Study of chondrogenesis of umbilical cord mesenchymal stem cells in curdlan- poly(vinyl alcohol) composite hydrogels and its mechanical properties of freezing-thawing treatments.

The curdlan gel is a natural material produced by bacteria. It utilizes chemical cross-linking reactions to form a 3D porous composite hydrogel, increasing its porosity and water content, and improving its mechanical properties. It can be used in tissue repair and regenerative medicine. Curdlan-Poly(vinyl alcohol) (PVA) composite hydrogel can rapidly swell within 1 min due to its porous structure. Compression tests confirmed that it still maintains its original mechanical strength, even after five repeated freeze-thaw (FT) processes, making it suitable for long-term cryopreservation. The purpose of this study is to transplant umbilical cord mesenchymal stem cells (UC-MSCs) on Curdlan-PVA composite hydrogel and observe the chondrocytes on the material. The results of using 4',6-diamidino-2-phenylindole (DAPI), hematoxylin and eosin (H&E), calcein-acetoxymethyl ester (calcein AM), and Collagen type II-Fluorescein isothiocyanate (FITC) staining, confirmed that UC-MSCs can attach and differentiate into chondrocytes on 3D Curdlan-PVA composite hydrogel.

Anti-Cancer Effects of CDK4/6 Inhibitor LEE011 and Chemotherapy Drugs on Lymphocytic Leukemia L1210 Cells.

BACKGROUND/AIM: Chronic lymphocytic leukemia is a slowly-progressing disease in which symptoms often do not manifest until years after disease onset. In advanced stages, infection and bleeding are common. Past studies have shown that the interaction between CDK4/6 inhibitors and chemotherapy drugs can enhance the anti-tumor efficacy of drugs and limit toxicity. Therefore, in this study, the treatment effects of combining the CDK4/6 inhibitor LEE011 with chemotherapy drugs bendamustine or hydroxyurea were investigated in vitro. MATERIALS AND METHODS: The mouse lymphocytic leukemia cell line L1210 was treated with LEE011 combined with hydroxyurea or bendamustine. Western blot and flow cytometry were performed to elucidate the mechanisms behind tumor suppression. RESULTS: LEE011 combined with hydroxyurea or bendamustine significantly inhibited proliferation of L1210 cell lines in a concentration- and time-dependent manner as well as increased the arrest of cells in G1 and S phases. The combination of LEE011 with hydroxyurea also reduced the phosphorylation of Rb while increased the expression of total Rb protein. Furthermore, reduced expression of GPX4, which is a key protein in ferroptosis, indicates that the tumor suppression effects of this drug combination could involve ferroptosis. CONCLUSION: CDK4/6 inhibitor LEE011 treatment alone may not be a suitable treatment option for lymphocytic leukemia; however, our findings in vitro support the combination of LEE011 with chemotherapy drugs to enhance anti-tumor activity in lymphocytic leukemia.

Structural and Mechanical Properties of Konjac Glucomannan Gels and Influence of Freezing-Thawing Treatments on Them.

Freezing has been widely used for long-term food preservation. However, freezing-thawing (FT) treatment usually influences the texture and structure of food gels such as konjac. For their texture control after FT treatment, it is important to clarify the structural change of food gels during the FT process. In this study, we investigated the aggregated structures of konjac glucomannan (GM) gels during the FT process using simultaneous synchrotron small-angle X-ray/wide-angle X-ray scattering (SAXS/WAXS) techniques. The FT treatment resulted in more crystallization of GM, and consequently, a large increase in compressive stress. In-situ SAXS/WAXS measurements revealed the following findings: on freezing, water molecules came out of the aggregated phase of GM and after the thawing, they came back into the aggregated phase, but the aggregated structure did not return to the one before the freezing; the gel network enhanced the inhomogeneity due to the growth of ice crystals during freezing. Furthermore, we examined the influence of additives such as polyvinyl (alcohol) (PVA) and antifreeze glycoprotein (AFGP) on the mechanical and structural properties of freeze-thawed GM gels. Although the addition of PVA and AFGP suppressed the crystallization of GM, it could not prevent the growth of ice crystals and the increase in the inhomogeneity of the gel network. As a result, the compressive stresses for freeze-thawed GM gels containing PVA or AFGP were significantly higher compared with those of GM gels without FT treatments, although they were lower than those of freeze-thawed GM gels. The findings of this study may be useful for not only the texture control of freeze-thawed foods but also the improvement of the mechanical performance of the biomaterials.

