A head-to-head comparison of [68Ga]Ga-DOTATATE and [68Ga]Ga-FAPI PET/CT in patients with nasopharyngeal carcinoma: a single-center, prospective study.

PURPOSE: We aimed to compare the staging efficiency of [68Ga]Ga-DOTATATE and [68Ga]Ga-FAPI PET/CT in nasopharyngeal carcinoma (NPC) patients. METHODS: Thirty-nine patients with pathologically confirmed NPC were enrolled in this prospective study. Each patient underwent paired [68Ga]Ga-DOTATATE and [68Ga]Ga-FAPI PET/CT on 2 successive days. The accuracy of two PET/CT for assessing T, N, and M stages was compared by using head-and-neck MRI, histopathologic diagnosis and follow-up results as reference standards. The radiotracer uptake derived from two PETs was also compared. RESULTS: For treatment-naïve patients, [68Ga]Ga-DOTATATE PET/CT showed identical sensitivity for the primary tumours but clearer tumor delineation induced by higher tumour-to-background (TBR) ratio (19.1 ± 8.7 vs. 12.4 ± 7.7, P = 0.003), compared with [68Ga]Ga-FAPI PET/CT. Regarding cervical lymph node (CLN) metastases, [68Ga]Ga-DOTATATE PET had significantly better sensitivity and accuracy based on neck sides (98% vs. 82%, P < 0.001; 99% vs. 88% P = 0.008), neck levels (98% vs. 78%, 99% vs. 97%; both P < 0.001) and individual nodes (89% vs. 56%, 91% vs. 76%; both P < 0.001), and higher TBR (8.1 ± 4.1 vs. 6.3 ± 3.7, P < 0.001). Additionally, [68Ga]Ga-DOTATATE PET/CT revealed higher sensitivity and accuracy for distant metastases (96% vs. 53%, 95% vs. 52%; both P < 0.001), particularly in bone metastases (99% vs. 49%, 97% vs. 49%; both P < 0.001). For post-treatment patients, [68Ga]Ga-DOTATATE PET/CT identified one more true-negative case than [68Ga]Ga-FAPI PET/CT. CONCLUSION: [68Ga]Ga-DOTATATE PET/CT performed better than [68Ga]Ga-FAPI PET/CT in visualizing the primary tumours, detecting the metastatic lesions and identifying the local recurrence, suggesting [68Ga]Ga-DOTATATE PET/CT may be superior to [68Ga]Ga-FAPI PET/CT for NPC staging.

Gut microbial signatures of patients with diarrhea-predominant irritable bowel syndrome and their healthy relatives.

AIMS: Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder, encompassing diarrhea-predominant irritable bowel syndrome (IBS-D). Here, we utilized 16S rDNA gene sequencing to identify potential microbial drivers of IBS-D. METHODS AND RESULTS: A total of 30 healthy relatives and 27 patients with IBS-D were recruited. Clinical data and fecal samples were collected from patients and controls. 16S rDNA gene sequencing was performed to obtain fecal bacterial data. Differences in community composition were evaluated utilizing analysis of similarity (ANOSIM) using Bray-Curtis dissimilarity. The Wilcoxon rank sum test was used to compare differences in taxa and functional pathways. Finally, the key gut microbiota was identified using the random forest algorithm. Gut microbiota diversity, estimated through the Observe, Chao1, and abundance-based coverage estimator (ACE) indices, was significantly lower in the IBS-D patients than in the healthy relatives. ANOSIM analysis further confirmed significant differences in the composition of the gut microbiota between IBS-D patients and healthy relatives, with an R value of 0.106 and a P-value of 0.005. Notably, the IBS-D patients exhibited a significant enrichment of specific bacterial genera, including Fusicatenibacter, Streptococcus, and Klebsiella, which may possess potential pathogenic properties. In particular, the bacterial genus Klebsiella demonstrated a positive correlation with irritable bowel syndrome severity scoring system scores. Conversely, healthy subjects showed enrichment of bacterial genera such as Alistipes, Akkermansia, and Dialister, which may be beneficial bacteria in IBS-D. Utilizing the random forest model, we developed a discriminative model for IBS-D based on differential bacterial genera. This model exhibited impressive performance, with an area under the curve value of 0.90. Additionally, our analysis did not reveal any gender-specific differences in the microbiota community composition among IBS-D patients. CONCLUSIONS: Our findings offer preliminary insights into the potential relationship between intestinal microbiota and IBS-D. The identification model for IBS-D, grounded in gut microbiota, holds promising prospects for improving early diagnosis of IBS-D.

