance in cardiac conduction31. A study of MMP deletion in a transgenic mouse model showed that MMP deletion decreased gap junction protein degradation and recovered cardiac conduction velocity in post-myocardial infarction mice17. Apparently, MMP plays an important role in the modification of cardiac conduction.AVB has been reported to be closely related to fibrosis over the conduction system33. Consistently, a close association was found between the −1607 1 G/2 G polymorphism and MMP1 serum levels34. The MMP1 − 1607 2 G allele, but not the 1 G allele, accounted for the increased levels of pre-mRNA and protein. In addition, variation in MMP1 haplotypes has been reported to affect serum MMP-1 levels35. A study of the secondary structures of pre-mRNA of the MMP1 −1607 1 G/2 G gene polymorphism has shown that the pre-mRNA secondary structure of the 2 G allele is more stable than the 1 G structure based on deduction of the minimum free energy34. The lower free energy and more stable pre-mRNA structure of the 2 G allele may cause the increased expression of the 2 G/2 G gene polymorphism with higher serum protein levels and therefore increased the risk of acquired AV block34.The 1 G/2 G polymorphism has been reported to affect an Ets binding site and increase MMP1 gene transcription38. Currently, most of meta-analyses of genetic association studies are usually conducted by comparing genotype frequencies between cases and controls under various genetic models. A study with meta-analysis of genetic association has been conducted to show the relationship between MMP gene polymorphisms and lung cancer by logistic regression analysis36. In addition, meta-analysis has also been used to investigate the relationship of steroid medication and outcome of congenital conduction disorder38. However, no meta-analysis of genetic association study has been reported for the relationship between acquired AVB and MMP gene polymorphisms. To our best knowledge, the present study is the first case-control genetic association study to demonstrate that there is a close association between MMP1 gene polymorphisms and acquired AVB and provide functional results to clarify the underlying mechanism of acquired AVB. Our results may provide biological insights for acquired AVB. Thus far no other case-control genetic association study is available for resolving the relationship between MMP1 gene polymorphisms and the acquired AVB. The future study with a well-designed meta-analysis is needed to reconfirm the role of MMP gene polymorphisms in the disease susceptibility to acquired AVB.A single case-control genetic study is of limited to reveal the true association between the genotypes and the disease susceptibility. Alternatively, meta-analysis is a useful statistical approach method to analyze the divergence among several independent studies by interrogating available data published. In addition, meta-analysis of genetic association study provide a better power to derive a more precise relationship between gene polymorphisms and disease susceptibilityIn the present study, we provided evidence obtained with the gene association study and with the functional measurement of serum MMP1 protein levels to support the crucial role of MMP1 gene polymorphism in the pathophysiology of acquired AVB. However, the direct impact of MMP1 gene polymorphism on cardiac cells located at the AV node still warrants further study. Another limitation is that the population of the present study is relatively small, and it needs to be further confirmed by large-scale studies.Patients with acquired AVB have a higher frequency of the 2 G allele and a higher distribution of 2 G2G genotype or 2G-dominant genotypes for the MMP1 1 G/2 G polymorphism. Consistently, the MMP1 serum protein level was significantly higher in the 2G2G genotypes than in the other genotypes in the study population. Taken together, these results suggest that the MMP1 1 G/2 G polymorphism is associated with the disease susceptibility of acquired AVB via linking higher serum MMP1 protein levels. Here, we provide the potential mechanism for the acquired AV block, which might be useful for disease prevention.