Glucose starvation induces a switch in the histone acetylome for activation of gluconeogenic and fat metabolism genes.

Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.

Chemopreventive Effect of Phytosomal Curcumin on Hepatitis B Virus-Related Hepatocellular Carcinoma in A Transgenic Mouse Model.

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC), a leading cause of cancer mortality worldwide. Hepatitis B X protein (HBx) and pre-S2 mutant have been proposed as the two most important HBV oncoproteins that play key roles in HCC pathogenesis. Curcumin is a botanical constituent displaying potent anti-inflammatory and anti-cancer properties without toxic side effects. Phytosomal formulation of curcumin has been shown to exhibit enhanced bioavailability, improved pharmacokinetics, and excellent efficacy against many human diseases. However, effectiveness of phytosomal curcumin for HCC treatment remains to be clarified. In this study, we evaluated chemopreventive effect of phytosomal curcumin on HBV-related HCC by using a transgenic mouse model specifically expressing both HBx and pre-S2 mutant in liver. Compared with unformulated curcumin, phytosomal curcumin exhibited significantly greater effects on suppression of HCC formation, improvement of liver histopathology, decrease of lipid accumulation and leukocyte infiltration, and reduction of total tumor volume in transgenic mice. Moreover, phytosomal curcumin exerted considerably stronger effects on activation of anti-inflammatory PPARγ as well as inhibition of pro-inflammatory NF-κB than unformulated curcumin. Furthermore, phytosomal curcumin showed a comparable effect on suppression of oncogenic mTOR activation to unformulated curcumin. Our data demonstrated that phytosomal curcumin has promise for HCC chemoprevention in patients with chronic HBV infection.

Liver regeneration accelerates hepatitis B virus-related tumorigenesis of hepatocellular carcinoma.

Although partial hepatectomy (PH) to remove tumors provides a potential cure of hepatocellular carcinoma (HCC), long-term survival of hepatitis B virus (HBV)-related HCC patients after PH remains a big challenge. Early recurrence within 2 years post-PH is associated with the dissemination of primary HCC. However, late recurrence after 2 years post-PH is supposed due to the de novo or a secondary tumor. Since PH initiates liver regeneration (LR), we hypothesize that LR may accelerate tumorigenesis through activation of pre-existing precancerous lesions in the remaining liver. In this study, we explored the potential role of several LR-related factors in the de novo recurrence in a HBV X protein (HBx) transgenic mouse model receiving PH to mimic human HCC development. Following PH, we observed that tumor development was significantly accelerated from 16.9 to 10.4 months in HBx transgenic mice. The expression of suppressor of cytokine signaling (SOCS) family proteins was remarkably suppressed in livers of HBx transgenic relative to non-transgenic mice from early to late stages after PH as compared with non-PH mice. The expression of transforming growth factor-ß (TGF-ß)/Smad pathway, hepatocyte growth factor (HGF), Myc, signal transducer and activator of transcription 3 (STAT3), and ß-Catenin also showed a significant difference between livers of HBx transgenic and non-transgenic mice at variable time points after PH in comparison with non-PH mice. Taken together, our results provide an explanation for the high de novo recurrence of HBV-related HCC after PH, probably through induction of the sequential changes of LR-related SOCS family proteins, growth factors, and transcription factors, which may promote growth on the precancerous remnant liver.

The Use of PET Imaging for Prognostic Integrin α2ß1 Phenotyping to Detect Non-Small Cell Lung Cancer and Monitor Drug Resistance Responses.

