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Background Neoadjuvant immunotherapy has shown promising clinical activity in the treatment of early non-small cell lung cancer (NSCLC); however, further clarification of the specific mechanism and identification of biomarkers are imperative prior to implementing it as a daily practice. Methods We investigated the impact of neoadjuvant chemoimmunotherapy (combining cisplatin with pembrolizumab) on an established preclinical NSCLC mouse model engrafted with human immune system, and explored its potential mechanism. We further explored potential biomarkers for neoadjuvant therapy and conducted in vivo validation using the model. In addition, both peripheral blood and tumor specimens from 14 patients undergoing neoadjuvant chemoimmunotherapy were analyzed to validate our findings. Results Neoadjuvant chemoimmunotherapy could prevent postoperative recurrence and metastasis by increasing the frequency and activation of CD8+ T cells in both peripheral blood and tumor immune microenvironment (TIME). The kinetics of peripheral CD8+PD-1+ T cells reflected the changes in the TIME and pathological responses, ultimately predicting survival outcome of mice. Further analysis of clinical specimens not only confirmed the reprogramming of TIME, but also validated CD8+PD-1+ T cells as rational biomarker. Conclusions The kinetics of peripheral CD8+ T cells can serve as a predictor for changes in TIME and overall antitumor immune responses, ultimately reflecting the outcomes of neoadjuvant chemoimmunotherapy in both preclinical and clinical setting. Our findings establish a theoretical foundation for the clinical translation of this promising biomarker.

Prostate cancer is the most common cancer among men and has a high incidence and associated mortality worldwide. It is an androgen-driven disease in which tumor growth is triggered via ligand-mediated signaling through the androgen receptor (AR). Recent evidence suggests that the widespread use of effective AR pathway inhibitors may increase the occurrence of neuroendocrine prostate cancer (NEPC), an aggressive and treatment-resistant AR-negative variant; however, mechanisms controlling NEPC development remain to be elucidated. Various preclinical models have recently been developed to investigate the mechanisms driving the NEPC differentiation. In the present study, we summarized strategies for the development of NEPC models and proposed a novel method for model evaluation, which will help in the timely and accurate identification of NEPC by virtue of its ability to recapitulate the heterogeneity of prostate cancer. Moreover, we discuss the origin and the mechanism of NEPC. The understanding of the regulatory network mediating neuroendocrine differentiation presented in this review could provide valuable insights into the identification of novel drug targets for NEPC as well as into the causes of antiandrogenic drug resistance.

Pancreatic adenocarcinoma (PAAD) is a highly malignant tumor of the digestive system with increasing morbidity and mortality. The lack of sensitive and reliable biomarkers is one of the main reasons for the poor prognosis. Volume-regulated anion channels (VRAC), which are ubiquitously expressed in the vertebrate cell membrane, are composed of leucine-rich repeat-containing 8A (LRRC8A) and four other homologous family members (LRRC8B–E). VRAC heterogeneous complex is implicated in each of the six “hallmarks of cancer” and represents a novel therapeutic target for cancer. In this study, LRRC8A was speculated to be a promising prognostic biomarker and therapeutic target for PAAD based on a series of bioinformatics analyses. Additional cell experiments and immunohistochemical assays demonstrated that LRRC8A can affect the prognosis of PAAD and is correlated to cell proliferation, cell migration, drug resistance, and immune infiltration. Functional analysis indicated that LRRC8A influences the progression and prognosis of patients with PAAD by the regulation of CD8+ T cells immune infiltration. Taken together, these results can help in the design of new therapeutic drugs for patients with PAAD.

Accumulating evidence suggests that the intestinal microbiota is associated with the antitumor efficacy of immune checkpoint inhibitors (ICIs) and the occurrence of immune-related adverse events (irAEs) following ICI treatment. However, the mechanisms underlying these interactions remain unclear. Recent technological advances have allowed more extensive investigation into the interplay between the intestinal microbiota and the tumor immune microenvironment. Breakthroughs by two research groups revealed that Bifidobacterium enhanced the efficacy of ICIs via the stimulator of interferon genes (STING) and adenosine 2A receptor (A2AR) signaling pathways, highlighting the molecular mechanisms through which the intestinal microbiota modulates immunotherapy. In this review, we summarize recent findings related to the potential role and mechanisms of the gut microbiota in ICI therapy, available microbiota-targeting strategies, and ongoing clinical trials. Further we discuss the associated challenges that remain in this field of research. The current review aims to evaluate the potential of the intestinal microbiota in maximizing the antitumor efficacy of ICIs while minimizing their toxic effects and guiding the development of more specific treatment regimens.

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A rapid colloidal gold immunochromatography assay (GICA) for the detection of pefloxacin (PEF) was established and optimized. The anti-PEF monoclonal antibody (mAb) was used to target PEF as a colloidal gold-mAb conjugate. The mAb belonged to the IgG2b subtype, lambda light chain, the affinity constant (Ka) was 5.21 × 10

Neuroendocrine transdifferentiation (NED) of prostate cancer (PCa) is the main cause of failure of androgen receptor inhibitor treatment. However, the molecular mechanisms underlying the development of NEPC, especially treatment-induced NEPC, remain unclear. Emerging evidence indicates that elevated monoamine oxidase A (MAOA) contribute to the proliferation, cell stemness, and bone metastasis in PCa. Here, we generated an enzalutamide-induced NED cell model to assess the role of MAOA during NED. Overall, MAOA expression was significantly increased upon Enz long-term exposure and was required for neuroendocrine marker expression. In particular, Enz was found to induce NED via the MAOA/mTOR/HIF-1α signaling axis. Further analyses revealed that the MAOA inhibitor clorgyline(CLG) may bring multiple benefits to CRPC patients, including better therapeutic effect and delays NED. These findings suggest that MAOA may be an important target for the development of anti-NED therapies, thereby providing a novel strategy for the combined application of CLG and AR inhibitors in the clinic.

