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Objective Lysosomal protein transmembrane 4 beta (LAPTM4B) is selectively expressed in hepatocellular carcinoma (HCC) cells and thus a potential biomarker for diagnosing HCC. In this study, we designed a novel 18F-labeled PET probe to non-invasively visualize LAPTM4B expression in mouse model of HCC tumor. Methods A PET targeting tracer named [18F]FP-LAP2H was radio-synthesized using a LAPTM4B targeting peptide, LAP2H, coupled with 4-nitrophenyl-2-[18F]fluoropropionate ([18F]NFP). Radio-stability, cell uptake, micro PET/CT imaging and ex vivo biodistribution were performed for determining its stability, cell binding specificity, and tumor targeting in vivo. Results [18F]FP-LAP2H was successfully synthesized with radiochemical yields of 6–14% (decay-corrected yield) and molar activity of 10–44 GBq/μmol. The tracer showed stable (~90%) in phosphate-buffered saline, pH 7.4, and in human serum (~80%) for 2 h. In vitro cell uptake studies indicated the radioactivity accumulation in HCC cells was LAPTM4B protein-specific. Micro PET/CT demonstrated that implanted LAPTM4B positive HepG2 and BEL7402 tumors could be clearly visualized. The ex vivo biodistribution studies demonstrated that the tumor/liver ratio were 1.80 ± 0.65 and 2.09 ± 0.68 in implanted HepG2 and BEL7402 tumors respectively. Negative control and blocking experiments revealed that the radioactivity uptake in the HCC tumor was LAPTM4B protein-specific. Conclusions [18F]FP-LAP2H appears to be a potential PET tracer for imaging LAPTM4B-positive HCC tumor. Further endeavors need to do to improve tumor/liver ratio.

Targeting metastatic esophageal squamous cell carcinoma (ESCC) has been a challenge in clinical practice. Emerging evidence demonstrates that C-X-C chemokine receptor 4 (CXCR4) highly expresses in ESCC and plays a pivotal role in the process of tumor metastasis. We developed a copper-64 (t1/2 = 12.7 h, 19% beta+) labeling route of NOTA-CP01 derived from LY2510924, a cyclopeptide-based CXCR4 potent antagonist, in an attempt to noninvasively visualize CXCR4 expression in metastatic ESCC. Precursor NOTA-CP01 was designed by modifying the C-terminus of LY2510925 with bis-t-butyl NOTA via a butane-1,4-diamine linker. The radiolabeling process was finished within 15 min with high radiochemical yield (>95%), radiochemical purity (>99%), and specific activity (10.5-21 GBq/μmol) (non-decay-corrected). The in vitro solubility and stability tests revealed that [64Cu]NOTA-CP01 had a high water solubility (log P = -3.44 ± 0.12, n = 5) and high stability in saline and fetal bovine serum. [64Cu]NOTA-CP01 exhibited CXCR4-specific binding with a nanomolar affinity (IC50 = 1.61 ± 0.96 nM, Kd = 0.272 ± 0.14 nM) similar to that of the parental LY2510924. The in vitro cell uptake assay indicated that the [64Cu]NOTA-CP01-selective accumulation in EC109 cells was CXCR4-specific. Molecular docking of the CXCR4/NOTA-CP01 complex suggested that the Lys, Arg, and NOTA of this ligand have a strong polar interaction with the key residues of CXCR4, which explains the tight affinity of [64Cu]NOTA-CP01 for CXCR4. To test the target engagement in vivo, prolonged-time positron emission computed tomography (PET) imaging was performed at 0.5, 4, 6, 8, 12, 16, and 24 h postinjection of [64Cu]NOTA-CP01 to the EC109 tumor-bearing mice. The EC109 tumors were most visible with high contrast to the contralateral background at 6 h postinjection. The tracer revealed receptor-specific tumor accumulation, which was illustrated by effective blocking via coinjection with a blocking dose of LY2510924. Quantification analysis of the prolonged-time images showed that there was obvious radioactivity accumulation in the tumor (1.27 ± 0.19%ID/g) with the best tumor-to-blood ratio (4.79 ± 0.06) and tumor-to-muscle ratio (15.44 ± 2.94) at 6 h postinjection of the probe. The immunofluorescence and immunohistochemistry confirmed the positive expression of CXCR4 in the EC109 tumor and ESCC and metastatic lymph nodes of patients, respectively. We concluded that [64Cu]NOTA-CP01 possessed a very high target engagement for CXCR4-positive ESCC and could be a potential candidate in the clinical detection of metastatic ESCC.

Jijin Yao,1,2,* Ying Wang,3,* Yujing Lin,4 Yingying Yang,5 Jingjing Wan,3 Xiaohua Gong,1 Fanwei Zhang,3 Wangjian Zhang,6 Tia Marks,7 Siyang Wang,1 Hongjun Jin,2 Hong Shan2,8 1The Cancer Center of the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, People’s Republic of China; 2Guangdong Provincial Key Laboratory of Biomedical Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, People’s Republic of China; 3Department of Nuclear Medicine, Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, People’s Republic of China; 4Department of Pathology, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong Province, 519001, People’s Republic of China; 5Department of Pharmacy, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, People’s Republic of China; 6Department of Medical Statistics, School of Public Health, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China; 7Department of Environmental Health Sciences, School of Public Health, University at Albany, State University of New York, Rensselaer, NY, 12144, USA; 8Department of Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hongjun JinGuangdong Provincial Key Laboratory of Biomedical Imaging, Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, People’s Republic of ChinaEmail jinhj3@mail.sysu.edu.cnHong ShanDepartment of Interventional Medicine, Guangdong Provincial Key Laboratory of Biomedical Imaging, Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, People’s Republic of ChinaEmail shanhong@mail.sysu.edu.cnIntroduction: To evaluate the role of maximal standardized uptake values (SUVmax) and total lesion glycolysis (TLG) from serial 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) for early prediction of neoadjuvant chemotherapy (NAC) response in locoregionally advanced nasopharyngeal carcinoma (LANPC).Methods: A total of 121 LANPC patients who completed pretreatment 18F-FDG PET/CT between June 2017 and July 2020 were retrospectively included. The median age of all the participants was 50 years old (range: 19– 74 years), with 94 (77.7%) males and 27 (22.3%) females. The SUVmax from the primary tumor site (SUVmax-PT) and the total lesion glycolysis from the primary tumor site (TLG-PT) were recorded. Tumor response was calculated according to the Response Evaluation Criteria in Solid Tumor (RECIST) 1.1 Criteria at two-week post-secondary NAC cycle. Patients who achieved an objectively partial or full reaction after two cycles of NAC were defined as ‘responders’, and patients who obtained stability or progression were classified as ‘non-responders’.Results: After two cycles of NAC, 96 patients were categorized as “responders” and 25 patients as “non-responders”. The optimal thresholds of the SUVmax-PT were 11.8 and 38.5 for the TLG-PT. Non-responders were significantly associated with high SUVmax-PT (HR, 3.49; 95% CI, 1.17– 10.36; p = 0.024) and TLG-PT (HR, 4.45; 95% CI, 1.44– 13.78; p = 0.010) in multivariate analysis. Recursive partitioning analysis (RPA) categorized patients into three prognostic groups based on SUVmax-PT and TLG-PT: high-response group, intermediate-response group, and low-response group, with corresponding favorable response rates of 94%, 80%, and 55%, respectively. Moreover, a nomogram was created based on metabolic parameters that precisely projected an individual’s response of NAC (C-index, 0.787; 95% CI, 0.533– 1.000).Conclusion: Pretreatment 18F-FDG PET/CT to measure SUVmax-PT and TLG-PT could be a useful non-invasive method for early indication of NAC efficacy. The nomogram based on PET/CT parameters may potentially provide direction for treatment decisions based on NAC levels.Keywords: nasopharyngeal carcinoma, neoadjuvant chemotherapy, tumor response, PET/CT, nomogram

Primary tumor (PT) and metastatic lymph node (MLN) status have a great influence on diagnosis and treatment of lung cancer. Our main purpose was to investigate the imaging characteristics of PT or MLN by applying the 18F-FDG PET dynamic modeling approach for non-small cell lung cancer (NSCLC). Dynamic 18F-FDG PET scans were performed for 76 lung cancer patients, and 62 NSCLC cases were finally included in this study: 37 with newly diagnosed early and locally advanced lung cancer without distant metastases (group M0) and 25 metastatic lung cancer (group M1). Patlak graphic analysis (Ki calculation) based on the dynamic modeling and SUV analysis from conventional static data were performed. For PT, both KiPT (0.050 ± 0.005 vs 0.026 ± 0.004 min−1, p 0.05). Both SUV PT and KiPT showed significant high values in squamous cell carcinoma than adenocarcinoma, but neither SUVPT nor KiPT showed significant differences between EGFR mutants versus wild types. The overall Spearman analysis for SUV and Ki from different groups showed variable correlation (r = 0.46–0.94). The dynamic modeling for MLN (KiMLN) showed more sensitive than the static analysis (SUV) to detect metastatic lymph nodes in NSCLC, although both methods were sensitive for PT. This methodology of non-invasive imaging may become an important tool to evaluate MLN and PT status for patients who cannot undergo histological examination. The clinical trial registration number is NCT03679936 (http://www.clinicaltrials.gov/).