PURPOSE: Growing evidence has demonstrated that aberrant expression of integrin α2ß1 might contribute to the invasion, metastasis and drug resistance of non-small cell lung cancer (NSCLC). Thus, the integrin α2ß1 targeting 68Ga-DOTA-A2B1 tracer was validated in NSCLC in contrast to accumulation of the clinically used 18F-FDG PET tracer to see if 68Ga-DOTA-A2B1-PET imaging can offer a valuable and critical diagnostic imaging criterion for the identification of phenotypes of aggressive lung cancer. METHODS: To verify the prognostic value of integrin α2ß1, several quantitative and functional in vitro assays were validated in different NSCLC cell lines (CL1-0, CL1-5, A549 and selected A549++ cells). Positron emission tomography (PET) imaging studies using both standard 18F-FDG and a newly developed 68Ga-labeled integrin α2ß1 (68Ga-DOTA-A2B1) tracer were sequentially performed on mice with lung tumor xenografts in different anatomic locations (subcutaneous, orthotopic and osseous) to validate the targeting capability of the 68Ga-DOTA-A2B1 tracers. Treatment responses were monitored by injecting animals with metastatic bone tumors with 5 mg/kg doxorubicin. All in vivo treatment responses in each treatment subgroup were monitored with a PET imaging system to evaluate the up-regulation of integrin expression at the earliest stage of treatment (6 h). RESULTS: The PET and computed tomography (CT) images from NSCLC xenograft animals unambiguously demonstrated accumulation of the integrin tracer 68Ga-DOTA-A2B1 in the tumor lesions at all locations. The average tumor uptake and tumor-to-normal (T/N) ratio were 2.51 ± 0.56 %ID/g and T/N = 2.82, 3.40 ± 0.42 %ID/g and T/N = 1.52, and 1.58 ± 0.108 %ID/g and T/N = 2.31 in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). The xenograft tumors were all clearly visible. In contrast, the accumulation of 18F-FDG reached 3.6 ± 0.76 %ID/g, 1.39 ± 0.075 %ID/g and 3.78 ± 0.73 %ID/g in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). However, due to the high background uptake by normal tissue, the T/N values were less than or close to 1, making the tumors almost indistinguishable in the PET imaging analysis. Furthermore, 68Ga-DOTA-A2B1-PET imaging of the treated osseous tumor model demonstrated more than 19% tracer uptake in A549 lesions (1.72 ± 0.95 %ID/g vs. pretreatment 1.44 ± 0.12 %ID/g,p = 0. 015) 6 h post-treatment with doxorubicin. The elevated intensity of tracer uptake was in accordance with the results of in vitroWestern blot and ex vivo integrin staining, demonstrating elevated integrin α2ß1 expression. CONCLUSION: In this study, integrin α2ß1 was identified as a biomarker of aggressive malignant NSCLC. Thus, efforts should be devoted to validating integrin α2ß1 as a potential target for non-invasive diagnosis and as a predictive marker for monitoring treatment responses using a preclinical PET imaging system.

Saikosaponin a Induces Apoptosis through Mitochondria-Dependent Pathway in Hepatic Stellate Cells.

Saikosaponin a (SSa) is one of the main active components of Bupleurum falcatum. It is commonly used to treat liver injury and fibrosis in traditional Chinese medicine. Our previous study showed that SSa induces apoptosis and inhibits the proliferation of rat hepatic stellate cell (HSC) line HSC-T6. The aim of the present study was to elucidate the mechanism of SSa-mediated apoptosis. Rat HSC cell line HSC-T6 and human HSC cell line LX-2 were used in this study. SSa triggered cell death mainly by apoptosis, as indicated by the typical morphological changes, sub-G1 phase of cell cycle increase, and activation of the caspase-9/caspase-3 cascade. In addition, SSa-induced apoptosis was partially inhibited by the caspase-3 inhibitor Z-DEVD-FMK, suggesting an involvement of caspase-3 dependent and independent pathways. Moreover, SSa upregulated pro-apoptotic proteins [BAK, Bcl-2-associated death promoter (BAD), and p53 upregulated modulator of apoptosis (PUMA)] and downregulated anti-apoptotic proteins (Bcl-2). In the mitochondria, SSa triggered the translocation of BAX and BAK from the cytosol to the outer membrane, resulting in a reduction of mitochondrial functions and membrane potential and subsequent release of apoptotic factors. Therefore, this study demonstrates that SSa induces apoptosis through the intrinsic mitochondrial-dependent pathway in HSCs.

Biocompatible testing and physical properties of curdlan-grafted poly(vinyl alcohol) scaffold for bone tissue engineering.

