SARM) GTx‐024. The histone deacetylase inhibitor (HDACi) AR‐42 exhibited anti‐cachectic activity in this model. We explored combined SARM/AR‐42 therapy as an improved anti‐cachectic treatment paradigm. A reduced dose of AR‐42 provided limited anti‐cachectic benefits, but, in combination with GTx‐024, significantly improved body weight, hindlimb muscle mass, and grip strength versus controls. AR‐42 suppressed the IL‐6/GP130/STAT3 signaling axis in muscle without impacting circulating cytokines. GTx‐024‐mediated β‐catenin target gene regulation was apparent in cachectic mice only when combined with AR‐42. Our data suggest cachectic signaling in this model involves catabolic signaling insensitive to anabolic GTx‐024 therapy and a blockade of GTx‐024‐mediated anabolic signaling. AR‐42 mitigates catabolic gene activation and restores anabolic responsiveness to GTx‐024. Combining GTx‐024, a clinically established anabolic therapy, with AR‐42, a clinically evaluated HDACi, represents a promising approach to improve anabolic response in cachectic patients.No approved therapy exists for cancer‐associated cachexia. The colon‐26 mouse model of cancer cachexia mimics recent late‐stage clinical failures of anabolic anti‐cachexia therapy and was unresponsive to anabolic doses of diverse androgens, including the selective androgen receptor modulator ( Cancer cachexia increases morbidity and mortality of cancer patients. Given the multifactorial pathogenesis of cachexia, effective treatment remains elusive. Here, a novel therapeutic strategy combining anti‐catabolic and anabolic activities is established in a murine model of cancer cachexia. The progressive loss of lean mass associated with cachexia results in decreased quality of life, decreased tolerance of chemotherapy, and reduced overall survival  in C‐26 tumor‐bearing mice  and compared favorably to the less potent structural analog S‐23  and female mice (Study 3). Our follow‐up study in males resulted in significantly larger tumors and more aggressive wasting relative to Study 1, though no differences within treatment groups were apparent Fig A. As a r and femaet al, et al, et al, Male mice are generally considered more sensitive to C‐26 tumor burden so the majority of reported C‐26 studies utilize male animals . Tumor‐mediated deficits in grip strength were not as apparent in this study, but the only treatment resulting in significantly improved grip strength was the combination of TFM‐4AS‐1 and AR‐42, which increased muscle function over baseline (114.6 ± 16.6.1% of tumor‐free controls) despite the presence of C‐26 tumors.et al, et al, et al, et al, Candidate gene expression analyses were performed on gastrocnemius tissue from Study 1 to characterize the effects of C‐26 tumors and treatment with GTx‐024, AR‐42, or both agents on genes whose function has been previously associated with C‐26 tumor‐mediated wasting Fig A. As expet al, To determine the effect of tumor burden and treatment on AR levels in skeletal muscle, which could influence response to androgen therapy, gastrocnemius AR levels were characterized. Neither tumor nor treatment had a significant impact on AR mRNA Fig B. AR proet al, n = 548) for transcripts critical to the anti‐cachectic efficacy of AR‐42, these data were intersected with previously published differentially regulated genes from the quadriceps of moderate and severely wasted C‐26 tumor‐bearing mice .In agreement with the present pathway analyses, we previously reported that the higher 50 mg/kg dose of AR‐42 reduced serum IL‐6 levels, as well as gastrocnemius IL‐6 receptor mRNA abundance in tumor‐bearing mice, suggesting AR‐42's efficacy may be related to its suppression of systemic IL‐6 activation, which is thought to drive muscle wasting in the C‐26 model 3  target gene sets, and revealed multiple over‐represented TF targets in cachectic versus control muscle . Analysis of overlap of the leading edge genes revealed a large number of CTNNB1 target genes regulated by both GTx‐024 and cachexia versus tumor‐free controls but in different directions  unique to combination‐treated muscle and not solely attributable to AR‐42 treatment Fig A. We hypet al, Expression of the canonical skeletal muscle WNT agonist Wnt5a (WNT5A), canonical WNT receptor Fzd1 (FZD1), and β‐catenin itself (CTNNB1) was all reduced with C‐26 tumor burden, whereas the negative regulator of β‐catenin, GSK3B, was up‐regulated Fig E. In eacet al, et al, et al, Our initial objective was to determine cachectic signaling occurring in common mouse models of cancer wasting that limited the effectiveness of anabolic SARM therapy. Upon determining that catabolic E3‐ligase expression in skeletal muscle was sensitive to androgen administration in hypogonadism‐ and dexamethasone‐induced wasting . Driven by tolerability concerns from recent clinical experience, we evaluated a fivefold AR‐42 dose reduction and found that elements of anti‐cachectic efficacy were retained across multiple studies. This allowed us to focus on key aspects of AR‐42's pharmacologic activity that contribute to its anti‐cachectic effects. We report that STAT3 activation was sensitive to AR‐42 treatment in cachectic skeletal muscle  and a potent steroidal androgen (DHT), which resulted in no detectable efficacy. This occurred despite sufficient circulating levels of drug, demonstrated anabolic capability at the dose administered, and evidence of systemic hormonal activity Figs B. Our reet al, et al, et al, Androgens have a well‐characterized ability to normalize skeletal muscle catabolic gene expression associated with either glucocorticoid (dexamethasone)‐ or hypogonadism (castration)‐induced atrophy  afforded by the C‐26 model in our hands also severely curtailed our ability to demonstrate overt anabolism following GTx‐024 treatment relative to other anabolic agents in less severe models of cachexia ‐N‐(4‐cyano‐3‐(trifluoromethyl)phenyl)‐3‐(4‐cyanophenoxy)‐2‐hydroxy‐2‐methylpropanamide] was synthesized as previously described , Tween‐20 , benzyl alcohol , and sesame oil . AR‐42 was formulated in 0.5% methylcellulose [w/v] and 0.1% Tween‐80 [v/v] in sterile water. GTx‐024  and tested for mycoplasma by PCR using a commercial testing service . For injection into mice, cells were harvested by trypsinization, pelleted in FBS‐supplemented culture medium, and then resuspended in sterile PBS at a concentration of 5 × 106 cells/ml. The C‐26 and LLC cell lines were generously provided by the laboratory of Denis Guttridge (while at The Ohio State University) who had obtained the cells through a Material Transfer Agreement with the NCI  in 2001. For all experiments, cells were cryopreserved at 7–10 passages and then passaged no more than four times after thawing from the same stock to propagate for injection into mice. This cell line has been maintained in the laboratories of co‐authors Coss and Chen and has consistently produced cachexia, demonstrating that these cells have maintained their identity over time.Cultured murine C‐26 adenocarcinoma cells and LLC cells were maintained in fetal bovine serum (FBS)‐supplemented (10%) RPMI 1640 medium or DMEM, respectively , at 37°C in a humidified incubator with 5% COad libitum access to water and standard pelleted chow. Mice were briefly anesthetized (isoflurane) during administration of drugs  by oral gavage. In experiments in which food consumption was determined, the food in each cage was weighed as frequently as once daily to once weekly and the decrease in food weight was divided by the number of mice in the cage. Tumor volumes were calculated from caliper measurements using a standard formula (length × width2 × π/6). Mice were euthanized by CO2 inhalation. All animal studies were conducted according to protocols approved by The Ohio State University Institutional Animal Care and Use Committee.Male CD2F1 mice (6–7 weeks of age), female CD2F1 mice (10–11 weeks of age), and male C57BL/6NHsd mice (7–8 weeks of age)  were group‐housed under conditions of constant photoperiod (12‐h light/12‐h dark), temperature, and humidity with et al, 6 cells in 0.1 ml). AR‐42, GTx‐024, and their vehicles were administered orally by gavage. TFM‐4AS‐1 , DHT, and vehicles were administered by subcutaneous injection.These studies were performed as previously described  and 20 mg/kg (n = 6), and every other day at 50 mg/kg  for 13 days. Upon sacrifice on study day 18, when the majority of tumor‐bearing control mice met euthanasia criteria, the left gastrocnemius muscle was excised, flash‐frozen in liquid nitrogen, and stored at −80°C for subsequent analyses. Carcass weights were corrected for tumor weight by assuming a tumor density equivalent to water (1 g/cm3). Demonstration of anti‐cachectic efficacy with the 10 mg/kg dose of AR‐42 in this model prompted a follow‐up experiment assessing the anti‐cachectic efficacy of lower doses of AR‐42. This study was performed as described above with animals treated with AR‐42 once daily at 1, 3, and 10 mg/kg or with vehicle control (n = 8).Emergent pharmacokinetic and tolerability data from humans receiving AR‐42 (Sborov n = 5), GTx‐024 , AR‐42 , or the AR‐42 + GTx‐024 combination . The remaining tumor‐free groups received either vehicles (n = 6) or GTx‐024 . Body weight, tumor volume, and food consumption were monitored every other day. Upon sacrifice on day 18, sera were collected and hindlimb skeletal muscles, heart, spleen, and epididymal adipose tissues were harvested, weighed, flash‐frozen, and stored for subsequent analyses.Male CD2F1 mice were stratified by body weight and then randomly assigned into six groups of six animals each. Historically, six animals per group provided sufficient power to detect treatment‐mediated differences in tumor‐bearing treated animals compared to controls. Tumors were established in four of the groups, while the fifth and sixth groups served as tumor‐free controls. Six days later, animals with palpable tumors were treated twice daily for 13 days. AR‐42 and its vehicle were administered in the mornings, and GTx‐024 and its vehicle in the afternoons. Treatments included vehicles for AR‐42 and GTx‐024 (n = 7), GTx‐024 (n = 10), AR‐42 (n = 9), or the combination (n = 9). Grip strength was measured on study days 0 (baseline) and 16. Due to rapid model progression, this study was terminated on day 17 after only 12 days of treatment.This confirmatory study was performed exactly as Study 1 with expanded animal numbers. Tumor‐free control groups were maintained at 6 animals each, whereas 10 animals were included in each of the tumor‐bearing groups. Six days after cell injection, animals with palpable tumors were treated as in Study 1 with vehicles , 4, 11, and 18. Body