[18F]FDG as a probe of PET/CT is a radiolabeled glucose analogue taken up by most cells, but its batch activity is limited. [68Ga]FAPI-04 is a promising alternative based on a fibroblast activation protein-specific inhibitor (FAPI) labeled with radiotracer FAP. Here, a series of databases suggested that FAP expression was significantly different in pancreatic cancer compared to normal tissue. The FAP-positive fibroblasts were evaluated around the tumor cells and the stroma. A patient-derived orthotopic xenograft (PDOX) model of pancreatic adenocarcinoma (PDAC) exhibits significantly higher quantitative uptake of [68Ga]FAPI-04 (P

Complex heterogeneity is an important characteristic in the development of prostate cancer (PCa), which further leads to the failure of known therapeutic options. PCa research has been hampered by the current in vitro model systems that cannot fully reflect the biological characteristics and clinical diversity of PCa. The tumor organoid model in three-dimensional culture retains the heterogeneity of primary tumor tissues in vitro well and enables high-throughput screening and genome editing. Therefore, the establishment of a PCa organoid model that recapitulates the diverse heterogeneity observed in clinical settings is of great significance for the study of PCa. In this review, we summarize the culture conditions, establishments, and limitations of PCa organoids and further review their application for the study of pathogenesis, drug screening, mechanism of drug resistance, and individualized treatment for PCa. Additionally, we look forward to other potential developmental directions of PCa organoids, such as the interaction between prostate cancer tumor cells and their microenvironment, clinical individualized treatments, heterogeneous transformation model, tumor immunotherapy, and organoid models combined with liquid biopsy. Through this, we provide more effective preclinical experimental schemes using the PCa organoid model.

S-trans–trans-Farnesylthiosalicylic acid (FTS) is a Ras inhibitor that exhibits desirable anticancer property and currently undergoing clinical trials for pancreatic cancer (PC). However, its poor ...

Background: Immunotherapy has dramatically changed the treatment landscape of cancer. Immunotherapies targeting the programmed death 1/programmed death ligand 1 pathway have been shown to lead to durable responses in patients with a variety of cancers. However, combination approaches are required to extend this benefit beyond a subset of patients. Studies have suggested that anti-angiogenic therapy can elicit or enhance tumor immunity response.Therefore, we hypothesised that combining immunotherapy with anti-angiogenic treatment may have a synergistic effect and may enhance the efficacy of both treatments.Methods: We evaluated the combination of bevacizumab (anti-vascular endothelial growth factor monoclonal antibody) and pembrolizumab (anti-programmed death 1 monoclonal antibody) in two mouse models of human non-small cell lung cancer cell lines (H1299 and A549). We monitored tumour growth and examined changes in the tumour vasculature, along with the frequency and phenotype of tumour-infiltrating lymphocytes.Results: The combination of bevacizumab and pembrolizumab synergistically inhibited tumour growth in vivo without overt toxicity in both cell lines. Combination therapy reprogrammed the immune microenvironment by increasing CD8+ cytotoxic T cell infiltration, thereby turning tumours with different phenotypes into inflamed (‘hot’) tumours. This is potentially mediated by vascular normalisation and endothelial cell activation, as demonstrated by a reduction in the number of microvessels and an increase in adhesion molecules.Conclusions: Taken together, our preclinical studies demonstrate that the combination of bevacizumab and pembrolizumab had a synergistic anti-tumour effect and provides a theoretical basis for translating basic research into clinical applications.

Tuberculosis (TB) is a major bacterial infectious disease worldwide that is predominantly caused by Mycobacterium tuberculosis (Mtb). The comorbidity of multiple drug-resistant TB strains with HIV and diabetes is widespread. In the presence of these diseases, host immunity is weakened, allowing the recovery of dormant bacilli and leading to recurrent TB infection. As an important component of the host innate and adaptive immune responses, macrophage autophagy plays a significant role in protecting the host against TB. However, dormant bacilli can escape from autophagosomes and/or suppress autophagy, thus surviving within the host for an extended period of time, although the underlying mechanism remains elusive. Heat shock protein 16.3 (Hsp16.3, HspX, Rv2031c, and Acr) is one of the immunodominant proteins expressed during latent TB infection (LTBI). It may help maintain the protein stability and long-term viability of Mtb by inhibiting macrophage autophagy, resulting in LBTI. In this review, we discuss how dormant bacilli escape from autophagosomes, and we focus on the role of Hsp16.3 in regulating macrophage autophagy in LTBI so as to provide a firm basis for further studies. Hsp16.3 may represent a potential biomarker of LTBI and novel pharmacological target for anti-tubercular drugs.

Bisphenol S (BPS) has been gradually used in all kinds of productions. Our previous study has demonstrated that BPS increases the obesogenic effects of a high-glucose diet through regulating lipid metabolism in Caenorhabditis elegans (C. elegans). Whether the effects pass on to the next generations remains uncovered. In the present study, C. elegans was selected as the model organism to investigate the effects of BPS on lipid metabolism in multiple generations. Oil Red O staining and triglyceride assays showed that multi-generational exposure to BPS in C. elegans significantly increased the fat accumulation in wild type worms, while not in the daf-16 gene-deficient worms. In addition, BPS affected the expressions of fat-7 and acs-2 in four generations of C. elegans. Furthermore, BPS promotes fat storage in C. elegans of multiple generations by the daf-16/nhr-49-mediated signaling pathway.