Curdlan was grafted to poly(vinyl alcohol) (PVA) to form a porous scaffold. The grafted PVA-curdlan 3D scaffold was then examined by Fourier transform infrared spectroscopy (FT-IR). Grafting increased the water absorbency of the scaffold by 280%. Scanning electron microscopic (SEM) observations of the material revealed that the 3D scaffold was highly porous when it was fabricated using a homogenizer at 300rpm. Compression testing revealed that, increasing the amount of curdlan increased the strength of the 3D scaffold to 8-16×10-3MPa. Over 28days, various enzymes degraded the 3D scaffold, causing a weight loss of up to 20-40%. In vivo tests revealed favorable cell proliferation and growth in a 3D scaffold.

Saikosaponin d induces cell death through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells.

BACKGROUND: Saikosaponin d (SSd) is one of the main active triterpene saponins in Bupleurum falcatum. It has a steroid-like structure, and is reported to have pharmacological activities, including liver protection in rat, cell cycle arrest and apoptosis induction in several cancer cell lines. However, the biological functions and molecular mechanisms of mammalian cells under SSd treatment are still unclear. METHODS: The cytotoxicity and apoptosis of hepatic stellate cells (HSCs) upon SSd treatment were discovered by MTT assay, colony formation assay and flow cytometry. The collage I/III, caspase activity and apoptotic related genes were examined by quantitative PCR, Western blotting, immunofluorescence and ELISA. The mitochondrial functions were monitored by flow cytometry, MitoTracker staining, ATP production and XF24 bioenergetic assay. RESULTS: This study found that SSd triggers cell death via an apoptosis path. An example of this path might be typical apoptotic morphology, increased sub-G1 phase cell population, inhibition of cell proliferation and activation of caspase-3 and caspase-9. However, the apoptotic effects induced by SSd are partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK, suggesting that SSd may trigger both HSC-T6 and LX-2 cell apoptosis through caspase-3-dependent and independent pathways. We also found that SSd can trigger BAX and BAK translocation from the cytosol to the mitochondria, resulting in mitochondrial function inhibition, membrane potential disruption. Finally, SSd also increases the release of apoptotic factors. CONCLUSIONS: The overall analytical data indicate that SSd-elicited cell death may occur through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian HSCs, and thus can delay the formation of liver fibrosis by reducing the level of HSCs.

A biphasic response pattern of lipid metabolomics in the stage progression of hepatitis B virus X tumorigenesis.

Metabolic syndrome has closely linked to the development of human hepatocellular carcinoma (HCC). By using the hepatitis B virus (HBV) X (HBx) transgenic mouse model, we studied the dynamic evolution of serum and liver profiles of lipids and global cDNA expression at different stages of HBx tumorigenesis. We observed that the lipid (triglycerides, cholesterol, and fatty acids) profiles revealed a biphasic response pattern during the progression of HBx tumorigenesis: a small peak at early phase and a large peak or terminal switch at the tumor phase. By analyzing cDNA microarray data, the early peak correlated to the oxidative stress and pro-inflammatory response, which then resolved at the middle phase and were followed by the terminal metabolic switch in the tumor tissues. Five lipid metabolism-related genes, the arachidonate 5-lipoxygenase, lipoprotein lipase, fatty acid binding protein 4, 1-acylglycerol-3-phosphate O-acyltransferase 9, and apolipoprotein A-IV were identified to be significantly activated in HBx transgenic HCCs and further validated in human HBV-related HCCs. Inhibition of these lipid genes could reverse the effect of HBx on lipid biosynthesis and suppress HBx-induced cell proliferation in vitro. Our results support the concept that metabolic syndrome plays an important role in HBV tumorigenesis. The dysregulation of lipid metabolic genes may predict the disease progression to HCC in chronic hepatitis B patients.

Hepatitis B Virus Pre-S2 Mutant Induces Aerobic Glycolysis through Mammalian Target of Rapamycin Signal Cascade.