weights were measured on days 0, 5, 11, 14, 16, and 18, and food consumption was monitored weekly. Similar to Study 1, sera, hindlimb skeletal muscles, spleen, and adipose were collected, weighed, and flash‐frozen at sacrifice 18 days after cell injection.n = 9), AR‐42 , TFM‐4AS‐1 , DHT , the combination of AR‐42 and TFM‐4AS‐1 , or the combination of AR‐42 (10 mg/kg) and DHT . Grip strength was measured, and tissues were collected as in the previous studies.Similar to Study 2, the tumor‐free control group was maintained at six animals, whereas 10 animals were included in each of the 6 tumor‐bearing groups. Six days after cell injection, animals with palpable tumors were treated once daily for 13 days with vehicles for AR‐42 and TFM‐4AS‐1/DHT . Similar to Study 3, the tumor‐free control group was maintained at six animals, whereas 12 animals were included in each of the tumor‐bearing groups. Six days after cell injection, animals were treated once daily with vehicle, AR‐42 (10 mg/kg), GTx‐024 (15 mg/kg), or the combination. Grip strength, body weight, and food consumption were measured, and tissues were collected as in the previous C‐26 studies. This study was repeated as described with a lower dose of GTx‐024 (0.5 mg/kg).This study was performed as previously described (Tseng n = 12) or vehicle (n = 13) by oral gavage once daily for 4 weeks. A sham‐castrated group was treated identically with vehicle (n = 14). Body weights were measured twice weekly. Forelimb grip strength was measured 1 day prior to first treatment (baseline) and at end of study. At terminal sacrifice, hindlimb muscles were removed and weighed.Eight‐week‐old, male, tumor‐free CD2F1 mice were surgically castrated under isoflurane anesthesia. Three weeks later, the mice were assigned to groups treated with GTx‐024 . Each mouse was held by the base of its tail and lowered over the apparatus until its forepaws grasped the metal pull bar. The mouse was then gently pulled horizontally in a straight line away from the grip strength meter until the mouse released the bar. The force applied to the bar at the moment of release was recorded as the peak force. Five measurements were taken from each mouse, the average of which was designated as the mouse's grip strength.et al, n = 3 per dose and time point) were administered single oral doses of 10, 20, and 50 mg/kg AR‐42 and then sacrificed 0.25, 4, and 24 h later. Fifty milligrams of gastrocnemius muscle tissue was flash‐frozen in 2 ml BeadBlaster™ 24  tubes and stored at −80°C. Samples were homogenized for 6 cycles using a BeadBlaster 24 with analytical standards in 1 mL methanol and then centrifuged at 15,600 g (4°C) for 10 min. Supernatants were transferred to glass tubes, dried under nitrogen, and then reconstituted in 200 μl 40% methanol/0.1% formic acid. Plasma preparation and LC‐MS/MS analyses were performed as previously described ].Pharmacokinetic studies were performed as previously described . Blood was collected by cardiac puncture into heparinized tubes  and immediately centrifuged at room temperature for 3 min at 4,200 RCF. Plasma was removed and placed on dry ice prior to storage at −80°C until analysis. Bioanalytical analyses were conducted as previously described . Substrate concentrations ranged from 20 to 400 μM, and incubation conditions ranged from 10 to 90 min (RT or 37°C), depending on isoform. Results are expressed as percent inhibition of control specific activity in the presence of 1 μM AR‐42.et al, Serum cytokine panel analyses were performed by a commercial vendor  as previously described  from whole blood samples collected from mice immediately post‐mortem, and then stored at −80°C until shipment on dry ice.Luteinizing hormone was measured in serum by a two‐site sandwich radioimmunoassay performed by the Ligand Assay and Analysis Core at the Center for Research in Reproduction, University of Virginia School of Medicine . Serum was isolated by centrifugation  mouse mAb #9139 ; α‐tubulin (B‐7), sc‐5286 ; androgen receptor (EP670Y), ab52615  and GAPDH (6C5), sc‐32233 .Western blots from all studies were performed on gastrocnemius muscle from representative animals lysed by Nonidet P‐40 isotonic lysis buffer . Equivalent amounts of protein from each sample, as determined by the Bradford assay (Bio‐Rad), were resolved by SDS–PAGE and then transferred (semi‐dry) onto Immobilon nitrocellulose membranes . Membranes were washed twice with TBST [Tris‐buffered saline (TBS) containing 0.1% Tween‐20], blocked with 5% non‐fat milk in TBST for 1 h, and then washed an additional three times. Membranes were incubated with specific primary antibody in TBST  at 4°C overnight, washed three times (TBST), and then incubated with appropriate goat anti‐rabbit or anti‐mouse IgG–horseradish peroxidase‐conjugated secondary antibodies  at room temperature (1 h). Following additional washes (TBST), immunoblots were visualized as appropriate by ECL chemiluminescence  or Duration HRP Chemiluminescence . Primary antibodies: phospho‐STAT3 (Tyr705) (D3A7) XP2−ΔCt values [ΔCt = ]. All values are normalized to the geometric mean tumor‐free control values. As transformed expression data are not normally distributed, statistical differences between treatment groups were determined by one‐way ANOVA followed by Dunnett's test on raw delta Ct values.To generate muscle tissue RNA, 15 mg of gastrocnemius muscle tissue was lysed in 10 volumes of lysis buffer per tissue mass in prefilled 2.0‐ml tubes with 3.0 mm zirconium beads . Tubes were loaded into BeadBlaster™ 24  and centrifuged for five cycles of 5 s with a 30‐s pause between cycles. Lysate was collected, and RNA was isolated using the mirVana™ miRNA Isolation Kit, with phenol . Samples were treated with DNA‐free DNA Removal Kit to eliminate any DNA contamination . Total RNA (0.5 μg) was reverse‐transcribed using high‐capacity cDNA reverse transcription kit  for 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C . Real‐time qPCR was performed on the QuantStudio 7 system  using the powerup SYBR Green Master Mix . Cycling was performed using the QuantStudio 7 real‐time PCR software—2 min at 50°C and 10 min at 95°C—followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. All real‐time qPCR assays were carried out using technical duplicates using β‐actin or GAPDH as the internal control genes. Reaction specificity was supported by the detection of a single amplified product in all reactions by a post‐cycling melt curve, the absence of non‐template control signal for 40 cycles, and confirmation of amplicon size using agarose electrophoresis. Primers for analyses are listed in et al  was determined and plotted using the Venn diagram web tool (http://bioinformatics.psb.ugent.be/webtools/Venn/) in the Bioinformatics and Evolutionary Genomics Suite . The lists of AR‐42‐regulated genes from Tseng et al, as well as genes regulated in common between Tseng et al and Bonetto et al, were tested for significant overlap with canonical pathway gene sets using the ‘Compute Overlaps’ function from the Molecular Signatures Database . Redundant results were collapsed to show only the gene set with the largest number of genes.Overlap between the gene sets in Tseng et al  . Samtools  was used to generate polyA‐enriched RNA‐seq libraries with the NEBNext Ultra II Directional RNA Library Prep Kit  and sequenced as PE‐150 reads on HiSeq 4000 . Raw fastq files were adaptor‐trimmed using Trimmomatic .Expression of phospho‐STAT3 and total STAT3 in C‐26 tumor tissues was measured using commercial sandwich ELISA kits according to the manufacturer's instructions . Briefly, 1,250 cells per well were plated on 96‐well plates and allowed to adhere overnight. Cells were treated with DHT or GTx‐024 (0.001–10 μM) for 24 or 48 h and then assayed according to the manufacturer's protocol.P‐values using either Tukey's or Dunnett's methodology  test, and multiple comparisons were performed using a Bonferroni‐corrected threshold. P < 0.016 were significant for multiple comparisons.Plotting and statistical analyses were performed using GraphPad Prism version 7 . The specific statistical tests employed are outlined in detail within the figure legends. Briefly, data were analyzed with a one‐way ANOVA, followed by multiplicity adjusted  Wright,  as notedConceived the study: CCC, C‐SC; Designed the experiments: CCC, C‐SC; Performed experiments and acquired data: SGL, Y‐CT, AMD, SKK, BCR, TV, SEH, Y‐CK; Data analysis: MGS, JAB, XZ, MC, MAP, CCC; Wrote, edited the manuscript: CCC, C‐SC, SGL, SKK, Y‐CT, TV, MAP, TB‐S.C.‐S. Chen is an inventor of AR‐42, which was licensed to Recursion Pharmaceuticals, Inc., for clinical development by The Ohio State University Research Foundation. C.C. Coss is a former employee of GTx, Inc., owner of GTx‐024, but has no financial relationship with or any equity in GTx, Inc. Recursion Pharmaceuticals, Inc. and GTx, Inc. were not involved in any way with the financing, design, or interpretation of the reported studies. All other authors declare that they have no conflict of interest.Cancer cachexia is a multifactorial syndrome characterized by involuntary muscle loss leading to decreased tolerance of chemotherapy, lowered quality of life, and reduced overall survival. Given the complex pathophysiology of this syndrome, there is an unmet need to develop multimodal intervention strategies in advanced cancer patients.The present study demonstrates: (i) consistent with clinical experience in NSCLC patients, anabolic therapy GTx‐024 is ineffective as a monotherapy in murine models of cancer wasting, (ii) combined treatment with AR‐42, a clinically evaluated HDAC inhibitor, and GTx‐024 demonstrated improved anti‐cachectic efficacy, (iii) the anti‐cachectic efficacy of AR‐42 is associated with limiting IL‐6 axis activation, (iv) GTx‐024‐mediated β‐catenin target gene regulation in skeletal muscle is abrogated in the presence of cachectic tumor burden, and (v) permissive anabolic WNT signaling in experimental cachexia is restored following combination treatment of AR‐42 and GTx‐024.Our data suggest anabolic signaling as a result of androgen administration cannot overcome the catabolic signaling associated with cachectic burden in the C‐26 murine model of cancer wasting. Furthermore, AR‐42 demonstrates anti‐cachectic efficacy through alleviating IL‐6 axis‐mediated catabolic signaling and restoring anabolic sensitivity in cachectic skeletal muscle. These data provide support for combined AR‐42/GTx‐024 administration as a potential therapeutic avenue to combat the complex mechanisms underlying wasting in advanced cancer patients.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 6Click here for additional data file.