X-ray-induced epithelial-mesenchymal transition in SW480 colorectal cancer cells and its potential mechanisms.

PURPOSE: The purpose of this study was to investigate the relation between X-ray irradiation and epithelial-mesenchymal transition (EMT), as well as the potential mechanisms of X-ray-induced EMT in SW480 colorectal cancer (CRC) cells. METHODS: It is well known that EMT plays a critical role in invasive and metastatic of colorectal cancer progression. However, the possible role of X-ray irradiation on EMT in colorectal cancer is widely disputed and its potential mechanisms are unclear. SW480 CRC were irradiated (0, 2, 4, 6, 8 Gy) and cultured for 48 hrs, and then the cellular morphology was observed. Protein and mRNA expressions were examined by Western blot and QRT-PCR. Cell migratory and invasive capacity was evaluated by Transwell assay. RESULTS: In the 2, 4, 6, 8 Gy groups, SW480 CRC exhibited a classical mesenchymal phenotype compared with the 0 Gy group. The expression of E-cadherin was significantly decreased, while the expression of vimentin and Smad3 was significantly increased in the 2, 4, 6, 8 Gy groups (p<0.05) compared with the 0 Gy group; still, the expression of K-ras decreased in the 4, 6, 8 Gy groups (p<0.05) compared with the 0 and 2 Gy groups. Furthermore, the cell migration and invasion capacity was significantly enhanced in the 4 and 8 Gy groups compared with the 0 Gy group (p<0.05). CONCLUSION: These results support the fact that X-ray irradiation can induce EMT through promoting Vimentin and Smad3 expression in SW480 CRC cells.

Association between long non-coding RNA HOTAIR polymorphism and lung cancer risk: A systematic review and meta-analysis.

Single nucleotide polymorphism (SNP) of long noncoding RNA (lnc)RNA has been reported to be an important factor in cancer development. Recently, lncRNA homeobox transcript antisense intergenic RNA (HOTAIR) was indicated to induce tumorigenesis of several cancer types, but the association between the SNP of lncRNA HOTAIR and lung cancer susceptibility has remained undetermined. The present meta-analysis aimed to investigate the effect of HOTAIR polymorphism on susceptibility to lung cancer. The PubMed, Ovid Medline, Embase and Cochrane Library databases were thoroughly searched. Studies containing data on the incidence of lung cancer in patients with different HOTAIR SNPs were included. The Hardy-Weinberg equilibrium was analyzed to determine genotype distribution and allele frequencies. The odds ratio (OR) was pooled to evaluate the association of different SNPs with the susceptibility to lung cancer. A total of six studies comprising 1,715 patients with lung cancer and 2,745 healthy controls were finally included. A total of 4 SNPs (rs12826786, rs1899663, rs920778 and rs4759314) were reported. Analyses for all of these SNPs individually indicated that the lncRNA HOTAIR rs1899663 C>A polymorphism was a risk factor for lung cancer (dominant mode, AA+CA vs. CC: OR=0.816, 95% CI=0.707-0.942, P=0.005). The present study was the first meta-analysis investigating the association between lncRNA HOTAIR and lung cancer susceptibility. The results indicated that the lncRNA HOTAIR rs1899663 C>A polymorphism is a risk factor for lung cancer. LncRNA HOTAIR may be of value in lung cancer screening, particularly for populations with high-risk factors, as well as prognosis prediction. Future investigations are required to further clarify the intrinsic mechanism of the role of HOTAIR in the oncogenesis of lung cancer.

ALDH1A3-Linc00284 Axis Mediates the Invasion of Colorectal Cancer by Targeting TGFß Signaling via Sponging miR-361-5p.