// Jiaze An 1, 2, * , Ningning Zhao 1, * , Caiqin Zhang 1 , Yong Zhao 1 , Dengxu Tan 1 , Ya Zhao 1 , Bing Bai 1 , Hai Zhang 1 , Boyang Jason Wu 3, ** and Changhong Shi 1, ** 1 Laboratory Animal Center, The Fourth Military Medical University, Xi’an, Shaanxi 710032, China 2 Department of Hepatobiliary and Pancreaticosplenic Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi 710032, China 3 Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Spokane, WA 99210, USA * These authors contributed equally to this work ** Joint corresponding authors Correspondence to: Changhong Shi, email: changhong@fmmu.edu.cn Boyang Jason Wu, email: boyang.wu@wsu.edu Keywords: heptamethine carbocyanine, hepatocellular carcinoma, HIF1α, near-infrared fluorescence, organic anion-transporting polypeptide Received: February 28, 2017 Accepted: April 17, 2017 Published: May 24, 2017 ABSTRACT Near-infrared fluorescence (NIRF) dyes have recently emerged as promising tools for non-invasive imaging of different types of cancers. Here, we explored the potential utility of a NIRF DZ-1 dye, with dual imaging and tumour targeting functions, in hepatocellular carcinoma (HCC). We showed the preferential uptake of DZ-1 by HCC cells in vitro and in derived subcutaneous/orthotopic tumour xenografts, accompanied by a minimal effect on normal cells. DZ-1 simplified tumour growth profiling as well, since we were able to correlate NIRF signals with tumour volume and/or tumour-emitting luminescence in mice. Using both orthotopic tumour transplantation and cirrhosis models in parallel, we demonstrated the ability of DZ-1 to differentiate liver tumour from cirrhosis. DZ-1 showed superiority in HCC imaging over indocyanine green by demonstrating significantly enhanced tumour-targeting specificity. At the cellular level, DZ-1 was mainly retained in mitochondria and lysosomes. Additionally, DZ-1 fluorescence spectroscopy has been used for the intraoperative navigation of rabbit liver cancer, to determine surgical margins. We showed that tumor hypoxia and select organic anion-transporting polypeptide genes mediate NIRF dye uptake in HCC, which was supported by clinical evidence. All these findings represent the first evidence that DZ-1 is an effective molecular probe for tumour-specific imaging in HCC, and provide insights into the development of a new generation of imaging agents for intraoperative guidance of cancer surgery.

Carbendazim, as a fungicide, was commonly used to control fungal diseases in agriculture, forestry, and veterinary medicines. In this study, the acute and reproductive toxicity of carbendazim was assessed using Caenorhabditis elegans (C. elegans) as a model in order to preliminarily evaluate the potential risks of this fungicide in agricultural production and application. The results showed that the growth of C. elegans was inhibited by 0.01 μg/L carbendazim. The treatment of 0.1 μg/L carbendazim caused a significant decrease in locomotion behavior and significant damage to the reproductive and antioxidant system, causing the lifespan of nematodes to be drastically shortened. These results provide a better understanding of the environmental risk of carbendazim and raise new concerns about safety.

Prostate cancer (PC) is a heterogeneous disease characterized by variable morphological patterns. Thus, establishing a patient-derived xenograft (PDX) model that retains the key features of the primary tumor for each type of PC is important for appropriate evaluation. In this study, we established PDX models of hormone-naive (D17225) and castration-resistant (B45354) PC by implanting fresh tumor samples, obtained from patients with advanced PC under the renal capsule of immune-compromised mice. Supplementation with exogenous androgens shortened the latent period of tumorigenesis and increased the tumor formation rate. The PDX models exhibited the same major genomic and phenotypic features of the disease in humans and maintained the main pathological features of the primary tumors. Moreover, both PDX models showed different outcomes after castration or docetaxel treatment. The hormone-naive D17225 PDX model displayed a range of responses from complete tumor regression to overt tumor progression, and the development of castrate-resistant PC was induced after castration. The responses of the two PDX models to androgen deprivation and docetaxel were similar to those observed in patients with advanced PC. These new preclinical PC models will facilitate research on the mechanisms underlying treatment response and resistance.

Bisphenol A (BPA) is a typical endocrine disruptor. Bisphenol S (BPS) has been widely used as a substitute for various plastic materials due to the limited application of BPA. However, it does not mean that BPS is a safe substitute due to the lack of effective evaluation of BPS. In this study, the clinical model of Caenorhabditis elegans (C. elegans) was used to study the effects of BPS on the locomotion behavior, growth, reproduction, lifespan and antioxidant system. Our study found that C. elegans exposed to 0.01 μM BPS could have significantly inhibited locomotion behavior and growth, as well as damaged reproductive and antioxidant systems and lifespan. It is interesting to note that in multi-generational exposure studies, we found that BPS exhibits complex genotoxicity. With the transmission to the offspring, BPS showed more significant inhibition of the head thrashes of the nematode, while the effect on the body bends and body length was gradually weakened. The effect of BPS on the brood size shows different rules according to different concentrations and offsprings. Therefore, the safety of BPS still needs further evaluation, especially the multi-generational genotoxicity.