Overactivation of androgen receptor (AR)-mediated signal has been extensively implicated in prostate cancer (CaP) development, progression, and recurrence, which makes it an attractive therapeutic target. Meanwhile, as an endogenous inhibitor of histone deacetylase 1 (HDAC 1), tumor-suppressive mammary serine protease inhibitor (maspin) was reported to sensitize drug-induced apoptosis with a better therapeutic outcome in CaP, but the relationship between AR and maspin remains unclear. In the current study, treatment of 5′-Aza or MS-275/enzalutamide induced poly (ADP-ribose) polymerase (PARP) cleavage and p-H2A.X in CaP cells with an increase of maspin expression but a decrease of AR. Then, treatment with protease inhibitor MG132 did not rescue the above drug-induced loss of AR. In addition, modulation of maspin expression by gene recombinant or siRNA technology showed an inverse correlation between expression of maspin and AR, consequently affecting the AR-regulated downstream gene transcription . Bioinformatics analysis of the data extracted from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database also revealed an inverse correlation between low maspin expression and high AR level in advanced CaP. Furthermore, chromatin immunoprecipitation (ChIP) assay using anti-maspin antibody identified that a portion of AR promoter sequence was co-precipitated and presented in the immunoprecipitated complex. Finally, maspin-mediated repression of AR was induced by treatment of MS-275, which promoted enzalutamide treatment efficacy with decrease of prostate-specific antigen (PSA) expression in LNCaP and 22RV1 cells. Taken together, the data not only demonstrated maspin-mediated repression of AR to augment drug anti-tumor activity but also provided in-depth support for combination of HDAC inhibitors with AR antagonist in CaP therapy. Mammary serine protease inhibitor (maspin) is encoded by the SERPIN B5 gene in humans and belongs to the serine protease inhibitor/non-inhibitor superfamily. As it does not undergo the S (stressed) to R (relaxed) conformational transition of active serpins, maspin protein exhibits no serine protease inhibitor activity . Maspin In fact, not only the loss of maspin expression was reported to be associated with epithelial malignant transformation and drug resistance in tumor but its allocation of expression in different cellular compartment was also documented with a discrepancy in cancer biology . Maspin,2 at 37°C and were tested for free-of-mycoplasma contamination periodically with LookOut Mycoplasma PCR Detection Kit from Sigma-Aldrich . DNA methyltransferase inhibitor 5′-Aza-1-(2-deoxy-β-D-ribofuranosyl)cytosine , and dihydrotestosterone (DHT) were also purchased from Sigma-Aldrich. AR antagonist enzalutamide (Enza), class I HDAC inhibitor entinostat (MS-275), and protease inhibitor MG132 were obtained from Selleck .Prostate cancer PC3 and LNCaP cells were purchased from the cell bank of the Chinese Academy of Sciences . Prostate cancer 22RV1 cell line was obtained from Nanjing Medical University . PC3 cells were maintained in F-12 medium  with 10% fetal bovine serum (FBS)  supplemented with 1 mmol/l L-glutamine  and 1% penicillin/streptomycin  according to the manufacturer’s description. LNCaP and 22RV1 were maintained in RPMI 1640 medium with 10% FBS and the same additives as above. All cells were cultured and maintained in a humidified incubator with 6% COE. coli strain DH5α cells and the positive clones for maspin expression were selected and used to amplify the plasmid DNA. The purified plasmid DNA were subsequently sequenced and verified. Then, maspin-encoding plasmid DNA was transfected into 22RV1 cells using Lipoteamine 2000  followed by western blotting assay for maspin expression. Then, the positive clones were named as M# clones. Control transfection was conducted with the empty vector plasmid DNA and the resulting control cell clones were designated as Neo clones . Individual plasmid DNA was extracted to transform host o clones .Both the recombinant EX-E2325-M02 plasmid with ectopic cytomegalovirus promoter (CMV)-driven AR gene (NM_000044) expression and GFP expression control EX-EGFP-M02 plasmid were obtained from GeneCopoeia . Then the individual EX-E2325-M02 plasmid DNA or EX-EGFP-M02 plasmid DNA was stably transfected into CaP PC3 cells respectively as described above. The AR overexpression positive clones were confirmed by western blot and designated as AR# clones. The GFP expression control cell clones were selected and named as Neo clone . The levFor maspin knockdown by siRNA, cells cultured in six-well plates were transfected with solution vehicle, a maspin-specific siRNA , or scramble oligo control(Scr-siRNA) at 15 mmol/l by using Lipoteamine 2000 . Cells wResults section.Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer  containing 1 mM phenylmethanesulfonyl fluoride (PMSF) and then protein concentration was measured by BCA kit . The protein in cell lysate was denatured by boiling in sample buffer and resolved by running SDS-PAGE. Then the protein in gel was transferred onto a polyvinylidene difluoride membrane  followed by blocking with 5% milk for 40 min at room temperature (RT). The membrane was incubated with primary antibodies with predetermined dilution overnight at 4°C. Then membrane was washed with Tris-buffered saline with 0.1% Tween-20 and incubated with HRP-conjugated secondary antibodies  for 1 h at RT. The expression of protein was detected by enhanced chemiluminescence (ECL) system and autoradiography using Tanon 5500  . The ant5) were seeded onto six-well plate and treated with enzalutamide (5 μM) and/or MS-275 (1 μM) for 48 h. Then, the cells were harvested and suspended in cold PBS. The cells were then stained with the annexin V-fluorescein isothiocyanate (FITC) and prodidium iodine (PI) kit  according to the manufacturer’s instructions followed by flow cytometer sorting . The apoptotic cells with annexin V-FITC staining were calculated as apoptotic index (%).CaP cells . The cDNA was generated by utilizing High-Capacity cDNA Reverse Transcription Kit  according to the manufacturers’ instruction. Quantitative real-time RT-PCR was performed using POWER SYBR Green Master Mix (Applied Biosystems/Thermo Fisher Scientific) . The fol® Enzymatic Chromatin IP (Magnetic Beads) Kit  according to the manufacturer’s instructions (The chromatin immunoprecipitation (ChIP) assay was performed as described previously using SimpleChIPructions . In brie1t test. The p values equal to or below 0.001 were considered significant.The microarray data of maspin and AR gene expression profile from patients with CaP specimens was downloaded from Gene Expression Omnibus (GEO) databaseIn general, PARP cleavage and p-H2A.X expression were the classic biomarkers for apoptotic cell death and cellular DNA damage. To investigate its cytotoxicity and therapeutic effect on CaP, demethylation agent 5′-Aza was applied to treat LNCaP and 22RV1 cells at the indicated dose, respectively, and PARP cleavage and p-H2A.X were tested by western blot (WB) analysis. The results showed that treatment with 5′-Aza induced both the PARP cleavage with an 89-kd fragment expression and p-H2A.X level in a dose-dependent manner . Meanwhip < 0.001, It was well documented that the AR antagonist enzalutamide or HDAC inhibitor MS-275 possessed anti-tumor activity. To investigate the synergistic effect of AR antagonist enzalutamide and MS-275 on apoptosis induction, LNCaP or 22RV1 cells were treated with enzalutamide (5 μM) and/or MS-275 (1 μM) followed by WB analysis. The results showed that treatment with MS-275, but not enzalutamide, induced PARP cleavage and p-H2A.X. Meanwhile, the treatment also increased maspin expression but inversely decreased AR expression . ImportaTo further evaluate the role of maspin in sensitizing drug-induced apoptosis in CaP, the maspin-expressed 22RV1-M7, -M11 cells, and the control Neo cells were treated with enzalutamide (5 μM) and/or MS-275 (1 μM) followed by WB analysis. The results showed that, in addition to showing the similar pattern of inverse expression of maspin with AR, M7 and M11 cells showed higher level of both PARP cleavage and p-H2A.X compared with Neo cells while treated with enzalutamide and/or MS-275. Taken together, the data suggested that the above drug-induced CaP cell apoptosis concomitantly increased maspin-associated downregulation of AR.To exclude the protein instability of AR induced by above drug treatment, LNCaP cells were treated with MS-275 (1 μM) or 5′-Aza with or without the presence of protease inhibitor MG132 followed by WB assay for AR and maspin levels. The results showed that MS-275 indeed induced maspin expression along with the decrease of AR in a dose-dependent manner, and MG132 did not rescue the loss of AR in both LNCaP and 22RV1 cells . NoticeaTo study whether the expression of maspin in CaP affects AR level, SERPIN 5 gene was transfected into 22RV1 cells through recombinant gene expression technique, and a few positive cell clones were selected and analyzed by WB assay for maspin and AR expression. The results showed that multiple M cell clones with variant levels of maspin expression concomitantly reduced AR level compared with Neo control .A study of the National Center for Biotechnology Information (NCBI) GEO data generated from clinical patient tissue specimens of normal prostate, benign prostate, and primary and metastatic CaP also found that there was a differential expression between maspin and AR. Also, downregulation of maspin expression in CaP, which is more significant in metastatic tumors of GDS1439 and GDS2545 groups than in primary and benign or normal tissues, was correlated with upregulation of AR mRNA level  rather than other AR primers was observed in the anti-maspin antibody-immunoprecipitated complex derived only from LNCaP cell lysate, but not from PC3-AR28 cell lysate. The negative control and positive control were also verified in both cells when AR promoter fragment was amplified using the primer 7 . These dTaken together, the evidence from both ChIP assay and GEO demonstrated that maspin indeed repressed AR expression at the