Non-small cell lung cancer is the most common type of cancer with a poor prognosis, and development of an effective diagnostic method is urgently needed. Exosomal lncRNAs, a class of transcripts longer than 200 nucleotides packaged into exosomes, have been defined as an ideal diagnostic biomarker for cancer. However, little is known about the clinical utility of exosomal lncRNAs in NSCLC. Here, we aimed to identify exosomal lncRNAs as promising biomarkers for NSCLC diagnosis. First, serum exosomes from NSCLC patients were successfully isolated by a polymer precipitation kit and then identified by TEM, NTA and western blot analysis. A total of nine candidate lncRNAs were detected by qRT-PCR in a training set. The two exosomal lncRNA TBILA and AGAP2-AS1 were screened out for the higher levels in NSCLC patients than that of healthy controls in a validation set. And there was a significant positive correlation between these exosomal lncRNAs levels and tumor size, lymph node metastasis and TNM stage. Additionally, we validated that these exosomal lncRNAs were stable in serum. Next, we evaluated the diagnostic efficiency of exosomal lncRNAs in NSCLC patients by ROC curve analysis. The data showed that individual TBILA or AGAP2-AS1 exhibited better diagnostic efficiency in NSCLC patients with different tumor pathologic subtypes and early stage, whereas the combination of lncRNAs did not provide better results than individual lncRNAs. Notably, the combination of two exosomal lncRNAs and the serum tumor biomarker Cyfra21-1 widely used in clinical practices further improved the diagnostic accuracy for NSCLC patients. This study suggests that exosomal lncRNA TBILA and AGAP2-AS1 may be promising biomarkers for diagnosis of NSCLC.

Rapid progress has been made to identify and study the causative agent leading to coronavirus disease 2019 (COVID‐19) but many questions including who is most susceptible and what determines severity remain unanswered. Angiotensin‐converting enzyme 2 (ACE2) is a key factor in the infection process of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). In this study, molecularly specific positron emission tomography imaging agents for targeting ACE2 are first developed, and these novel agents are evaluated in vitro, in preclinical model systems, and in a first‐in‐human translational ACE2 imaging of healthy volunteers and a SARS‐CoV‐2 recovered patient (NCT04422457). ACE2 expression levels in different organs in live subjects are quantitatively delineated and observable differences are measured in the patient recovered from COVID‐19. Surprising sites of uptake in the breast, reproductive system and very low uptake in pulmonary tissues are reported. This novel method can add a unique tool to facilitate SARS‐CoV‐2 related research and improve understanding of this enigmatic disease. Molecular imaging provides quantitative annotation of ACE2, the SARS‐CoV‐2 entry receptor, to noninvasively monitor organs impacted by the COVID‐19., Severe acute respiratory syndrome coronavirus‐2 gains infect cells through binding to the angiotensin‐converting enzyme 2 (ACE2) receptor. Using a high affinity radiolabeled peptide to the receptor, the whole body and quantitative determination of ACE2 expression can be imaged. This molecular imaging approach enables animal model and clinical evaluation of this critical receptor, noninvasively.

BackgroundMost esophageal squamous cell carcinoma (ESCC) imaging diagnoses can be performed by routine CT and ultrasound, but it is difficult to detect metastatic lymph nodes or minor lesions. Functional imaging diagnosis based on 18F-FDG PET/CT has potential advantages for detection of metastatic lymph nodes, or differentiation of benign from malignant lymph nodes, and for typing and staging of ESCC. The purpose of this study is to provide 18F-FDG PET/CT imaging for ESCC patient to quantify the difference between malignant lymph nodes (MLN) and benign lymph nodes (BLN) for ESCC. MethodsDynamic 18F-FDG PET/CT was performed in 46 patients (26 patients without MLN (N0 stage) and 20 with MLN (non-N0 stage) who were pathologically confirmed for ESCC. Visual and quantitative differences were measured in primary tumor (PT), MLN and BLN regions of interest (ROIs). Finally, 52 MLN and 133 BLN (83 from N0 stage and 50 from non-N0 stage) were included for analysis. Pharmacokinetic analysis was performed by a Patlak model using Matlab program to obtain the influx constant (Ki). Maximum standardized uptake value (SUVmax) was also determined from the static and dynamic PET/CT scans. Based on the receiver operator characteristic (ROC) curve, the sensitivity and specificity for each parameter in differentiation diagnosis were evaluated. ResultsKi and SUVmax in PT non-N0 group was slightly higher than in N0 groups (0.04 ± 0.02 vs 0.03 ± 0.03, 8.01 ± 3.90 vs 7.08 ± 5.39, respectively), but with no significant difference (p > 0.05). And Ki and SUVmax in MLN were higher than BLN with statistically significant difference (KiMLN vs KiBLN ( 0.021 ± 0.014 vs 0.006 ± 0.004, p < 0.0001); (SUVmaxMLN vs SUVmaxBLN (4.35 ± 2.27 vs 1.89 ± 0.85, p < 0.0001); The sensitivity both Ki and SUVmax were 80.77 %, the specificity for Ki was 89.47%, and SUVmax 87.22% respectively. And the diagnostic accuracy Ki (90.61%) was slightly better than SUVmax (88.16%). Conclusions Quantitative parameters (both Ki and SUVmax) of 18F-FDG in ESCC patients are sensitive diagnostic measurements capable to identify MLNs from BLNs.

Rationale: Current therapies for hepatocellular carcinoma (HCC) are hampered by treatment failure and recurrence due to the remaining treatment-resistant liver cancer stem cells (CSCs). Stemness and epithelial-mesenchymal transition (EMT) are regarded as two fundamental characteristics of liver CSCs necessary for cancer progression; thus, drugs that simultaneously target both characteristics should prove effective in eliminating HCC and impeding recurrence. In this study, we developed new arsenic trioxide (ATO)-based nanoparticles (NPs), which are expected to be more effective than the current HCC therapy, and explored their potential mechanism. Methods: A “one-pot” reverse emulsification approach was employed to prepare the ZnAs@SiO2 NPs. HCC cell lines, MHCC97L and Hep3b, were used to analyze the antitumor activity of ZnAs@SiO2 NPs in vitro and in vivo by quantifying cell growth and metastasis as well as to study the effect on stemness and EMT. SHP-1 siRNA was used to validate the role of the SHP-1/JAK2/STAT3 signaling pathway in mediating inhibition of stemness and EMT by ZnAs@SiO2. Results: Compared with the current ATO treatment, ZnAs@SiO2 NPs promoted apoptosis and significantly inhibited proliferation, migration, and invasion of both MHCC97L and Hep3b cells. In the in vivo assay, ZnAs@SiO2 NPs inhibited tumor growth by 2.2-fold and metastasis by 3.5-fold as compared to ATO. The ZnAs@SiO2 NPs also inhibited tumor spheroid formation in vitro and tumor initiation in vivo and induced significant changes in the expression of stemness markers (CD133, Sox-2, and Oct-4) and EMT markers (E-cadherin, Vimentin, and Slug) both in vitro and in vivo. These effects of ZnAs@SiO2 that correlated with prognosis of HCC were mediated by the SHP-1/JAK2/STAT3 signaling. Conclusions: ZnAs@SiO2 NPs can effectively suppress tumor initiation, growth, metastasis, and inhibit stemness and EMT through regulation of SHP-1/JAK2/STAT3 signaling pathway in liver cancer cells in vitro and in vivo. Thus, ZnAs@SiO2 NPs have immense potential for HCC treatment in the future.

