We evaluated all 5 somatostatin receptors (SSTR1-5) with immunohistochemistry and prospectively compared the results with both [68Ga]Ga-DOTANOC and [18F]F-FDG PET/CT in a cohort of 21 non-functional (NF) PNENs. SSTR2 was the only SSTR subtype to correlate with [68Ga]Ga-DOTANOC PET/CT. High SSTR5 expression correlated with a low Ki-67 proliferation index, suggesting a better prognosis for these patients. Thus, our results confirm that SSTR2 has the highest impact on SSTR PET signaling of PNENs.The need for prognostic and predictive biomarkers in pancreatic neuroendocrine neoplasms (PNENs) is great. Overexpression of somatostatin receptors (SSTRs) provides a molecular basis for imaging these tumors with 68Ga]Ga-DOTANOC uptake in a prospective series of NF-PNENs. Methods: Twenty-one patients with a total of thirty-five NF-PNEN-lesions and twenty-one histologically confirmed lymph node metastases (LN+) were included in this prospective study. Twenty patients were operated on, and one underwent endoscopic ultrasonography and core-needle biopsy. PET/CT with both [68Ga]Ga-DOTANOC and [18F]F-FDG was performed on all patients. All histological samples were re-classified and IHC-stained with monoclonal SSTR1-5 antibodies and Ki-67 and correlated with [68Ga]Ga-DOTANOC and [18F]F-FDG PET/CT. Results: Expression of SSTR1-5 was detected in 74%, 91%, 80%, 14%, and 77% of NF-PNENs. There was a concordance of SSTR2 IHC with positive/negative [68Ga]Ga-DOTANOC finding . All [68Ga]Ga-DOTANOC-avid tumors expressed SSTR2 or SSTR3 or SSTR5. Expression of SSTR5 was higher in tumors with a low Ki-67 proliferation index (PI) . The mean Ki-67 PI for SSTR5 positive tumors was 2.44  and 6.38  for negative tumors. Conclusion: SSTR2 was the only SSTR subtype to correlate with [68Ga]Ga-DOTANOC PET/CT. Our prospective study confirms SSTR2 to be of the highest impact for SST PET/CT signal.Purpose: The aim of this study was to correlate immunohistochemical (IHC) tissue levels of SSTR1-5 with the receptor density generated from [ Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) exhibit heterogeneous phenotypes with a variable clinical course ranging from very indolent tumors to highly aggressive carcinomas. Pancreatic neuroendocrine neoplasms (PNENs) have the lowest five-year survival rate 23%) of all GEP-NEN sites . However3% of all18F]F-FDG-PET/CT helps to predict the tumor grade better [68Ga]Ga-DOTANOC and [18F]F-FDG can predict the proliferation index, thus the aggressiveness of NF-PNENs Ga-DOTANOC-PET/CT and [18F]F-FDG-PET/CT. None of these patients had classical symptoms indicating a functioning PNEN, and all patients were evaluated by an endocrinologist at the corresponding university hospital. Patient selection is described in The SSTR analysis was performed as part of a prospective, controlled, two-center clinical trial at Turku and Helsinki University Hospitals in Finland. From 1/2016 to 1/2018, 35 patients suspected of having NF-PNEN after primary CT were prospectively imaged using Ga-DOTANOC and 54 ± 9 min after [18F]F-FDG. Analyses of PET/CT images were interpreted by a nuclear medicine physician (J.K), with short referral information but blinded to histopathological reports. The maximum standardized uptake values (SUVmax) were determined for every tumor or abnormal region for both [68Ga]Ga-DOTANOC and [18F]F-FDG PET/CT. PET/CT study areas with a focal activity greater than the background that could not be identified as physiological activity were considered to indicate tumor tissue. Lesions were graded on [68Ga]Ga-DOTANOC-PET/CT with the Krenning score by visually and semi-quantitatively comparing the SUVmax of the tumors to the liver and spleen reference organs [68Ga]Ga-DOTANOC was 142.7 ± 18.9 MBq, and [18F]F-FDG was 331.1 ± 58.5 MBq. The PET/CT protocol and data analysis have been described previously Ga-DOTANOC and immunohistochemical SSTR2 expression  (shown in the There was a correlation between positive/negative [68Ga]Ga-DOTANOC-avid tumors (n = 22/23) expressed membranous SSTR2 and the only [68Ga]Ga-DOTANOC-negative tumor did not express membranous SSTR2 . However, there was no association between SSTR2 expression and uptake intensity of [68Ga]Ga-DOTANOC, expressed as SUVmax (p = 0.043). We also found a correlation between SSTR2 (positive/negative) and the Krenning score. Of the tumors that expressed SSTR2, 9 had a Krenning score of 4, 13 tumors had a Krenning score of 3, and only the tumor with a Krenning score of 2 did not express SSTR2 .When SSTR2 immunohistochemical expression was scored either positive or negative, all Ga-DOTANOC PET/CT and SSTR1 or SSTR3-5 immunohistochemical expression. There was no correlation between SSTR1 or SSTR3-5 expression profile and NETPET score or between SSTR1 or SSTR3-5 expression profile and Krenning score. Immunohistochemical SSTR1-5 membrane expression profiles versus [68Ga]Ga-DOTANOC uptake of all tumors are presented in There was no correlation between [68Ga]Ga-DOTANOC-avid tumors expressed SSTR2, SSTR3 or SSTR5, in any combination: 12 tumors expressed all 3 receptors, 7 expressed 2 of these receptors, and 4 expressed 1 receptor. However, the [68Ga]Ga-DOTANOC-negative tumor expressed only SSTR5. Eventually, it metastasized to the lymph nodes.All 23 [18F]F-FDG-negative. There was a negative correlation between the uptake of [18F]F-FDG and SSTR5 membranous expression (score 0–4); SSTR5 score 0 median SUVmax 5.2, range 3.4–12.6 (n = 3), score 1 median SUVmax 2.8, IQR 1.5–6.5 (n = 5), score 2 SUVmax 8.7 (n = 1), score 3 median SUVmax 2.6, IQR 1.7–3.1 (n = 13), score 4 SUVmax 1.4 (n = 1) (p = 0.033). No correlation between [18F]F-FDG PET/CT imaging and immunohistochemical SSTR1-4 expression was apparent.Thirteen tumors (59%) were Ga-DOTANOC PET/CT [68Ga]Ga-DOTANOC PET/CT is supported by our findings.Previous studies on correlations between SSTR1-5 expression and modern SSTR PET/CT in GEP-NENs are scarce, and all are retrospective ,24,25,26n by IHC . Miedererelation . Olsen e68Ga]Ga-DOTANOC and SSTR1-5. To our knowledge, this is the first study to correlate SSTR PET/CT with all SSTR1-5 IHC in PNEN patients. All the [68Ga]Ga-DOTANOC-avid tumors expressed SSTR2, and the only [68Ga]Ga-DOTANOC negative tumor did not express SSTR2. Nonetheless, no statistically significant correlation between SSTR immunoactivity scores and [68Ga]Ga-DOTANOC uptake (SUVmax) was detected. This may be due to our small cohort size and to the more heterogenic patient cohorts of the former studies. Further, some [68Ga]Ga-DOTANOC to different SSTR subtypes compared to other [68Ga]Ga-SSTR-ligands affected our results, too.However, we found no correlation between the uptake of F-FDG PET/CT imaging; both results suggest a better prognosis. We previously showed [18F]F-FDG PET/CT predicts better outcomes for the PNEN patients. Here, we show that SSTR5 expression also correlates with a better prognosis. It is known that SSTR2 and SSTR5 downregulate pancreatic carcinogenesis and angiogenesis and initiate apoptosis Ga-DOTANOC and [18F]F-FDG. Another strength is the detailed morphological analysis using novel monoclonal antibodies and optimized IHC staining protocols Ga-DOTANOC PET/CT. SSTR5 expression is associated with low Ki-67 PI and might thus associate with a better prognosis. Further prospective studies, in larger tumor series, are needed to study the correlation of SSTR expression profile with 68Ga-labeled SST and [18F]F-FDG PET/CT for the personalized management of PNEN patients.In conclusion, our study offers data on immunohistochemical SSTR1-5 expression patterns in NF-PNENs in a prospective setting and confirms that SSTR2 correlates with positive/negative [


There are limited data regarding SARS-CoV-2 (SC2) environmental contamination in staff areas of healthcare settings. We performed environmental sampling of staff areas in wards where coronavirus disease 19 (COVID-19) patients received care and compared findings to surfaces within COVID-19 patient rooms.2 surfaces in staff common areas , nurse workstations (computer mice and floors), and COVID-19 patient rooms  was performed using flocked swabs one or more times per week. Specimens underwent RNA extraction and quantitative real-time polymerase chain reaction to detect the SC2 N1 region. Median comparisons were performed using Wilcoxon rank sum test. Trends in odds were evaluated using Score test.The study was conducted at the Hospital of the University of Pennsylvania  from 9/15/20-1/26/21. Sampling of 20cmProportions of surface specimens with detectable SC2 RNA are summarized in Table 1. Median copy numbers were lower among staff toilets compared to COVID-19 patient toilets , lower among staff breakroom compared to patient room high-touch surfaces , and similar between staff and patient room samples from sinks and floors. At nurse workstations, SC2 RNA was detected among 22/177 (12.4%) computer mouse and 147/178 (82.6%) floor samples. Odds of SC2 detection increased by study week among common area (p< 0.001) and nurse workstation samples (p< 0.001) .Table 1. SARS-CoV-2 (SC2) RNA detection on staff common area and coronavirus disease 19 (COVID-19) patient room surfaces at the Hospital of the University of Pennsylvania, 9/15/20-1/26/21.Figure 1. Proportion of environmental surface specimens with detectable SARS-CoV-2 RNA from a) staff common areas and b) nurse workstations of inpatient wards where coronavirus disease-19 patients received care at the Hospital of the University of Pennsylvania, 9/15/20-1/26/21.Figure 2. Proportion of environmental surface specimens with detectable SARS-CoV-2 RNA in staff common areas of inpatient wards where coronavirus disease-19 patients received care at the Hospital of the University of Pennsylvania, 9/15/20-1/26/21, by surface type: a) staff breakroom surfaces, b) staff bathroom surfaces, c) staff common area floors.A low prevalence of detectable SC2 RNA was observed among staff area high-touch surfaces; however, the likelihood of detection increased over time. Environmental SC2 RNA detection may reflect primary contamination from infected healthcare workers or secondary contamination from contact with infected patients, though a direct relationship between surface SC2 RNA viral detection and transmission risk has not been established.Michael Z. David, MD PhD, GSK (Board Member) Ebbing Lautenbach, MD, MPH, MSCE, Merck )


