Identification of a biomarker to predict doxorubicin/cisplatin chemotherapy efficacy in osteosarcoma patients using primary, recurrent and metastatic specimens.

BACKGROUND: Doxorubicin and cisplatin are both first-line chemotherapeutics for osteosarcoma (OS) treatment. However, the efficacy of doxorubicin/cisplatin chemotherapy varies considerably. Thus, identifying an efficient diagnostic biomarker to distinguish patients with good and poor responses to doxorubicin/cisplatin chemotherapy is of paramount importance. METHODS: To predict the efficacy of doxorubicin/cisplatin chemotherapy, we analyzed the differentially expressed proteins in 37 resected OS samples, which were categorized into the primary group (PG), the recurrent group (RG) and the metastatic group (MG). The characteristics of the enriched differentially expressed proteins were assessed via GO and KEGG analyses. Protein‒protein interactions were identified to determine the relationships among the differentially expressed proteins. Receiver operating characteristic (ROC) curve analyses were performed to explore the clinical significance of the differentially expressed proteins. Parallel reaction monitoring (PRM) was used to validate the candidate proteins. Immunohistochemical (IHC) staining was performed to confirm the expression of cathepsin (CTSG) in patients with good and poor response to doxorubicin/cisplatin. RESULTS: A total of 9458 proteins were identified and quantified, among which 143 and 208 exhibited significant changes (|log2FC|>1, p < 0.05) in the RG and MG compared with the PG, respectively. GO and KEGG enrichment led to the identification of neutrophil extracellular traps (NETs). ROC curve analyses revealed 74 and 86 proteins with areas under the curve greater than 0.7 in the RG and MG, respectively. PRM validation revealed the statistical significance of CTSG, which is involved in NET formation, at the protein level in both the RG and MG. IHC staining of another cohort revealed that CTSG was prominently upregulated in the poor response group after treatment with doxorubicin/cisplatin. CONCLUSION: CTSG and its associated NETs are potential biomarkers with which the efficacy of doxorubicin/cisplatin chemotherapy could be predicted in OS patients.

RICH2 decreases the mitochondrial number and affects mitochondrial localization in diffuse low-grade glioma-related epilepsy.

Epilepsy, a common complication of diffuse low-grade gliomas (DLGGs; diffuse oligodendroglioma and astrocytoma collectively), severely compromises the quality of life of patients. DLGG epileptogenicity may primarily be generated by interactions between the tumor and the neocortex. Neuronal uptake of dysfunctional mitochondria from the extracellular environment can lead to abnormal neuronal discharge. Mitochondrial dysfunction is frequently observed in gliomas that can transmigrate across the plasma membranes. Here, we examined the role of the Rho GTPase-activating protein 44 (RICH2) in mitochondrial dynamics and DLGG-related epilepsy. We investigated the association between mitochondrial and RICH2 expression in human DLGG tissues using immunohistochemistry. We examined the association between RICH2 and epilepsy in nude mouse glioma models by electrophysiology. The effect of RICH2 on mitochondrial morphology and calcium motility were assessed by single cell fluorescence microscopy. Quantitative RT-PCR (qRT-PCR) and Western blot analysis were performed to characterize RICH2 induced expression changes in the genes related to mitochondrial dynamics, mitogenesis and mitochondrial function. We found that RICH2 expression was higher in oligodendroglioma than in astrocytoma and was correlated with better prognosis and higher epilepsy rate in patients. The expression of mitochondria may be associated with clinical DLGG-related epilepsy and reduced by RICH2 overexpression. And RICH2 could promote DLGG-related epilepsy in tumorigenic nude mice. RICH2 overexpression decreased calcium flow and the mitochondria released from glioma cells (SW1088 and U251) into the extracellular environment, potentially via downregulation of MFN-1/MFN-2 levels which suggests reduced mitochondrial fusion. In addition, we observed decreased mitochondrial trafficking into neurons (released from glioma cells and trafficked into neurons), which could explain the higher incidence of DLGG-related epilepsy due to reduced neuroprotection. Furthermore, RICH2 downregulated MAPK/ERK/HIF-1 pathway. In conclusion, these results suggest that RICH2 could promote epilepsy by (i) inhibiting mitochondrial fusion via MFN downregulation and Drp-1 upregulation; (ii) altering the MAPK/ERK/Hif-1 signaling axis. RICH2 may be a potential target in the treatment of DLGG-related epilepsy.