The purpose of this study was to establish the quality control and quantify the novel 64Cu-NOTA-Trastuzumab in gastric cancer patient-derived xenografts (PDX) mice models and patients by applying the molecular imaging technique. Trastuzumab was labeled with 64Cu using NCS-Bz-NOTA as bifunctional chelator, and hIgG1 was labeled with the same procedures as a negative control agent. HER2-positive (case 176, n = 12) and HER2-negative (case 168, n = 3) PDX models were established and validated by Western blot, DNA amplification, and immunohistochemistry (IHC). Both models were conducted for micro-PET imaging by tail injection of 18.5 MBq of 64Cu-NOTA-Trastuzumab or 64Cu-NOTA-hIgG1. Radioprobe uptake in tumor and main organs was quantified by region of interested (ROI) analysis of the micro-PET images and autoradiography. Finally, gastric cancer patients were enrolled in preliminary 64Cu-NOTA-Trastuzumab PET/CT scans. NOTA-Trastuzumab was efficiently radiolabeled with 64Cu over a 99% radiochemical purity and 17.5 GBq/μmol specific activity. The immune activity was preserved as the nonmodified antibody, and the radiopharmaceutical proved to be stable for up to 5 half-decay lives of 64Cu both in vitro and in vivo. Two serials of PDX gastric cancer models were successfully established: case 176 for HER2 positive and case 168 for HER2 negative. In micro-PET imaging studies, 64Cu-NOTA-Trastuzumab exhibits a significant higher tumor uptake (11.45 ± 0.42 ID%/g) compared with 64Cu-NOTA-IgG1 (3.25 ± 0.28 ID%/g, n = 5, p = 0.0004) at 36 h after intravenous injection. Lower level uptake of 64Cu-NOTA-Trastuzumab (6.35 ± 0.48 ID%/g) in HER2-negative PDX tumor models further confirmed specific binding of the radioprobe. Interestingly, the coinjection of 2.0 mg of Trastuzumab (15.52 ± 1.97 ID%/g) or 2.0 mg of hIgG1 (15.64 ± 3.54 ID%/g) increased the 64Cu-NOTA-Trastuzumab tumor uptake in PDX tumor (HER2+) models compared with 64Cu-NOTA-Trastuzumab alone ( p < 0.05) at 36 h postinjection. There were good correlations between micro-PET images and IHC ( n = 4) and autoradiography in PDX (HER2+) tumor tissues. Therefore, 64Cu-NOTA-Trastuzumab successfully translated to clinical PET imaging, and 64Cu-NOTA-Trastuzumab PET/CT scan in gastric cancer patients showed good detection ability. In conclusion, we reported quality control and application of novel 64Cu-NOTA-Trastuzumab for HER2 expression in PDX gastric cancer mice models and gastric cancer patients. Moreover, 64Cu-NOTA-Trastuzumab holds great potential for noninvasive PET detection, staging, and follow-up of HER2 expression in gastric cancer.

Cholinergic dysfunction in the central nervous system is an important characteristic of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). By using a rat EAE model, upregulation of vesicular acetylcholine transporter (VAChT) level in the EAE rat lumbar spinal cord was detected by western blot and immunostaining, and was associated with lymphocyte filtration and glial activation. Ex vivo and in vitro autoradiography studies with [(18)F]VAT, a VAChT-specific radioligand, also revealed increased tracer uptake in EAE rat lumbar spinal cord compared with shams. These studies on VAChT expression suggest central cholinergic imbalance during EAE progression.

Molecular imaging of vesicular acetylcholine transporter (VAChT) in the brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease. In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VAChT-specific positron emission tomography radiotracer, ((-)-(1-(-8-(2-fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)-methanone) ([18 F]VAT) in macaques. Total volume distribution (VT ) estimates were compared for one-tissue compartment model (1TCM), two-tissue compartment model (2TCM), Logan graphic analysis (LoganAIF) and multiple linear analysis (MA1) with arterial blood input function using data from three macaques. Using the cerebellum-hemispheres as the reference region with data from seven macaques, three additional models were compared: reference tissue model (RTM), simplified RTM (SRTM), and Logan graphic analysis (LoganREF). Model selection criterion indicated that a) 2TCM and SRTM were the most appropriate kinetics models for [18 F]VAT; and b) SRTM was strongly correlated with 2TCM (Pearson's coefficients r > 0.93, p 0.72). These studies demonstrate [18 F]VAT is a promising VAChT positron emission tomography tracer for quantitative assessment of VAChT levels in the brain of living subjects.

Positron emission tomography (PET) with phosphodiesterase 10A (PDE10A) specific radioligands provides a noninvasive and quantitative imaging tool to access the expression of this enzyme in vivo under normal and diseased conditions. We recently reported two potent 18F-labeled PDE10A radioligands (18F-TZ19106B and 18F-TZ8110); initial evaluation in rats and nonhuman primates indicated stable metabolic profiles and excellent target-to-nontarget ratio (striatum/cerebellum) for both tracers. Herein, we focused on in vivo characterization of 18F-TZ19106B and 18F-TZ8110 to identify a suitable radioligand for imaging PDE10A in vivo. We directly compared microPET studies of these two radiotracers in adult male Macaca fascicularis nonhuman primates (NHPs). 18F-TZ19106B had higher striatal uptake and tracer retention in NHP brains than 18F-TZ8110, quantified by either standardized uptake values (SUVs) or nondisplaceable binding potential (BPND) estimated using reference-based modeling analysis. Blocking and displace...

Objective The P2X7 receptor (P2X7R) is a key regulatory element in the neuroinflammatory cascade that provides a promising target for imaging neuroinflammation. GSK1482160, a P2X7R modulator with nanomolar binding affinity and high selectivity, has been successfully radiolabeled and utilized for imaging P2X7 levels in a mouse model of lipopolysaccharide-induced systemic inflammation. In the current study, we further characterized its binding profile and determined whether [C]GSK1482160 can detect changes in P2X7R expression in a rodent model of multiple sclerosis. Methods [C]GSK1482160 was synthesized with high specific activity and high radiochemical purity. Radioligand saturation and competition binding assays were performed for [C]GSK1482160 using HEK293-hP2X7R living cells. Micro-PET studies were carried out in nonhuman primates. In vitro autoradiography and immunohistochemistry studies were then carried out to evaluate tracer uptake and P2X7 expression in experimental autoimmune encephalomyelitis (EAE) rat lumbar spinal cord at EAE-peak and EAE-remitting stages compared with sham rats. Results [C]GSK1482160 binds to HEK293-hP2X7R living cells with high binding affinity (Kd=5.09±0.98 nmol/l, Ki=2.63±0.6 nmol/l). Micro-PET studies showed high tracer retention and a homogeneous distribution in the brain of nonhuman primates. In the EAE rat model, tracer uptake of [C]GSK1482160 in rat lumbar spinal cord was the highest at the EAE-peak stage (277.74±79.74 PSL/mm), followed by the EAE-remitting stage(149.00±54.14 PSL/mm) and sham (66.37±1.48 PSL/mm). The tracer uptake correlated strongly with P2X7-positive cell counts, activated microglia numbers, and disease severity. Conclusion We conclude that [C]GSK1482160 has the potential for application in monitoring neuroinflammation.

The overall prognosis for hepatocellular carcinoma (HCC) patients is poor but immunotherapeutic strategies may represent a novel and effective tool for HCC. However, the prediction of the early response for the immunotherapeutic effect of HCC remains a big challenge. We developed a novel near-infrared fluorescence (NIRF) probe (IRDye800-AbOX40) for OX40-targeted imaging. The H22 dual-tumor-bearing mice models were established and treated with CpG ODN intratumoral vaccination. Sixteen hours after vaccination, the mice were injected with the probe via the tail vein and conducted with NIRF imaging. The uptake of this probe in HCC tumors was greatly increased as early as 40 h post vaccination and reached a plateau between 54 and 112 h, while the untreated tumors showed a lower uptake, which was further confirmed by ex vivo imaging and flow cytometry. Immunofluorescence staining identified the colocalization of CD3 and OX40 in the tumor microenvironment. Moreover, immunohistochemistry analysis showed that OX40 expression level on tumor infiltrating lymphocytes (TILs) was associated with the fluorescence signal of the H22 tumors. IRDye800-AbOX40 could be used as a specific NIRF probe for noninvasive imaging of OX40 expression on TILs, which may aid in predicting the early response to immunotherapy of HCC.