The molecular mechanisms underlying post-operative pericardial adhesions remain poorly understood. We aimed to unveil the temporal molecular and cellular mechanisms underlying tissue dynamics during adhesion formation, including inflammation, angiogenesis, and fibrosis.We visualized cell-based tissue dynamics during pericardial adhesion using histological evaluations. To determine the molecular mechanism, RNA-seq was performed. Chemical inhibitors were administered to confirm the molecular mechanism underlying adhesion formation. A high degree of adhesion formation was observed during the stages in which collagen production was promoted. Histological analyses showed that arterioles excessively sprouted from pericardial tissues after the accumulation of neutrophils on the heart surface in mice as well as humans. The combination of RNA-seq and histological analyses revealed that hyperproliferative endothelial and smooth muscle cells with dedifferentiation appeared in cytokine-exposed sprouting vessels and adhesion tissue but not in quiescent vessels in the heart. SMAD2/3 and ERK activation was observed in sprouting vessels. The simultaneous abrogation of PI3K/ERK or TGF-β/MMP9 signaling significantly decreased angiogenic sprouting, followed by inhibition of adhesion formation. Depleting MMP9-positive neutrophils shortened mice survival and decreased angiogenic sprouting and fibrosis in the adhesion. Our data suggest that TGF-β/matrix metalloproteinase-dependent tissue remodeling and PI3K/ERK signaling activation might contribute to unique angiogenesis with dedifferentiation of vascular smooth muscle cells from the contractile to the synthetic phenotype for fibrosis in the pericardial cavity.Our findings provide new insights in developing prevention strategies for pericardial adhesions by targeting the recruitment of vascular cells from heart tissues. Pericardial adhesions are frequently recognized in repeat open-heart surgeries and complicate the procedure , 2. Growvia serous fluid secretion , vascular endothelial cells, and mesothelial cells . Mesotheecretion . Of the ecretion . Previouecretion –8. Folloecretion . Finallyecretion . AlthougBlood vessels support the maintenance and survival of various tissues or organs by delivering nutrition and oxygen . HypoxicMolecular-based analyses of inflammatory signaling in pericardial fibrosis have been previously reported. Transforming growth factor-beta (TGF-β) is reportedly a central player in the activation of inflammatory reactions and the production of the extracellular matrix for fibrosis . The actWe have previously established a simple model of pericardial adhesion in mice . In the Rosa26-EGFP mice  were crossed with Cdh5CreERT2 mice  via intraperitoneal injection (5 mg/40 g mouse body weight) for 3 consecutive days before talc administration. The induction of pericardial adhesion in mice was conducted as per a previously reported method  and performed in accordance with the standards of the committee using the approved animal protocols. C57/Black6 (B6) mice were obtained from CLEA Japan . To generate endothelial cell-specific reporter mice, RT2 mice . To labed method . Brieflyd method . The ched method , defined® 488 anti-mouse/human CD11b Antibodies  and APC anti-mouse Ly-6G Antibody  were obtained from BioLegend . Anti-Ly6G/Ly6C (Gr-1) neutralizing antibodies  and Rat IgG2b Isotype control anti-KLH InVivoPlus antibodies (Clone: LTF-2)  were obtained from Bio X Cell, West Lebanon, NH). Anti-fibrinogen β chain (FGβ) rabbit antibodies  were obtained from Atlas Antibodies, Inc. . The Histofine® Mouse Stain (peroxidase-conjugated anti-mouse IgG), Histofine® Simple Stain™ Mouse MAX PO (anti-rabbit IgG), Histofine® Simple Stain™ Mouse MAX PO (anti-rat IgG), and Histofine® DAB substrate kits were obtained from Nichirei Bioscience Inc. . Alexa Fluor 488-conjugated anti-rat IgG (H+L) donkey , anti-mouse IgG (H + L) goat , and anti-rabbit IgG (H + L) goat  antibodies, and Alexa Fluor 568-conjugated anti-rabbit IgG (H + L) goat , and anti-mouse IgG (H + L) goat  antibodies were purchased from Thermo Fisher Scientific . Hoechst 33342 solution was purchased from Molecular Probes, Inc. . ISOGEN II was obtained from Nippon Gene, Inc. . Gelatin Zymography Kit (Cosmo Bio type)  was obtained from Cosmo Bio Co., Ltd. . Monoclonal Anti-β-Actin antibody , SB-431542 , PD98059 , and LY294002  were obtained from Sigma . Marimastat  was purchased from Selleckchem .Hydrogen peroxide solution was obtained from Wako Pure Chemical Industries . Anti-alpha smooth muscle actin (αSMA) mouse  and anti-proliferating cell nuclear antigen (PCNA) rabbit  antibodies were purchased from Santa Cruz Biotechnology, Inc. . Anti-CD31 rat antibodies  were obtained from Dianova . Anti-MMP9 rabbit , anti-TAGLN goat , anti-CD31 rabbit , and anti-CD34 mouse  antibodies were obtained from Abcam . Anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204)  and anti-Phospho-Smad2 (Ser465/467) / Smad3 (Ser423/425) (D6G10) rabbit  antibodies were purchased from Cell Signaling Technology . Anti-green fluorescent protein (GFP) rabbit antibodies  and anti-Glycoprotein M6a (GPM6A) monoclonal antibodies  were purchased from MBL Life Science, Inc. . Anti-myeloperoxidase (MPO) Antibodies  were obtained from R&D Systems . Alexa FluorMouse chest cavities were excised and fixed using 10% neutral buffered formalin for 24 h. After decalcification with 10% ethylenediaminetetraacetic acid for 3 weeks, the tissues were embedded in paraffin and sliced into 5 μm sections. Paraffin-embedded heart tissue from the autopsy was used for comparative investigations of fibrosis and structure comparison between mice and humans. All clinical experiments were approved by the Ehime University Internal Review Board (protocol no.2105012), and informed consent was obtained based on the opt-out principle. Hematoxylin and eosin (H and E) and Masson's trichrome staining were performed to visualize tissue structures and collagen deposition, respectively. After the specimens were deparaffinized with xylene, samples were rehydrated and stained as previously described . For H aTM Antibody Diluent  for 30 min. Anti-αSMA , CD31 (1:200), PCNA , MMP9 , GPM6A (1:400), phosphor-ERK (1:200), or phosho-SMAD2/3 (1:200) antibodies were probed onto the tissues and incubated overnight at 4°C. After washing with phosphate-buffered saline (PBS), the sections were treated with HRP-conjugated goat anti-mouse (for αSMA and PCNA), rat (for CD31 and GPM6A), and rabbit  antibodies  for 45 min at room temperature. The antigens were colored through treatment with the DAB substrate solution. After staining the nuclei with the hematoxylin solution, the sections were sealed with a cover glass. Tissue images were visualized as described above.To visualize the localization of FGB, anti-rabbit IgG HRP conjugate , αSMA, CD31, PCNA, MMP9, GPM6A phosphor-ERK, and phosphor-SMAD2/3, the tissues were deparaffinized with xylene. For antigen activation, the samples were heated for 15 min at 120°C in 10 mM sodium citrate buffer (pH 6.0) using an autoclave. Following inactivation of endogenous peroxidase with 3% hydrogen peroxide solution, samples were treated with blocking reagent Dako REALTM Antibody Diluent, followed by reaction with the following primary antibody mixtures overnight at 4 °C: anti-αSMA (1:200)/CD31 (1:200), anti-αSMA (1:200)/TAGLN (1:200), anti-αSMA (1:200)/GFP (1:200), anti-αSMA (1:200)/PCNA (1:200), anti-CD31 (1:200)/PCNA (1:200), anti-MMP9 (1:500)/CD31 (1:200), anti-MMP9 (1:500)/CD31 (1:200), anti-MPO (1:400)/MMP9 (1/500), anti-CD34 (1:200)/CD31 (1:200), anti-CD34 (1:200)/αSMA (1:200), anti-pERK (1:200)/αSMA (1:200)/CD31 (1:200), or anti-pSMAD2/3 (1:200)/αSMA (1:200)/CD31 (1:200). After washing with PBS three times, the specimens were probed with the following fluorescent secondary antibody mixtures for 2 h at RT: Alexa Fluor 488-conjugated goat anti-rat IgG /Alexa Fluor 568-conjugated goat anti-mouse IgG  for αSMA/CD31; Alexa Fluor 488-conjugated goat anti-rabbit IgG /Alexa Fluor 568-conjugated goat anti-mouse IgG  for αSMA/GFP; Alexa Fluor 488-conjugated goat anti-mouse IgG /Alexa Fluor 568-conjugated goat anti-rabbit IgG  for αSMA/PCNA and αSMA/CD34; Alexa Fluor 488-conjugated goat anti-rat IgG /Alexa Fluor 568-conjugated goat anti-rabbit IgG  for CD31/PCNA, CD31/CD34, and MMP9/CD31; Alexa Fluor 488-conjugated donkey anti-rabbit IgG /Alexa Fluor 568-conjugated donkey anti-goat IgG  for MMP9/MPO; Alexa Fluor 488-conjugated donkey anti-mouse IgG /Alexa Fluor 568-conjugated donkey anti-goat IgG  for αSMA/TAGLN; and Alexa Fluor 488-conjugated goat anti-rat IgG /Alexa Fluor 568-conjugated goat anti-rabbit IgG  for CD31/CD34. Following nuclear staining with Hoechst 33342, immunofluorescence signals were detected using an A1 confocal laser microscope .To investigate tissue co-localization, immunofluorescence assays were performed using multiple antibodies. After deparaffinization and antigen activation, samples were blocked using Dako REALhttp://david.abcc.ncifcrf.gov).Pericardial adhesion tissues and the myocardium were separately harvested from euthanized mice, homogenized with the ISOGEN II reagent, and stored at −20°C until further use. Total RNA was isolated according to the manufacturer's instructions for the ISOGEN II reagent. Purified total RNA was quantified using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and RNA integrity score was evaluated using the Bioanalyzer RNA 6000 Nano Assay Kit . RNA sequencing using the MiSeq system  was performed as previously described , MEK/ERK pathway inhibitor , PI3K-kinase inhibitor , MMPs inhibitor  were simultaneously injected into the pericardial cavity. To evaluate the inhibitory activity of TGF-β/SMAD/MMP signaling or PI3K/Akt/MEK/ERK signaling during adhesion formation, a combination of TGF-β type1 receptor inhibitor  and MMPs inhibitor  or MEK/ERK pathway inhibitor  and PI3K-kinase inhibitor  was injected into the pericardial cavity. The chemical compounds were intraperitoneally administered on days 2 and 4. All compounds were dissolved with 5% DMSO in corn oil . Mice were euthanized on day 6, and the adhesion severity was scored as described above.via mixed injection of ketamine (80 mg/kg) and xylazine (10 mg/kg), and blood was collected using heparin-treated 23G needles. After perfusion with PBS, heart tissues were excised and digested with PBS-based collagenase  solution for 3 h at 37°C. After digestion, the samples were filtered through 40 μm cell strainers , and the cells were labeled with Alexa Fluor® 488 anti-mouse/human CD11b antibodies and APC anti-mouse Ly-6G antibody. Peripheral blood treated with the antibodies were hemolyzed using OptiLyse C Lysing Solution according to the manufacturer's instructions. The samples were then washed with 10% fetal bovine serum and analyzed using a FACS Calibur flow cytometer .To quantify the cell population of neutrophils in the peripheral blood and heart tissue of adhesion-induced mice, Ly6g and CD11b double-positive cells were selectively collected using a flow cytometer. TALC-injected and sham mice were anesthetized The heart tissues from talc-injected mice at each time point (day 0-7) were homogenized with Laemmli SDS sample buffer. After centrifugation, the protein concentration of the supernatant was measured using DC/RC Protein Assay Kit , and 20 μg of the extracted protein was subjected to 10-20% gradient polyacrylamide gels . Proteins were transferred to the polyvinylidene fluoride membrane and probed with anti-MMP9, TAGLN, and β-actin antibodies, followed by treatment with anti-rabbit IgG HRP conjugate  for MMP9, anti-goat IgG HRP conjugate  for TAGLN, and anti-mouse IgG HRP conjugate  for β-actin. Chemiluminescent western blot images were obtained using a LAS-4000 .The enzymatic activities of MMP9 were measured using a Gelatin Zymography Kit following the manufacturer's instructions. Forty micrograms of the extracted proteins from heart tissues were separated by SDS polyacrylamide gel electrophoresis, and the gel was incubated in the reaction buffer for 36 h at 37°C. The bands were visualized by staining with coomassie blue staining solution.To eliminate neutrophils, mice were injected intraperitoneally with 387 μg of Ly6g neutralizing antibodies or isotype control antibodies in 200 μl PBS, as previously reported . After 2t-tests (parametric variables) and Mann-Whitney U-test (non-parametric variables). The normality of the obtained experimental data was assessed using the Shapiro-Wilk normality test. ImageJ software (NIH) was used for quantification histological data. ns.: not significant. ND.: not detected.All data are presented as mean ± standard error (SE). GraphPad Prism Ver. 7  was used to conduct a one-way analysis of variance (ANOVA) for normal distributions and the Kruskal-Wallis test for non-normal distributions. Statistical significances between two groups were calculated using Student's p < 0.001) at the third hour after injection  on day 7 in proportion to collagen deposition (p = 0.0005) on day 7  around layers of cardiomyocytes, while adipose tissues were partially observed within myocardial and adhesion tissues  and myofibroblasts (p < 0.0001) between sprouting vessels and adhesion tissues (p < 0.0001), rather than in cardiac arterioles. Our data indicate that proliferative dedifferentiated smooth muscle cells in arterioles of the heart are one of the origins of fibrosis in the pericardial cavity.Based on the RNA sequencing results regarding cell cycle regulators, to visualize proliferative cells during adhesion formation, we performed immunohistochemical staining using anti-PCNA antibodies. A few PCNA-positive cells were observed around the injured surface of the heart on day 1, and the cell population gradually increased for 7 days . From da tissues . Cell pr tissues , suggestMMP9   . Additio 0.0006) . Finally 0.0006) . Histolo 0.0006) . These dSeveral prevention methods have been developed for post-operative pericardial adhesions based on the observations in humans and animal models that bleeding, inflammation, and fibrosis are involved in the adhesion process . HoweverAngiogenic development has been well characterized using a retinal model of angiogenesis in mice –13. ThesMYOCD, ACTA2, Tpm1, Tagln, Cnn1, ltga7, Des, and MYH11, and upregulated expression of Vim, Myh9, CD44, MMP3, and collagen, which contribute to the development of vascular fibrosis via promotion of conversion to the synthetic phenotype (TGFB1 and TGFB-induced (TGFBI) expression  can be found below: NCBI [accession: The studies involving human participants were reviewed and approved by Ehime University Internal Review Board (protocol no.2105012). Informed consent for participation was obtained based on the opt-out principle, as approved by the Ehime University Internal Review Board. The animal study was reviewed and approved by Ehime University Animal Care Committee (Project number: 05-RO-7-1 and 05-RO-42).KN and TS wrote the manuscript. HI, KN, and TS designed the study. KK, KN, MK, TS, and YKo conducted the experiments and acquired the data. FS, HI, JM, KK, KN, MK, MO, TN, HK, NO, SH, TS, YKo, and YKu analyzed the data and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Grants-in-Aid for Scientific Research (grant no.18K16396 to KN) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