[IgG Fc binding protein (FCGBP) as a prognostic marker of low-grade glioma and its correlation analysis with immune infiltration].

Objective To identify the possibility of IgG Fc binding protein (FCGBP) acting as a prognostic marker of low-grade glioma (LGG) and its correlation with immune infiltration. Methods The expression of FCGBP was analyzed in pan-cancer using The Cancer Genome Atlas (TCGA), Genotypic tissue expression (GTEX), and China Glioma Genome Atlas (CGGA) database. Then, GSE15824 and GSE68848 datasets were selected for further verification. And gene expression Profile Interaction analysis (GEPIA) database and R language were used to analyze the relationship between FCGBP and survival prognosis. Metascape and GSEA were used for functional annotation and enrichment analysis. Finally, the expression of FCGBP gene in LGG immune microenvironment and its correlation with immune cells were analyzed by TIMER database. Results FCGBP was highly expressed in LGG tissues, indicating poor prognosis of LGG patients. Receiver operating characteristic (ROC) curve analysis and COX analysis showed that FCGBP was an independent risk factor for the prognosis of LGG. Moreover, Gene Ontology (GO) demonstrated that FCGBP was involved in cell metabolism, localization, positive, and negative regulation of biological processes, as well as biological adhesion, response to viral and microbial stimulation, and inflammation. GSEA pathway enrichment analysis showed that FCGBP was significantly correlated with Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, Toll-like receptor (TLR) pathway, chemokine pathway, and P53 pathway. In addition, FCGBP expression was positively correlated with the expression of most immune cells in the immune microenvironment of LGG. Conclusion The high expression of FCGBP in LGG is a risk factor for survival and prognosis, and it is positively correlated with the expression of immune cells.

Photocatalytic Alkynylation of Hydrosilanes via Hydrogen Atom Transfer.

Alkynylsilanes are significant structural units frequently used in synthetic chemistry, medicinal chemistry, functional materials, and life sciences. Herein, we report a method for using a hydrogen atom transfer (HAT) strategy in combination with visible-light-driven photocatalysis to achieve a direct coupling reaction between benzene sulfonyl acetylene and tertiary silanes, and a diverse alkynylation of hydrosilanes in the presence of reactive groups was achieved with this strategy. It is important to note that dihydroalkyl/aryl silanes are also suitable for the protocol of HAT photocatalytic of 4CzIPN and quinuclidine.

Entrepreneurial role models and college students' entrepreneurial calling: A moderated mediation model.

Introduction: College entrepreneurship education should not only cultivate a group of college students who have strong willingness to start a business immediately after graduation, but also pay attention to future entrepreneurship success of college students. Correspondingly, in addition to attaching importance to improving college students' entrepreneurial intention, college entrepreneurship education should pay attention to improving college students' entrepreneurial calling. At present, there is insufficient research on the association between entrepreneurial role models and entrepreneurial calling. We aim to study the mechanism and boundary condition of the association between entrepreneurial role models and entrepreneurial calling. Methods: A longitudinal survey was distributed among 519 students from 16 colleges and universities in China. In the survey, the college students answered questions on entrepreneurial role models, entrepreneurial calling, entrepreneurial perceived behavioral control and entrepreneurial hands-on practice. Hierarchical regression was conducted, testing the association between entrepreneurial role models and entrepreneurial calling of college students, mediated by entrepreneurial perceived behavioral control and moderated by entrepreneurial hands-on practice. Results: Therefore, based on the social learning theory, the theory of planned behavior and the entrepreneurial event model, and by hierarchical regression of the data, this study confirmed that entrepreneurial role models were positively associated with college students' entrepreneurial calling by partially mediating with entrepreneurial perceived behavioral control. Moreover, Entrepreneurial hands-on practice positively moderated not only the relationship between entrepreneurial perceived behavioral control and entrepreneurial calling, but also the mediating association of entrepreneurial perceived behavioral control between entrepreneurial role models and entrepreneurial calling. Discussion: This study not only enriches the theoretical research on entrepreneurial calling and entrepreneurial role models, but also provides valuable educational enlightenment for colleges and universities to improve the students' entrepreneurial calling.