Background Esophageal squamous carcinoma (ESCC) is one of the most common cancers in developing countries. However, currently there are no specific biomarkers for ESCC. This study evaluated the expression of proliferating cell nuclear antigen (PCNA), tumor suppressor protein p53, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) as biomarkers for ESCC. Methods This study included 60 clinical cases (30 ESCC and 30 non-ESCC cases that were confirmed pathologically). The expression of PCNA, p53, EGFR, and VEGF were investigated using a quantitative computerized immunohistochemistry (IHC) method. The expression level of each protein was indicated by a H-score from the quantitative analysis. Receiver operating characteristic curve (ROC) and area under curve (AUC) analyses were performed. The sensitivity and specificity of each individual protein and combinations of the proteins were calculated. Results The H-score analysis indicated that expressions of EGFR, PCNA, and VEGF were statistically significantly higher in ESCC than non-ESCC patients; however, p53 was not. The panels of combinations of these proteins were more sensitive than that of any single protein. In the triplicate combination, the AUC prediction probability increased to 0.86, while the single protein AUC prediction probabilities were 0.74 (EGFR), 0.80 (PCNA), and 0.70 (VEGF). Conclusions The high expression of PCNA, EGFR, and VEGF suggests that they are potential biomarkers for ESCC. The combination of these biomarkers may provide targets for molecular therapy and molecular imaging.

Accumulating evidence has defined nucleophosmin 1 (NPM1) mutation as a driver genetic event in acute myeloid leukemia (AML), whereas the pathogenesis of NPM1-mutated AML remains to be fully elucidated. In this study, we showed that mutant NPM1 elevated autophagic activity and autophagic activation contributed to leukemic cell survival in vitro. Meanwhile, we also found high expression of promyelocytic leukemia gene (PML) and its cytoplasmic dislocation in primary NPM1-mutated AML blasts and NPM1-mA positive OCI-AML3 cells. Mechanically, mutant NPM1 interacted with PML and mediated it delocalization as well as stabilization. Notably, NPM1-mA knockdown impaired autophagic activity, while induced expression of PML reversed this effect. Finally, we confirmed that PML modulated autophagic activity via AKT signal. These findings suggest that aberrant PML expression and autophagy are beneficial to the leukemic transformation driven by NPM1 mutations. This indicates an attractive therapeutic avenue for PML targeting and/or autophagy inhibition in the treatment of NPM1-mutated AML.

// Qin Zou 1 , Shi Tan 2 , Zailin Yang 3 , Juan Wang 4 , Jingrong Xian 1 , Shuaishuai Zhang 1 , Hongjun Jin 1 , Liyuan Yang 1 , Lu Wang 1 , Ling Zhang 1 1 Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, College of Laboratory Medicine, Chongqing Medical University, Chongqing, China 2 Department of Clinical Laboratory, Chongqing Health Center for Women and Children, Chongqing, China 3 Center for Hematology, Southwest Hospital, Third Military Medical University, Chongqing, China 4 Children’s Medical Laboratory Diagnosis Center, Qilu Children’s Hospital of Shandong University, Jinan, China Correspondence to: Ling Zhang, email: lingzhang@cqmu.edu.cn Keywords: nucleophosmin 1, mutation, microRNA, leukemia, differentiation Received: May 30, 2016 Accepted: September 16, 2016 Published: September 23, 2016 ABSTRACT Mutations in the nucleophosmin 1 (NPM1) gene are the most frequent genetic alteration in acute myeloid leukemia (AML). Here, we showed that enforced expression of NPM1 mutation type A (NPM1-mA) inhibits myeloid differentiation of leukemia cells, whereas knockdown of NPM1-mA has the opposite effect. Our analyses of normal karyotype AML samples from The Cancer Genome Atlas (TCGA) dataset revealed that miR-10b is commonly overexpressed in NPM1-mutated AMLs. We also found high expression of miR-10b in primary NPM1-mutated AML blasts and NPM1-mA positive OCI-AML3 cells. In addition, NPM1-mA knockdown enhanced myeloid differentiation, while induced expression of miR-10b reversed this effect. Finally, we showed that KLF4 is downregulated in NPM1-mutated AMLs. These results demonstrated that miR-10b exerts its effects by repressing the translation of KLF4 and that NPM1-mA inhibits myeloid differentiation through the miR-10b/KLF4 axis. This sheds new light on the effect of NPM1 mutations’ on leukemogenesis.

Upregulation of sphingosine-1-phosphate receptor 1 (S1PR1) expression in multiple sclerosis (MS) lesions is associated with neuroinflammatory response. This study investigated the correlation between neuroinflammation and S1PR1 expression in the spinal cord of an experimental autoimmune encephalomyelitis (EAE) rat model of MS, using the S1PR1 positron emission tomography (PET) radiotracer [(11)C]TZ3321.MicroPET imaging studies of [(11)C]TZ3321 were performed to measure uptake of [(11)C]TZ3321 in the spinal cord of EAE rats. Immunohistochemical staining was performed to confirm the overexpression of S1PR1 and other inflammatory biomarkers.MicroPET imaging demonstrated a 20-30 % increase in [(11)C]TZ3321 uptake in the lumbar spinal cord of EAE rats versus sham controls at 35-60 min post injection. The increased uptake of [(11)C]TZ3321 was correlated with the overexpression of S1PR1 in the lumbar spinal cord of EAE rats that was confirmed by immunohistochemical staining. Upregulated S1PR1 expression was associated with glial cell activation and immune cell infiltration.MicroPET imaging modality with a specific radioligand [(11)C]TZ3321 is able to assess the expression of S1PR1 in EAE rat lumbar spinal cord. This may provide a new approach to the assessment of neuroinflammatory response in MS and other inflammatory diseases.

BACKGROUND: To summarise the ultrasound manifestations of coronavirus disease-19 (COVID-19) patients with lung lesions and explore the clinical value of bedside ultrasound in the identification of patients at risk of progression to severe disease. METHODS: This retrospective study enrolled 31 patients with COVID-19 who were admitted to our hospital from January 18 to February 5, 2020. Lung ultrasounds were performed in all cases to evaluate the ultrasound manifestations of the patient’s lung lesions and to determine the lung ultrasound scores (LUS). The Cox proportional hazards regression model was used for the multifactor analysis of 7 candidate parameters, including the LUS and the oxygenation index (PaO(2)/FiO(2)). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive value of the LUS. RESULTS: Lung ultrasound images of COVID-19 patients mainly reflected the presence of interstitial pulmonary lesions (90.3%, 28/31). The lung lesions were primarily distributed in the subpleural and peripheral pulmonary zones. Multivariate analyses identified the oxygenation index, the LUS, and the lymphocyte count as factors related to the progression to severe-critical disease in COVID-19 patients (P

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a distinct leukemia entity in the 2016 updated WHO classification of myeloid neoplasm. Our previous report showed that autophagic activity was elevated in NPM1-mutated AML, but the underlying molecular mechanisms remain elusive. Mount of study provides evidence that glycometabolic enzymes are implicated in the autophagic process. Pyruvate kinase isoenzyme M2 (PKM2), a key glycolytic enzyme, has been recently reported as a tumor supporter in leukemia. However, little is known about the roles of PKM2 in autophagic activity in NPM1-mutated AML. In this study, PKM2 highly expressed in NPM1-mutated AML, and partially, high levels of PKM2 were upregulated by PTBP1. Further experiments demonstrated that PKM2 mediated autophagic activation and increased the phosphorylation of key autophagy protein Beclin-1. Importantly, functional experiments demonstrated that PKM2 contributed to cell survival via autophagic activation. Ultimately, high PKM2 expression was associated with short overall and event-free survival time in NPM1-mutated AML patients. Our findings indicate for the first time that glycolytic enzyme PKM2 mediates autophagic activation and further contributes to cell survival in NPM1-mutated AML, suggesting that PKM2 may serve as a promising target for treatment of NPM1-mutated AML.