T-cell responses to insulin and its precursor proinsulin are central to islet autoimmunity in humans and non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes. Mice have two proinsulin genes proinsulin -1 and 2 that are differentially expressed, with predominant proinsulin-2 expression in the thymus and proinsulin-1 in islet beta-cells. In contrast to proinsulin-2, proinsulin-1 knockout NOD mice are protected from autoimmune diabetes. This indicates that proinsulin-1 epitopes in beta-cells maybe preferentially targeted by autoreactive T cells. To study the contribution of proinsulin-1 reactive T cells in autoimmune diabetes, we generated transgenic NOD mice with tetracycline-regulated expression of proinsulin-1 in antigen presenting cells (TIP-1 mice) with an aim to induce immune tolerance. TIP-1 mice displayed a significantly reduced incidence of spontaneous diabetes, which was associated with reduced severity of insulitis and insulin autoantibody development. Antigen experienced proinsulin specific T cells were significantly reduced in in TIP-1 mice indicating immune tolerance. Moreover, T cells from TIP-1 mice expressing proinsulin-1 transferred diabetes at a significantly reduced frequency. However, proinsulin-1 expression in APCs had minimal impact on the immune responses to the downstream antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and did not prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, boosting immune tolerance to proinsulin-1 partially prevents islet-autoimmunity. This study further extends the previously established role of proinsulin-1 epitopes in autoimmune diabetes in NOD mice. Ins gene and is strongly associated with susceptibility to type 1 diabetes (T1D) in humans  and six amino acids in the leader peptide (Recognition of proinsulin by the immune system is a major determinant in the pathogenesis of autoimmune diabetes in both humans and non-obese diabetic (NOD) mice , 2. A pon humans –5. Mice  peptide . The two peptide –9.Ins2 gene develop accelerated diabetes, ascribed to loss of central tolerance to insulin peptides; however, development of insulin autoantibodies (IAA) in Ins2 -/- mice suggests that immune responses against PIns1 epitopes are intact (Ins1 or replacement of murine Ins1 with human insulin gene (INS) in NOD mice provides significant protection from diabetes , are essential for autoimmune diabetes in NOD mice , 11. Thee intact . In contdiabetes , 17. Proreported , and T creported . Thus, eIns2 globally . A 1100 bp transgene cassette comprising of the TetO-minimal CMV promoter, followed by the PIns1 gene and a polyA signal was excised between Xho I sites and purified for injection into fertilized NOD/Lt ova using standard techniques. Founders and transgene positive offspring were screened by PCR using primers spanning the PIns1 gene (5’-TTAAGATATCTTCATTCATTATAGAACTC -3’) and the tetO-CMV promoter (5’-TCAGTGATAGAGAACGTATGTCG -3’).NOD/Lt mice were bred and housed at the bioresources center St. Vincent’s Hospital, Fitzroy. The NOD-IEα-tTA mice that drive the expression of tetracycline transactivator (tTA) under the control of MHC class II IEα promoter have been previously described  and werevia drinking water at concentration of 2mg/ml. Water bottles were changed thrice weekly.Untreated TIP-1 mice constitutively express proinsulin-1 in antigen presenting cells (APCs). To turn-off proinsulin-1 expression, doxycycline hyclate (Dox) (Sigma-Aldrich) was administered Ins1; Mm01950294_s1), murine β-actin  and murine Glyceraldehyde 3-phosphate dehydrogenase  were purchased from Applied Biosystems. To determine relative expression, Ct values of Insulin were subtracted from Ct values of reference genes for each sample and the difference (dCt) was plotted to determine the abundance of the gene of interest.For total RNA extraction, whole spleen and thymus were harvested in cold Phosphate Buffered Saline (PBS). Tissue homogenates were prepared in RNA lysis buffer RA1 (Macherey-Nagel) from a 15mg slice of tissue using a tissue homogenizer. RNA was isolated using Nucleospin RNA II-isolate kits (Macherey-Nagel), and first strand cDNA was generated from 500ng RNA using High Capacity cDNA Reverse Transcription kits (Applied Biosystem) according to the manufacturers’ instructions. cDNA was diluted (1:20) and Real-time PCR analysis was performed using Rotor-Gene-RG-3000 cycler . Taqman gene expression primers murine insulin 1  and stored at −80°C. For histological analysis 5-µm frozen sections of pancreas were prepared from three levels (200 µm apart), acetone fixed, stained with guinea pig anti-insulin followed by horseradish peroxidase–conjugated anti–guinea pig Ig  and counterstained with hematoxylin. Insulitis was graded using the following scale: 0 = no infiltrate, 1 = peri-islet infiltrate, 2 = extensive (>50%) peri-islet infiltrate, 3 = intraislet infiltrate, and 4 = extensive intraislet infiltrate (>80%) or total β-cell loss. The percentage of islets with each grade per pancreas was calculated by addition of the grades for the three sections. Individual insulitis scoring for each mouse was performed as previously described .7 splenocytes from 13-17 week old pre-diabetic TIP-1 mice or control NOD mice were transferred (i.v.) into 9-12week old NOD Rag-/- recipients and diabetes development was monitored as above.Diabetes onset was monitored by weekly measurement of urine glucose levels using Diastix (Bayer Diagnostics). Blood glucose levels were measured in mice with glycosuria using Advantage II Glucose strips (Roche). Animals displaying two consecutive blood glucose measurements of ≥ 15mmol/L were considered diabetic. For adoptive transfer of diabetes, 2 x10Antibodies used were anti-CD4 (RM4-5) conjugated to PerCpCy5.5, anti-CD3 (145-2C11) conjugated to FITC or anti-CD3 (500 A2) V500, anti-CD44 (1M7) conjugated to AlexaFlour700 , anti-CD11c (N418), anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-F4/80 (BM8) conjugated to eFlour450 and anti-FoxP3 (FJK-16S) conjugated to APC , anti-CD8a (5H10) conjugated to Pacific Orange (Invitrogen) or anti-CD8a (53-6.7) conjugated to PE-Cy7, anti-CD62L (MEL-14) conjugated to APC-Cy7 (BD Biosciences). FoxP3 was stained intracellularly using FoxP3/Transcription Factor Fixation/Permeabilization kit (eBiosciences). Data were collected on an LSR Fortessa flow-cytometer (BD) and analyzed using FlowJo (Treestar) software.10-23 (HLVERLYLVCGGEG) tetramer for 1 hour at room temperature. The Ins B10-23 peptide in the I-Ag7-INSB10-23 tetramer has been mutated (Glutamic acid to Glycine (E-G) at position 20 and Arginine to Glycine (R-G) at position 21) to improve its binding to the I-Ag7 molecule, which allows for better detection of insulin-specific CD4+ T cells (15-23 (LYLVCGGEG) tetramer for 1 hour on ice. Hen Egg Lysozyme I-Ag7-HEL (AMKRHGLDNYRGYSL) tetramer or H-2Kd-TUM (KYQAVTTTL) were used as controls. Cells were then washed and stained with anti-PE or anti-APC microbeads (Miltenyi Biotec) followed by magnetic separation using an AutoMACSpro (Miltenyi Biotec) according to manufacturer’s instructions. IGRP206-214 specific CD8+ T cells  were stained and enriched as previously described  test. Statistical significance was defined as P < 0.05.Statistical analysis was performed using GraphPad Prism 8 Software . Pooled data are shown as dot-plots with individual mice and the mean ± SEM. Data were tested for normal distribution using D’Agostino-Pearson omnibus normality test or Shapiro-Wilks test. Comparisons between two groups were performed using two-tailed unpaired student To test whether inducing immune tolerance to proinsulin-1 (PIns1) influenced autoimmune diabetes we generated transgenic NOD mice to facilitate conditional expression of PIns1 in the antigen presenting cells (APCs). Reporter NOD mice expressing PIns1 under the control of the tetracycline-responsive CMV promoter (TetO-Ins1 mice) were bred with previously described driver NOD mice expressing TetR-VP16 tetracycline transactivator protein (tTA) under the control of IEα-MHC-II promoter referred to as TA-NOD mice . Bi-tranWe recently reported that constitutive or temporal expression of PIns2 (PIns2) in the APCs limited to the perinatal period prevented insulitis and diabetes in NOD mice . To testReduced insulitis and IAA suggest that diabetes development may be altered in TIP-1 mice. A cohort of female TIP-1 mice expressing PIns1 continuously and control NOD mice were observed for incidence of spontaneous diabetes. TIP-1 mice developed diabetes but at a significantly reduced incidence compared to non-transgenic control NOD mice. By 300 days of age 40% of TIP-1 mice and 65% of the control mice developed diabetes  (pooled spleen and non-draining lymph nodes) of 20-25 weeks old non-diabetic TIP-1 mice and age matched control mice using respective I-A (g7) and Kd tetramers. There was a significant reduction in the absolute number of insulin B:9-23 specific CD4+ T cells binding to insulin B:10-23/I-A (g7) tetramer  are dependent upon immune response to PIns2 . To inveAutoreactivity to PIns2 is required for diabetes development in NOD 8.3 mice that have a pre-existing repertoire of IGRP specific T cells . Since wIn this study we generated transgenic NOD mice to induce PIns1 expression in the APCs and examined the impact of antigen specific tolerance on autoimmune diabetes. The main findings of this study are 1) TIP-1 mice expressing PIns1 in the APCs show significantly reduced incidence of diabetes, which is associated with reduced insulitis and insulin autoantibody (IAA) expression. 2) Proinsulin specific CD4+ T cells are detectable in TIP-1 mice at a reduced frequency and are not activated. 3) Immune responses to downstream antigen IGRP are delayed but not absent in TIP-1 mice.9-23 we expected to achieve robust protection from diabetes onset in TIP-1 mice, similar to previously described proinsulin-2 tolerant NOD mice  as compared to thymus (~20pmol/L) in the partially protected Pins2 tolerant mice previously described by Jaeckel et al. , whereas10-23 and Insulin B:15-23 specific tetramers used in our study are likely to detect both PIns1 and 2 reactive CD4+ and CD8 +T cells, due to the invariant nature of the Insulin B:9-23 peptide between the two isoforms. Immune responses to Insulin B:9-23 epitope are required for both priming and effector phase of islet autoimmunity in NOD mice. Moreover, Insulin B:9-23 primed CD4+ T cells are able to induce islet autoimmunity evidenced by IAA production  and fellowship from Juvenile Diabetes Research Foundation (BK). The St Vincent’s Institute receives support from the Operational Infrastructure Support Scheme of the Government of Victoria.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.


