Expression of clMagR/clCry4 protein in mBMSCs provides T2-contrast enhancement of MRI.

Here, we propose for the first time the evaluation of magnetosensitive clMagR/clCry4 as a magnetic resonance imaging (MRI) reporter gene that imparts sensitivity to endogenous contrast in eukaryotic organisms. Using a lentiviral vector, we introduced clMagR/clCry4 into C57BL/6 mice-derived bone marrow mesenchymal stem cells (mBMSCs), which could specifically bind with iron, significantly affected MRI transverse relaxation, and generated readily detectable contrast without adverse effects in vivo. Specifically, clMagR/clCry4 makes mBMSCs beneficial for enhancing the sensitivity of MRI-R2 for iron-bearing granules, in which cells recruit exogenous iron and convert these stores into an MRI-detectable contrast; this is not achievable with control cells. Additionally, Prussian blue staining was performed together with ultrathin cell slices to provide direct evidence of natural iron-bearing granules being detectable on MRI. Hence, it was inferred that the sensitivity of MRI detection should be correlated with clMagR/clCry4 and exogenous iron. Taken together, the clMagR/clCry4 has great potential as an MRI reporter gene. STATEMENT OF SIGNIFICANCE: In this study, we propose the evaluation of magnetosensitive clMagR/clCry4 as an MRI reporter gene, imparting detection sensitivity to eukaryotic mBMSCs for endogenous contrast. At this point, the clMagR and clCry4 were located within the cytoplasm and possibly influence each other. The clMagR/clCry4 makes mBMSCs beneficial for enhancing the sensitivity of MRI-R2 for iron-bearing granules, in which protein could specifically bind with iron and convert these stores into MRI-detectable contrast; this is not achieved by control cells. The viewpoint was speculated that the clMagR/clCry4 and exogenous iron were complementary to each other. Additionally, Prussian blue staining was performed together with TEM observations to provide direct evidence that the iron-bearing granules were sensitive to MRI.

[18F]FDG PET/MRI combined with chest HRCT in early cancer detection: a retrospective study of 3020 asymptomatic subjects.

PURPOSE: PET/MRI has become an important medical imaging approach in clinical practice. In this study, we retrospectively investigated the detectability of fluorine-18 (18F)-fluorodeoxyglucose positron emission tomography/magnetic resonance imaging ([18F]FDG PET/MRI) combined with chest computerized tomography (CT) for early cancer in a large cohort of asymptomatic subjects. METHODS: This study included a total of 3020 asymptomatic subjects who underwent whole-body [18F]FDG PET/MRI and chest HRCT examinations. All subjects received a 2-4-year follow-up for cancer development. Cancer detection rate, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the [18F]FDG PET/MRI with or without chest HRCT were calculated and analyzed. RESULTS: Sixty-one subjects were pathologically diagnosed with cancers, among which 59 were correctly detected by [18F]FDG PET/MRI combined with chest HRCT. Of the 59 patients (32 with lung cancer, 9 with breast cancer, 6 with thyroid cancer, 5 with colon cancer, 3 with renal cancer, 1 with prostate cancer, 1 with gastric cancer, 1 with endometrial cancer, and 1 with lymphoma), 54 (91.5%) were at stage 0 or stage I (according to the 8th edition of the tumor-node-metastasis [TNM] staging system), 33 (55.9%) were detected by PET/MRI alone (27 with non-lung cancers and 6 with lung cancer). Cancer detection rate, sensitivity, specificity, PPV, and NPV for PET/MRI combined with chest CT were 2.0%, 96.7%, 99.6%, 83.1%, and 99.9%, respectively. For PET/MRI alone, the metrics were 1.1%, 54.1%, 99.6%, 73.3%, and 99.1%, respectively, and for PET/MRI in non-lung cancers, the metrics were 0.9%, 93.1%, 99.6%, 69.2%, and 99.9%, respectively. CONCLUSIONS: [18F]FDG PET/MRI holds great promise for the early detection of non-lung cancers, while it seems insufficient for detecting early-stage lung cancers. Chest HRCT can be complementary to whole-body PET/MRI for early cancer detection. TRIAL REGISTRATION: ChiCTR2200060041. Registered 16 May 2022. Public site: https://www.chictr.org.cn/index.html.

Transfection of clMagR/clCry4 imparts MR-T2 imaging contrast properties to living organisms (E. coli) in the presence of Fe3+ by endogenous formation of iron oxide nanoparticles.