Introduction Deficits in cholinergic function have been found in the aged brain and in neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for the cholinergic system. We previously reported the initial in vitro and ex vivo characterization of (−)-[ 11 C] TZ659 as a VAChT specific ligand. Here, we report the in vivo specificity, tracer kinetics, and dose-occupancy studies in the nonhuman primate brain. Methods MicroPET brain imaging of (−)-[ 11 C] TZ659 was performed under baseline conditions in two male macaques. Tracer kinetic modeling was carried out using a two-tissue compartment model (2TCM) and Logan plot with arterial blood input function and using a simplified reference tissue model (SRTM) and Logan plot (LoganREF) without blood input. Specificity for VAChT was demonstrated by pretreatment with (+)-pentazocine, (−)-vesamicol, or S -(−)-eticlopride. Target occupancy (Occ) was calculated following pretreatment with escalating doses of (−)-vesamicol. Results Baseline PET imaging revealed selective retention in the striatum with rapid clearance from the cerebellar hemispheres as a reference region. Total volume of distribution (V T ) values derived from both 2TCM and Logan analysis with blood input revealed ~ 3-fold higher levels of (−)-[ 11 C] TZ659 in the striatum than the cerebellar hemispheres. Injection of (−)-vesamicol either as a blocking or displacing agent significantly reduced striatal uptake of (−)-[ 11 C] TZ659 . In contrast, pretreatment with the sigma-1 ligand (+)-pentazocine had no impact. Pretreatment with the S -(−)-eticlopride, a dopamine D 2 –like receptor antagonist, increased striatal uptake of (−)-[ 11 C] TZ659 . Striatal binding potential (BP ND, range of 0.33–1.6 with cerebellar hemispheres as the reference region) showed good correlation (r 2 = 0.97) between SRTM and LoganREF. Occupancy studies found that ~ 0.0057 mg/kg of (−)-vesamicol produced 50% VAChT occupancy in the striatum. Conclusion (−)-[ 11 C] TZ659 demonstrated specific and reversible VAChT binding and favorable pharmacokinetic properties for assessing the density of VAChT in the living brain.

Phosphodiesterase 10A (PDE10A) plays a key role in the regulation of brain striatal signaling. A PET tracer for PDE10A may serve as a tool to evaluate PDE10A expression in vivo in central nervous system disorders with striatal pathology. Here, we further characterized the binding properties of a previously reported radioligand we developed for PDE10A, [(11)C]TZ1964B, in rodents and nonhuman primates (NHPs). The tritiated counterpart [(3)H]TZ1964B was used for in vitro binding characterizations in rat striatum homogenates and in vitro autoradiographic studies in rat brain slices. The carbon-11 labeled [(11)C]TZ1964B was utilized in the ex vivo autoradiography studies for the brain of rats and microPET imaging studies for the brain of NHPs. MicroPET scans of [(11)C]TZ1964B in NHPs were conducted at baseline, as well as with using a selective PDE10A inhibitor MP-10 for either pretreatment or displacement. The in vivo regional target occupancy (Occ) was obtained by pretreating with different doses of MP-10 (0.05-2.00 mg/kg). Both in vitro binding assays and in vitro autoradiographic studies revealed a nanomolar binding affinity of [(3)H]TZ1964B to the rat striatum. The striatal binding of [(3)H]TZ1964B and [(11)C]TZ1964B was either displaced or blocked by MP-10 in rats and NHPs. Autoradiography and microPET imaging confirmed that the specific binding of the radioligand was found in the striatum but not in the cerebellum. Blocking studies also confirmed the suitability of the cerebellum as an appropriate reference region. The binding potentials (BPND) of [(11)C]TZ1964B in the NHP striatum that were calculated using either the Logan reference model (LoganREF, 3.96 ± 0.17) or the simplified reference tissue model (SRTM, 4.64 ± 0.47), with the cerebellum as the reference region, was high and had good reproducibility. The occupancy studies indicated a MP-10 dose of 0.31 ± 0.09 mg/kg (LoganREF)/0.45 ± 0.17mg/kg (SRTM) occupies 50% striatal PDE10A binding sites. Studies in rats and NHPs demonstrated radiolabeled TZ1964B has a high binding affinity and good specificity for PDE10A, as well as favorable in vivo pharmacokinetic properties and binding profiles. Our data suggests that [(11)C]TZ1964B is a promising radioligand for in vivo imaging PDE10A in the brain of living subject.

Nine fluorine-containing vesicular acetylcholine transporter (VAChT) inhibitors were synthesized and screened as potential PET tracers for imaging the VAChT. Compound 18a was one of the most promising carbonyl-containing benzovesamicol analogs; the minus enantiomer, (−)- 18a displayed high potency (VAChT K i = 0.59 ± 0.06 nM) and high selectivity for VAChT versus σ receptors (>10,000-fold). The radiosynthesis of (−)-[ 18 F] 18a was accomplished by a two-step procedure with 30–40% radiochemical yield. Preliminary biodistribution studies of (−)-[ 18 F] 18a in adult male Sprague-Dawley rats at 5, 30, 60 and 120 min post-injection (p.i.) were promising. The total brain uptake of (−)-[ 18 F] 18a was 0.684 %ID/g at 5 min p.i. and by 120 min p.i. slowly washed out to 0.409 %ID/g; evaluation of regional brain uptake showed stable levels of ∼0.800 %ID/g from 5 to 120 min p.i in the VAChT-enriched striatal tissue of rats, indicating the tracer had crossed the blood brain barrier and was retained in the striatum. Subsequent microPET brain imaging studies of (−)-[ 18 F] 18a in nonhuman primates (NHPs) showed high striatal accumulation in the NHP brain; the standardized uptake value (SUV) for striatum reached a maximum value of 5.1 at 15 min p.i. The time–activity curve for the target striatal region displayed a slow and gradual decreasing trend 15 min after injection, while clearance of the radioactivity from the cerebellar reference region was much more rapid. Pretreatment of NHPs with 0.25 mg/kg of the VAChT inhibitor (−)-vesamicol resulted in a ∼90% decrease of striatal uptake compared to baseline studies. HPLC metabolite analysis of NHP plasma revealed that (−)-[ 18 F] 18a had a good in vivo stability. Together, these preliminary results suggest (−)-[ 18 F] 18a is a promising PET tracer candidate for imaging VAChT in the brain of living subjects.

Background Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been recognized as a distinct leukemia entity in the 2016 World Health Organization (WHO) classification. The genetic events underlying oncogenesis in NPM1-mutated AML that is characterized by a normal karyotype remain unclear. Inositol polyphosphate 4-phosphatase type II (INPP4B), a new factor in the phosphoinositide-3 kinase (PI3K) pathway-associated cancers, has been recently found a clinically relevant role in AML. However, little is known about the specific mechanistic function of INPP4B in NPM1-mutated AML. Methods The INPP4B expression levels in NPM1-mutated AML primary blasts and AML OCI-AML3 cell lines were determined by qRT-PCR and western blotting. The effect of INPP4B knockdown on OCI-AML3 leukemia cell proliferation was evaluated, using the Cell Counting Kit-8 and colony formation assay. After INPP4B overexpression or knockdown, the activation of serum and glucocorticoid-regulated kinase 3 (SGK3) and AKT was assessed. The effects of PI3K signaling pathway inhibitors on the levels of p-SGK3 in OCI-AML3 cells were tested. The mass of PI (3,4) P2 and PI (3) P was analyzed by ELISA upon INPP4B overexpression. Knockdown of SGK3 by RNA interference and a rescue assay were performed to confirm the critical role of SGK3 in INPP4B-mediated cell survival. In addition, the molecular mechanism underlying INPP4B expression in NPM1-mutated leukemia cells was explored. Finally, Kaplan–Meier survival analysis was conducted on the NPM1-mutated AML cohort stratified into quartiles for INPP4B expression in The Cancer Genome Atlas (TCGA) dataset. Results High expression of INPP4B was observed in NPM1-mutated AML. Knockdown of INPP4B repressed cell proliferation in OCI-AML3 cells, whereas recovered INPP4B rescued this inhibitory effect in vitro. Mechanically, INPP4B enhanced phosphorylated SGK3 (p-SGK3) status, but did not affect AKT activation. SGK3 was required for INPP4B-induced cell proliferation in OCI-AML3 cells. High levels of INPP4B were at least partially caused by the NPM1 mutant via ERK/Ets-1 signaling. Finally, high expression of INPP4B showed a trend towards lower overall survival and event-free survival in NPM1-mutated AML patients. Conclusions Our results indicate that INPP4B promotes leukemia cell survival via SGK3 activation, and INPP4B might be a potential target in the treatment of NPM1-mutated AML.