New strategies to treat advanced bladder cancer are urgently required. A patient-derived xenograft (PDX) model has become a useful tool to evaluate chemotherapeutics and investigate personalized cancer treatment options. In this study, fresh human bladder cancer specimens (C09303 and C21391) were transplanted subcutaneously into nude mice to establish PDX models. These models well retained pathological characteristics and molecular markers of the original tumor. Primary cells derived from C09303 were cultured and perfused into the bladders of nude mice to establish orthotopic xenograft models. Evident signals of lung and kidney metastases were detected by whole-body optical imaging. Gemcitabine displayed a significant therapeutic effect on the subcutaneous or orthotopic xenograft tumors of C09303. In vitro, matrigel invasion assays showed that C09303 primary culture cells are remarkably invasive. To study the metastatic properties of C09303, we sequenced the genomes of C09303 and C21391, and found that the expression of VEGF and CDK4 was significantly upregulated, and VEGF-knockdown or CDK4-knockdown C09303 cells showed attenuated invasiveness and migration. This PDX model revealed that VEGF and CDK4 enhanced tumor metastasis behavior, suggesting them as novel molecular therapeutic targets. This framework provides a tool for research on metastasis mechanism and individualized treatment for bladder cancer.

Bisphenol S (BPS), a structural analog of Bisphenol A (BPA), has been widely used as a substitute for epoxy resin, food packaging materials, and other products due to the limited application of BPA. Studies in vivo and in vitro have indicated that BPA could induce fat accumulation like an obesogen. The main goal of this study was to investigate the role and mechanism of BPS in lipid metabolism using Caenorhabditis elegans (C. elegans) as a model. Results showed that both the overall fat deposition and the triglyceride level were significantly increased in a non-monotonically increasing trend, and the low dose of BPS (0.01 μM) exhibited a stronger influence. Additionally, BPS enhanced fat synthesis depending on daf-16, fat-5, fat-6 and fat-7, and inhibited fatty acid oxidation via nhr-49 and acs-2. This study further indicate that fat accumulation induced by BPS requires nhr-49, which also mediated the nuclear hormone signaling pathway.

Cabazitaxel has been used to treat castration-resistant prostate cancer since its approval by the US Food and Drug Administration in 2010. However, whether cabazitaxel may inhibit the proliferation of other tissue‑derived cancer cells, and its underlying mechanism, remains unknown. In the present study, the A549 lung adenocarcinoma cancer cell line was exposed to cabazitaxel, in order to investigate its cytotoxic effect and determine the underlying mechanism. The results demonstrated that cabazitaxel was able to induce autophagy in A549 cells, as evidenced by the formation of autophagosomes, upregulated LC3‑II expression and increased LC3 puncta. Cabazitaxel‑induced autophagy had a cytotoxic effect on A549 cells, as evidenced by the induction of cell death and cell cycle arrest at G2/M phase, which was independent of the apoptotic pathway. Furthermore, transfection with Beclin1 small interfering RNA and treatment with the autophagy inhibitor 3‑methyladenine protected cells from cabazitaxel‑induced cell death, thus confirming that cabazitaxel‑induced autophagy contributed to A549 cell death. In addition, cabazitaxel targeted the phosphoinositide 3‑kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway to induce autophagy, as indicated by reduced phosphorylation of Akt and mTOR. In conclusion, the present study demonstrated that cabazitaxel exerts a cytotoxic effect on A549 cells by acting on the PI3K/Akt/mTOR pathway to promote autophagic cell death. This result supports the potential use of cabazitaxel as a chemotherapeutic agent for the treatment of lung cancer.

// Ningning Zhao 1, * , Caiqin Zhang 1, * , Yong Zhao 1 , Bing Bai 1 , Jiaze An 1 , Hai Zhang 1 , Jason Boyang Wu 2 , Changhong Shi 1 1 Laboratory Animal Center, the Fourth Military Medical University, Xi’an, Shaanxi 710032, China 2 Uro-Oncology Research Program, Samuel Oschin Comprehensive Cancer Institute, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA * These authors have contributed equally to this work Correspondence to: Changhong Shi, email: changhong@fmmu.edu.cn Jason Boyang Wu, email: BoyangJason.Wu@cshs.org Keywords: near-infrared fluorescence, heptamethine carbocyanine dyes, gastric cancer, patient-derived tumor xenograft, HIF1α Received: February 18, 2016 Accepted: May 26, 2016 Published: June 14, 2016 ABSTRACT Near-infrared fluorescence (NIRF) imaging agents are promising tools for noninvasive cancer imaging. Here, we explored the tumor-specific targeting ability of NIRF heptamethine carbocyanine MHI-148 dye in cultured gastric cancer cells, gastric cancer cell-derived and patient-derived tumor xenograft (PDX) models. We show that the NIRF dye specifically accumulated in tumor regions of both xenograft models, suggesting the potential utility of the dye for tumor-specific imaging and targeting in gastric cancer. We also demonstrated significant correlations between NIRF signal intensity and tumor volume in PDX models. Mechanistically, the higher cellular uptake of MHI-148 in gastric cancer cells than in normal cells was stimulated by hypoxia and activation of a group of organic anion-transporting polypeptide (OATP) genes. Importantly, this NIRF dye was not retained in inflammatory stomach tissues induced by gastric ulcer in mice. In addition, fresh clinical gastric tumor specimens, when perfused with NIR dye, exhibited increased uptake of NIR dye in situ . Together, these results show the possibility of using NIRF dyes as novel candidate agents for clinical imaging and detection of gastric cancer.

Metal-organic framework (MOF) UiO-66 thin films are solvothermally grown on conducting substrates. The as-synthesized MOF thin films are subsequently dried by a supercritical process or treated with polydimethylsiloxane (PDMS). The obtained UiO-66 thin films show excellent molecular sieving capability as confirmed by the electrochemical studies for redox-active species with different sizes.

Heat shock protein Hsp16.3 is closely related to latent Mycobacterium tuberculosis (MTB) infection and plays an important role in sustained survival when MTB is dormant. In this study, the Hsp16.3 gene mutant MTB H37Rv strain (Hsp16.3ΔMTB) was obtained through gene recombination and infected into murine RAW 264.7 macrophages. Western blotting and immunofluorescence showed increased expression of the autophagy-related protein LC3, and transmission electron microscopy showed significantly increased macrophage autophagosomes, suggesting that Hsp16.3ΔMTB facilitates murine macrophage autophagy. These findings have implications for preventing and controlling tuberculosis.