ALDH1A3 and Linc00284 involve in colorectal cancer (CRC) development; however, the regulatory mechanism is still unclear. In this study, we collected clinicopathological characteristics and tissue samples from 73 CRC patients to analyze the expression of ALDH1A3, Linc00284, TGFß signaling and miR-361-5p using qPCR, Western blotting, and ELISA. Multiple CRC cell lines were evaluated in this study, and the highest level of ALDH1A3 was observed in SW480 cells. To investigate the regulatory mechanism, RIP and luciferase assays were used to validate the interaction between Linc00284, miR-361-5p, and TGFß. Proliferation, viability, migration, and invasion assays were performed to profile the effects of the ALDH1A3-Linc00284 axis in CRC cell functions, which was upregulated in CRC tissues. Knockdown ALDH1A3 or Linc00284 significantly reduced TGFß expression and suppressed the EMT process, while overexpression had opposite effects. miR-361-5p targeted TGFß directly, which negatively correlated with ALDH1A3-Linc00284 expression and CRC progression. Mechanistically, upregulation of ALDH1A3-Linc00284 promotes colorectal cancer invasion and migration by regulating miR-361-5p/TGFß signaling pathway. Dysregulation of the ALDH1A3-Linc00284-miR-361-5p-TGFß axis causes CRC invasion, which might provide a new insight into the treatment of CRC.

Treatment outcomes of induction chemotherapy combined with intensity-modulated radiotherapy and adjuvant chemotherapy for locoregionally advanced nasopharyngeal carcinoma in Southeast China.

ABSTRACT: Induction chemotherapy (IC) and adjuvant chemotherapy (AC) are used to enhance tumor locoregional control and support early treatment for distant metastases. However, optimum combinatorial treatment of these chemoradiotherapy regimens with radiotherapy in curing locoregionally advanced nasopharyngeal carcinoma (NPC) remains unclear. Here, we evaluate the efficacy and therapeutic outcome of a combinatorial treatment strategy involving IC, intensity-modulated radiotherapy (IMRT), and AC, by retrospectively analyzing 243 NPC patients who were treated by IC followed by IMRT and AC. The rates of 3-/5-year local-regional control rate, distant failure-free rate (DFFR), progression-free survival (PFS), and overall survival (OS) were 93.3%/90.3%, 84.2%/79.4%, 79.6%/74.4%, and 84.0%/72.6%, respectively. The 3-/5-year OS rates of patients in stage III or IVA were 91.5%/75.1% and 86.5%/56.5%, respectively. Combination cisplatin with paclitaxel showed no significance in OS as compared to cisplatin plus 5-fluorouracil (P-value = .17). Total four-cycle IC and AC was significantly beneficious versus three-cycle in DFFR (P-value = .04), as well as total 6 chemotherapy cycles compared to 4 in DFFR and PFS (P-value = .03 and P-value = .01, respectively). All survival indicators were adversely affected by T-category, while N-category could only predict DFFR and PFS. Radiation dosage represented as a second prognostic factor for local control. We propose that IC combined with IMRT and AC for locoregionally advanced NPC shows effective treatment outcomes.

Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in colorectal cancer.

Emerging evidences suggest that long noncoding RNA (lncRNA) plays a vital role in tumorigenesis and cancer progression. Here, the aim of this study is to investigate the biological function of long intervening noncoding RNA Linc00284 in colorectal cancer (CRC). The expression levels of Linc00284, miR-27a and c-Met were evaluated by qPCR and/or Western blotting. Immunohistochemistry was used to detect the expression of Ki67 and Phh3 in tumor tissues. The interaction between Linc00284, miR-27a and c-Met was validated by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Cell function experiments, including CCK-8, wound-healing and transwell invasion assays, were conducted. The in vivo studies were performed with the subcutaneous tumor xenograft mouse models. Our findings reveal that Linc00284 is upregulated in CRC tissues and colorectal cancer cell lines HCT116 and SW480 in comparison with corresponding para-carcinoma tissues and human fetal colonic mucosa cells FHC. High expression of Linc00284 in tumor tissues is associated with tumor metastasis and predicts a poor clinical outcome in CRC patients. Serum Linc00284 is increased, while miR-27a is decreased in CRC patients compared to healthy controls. ROC curve analysis indicates that serum Linc00284 and miR-27a produce the area under the curve (AUC) value of at 0.8151 and 0.7316 in patients with colorectal cancer compared to healthy individuals, respectively. Additionally, results in vitro and in vivo experiments suggest that Linc00284 silencing significantly suppresses CRC cell proliferation and/or invasion. Mechanistically, Linc00284 promotes c-Met expression by acting as miR-27a sponge, leading to the activation of downstream signaling pathways, thereby causing malignant phenotypes of CRC cells. Taken together, Linc00284 exhibits oncogenic function and the disturbance of Linc00284/miR-27a/c-Met regulatory axis contributes to CRC progression, providing new insight into the pathogenesis of colorectal cancer. Importantly, the expression levels of serum Linc00284 and miR-27a may serve as clinical biomarkers for CRC diagnosis.