C-phycocyanin (C-PC) produced from cyanobacterial species finds application in drug development. Therefore, it is of interest to document the molecular binding features of C-PC with the vascular endothelial growth factor receptor 2 (VEGFR2). C-PC showed H-bondinteractions with residues on both sides of the Deusche Forschugsgemein-Schalt (DFG) loop (Asp1046-Phe1047-Gly1048). A hydrophobic association between the activation loop and the DFG residue (Gly1048) helps to inhibit the activity of VEGFR2 kinases. Thus, C-PC isreported as a potential angiogenesis inhibitor for VEGFR2 in combating cancer. C-Phycocyanin (C-PC) is a light yielding protein pigment produced from Cyanobacterial species. We recently reviewed therapeutic applications of C-PC . C-PC shVEGFR2 (target) is docked with C-PC by using Auto Dock 4.2. The dynamics analysis was done using Desmond V3.2 (Table 1 - see PDF) . PDB is Complications such as loops, missing hydrogen atoms with flipped residues. The incomplete side chains were enhanced using Auto Dock. Energy minimization, refinement and minimization of the protein were performed in this study. Affinity maps (grid) with a spacingof 0.375 Å were generated using Auto grid program around the receptor-ligand complex binding site . The parThe stability of the VEGFR2 and C-PC docked complex was crosschecked using the force field Optimized Potentials for Liquid Simulations (OPLS)-2005 via the 100ns MD simulation at Desmond (v3.8). Desmond was unified with molecular modeling to simulate chemicaland biological systems, which compares with widely used analytical and trajectory viewing tools. The Desmond based simulations follow the systematic workflow for system building with the six steps relaxation protocol followed by MD simulations. The system builderallows building a solvated system for protein-ligand complex by choosing parameters for water model, force field, ensemble type, thermostats, barostats algorithms, adding ions, salt and defining periodic boundary conditions, etc. The six steps equilibration processwas performed as described (1) Minimization of the solvated system with solute was restrained by hybrid algorithm method of steepest descent and limited memory Broyden Fletcher Goldfarb Shanno (LBFGS) (2) Minimization without solute restraints by the steepest descentand LBFGS algorithms (3) minimization of system for 12 ps simulation with NVT ensemble at temperature of 10K with restrained non-hydrogen solute atoms, (4) Again system was minimized in the NPT ensemble, 12 ps at 10K (5) System minimization was carried out for 24ps simulations in NPT ensemble temperature of 300K restraining the non-hydrogen solute atoms. (6) At temperature 300K no restraints in NPT ensemble at 24 ps simulation were used . MD simuConsidering the VEGFR2 as valid target for docking studies revealed that C-PC was binding accurately by forming the well-established hydrogen bonds with the active site residues. Grid was formed around the co-crystal ligand quinolin-4-yl]oxy-1-benzofuran-3-carboxamide) binding site residues Leu840, Val848, Glu885, LYS868, Val916, Leu889, Val914, Cys919, Phe918, Glu917, Lys920, Phe921, Gly922, Leu1035, Cys1045, Asp1046, Phe1047 . The docStability and detailed interactions between the C-PC with VEGFR2 were explored. Total energy, RMSD, RMSF, ligand binding interactions and gyration energy was analyzed in 10416 trajectories. Total energy of the system was observed of -161100 kcal/mol. AverageRMSD was observed for back bone is 2.4 Å; C-alpha is 2.4Å; Side chain 3.6 Å; heavy atoms is 3 Å; ligand with protein is 2.6 Å and ligand with ligand was observed below 1.4 Å. RMSD reveals that back bone and C-alpha was observedbelow the 3Å . Where aOne more crucial step is to analyze the H-bond interaction profiles of VEGFR2-C-PC docked complex. The H-bonds was key property in the computational drug design because of their strong binding influence on drug specificity, metabolism and adsorption. During the100ns simulations the observed H-bond interactions VEGFR2 with C-PC were shown in C-PC is reported as a potential angiogenesis inhibitor for VEGFR2 in combating cancer. C-PC showed H-bond interactions with residues on both sides of the Deusche Forschugsgemein-Schalt (DFG) loop (Asp1046-Phe1047-Gly1048). A hydrophobic association between theactivation loop and the DFG residue (Gly1048) helps to inhibit the activity of VEGFR2 kinases.


Pseudomonas aeruginosa (PSA). During treatment, PSA can develop subpopulations exhibiting variable in vitro antimicrobial susceptibility patterns. Heteroresistance may underlie the reported discordant in vitro results and clinical responses to various antimicrobials. Here, we sought to examine the presence and nature of PSA heteroresistance to ceftolozane-tazobactam (C-T) in isolates originating from CF pulmonary exacerbations.Cystic fibrosis (CF) patients are often colonized with Respiratory cultures from 26 adult CF patients were collected. From each sample, 5-10 PSA colonies were selected. Susceptibility testing was conducted via E-test for C-T, ceftazidime-avibactam (CZA), and imipenem-relebactam (I-R). Polyclonal-heteroresistance (PHR) was defined as the presence of different susceptibility profiles among the colonies that originated from a single patient specimen. Population analysis profile (PAPs) were performed to assess the presence of monoclonal-heteroresistance (MHR), defined as ≥ 4 fold change in the C-T MIC from a single colony over 24-48 hours.50 and MIC90 were 1/4 and ≥ 256/4 µg/mL, respectively . Sixteen of the 26 patients (62%) demonstrated ≥ 2 fold change in C-T MIC between isolates from the same culture. Of these 16 isolates, the fold change in C-T MIC was >2 fold for 7 isolates (27%) and resulted in a susceptibility interpretation change in 6 of the isolates (23%). Of the 32 isolates that underwent PAP testing, 7 grew on MH plates at 2-fold the C-T MIC concentration. One isolate, PSA 1311, demonstrated growth on PAPs up to 4 fold the MIC (16/4 µg/mL) .246 PSA isolates from 26 adult CF patients were included. The C-T MICFigure 1. Ceftolozane-tazobactam, ceftazidime-avibactam, and imipenem-relebactam MIC distributions against PSA isolates from 26 adult CF patientsFigure 2. Monoclonal heteroresistance to C-T .in vitro potency. C-T PHR exists among PSA isolates in the majority of our CF patients. Approximately 25% of these PHR isolates resulted in susceptibility interpretation changes supporting concerns surrounding the utility of traditional susceptibility testing methodology for CF isolates. These data suggest MHR also exists, albeit rare in this small subset. Additional data are needed to better understand these results in clinical context. Susceptibilities to C-T and CZA were similar across our CF PSA isolates. Comparatively, I-R retained better David E. Greenberg, MD, Shionogi (Grant/Research Support)Solenic Medical (Shareholder)


Dysregulation of Toll-like receptor (TLR) signaling contributes to the pathogenesis of autoimmune diseases. Here, we provide genetic evidence that tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, negatively regulates TLR2 signaling. We show that mice lacking tankyrase in myeloid cells developed severe systemic inflammation with high serum inflammatory cytokine levels. We provide mechanistic evidence that tankyrase deficiency resulted in tyrosine phosphorylation and activation of TLR2 and show that phosphorylation of tyrosine 647 within the TIR domain by SRC and SYK kinases was critical for TLR2 stabilization and signaling. Last, we show that the elevated cytokine production and inflammation observed in mice lacking tankyrase in myeloid cells were dependent on the adaptor protein 3BP2, which is required for SRC and SYK activation. These data demonstrate that tankyrase provides a checkpoint on the TLR-mediated innate immune response. The Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) derived from microbes to drive the innate immune response –3. TLR a+ to its substrates. Tankyrase-mediated ADP-ribosylation of substrates such as AXIN, AMOT, SH3BP5, and 3BP2 creates a recognition site for the E3 ubiquitin ligase RNF146, leading to substrate ubiquitylation and subsequent proteasomal degradation (Tankyrase (tankyrase 1) and its paralog tankyrase 2 are members of the poly(ADP-ribose) polymerase (PARP) family, which catalyzes the addition of ADP-ribose from NADradation .SH3BP2 gene are associated with cherubism, a rare hereditary syndrome characterized by severe craniofacial developmental defects in children . Notably, we found that 3BP2 coimmunoprecipitated with TLR2 that was further induced by HKSA in primary murine macrophages  domain is a region of common homology between the IL-1 receptor and TLR family members , 26. TheWe next examined the dependency of TLR2-induced NF-κB transcriptional activity on the expression of SRC and SYK. SYK expression doubled the level of TLR2-induced NF-κB transcriptional activity, while the combination of both SYK and constitutively active c-SRC (Y529F) expression increased TLR2-dependent NF-κB activity 5-fold . The incWe noted that the protein expression of the TLR2 (6YF) mutant was significantly reduced . To deteWe next investigated whether SRC and SYK kinases potentiate TLR2 signaling. The combined expression of SRC and SYK enhanced TRAF6-mediated NF-κB stimulation 4-fold , showingWe investigated the molecular mechanism by which SRC- and SYK-mediated phosphorylation of tyrosine residues within the TLR2 cytoplasmic domain promotes TLR2 activation. We first created tyrosine add-back variants of the TLR2 (6YF) mutant to determine which tyrosine(s) were sufficient to mediate TLR2 activation of NF-κB. We found that Y647 in the TIR domain was sufficient to fully restore NF-κB activation  as well Last, we examined the role of tyrosine 647 in recruiting the adaptor protein 3BP2 to TLR2. The interaction of 3BP2 with TLR2 was disrupted in the TLR2 (6YF) mutant but restored in the Y647 add-back variant . MoreoveTnks) and tankyrase 2 (Tnks2) in mice is embryonic lethal while knockout of either Tnks or Tnks2 shows only mild phenotypes (–/–Tnksfl/fl LysM-CreTnks2) in which endogenous tankyrase 1 was excised in all tissues including germ cells while tankyrase 2 was deleted in the myeloid monocytic lineage (–/–Tnksfl/fl LysM-CreTnks2 macrophages compared with +/+Tnksfl/flTnks2 (WT) cells  cells . Tnks–/–nomegaly  and lympnomegaly . Infiltrnomegaly . The stond villi . PerivasCre mice .