Hepatitis B virus (HBV) pre-S2 mutant can induce hepatocellular carcinoma (HCC) via the induction of endoplasmic reticulum stress to activate mammalian target of rapamycin (MTOR) signaling. The association of metabolic syndrome with HBV-related HCC raises the possibility that pre-S2 mutant-induced MTOR activation may drive the development of metabolic disorders to promote tumorigenesis in chronic HBV infection. To address this issue, glucose metabolism and gene expression profiles were analyzed in transgenic mice livers harboring pre-S2 mutant and in an in vitro culture system. The pre-S2 mutant transgenic HCCs showed glycogen depletion. The pre-S2 mutant initiated an MTOR-dependent glycolytic pathway, involving the eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), Yin Yang 1 (YY1), and myelocytomatosis oncogene (MYC) to activate the solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1), contributing to aberrant glucose uptake and lactate production at the advanced stage of pre-S2 mutant transgenic tumorigenesis. Such a glycolysis-associated MTOR signal cascade was validated in human HBV-related HCC tissues and shown to mediate the inhibitory effect of a model of combined resveratrol and silymarin product on tumor growth. Our results provide the mechanism of pre-S2 mutant-induced MTOR activation in the metabolic switch in HBV tumorigenesis. Chemoprevention can be designed along this line to prevent HCC development in high-risk HBV carriers.

Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis.

Although hepatitis B virus (HBV) has been established to cause hepatocellular carcinoma (HCC), the exact mechanism remains to be clarified. Type II ground glass hepatocytes (GGHs) harbouring the HBV pre-S2 mutant large surface protein (LHBS) have been recognized as a morphologically distinct hallmark of HCC in the advanced stages of chronic HBV infection. Considering its preneoplastic nature, we hypothesized that type II GGH may exhibit high genomic instability, which is important for the carcinogenic process in chronic HBV carriers. In this study we found that pre-S2 mutant LHBS directly interacted with importin α1, the key factor that recognizes cargos undergoing nuclear transportation mediated by the importin α/ß-associated nuclear pore complex (NPC). By interacting with importin α1, which inhibits its function as an NPC factor, pre-S2 mutant LHBS blocked nuclear transport of an essential DNA repair and recombination factor, Nijmegen breakage syndrome 1 (NBS1), upon DNA damage, thereby delaying the formation of nuclear foci at the sites of DNA double-strand breaks (DSBs). Pre-S2 mutant LHBS was also found to block NBS1-mediated homologous recombination repair and induce multi-nucleation of cells. In addition, pre-S2 mutant LHBS transgenic mice showed genomic instability, indicated by increased global gene copy number variations (CNVs), which were significantly higher than those in hepatitis B virus X mice, indicating that pre-S2 mutant LHBS is the major viral oncoprotein inducing genomic instability in HBV-infected hepatocytes. Consistently, the human type II GGHs in HCC patients exhibited increased DNA DSBs representing significant genomic instability. In conclusion, type II GGHs harbouring HBV pre-S2 mutant oncoprotein represent a high-risk marker for the loss of genome integrity in chronic HBV carriers and explain the complex chromosome changes in HCCs. Mouse array CGH raw data: GEO Accession No. GSE61378 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61378).

Activation of ATP citrate lyase by mTOR signal induces disturbed lipid metabolism in hepatitis B virus pre-S2 mutant tumorigenesis.

UNLABELLED: The development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) has been found to be associated with disturbed lipid metabolism. To elucidate the role of lipid metabolism in HBV tumorigenesis, we investigated the dynamic pattern of lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis. Lipid and gene expression profiles were analyzed in an in vitro culture system and in transgenic mouse livers harboring HBV pre-S2 mutant. The pre-S2 mutant transgenic livers showed a biphasic pattern of lipid accumulation, starting from mild fatty change in early (1 month) transgenic livers, which subsided and then, remarkably, increased in HCC tissues. This biphasic pattern was synchronized with ATP citrate lyase (ACLY) activation. Further analyses revealed that the pre-S2 mutant initiated an endoplasmic reticulum (ER) stress-dependent mammalian target of rapamycin (mTOR) signalling cascade. The pre-S2 mutant-induced mTOR signal activated the sterol regulatory element binding transcription factor 1 (SREBF1) to upregulate ACLY, which then activated the fatty acid desaturase 2 (FADS2), mediated through ACLY-dependent histone acetylation. Such an ER stress-dependent mTOR signal cascade also is important for the proliferation of hepatocytes in vitro and is further validated in HBV-related HCC tissues. IMPORTANCE: Aberrations of lipid metabolism frequently occur in chronic HBV infection. Our results provide a potential mechanism of disturbed lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis, which should be valuable for the design of HCC chemoprevention in high-risk HBV carriers.