The efficacy and toxicity of induction chemotherapy plus concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma: A meta-analysis of randomized controlled trials.

BACKGROUND: A systemic review and meta-analysis of randomized controlled trials (RCTs) was performed to compare the efficacy, toxicity and safety of concurrent chemoradiotherapy (CCRT) with or without induction chemotherapy (IC) for locoregionally advanced nasopharyngeal carcinoma (NPC). METHODS: Research searching was performed in Web of Science, PubMed, The Cochrane Library, Embase, Chinese Biomedical Database, Chinese National Knowledge Infrastructure, Chongqing VIP Database for Chinese Technical Periodicals and Wanfang Database. RCTs including patients diagnosed with locoregionally advanced NPC without metastasis and randomly treated with IC plus CCRT and CCRT alone were included. Survival and outcome data were extracted and meta-analysis was performed using the Revman 5.3.0 software. RESULTS: Ten RCTs (2280 patients) were selected and used for pooled meta-analysis. In comparison with CCRT, IC plus CCRT treatment significantly improved the overall survival (OS; HR = 0.70, 95%CI 0.56-0.87, P = .002), progression-free survival (PFS; HR = 0.75, 95%CI 0.65-0.87, P < .0001), distant metastasis failure-free survival (DMFS; HR = 0.71, 95%CI 0.58-0.85, P = .0003) and loco-regional failure-free survival (LFES; HR = 0.72, 95%CI 0.59-0.88, P = .002) of patients with locoregionally advanced NPC. Patients treated with IC and CCRT had higher incidence of grade 3-4 leucopenia and thrombocytopenia than patients treated with CCRT alone (P < .0001). No significant difference in other grade 3-4 adverse events and radiation toxicity was observed between the two groups. IC combined with CCRT improved the survival of patients with locoregionally advanced NPC. CONCLUSIONS: Combined IC and CCRT therapy was an efficacy treatment regimen for locoregionally advanced NPC.

Asiaticoside suppresses cell proliferation by inhibiting the NF­κB signaling pathway in colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer­associated mortality. Asiaticoside (AC) exhibits antitumor effects; however, to the best of our knowledge, the biological function of AC in CRC cells remains unclear. Therefore, the aim of the present study was to investigate the effect of AC on CRC cells. In the present study, CCK­8 and colony formation assays were performed to assess the effects of AV on human CRC cell lines (HCT116, SW480 and LoVo). Mitochondrial membrane potential was examined by JC­1 staining. Cell apoptosis and cell cycle were monitored by flow cytometry, and the expression of genes was evaluated using RT­qPCR and western blot analysis. Furthermore, the biological effect of AC in vivo was detected using a xenograft mouse model. The findings revealed that 2 µM AC suppressed the proliferation of CRC cells in a time­ and dose­dependent manner, but had no adverse effects on normal human intestinal FHC cells at a range of concentrations. AC decreased the mitochondrial membrane potential and increased the apoptosis of CRC cells in a dose­dependent manner. Furthermore, AC induced cell cycle arrest at the G0/G1 phase. AC attenuated IκBα phosphorylation in a dose­dependent manner, thereby preventing P65 from entering the nucleus, and resulting in inhibition of the NF­κB signaling pathway. In addition, AC significantly reduced the expression of CDK4 and Cyclin D1 in a dose­dependent manner, significantly upregulated the activation of caspase­9 and caspase­3, and decreased the Bcl­2/Bax mRNA ratio. Furthermore, treatment with the NF­κB signaling pathway inhibitor JSH­23 significantly increased the cytotoxicity of AC in CRC cells. Findings of the xenograft mice model experiments revealed that AC significantly inhibited colorectal tumor growth in a dose­dependent manner. Overall, AC suppressed activation of the NF­κB signaling pathway by downregulating IκBα phosphorylation. This resulted in inhibition of CRC cell viability and an increase of cell apoptosis, which may form the basis of AC use in the treatment of patients with CRC.

Inhibition of esophageal-carcinoma cell proliferation by genistein via suppression of JAK1/2-STAT3 and AKT/MDM2/p53 signaling pathways.