–/–Tnksfl/fl LysM-CreTnks2 mice, we performed immunohistochemistry (IHC) staining of F4/80 (macrophage), Mac2 (macrophage), CSF-1R (monocyte), CD45R (B lymphocyte), myeloperoxidase , and eosinophil peroxidase  of spleen, lymph node, colon, lung, and liver from 12-week-old +/+Tnksfl/flTnks2 or –/–Tnksfl/fl LysM-CreTnks2 mice. We observed that F4/80- and Mac2- as well as CSF-1R–positive activated monocytes/macrophages predominantly infiltrated these organs in –/–Tnksfl/fl LysM-CreTnks2 mice, leading to destruction of lymphoid follicles in spleen and lymph node and reactive recruitment of CD45R-positive B lymphocytes in the enlarged colon and the perivascular regions of lung and liver .We next measured serum cytokine levels in the /fl mice , whereasCre mice . GM-CSF,–/–Tnksfl/fl LysM-CreTnks2 mice and observed elevated transcripts of Tnfa and Il6 and the IL-6 protein level in the culture supernatant under basal conditions as well as cell lysates of macrophages in comparison with +/+Tnksfl/flTnks2 mice  and –/–Tnksfl/fl LysM-CreTnks2 (KO) macrophages with the TLR2 ligand HKSA and monitored total cellular phosphotyrosine levels , which resulted in an increase of the TLR2 protein levels  mice (–/–Tnksfl/fl Sh3bp2fl/fl LysM-CreTnks2)  . ConsistCre mice . MoreoveCre mice . ActivatCre mice . These dCre mice .SH3BP2 gene stabilize 3BP2 proteins, which results in a genetic inflammatory disorder, cherubism  but also other TLR ligands, LPS (TLR4) or ssRNA (TLR7), induced elevated IL-6 protein levels in the culture supernatant as well as the Il6 transcript levels compared with WT cells  repository at the University of California, Davis (project ID CSD46418). Chimeric mice were generated by diploid aggregation of embryonic stem cells, and the Institute of Cancer Research (ICR) embryos and heterozygous F1 mice harboring a targeted Tnks2 allele were obtained by crossing of chimeric males with C57BL/6 female mice. These mice were crossed with LysM-Cre mice expressing Cre recombinase in the myeloid cell lineage including monocytes, macrophages, and granulocytes (The Jackson Laboratory)  .Tnks (WT), forward primer 5′-GGATGTGGGCATGCTTAAAA-3′, reverse primer 5′-CACAATGGACCTCAAAGCTG-3′; Tnks (KO), forward primer 5′-ACGTAAACTCCTCTTCAGACCTAATAAC-3′, reverse primer 5′-CACAATGGACCTCAAAGCTG-3′; Tnks2, forward primer 5′-CAGTGTCGTCTTCAGCTTGG-3′, reverse primer 5′-TGTACACATGAGCTCTGGGT-3′; Sh3bp2, forward primer 5′-ACAGCTCAGTGTTGGATTCCTGGCTC-3′, reverse primer 5′-ACCCTGACTGGTCGGTGTTTCAGAAC-3′.Tail DNA was extracted using a Wizard SV Genomic DNA Purification System (Promega), and PCR was performed using GoTaq Green Master Mix (Promega) and the following primers: 2 environment at 37°C. Bone marrow cells were cultured in α-MEM  supplemented with 10% FBS (Wisent) and 2% CMG (conditioned medium supernatant containing recombinant M-CSF) (All cultures were maintained in a 5% COt M-CSF) . HEK293TPrimary murine macrophages grown on glass coverslips were fixed with 4% formaldehyde for 10 minutes, permeabilized, and incubated with the indicated antibody. Following incubation with secondary antibody, nuclei were stained with DAPI . Slides were mounted using Aqua Mount (Thermo Fisher Scientific). Confocal imaging was performed with an Olympus IX81 inverted microscope and FluoView software (Olympus).Mouse cytokines and chemokines in the serum were analyzed using the Mouse Magnetic Luminex Screening Assay (R&D Systems) or the Mouse IL-6 ELISA Kit (MilliporeSigma or RayBiotech).Unless stated otherwise, all chemicals were purchased from MilliporeSigma. Antibodies were obtained from the following sources: anti–p-Tyr , anti–p-SRC (Y416) , anti-SRC , anti–p-SYK (Y525/526) , anti-SYK , anti–p-IκBα , anti-IκBα , anti-GAPDH , anti-TLR2 , anti–NF-κB , and anti–NF-κB  from Cell Signaling Technology; anti-MyD88  and anti-FLAG M2  from MilliporeSigma; anti-Myc  and anti-actin  from Santa Cruz Biotechnology; anti-HA (M180-3) from MBL International; anti–p-Tyr (4G10)  from Millipore; anti-PPP1R11 (ab171960) from Abcam; and anti-3BP2  from Abnova. Halt Protease and Phosphatase Inhibitor Cocktail was from Thermo Fisher Scientific. SYK inhibitor was from Millipore. PP2 and BMS 345541 were from Selleckchem.Rpl19, forward primer 5′-CTGAAGGTCAAAGGGAATGTG-3′, reverse primer 5′-GGACAGAGTCTTGATGATCTC-3′; Tnfa, forward primer 5′-TTGACCTCAGCGCTGAGTTG-3′, reverse primer 5′-CCTGTAGCCCACGTCGTAGC-3′; Il6, forward primer 5′-TGATGCACTTGCAGAAAACA-3′, reverse primer 5′-ACCAGAGGAAATTTTCAATAGGC-3′; and Tnks, forward primer 5′-CCCACTTCATAATGCCTGCT-3′, reverse primer 5′-GCTTTTGCTGAAGGATCTGC-3′. The relative expression of each mRNA normalized by the expression of Rpl19 was calculated by the ΔCt method.Total cellular RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN). The ImProm-II Reverse Transcription System (Promega) was used for reverse transcription, and quantitative real-time PCR was performed on the Step One Plus Real-Time PCR System (Applied Biosystems). The sequences of primers were as follows: g and 4°C. Immunoprecipitation was performed at 4°C with the indicated antibodies, and the products were collected on Dynabeads Protein A or G . GST-tagged protein was purified with Glutathione Sepharose 4B . For Western blotting, protein in whole-cell lysates was resolved by SDS-PAGE and transferred to PVDF membranes . Membranes were blocked in 5% BSA or 5% nonfat dried milk in PBST (PBS plus 0.1% Tween-20). The images are representative of 3 independent experiments. The relative integrated densities of each protein band were digitized by ImageJ (NIH) and are shown in the figures.Cells were lysed with Nonidet P-40 (NP-40) buffer  or RIPA buffer  supplemented with protease and phosphatase inhibitors as described previously . LysatesTissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections were stained with H&E. IHC was performed using anti-F4/80 , anti-Mac2 , anti–CSF-1R , anti-CD45R , anti-MPO , and anti-EPX  antibodies, and the antigen-antibody binding was detected using Simple Stain MAX PO (Nichirei) and the DAB Substrate Kit (Nichirei).In order to immunoprecipitate TLR2 protein complexes, HEK293T cells were lysed in NP-40 buffer  and subjected to immunoaffinity purification using immobilized anti-Myc affinity agarose beads as described previously , 55. Forg and 4°C, immunoprecipitation and Western blot were performed with the indicated antibodies.In vivo ubiquitin assay was performed as described previously , 58. BriTo assess the NF-κB transcriptional activity, we used the NF-κB–luciferase reporter construct . HEK293T cells were transiently cotransfected with NF-κB-luc reporter plasmid and phRL-CMV plasmid (Promega) with or without the indicated constructs using the LipoD293 DNA In Vitro Transfection Reagent (SignaGen Laboratories). Cells were lysed and assayed for firefly and Renilla luciferase activity using the Dual-Glo Luciferase Assay System (Promega). The data were expressed as the ratio of firefly to Renilla activity.Newly synthesized protein was labeled with Click-IT Metabolic Labeling Reagents for Proteins  according to the manufacturer’s instructions as described previously . PrimaryMyd88 (MGC clone) and cloning it into the BamHI/NotI sites of pEBG. GST-PPP1R11 plasmid was generated by amplifying human PPP1R11 from pCMV6 XL5 human PPP1R11 (OriGene) and cloning it in frame into the NotI/BamHI sites of pEBG. Flag-3BP2 plasmid (p3Xflag-CMV-10) was generated by amplifying mouse Sh3bp2 and cloning it into the EcoRI site of p3Xflag-CMV-10. HA-TRAF6 plasmid was generated by amplifying mouse Traf6 (MGC clone) and cloning it into the BamHI site of pSGT .Myc-TLR2 (WT) plasmid was generated by amplifying human TLR2  and cloning it into the XhoI site of pEF-BOS. Myc-TLR2 (6YF) plasmid was generated by amplifying gBlock fragments (Integrated DNA Technologies) and cloning them into the XhoI site of pEF-BOS . Myc-TLR2 (add-back) plasmids were generated by overlap extension PCR using primers with the desired mutations and cloning into the XhoI site of pEF-BOS. GST-MyD88 plasmid was generated by amplifying mouse Single-cell suspensions of the colon, lung, spleen, lymph node, and peripheral blood from mice were stained with a combination of fluorescently labeled antibodies on ice. All samples were analyzed by the LSRFortessa X-20 or FACSAria III Cell Sorter (BD Biosciences) and FlowJo software.q values less than 0.05) (Antigen microarrays were generated using 153 antigens as described previously . Brieflyan 0.05) .t test with JMP 7 (SAS Institute) to determine differences. P values of less than 0.05 were accepted as statistically significant.All results are shown as means ± SEM of data from at least 3 separate experiments. The data were subjected to ANOVA with Tukey-Kramer post hoc test or unpaired, 2-tailed All animal studies were approved by the Animal Research Council at University Health Network, Toronto, Canada, and by the IRB of the Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.YM and RR designed the experiments. YM performed experiments and analyzed the results. IDD, JLR, ML, SC, NL, HAA, JT, MFM, AC, FH, YA, TK, KS, and JW performed specific experiments and analyzed the results. YM and RR wrote the manuscript with helpful revision by HAA.


Anti-glioblastoma GBM) immunotherapy poses a great challenge due to immunosuppressive brain tumor environments and the blood brain barrier (BBB). Programmed death ligand 1 (PD-L1) is an immune checkpoint that mediated the immune resistance. Inhibition of PD-L1 by antibodies was widely studied to treat many type of cancers. However, the inefficient therapeutic immune response became a significant barrier for treatment of GBM. CRISPR/Cas9 gene editing can be used to knockout both membrane and cytoplasmic PD-L1, leading to an enhanced immunotherapeutic strategy. It is extremely difficulty to deliver CRISPR/Cas9 containing plasmid for translational and clinic applications. We have been developed a core-shell nanoparticle (NP) to carry CRISPR/Cas9 plasmid for PD-L1 knockout. The different NP formulations were made and optimized to deliver CRISPR/Cas9 plasmid. NPs were prepared by modifying the water temperature, sonication power and time and formulation time. The obtained NPs had a size of 115-160nm and a charge of 40-50mV. The size and charge were significantly altered after CRISPR/Cas9 plasmids were loaded into NPs (Cas9-NPs). Agarose gel electrophoresis showed that CRISPR/Cas9 plasmids were fully encapsulated by NPs with 1 and 2 ug. The positive DNA bands occurred with 4ng, indicating the overloaded CRISPR/Cas9 plasmid. Fluorescence microscopy determined Cas9-NPs uptake by U87 cells under a time-dependent manner. GFP tagged Cas9-NPs were treated to U87 cells for transfection evaluation. The obtained different NPs delivery of CRISPR/Cas9 exhibited various transfection efficiencies in U87 cells. Visualization of intracellular Cas9-NPs showed increases in uptake by U87 cells from 0.5, 1, 2, and 4 hours. The greater nuclear accumulation of Cas9-NPs was seen at 24 hours. A western blot assay determined the success of PD-L1 deletion by Cas9-NPs in human GBM U87 cells. NPs-based CRISPR/Cas9 gene-editing system has great potential as an immunotherapeutic platform to treat GBM.