Combining near-infrared fluorescence (NIRF) and nuclear imaging techniques provides a novel approach for hepatocellular carcinoma (HCC) diagnosis. Here, we report the synthesis and characteristics of a dual-modality NIRF optical/positron emission tomography (PET) imaging probe using heptamethine carbocyanine dye and verify its feasibility in both nude mice and rabbits with orthotopic xenograft liver cancer. This dye, MHI-148, is an effective cancer-specific NIRF imaging agent and shows preferential uptake and retention in liver cancer. The corresponding NIRF imaging intensity reaches 109/cm2tumor area at 24 h after injection in mice with HCC subcutaneous tumors. The dye can be further conjugated with radionuclide68Ga (68Ga-MHI-148) for PET tracing. We applied the dual-modality methodology toward the detection of HCC in both patient-derived orthotopic xenograft (PDX) models and rabbit orthotopic transplantation models. NIRF/PET images showed clear tumor delineation after probe injection (MHI-148 and68Ga-MHI-148). The tumor-to-muscle (T/M) standardized uptake value (SUV) ratios were obtained from PET at 1 h after injection of68Ga-MHI-148, which was helpful for effectively capturing small tumors in mice (0.5 cm × 0.3 cm) and rabbits (1.2 cm × 1.8 cm). This cancer-targeting NIRF/PET dual-modality imaging probe provides a proof of principle for noninvasive detection of deep-tissue tumors in mouse and rabbit and is a promising technique for more accurate and early detection of HCC.

// Changhong Shi 1, 2 , Jason Boyang Wu 2 , Gina C-Y. Chu 2 , Qinlong Li 2 , Ruoxiang Wang 2 , Caiqin Zhang 1 , Yi Zhang 3 , Hyung L. Kim 4 , Jing Wang 5 , Haiyen E. Zhau 2 , Dongfeng Pan 3 , Leland W.K. Chung 2 1 Laboratory Animal Center, the Fourth Military Medical University, Xi’an, Shaanxi 710032, China 2 Uro-Oncology Research Program, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA 3 Department of Radiology, the University of Virginia, Charlottesville, VA 22908, USA 4 Department of Surgery, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA 5 Department of Nuclear Medicine, Xijing Hospital, the Fourth Military Medical University, Xi’an, Shaanxi 710032, China Correspondence to: Leland W.K. Chung, e-mail: Leland.Chung@cshs.org Changhong Shi, e-mail: changhong@fmmu.edu.cn Dongfeng Pan, e-mail: DP3R@hscmail.mcc.virginia.edu Keywords: HIF-1α, imaging canine cancer, imaging human prostate cancer, near-infrared dye, organic anion-transporting polypeptides Received: July 13, 2014 Accepted: September 06, 2014 Published: October 25, 2014 ABSTRACT Near-infrared (NIR) fluorescence imaging agents are promising tools for noninvasive cancer imaging. This study explored the specific uptake and retention of a NIR heptamethine carbocyanine MHI-148 dye by canine cancer cells and tissues and human prostate cancer (PCa) specimens and also the dye uptake mechanisms. The accumulation of MHI-148 was detected specifically in canine cancer cells and tissues and freshly harvested human PCa tissues xenografted in mice by NIR fluorescence microscopy and whole-body NIR optical imaging. Specific dye uptake in canine spontaneous tumors was further confirmed by PET imaging. Higher hypoxia-inducible factor-1α (HIF-1α) and organic anion-transporting polypeptide (OATP) protein and mRNA expression was demonstrated by multiplex quantum dots labeling and qPCR in tumors over that of normal tissues. Treating cancer cells with HIF-1α stabilizers activated HIF-1α downstream target genes, induced OATP superfamily gene expression and enhanced cellular uptake and retention of NIR dyes. Moreover, silencing HIF-1 α by siRNA significantly decreased OATP mRNA expression and blocked NIR dye uptake in cancer cells. Together, these results demonstrated the preferential uptake of NIR dyes by canine and human cancer cells and tissues via the HIF-1α/OATPs signaling axis, which provides insights into future application of these dyes for cancer detection and treatment.

Background The detection of Mycobacterium tuberculosis (Mtb) specific human antibodies has been an important diagnostic aid in the diagnosis of tuberculosis (TB) cases with smear-negative sputum samples, especially for the screening of high-risk population. This study focused on the analysis and comparison of the four potential Mtb-secreted proteins (ESAT6, CFP10, Ag85B, Hsp16.3) and the fusion protein Ag85B-Hsp16.3 as new markers in the serodiagnosis between active TB and latent TB infection (LTBI). Methods These five recombinant proteins were produced and used in optimized ELISA to detect IgG serum antibodies against the four secreted proteins. The capacity of identifying infection was evaluated either in active TB patients or LTBI individuals, which was compared with the control groups consisting of hospitalized non-TB individuals. Results The results showed that Ag85B-Hsp16.3/ESAT6 and Hsp16.3/ESAT6 were the best-associated antigens for serology diagnosis of the active TB and LTBI individuals because of their specificity, sensitivity, YI values, and positive rates, respectively. ELISA test demonstrated that 41.67% (25/60) of blood donors respond to Ag85B-Hsp16.3/ESAT6. The consistency of this positive respond with clinical diagnosis almost reached 84% (21/25). Conclusion Thus, a combined test of multiple Mtb-secreted proteins Ag85B, Hsp16.3, and ESAT6 may be the ascendant preliminary screening antigens for active TB or LTBI patients.