transcription level.To investigate the synergistic effect of MS-275 with AR antagonist enzalutamide on the treatment of CaP cells, LNCaP cells were treated with enzalutamide (5 μM), MS-275 (1 μM), and indicated dose of MG132 for 48 h followed by WB analysis. The results showed that the expression of maspin, AR, and β-catenin had no change after treatment with enzalutamide and/or MG132. Treatment with MS-275 alone induced maspin expression along with downregulation of AR expression, which was rescued partially by addition of MG132. This MG132-mediated partial rescue of AR was diminished by the addition of enzalutamide . NoticeaWhen LNCaP or 22RV1 cells were treated with an agonist of the AR DHT alone or in the presence of enzalutamide and/or MS-275 for 24 h followed by qRT-PCR analysis for prostate-specific antigen (PSA) expression, the results showed that treatment with enzalutamide or MS-275 reduced PSA transcription in LNCaP but not in 22RV1 cells; however, the combination treatment with enzalutamide and MS-275 did reduce PSA significantly in 22RV1 cells but has no synergistic effect on LNCaP cells. Interestingly, both MS-275 and enzalutamide alone significantly inhibited DHT-induced PSA expression. The combination treatment with MS-275 and enzalutamide showed additive or synergistic effect on reduction of DHT-induced PSA expression in both LNCaP and 22RV1 cells . These dThe human AR gene is located on the X chromosome at Xq11-12 , and itsTumor suppressive maspin was reported to sensitize tumor cells to drug-induced apoptosis . In the It was also well known that abnormal and persistent activations of AR-mediated signaling pathway were the major mechanism of CaP development and castration-resistant prostate cancer (CRPC) formation . Thus, aHDAC 1 was reported previously to be involved in the regulation of AR gene transcription, and the outcomes of its mediated AR activity was reported controversially . GaughanThe drugs enzalutamide, MS-275, 5′-Aza, and MG132 showed their potent anti-tumor activity of inducing DNA damage and promoting apoptosis, but translational implementation of these agents into clinical practice actually faced variant challenges. For example, enzalutamide was commonly used to treat CaP but is only effective for a certain period of time of disease followed by drug-induced resistance. Thus, looking for a long-term and effective treatment strategy for CaP is desirable. In this study, the class I HDAC inhibitor MS-275 was found to not only promote the therapeutic effect of enzalutamide in the drug-sensitive LNCaP cell , we propAs one of CaP master regulators, AR expression and its activity dominate the disease development and progression. Currently, androgen deprivation therapy (ADT) remains the foundation but not the cure of treatment for CaP. However, after initial regression, the disease comes back in a more progressive form of CRPC with molecular alterations of AR. The second generation of AR pathway inhibitors, such as enzalutamide and abiraterone, were commonly used for high-risk locally or systemically advanced CaP . Thus, tTaken together, these data not only demonstrated maspin-mediated repression of AR expression to augment epi-drug’s anti-tumor activity but also provided in-depth support for combination treatment strategy with AR antagonist and HDAC inhibitors in CaP therapy.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ ST and XuL generated most of the data. JJ generated data from bioinformatics analysis. HC, JG, and CH helped to generate the western blot and RT-PCR data. HM helped to design the experiment and discussed the data. XiL designed the project, directed the research, evaluated the data, and finalized the manuscript. All authors participated in data discussion, 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.


The prostate cancer (PCa) has been a global problem to men health. Notably, the androgen receptor (AR) is essential for both normal development of prostate and prostate cancer progression.The RNA sequencing was used to identify the novel long non‐coding RNA (lncRNA) termed PCAL7. The RT‐qPCR was performed to quantify PCAL7 expression. Migration and proliferation assays were used to examine the function of PCAL7. Fluorescence in situ hybridization (FISH) was used to determine subcellular localization.By RNA sequencing, the differentially expressed lncRNAs were identified . PCAL7 was the lncRNA with the highest fold upregulation and significantly correlated with AR signaling during prostate cancer progression. The AR‐regulated PCAL7 was abundantly overexpressed in prostate cancer tissues and AR‐dependent cell lines. PCAL7 knockdown inhibited cell migration and proliferation. Consistently, the migration and proliferation were promoted by PCAL7 overexpression. PCAL7 depletion via antisense oligonucleotides (ASOs) markedly suppressed AR signaling and tumor growth. Mechanistically, PCAL7 interacted with Huntingtin‐interacting protein 1 (HIP1) to stabilize HIP1. Therefore, PCAL7 could advance AR signaling via a novel positive feedback loop.Our experiments support an oncogenic role for PCAL7 which promotes prostate cancer progression suggesting PCAL7 may serve as a potential therapeutic target. The novel lncRNA PCAL7 can promote prostate cancer progression via stabilizing HIP1. The androgen receptor (AR) can induce PCAL7 expression. Since HIP1 decreases androgen receptor degradation, the AR‐PCAL7‐HIP1 axis creates a novel positive feedback loop to advance prostate cancer development. We found that PCAL7 expression is increased in prostate cancer tissues and primarily distributed in cytoplasm. PCAL7 overexpression promoted migration and proliferation of prostate cancer cells. AR directly transcriptionally induces PCAL7 expression. PCAL7 can interact with HIP1 via RNA immunoprecipitation followed by mass spectrometry (MS). The PCAL7‐HIP1 association stabilizes HIP1 proteins to augment AR signaling. We designed several antisense oligonucleotides (ASOs) to reduce PCAL7 expression and ASOs targeting PCAL7 strongly impairs in vivo tumor growth. Taken together, we identified a novel AR‐dependent lncRNA PCAL7 and PCAL7 may serve as a potentially therapeutic target against prostate cancer development.By lncRNA sequencing, we explored a novel lncRNA related to prostate cancer progression named as 22.1All cell lines were obtained from Shanghai Institute of Cell Biology and maintained in DMEM (Sigma) with 8% fetal bovine serum (FBS) and 100 μg/mL streptomycin. All cells were mycoplasma negative. Cells in androgen stimulation experiments were first staved with charcoal‐deprived serum for 36 hours and stimulated with 10 nmol/L dihydrotestosterone (DHT) as indicated.2.2The human prostate cancer and normal adjacent tissue samples were collected from January 2017 to June 2019 by prostatectomy (N = 104). Samples were stored at −80°C immediately after resection before use. Written informed consent forms were obtained from all patients for academic use. Experiments related to human samples were formally approved by Institutional Review Board at the Seventh Medical Center of PLA General Hospital and conform to Declaration of Helsinki.2.3Extracted RNAs were obtained using the TRIzol kit (Invitrogen). The PrimeScript RT Reagent Kit with gDNA Eraser (Takara) was utilized for synthesizing the first strand cDNA. All primers used in current work were designed and purchased from Takara Table . ABI 3002.4P < .05 was considered significant.Statistical analyses were performed using SPSS . Data were represented by mean ± SD. Statistical significance was determined by Mann‐Whitney (two groups) or Kruskal‐Wallis rank sum test (multiple groups) as specified. 33.1prostate cancer‐associated lncRNA on chromosome 7 (PCAL7). PCAL7 was located on chromosome 7:143,379,692‐143,380,495 reverse strand with one annotated transcript (www.ensembl.org). The transcript (ENST00000429630.1) was 468 bp in length. Evaluating the coding potential suggested that it had low coding probability . PCAL7 was also significantly elevated in prostate cancer compared with normal adjacent tissues  on AR‐dependent VCaP and LNCaP cells. VCaP and LNCaP cells were stimulated with AR ligand (10 nmol/L DHT) for 12 hours Figure . 5208 lns Figure . Indeed,s Figure . PCAL7 ws Figure . We idens Figure . PCAL7 fs Figure , top. Sus Figure , bottom.3.2AR oligos . Results showed that the N‐terminal arm may contribute to PCAL7 and HIP1 binding . Instead, PCAL7 levels positively regulated HIP1 protein abundance . As expected, ectopic PCAL7 overexpression significantly facilitated LNCaP and VCaP cell migration . PCAL7 knockdown also substantially decreased LNCaP and VCaP cell proliferation, whereas PCAL7 overexpression advanced cell proliferation  to target PCAL7 and deplete intrinsic PCAL7 expression.g Figure . Mice hag Figure . Importag Figure A,B. Thes4Even castration resistance prostate cancer remains dependency on AR signaling; therefore, it is highly important to identify potential targets for therapeutic interventions in prostate cancer. Enzalutamide or abiraterone have shown its effectiveness for prostate cancer patients,By comprehensive profiling, we identified a novel AR‐regulated lncRNA PCAL7. PCAL7 stabilizes HIP1 protein via PCAL7‐HIP1 interaction. Since HIP1 has a defined positive role to activate AR signaling pathway,Rx has been used to regulate apolipoprotein expression during calcific aortic valve stenosis (CAVS) treatment.We found that PCAL7 interacts with HIP1, which suggests that PCAL7 might be an upstream regulator for AR signaling pathway. Therefore, PCAL7 regulation could provide an alternative regime in addition to direct AR targeting. Meanwhile, manipulation of PCAL7 expression might be required in specific patients with resistance to AR‐targeted therapies. Conventional RNA interference has many drawbacks such as inefficient cellular uptake and decreased stability.Furthermore, PCAL7 can interact with HIP1 to stabilize HIP1 proteins. Notably, RNAs have shown to interact with target mRNAs through RNA‐RNA interaction.Previous work has demonstrated that HIP1 protein is abundantly overexpressed in prostate cancer.App S1Click here for additional data file.