Primary pulmonary hyalinizing clear cell carcinoma with pseudopapillary structures and abundant cysts filled with mucus.

Pulmonary hyalinizing clear cell carcinoma (HCCC) is a new and rare form of lung salivary gland tumor. Only twenty-two cases have been reported in the literature to date. Furthermore, their clinicopathological features have not been fully characterized. In this paper, we describe the clinicopathological characteristics, immunohistochemical features, and molecular genetic changes in two HCCC cases. We also simultaneously reviewed related literature on similar cases reported. Of the two cases, one was of a 58-year-old man with a 4.3 cm lung tumor, which was the largest among all previously reported cases. The tumor showed an infiltrative growth pattern and perineural and vascular invasion microscopically. Moreover, nuclear grooves, high mitotic figures, and comedo necrosis were observed in addition to classic morphological features. More importantly, rare pseudopapillary structures were observed. The second case was of a 60-year-old woman in whom the tumor was mainly composed of multiple cysts filled with mucus. The remaining focal solid areas of the tumor comprised clear and acidophilic cells embedded in the hyalinizing stroma. Immunohistochemical analysis revealed that the tumor cells of both cases were positive for CK5/6, p40, and p63 expression, but negative for napsin A, TTF-1, and SOX10 expression. The HCCC diagnosis in both cases was validated by fluorescence in-situ hybridization (FISH) examination, which showed Ewing sarcoma breakpoint region 1-activating transcription factor 1 (EWSR1-ATF1) gene fusion. Primary pulmonary HCCC is a rare lung tumor originating from the bronchial mucosa, and its histological features may vary, such as rare pseudopapillary structures and abundant cysts. Thus, the diagnosis should be a combined analysis of histopathological characteristics with immunophenotype and molecular examination, including EWSR1-ATF1 gene fusion detection.

Human Parvovirus B19 May Be a Risk Factor in Myasthenia Gravis with Thymoma.

PURPOSE: Our previous studies have demonstrated that human parvovirus B19 (B19V) is involved in the pathogenesis of thymic hyperplasia-associated myasthenia gravis (MG). However, more cases need to be assessed to further elucidate the relationship between this virus and thymoma-associated MG. MATERIALS AND METHODS: The clinicopathological characteristics, presence of B19V DNA, and B19V VP2 capsid protein expression of 708 cases of thymomas were investigated using nested polymerase chain reaction (PCR), TaqMan quantitative (q) PCR, immunohistochemistry, fluorescent multiplex immunohistochemistry, and electron microscopy. RESULTS: Patients with MG or ectopic germinal centers (GCs) were significantly younger than those without MG (P < 0.0001) or GCs (P = 0.0001). Moreover, significantly more GCs were detected in thymomas associated with MG than in those without MG (P < 0.0001). The results of nested PCR and TaqMan qPCR were consistent, and B19V DNA positivity was only associated with presence of GCs (P = 0.011). Immunohistochemically, positive staining was primarily detected in neoplastic thymic epithelial cells (TECs) and ectopic GCs. The positive rate of B19V VP2 was significantly higher in thymoma with MG or GCs than in thymoma without MG (P = 0.004) or GCs (P = 0.006). Electron microscopy showed B19V particles in the nuclei of neoplastic TECs and B cells from GCs. CONCLUSIONS: We conclude that the pathogenesis of MG is closely associated with the presence of GCs, and B19V infection is plausibly an essential contributor to formation of ectopic GCs in thymoma. To the best of the authors' knowledge, this is the first study to elucidate the role of B19V in thymoma-associated MG and provide new ideas for exploring the etiopathogenic mechanism of MG.