Dehydrins (DHNs) are late embryonic abundant proteins characterized by the dehydrin domains that are involved in plant abiotic stress tolerance. In this study, fifty-four wheat DHN unigenes were identified in the expressed sequence tags database. These genes encode seven types of dehydrins (KS, SK3, YSK2, Y2SK2, Kn, Y2SK3, and YSK3) and separate in 32 homologous clusters. The gene amplification differed among the dehydrin types, and members of the YSK2- and Kn-type DHNs are more numerous in wheat than in other cereals. The relative expression of all of these DHN clusters was analyzed using an in silico method in seven tissue types (i.e. normal growing shoots, roots, and reproductive tissues; developing and germinating seeds; drought- and cold-stressed shoots) as well as semi-quantitative reverse transcription polymerase chain reaction in seedling leaves and roots treated by dehydration, cold, and salt, respectively. The role of the ABA pathway in wheat DHN expression regulation was analyzed. Transcripts of certain types of DHNs accumulated specifically according to tissue type and treatment, which suggests their differentiated roles in wheat abiotic stress tolerance.

Itraconazole is a common antifungal which may have promise for treating various human cancers. We report that itraconazole was cytotoxic to MCF-7 and SKBR-3 breast cancer cell lines via apoptosis by altering mitochondria membrane potential, reducing BCL-2 expression and elevating caspase-3 activity. Itraconazole also induced autophagic cell death via LC3-II expression upregulation, P62/SQSTM1 degradation, autophagosome formation and increases in autophagic puncta. Itraconazole treatment inhibited hedgehog pathway key molecular expression, such as SHH and Gli1, resulting in promotion of apoptosis and autophagy. The anti-proliferation effect of itraconazole-induced apoptosis and autophagy via hedgehog pathway inhibition was confirmed with Gli1 inhibitor GANT61 and SHH siRNA, GANT61 and SHH siRNA synergistically enhanced cytotoxicity induced by itraconazole. A human xenograft nude mouse model corroborated the anti-breast cancer activity as evidenced by reduced tumor size, and increased tumor tissue apoptosis and autophagy. Thus, itraconazole has a potent anti-breast cancer activity that may be improved when combined with hedgehog pathway inhibitors.

The secreted Mycobacterium tuberculosis (MTB) proteins, Ag85B and Hsp16.3, have been the focus of intensive research in recent years. These proteins have high sensitivity in bacterium-negative tuberculosis (TB) patients, and are valuable for the rapid diagnosis of bacterium-negative TB. Fusion proteins including multiple antigens such as Ag85B and Hsp16.3 provide improved sensitivity and specificity for serological diagnosis of active TB compared with a single antigen. Many studies have shown that the production of MAbs recognizing a specific repertoire of M. tuberculosis antigens and the tests based on monoclonal antibodies have been found to be valuable in positive detection of TB, particularly for smear-positive pulmonary TB. A number of MAbs are currently used for serodiagnosis of TB. Therefore, an Ag85B-Hsp16.3 fusion protein was expressed and purified using an E. coli system in this study. Three Ag85B-Hsp16.3 fusion protein-specific MAbs were generated by routine murine hybridoma techniques. The titer, specificity, and relative affinity of all three MAbs were determined by ELISA and the serological responses were analyzed. The levels of antigens in a proportion of TB patients were shown to be significantly higher than those in healthy controls. The sensitivity and specificity of the currently available detection systems is likely to be improved by the employment of a combination of these MAbs with others that are already in use.

Vacuolar H + -translocating pyrophosphatase (V-PPase) is a key enzyme related to plant growth as well as abiotic stress tolerance. In this work, wheat V-PPase genes TaVP1, TaVP2 and TaVP3 were identified. TaVP1 and TaVP2 are more similar to each other than to TaVP3 . Their deduced polypeptide sequences preserve the topological structure and essential residues of V-PPases. Phylogenetic studies suggested that monocot plants, at least monocot grasses, have three VP paralogs. TaVP3 transcripts were only detected in developing seeds, and no TaVP2 transcripts were found in germinating seeds. TaVP2 was mainly expressed in shoot tissues and down-regulated in leaves under dehydration. Its expression was up-regulated in roots under high salinity. TaVP1 was relatively more ubiquitously and evenly expressed than TaVP2 . Its expression level in roots was highest among the tissues examined, and was inducible by salinity stress. These results indicated that the V-PPase gene paralogs in wheat are differentially regulated spatially and in response to dehydration and salinity stresses.

The UFD1 protein is an important ubiquitin recognition component in the ubiquitin-mediated degradation pathway. To investigate the conservation of UFD1 genes among eukaryotes and their differentiation, two UFD1 paralogs from wheat were identified and mapped to homoeologous chromosome groups 6 and 2, respectively. TaUFD1a-6B and TaUFD1b-2D were cloned, and both genes consist of eight introns and of the same intron phases. These genes were compared with those in Arabidopsis, rice, polar, yeast, and mammals for their sequence, chromosome organization, and primary protein structure. The sequence structure, especially those corresponding to the fourth, fifth, and sixth exons of UFD1 genes, is highly conserved across these taxa. However, unlike yeast and mammals having a single UFD1 gene, higher angiosperm species have two ancient UFD1 paralogs. Besides the evolutionarily conserved ubiquitin-binding domain at the N-terminus, plant UFD1 proteins have three conserved C-terminal motifs. Motif I, near the UFD1 domain, displays a high level of similarity to the mammalian p97-binding site, and motif III is likely responsible for endoplasmic reticulum membrane retention. TaUFD1a-6B and TaUFD1b-2D are ubiquitously expressed in different plant tissues. A green fluorescent protein-transient expression assay in epidermal cells of onion demonstrated that TaUFD1 proteins primarily accumulate in the nucleus.