The emerging role of hepatitis B virus pre-S2 deletion mutant proteins in HBV tumorigenesis.

Chronic hepatitis B virus (HBV) infection can cause hepatocellular carcinoma (HCC). Several hypotheses have been proposed to explain the mechanisms of HBV tumorigenesis, including inflammation and liver regeneration associated with cytotoxic immune injuries and transcriptional activators of mutant HBV gene products. The mutant viral oncoprotein-driven tumorigenesis is prevailed at the advanced stage or anti-HBe-positive phase of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH). The retention of pre-S2 mutant protein in ER can induce ER stress and initiate an ER stress-dependent VEGF/Akt/mTOR and NFκB/COX-2 signal pathway. Additionally, the pre-S2 mutant large surface protein can induce an ER stress-independent pathway to transactivate JAB-1/p27/RB/cyclin A,D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress can also cause DNA damage, centrosome overduplication, and genomic instability. In 5-10% of type II GGHs, there is co-expression of pre-S2 mutant protein and HBx antigen which exhibited enhanced oncogenic effects in transgenic mice. The mTOR signal cascade is consistently activated throughout the course of pre-S2 mutant transgenic livers and in human HCC tissues, leading to metabolic disorders and HCC tumorigenesis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develop resistance to nucleoside analogues anti-virals and predict HCC development. The pre-S2 deletion mutants and type II GGHs therefore represent novel biomarkers of HBV-related HCCs. A versatile DNA array chip has been developed to detect pre-S2 mutants in serum. Overall, the presence of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development. Targeting at pre-S2 mutant protein-induced, ER stress-dependent, mTOR signal cascade and metabolic disorders may offer potential strategy for chemoprevention or therapy in high risk chronic HBV carriers.

Evaluation of prognostic integrin α2ß1 PET tracer and concurrent targeting delivery using focused ultrasound for brain glioma detection.

The ability to early detect and assess the treatment response of recurrent and/or disseminated metastatic glioblastoma is critical for the effective management of this group of patients. Accumulating experimental evidence indicates that integrin α2ß1 might be a prognostic biomarker for advanced phenotype of cancers. In this study, a novel (68)Ga-labeled integrin α2ß1-targeted PET tracer (68)Ga-NOTA-PEG4-cyclo (GDGEAyK) ((68)Ga-A2B1) was designed and evaluated for the potential prognostic imaging of glioblastoma tumor in preclinical model. To prospectively verify the prognostic value of integrin α2ß1, the in vitro Western blot and flow cytometry studies were performed to validate the integrin expression level of human glioblastoma (U87MG) cells. Extremely high expression level of integrin α2ß1 justifies its role as a potential targeting marker. Thus, (68)Ga-A2B1 positron emission tomography was performed in subcutaneous U87MG tumor bearing athymic mice at 15 min postinjection after injection of 7-8MBq tracers. The receptor targeting specificity was confirmed in a competition blocking experiment. The tumor uptake of (68)Ga-A2B1 in the control and blockage groups was 1.57 ± 0.13 %ID/g (n = 3) and 0.96 ± 0.23 %ID/g** (n = 3), respectively. However, because of the quick renal washout rate and labile nature of peptide tracers in circulation conditions, the focus ultrasound (FUS) mediated delivery method was adopted to enhance tumor uptake and retention of tracers. To test the FUS delivery efficacy in vivo, three experimental arms were designed as follows: tumor bearing mice were administrated with (68)Ga-A2B1 only or microbubbles (MBs) with FUS treatment ((68)Ga-A2B1 + FUS + MBs) or embedded (68)Ga-A2B1-microbubbles ((68)Ga-A2B1-MBs + FUS) followed with FUS sonication. The average radioactivity accumulation within a tumor was quantified from the multiple region of interest volumes using the %ID/g value and was analyzed in accordance with the ex vivo autoradiographic and pathologic data. The significant tumor uptake in (68)Ga-A2B1 + FUS +MBs group (n = 6) and (68)Ga-A2B1-MBs + FUS group (n = 4) following FUS treatment were calculated as 2.25 ± 0.50 %ID/g* and 2.6 ± 0.49 %ID/g**, comparing with (68)Ga-A2B1 only group 1.48 ± 0.42 %ID/g (n = 10). These results suggest that there is significant difference in (68)Ga-A2B1 tumor uptake by FUS treatment either with or without tracer integration with microbubbles, which demonstrate a promising delivery strategy and critical multimodal setting for phenotyping imaging of aggressive glioma tumor. In conclusion, (68)Ga labeled (68)Ga-A2B1 allows noninvasive imaging of tumor-associated α2ß1 expression and can be embedded in MB lipid shell for enhanced delivery and controlled release by sonoporation.