These findings lay a framework for the further development of peptides to target 14-3-3ε-CDC25A interactions for skin cancer treatment.Non-melanoma skin cancer is the most common form of cancer worldwide. We previously documented an anti-apoptotic role for CDC25A in cutaneous squamous cell carcinoma (SCC), an activity dependent on its association with 14-3-3 proteins. We hypothesized that targeting CDC25A-14-3-3ε interactions may be an effective strategy for inducing skin cancer cell apoptosis. Co-immunoprecipitation revealed that CDC25A associated with 14-3-3ε, 14-3-3γ and 14-3-3ζ in SCC cells but not normal keratinocytes. 14-3-3ε and CDC25A activated Akt/BAD/Survivin pro-survival signaling. To target the interaction of 14-3-3ε with CDC25A for cancer therapy, we developed two novel phospho-peptides, pS and pT, corresponding to each of the 14-3-3 binding sites of CDC25A, to specifically interfere with 14-3-3ε binding to CDC25A. Peptides pT (IC Skin cancer is the most common malignancy in humans, with squamous cell carcinoma (SCC) representing 20% of cases . SCCs acWe previously documented how the phosphatase CDC25A is overexpressed and mislocalized to the cytoplasm in SCC where it inhibits apoptosis . 14-3-3 14-3-3ε, a member of the 14-3-3 family, has been implicated as a tumor promoter in various cancers where it can increase proliferation, inhibit cell death or promote invasion –22. In cHere, we investigate CDC25A binding to 14-3-3 isoforms, revealing its interaction with 14-3-3ε, -γ and -ζ. CDC25A and 14-3-3ε similarly activated a Akt/BAD/Survivin pro-survival signaling pathway in SCC cells and suppressed cell death. From these data and our published work we hypothesized that interfering with 14-3-3ε binding to CDC25A would be a useful strategy for killing skin cancer cells. We characterized two phospho-peptide fragments of CDC25A phospho-Ser178 (pS) and phospho-Thr507 (pT), which are part of the region of CDC25A’s 14-3-3ε binding sites, in order to disrupt 14-3-3ε-CDC25A binding. Both peptides reduced 14-3-3ε and CDC25A interactions in SCC cells and also increased apoptosis. Xenograft tumors treated with either pS or pT displayed increased apoptosis, and reduced P-Akt (S473) and Survivin compared to vehicle-treated tumors. These data present a strategy for targeting the 14-3-3ε-CDC25A interaction in SCC cells to induce skin cancer cell death.In order to develop a strategy for targeting CDC25A binding to 14-3-3ε, we first needed to determine which 14-3-3 isoforms are expressed in skin cancer and also associate with CDC25A in skin cancer cells. Immunoblotting for 14-3-3 isoforms revealed increased levels of 14-3-3ε, -ζ, -γ, -θ and -β in SCC cells compared to normal keratinocytes . Associa14-3-3ε was silenced in SCC12B.2 cells, revealing significantly increased apoptosis in a Caspase activity assay , inhibitory phosphorylation of BAD (S136) and total Survivin (14-3-3ε decreased P-Akt (S473), P-BAD (S136) and Survivin levels (We previously documented an anti-apoptotic role for CDC25A in SCC that is dependent on its ability to interaction with 14-3-3 [ty assay . An antiSurvivin . Conversn levels . Inhibitn levels . From thThe -Arg-Gln-Asn-pSer-Ala-Pro- sequence around pSer178 of CDC25A corresponds to the binding motif 1 (-RSXpSXP-) of 14-3-3 client proteins  proposed50) of each peptide using a neutral red cell viability assay [50 values of 29 μM and 22.6 μM, respectively (On the basis of MD simulation results we hypothesized that blocking interactions between CDC25A and 14-3-3ε using synthetic phospho-peptide fragments (pS and pT) from the putative binding sites (Ser178 and Thr507) would decrease SCC cell survival. Therefore, SCC12B.2 cells were treated with increasing concentrations of either pS or pT for 96 h to determine the half maximal inhibitory concentration -induced DNA damage 14-3-3γ delivers CDC25A to Chk1 leading to phosphorylation of CDC25A and subsequent ubiquitin mediated degradation [We showed that CDC25A interacts with 14-3-3ε, -γ and -ζ in human SCC cells, but does not show a detectable association with any 14-3-3 isoform in normal skin keratinocytes. CDC25A has previously been shown to interact with 14-3-3ε, β and γ where they can sequester CDC25A into the cytoplasm , 31–33. radation . We showradation , 31, 33.CDC25A expression show decreased levels of 14-3-3ε protein and 14-3-3ε transcripts compared to wild type skin and tumors (not shown). The difference observed between HeLa and keratinocyte cells may be linked to differing localization patterns of CDC25A and 14-3-3ε in the cell lines. Modes of CDC25A and 14-3-3ε transcriptional regulation may also differ between cell types. Understanding how CDC25A can regulate 14-3-3ε expression, whether it can affect the expression of other 14-3-3 isoforms, and how this affects the skin’s response to UV-induced DNA damage and tumorigenesis may provide more insight into the role of CDC25A and 14-3-3ε in skin cancer development.In HeLa cells 14-3-3ε can stabilize CDC25A, while at the same time blocking its ability to activate cyclin-dependent kinase 1 (CDK1) to induce cell cycle progression . Data frWe have shown that in cutaneous SCC, 14-3-3 associates with CDC25A in the cytoplasm, a location that prevented CDC25A from promoting cell proliferation in the nucleus, but allowed it to inhibit apoptosis in the cytoplasm , 30. Thein vitro and in vivo. In all, we have demonstrated the potential of blocking CDC25A binding to 14-3-3ε in targeting skin cancer cells.In conclusion, we have demonstrated that CDC25A associated with 14-3-3ε, 14-3-3γ and 14-3-3ζ in SCC cells. CDC25A and 14-3-3ε inhibited apoptosis by promoting the activation of Akt, inhibition of BAD and an increase in Survivin, the same pathway that we have shown to be regulated by 14-3-3ε. Also, to the best of our knowledge, this is the first time that P-CDC25A (Ser178) and P-CDC25A (Ser507) peptide fragments have been shown to directly interact with 14-3-3ε in a cellular environment. Treatment of SCC cells with the peptide fragments reduced the interaction of 14-3-3ε with CDC25A, decreased SCC cell viability and increased apoptosis both 2. Primary normal human epidermal keratinocytes (nHEK)  isolated from neonatal foreskin were maintained in EpiLife medium (Invitrogen) supplemented with human keratinocyte growth supplement (HKGS) and 1% PenStrep. nHEK cells were never passaged more than twice before experiments. Cells were treated with CDC25A inhibitor NSC663284  suspended in DMSO, transfected with wild type CDC25A [Previously characterized human carcinoma cell lines (SCC12B.2 and SCC13) , 35 wereThe pS and pT peptides consisting of the 173-186 and 501-515 fragments of human CDC25A, respectively, were generated by the YASARA program . Peptide-1. The system was then equilibrated during 10 ns unrestrained constant number of molecules, pressure and temperature (NPT) simulation at 1 bar pressure and 300 K temperature. The integration step was 2 fs, the LINCS algorithm [−5 bar-1 isothermal compressibility [T of 2 ps and the system was coupled to 1 bar Parrinello-Rahman barostat [The structures of the 14-3-3ε-peptide complexes were solvated in a dodecahedron with TIP3P water molecules  and 0.15lgorithm  was usedsibility . The temsibility . The lonbarostat .covar module of GROMACS, the eigenvectors corresponding to the 150 highest eigenvalues were used to calculate the backbone configurational entropy [Configurational entropy was calculated to determine if thermodynamic equilibrium was reached. The covariance matrix for the Cα-atoms was calculated using the  entropy . Stabili entropy  with Cα-Peptides were synthesized and purified to greater than 95% purity by Bachem  and EZBiolabs .Immunostaining was performed using antibodies recognizing P-Akt (S473) (Thermo Fisher) and Survivin , with alexa-fluor488-conjugated secondary antibody (IgG rabbit) (Invitrogen) with 4′,6-diamidino-2-phenylindole (DAPI)  to identify the nuclei. Slides were digitalized and analyzed using an Olympus VS120 Slide Scanner .Immunohistochemistry was performed using antibodies to detect 14-3-3ε, β, η, γ, σ, θ or ζ , with biotinylated secondary antibody (IgG rabbit) . HRP-streptavidin  and 3,3 diaminobenzidine (DAB) substrate kit  were used for signal development.50 concentration for 48 h in Opti-MEM I Reduced Serum Medium (Invitrogen) and collected by trypsinization. One hundred mg of cells were lysed in 1× IP buffer (provided by supplier) supplemented with 100 mM sodium chloride, 2 mM dithiothreitol, 1 mM magnesium chloride and protease/phosphatase inhibitors. Immunoprecipitation reactions were carried out using 1.5 mg of antibody-coupled beads incubated with 100 mg of cell lysate at 4°C for 30 minutes. Negative controls were immunoprecipitation reactions using beads coupled to IgG isotype control antibody (rabbit), and whole lysate from immunoprecipitation input loaded directly onto a gel was used as a positive control.Co-Immunoprecipitation (Co-IP) was performed using the Dynabeads Protein G Immunoprecipitation kit . Beads were coupled to antibodies targeting 14-3-3ε  or CDC25A  at a ratio of 7 μg of antibody for every 1 mg of beads according to the manufacturers protocol. SCC12B.2 cells were treated with vehicle  or fresh peptide daily at the IC4, 150 mM NaCl, 1% triton X) supplemented with protease (Roche Life Scientific) and phosphatase inhibitors  or by nuclear/cytosolic extraction using the NE-PER Nuclear and Cytoplasmic Protein Extraction Reagent Kit according to the manufacturer’s protocol . Tissue protein was extracted by using a tissue grinder to lyse cells in 100 μL of RIPA lysis buffer for every 1 mg of tissue. Samples were centrifuged for 5 minutes to remove depleted tissue debris. Protein concentration was assessed with the Bradford assay. Protein lysates (30 μg/lane) were resolved on a 10% SDS-PAGE denaturing gel, transferred to a nitrocellulose membrane and blocked in 5% non-fat dry milk in 1× Tris buffered saline supplemented with 0.01% Tween 20. Immunoblotting was performed by incubating membranes overnight at 4°C using antibodies to detect 14-3-3ε, β, η, γ, σ, θ or ζ , CDC25A (Santa Cruz Biotechnology), P-Akt (S473) , total Akt , P-Bad (S136) , total BAD  Survivin  or GAPDH . Bands were detected using horseradish peroxidase-conjugated secondary antibodies (IgG mouse or rabbit)  and visualized using western lightning plus chemiluminescent reagent . The signal was read, and densitometry was performed using a Chemidoc XRS Molecular Imager and accompanying software .Protein was collected by either standard whole-cell lysis using ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (10 