Engineering the Near-Surface Structure of WO3 by an Amorphous Layer with Trivalent Ni and Self-Adapting Oxygen Vacancies for Efficient Photocatalytic and Photoelectrochemical Acidic Oxygen Evolution Reaction.

Exploiting an effective strategy to tailor the construction, composition, and local electronic structure of the photocatalyst surface is pivotal to photocatalytic activity, but remains challenging. Transition metal elements can boost the oxygen evolution reaction activity especially one like Ni in high oxidation states, whereas it is uneasy to prepare Ni3+ under mild conditions or play to their strengths in acidic conditions. In this article, we report a facile "etch and dope" synthesis of Ni3+-doped WO3 nanosheets with oxygen vacancies. Through detailed experimental and theoretical studies, it is established that the abundant oxygen vacancies and the doped Ni3+ ions in the near-surface amorphous layer can synergistically optimize the surface electronic structure of WO3 and the adsorption and desorption of intermediates. Impressively, the etched WO3 nanosheets coupled with Ni3+ offer a greatly promoted photocatalytic performance of 1.78 mmol g-1 h-1, and the photoanode achieves a photocurrent density of 2.11 mA cm-2 at 1.23 V versus reversible hydrogen electrode (VRHE). This work provides a new inspiration for rational manufacture of defects and high-valence metal ions in catalysts for photocatalytic and photoelectrochemical reactions.

Umpolung Reactivity of Imine Ester: Visible-Light Mediated Transfer Hydrogenation of α-Aryl Imino Esters by Phenylsilane and Water.

A visible-light mediated chemoselective transfer hydrogenation of α-aryl imino esters was demonstrated. The methodology allowed the efficient and practical preparation of α-amino acid esters. The mechanism of the reaction was probed by DFT calculations, and deuteration experiments indicated deuterium was introduced into amino acid esters efficiently (up to 99 % D ratio), enabling a feasible way to obtain deuterated amino acids using D2 O as a cheap deuterium source.

Primary collision tumors of the sellar region: Experience from a single center.

Collision tumors are extremely rare in the sellar region, and their features have not been fully characterized. Here, we report our single-center experience in the diagnosis and management of these tumors, focusing primarily on their clinicopathological features. We first performed a retrospective study of pathological reports from patients who had undergone surgery for pituitary adenoma (PA) or craniopharyngioma (CP) at our hospital. Next, to identify collision tumors, patients with a second pathological diagnosis-such as Rathke's cleft cyst (RCC), gangliocytoma (GC), meningioma, or atypical teratoid/rhabdoid tumor (AT/RT)-were considered. Finally, the clinicopathological characteristics of these tumors were reviewed and analyzed. The results demonstrated that eleven of 2359 PA or CP cases (0.47 %) were found to exhibit sellar collision tumors; the patient cohort had a median age of 52 years (23-71) and was predominantly female (63.6 %, 7/11). In details, of the 2092 cases of PA, 10 were diagnosed with concurrent lesions (seven of RCC and one each of CP, meningioma, and GC). Of the 267 CP cases, a single patient presented with associated AT/RT. To our knowledge, this is the first reported adult case of this subtype. Notably, the preoperative CT and/or MRI of each patient revealed solely PA or CP. The endoscopic endonasal approach was the preferred surgery. In conclusion, the sellar collision tumors occur with low incidence, and the primary subtype is PA and RCC. Their definitive diagnosis depends primarily on pathological findings.

Does high-speed rail improve China's urban environmental efficiency? Empirical evidence from a quasi-natural experiment.