Esophageal carcinoma (EsC) is a clinically challenging neoplastic disease. Genistein, a natural isoflavone product, has anti-tumor properties. Through in vitro and in vivo studies, we found that genistein suppressed EsC cell proliferation in a time- and concentration-dependent manner. In addition, genistein markedly promoted apoptosis and arrested cell cycle at the G0/G1 phase in a concentration-dependent manner. Furthermore, high concentrations of genistein have no adverse effect on normal esophageal epithelial cells. Mechanistically, genistein treatment strikingly reduced the expression of cell cycle-associated genes, and up-regulated the expression of cell apoptosis-related genes in EsC cells. Additionally, genistein dramatically decreased epidermal growth factor receptor (EGFR) expression and attenuated its down-stream signaling molecules STAT3, MDM2, Akt and JAK1/2 phosphorylation, resulting in inhibited nuclear translocation of STAT3 and MDM2, thereby inhibiting the JAK1/2-STAT3 and AKT/MDM2/p53 signaling pathways. In xenograft nude mice, genistein administration strikingly impaired tumor growth in a dose-dependent manner. Moreover, similar disturbances in molecular mechanisms were observed in vivo. Taken together, genistein suppressed the JAK1/2-STAT3 and AKT/MDM2/p53 signaling pathways by decreasing EGFR expression, leading to cell apoptosis, cell cycle arrest, and proliferation inhibition in EsC cells. Our findings suggest that genistein may be a promising alternative adjuvant therapy for patients with EsC.

Effects of Hyriopsis cumingii polysaccharides on angiogenesis, macrophage chemotaxis, proliferation and phagocytosis.

Anti-angiogenic activities of crude Hyriopsis cumingii polysaccharides (HCPS) and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) were evaluated in vivo using the chicken embryo chorioallantoic membrane (CAM) assay. The promoting effects of crude HCPS and its purified fractions on the chemotaxis, proliferation and phagocytosis of peritoneal macrophage were tested by cell model in vitro and cyclophosphamide-induced immuno-suppression animal model in vivo. The results showed that HCPS could significantly suppress the neovascularization of chicken embryo CAM and promote peritoneal macrophage migrating to monocyte chemotactic protein-1 (MCP-1), propagating and devouring sheep red blood cell (SRBC) in a dose-dependent manner. In addition, HCPS-3 showed stronger immunostimulatory activities in vitro than crude HCPS, HCPS-1 and HCPS-2. The beneficial effects of HCPS on the immune system might be, at least in part, attributed to the improvement of chemotaxis, proliferation and phagocytosis of peritoneal macrophage. All these results suggest that HCPS is a potential immunoenhancing and anti-tumor agent.

Immunostimulatory and antiangiogenic activities of low molecular weight hyaluronic acid.

The immunostimulatory activities of two low molecular weight hyaluronic acids (LMWHA-1 and LMWHA-2 with MW of 1.45×10(5) and 4.52×10(4) Da, respectively) and HA (MW, 1.05×10(6) Da) were evaluated by using in vitro cell models and in vivo animal models, and their effects on angiogenesis were measured in vivo by using the chick embryo chorioallantoic membrane (CAM) assay. The results demonstrated that LMWHA-1, LMWHA-2 and HA could promote the splenocyte proliferation, increase the activity of acid phosphatase in peritoneal macrophages and strengthen peritoneal macrophages to devour neutral red in vitro in a dose-dependent manner. Furthermore, LMWHA-1 and LMWHA-2 exhibited much stronger immunostimulatory activity than HA. For assay in vivo, LMWHA-1 and LMWHA-2 significantly increased the indices of spleen and thymus, the activity of lysozyme in serum and the swelling rate of earlap in delayed-type hypersensitivity in a dose-dependent manner. In the CAM model, the results showed that LMWHA-1, LMWHA-2 and HA suppressed angiogenesis in chicken embryos. Moreover, LMWHA-1 exhibited higher antiangiogenesis activity than LMWHA-2 and HA. All these results suggested that LMWHA might be a potential natural immunomodulator and a potential candidate compound for antiangiogenic.

Immunostimulatory activity and structure of polysaccharide from Streptococcus equi subsp. zooepidemicus.