mM NaPOProtein was extracted from SCC12B.2 cells using lysis buffer supplemented with protease and phosphatase inhibitors. SCC12B.2 cells were transfected with empty vector DNA or wild-type CDC25A  for 48 hNOD CRISPR Prkdc Il2r gamma/NjuCrl)  were injected s. c. with 5 × 105 human SCC13 cells suspended in 1:1 mixture of 1× PBS (pH = 7.5): Matrigel . Once the tumors reached 150 mm3 in volume they were treated intratumorally with either vehicle  (N = 3), 2.5 nmol/50 μL of pS (500 nM) or 2.5 nmol/50 μL pT (500 nM) (N = 3 for pS and pT) daily for 2 days and then the mice were euthanized 24 h after the second treatment. Tumors were fixed overnight in 10% neutral buffered formalin (Thermo Fisher) and then switched to 70% ethanol the following day before embedding and sectioning. All procedures were performed in accordance with the guidance of the Creighton University Institutional Animal Care and Use Committee.Groups of male NCG mice  assays were carried out using the Promega DeadEnd Fluorometric TUNEL system in accordance with the manufacturer’s instructions . Cells were treated with DAPI to locate the nuclei within cells. Slides were scanned and analyzed using an Olympus VS120 Slide Scanner (Olympus).3 cells/well and Caspase-3/7 glo was performed 24 h post-treatment according to the manufacturer’s instructions and read using a Cytation 5 multi-mode plate reader .Apoptosis was assessed using a Caspase-3/7 Glo assay (Promega) in a 96-well white-bottom plate . Cells were seeded at a density of 8 × 1050) for pS and pT in SCC12B.2 cells, cells were plated in a 96-well plate at a density of 6 × 103 cells/well. Once cells reached 50–60% confluency they were treated for 4 consecutive days with increasing concentrations of pS and pT peptides  diluted in Opti MEM media (no additions) (Invitrogen). The peptides were resuspended fresh daily in 20 mM Tris buffer . The Neutral Red Cell Viability assay was previously described in [50 value using GraphPad Prism Software .To determine the half maximal inhibitory concentration (ICribed in . Absorbapost-hoc test for comparison of more than two groups, where P ≤ 0.05. Comparisons between two groups were performed using a two-tailed Student’s t-test, where P ≤ 0.05.Significance was determined using one-way ANOVA with the Dunnett’s


The aim of the present work is to evaluate the effect of NaOH solution as a stress cracking agent on the thermal and tensile properties of PET and PET/ZnO composites. The solutions were applied during tensile testing and the effects were monitored by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and testing the actual mechanical properties. The rate of crystallization was increased when the samples were exposed to NaOH, as observed by both cold and melt crystallization; this is possibly due to the reduction in molar mass of the PET molecules caused by NaOH. During melting, the DSC peaks became more complex, which is probably due to the distinct macromolecular mass, as well as crystallites with different sizes and levels of perfection. According to TGA analyses, no drastic changes were observed on the thermal stability of PET due to the action of NaOH. The tensile properties were shown to decrease drastically upon exposure to NaOH, which is the main symptom of stress cracking, leading to increased fragility, as also observed in the scanning electron microscopy (SEM) images. The presence of ZnO improved PET crystallization and provided some protection against the harmful effects of NaOH. Poly  (PET) is a relatively low cost thermoplastic with excellent physical and mechanical properties, good processability and reasonable thermal stability, and ideal characteristics for packaging food products such as soft drinks, water and juices. PET is also widely used for durable articles in the electronics and automobile industries ,2,3. DesESC is generally used to describe the product cracking phenomenon due to contact with aggressive fluids. This is not yet fully understood, but it happens when there is the simultaneous action of a chemical agent (liquid or vapor) and mechanical stress , leading to cracking. It has been one of the most common causes of premature failure in plastic products, accounting for approximately 25% of reported cases ,6. Both In its various applications, PET may be subject to the simultaneous action of aggressive fluids and mechanical stresses and, hence, ESC investigation is fundamentally important. Studies on the stress cracking of PET were done with several types of fluids, including isopropanol, methanol, ethanol ,10, and The properties of polymers can also be changed when inorganic fillers are added, which has been the focus of a number of publications, due to the diversified properties achieved in the final product. Among these additives, nanofillers may improve thermal stability, flame retardancy, the mechanical properties, vapor permeability, chemical resistance, and antimicrobial action . As an oBased on the aforementioned observations, the present work investigates the ESC phenomenon in PET and PET/ZnO composites in contact with NaOH solutions in order to better understand their tensile and thermal behavior.2/g. Raw materials were used as received without any further purification.Poly  CLEARTUF MAX, with an intrinsic viscosity of 0.84 dL/g, was supplied by M&G Chemicals as pellets. The active fluid was sodium hydroxide (NaOH), purchased from Labsynth Produtos para Laboratórios LTDA. The nanofiller was zinc oxide (ZnO), purchased from Acros Organics with purity 99.5% and a specific area of 28 mPET and ZnO were dried at 120 °C for 24 h prior to the mixing procedure. Firstly, a PET/ZnO masterbatch was prepared at a 7:1 ration using a Haake torque rheometer, operating with roller rotors at 260 °C, 60 rpm, for 10 min. The output was milled, and then compounded with PET in a Coperion Werner-Pfleiderer  model ZSK (D = 18 mm and L/D = 40) modular corotational twin screw extruder with a temperature profile between of 240–260 °C, a screw rate of 250 rpm, and a feed rate of 2.5 kg/h. The screw profile was configured with distributive and dispersive mixing elements, as illustrated in Tensile test bars were injection molded in an Arburg Injector, Model Allrounder 207C Golden Edition , according to ASTM D638 . The injThe injected specimens presented some whitening regions due to crystallization that developed during cooling. Aiming to avoid testing specimens with uncontrolled microstructures which could lead to unreliable results, all specimens were heated at 120 °C for 4 h to promote cold crystallization. α radiation, 40 KV voltage, 30 mA current, and a scanning rate of 2/min with 2θ ranging from 5 to 80°. X-ray diffraction (XRD) analysis was conducted with injection molded samples in a SHIMADZU XRD-7000  using copper KDifferential Scanning Calorimetry (DSC) analyses were performed in a DSC-Q20 from TA Instruments  using approximately 5 mg in sealed aluminum pans. Specimens were heated/cooled/reheated from 30 to 300 °C with a heating rate of 10°/min in nitrogen atmosphere with a gas flow rate of 50 mL/min.J0 is the virtual base line during the event (straight line in the present case) and E0 is the total latent heat of the phase change:t1 and t2 are the initial and final times. The rate of the phase change  c is:T), knowing the linear relationship between time and temperature during the event: T = T1 + ϕ (t − t1), where T1 is the sample temperature at the starting point t1, τ = t − t1 is the time measured from the onset of the event, and ϕ = dT/dt is the (constant) rate of heating or cooling during the event. The effect of NaOH and ZnO on the fractional crystallization  and fractional fusion (or relative fusion) of PET was computed as a function of time by integration:Thermogravimetry (TG) was performed using a Q20 calorimeter from TA Instruments. Specimens were heated from 30 to 600 °C with a heating rate of 10 °C/min in nitrogen atmosphere with a gas flow rate of 50 mL/min.Tensile testing was performed with injected specimens, according to ASTM D638, using a universal test machine EMIC DL 2000  with a crosshead speed of 50 mm/min, at room temperature (~23 °C). An aggressive fluid NaOH solution with a concentration of 3M was applied to the molded surfaces of the specimens during the test using a cotton pad. Reported data are averages of six specimens.Scanning electron microscopy (SEM) images were captured from the specimen fractured surfaces. A Shimadzu SSX-550 Superscan  scanning electron microscope was used with a voltage of 30 kV under high vacuum. Prior to SEM analysis, the fracture surfaces were gold coated to avoid charge accumulation.The X-ray diffractograms of the as-molded PET and PET/ZnO 1% samples given in Even though the injection molding conditions were chosen to produce only amorphous material (like the injection mold temperature set at 10 °C), some ordering did occur, possibly due to a nucleating action of the ZnO particles ,20. ThisThe presence of NaOH may cause a chemical reaction in the polymer chains of PET, as shown in From the DSC scans, the relative crystallinities were calculated; the results are given in Plots of crystallization and fusion rates, calculated using Equation (3), are graphically presented in Tmax ~435 °C [ ~435 °C .NaOH in aqueous solution is a stress cracking agent of PET ,10 that Related to the action of NaOH, it is clearly seen that PET lost its ability to withstand high stress, fracturing at lower strain; see the PET and PET-NaOH plots.Related to the action of ZnO, PET presented not only higher stress but also higher strain ability; see the PET and PET/ZnO plots. Upon exposure to NaOH, ZnO provided the PET with the ability to withstand higher stress and strain, as presented in the PET–NaOH and PET/ZnO (1%)–NaOH plots.According to The present study aimed to evaluate the stress cracking effects caused by NaOH solutions on the thermal and tensile properties of PET and PET/ZnO composites. The results showed that NaOH did not significantly change the thermal stability PET, but that the cold and melt crystallization, as well as the melting behavior, changed by its influence. Cold crystallization was observed at lower temperatures, whereas more complex peaks were obtained during melting, suggesting the presence of smaller and less perfect crystals. The mechanical properties of PET were reduced when NaOH was applied during tensile testing, indicating failure by stress cracking. On the other hand, ZnO not only improved PET crystallization, mostly due to heterogeneous centers improving crystallinity, but also offered some protection against the harmful effects of NaOH.