Whether high-speed rail (HSR) can promote the coordination between the economy and environment is a critical issue that needs to be investigated. We used balanced panel data of 281 prefecture-level or above cities in China from 2005 to 2017 to consider the opening of HSR as a quasi-natural experiment. We integrated the difference-in-differences (DID) model, the spatial difference-in-differences (SDID) model, and social network analysis (SNA) to empirically investigate the impact of HSR on urban environmental efficiency (UEE). The results showed that HSR significantly improved UEE by 4.6% annually during the study period, although the effect of HSR on UEE exhibited a time lag and varied dramatically in different cities. An analysis of the mechanism showed that the effect of technological innovation and the structural effect brought by the opening of HSR were the main contributors to the improved UEE. Further analysis showed that HSR service centrality also significantly improved UEE and HSR opening and HSR service centrality both had positive spatial spillover effects on the UEE of neighboring cities. Several policy implications are proposed accordingly to make full use of the advantages of HSR to improve UEE for China.

Noble-metal-free Cd0.3Zn0.7S-Ni(OH)2 for high efficiency visible light photocatalytic hydrogen production.

Heterogeneously structured materials with supported precious metals, such as Pd, Pt, and Ru, as co-catalysts are important catalysts for efficient photocatalytic water splitting. However, the high costs and low reserves of precious metals have been an obstacle to their application in hydrogen production. In this work, the noble-metal-free Cd0.3Zn0.7S solid solution was designed and synthesized with an optimized molar ratio of Cd/Zn for the best visible light photocatalytic performance. In addition, a heterojunction hybrid material formed between the Cd0.3Zn0.7S and Ni(OH)2 nanosheet was engineered to improve the utilization of light and to inhibit the recombination of holes and electrons. Ni(OH)2 nanosheets assisted the transfer of the photoexcited electrons to participate in the reduction reactions which is critical for efficient and rapid catalytic hydrogen production. The photoelectrochemical property of the hybrid material was investigated with UV-vis absorption, photoluminance (PL) and electrochemical impedance spectroscopy measurements. The mechanism of the high-efficiency and low-cost photocatalytic hydrogen production was established by analyzing the hydrogen evolution kinetics. With the success of replacing precious metal with nickel-based surface heterostructure, this work is expected to provide a new type of photocatalyst for the application of photocatalytic hydrogen production.

Relationship between FDI, fiscal expenditure and green total-factor productivity in China: From the perspective of spatial spillover.

Deeply investigating the relationship between foreign direct investment (FDI), fiscal expenditure and green total-factor productivity (GTFP) is beneficial to formulating effective policies to promote the high-quality development in China. Based on theoretical mechanism analysis, with panel data of China's mainland 30 provinces during 2003-2017, this paper utilizes spatial econometric model to empirically explore the effects of FDI, fiscal expenditure and their interaction item on the growth of GTFP in China. The results show that FDI significantly promote the growth of the local and its neighboring GTFP, and both fiscal expenditure and the interaction between FDI and fiscal expenditure exert significantly negative effects on the growth of GTFP in the local and its neighboring regions. A series of robustness checks and the endogeneity test can ensure the reliability of these results. In addition, great heterogeneity can be found across China's different regions in the relationship between FDI, fiscal expenditure and GTFP. The conclusions suggest that it is necessary to give fully play to the synergy between FDI and fiscal expenditure and formulate regionally targeted policies to improve GTFP and promote high-quality development in China.

Detection of IgM and IgG Antibodies to Human Parvovirus B19 in Sera of Patients with Thymoma-Associated Myasthenia Gravis.

Much uncertainty still exists about the viral etiology of myasthenia gravis (MG). To address this, we explored the relationship between human parvovirus B19 (PVB19) infection and MG by investigating the presence of PVB19-specific antibodies in serum. A total of 131 patients with MG (including 47 with thymoma-associated MG, 14 with hyperplasia-associated MG, and 70 with unknown thymic lesions) and 172 healthy volunteers were enrolled in this study. Enzyme linked immunosorbent assay was conducted to detect virus-specific antibodies in cell-free serum. The data were analyzed using Pearson chi-square (χ2) and Fisher's exact tests. In the 131 patients with MG, there was no significant difference between male (53.41 ± 14.65 years) and female (50.19 ± 15.28 years) groups regarding mean age (p > 0.05). Among all MG subgroups, the largest age group comprised participants aged 30-60 years. We found that the frequency of detecting immunoglobulin G (IgG) antibodies against PVB19 VP1 and VP2 was significantly higher among patients with MG (68.70%) than in healthy controls (41.86%) (p < 0.001). In particular, the positive rate for anti-PVB19 IgG in patients with thymoma-associated MG (35/47, 74.47%) was significantly higher than that in healthy participants (72/172, 41.86%; p < 0.001). The findings of this study indicate that PVB19 infection may play a role in the etiopathogenesis of MG, particularly in patients with thymoma-associated MG. The study protocol was registered at ClinicalTrials.gov with the identifier ChiCTR-1900023338.