Flowering time is an important trait for the adaptation of wheat to its target environments. To identify chromosome regions associated with flowering time in wheat, a whole genome scan was conducted with five sets of field trial data on a recombinant inbred lines (RIL) population derived from the cross of spring wheat cultivars ‘Nanda 2419’ and ‘Wangshuibai’. The identified QTLs involved seven chromosomal regions, among which QFlt.nau-1B and QFlt.nau-2B were homoeologous to QFlt.nau-1D and QFlt.nau-2D, respectively. Nanda 2419, the earlier flowering parent, contributed early flowering alleles at five of these QTLs. QFlt.nau-1B and QFlt.nau-7B had the largest effects in all trials and were mapped to the Xwmc59.2–Xbarc80 interval on chromosome 1BS and the Xgwm537–Xgwm333 interval on 7BS. Most of the mapped QTL intervals were not coincident with known vernalization response or photoperiod sensitivity loci and QFlt.nau-1B seems to be an orthologue of EpsAm1. Four pairs of loci showed significant interactions across environments in determining flowering time, all of which involved QFlt.nau-1B. These findings are of significance to wheat breeding programs.

Triticum monococcum accession TA2026 showed resistance to wheat powdery mildew. To identify the resistance gene and transfer it to common wheat, genetic analysis and molecular mapping were conducted using an F2 population and derived F3 families from the cross of TA2026 × M389. The results indicated that TA2026 possessed a recessive powdery mildew resistance gene. This gene was mapped to the terminal portion of chromosome 5AmL and flanked by SSR marker loci Xcfd39 and Xgwm126. Eight RFLP markers previously mapped to the terminal chromosome 5AmL were converted into STS markers. Three loci, detected by MAG1491, MAG1493 and MAG1494, the STS markers derived from RFLP probes CDO1312, PSR164 and PSR1201, respectively, were linked to this resistance gene with Xmag1493 only 0.9 cM apart from it. In addition, the STS marker MAG2170 developed from the tentative consensus wheat cDNA encoding the Mlo-like protein identified a locus co-segregating with Xmag1493. This is the first recessive powdery mildew resistance gene identified on chromosome 5Am, and is temporarily designated pm2026. We have successfully transferred it to a tetraploid background, and this resistance stock will now be used as the bridge parent for its transfer to common wheat.

Bread wheat (Triticum aestivum L.) is a hexaploid species with a large and complex genome. A reference genetic marker map, namely the International Triticeae Mapping Initiative (ITMI) map, has been constructed with the recombinant inbred line population derived from a cross involving a synthetic line. But it is not sufficient for a full understanding of the wheat genome under artificial selection without comparing it with intervarietal maps. Using an intervarietal mapping population derived by crossing Nanda2419 and Wangshuibai, we constructed a high-density genetic map of wheat. The total map length was 4,223.1 cM, comprising 887 loci, 345 of which were detected by markers derived from expressed sequence tags (ESTs). Two-thirds of the high marker density blocks were present in interstitial and telomeric regions. The map covered, mostly with the EST-derived markers, approximately 158 cM of telomeric regions absent in the ITMI map. The regions of low marker density were largely conserved among cultivars and between homoeologous subgenomes. The loci showing skewed segregation displayed a clustered distribution along chromosomes and some of the segregation distortion regions (SDR) are conserved in different mapping populations. This map enriched with EST-derived markers is important for structure and function analysis of wheat genome as well as in wheat gene mapping, cloning, and breeding programs.

Fusarium head blight (FHB), caused by Gibberella zeae (Schw.) Petch, is a serious disease in many wheat (Triticum aestivum L.) growing regions worldwide. To study the inheritance of FHB resistance against fungal penetration (type I resistance), an “Immortalized F2” (IF) population containing 198 lines was constructed by crossing recombinant inbred lines chosen with random permutation of 132 RILs. The 132 RILs were chosen from the RIL population derived from the cross between FHB-susceptible cultivar Nanda 2419 and FHB-resistant cultivar Wangshuibai. The population was then evaluated for the percentage of infected spikes (PIS) across 2 years, and 6 chromosome regions were detected as being associated with type I resistance through interval mapping, among which Qfhi.nau-4B and Qfhi.nau-5A with the resistance alleles originating from Wangshuibai and Qfhi.nau-2B with the resistance allele from Nanda 2419 were consistently detected. Qfhi.nau-4B and Qfhi.nau-5A had the largest effects among the detected QTLs and for the most part, both showed additive allelic effects. The former was also partially dominant. In addition, 4 pairs of significant interaction loci were identified. These results demonstrated that wheat scab resistance was under complex genetic control and also implied that early generation selection for type I resistance in scab resistance breeding was feasible with Wangshuibai as the parent.

Fusarium head blight (FHB), or scab, is a devastating wheat disease worldwide, reducing both grain yield and quality. The percentage of Fusarium-damaged kernels (FDK) directly reflects the damage level caused by scab on wheat grains and its variation represents the so-called type IV scab resistance in germplasm. To identify genes governing type IV resistance and investigate its relationship with other scab resistance types, we mapped QTLs associated with percent FDK using data from three different field evaluations of the recombination inbred line (RIL) population derived from the susceptible cultivar Nanda 2419 × the scab-resistant cultivar Wangshuibai. Five QTLs related to percent FDK were identified in at least two different trials, for which Wangshuibai contributed four of the resistance alleles. Most of the FDK-related QTLs, including the three with larger effects, QFdk.nau-2B, QFdk.nau-3B and QFdk.nau-4B, mapped to intervals associated with either type IV resistance or type II resistance. Moreover, most of the major type I and type II resistance QTLs detected previously were associated with type IV resistance, suggesting that resistance to initial infection and disease spread play major roles in conditioning less FDK. Therefore, breeders have options to choose inoculation methods based on their expertise and resources without risking significant loss of information when using percent FDK as the disease index. The most useful scab resistance QTLs for breeding would be those with stable influences on FDK and/or deoxynivalenol (DON) accumulation besides the initial infection and disease spread.