Sphingosine-1-phosphate receptor (S1PR) activation plays a key role in vascular inflammatory response. Here, we report in vivo validation of [ 11 C]TZ3321, a potent S1PR1 radioligand, for imaging vascular inflammation in a rat model of carotid injury. The right common carotid artery of male adult Sprague-Dawley rats was injured by balloon overinflation that denuded the endothelium and distended the vessel wall. Animals received a 60-minute micro-positron emission tomography (micro PET) scan with [ 11 C]TZ3321 at 72 hours after injury. Ex vivo autoradiography was also conducted. The expression and cellular location of S1PR1 were examined by immunohistological analysis. Three-dimensional (3D) reconstruction of the first 100-second microPET/computed tomography (CT) image indicated the location of bilateral common carotid arteries. [ 11 C]TZ3321 displayed significantly higher accumulation (standardized uptake values: 0.93 ± 0.07 vs 0.78 ± 0.09, n = 6, P = .001) in the injured carotid artery than in the contralateral side. Increased tracer uptake in the injured artery was confirmed by autoradiography (photostimulated luminescence measures: 85.5 ± 0.93 vs 71.48 ± 6.22, n = 2). Concordantly, high S1PR1expression was observed in infiltrated inflammatory cells in the injured artery. Our studies demonstrate [ 11 C]TZ3321 microPET is able to detect the acute upregulation of S1PR1 expression in inflamed carotid artery. Therefore, [ 11 C]TZ3321 has potential to be a PET radiotracer for detecting early inflammatory response and monitoring therapeutic efficacy of vascular inflammation.

PurposeThe vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. Herein, two potent and selective (11)C-labeled VAChT inhibitors were evaluated in rodents and nonhuman primates for imaging VAChT in vivo.ProceduresFor both (-)-[(11)C]2 and (-)-[(11)C]6, biodistribution, autoradiography, and metabolism studies were performed in male Sprague Dawley rats. Positron emission tomography (PET) brain studies with (-)-[(11)C]2 were performed in adult male cynomolgus macaques; 2 h dynamic data was acquired, and the regions of interest were drawn by co-registration of the PET images with the MRI.ResultsThe resolved enantiomers (-)-2 and (-)-6 were very potent and selective for VAChT in vitro (K i 35-fold selectivity for VAChT vs. σ receptors); both radioligands, (-)-[(11)C]2 and (-)-[(11)C]6, demonstrated high accumulation in the VAChT-enriched striatum of rats. (-)-[(11)C]2 had a higher striatum to cerebellum ratio of 2.4-fold at 60 min; at 30 min, striatal uptake reached 0.550 ± 0.086 %ID/g. Uptake was also specific and selective; following pretreatment with (±)-2, striatal uptake of (-)-[(11)C]2 in rats at 30 min decreased by 50 %, while pretreatment with a potent sigma ligand had no significant effect on striatal uptake in rats. In addition, (-)-[(11)C]2 displayed favorable in vivo stability in rat blood and brain. PET studies of (-)-[(11)C]2 in nonhuman primates indicate that it readily crosses the blood-brain barrier (BBB) and provides clear visualization of the striatum; striatal uptake reaches the maximum at 60 min, at which time the target to nontarget ratio reached ~2-fold.ConclusionsThe radioligand (-)-[(11)C]2 has high potential to be a suitable PET radioligand for imaging VAChT in the brain of living subjects.

The biological properties of the novel monoclonal antibody (mAb) H6-11 and its potential utility for oncological imaging studies were evaluated using in vitro and in vivo assays. Immunoreactivity of H6-11 to the human prostate cancer PC-3 cell line and solid tumor xenografts was initially demonstrated using immunofluorescence staining; the specificity of H6-11 for prostate cancer was further evaluated using a commercial array of human prostate cancer and normal tissue samples (n=49) in which H6-11 detected 95% of prostate adenocarcinomas. The Kd value of 61.7±30 nM was determined using 125I-labeled H6-11. Glycosylation analysis suggested the antigenic epitope of the glycan is an O-linked β-N-acetylglucoside (O-GlcNAc) group. Imaging studies of PC-3 tumor-bearing mice were performed using both optical imaging with NIR fluorescent dye-labeled H6-11 and microPET imaging with 89Zr-labeled H6-11. These in vivo studies revealed that the labeled probes accumulated in PC-3 tumors 48-72 h postinjection, although significant retention in liver was also observed. By 120 h postinjection, the tumors were still evident, although the liver showed significant clearance. These studies suggest that the mAb H6-11 may be a useful tool to detect prostate cancer in vitro and in vivo.

Background Post-translational protein modifications (PTMs) are increased in breast tumors. Objective We explored whether PTMs on proteins secreted by the breast could be detected in plasma and potentially used for the early detection of breast cancer. Methods We used a custom ELISA microarray platform to measure 4-hydroxynonenal (HNE), glutathione (GSH), nitrotyrosine and halotyrosine adducts in 27 secreted proteins, for a total of 108 candidate biomarkers. Two independent sets of human plasma samples were measured, for a total of 160 samples. The results were analyzed for consistent cancer-associated changes across the two sample sets. Plasma samples for both cases and benign controls were collected at the time of tissue diagnosis after referral from a positive screen (such as mammography). The results from both studies were evaluated using ANOVA and t-tests or receiver operator curves (ROC). Results Levels of GSH-modified ceruloplasmin and HNE-modified PDGF were significantly altered in plasma samples from cancer patients relative to benign controls. Healthy controls, which were only included in the first set of samples, were similar to the benign controls for both of these markers. A combination of three glutathionylated proteins produced the best area under the ROC curve, with a value of 76%. Conclusions Specific PTMs in individual proteins may be useful for distinguishing between women with breast cancer and those with benign breast disease. These oxidative changes in plasma proteins may reflect redox changes in breast cancer. Additional studies on oxidative modifications in individual proteins are warranted.

Nucleophosmin (NPM1) - a gene that encodes for a nuclear protein with multiple functions. Mutations in NPM1 are seen in approximately one-third of acute myeloid leukemia (AML) and are generally associated with good response to induction chemotherapy. However, the mechanisms underlying this chemosensitivity are still unknown. Recent studies have established that nuclear factor-κB (NF-κB) activation is a key response of leukemia cell to chemotherapy. In this study, we transfected human monocytic leukemia THP-1 cells with the vector expressing NPM1 mutation variant (NPM1mA), and confirmed overexpression of NPM1mA at mRNA and protein levels by reverse transcription PCR (RT-PCR) and immunohistochemistry, respectively. The effects of NPM1 mutations on chemotherapeutical agents induced apoptosis, NF-κB activity and gene expression were examined using flow cytometry, luciferase reporter assays, quantitative real time PCR (qRT-PCR) and Western blot. We found that overexpression of NPM1mA in THP-1 cells sensitized these cells to apoptosis induced by chemotherapeutical agents such as daunorubicin (DNR) and cytarabine (Ara-C). Moreover, we demonstrated that expression of NPM1 mA reduced the NF-κB transcription activity of THP-1 cells upon drug treatment. In addition, restoration of NF-κB activity via TNF-α stimulation could attenuate the effect of NPM1mA overexpression on DNR-and Ara-C-induced apoptosis. Interestingly, expression of NPM1mA could upregulate Bax and downregulate Bcl-2 at mRNA and protein levels in THP-1 cells when treated with DNR or Ara-C. We also demonstrated that restoration of NF-κB activity via TNF-α pre-treatment reversed the effect of NPM1mA on the Bax/Bcl-2 expression. Furthermore, evaluation of gene expression data from The Cancer Genome Atlas (TCGA) dataset revealed that NPM1-mutated patients showed a higher expression of Bax and a lower expression of Bcl-2. These results suggest that the NPM1 gene mutations could confer increased sensitivity to chemotherapeutic agents, at least in part, by suppressing NF-κB activity and regulating Bax/Bcl-2 expression.

Recent studies revealed that micro RNA-10b (mir-10b) is highly expressed in metastatic breast cancer cells and positively regulates breast cancer cell migration and invasion through inhibition of HOXD10 target synthesis. In this study we designed anti-mir-10b molecules and combined them with poly L-lysine (PLL) to test the delivery effectiveness. An RNA molecule sequence exactly matching the mature mir-10b minor antisense showed strong inhibition when mixed with PLL in a wound-healing assay with human breast cell line MDA-MB-231. The resulting PLL-RNA nanoparticles delivered the anti-microRNA molecules into cytoplasm of breast cancer cells in a concentration-dependent manner that displayed sustainable effectiveness.