RICH2, a potential tumor suppressor in hepatocellular carcinoma.

Rho GTPase-activating proteins (RhoGAPs) are implicated in the development and progression of hepatocellular carcinoma (HCC). We tested the idea that RICH2 is a tumor suppressor in HCC. Consistent with this, RICH2 was downregulated in HCC and HCC cell lines and RICH2 expression negatively correlated with the tumor size, TNM stage and metastasis in HCC. RICH2, in a Cdc42 dependent manner, regulated the formation of filopodia in HCC and stable overexpression of RICH2 significantly inhibited the clone formation, proliferation and invasion of HCC cells in vitro. Gene set enrichment analysis (GSEA) showed that the expression of RICH 2 positively correlated with the expression of WNT5a, that exert antagonistic effect on canonical WNT signalling whereas RICH2 expression inversely correlated with the expression of ß-catenin (CTNNB1), that is involved in the proliferation and invasion HCC. These findings concurred with co-immunoprecipation of RICH2 with endogenous Cdc42, Rac1, and ß-catenin. Finally, RICH2 overexpression suppressed tumor growth in vivo. The findings support the idea that RICH2 might act as a tumor suppressor in HCC.

Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation.

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

Hepatocellular carcinoma metastasis to the gingival soft tissues: A case report and review of the literature.

Metastases to the gingival soft tissues are rare in hepatocellular carcinoma (HCC). To the best of our knowledge, only 13 cases have been reported in English literature to date. The present study described the case of a 43-year-old Chinese man who was admitted to Tangdu Hospital (Xi'an, China) due to the presence of a gingival tumor that was initially diagnosed as granulation tissue by a dental surgeon. Examination of the patient's medical history revealed that a solid mass, measuring 1.5 cm in diameter, was identified in the right lobe of the liver 2 years prior to presentation at the current hospital; however, no biopsy was performed. Thus, the tumor was resected and histological examination resulted in an initial diagnosis of atypical squamous cell carcinoma. However, the histopathological characteristics, immunohistochemical features and serum α-fetoprotein expression levels supported a diagnosis of metastatic HCC. In conclusion, the present case study highlights the difficulties in diagnosing metastatic HCC without a history of primary HCC, and the importance of excluding a diagnosis of metastatic tumor when a lesion is identified in the gingival. Furthermore, it was determined that a final diagnosis of gingival metastasis of HCC predominantly depends on pathological characteristics and immunohistochemical features.

Expression of porcine respiratory and reproductive syndrome virus membrane-associated proteins in Listeria ivanovii via a genome site-specific integration and expression system.

Listeria ivanovii (LI) possesses a facultative intracellular life cycle in macrophages and non-professional phagocytes thus making it a potential novel vaccine vector. We have developed a genome site-specific integration system as a universal tool for constructing the live LI-based vaccines. Using this system, the porcine reproductive and respiratory syndrome virus (PRRSV) orf5 and orf6 genes, coding for PRRSV membrane-associated protein GP5 and M, respectively, were integrated into the LI genome. Genome PCR and sequencing results showed that the orf5 or orf6 gene had been integrated into the LI genome downstream of orfXYZ or mpl gene. Western blot analysis of the non-secreted and secreted bacterial protein samples showed the expression and secretion of GP5 or M protein from the recombinant LI strains. The secretion of the heterologous proteins is a key step for bacterial vaccine vectors to deliver the antigens to the immune system to stimulate antigen-specific immune responses. Therefore, this integration system may be a potential tool for constructing novel vaccines using live LI as vector.