Rapid development of medical imaging, such as cellular tracking, has increased the demand for "live" contrast agents. This study provides the first experimental evidence demonstrating that transfection of the clMagR/clCry4 gene can impart magnetic resonance imaging (MRI) T2-contrast properties to living prokaryotic Escherichia coli (E. coli) in the presence of Fe3+ through the endogenous formation of iron oxide nanoparticles. The transfected clMagR/clCry4 gene markedly promoted uptake of exogenous iron by E. coli, achieving an intracellular co-precipitation condition and formation of iron oxide nanoparticles. This study will stimulate further exploration of the biological applications of clMagR/clCry4 in imaging studies.

Characterization of neuroinflammation pattern in anti-LGI1 encephalitis based on TSPO PET and symptom clustering analysis.

PURPOSE: TSPO PET with radioligand [18F]DPA-714 is an emerging molecular imaging technique that reflects cerebral inflammation and microglial activation, and it has been recently used in central nervous system diseases. In this study, we aimed to investigate the neuroinflammation pattern of anti-leucine-rich glioma-inactivated 1 (LGI1) protein autoimmune encephalitis (AIE) and to evaluate its possible correlation with clinical phenotypes. METHODS: Twenty patients with anti-LGI1 encephalitis from the autoimmune encephalitis cohort in Huashan Hospital and ten with other AIE and non-inflammatory diseases that underwent TSPO PET imaging were included in the current study. Increased regional [18F]DPA-714 retention in anti-LGI1 encephalitis was detected on a voxel basis using statistic parametric mapping analysis. Multiple correspondence analysis and hierarchical clustering were conducted for discriminate subgroups in anti-LGI1 encephalitis. Standardized uptake value ratios normalized to the cerebellum (SUVRc) were calculated for semiquantitative analysis of TSPO PET features between different LGI1-AIE subgroups. RESULTS: Increased regional retention of [18F]DPA-714 was identified in the bilateral hippocampus, caudate nucleus, and frontal cortex in LGI1-AIE patients. Two subgroups of LGI1-AIE patients were distinguished based on the top seven common symptoms. Patients in cluster 1 had a high frequency of facio-brachial dystonic seizures than those in cluster 2 (p = 0.004), whereas patients in cluster 2 had a higher frequency of general tonic-clonic (GTC) seizures than those in cluster 1 (p < 0.001). Supplementary motor area and superior frontal gyrus showed higher [18F]DPA-714 retention in cluster 2 patients compared with those in cluster 1 (p = 0.024; p = 0.04, respectively). CONCLUSIONS: Anti-LGI1 encephalitis had a distinctive molecular imaging pattern presented by TSPO PET scan. LGI1-AIE patients with higher retention of [18F]DPA-714 in the frontal cortex were more prone to present with GTC seizures. Further studies are required for verifying its value in clinical application.

Magnetoferritin: Process, Prospects, and Their Biomedical Applications.

Ferritin is a spherical iron storage protein composed of 24 subunits and an iron core. Using biomimetic mineralization, magnetic iron oxide can be synthesized in the cavity of ferritin to form magnetoferritin (MFt). MFt, also known as a superparamagnetic protein, is a novel magnetic nanomaterial with good biocompatibility and flexibility for biomedical applications. Recently, it has been demonstrated that MFt had tumor targetability and a peroxidase-like catalytic activity. Thus, MFt, with its many unique properties, provides a powerful platform for tumor diagnosis and therapy. In this review, we discuss the biomimetic synthesis and biomedical applications of MFt.

Directed weighted network structure analysis of complex impedance measurements for characterizing oil-in-water bubbly flow.

Characterizing the flow structure underlying the evolution of oil-in-water bubbly flow remains a contemporary challenge of great interests and complexity. In particular, the oil droplets dispersing in a water continuum with diverse size make the study of oil-in-water bubbly flow really difficult. To study this issue, we first design a novel complex impedance sensor and systematically conduct vertical oil-water flow experiments. Based on the multivariate complex impedance measurements, we define modalities associated with the spatial transient flow structures and construct modality transition-based network for each flow condition to study the evolution of flow structures. In order to reveal the unique flow structures underlying the oil-in-water bubbly flow, we filter the inferred modality transition-based network by removing the edges with small weight and resulting isolated nodes. Then, the weighted clustering coefficient entropy and weighted average path length are employed for quantitatively assessing the original network and filtered network. The differences in network measures enable to efficiently characterize the evolution of the oil-in-water bubbly flow structures.

Significance of resistin expression in acute pancreatitis.