Background: Nitric oxide is a physiological regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of smoking. Even so, it is unclear if this effect results from decreased nitric oxide production or increased oxidative degradation of nitric oxide to reactive nitrating species. These two processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. Objective: In this study, we evaluated associations of cigarette smoking and chronic obstructive pulmonary disease (COPD) with nitrotyrosine modifications of specific plasma proteins to gain insight into the processes regulating nitrotyrosine formation. Methods: A custom antibody microarray platform was developed to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. In a cross-sectional study, plasma samples from 458 individuals were analyzed. Results: Average nitrotyrosine levels in plasma proteins were consistently lower in smokers and former smokers than in never smokers but increased in smokers with COPD compared with smokers who had normal lung-function tests. Conclusions: Smoking is associated with a broad decrease in 3-nitrotyrosine levels of plasma proteins, consistent with an inhibitory effect of cigarette smoke on endothelial nitric oxide production. In contrast, we observed higher nitrotyrosine levels in smokers with COPD than in smokers without COPD. This finding is consistent with increased nitration associated with inflammatory processes. This study provides insight into a mechanism through which smoking could induce endothelial dysfunction and increase the risk of cardiovascular disease.

A variety of post-translational protein modifications (PTMs) are known to be altered as a result of cancer development. Thus, these PTMs are potentially useful biomarkers for breast cancer. Mass spectrometry, antibody microarrays and immunohistochemistry techniques have shown promise for identifying changes in PTMs. In this review, we summarize the current literature on PTMs identified in the plasma and tumor tissue of breast-cancer patients or in breast cell lines. We also discuss some of the analytical techniques currently being used to evaluate PTMs.

Background The objective of this study is to determine the radiation dosimetry of a novel radiotracer for vesicular acetylcholine transporter (−)-(1-((2R,3R)-8-(2-[18F]fluoro-ethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)-methanone ([18F]VAT) based on PET imaging in nonhuman primates. [18F]VAT has potential for investigation of neurological disorders including Alzheimer’s disease, Parkinson’s disease, and dystonia. Methods Three macaque fascicularis (two males, one female) received 185.4–198.3 MBq [18F]VAT prior to whole-body imaging in a MicroPET-F220 scanner. Time activity curves (TACs) were created from regions of interest (ROIs) that encompassed the entire small organs or samples with the highest activity within large organs. Organ residence times were calculated based on the TACs. We then used OLINDA/EXM 1.1 to calculate human radiation dose estimates based on scaled organ residence times. Results Measurements from directly sampled arterial blood yielded a residence time of 0.30 h in agreement with the residence time of 0.39 h calculated from a PET-generated time activity curve measured in the left ventricle. Organ dosimetry revealed the liver as the critical organ (51.1 and 65.4 μGy/MBq) and an effective dose of 16 and 19 μSv/MBq for male and female, respectively. Conclusions The macaque biodistribution data showed high retention of [18F]VAT in the liver consistent with hepatobiliary clearance. These dosimetry data support that relatively safe doses of [18F]VAT can be administered to obtain imaging in humans.

A cloud-point extraction (CPE) technique for the determination of a cyanobacterial hepatotoxin, nodularin-R, in aqueous media using a cationic surfactant, tricaprylmethylammonium chloride (Aliquat-336), was developed. Cloud-point phase separation of Aliquat-336 at ambient temperature was induced by the addition of sodium sulfate. The Aliquat-336/Na 2 SO 4 CPE system displayed large preconcentration factor, F C , for nodularin-R. At the operational CPE conditions, F C of 709.2 was achieved. Distribution constant, K D , of the distribution of nodularin-R between the surfactant-rich and aqueous phases of the CPE system was estimated to be (4.94±0.8)×10 3 . Coupled to liquid chromatography with UV detection, the CPE method offered a detection limit of 330 pg ml −1 (in freshwater)/1.3 ng ml −1 (in seawater) and a repeatability of 6.4% (in freshwater) ( n =7, P

Sphingosine-1-phosphate receptor 1 (S1PR1) is highly expressed in vascular smooth muscle cells from intimal lesions. PET imaging using S1PR1 as a biomarker would increase our understanding of its role in vascular pathologies including in-stent restenosis. The S1PR1 compound TZ3321 was synthesized for in vitro characterization and labeled with Carbon-11 for in vivo studies. The biodistribution of [11C]TZ3321 was evaluated in normal mice; microPET and immunohistochemistry (IHC) studies were performed using a murine femoral artery wire-injury model of restenosis. The high potency of TZ3321 for S1PR1 (IC 50 = 2.13 ± 1.63 nM), and high selectivity (>1000 nM) for S1PR1 over S1PR2 and S1PR3 were confirmed. Biodistribution data revealed prolonged retention of [11C]TZ3321 in S1PR1-enriched tissues. MicroPET imaging of [11C]TZ3321 showed higher uptake in the wire-injured arteries of ApoE−/− mice than in injured arteries of wild-type mice (SUV 0.40 ± 0.06 vs 0.28 ± 0.04, n = 6, P .05). Post-PET autoradiography showed >4-fold higher [11C]TZ3321 retention in the injured artery of ApoE−/− mice than in wild-type mice. Subsequent IHC staining confirmed higher expression of S1PR1 in the neointima of the injured artery of ApoE−/− mice than in wild-type mice. This preliminary study supports the potential use of PET for quantification of the S1PR1 expression as a biomarker of neointimal hyperplasia.

The σ1 receptor is an important target for CNS disorders. We previously identified a σ1 ligand TZ3108 having highly potent (Ki-σ1 = 0.48 nM) and selective affinity for σ1 versus σ2 receptors. TZ3108 was 18F-labeled with F-18 for in vivo evaluation. Biodistribution and blocking studies of [18F]TZ3108 in male Sprague-Dawley rats demonstrated high brain uptake, which was σ1-specific with no in vivo defluorination. MicroPET studies in cynomolgus macaques showed high brain penetration of [18F]TZ3108; the regional brain distribution was consistent with that of the σ1 receptor. Pseudo-equilibrium in the brain was reached ~ 45 min post-injection. Metabolite analysis of [18F]TZ3108 in NHP blood and rodent blood and brain revealed that ~ 70% parent remained in the plasma of NHPs 60 min post-injection and the major radiometabolite did not cross the blood-brain barrier in rats. In summary, the potent, selective and metabolically stable σ1 specific radioligand [18F]TZ3108 represents a potentially useful PET radioligand for quantifying the σ1 receptor in the brain.

The radiosyntheses and in vivo evaluation of four carbon-11 labeled quinoline group-containing radioligands are reported here. Radiolabeling of [ 11 C] 1 – 4 was achieved by alkylation of their corresponding desmethyl precursors with [ 11 C]CH 3 I. Preliminary biodistribution evaluation in Sprague-Dawley rats demonstrated that [ 11 C] 1 and [ 11 C] 2 had high striatal accumulation (at peak time) for [ 11 C] 1 and [ 11 C] 2 were 6.0-fold and 4.5-fold at 60 min, respectively. Following MP-10 pretreatment, striatal uptake in rats of [ 11 C] 1 and [ 11 C] 2 was reduced, suggesting that the tracers bind specifically to PDE10A. MicroPET studies of [ 11 C] 1 and [ 11 C] 2 in nonhuman primates (NHP) also showed good tracer retention in the striatum with rapid clearance from non-target brain regions. Striatal uptake (SUV) of [ 11 C] 1 reached 1.8 at 30 min with a 3.5-fold striatum:cerebellum ratio. In addition, HPLC analysis of solvent extracts from NHP plasma samples suggested that [ 11 C] 1 had a very favorable metabolic stability. Our preclinical investigations suggest that [ 11 C] 1 is a promising candidate for quantification of PDE10A in vivo using PET.

Two α-synuclein ligands, 3-methoxy-7-nitro-10H-phenothiazine (2a, Ki = 32.1 ± 1.3 nM) and 3-(2-fluoroethoxy)-7-nitro-10H-phenothiazine (2b, Ki = 49.0 ± 4.9 nM), were radiolabeled as potential PET imaging agents by respectively introducing 11C and 18F. The syntheses of [11C]2a and [18F]2b were accomplished in a good yield with high specific activity. Ex vivo biodistribution studies in rats revealed that both [11C]2a and [18F]2b crossed the blood-brain barrier (BBB) and demonstrated good brain uptake 5 min post-injection. MicroPET imaging of [11C]2a in a non-human primate (NHP) confirmed that the tracer was able to cross the BBB with rapid washout kinetics from brain regions of a healthy macaque. The initial studies suggested that further structural optimization of [11C]2a and [18F]2b is necessary in order to identify a highly specific positron emission tomography (PET) radioligand for in vivo imaging of α-synuclein aggregation in the central nervous system (CNS).