In the present study, crude capsule polysaccharides from Streptococcus equi subsp. zooepidemicus C55129 (CP) were purified by DEAE cellulose-52 column and Sephadex G-100 column chromatography to afford purified fractions of CP-1, CP-2 and CP-3. The relative molecular weights of CP-1, CP-2 and CP-3 were 29.0, 891.0 and 1338.0 kDa, respectively. CP-1 was composed of mannose, glucose, arabinose and galactose in a percentage ratio of 66.15:28.97:2.43:2.45. CP-2 was composed of mannose, glucose, galactose and glucuronic acid in a percentage ratio of 40.94:27.71:5.96:25.39. In CP-1, there were pyranose rings, α-configuration glycosidic bond residues, one backbone chain (1→6 glycosidic bonds) and two branch chains (1→3 and 1→4 glycosidic bonds) in one repeating unit. In vitro immunostimulatory assay, it was found that CP could promote the splenocyte proliferation and increase the activity of acid phosphatase in peritoneal macrophages. The immunostimulatory activity of CP-1 might be related to its monosaccharide composition, molecular weight and α-configuration glycosidic bond.

[Kiss-1 gene expression after radiation and its association with proliferation and apoptosis in colorectal cancer cells].

OBJECTIVE: To investigate the change of expression level of metastasis suppressor gene Kiss-1 in the colorectal cancer cell line SW480 after radiation, and to determine its association with the proliferation and apoptosis of SW480 cells. METHODS: SW480 cells were divided into control group (0 Gy) and study groups (2, 4, 6, 8 Gy). Cells in the study groups were irradiated by 6-MV X-ray radiation for 48 hours. Immunohistochemistry and real-time PCR methods were used to investigate the influence of radiation on Kiss-1 gene expression of SW480. Colony formation assay was used to detect the proliferation of SW480. Flow cytometry-Annexin- V/PI assay was used to observe the change of the apoptosis rate. RESULTS: Compared with the control group, Kiss-1 protein expression increased after radiation of 6, 8 Gy (P<0.05), but no significant changes were observed after radiation of 2, 4 Gy(P>0.05). Kiss-1 gene mRNA level increased after radiation of 2, 4, 6 Gy, while no obvious change was observed for 8 Gy radiation. The apoptosis rates increased for 4, 6, 8 Gy radiation(P<0.05), however, there was no significant difference for 2 Gy radiation (P<0.05). CONCLUSION: Radiation may increase Kiss-1 gene expression in SW480 cells, which results in decreases proliferation and increases apoptosis in residual surviving cells.

Antioxidant acitivity of low molecular weight hyaluronic acid.

Two polysaccharides, low molecular weight hyaluronic acid-1 (LMWHA-1) and LMWHA-2, with their molecular weight of 1.45×10(5) and 4.52×10(4)Da, respectively, were prepared from high molecular weight hyaluronic acid (HA,1.05×10(6)Da). LMWHA-1, LMWHA-2 and HA were studied for their antioxidant activities. In vitro antioxidant assay, LMWHA showed strong inhibition of lipid peroxidation and scavenging activities of hydroxyl radical, moderate 1,1-diphenyl-2-picryldydrazyl radical and superoxide anion scavenging activity. In addition, the LMWHA-1 exhibited much stronger antioxidant activity than LMWHA-2 and HA. For antioxidant testing in vivo, LMWHA-1, LMWHA-2 and HA were orally administrated over a period of 7days in a cyclophosphamide(CY) induced immunosuppressed mice model. As results, administration of LMWHA was able to overcome CY-induced immunosuppression and significantly raised the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant capacity (TAOC) in immunosuppressed mice. The results showed that the LMWHA, possessing pronounced free radical scavenging and antioxidant activities.

Listeria monocytogenes mutants carrying Newcastle disease virus F gene fused to its actA and plcB: in vitro expression and immunogenicity in chickens.

Recombinant Listeria monocytogenes mutants carrying Newcastle disease virus (NDV) fusion protein gene F were constructed by homologous recombination. NDV F or its truncated fragment Fa was used as the model heterologous gene to be integrated into actA or plcB downstream of their signal sequences. Correct orientation of the inserted genes was verified by polymerase chain reaction amplification of F or Fa. The inserted F and Fa were expressed in the two recombinants Lm-DeltaactA-F and Lm-DeltaplcB-Fa as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Both recombinants exhibited reduced virulence to embryonated eggs and mice by about 1.5-2.5 logs as compared with the parent wild strain 10403S. They were also less invasive than strain 10403S (P<0.05). Chickens receiving the recombinant strains orally or intraperitoneally were partially protected from virulent NDV challenge possibly due to enhancement of non-specific immunity because the antibody titers against the homologous virus strain or the recombinant truncated fusion protein were marginal. Further research is needed in other animal models to see if the low antibody response results from insufficient expression of the heterologous genes as a result of failure of L. monocytogenes or its recombinants to persist or replicate in chickens.

Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein.

To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogenes could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.