The aim of the present study was to detect the expression of resistin in rats with acute pancreatitis (AP) and investigate its significance in the pathogenesis of AP. In total, 40 Sprague-Dawley rats were randomly divided into four groups (n=10), including the normal control, sham-operated, acute edematous pancreatitis (AEP) and acute necrotizing pancreatitis (ANP) groups. Following the establishment of animal models, the levels of serum resistin, C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß were measured using ELISA. Resistin expression in the pancreatic tissues was detected using an immunohistochemical method. In addition, the mRNA expression of resistin in the pancreatic tissues was analyzed with quantitative polymerase chain reaction. The levels of serum amylase, serum resistin, TNF-α, IL-1ß and CRP were all significantly higher in the AEP and ANP groups when compared with the control and sham-operated groups (P<0.01), as were the pancreas/body weight ratios and pathological scores of the pancreas. These increases were more significant in the ANP group than in the AEP group (P<0.05). The mRNA expression levels of resistin in the pancreatic tissues were markedly higher in the AEP and ANP groups when compared with the control and sham-operated groups (P<0.01), particularly in the pancreatic tissues of the ANP group, which exhibited notably higher levels compared with the AEP group. The serum resistin level was found to positively correlate with the serum levels of CRP, TNF-α and IL-1ß, and the pathological scores of the pancreatic tissues. In conclusion, the results indicated that resistin may be associated with the occurrence and development of AP; thus, the protein may be a valuable indicator for assessing the severity of AP.

Associations between vitamin D receptor polymorphisms and susceptibility to ulcerative colitis and Crohn's disease: a meta-analysis.

BACKGROUND: Several polymorphisms have been identified in the vitamin D receptor (VDR) gene, while their roles in the incidence of ulcerative colitis (UC) and Crohn's disease (CD) are conflicting. This meta-analysis was designed to clarify the impact of these polymorphisms on UC and CD risk. METHODS: The PubMed, Embase, and Cochrane electronic databases were searched from February 1995 to August 2011 for studies on the four VDR polymorphisms: TaqI, BsmI, FokI, and ApaI. Data were extracted and pooled odd ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: Nine studies were included. In Asians, the ff genotype of FokI was associated with increased UC risk (OR = 1.65; 95% CI, 1.11- 2.45). The "a" allele carrier status of ApaI appeared to be a protective factor for CD (OR = 0.81; 95% CI, 0.67-0.97). The tt genotype increased the risk of CD in Europeans (OR = 1.23; 95% CI, 1.02-1.49). Moreover, the tt genotype of TaqI in males had a moderate elevated risk of UC (OR = 1.56; 95% CI, 1.02-2.39) and CD (OR = 1.84; 95% CI, 1.19-2.83). CONCLUSIONS: The meta-analysis reveals a significant increase in CD risk for Europeans carrying TaqI tt genotype and a significant decrease in CD risk for all carriers of the Apal "a" allele. For Asians, the VDR FokI polymorphism appears to confer susceptibility to UC. For males, the TaqI tt genotype is associated with susceptibilities to both UC and CD. Our study explored the genetic risk prediction in UC and CD, and may provide valuable insights into IBD therapy.

[Curcumin promotes apoptosis of esophageal squamous carcinoma cell lines through inhibition of NF-kappaB signaling pathway].

BACKGROUND & OBJECTIVE: Activation of NF-kappaB signaling pathway plays a critical role in the initiation and progression of carcinogenesis. However, the role of NF-kappaB pathway in esophageal squamous cell carcinoma (ESCC) has not been fully elucidated. Studies have shown that curcumin possesses anti-infection and anti-oxidation effects. This study was to evaluate whether curcumin could induce apoptosis through inhibition of NF-kappaB signaling pathway in ESCC cells. METHODS: Expressions of pIkappaBalpha and Bcl-2 were detected using Western blott after incubation of ESCC cells with curcumin (50 micromol/L) at different time points. Apoptosis and the number of viable ESCC cells were analyzed using flow cytometry and MTT, respectively, after the treatment of curcumin, 5-FU, or the combination of curcumin and 5-FU. RESULTS: In two ESCC cell lines, EC9706 and Eca109,curcumin inhibited IkappaBalpha phosphorylation and Bcl-2 in a time-dependent manner; curcumin alone increased cell apoptosis (P<0.05), and the effect became more prominent when it was combined with 5-FU (P<0.05); curcumin plus 5-FU exerted a stronger inhibition effect on cell proliferation than curcumin alone (P<0.05) or 5-FU alone (P<0.05). CONCLUSION: Curcumin inhibits the phosphorylation of IkappaBalpha, leading to suppression of proliferation, induction of apoptosis and an increase of the sensitivity of ESCC cell lines towards 5-FU.

[Expression of Notch1 gene in esophageal squamous cell carcinoma cell line EC9706 and its effect on cell apoptosis].