Allergic rhinitis (AR) is an inflammatory disease of the nasal mucosa impacting up to 25% of Canadians. The standard of care for AR includes a treatment plan that takes into account patient preferences, the severity of the disease, and most essentially involves a shared decision-making process between patient and provider.Since their introduction in the 1940s, antihistamines (AHs) have been the most utilized class of medications for the treatment of AR. First-generation AHs are associated with adverse central nervous system (CNS) and anticholinergic side effects. On the market in the 1980s, newer generation AHs have improved safety and efficacy. Compared to antihistamines, intranasal corticosteroids (INCS) have significantly greater efficacy but longer onset of action. Intranasal AH and INCS combinations offer a single medication option that offers broader disease coverage and faster symptom control. However, cost and twice-per-day dosing remain a major limitation. Allergen immunotherapy (AIT) is the only disease-modifying option and can be provided through subcutaneous (SCIT) or sublingual (SLIT) routes. While SCIT has been the definitive management option for many years, SLIT tablets (SLIT-T) have also been proven to be safe and efficacious.There is a range of available treatment options for AR that reflect the varying disease length and severity. For mild to moderate AR, newer generation AHs should be the first-line treatment, while INCS are mainstay treatments for moderate to severe AR. In patients who do not respond to INCS, a combination of intranasal AH/INCS (AZE/FP) should be considered, assuming that cost is not a limiting factor. While SCIT remains the option with the most available allergens that can be targeted, it has the potential for severe systemic adverse effects and requires weekly visits for administration during the first 4 to 6 months. SLIT-T is a newer approach that provides the ease of being self-administered and presents a reduced risk for systemic reactions. In any case, standard care for AR includes a treatment plan that takes into account disease severity and patient preferences. Allergic rhinitis (AR) is an IgE-mediated inflammatory disease of the nasal mucosa, triggered by exposure to airborne allergens. It is estimated to afflict almost 25% of Canadians  and has Standard of care for AR includes a treatment plan that considers patient preferences, the severity of the disease, and most essentially involves a shared decision-making process between patient and provider. Diagnosing AR and finding a care plan should consist of in-depth patient history, physical exam, and skin test to confirm allergies . The patThe standard treatment algorithm for AR begins with allergen avoidance . PatientFor decades, AHs have been the most utilized class of medications for the treatment of AR. AHs are inverse agonists; that is, they target H1 receptors (H1 antihistamines) at binding sites that are different from those of histamine . There aThe adverse effects associated with first-generation AHs have been reported since their introduction in the 1940s . CurrentFirst-generation AHs can also cross the blood–brain barrier (BBB) and bind H1-receptors on neurons throughout the CNS and, therefore, may cause drowsiness, sedation, somnolence, and fatigue leading to impairment of cognitive function, memory, and psychomotor performances . The strLong-term, randomized, controlled studies of the safety of first-generation antihistamines are limited. However, many studies outline the association of these drugs with transportation-related injuries and fatalities. A recent review of toxicology tested profiles from 6677 fatally injured civil aviation pilots in the US from 1990 to 2012. In this study diphenhydramine was the most common drug found on autopsy capable of causing impairment (7.3%) . As a reThe CNS effects of first-generation AHs resemble and exacerbate those produced by alcohol and by other CNS-active chemicals. It may not be surprising, then, that diphenhydramine and other first-generation antihistamines are common drugs of abuse . InfantsCardiac toxicity was previously an under-recognized risk of first-generation AHs. Diphenhydramine and hydroxyzine interfere with cardiac potassium channels involved in action potential repolarization. As a consequence, these drugs may cause dose-related prolongation and a form of polymorphic ventricular dysrhythmia called ‘torsade de pointes’ –22.Newer generation antihistamines came on the Canadian market in the 1980s. These newer non-sedating AHs were developed to be less soluble, limiting their ability to penetrate the BBB . Since tIt is worth mentioning that two second-generation AHs, astemizole and terfenadine, have been associated with prolonged cardiac AT intervals and “torsade de pointes” at high doses. This was a specific issue with these compounds and are not representative of a class effect of second-generation agents. Both drugs have been off the market for over 20 years .It is a misconception that older AHs have a faster onset of action than newer agents. In a double-blind placebo-controlled trial comparing cetirizine and loratadine to chlorpheniramine (a first-generation AH), both cetirizine and loratadine were found to have a significantly faster onset and a longer duration of action .Many well-powered randomized, double-blind, placebo-controlled clinical trials have compared the efficacy of newer generation antihistamines –33. StudNew advances in antihistamines available on the Canadian market have focused on rupatadine and bilastine. Rupatadine is a novel substance which, in addition to being an H1 antagonist, is also a potent platelet-activating factor (PAF) inhibitor . A randoBilastine is another newer generation antihistamine that is highly selective for the H1 receptor, has a fast onset of action and a long duration of action. In a double-blind, randomized, placebo-controlled, balance four-treatment, four-period crossover phase II study using the Vienna Challenge Chamber, the efficacy of bilastine (20 mg), cetirizine (10 mg) and fexofenadine (120 mg) to relieve SAR symptoms were compared . All treIn summary, first generation antihistamines are no longer recommended for the treatment of allergic rhinitis . Newer aOne concern regarding oral antihistamines (OAHs) is the possibility that OAHs cannot reach high enough concentrations in the nasal mucosa following oral administration to inhibit histamine-stimulated cytokine release and other mediators of early- and late-phase allergic reactions.  IntranasIn short, while intranasal antihistamines are safe and effective, only one is available in Canada and is often hard to obtain currently.ARIA guidelines recommend INCS as the best option for both mild and moderate to severe AR in both children and adults . INCS inINCS are less likely to display the systemic effects of oral steroids such as growth suppression, and ocular effects, due to reduced exposure and lower bioavailability. However, INCS are associated with mild to moderate local adverse effects. These include, epistaxis, nasal drying, burning, and stinging sensations .The ability of INCS to suppress bone growth is controversial. Measurement of the hypothalamic–pituitary–adrenal (HPA) axis function is a sensitive way to evaluate the potential systemic effects of INCS. Using this method, a 1-year study showed that the use of beclomethasone dipropionate aqueous nasal spray twice daily resulted in significant suppression of growth in children compared with placebo . SimilarThe literature examining the risk of development of glaucoma and/or cataracts from the use of INCS is also complex and controversial. While it is clear that inhaled and oral corticosteroid use is associated with high long-term risks of cataract development , the potCompared to placebo and antihistamines, INCS have significantly greater efficacy . This isTo summarize, INCS are mainstay treatments for moderate to severe allergic rhinitis. All Health Canada approved products are generally safe and effective, and should be used with consideration to formulation, delivery device preferences and out of pocket costs to the patient (summarized in Table The other major therapeutic class of drug indicated for AR therapy are the leukotriene-receptor antagonists (LTRAs). LTRAs block the activity of cysteinyl leukotrienes (CysLTs), a potent inflammatory mediator associated with nasal congestion, mucus production, and inflammatory cell recruitment responsible for AR symptoms . CurrentIt is evident that no single medication class is without limitations Table . The 201Combining an INAH and an INCS, AZE/FP is a novel formulation in a single spray. There are many benefits to AZE/FP. Patients benefit from the additive effects that result from the different primary mechanisms of action of each drug (AZE and FP) and there is possible improvement in adherence to therapy by delivering the two agents in a single device . Moreove®), respectively. The treatment difference was more considerable. When nasal and ocular symptoms were combined, AZE/FP was more than twice as effective as either FP or AZE. Likewise, patients reached a 50% reduction in their overall nasal symptom burden one week faster than those treated with FP or AZE . Th. Th® . ILIT proposes the injection of allergens directly into lymph nodes guided by ultrasound. In the literature, this technique thus far appears to be safe, effective, and requires a lesser time commitment, however, it has not yet been standardized or approved for clinical use. .There is a range of available treatment options for AR that reflect the varying disease length and severity. The standard treatment algorithm begins with allergen avoidance, followed by pharmacologic agents. For mild to moderate AR, newer generation AHs should be first-line treatments and preferred over older AHs, due to better safety profiles. INCS are mainstay treatments for moderate to severe AR, with the Health Canada approved products proven to be safe and effective. Therefore, the formulation, delivery device preferences, and out of pocket costs to patients must be weighed. In patients who do not respond to INCS, a combination of intranasal AH/INCS (AZE/FP) should be considered, assuming that cost is not a limiting factor. The only disease-modifying option for patients who do not respond to or wish to avoid long-term use of pharmacotherapy is AIT. SCIT and SLIT-T both require a minimum three-year treatment period to provide long-term symptom relief; however, the mode of delivery and possible adverse effects do differ. While SCIT remains the option with the most available allergens that can be targeted, it has the potential for severe systemic adverse effects and requires weekly visits for administration during the first 4 to 6 months. SLIT-T is a newer approach that provides the ease of being self-administered and presents a reduced risk for systemic reactions. In any case, standard care for AR includes a treatment plan that takes into account patient preferences, disease severity, and is a shared decision-making process between patient and provider.