Ground-glass hepatocytes co-expressing hepatitis B virus X protein and surface antigens exhibit enhanced oncogenic effects and tumorigenesis.

Hepatitis B virus (HBV) X protein (HBx) and pre-S2 deletion mutant large surface antigens are oncoproteins that induce hepatocellular carcinoma (HCC). The interaction of these two oncoproteins in hepatocytes and its significance in tumorigenesis remain to be elucidated. In this study, we observed the co-expression of HBx with surface antigens in ground-glass hepatocytes in 5 of 20 hepatitis B surface antigen-positive livers. In vitro, hepatocytes co-expressing HBx and a pre-S2 mutant showed enhanced expression of vascular endothelial growth factor-A, phosphorylated Akt 1/2/3, phosphorylated extracellular signal-regulated kinase 1/2, and phosphorylated mammalian target of rapamycin signals. Transgenic mice harboring both HBx and pre-S2 mutant construct plasmids developed HCCs at an average of 15.1 months, earlier than animals carrying either HBx (16.9 months) or pre-S2 mutant (24.5 months) alone. The oncogenic signals of vascular endothelial growth factor-A, phosphorylated Akt 1/2/3, phosphorylated extracellular signal-regulated kinase 1/2, and phosphorylated mammalian target of rapamycin were sequentially and differentially activated at different stages in tumorigenesis. Phosphorylated mTOR was consistently activated in transgenic and human HCCs. We conclude that ground-glass hepatocytes co-expressing HBx and surface antigens exhibit enhanced oncogenic effects and tumorigenesis in chronic HBV infections. The mTOR signal cascade may be the key regulator in HBV tumorigenesis and may be useful targets in the design of HCC therapy.

Cell culture and characterization of cross-linked poly(vinyl alcohol)-g-starch 3D scaffold for tissue engineering.

The research goal of this experiment is chemically to cross-link poly(vinyl alcohol) (PVA) and starch to form a 3D scaffold that is effective water absorbent, has a stable structure, and supports cell growth. PVA and starch can be chemically cross-linked to form a PVA-g-starch 3D scaffold polymer, as observed by Fourier transform infrared spectroscopy (FTIR), with an absorbency of up to 800%. Tensile testing reveals that, as the amount of starch increases, the strength of the 3D scaffold strength reaches 4×10(-2) MPa. Scanning electron microscope (SEM) observations of the material reveal that the 3D scaffold is highly porous formed using a homogenizer at 500 rpm. In an enzymatic degradation, the 3D scaffold was degraded by various enzymes at a rate of up to approximately 30-60% in 28 days. In vitro tests revealed that cells proliferate and grow in the 3D scaffold material. Energy dispersive spectrometer (EDS) analysis further verified that the bio-compatibility of this scaffold.

Detection and characterization of R-loops at the murine immunoglobulin Sα region.

IgA is the most abundant antibody in mammals. However, the mechanism of its class switching is still not clear. The formation of the R-loops, as the target for AID, has been proposed to play a crucial role during mammalian class switch recombination. Here, we provide a systematic evaluation of R-loops at Sα (IgA) in CH12F3-2A cells, which is a unique cell model system for class switch recombination because of its consistent switching to IgA upon stimulation. The results of R-loop analysis demonstrate distinct features specific to Sα. Some R-loops may initiate from the end of Iα, but all terminate exclusively within Sα. Time-course analysis also indicates that the percentage of R-loops peaks prior to the occurrence of class switch recombination. This is the first demonstration that R-loops form at Sαin vitro and in situ, despite variable G density and relatively few GGGG clusters in Sα. The short distance from the promoter to Sα may compensate for the less robust R-loop-forming factors at Sα relative to other switch regions. In conclusion, R-loops at the Sα region further support R-loop formation as a general feature of all stimulated switch regions.