Nutrient-responsive protein kinases control the balance between anabolic growth and catabolic processes such as autophagy. Aberrant regulation of these kinases is a major cause of human disease. We report here that the vertebrate nonreceptor tyrosine kinase Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) inhibits autophagy and promotes growth in a nutrient-responsive manner. Under nutrient-replete conditions, SRMS phosphorylates the PHLPP scaffold FK506-binding protein 51 (FKBP51), disrupts the FKBP51-PHLPP complex, and promotes FKBP51 degradation through the ubiquitin-proteasome pathway. This prevents PHLPP-mediated dephosphorylation of AKT, causing sustained AKT activation that promotes growth and inhibits autophagy. SRMS is amplified and overexpressed in human cancers where it drives unrestrained AKT signaling in a kinase-dependent manner. SRMS kinase inhibition activates autophagy, inhibits cancer growth, and can be accomplished using the FDA-approved tyrosine kinase inhibitor ibrutinib. This illuminates SRMS as a targetable vulnerability in human cancers and as a new target for pharmacological induction of autophagy in vertebrates. This study describes the discovery and characterization of a nutrient-sensitive signaling pathway that drives growth and inhibits autophagy in mammalian cells. This pathway, which involves the non-receptor tyrosine kinase SRMS and the PHLPP scaffold protein FKBP51, promotes tumor growth and is amenable to pharmacological inhibition. Evolutionarily ancient nutrient-sensitive kinases such as mTORC1 and AMPK control the metabolic switch between cell growth (when conditions are favorable) and adaptive survival (under times of scarcity) . PowerfuNonreceptor tyrosine kinases phosphorylate various substrates to regulate significant molecular pathways in mammalian development and cell growth . SRMS isWe previously identified the nonreceptor tyrosine kinase SRMS as a negative regulator of autophagy . SRMS ovFK506-binding protein 51  is a stress response protein that scaffolds specific protein–protein interactions . FKBP51 We report here the identification of FKBP51 as a bona fide endogenous substrate of SRMS. We demonstrate that SRMS phosphorylates FKBP51 to inhibit its scaffolding activity and promote its degradation through the ubiquitin-proteasome pathway. Phosphorylation of FKBP51 by SRMS prevents the interaction of AKT with the PHLPP phosphatase, which leads to persistent activation of AKT under nutrient-replete conditions. Therefore, SRMS inhibits autophagy and accelerates tumor growth by direct phosphorylation of FKBP51.+-ATPase inhibitor bafilomycin A1, which inhibits lysosomal degradation of autophagosomes [We previously reported that SRMS depletion activates autophagy as evidenced by increased formation of LC3-positive puncta accompanied by the lysosome-dependent degradation of LC3 and p62 in an ULK1- and ATG5-dependent manner . To deteagosomes . We founWe next tested whether the catalytic lysine of SRMS (K258) is required for inhibition of autophagy by SRMS. Catalytic SRMS mutant (K258A) rendered SRMS enzymatically inactive as evidenced by loss of autophosphorylation . OverexpWe next asked whether acute pharmacological inhibition of SRMS activates autophagy. The tyrosine kinase inhibitor (TKI) ibrutinib was developed as a covalent inhibitor of bruton tyrosine kinase (BTK) but has also been reported to bind SRMS ,18. SRMSTo confirm this, we generated an ibrutinib-resistant version of SRMS for rescue experiments. We designed the mutant by homology modeling the SRMS kinase domain and using unbiased docking to propose a structural model through which ibrutinib may bind and inhibit SRMS . This moReconstitution of SRMS knockout U2OS cells with either wild-type SRMS or SRMS(T302M) reduced the number of LC3-positive puncta per cell to an equivalent degree . This reFKBP5 gene , Flag-SRMS(WT), or empty Flag vector in HEK293 cells and identified interacting proteins using anti-Flag IP followed by mass spectrometry . We sougBP5 gene . We confBP5 gene . Direct BP5 gene . We obseBP5 gene . We nextBP5 gene . The regBP5 gene . We founBP5 gene , S3F FigBP5 gene . NotablyBP5 gene . CollectBP5 gene . TyrosinBP4 gene . FKBP51 We next asked whether SRMS controls the activity of FKBP51. One reported function of FKBP51 is to inhibit the PI3K-AKT signaling pathway, which itself plays key roles in promoting cell growth and proliferation and inhibiting apoptosis and autophagy ,22,23. SWe next observed that overexpression of SRMS reduced the interaction between ectopic FKBP51 and PHLPP1 . ConversWe also observed an SRMS-dependent reduction in FKBP51 abundance in response to nutrient stimulation , S4F FigThese mechanistic studies suggest a specific molecular pathway through which SRMS may inhibit autophagy . Nutrienp < 0.0001). The most common oncogenic driver in the METABRIC cohort was activation of the PIK3CA gene by mutation and/or amplification, which occurred in 42% of patients. Co-occurrence between SRMS amplification and PIK3CA activation was significantly less than would be expected by chance . These data are consistent with the possibility that SRMS amplification and overexpression may be previously unrecognized drivers of unrestrained PI3K-AKT signaling in human cancers.Human cancer cells from diverse tissues of origin commonly harbor mutations and copy number variations that drive unrestrained PI3K-AKT signaling through enhanced activation or impaired inactivation of the pathway ,25. We uTo investigate whether SRMS signaling contributes to cancer growth, we inactivated SRMS in human cancer cells and measured the effects on growth in culture and in vivo. SRMS knockout MDA-MB-231 cells were profoundly impaired in anchorage-independent growth . SRMS knConstitutively active AKT rescued anchorage-independent growth of SRMS knockout MDA-MB-231 cells, indicating that AKT acts downstream of SRMS to promote growth . To invePI3K-AKT signaling promotes tumor growth through phosphorylation of myriad substrates that control diverse metabolic and cell survival pathways. Whether autophagy inhibition per se plays any role in promotion of tumor growth by SRMS-FKBP51-AKT signaling is an important question. To address this, we measured the growth of human breast cancer cells after depletion of SRMS alone versus co-depletion of SRMS and core autophagy genes. We found that depletion of SRMS impaired short-term two-dimensional growth in an autophagy-independent manner , whereasIbrutinib inhibited anchorage-independent growth in parental MDA-MB-231 cells in a dose-dependent manner . In contSRMS is a nonreceptor tyrosine kinase of the BRK family and is structurally similar to BRK and FRK. It is expressed in multiple tissues and at multiple developmental stages within the vertebrate lineage ,33. AnalBoth SRMS and FKBP51 are evolutionarily restricted to the bony vertebrate lineage . Mice laWhereas the role of FKBP51 in preventing Paget’s disease of bone appears to be independent of autophagy regulation, the ability of the FKBP51-PHLPP-AKT-BECN1 complex to induce autophagy is determinant of outcome in other settings including antidepressant treatment efficacy and coronavirus infection ,22,48. M+ breast cancer cells [We have shown here that ibrutinib PCI-32765) can inhibit SRMS in vitro and in cells. Ibrutinib is an inhibitor of BTK, which is in the TEC family of nonreceptor tyrosine kinases. Since 2013, ibrutinib was approved by the FDA for treating B cell malignancies. Genetic ablation or knockdown of BTK significantly reduces tumor growth in mouse model of chronic lymphocytic leukemia (CLL) or lymphoma cells ,54. Ibruer cells . Moreoveer cells . Interes65 can iner cells –62, we cer cells . WhetherIn conclusion, the vertebrate tyrosine kinase SRMS restrains autophagy and drives cell growth by phosphorylating FKBP51 to prevent PHLPP-mediated inactivation of AKT. SRMS is amenable to pharmacological inhibition and may represent a previously unrecognized target for manipulating cell growth and autophagy for therapeutic benefit.Srms heterozygous mice [http://www.clst.riken.jp/arg/mutant%20mice%20list.html). MEFs, MDA-MB-231, HeLa, HEK293FT, and U2OS cells were cultured at 37°C in a 5% CO2 atmosphere in DMEM high-glucose GlutaMAX supplemented with 10% FBS, 100 units/mL penicillin, 100 units/mL streptomycin. MDA-MB-435 and MCF7 cells were grown in RPMI supplemented with 10% FBS, 100 units/mL penicillin, 100 units/mL streptomycin. siRNA and plasmid transfections were performed using Lipofectamine RNAiMAX, Lipofectamine 3000  and Metafectene  according to the manufacturers’ protocol. Starvation was performed by rinsing the cells and replacing the media with EBSS (Invitrogen) for 4 hours. Feeding was performed by rinsing the cells and replacing the media with fresh complete growth media for 4 hours.SRMS knockout and wild-type MEFs were isolated from embryonic offspring of ous mice  . Cells were incubated at 27°C for 5 to 7 days, and virus was collected and further amplified to achieve a high multiplicity of infection. For protein production, virus was added to Hi5 cells and the cells harvested after 72 hours. Cells were lysed in buffer containing 50 mM HEPES (pH 7.5 to 8.0), 200 mM NaCl, 5 mM DTT, 10% Glycerol and protease inhibitor cocktail. Purification was by GST affinity chromatography followed by size-exclusion chromatography in 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 5% Glycerol. The purified SRMS protein was activated by addition of 100 μM ATP and 10 mM magnesium and incubation at 4°C overnight.Codon optimized sequence for expression of human SRMS kinase domain (residues 221 to 486) in SRMS activity was measured using a peptide substrate mobility shift assay assembled from purified components. The SRMS substrate was 5-FAM-EFPIYDFLPAKKK-CONH2. Final reaction conditions were 25 nM SRMS protein, 100 mM HEPES (pH 7.5), 0.015% Brij-35, 5.0% DMSO, 0.004% Tween 20, 10 mM MgCl2 and 2 mM DTT, 1 uM peptide 27, and 100 uM ATP. If inhibitors or DMSO were used, they were allowed to incubate with SRMS for an hour at room temperature prior to initiation of the assay by adding peptide and ATP. Endpoint assays were run for 3 hours at which time the reaction was quenched with 40 mM EDTA. Data were collected using a LabChip EZ Reader  and analyzed using GraphPad (V8.2.1).A homology model of SRMS kinase domain was generated using SWISS-MODEL  using thHEK293 cells stably expressing 3xFLAG-SRMS K258A or 3xFLAG-vector were cultured in ten 15-cm plates. Cells were washed in cold PBS and scraped in cold PBS, spun down, and resuspended in FLAG Buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 1X protease inhibitor cocktail) for 15 minutes on ice. Lysate were spun down and filtered through a 0.45-μm filter and incubated with anti-FLAG beads (Sigma-Aldrich A2220) for 4 hours rotating at 4°C. And then, beads were washed by 1xTBS 5 times and eluted for 4 fractions by elution buffer (200 pg/ul FLAG peptide in 1xTBS) at 4°C rotator, 30 minutess each fraction. Eluted proteins were prepared with SDS sample buffer and run on SDS-PAGE gel to submit entire sample for mass-spec analysis.Cells were lysed in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 