The resuscitation-promoting factor (Rpf), a secretory protein first reported in Micrococcus luteus, plays a critical role in mycobacterial survival and infection. There are five functionally redundant Rpf-like proteins identified in M. tuberculosis (Mtb). All these Rpfs share a conserved Rpf domain (Rpfd) composed of approximately 70 amino acids, which possesses the same biological functions as the full-length Rpf protein. Glutamic acid at position 54 in Rpfd (E54) has been implicated in mediating multiple physiological processes, and a single amino acid substitution at residue E54 can affect the protein biological activity. In order to determine the effects of different amino acid substitutions of E54 in Rpfd on its immunogenic activity, we generated three recombinant Rpfd mutants, Rpfd1 (E54K), Rpfd2 (E54A) and Rpfd3 (E54K and D48A), based on T-cell epitope prediction and tested their potential protective/therapeutic effects against Mtb in mice. Our results demonstrated that replacement of E54 by different amino acids in Rpfd distinctively influenced its resuscitation-promoting activities and Th1-type immune responses induced in mice. Administration of Rpfd2 mutant enhanced Th1-type cellular responses (IFN-γ and IL-2) in mice (P < 0.05, Rpfd2 versus control) and provided effective protection against Mtb in mice by significantly inhibiting the growth of Mtb during the initial stage of infection. Four weeks after the challenge, the slightest pathological injury in lung was observed in the Rpfd2-immunized group among all three Rfpd mutant-immunized groups. Furthermore, Rpfd2 therapy significantly decreased the bacterial load in lung and alleviated histopathological damage in Mtb-infected mice. Together, our results suggest Rpfd2 as a novel effective vaccine candidate against Mtb.

Near infrared fluorescence (NIRF) imaging has strong potential for widespread use in noninvasive tumor imaging. Indocyanine green (ICG) is the only Food and Drug Administration (FDA) -approved NIRF dye for clinical diagnosis; however, it is unstable and poorly targets tumors. DZ-1 is a novel heptamethine cyanine NIRF dye, suitable for imaging and tumor targeting. Here, we compared the fluorescence intensity and metabolism of DZ-1 and ICG. Additionally, we assayed their specificities and abilities to target tumor cells, using cultured hepatocellular carcinoma (HCC) cell lines, a nude mouse subcutaneous xenograft model of liver cancer, and a rabbit orthotopic transplantation model. We found that DZ-1 accumulates in tumor tissue and specifically recognizes HCC in subcutaneous and orthotopic models. The NIRF intensity of DZ-1 was one order of magnitude stronger than that of ICG, and DZ-1 showed excellent intraoperative tumor targeting in the rabbit model. Importantly, ICG accumulated at tumor sites, as well as in the liver and kidney. Furthermore, DZ-1 analog-gemcitabine conjugate (NIRG) exhibited similar tumor-specific targeting and imaging properties, including inhibition of tumor growth, in HCC patient-derived xenograft (PDX) mice. DZ-1 and NIRG demonstrated superior tumor-targeting specificity, compared to ICG. We show that DZ-1 is an effective molecular probe for specific imaging, targeting, and therapy in HCC.

Current tuberculosis control measures are focused on the prompt detection and treatment of active tuberculosis. Despite the measured success of this strategy, tuberculosis continues to be a public health issue of major significance around the world. This unwanted situation suggests the need to expand our control efforts by exploring specific markers for the disease. Insulin resistance is one such marker. Although insulin resistance has been implicated in various diseases, thus far, no attempt has been made to analyze what has proved to be a direct relationship between insulin resistance and Mycobacterium tuberculosis susceptibility. Several studies have shown the role of insulin not only in cellular metabolism but also, more importantly, in phagocytosis of M. tuberculosis. Therefore, we hypothesize that insulin resistance can be considered a potential risk factor for active M. tuberculosis infection.

Tryptanthrin, a kind of indole quinazoline alkaloid, has been shown to exhibit anti-microbial, anti-inflammation and anti-tumor effects both in vivo and in vitro. However, its biological activity on human chronic myeloid leukemia cell line K562 is not fully understood. In the present study, we investigated the proliferation-attenuating and apoptosis-inducing effects of tryptanthrin on leukemia K562 cells in vitro and explored the underlying mechanisms. The results showed that tryptanthrin could significantly inhibit K562 cells proliferation in a time- and dose-dependent manner as evidenced by MTT assay and flow cytometry analysis. We also observed pyknosis, chromatin margination and the formation of apoptotic bodies in the presence of tryptanthrin under the electron microscope. Nuclei fragmentation and condensation by Hoechst 33258 staining were detected as well. The amount of apoptotic cells significantly increased whereas the mitochondrial membrane potential decreased dramatically after tryptanthrin exposure. K562 cells in the tryptanthrin treated group exhibited an increase in cytosol cyt-c, Bax and activated caspase-3 expression while a decrease in Bcl-2, mito cyt-c and pro-caspase-3 contents. However, the changes of pro-caspase-3 and activated caspase-3 could be abolished by a pan-caspase inhibitor ZVAD-FMK. These results suggest that tryptanthrin has proliferation-attenuating and apoptosis-inducing effects on K562 cells. The underlying mechanism is probably attributed to the reduction in mitochondria membrane potential, the release of mito cyt-c and pro-caspase-3 activation.