Airway inflammation has a pathophysiological role in asthma. Eosinophils, which are commonly increased in asthmatic airways, express eosinophil peroxidase and thereby produce hypobromite and bromotyrosine. Bromotyrosine is believed to be a specific marker for eosinophil activity, but developing an antibody against monobromotyrosine, the predominant brominated tyrosine residue found in vivo has proven difficult. We evaluated whether a 3-bromobenozoic acid hapten antigen produced antibodies that recognized halogenated tyrosine residues. Studies with small-molecule inhibitors or brominated or chlorinated protein suggested that a mouse monoclonal antibody (BTK-94C) selectively bound free and protein mono- and dibromotyrosine and, to a lesser degree, chlorotyrosine, and thus was designated a general halotyrosine antibody. We evaluated if this antibody had potential for characterizing human asthma using an enzyme-linked immunosorbent assay (ELISA) microarray platform to examine the halogenation of 23 proteins in three independent sets of sputum samples (52 samples total). In 15 healthy control or asthmatic subjects, ICAM, PDGF and RANTES had greater proportional amounts of halogenation in asthmatic subjects and the halogenation signal was associated with the severity of exercise-induced airway hyperresponsiveness. In 17 severe asthma patients treated with placebo or mepolizumab to suppress eosinophils, drug-related decreases in halogenation were observed with p values ranging from 0.006 to 0.11 for these 3 proteins. Analysis of 20 subjects that either had neutrophilic asthma or were healthy controls demonstrated a broad increase in halotyrosine (possibly chlorotyrosine) in neutrophilic asthmatics. Overall, these results suggest that an ELISA utilizing BTK-94C could prove useful for assessing airway inflammation in asthma patients.

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are derived as a result of inflammation and oxidative stress and can result in protein modifications. As such, these protein modifications are used as biomarkers for inflammation and oxidative stress. In addition, modifications in single-tissue-associated proteins released into blood can provide insight into the tissue localization of the inflammation or oxidative stress. We have developed an enzyme-linked immunosorbent assay antibody microarray platform to analyze the levels of 3-nitrotyrosine in specific proteins in a variety of biological samples, including human plasma and sputum. Selective-capture antibodies are used to immunoprecipitate individual proteins from samples onto isolated spots on the microarray chips. Then, a monoclonal antibody for 3-nitrotyrosine is used to detect the amount of 3-nitrotyrosine on each spot. Our studies suggest that this approach can be used to detect trace amounts of 3-nitrotyrosine in human plasma and sputum. In this paper, we describe our antibody microarray protocol for detecting 3-nitrotyrosine in specific proteins. Curr. Protoc. Toxicol. 51:17.15.1-17.15.16. © 2012 by John Wiley & Sons, Inc. Keywords: 3-nitrotyrosine; ELISA microarray; multiplex; biomarker

In this project, we focus on oxidation and glycosylation PTMs on proteins known to be secreted by the breast as candidate biomarkers for the early detection of breast cancer. ELISA microarray technology is employed to evaluate assays that have potential be used as breast cancer biomarkers. During the second year of this project we have continued the validation for our PTM-ELISA microarray. Overall, our data suggest that the PTM ELISA microarray platform is a promising tool for discovery and evaluation of biomarkers that have potential for the early detection of breast cancer. Besides the post-translational modification, we also investigated post-transcription factors (microRNAs) for breast cancer studies. Our studies suggested that microRNAs may be a potential promising biomarkers for breast cancer.

In this project, we focus on oxidation and glycosylation PTMs on proteins known to be secreted by the breast as candidate biomarkers for the early detection of breast cancer. ELISA microarray technology is employed to evaluate assays that have potential be used as breast cancer biomarkers. During the first year of this project we have validated a panel of ELISA capture antibodies for our PTM-ELISA microarray. And we tested 8 proposed PTM detection antibodies with clinical breast cancer plasma samples. Nitrotyrosine, bromotyrosine, chlorotyrosine, TF antigens, and AEG do not give significant difference in our test with clinical breast cancer samples. GSH, GloboH and 4-HNE were found to be altered with breast cancer plasmas samples. The sensitivity and the specificity of each assay and the combined assays were evaluated using the ROC analysis. In the best multivariate analysis, the AUC values ranged from 78% to 89% for 3 different PTMs (GSH, GloboH and 4-HNE). Overall, our data suggest that the PTM ELISA microarray platform is a promising tool for discovery and evaluation of biomarkers that have potential for the early detection of breast cancer.

In the chemokine family, we characterize two examples of evolutionarily conserved unfavorable sequence motifs that affect quaternary structure. In contrast to the straightforward action of favorable sequences, these unfavorable motifs produce interactions disfavoring one outcome to indirectly promote another one but should not be confused with the broad sampling produced by negative selection and/or design. To identify such motifs, we developed a statistically validated computational method combining structure and phylogeny. This approach was applied in an analysis of the alternate forms of homodimerization exhibited in the chemokine family. While the chemokine family exhibits the same tertiary fold, members of certain subfamilies, including CXCL8, form a homodimer across the beta1 strand whereas members of other subfamilies, including CCL4 and CCL2, form a homodimer on the opposite side of the chemokine fold. These alternate dimerization states suggest that CCL4 and CCL2 contain specific sequences that disfavor CXCL8 dimerization. Using our computational approach, we identified two evolutionarily conserved sequence motifs in the CC subfamilies: a drastic two-residue deletion (DeltaRV) and a simple point mutation (V27R). Cloned into the CXCL8 background, these two motifs were experimentally proven to confer a monomeric state. NMR analyses indicate that these variants are structured in solution and retain the chemokine fold. Structurally, the motifs retain a chemokine tertiary fold while introducing unfavorable quaternary interactions that inhibit CXCL8 dimerization. In demonstrating the success of our computational method, our results argue that these unfavorable motifs have been evolutionarily conserved to specifically disfavor one dimerization state and, as a result, indirectly contribute to favoring another.

This article, published ahead of print on 28 July 2008, has been withdrawn by the authors. Although moderate synergy between P2-RANTES and C peptides can be observed with high statistical significance in cell fusion assays, this synergy was not able to be verified in HIV viral assays. The authors regret the overstatement of synergy and will revise the paper for publication at a later date.

Altered post-translational modification (PTM) of glycosylations to various nuclear and cytosolic proteins is a universal feature of cancer cells and these modified glycan plan significant role in immune surveillance, tumor invasion, and increase malignancy. In this study, we reported a novel monoclonal antibody reacting to the tumor glycoproteins with molecular weight around 40-60 kDa. Deglycosylation examination suggested the antigenic isotope of the glycan is O-linked β-N-acetylglucosamine (O-GlcNAc) group. We examined the reactivity of H6-11 to cancer cells including PC-3, MCF-7 cells and implanted PC-3 and EMT-6 tumor tissues using immunofluorescence staining and autoradiography technique. The Zr-89 labeled H6-11 was successfully synthesized and the microPET study was performed in PC-3 xenograft prostate model in nude mice. microPET study showed that significant amount of radioactivity accumulation was observed in the excretory organs during the early period post-injection of radioactivity. At 24 hrs post-injection of radioactivity, significant amount of radioactivity was observed in the xenograft prostate tumor, while the radioactivity intensity was decreased in the liver and kidney. Radioactivity uptake in the tumor was still evident at 120 hrs post-injection of radioactivity. Our work suggested that the H6-11 antibody is capable of detecting prostate tumors both in vitro and in vivo. Citation Format: Hongjun Jin, Mai Xu, Prashanth K. Padakanti, Zhude Tu. Preclinical evaluation of monoclonal antibody H6-11 for prostate cancer imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2013-318

Commonly used in biotechnology applications, filamentous M13 phage are nonlytic viruses that infect E. coli and other bacteria, with the potential to promote horizontal gene transfer in natural populations with synthetic biology implications for engineering community systems. Using the E. coli strain TG1, we have investigated how a selective pressure involving elevated levels of toxic chromate, mimicking that found in some superfund sites, alters population dynamics following infection with either wild-type M13 phage or an M13-phage encoding a chromate reductase (Gh-ChrR) capable of the reductive immobilization of chromate (ie, M13-phageGh-ChrR). In the absence of a selective pressure, M13-phage infection results in a reduction in bacterial growth rate; in comparison, in the presence of chromate there are substantial increases in both cellular killing and biomass formation following infection of E. coli strain TG1with M13-phageGh-ChrR that is dependent on chromate-reductase activity. These results are discussed in terms of community structures that facilitate lateral gene transfer of beneficial traits that enhance phage replication, infectivity, and stability against environmental change.