0.5% NP-40, 1 mM PMSF, tyrosine protein phosphatase inhibitor , and 1x protease inhibitor cocktail (Sigma). Cell lysates were incubated with indicated antibodies for 2 hours at 4°C and then with 30 μl of 50% slurry of protein A-conjugated Sepharose for the next 1 hour. For pull-down analysis, cell lysates were prepared as above and treated with NTA resins . Cell lysates were incubated with indicated antibodies for 2 hours at 4°C and then with 50 μl of 50% slurry of protein A/G-conjugated dynabeads for the next 10 minutes.Escherichia coli (BL21 strain) and cultured in YT media. Protein induction was initiated with 1 mM isopropylthio-β-D-galactoside to bacterial cultures at D0.6. Cell extracts were subjected to column chromatography using glutathione resins. Proteins bound to the columns were eluted as recommended by the manufacturers. GST pull-down assay was performed using purified GST and GST-FKBP51 protein incubating with purified Myc-SRMS in vitro transcribed and translated protein (TnT Quick Coupled Transcription/Translation Systems from Promega). In vitro binding assay was performed using purified GST-FKBP51 bound to glutathione Sepharose beads for 1 hour in binding buffer (25 mM Tris (pH 8.0), 2.7 mM KCl, 137 mM NaCl, 0.05% v/v Tween-20, and 10 mM 2-mercaptoethanol) and then blocked for 1 hour in binding buffer containing 5% (w/v) milk powder. In vitro translated Myc-SRMS or Myc-SRMS K258A proteins were then incubated with the bound beads for 1 hour, washed, and the proteins were eluted in SDS-sample buffer and subjected to SDS-PAGE and immunoblotting.GST and GST-FKBP51 constructs were expressed in 5 cells per well in 12-well plates (Corning #3513) and cultured at 37°C in a 5% CO2 atmosphere in DMEM high-glucose GlutaMAX supplemented with 10% FBS, 100 units/mL penicillin, and 100 units/mL streptomycin for 24 hours. Cells were treated as indicated and transferred to an IncuCyte S3 . The cells were imaged using the 20x objective at 440 to 480 nm to detect GFP and at 565 to 605 nm to detect RFP signal. Image analysis was performed using IncuCyte S3 software, and puncta were counted by Image J.Autophagosomes were visualized using either U2OS cells stably expressing the GFP-LC3 reporter or immunocytochemistry to detect endogenous LC3. In both cases, cells were seeded on coverslips and treated as indicated. Cells were then fixed by incubation with 3.7% paraformaldehyde in PBS for 10 to 15 minutes, washed 3 times with 0.1% (v/v) Triton X-100 or PBS, permeabilized with 0.5% Triton X-100 for 5 minutes or 100 μg/ml digitonin for 10 minutes, and treated with 3% BSA in PBS. For endogenous LC3 detection, primary antibody incubation was carried out at 1:200 dilution for 1 hour, and secondary antibody incubation was carried out 1:500 dilution for 30 minutes (with 3 washes after each incubation). Cells were then mounted, observed, and imaged using a confocal laser scanning microscope . Images were either blinded and the number of LC3-positive puncta per cell counted manually, or LC3-positive puncta were identified and counted in an automated fashion using Photoshop (Adobe). Autolysosomes were visualized using either acridine orange or the tandem monomeric RFP-GFP-LC3 reporter. For acridine orange, cells were seeded on coverslips and stained with 1 μg/mL acridine orange for 20 minutes. Cells were then observed and imaged using a confocal laser scanning microscope  at 609 to 695 nm to detect acidic vesicles and at 511 to 566 nm to detect the nonacidic acridine orange signal. Image analysis was performed using Cell Profiler. For the tandem monomeric RFP-GFP-LC3 reporter, U2OS cells stably expressing RFP-GFP-LC3 were plated at 2 × 102 atmosphere in DMEM high glucose GlutaMAX supplemented with 10% FBS, 100 units/mL penicillin, 100 units/mL streptomycin, and 250 g/mL G418 (ThermoFisher #10131). Cells were plated in G418-free media at 8 × 103 cells per 80 μl into each well of clear-bottom 96-well white plates (Costar #3903). Twenty-four hours later, cells were transfected with the indicated siRNA oligonucleotides using RNAiMax (Invitrogen #13778) or treated with DMSO, ibrutinib, or acalabrutinib as indicated. Seventy-two hours after transfection or 24 hours after compound treatment, the plate was equilibrated to room temperature for 15 minutes. A volume of Nano-Glo HiBiT Lytic Reagent (Promega #N3030) equal to the volume in each well was added and mixed by orbital shaker (300 rpm) for 5 minutes. Luminescence was measured using a PerkinElmer plate reader and normalized to either siControl-transfected wells or DMSO-treated wells as appropriate.Autophagic flux was measured using the Autophagy LC3 HiBiT Reporter Assay System from Promega. U2OS HiBiT-HaloTag-LC3 cells (Promega #GA1050) were cultured at 37°C in a 5% COMyc-SRMS was produced using TNT coupled reticulocyte lysate system (Promega). In vitro kinase assay was performed using GST-FKBP51 and Myc-SRMS in a reaction volume 50 μl compromising 20 μl kinase buffer  with or without 200 μM ATP. The reaction mixture was incubated at 30°C for 30 minutes. To terminate the reaction, 2X SDS sample buffer was added, and the samples were boiled at 100°C. The samples were then resolved via SDS/PAGE.http://crispor.tefor.net and was cloned in pSpCas9(BB)-2A-Pro(PX495) V2.0 . We transfected 2 μg of gRNA using Effectene (Qiagen) for 24 hours, changed to fresh media, and incubated for an additional 24 hours. Cells were then treated with 1 μg/ml of puromycin for 3 days. Single cells were sorted by flow cytometry into 96-well plates, clonally expanded, and verified for the desired modification via targeted next-generation sequencing and analysis with CRIS.py [For the generation of U2OS or MDA-MB-231 SRMS knockout cells, gRNA of SRMS-CACCGTATGACTTCACGGCGCGGTG was designed using the CRISPR design tool at  CRIS.py . Briefly2, 0.34 M sucrose, 10% (v/v) glycerol, 1 mM DTT, 0.1% (v/v) Triton X-100, and 1x protease inhibitor cocktail. After incubation for 5 minutes on ice, the samples were subjected to centrifugation for 5 minutes at 3,000g. The supernatants were again centrifuged at 15,000g for 15 minutes, and the soluble fractions were used as the cytosolic fractions. The pellets from the low-speed centrifugation were resuspended in 3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and 1x protease inhibitor cocktail, incubated on ice for 30 minutes, and centrifuged for 5 minutes at 3,000g. The insoluble fractions were used as the nuclear fractions.Subcellular fractionation was performed as described with minor modifications . BrieflyCg-PrkdcscidIl2rgtm1Wjl/SzJ (NOD scid gamma) mice. MDA-MB-231 SRMS KO cells (clone G10) that stably express Flag empty vector, Flag-SRMS wild-type, or Flag-SRMS K258A (kinase-dead) mutant were subcutaneously injected into the upper thigh of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NOD scid gamma) mice. Tumor volumes were calculated as (axaxb)/2, in which a is the smallest diameter and b the largest. Animal studies were approved by the St. Jude Institutional Animal Care and Use Committee (IACUC) under approval number 599-100565-10/18.MDA-MB-231 cells and MDA-MB-231 SRMS KO cells were subcutaneously injected into the upper thighs of NOD.For clonogenic growth assays on plates, wild-type, knockout, or rescued cells were plated in 6-well plates in triplicate. After 2 to 3 weeks, cells were fixed and stained with 0.05% (w/v) crystal violet and counted. For anchorage-independent growth soft agar assays, cells were suspended in 0.375% (w/v) Noble agar  supplemented with standard growth medium and overlaid on 0.5% (w/v) Noble agar. Cells were incubated for 3 to 4 weeks before colonies ≥100 μm in size were counted.siRNA transfections were performed using Lipofectamine RNAiMAX or Dharmafect 1 according to the manufacturer’s protocol. The siRNA oligonucleotides were purchased from Dharmacon  or SigmaCommercial antibodies used: anti-SRMS , anti-Xpress , anti-GAPDH , anti-HA , anti-FLAG , anti-Myc , anti-β-Actin , anti-Tubulin , anti-FKBP51 , anti-GSK3β , anti-phospho-GSK3β , anti-Akt , anti-phospho-Akt S473 , anti-phospho-Akt T308 , anti-SQSTM1/p62 , anti-LC3 , anti-LC3 , anti-p-Tyrosine , anti-p-Y-1000 Multimab , anti-Beclin-1 , anti-Fox01 , anti-p-Fox01 S256 , anti-mCherry , anti-TFIIH p89 .S1 FigA) The efficiency of depletion of endogenous SRMS protein by RNAi vs. CRISPR/Cas9-mediated gene editing was compared in U2OS cells by western blot. (B) Autophagosome biogenesis and autophagic flux were compared between SRMS KO and WT MEFs using LC3 immunocytochemistry. Representative images are shown. (C) Number of LC3-positive puncta  per cell was counted from n = 5 images (205–276 MEF cells) per condition. **p < 0.01, t test. (D) Autophagic flux was compared between SRMS WT and KO MEFs. The average number of LC3-positive puncta degraded per hour per cell was calculated from the data presented in n = 5 images (>200 cells) per condition. **p < 0.01, t test. (E) Parental and SRMS KO MDA-MB-231 cells were treated with DMSO or the indicated concentration of Bafilomycin A1 for 2 hours. Lysates were collected and analyzed by western blot. LC3 accumulation is heightened in SRMS KO cells relative to parental controls. (F) SRMS(K258A) is enzymatically inactive. Myc-SRMS(WT) and Myc-SRMS(K258A) were expressed in SRMS KO U2OS cells. Cell lysates were immunoprecipitated with anti-Myc antibody and blotted with anti-p-Tyr antibody to detect SRMS autophosphorylation. (G) Myc-SRMS(WT) and Myc-SRMS(K258A) were transiently expressed in U2OS cells stably expressing GFP-LC3. Cells were fixed and stained with anti-Myc antibody to detect transfected cells. Representative images are shown. For quantitation, see H) MS/MS fragmentation data for human SRMS AA 374–387 sequence LLKDDIY(+79.97)SPSSSK, M/z 810.3703, z2, showing b/y ions. MS/MS fragment ions at M/z 941.42 (b7) and M/z 922.34 (y8) represent characteristic ions that unambiguously identify Y380 phosphorylation. The data underlying the figure can be found in ((PDF)Click here for additional data file.S2 FigA) SRMS overexpression increases p-Tyr immunoreactivity. HeLa cells were transiently transfected with the indicated constructs. Twenty-four hours later, lysates were collected and analyzed by western blot using the indicated antibodies. (B) Ibrutinib inhibits SRMS activity in a dose-dependent manner. HEK293 cells stably expressing Myc-SRMS(WT) were treated with the indicated compounds at the indicated concentrations for 2 hours. Cell lysates were subjected to immunoblotting with indicated antibodies. (C) Ibrutinib activates LC3 lipidation in a dose-dependent manner. Parental MDA-MB-231 cells were treated with ibrutinib at the indicated concentrations for 4 hours. Cell lysates were immunoblotted with anti-LC3 antibody. (D) Ibrutinib activates autophagosome formation in a dose-dependent manner. U2OS cells stably expressing GFP-LC3 were treated with ibrutinib at the indicated concentrations for 4 hours. GFP-LC3 puncta were detected by confocal microscopy. Representative images are shown. (E–G) Ibrutinib activates autophagy in an SRMS-dependent manner as measured by acridine orange. Parental or SRMS KO U2OS cells were treated with DMSO or ibrutinib (0.5 μM or as indicated) for 8 hours. Cells were then stained with 1 μg/mL acridine orange for 20 minutes and imaged at the indicated wavelengths. Representative images are shown (E) along with quantitation . For panel F, n = 10 images (123 cells) for parental and n = 8 images (130 cells) for SRMS KO. G shows mean +/− standard deviation of n = 10 images (123 cells), n = 11 images (131 cells), n = 8 images (100 cells), and n = 9 images (79 cells) for parental and n = 8 images (130 cells), n = 8 images (139 cells), n = 8 images (128 cells), and n = 9 images (181 cells) for SRMS KO (left to right). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t test. (H) Ibrutinib induces autophagosome biogenesis and autophagosome–lysosome fusion. U2OS cells stably expressing RFP-GFP-LC3 were treated with 1 μM ibrutinib for 12 hours. Cells were imaged by IncuCyte. Cells having more than 3 puncta were counted. Mean + SD of n = 15 cells per condition is shown. **p < 0.01, ***p < 0.001, ****p < 0.0001, t test. (I) Ibrutinib induces autophagic flux, but acalabrutinib does not. U2OS Autophagy LC3 HiBiT Reporter cells were plated at 8,000 cells per well. (J) SRMS kinase activity restrains LC3 lipidation. Myc-SRMS(WT) and Myc-SRMS(T302M) were expressed in SRMS KO U2OS cells and treated with 0.5 μM ibrutinib for 4 hours. Cell lysates were immunoprecipitated with anti-Myc antibody and blotted with indicated antibodies. The data underlying the figure can be found in ((TIF)Click here for additional data file.S3 FigA, B) SRMS interacts with FKBP51 through its kinase domain. SRMS truncated constructs (schematized in A) were transfected with Flag-FKBP51 in HEK293FT cells. Cell lysates were subjected to IP with anti-Myc and blotted with indicated antibodies (B). (C) FKBP51 interacts with SRMS through its TPR1 domain. Schematic representation of the 9 truncated FKBP51 constructs.  Each FKBP51 truncated construct was transfected with Myc-SRMS in HEK293FT cells. Cell lysates were subjected to anti-Flag IP and blotted with indicated antibodies. (F) SRMS directly phosphorylates FKBP51. An in vitro kinase assay was performed using GST-FKBP51 and in vitro transcribed/translated Myc-SRMS(WT) or Myc-SRMS(K258A) proteins, in presence or absence of λ-phosphatase as indicated. Tyrosine phosphorylation of FKBP51 was probed via anti-pTyr antibody, and GST-FKBP51 proteins were detected via anti-GST antibody. (G) Tyrosine 54 of FKBP5/FKBP51 is evolutionarily conserved, while the paralog FKBP4/FKBP52 has phenylalanine or threonine at the corresponding residue. FKBP51, FK506-binding protein 51; GST, glutathione S-transferase; IB, immunoblot; IP, immunoprecipitation; SRMS, Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites; WT, wild-type.((TIF)Click here for additional data file.S4 FigA) SRMS promotes accumulation of cytosolic p-AKT(S473) in a kinase-dependent manner. SRMS KO U2OS cells were reconstituted with empty vector, Flag-SRMS(WT), or Flag-SRMS(K258A) as indicated. Lysates were collected, subjected to subcellular fractionation, and blotted with the indicated antibodies. Lamin B was used as a marker for nuclear fraction and α-tubulin for cytosolic fraction. (B) SRMS blocks the interaction between FKBP51 and PHLPP. FKBP51 was transfected with PHLPP and SRMS in 293T cells. Cell lysates were subjected to IP by anti-Flag antibody and blotted with indicated proteins. *nonspecific band. (C) WT SRMS or SRMS(K258A) mutant was expressed in SRMS KO U2OS cells. Lysates were collected and subjected to IP with anti-FKBP51 antibody or IgG control as indicated. Input (whole cell lysates) and immunoprecipitates were analyzed by western blot with the indicated antibodies. Note that FKBP51 interacts more strongly with AKT and PHLPP in cells expressing SRMS(K258A) compared to cells expressing WT SRMS. Also note that dephosphorylation of p-AKT(S473) is more prevalent in the whole cell lysate of cells expressing SRMS(K258A) mutant than WT SRMS. (D) U2OS cells in which all endogenous FKBP51 alleles were converted to the nonphosphorylatable FKBP51 Y54F mutant by CRISPR/Cas9 exhibit decreased accumulation of p-AKT at Ser 473 and increased LC3 lipidation relative to parental U2OS cells. Cell lysates were immunoblotted with the indicated antibodies. (E) The proposed signaling pathway through which SRMS promotes AKT signaling and restrains autophagy. (F) Nutrient-dependent FKBP51 reduction is not unique to MDA-MB-231 cells. MDA-MB-435 cells were incubated under starved or fed conditions for 4 hours. Cell lysates were immunoblotted with indicated antibodies. (G) FKBP51 is poly-ubiquitinated under fed condition. MDA-MB-231 cells were incubated under starved or fed conditions and treated with 10 nM MG132 for 4 hours. Cell lysates were subjected to IP by anti-FKBP51 antibody and blotted with indicated antibodies. (H) Poly-ubiquitination of FKBP51 requires SRMS. Parental or SRMS KO MDA-MB 231 cells (clone 15) were treated with 10 nM MG132 for 4 hours. Cell lysates were subjected to IP with anti-FKBP51 antibody and blotted with indicated antibodies. (I) FKBP51 protein stability increases with treatment of MG132 or ibrutinib. MDA-MB-231 cells were treated with 100 μg/mL cycloheximide in the presence of DMSO, 10 nM MG132, or 0.5 μM of ibrutinib for the indicated times. Lysates were immunoblotted with the indicated antibodies. FKBP51, FK506-binding protein 51; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IgG, immunoglobulin G; IP, immunoprecipitation; KO, knockout; SRMS, Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites; WT, wild-type.((TIF)Click here for additional data file.S5 FigA) KO of FKBP51 increases abundance of p-AKT at Ser 473. Lysates were analyzed by western blot with the indicated antibodies.  SRMS restrains Beclin 1-dependent autophagy. Parental U2OS cells were transfected with the indicated siRNA oligonucleotides alone or in combination, as indicated. Seventy-two hours later, the cells were fixed and stained with anti-LC3 antibody for imaging (B) or lysed and processed for western blot with the indicated antibodies (C). From left to right, mean +/− standard deviation of n = 3 images (60 cells), n = 3 images (48 cells), and n = 4 images (113 cells) are shown. *p < 0.1, ***p < 0.001, t test. (D) Parental and SRMS KO MDA-MB-231 cells were lysed and subjected to IP with anti-Beclin 1 antibody or control anti-IgG as indicated. Input (whole cell lysates) and immunoprecipitates were subjected to western blot analysis with the indicated antibodies. Note that Beclin 1 interacts more strongly with VPS34, UVRAG, and ATG14 (but not Rubicon) in the SRMS KO cells relative to parental control cells. (E) Parental MDA-MB-231 cells were treated with DMSO or ibrutinib (0.5 μM concentration for 4 hours). Anti-Beclin 1 IP was performed, and both input (whole cell lysates) and immunoprecipitates were analyzed by western blot with the indicated antibodies. Note that Beclin 1 interacts more strongly with VPS34, UVRAG, and ATG14 (but not Rubicon) in cells treated with ibrutinib relative to DMSO control. The data underlying the figure can be found in ((TIF)Click here for additional data file.S6 FigA) SRMS expression is elevated in human breast and colorectal tumors (red) relative to corresponding normal tissues (gray). *p < 0.05. (B) SRMS supports clonogenic growth of U2OS osteosarcoma cells. Parental and SRMS KO U2OS cells were plated sparsely and allowed to grow for 12 days. Colonies were stained with crystal violet, imaged, and counted. Mean + standard deviation of n = 3 replicates is shown along with representative images. **p < 0.01, t test. (C) SRMS supports clonogenic growth of MDA-MB-231 triple-negative breast cancer cells. Parental or SRMS KO MDA-MB-231 cells were seeded sparsely in 6-well plates and allowed to grow for 17 days. Colonies were stained with crystal violet, imaged, and counted. Mean + standard deviation of n = 3 replicates is shown along with representative images. **p < 0.01, t test. (D) The ability of WT SRMS vs. kinase-dead SRMS to rescue the clonogenic growth defect of SRMS KO U2OS cells were compared. Mean + standard deviation of n = 3 replicates is shown along with representative images. ns p > 0.05, ***p < 0.001, t test. (Relates to E) CRISPR-Cas9-mediated mutation of all FKBP51 alleles to nonphosphorylatable FKBP51 Y54F mutant into U2OS cells impairs growth. Clonogenic growth was compared between parental and FKBP51 Y54F edited cells. Mean + standard deviation of n = 3 replicates is shown along with representative images. *p < 0.05. (F) Constitutively active AKT rescues the growth defect of SRMS KO cells without conferring sensitivity to ibrutinib. EV or Myr-AKT was transfected in SRMS KO MDA-MB-231 cells. Cells were sparsely seeded in soft agar and treated with DMSO or 0.5 μM ibrutinib for 20 days. Mean + standard deviation of n = 3 replicates is shown along with representative images. ns p > 0.05, **p < 0.01, t test. (G–I) SRMS antagonizes the tumor suppressive function of WT Beclin 1 but not of Beclin 1 , which is not phosphorylated by AKT [G, H) or inhibited by 0.5 μM ibrutinib (I). Lysates were analyzed by western blot using the indicated antibodies (G), or cells were plated sparsely in soft agar and allowed to grow for 17 days before being imaged and counted . Mean + standard deviation of n = 3 replicates is shown along with representative images . ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, t test. The data underlying the figure can be found in p>0.05); RNAi, RNA interference; SRMS, Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites; WT, wild-type.Click here for additional data file.S7 FigA) MCF7 breast cancer cells were transfected with the indicated siRNA oligonucleotides alone or in combination in a standard 96-well proliferation assay. siLONRF1 served as a negative control. Seventy-two hours later, growth was measured by Cell Titer Glo assay and normalized to siLONRF1 control (top), and efficiency of knockdown was measured by western blot (bottom). (B) The same experiment as described in (A) was performed using T47D breast cancer cells. Means + standard deviation of n = 3 replicates are shown; ns p > 0.05, *p < 0.05, **p < 0.01, t test. The data underlying the figure can be found in p>0.05); siRNA, small interfering RNA; SRMS, Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites.((JPG)Click here for additional data file.S8 FigA) The ability of WT SRMS vs. SRMS(T302M) to rescue the clonogenic growth defect and ibrutinib resistance of SRMS KO U2OS cells was compared. Mean + standard deviation of n = 3 replicates is shown along with representative images. ***p < 0.001, t test. (B) The ability of WT SRMS vs. SRMS(T302M) to rescue the clonogenic growth defect and ibrutinib resistance of SRMS KO MDA-MB-231 cells was compared. Mean + standard deviation of n = 3 replicates is shown along with representative images. n.s, ***p < 0.001, t test. The data underlying the figure can be found in p>0.05); SRMS, Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites; WT, wild-type.((TIF)Click here for additional data file.S1 TableSpectral counts from each experiment are given.(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.