NRP1 and NRP2 genes that could be linked to the development of lymphatic malformations in patients diagnosed with lymphedema. Two hundred and thirty-five Italian lymphedema patients, who tested negative for variants in known lymphedema genes, were screened for variants in NRP1 and NRP2. Two probands carried variants in NRP1 and four in NRP2. The variants of both genes segregated with lymphedema in familial cases. Although further functional and biochemical studies are needed to clarify their involvement with lymphedema and to associate NRP1 and NRP2 with lymphedema, we suggest that it is worthwhile also screening lymphedema patients for these two new candidate genes.Neuropilins are transmembrane coreceptors expressed by endothelial cells and neurons. NRP1 and NRP2 bind a variety of ligands, by which they trigger cell signaling, and are important in the development of lymphatic valves and lymphatic capillaries, respectively. This study focuses on identifying rare variants in the  NRP1 and NRP2 genes. They were first identified in the nervous system of Xenopus laevis embryos ..nrp2+/− Nrp1−/− mice showed deficient neuronal vascularization, aortic arch malformations and impaired yolk sac vascularization [Nrp1 gene was also observed [Most information about neuropilin function comes from studies with mice. Early studies of rization ,15. Abnoobserved , althougNrp1 was recently implicated in lymphatic valve development via Sema3a/Nrp1 signaling [ignaling . Sema3a ignaling ,17,18.Nrp2−/− mouse model provided evidence of the selective role of Nrp2 in the development of lymphatic vessels. Nrp2-deficient mice showed significantly fewer or a complete absence of small lymphatic vessels and capillaries in many organs. The small number of vessels that did develop were wrongly positioned, and in some cases, small vessels and capillaries were larger in size. A decrease in LEC proliferation was also observed, indicating a role of Nrp2 in the proliferation of LECs in selected types of lymphatic vessels and implying the control of positional guidance by Nrp2 [In comparison, the  by Nrp2 .Nrp1 and Nrp2 separately has evident effects on the mouse phenotype, and the joint deletion of Nrp1 and Nrp2 has much graver consequences. Takashima et al. [−/−/Nrp2−/−Nrp1 mice died at an early stage of embryogenesis (E8.5) and displayed severely defective blood vessel development in the yolk sac. In the study, the authors observed that double-knockout mice had severer vascular malformations than Nrp1- or Nrp2-deleted animals. Surprisingly, heterozygous mice with +/−/Nrp2−/−Nrp1 or −/−/Nrp2+/Nrp1− genotypes did not survive embryo development either but lived longer than double-knockout mice (E10–E10.5) [Nrp1 and Nrp2 deletions caused severe lymphatic system malformations suggested that a double knockout also results in impaired lymphatic system development.Knocking out a et al.  reported0–E10.5) . AlthougNRP2, are indispensable for normal drainage of interstitial fluid into the lymphatic system. Lymphatic capillaries lack a basement membrane and feature endothelial cell junctions that grant the passage of interstitial fluid into the lymphatic system [The development of small lymphatic vessels, capillaries and valves is important, and its impairment may lead to an overaccumulation of lymph in the tissues and lymphedema. Lymphedema is a pathological condition characterized by swelling, usually at the extremities, accompanied by pain and inflammation . Lymphatc system . When lyc system . The bloNRP1 and NRP2 genes. A segregation analysis in familial lymphedema cases added evidence that NRP1 and NRP2 qualify as candidate genes to include in the genetic testing of lymphedema patients.In our study, a cohort of 235 Italian lymphedema patients, who tested negative for variants in known lymphedema-associated genes, was screened by a next-generation sequencing (NGS)-targeted panel for variants the in We retrospectively enrolled 235 Caucasian lymphedema patients in this study, which was conducted in accordance with the Declaration of Helsinki, and whose protocol was approved by the Ethics Committee of Azienda Sanitaria dell’Alto Adige, Italy . No consanguinity was reported in any family. Clinical diagnosis of lymphedema was made in hospitals across Italy, according to generally accepted criteria. All subjects gave their informed consent for inclusion before they participated in the study.NRP1 and NRP2 to our panel. DNA from probands was analyzed for germline variants. Variants were confirmed by bidirectional Sanger sequencing on a CEQ8800 Sequencer .Genetic testing was performed on genomic DNA extracted from saliva or peripheral blood of probands, as previously described . Brieflyhttps://gnomad.broadinstitute.org/). All variants were evaluated according to the American College of Medical Genetics and Genomics guidelines [We searched the international Single Nucleotide Polymorphism Database (dbSNP) and the Human Gene Mutation Database professional for all nucleotide changes. In-silico evaluation of the pathogenicity of sequence changes in NRP1 and NRP2 was performed using the Variant Effect Predictor tool and MutationTaster. Minor allele frequencies were checked in the Genome Aggregation Database  with a GnomAD-reported frequency of 0.0001178.Regarding variants in the NRP1 variant, female, 54 years, has stage 2 lymphedema of the left foot and ankle, diagnosed at age 27. The case is familial: the proband’s daughter also has stage 2 lymphedema of the lower limbs . Indeed, upper limb lymphoscintigraphy failed to visualize the left axillary lymph nodes. She carries a missense variant c.580T > C (rs755679361), which has an allele frequency in healthy control populations of 0.000008122, according to GnomAD. The segregation analysis in available family members showed that the proband inherited the variant from her father. The father was apparently healthy; however, lymphoscintigraphy showed clear bilateral lymph transport problems, especially on the left side  (dbSNP rs746130411), with a GnomAD frequency of 0.000004068.The second proband with an The third proband, female, 45 years, was diagnosed with edema of the right foot at age 32. The heterozygous missense variant is c.838C >T (dbSNP rs79750907) and has a frequency of 0.001783 (GnomAD). This is a familial case, since the proband’s son was also diagnosed with left foot lymphedema at age nine. The son was found to carry the same variant as the proband.The fourth proband, female, 50 years, was diagnosed with swelling of the lower limbs at age 42. She carries a heterozygous single-nucleotide missense variant c.1000C > T (dbSNP rs114144673), with a frequency of 0.001525 (GnomAD). This is a sporadic case without family history, and no other family members were tested.NRP1 and NRP2 genes, respectively, with different sequence identity and quality percentages. Details of the top 10 templates are shown in In an attempt to study the possible role of the variants found in our cohort compared to the wildtype, we performed an in-silico study. A wildtype template  search wBased on the percentage of sequence identity, similarity and best quality square, the 4gz9.1.A and 2qql.1.A chains were selected to align the template and query sequences in order to build models of NRP1 and NRP2 .Then, we entered the model in the Discovery studio visualizer to generate versions of the NRP1 structure with Arg552Gln and versions of the NRP2 structure with Phe194Val, Pro280Ser, Arg334Cys and Ile583Thr. Unfortunately, the template to generate the model for Ile853 was not available, so we could not study its interaction with adjacent residues. A molecular-level interaction analysis between native/variant residues was performed  to a polar residue (Ser). Differences in the amino acid side chain greatly alter the interaction of the residues with the phenylalanine at residue 425, leading to a loss of Pi interaction. These results suggest that the overall protein structure is altered by these different interactions with nearby residues and is functionally defective .In the case of Arg334Cys, Arg334 has six interactions with adjacent residues, whereas Cys334 has only three similar to those of Arg334, and the interaction with Ile226 has a different hydrophobic interaction bond length .Likewise, in the case of Ile583Thr, Ile583 has six interactions with adjacent residues, whereas the Thr583 variant has only four that are the same, but the Pi interaction with Trp578 varies slightly in bond length .NRP1 and NRP2.Lymphedema is a progressive disease caused by lymphatic system malformations and impaired lymph flow. A panel consisting of 29 known lymphedema genes is currently used for the genetic testing of patients  but onlyNeuropilins are highly conserved transmembrane receptors that perform various functions in vessels, neurons and tumors, because they bind structurally to different ligands and coreceptors. Although NRP1 and NRP2 share basic structural features, they are implicated in different biological processes, such as the development of blood and lymphatic endothelial cells . They haNRP1 and NRP2, respectively. In four out of six cases, the proband was a female.Our screening detected two  and four  distinct probands with missense variants in NRP1 and NRP2 in patients who tested negative for variants in the 29 genes already associated with lymphedema. In three out of six cases, family members were screened, highlighting segregation of the variant with lymphedema or with subclinical signs of abnormal lymph flow. Bioinformatic modeling added evidence of a possible impact of the variant on the stability of the wildtype protein. Further biochemical studying or in-vitro studying is necessary to verify the altered function of coreceptors carrying the variant. In addition, other studies aiming at evaluating the effect of the interaction of the altered NRP1 with its principal ligand SEMA3A, or of the altered NRP2 with its coreceptor VEGFR3, could be important to define whether the variants also have a role in such signaling. Moreover, the loss of SEMA3A function by the specific inhibition of its interaction with NRP1 leads to impairment of lymphatic valve development [Overall, our results showed rare variants in elopment , while velopment .Additional studies could shed light on altered pathways, leading to a better understanding of the mechanism of action of the alteration; in particular, it could help determine whether the phenotype seen in our patients is due to haploinsufficiency or to a dominant negative effect of the coreceptor carrying the variant.NRP1 and NRP2, especially in large families with more than one affected subject, and performing a further functional analysis of the variants identified by us in in vitro and in vivo models to confirm their involvement in the development of lymphedema.Although these results are not sufficient to associate the two genes, known to be involved in the development of lymphatic capillaries and valves, with lymphedema, they suggest that it would be worthwhile screening a larger cohort of patients for variants in NRP1 and NRP2 should be considered candidate genes for inclusion in the gene panel for genetic testing of lymphedema patients.A comprehensive review of the literature strongly suggests that neuropilins NRP1 and NRP2 play a role in the development of lymphatic capillaries and lymphatic valves, crucial components of the lymphatic system. If these components do not function normally, the lymph does not drain, and fluid may accumulate in tissues, eventually leading to lymphedema. Given their functional role and our findings, we suggest that


Falling is a serious health concern among older wheelchair users as falling may result in functional impairment, morbidity, and mortality. Researchers have examined prevention strategies of falls. However, little is known about the prevalence of falls and rehabilitation service use among wheelchair users over age 65. We analyzed a population-based data set – the National Health and Aging Trends Study– to describe trends over time of wheelchair use among older adults who completed a sample interview in this study; we further identified 417 community-dwelling older wheelchair users in wave 5, described prevalence of falls and rehabilitation service use among them and investigated indicators of fall-related rehabilitation service utilization using multinomial logistic regression. The results indicated that there was an increasing trend of wheelchair use from 2011 (7.2%) to 2019 (10.6%). Falling was a common problem (56.1%); however, only 12.1% received rehabilitation service addressing the problem of falls. Around half of rehabilitation service users reported that their functioning improved, but the effect was not lasting. Age  and balance or coordination problem  were associated with receiving fall-related rehabilitation. These data suggest that we need to understand more about the underuse of the rehabilitation services, ensure that older wheelchair users are equally utilizing the services to prevent falling and improve the long-lasting effect of the services.