BACKGROUND & OBJECTIVE: Notch1 signaling pathway is closely associated with carcinogenesis and plays a key role in cell growth, proliferation, differentiation, and apoptosis. This study was to investigate the expression of Notch1 gene in esophageal squamous cell carcinoma (ESCC) cell line EC9706 and its effects on cell apoptosis. METHODS: The expression of Notch1 gene in EC9706 cells was detected by immunocytochemistry. Notch1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). A eukaryotic expression vector pcDNA3.1-Notch1 (termed pcNICD) was constructed and transfected into EC9706 cells; pcDNA3.1 was also transfected as control. After transfection, the expression of Notch1 in EC9706 cells was detected by RT-PCR and Western blot, cell apoptosis was measured by flow cytometry (FCM). RESULTS: The expression of Notch1 gene was detected in EC9706 cells. The mRNA and protein expression of Notch1 gene in pcNICD-transfected EC9706 cells were significantly increased by about 3 folds of those in untreated and pcDNA3.1-transfected cells (P<0.01). However, there was no difference in Notch1 expression between untreated and pcDNA3.1-transfected EC9706 cells (P>0.05). The apoptosis rate was significantly higher in pcNICD-transfected EC9706 cells than in untreated and pcDNA3.1-transfected cells (P<0.01); there was no prominent difference between untreated and pcDNA3.1-transfected EC9706 cells (P>0.05). CONCLUSION: The activated Notch1 signaling pathway gives rise to the apoptosis of EC9706 cells, suggesting that Notch1 gene may be a new therapeutic target for ESCC.

Nuclear factor-kB signaling pathway constitutively activated in esophageal squamous cell carcinoma cell lines and inhibition of growth of cells by small interfering RNA.

Although constitutive nuclear factor (NF)-kappaB activation has been reported in many human tumors, the role of the NF-kappaB pathway in esophageal squamous cell carcinoma (ESCC) has not been known. In this study, NF-kappaB pathway in two ESCC cell lines was investigated using immunocytochemistry, Western blot and reverse transcription-polymerase chain reaction. The activation of NF-kappaB DNA binding was determined by electrophoretic mobility-shift assay. RNA interference was used to specifically inhibit the expression of p65. Growth of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that p50, p65, IkappaBalpha p-IkappaBalpha and IkappaB kinase beta were expressed and mainly localized in the cytoplasm. Reverse transcription-polymerase chain reaction results showed the constitutive expressions of p50, p65 and IkappaBalpha mRNA in the two ESCC cell lines. Furthermore, the nuclear extracts revealed that p50 and p65 translocated to the nucleus had DNA-binding activity. Finally, small interfering RNA of p65 decreased the expression of p65, and the viability of cells transfected with p65 small interfering RNA was significantly suppressed at the same concentration of 5-fluorouracil (P < 0.05) compared to untransfected cells. The results of this study showed that there was the constitutively activated NF-kB signaling pathway in the ESCC cell lines. RNA interference targeting at p65 increased the sensitivity of the ESCC cell lines to 5-fluorouracil, suggesting that NF-kappaB might be a good target for cancer treatment.

Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system.

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.

[Preparation of human recombinant kringle 1-5 and its bioactivity].

To investigate antiangiogenesis activity and effects on endothelial cell proliferation of human recombinant K1-5 expressed in E.coli BL21, the cDNA of human K1-5 obtained from a cloning vector pUC57K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E.coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed and purified to a purity of 96% by the nickel affinity chromatography. Refoled K1-5 protein was tested on chicken CAMs, and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, human recombinant K1-5 potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrated that human recombinant K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell proliferation.

Human canstatin, a 24 kD fragment of the alpha2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

[Expression of mouse canstatin and its N-domain in E. coli BL21].

The mouse canstatin and its N-domain cDNA were amplified from total RNA of mouse liver by RT- PCR and cloned into vector pMD18-T for sequencing. Prokaryotic expression vectors pET/Can and pET/Can-N were constructed and expressed in E.coli BL21(DE3) with induction of IPTG.. Mouse canstatin cDNA is 684bp in length encoding 227 amino acids. The sequences of both cDNA and amino acids share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. N-domain of mouse canstatin is the same amino acid sequence as that of human canstatin. In the present study, prokaryotic expression vector pET/Can and pET/Can-N were expressed in E.coli BL21 with amount of 35% and 18% of the total bacterial proteins after being induced by IPTG for 4h. The expressed products existed mainly as inclusion bodies. This work has laid down the basis for further study of its angiogenic activity and potential application for tumor dormancy therapy.