Tissue microarray study for classification of breast tumors

Clinical and pathological heterogeneity of breast cancer hinders selection of appropriate treatment for individual cases. Molecular profiling at gene or protein levels may elucidate the biological variance of tumors and provide a new classification system that correlates better with biological, clinical and prognostic parameters. We studied the immunohistochemical profile of a panel of seven important biomarkers using tumor tissue arrays. The tumor samples were then classified with a monothetic (binary variables) clustering algorithm. Two distinct groups of tumors are characterized by the estrogen receptor (ER) status and tumor grade (p = 0.0026). Four biomarkers, c-erbB2, Cox-2, p53 and VEGF, were significantly overexpressed in tumors with the ER-negative (ER-) phenotype. Eight subsets of tumors were further identified according to the expression status of VEGF, c-erbB2 and p53. The malignant potential of the ER-/VEGF+ subgroup was associated with the strong correlations of Cox-2 and c-erbB2 with VEGF. Our results indicate that this molecular classification system, based on the statistical analysis of immunohistochemical profiling, is a useful approach for tumor grouping. Some of these subgroups have a relative genetic homogeneity that may allow further study of specific genetically-controlled metabolic pathways. This approach may hold great promise in rationalizing the application of different therapeutic strategies for different subgroups of breast tumors.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Mesenchymal Stem Cell Transplantation Enhancement in Myocardial Infarction Rat Model under Ultrasound Combined with Nitric Oxide Microbubbles

Objective

This study evaluated the effects of ultrasound combined with the homemade nitric oxide (NO) micro-bubble destruction on the in vitro proliferation, apoptosis, and migration of mesenchymal stem cells (MSCs). Furthermore, we studied whether or not irradiation of the NO micro-bubble combined with bone-marrow derived MSC infusion had a better effect on treating myocardial infarction. The possible mechanism of MSC delivery into the infarcted myocardium was also investigated.

Methods

The murine bone marrow-derived MSCs were isolated, cultured, irradiated, and combined with different concentrations of NO microbubbles. MTT proliferation assay, annexin V-FITC apoptosis detection, migration assay, and RT-PCR were performed 24 h after the irradiation. The NO micro-bubbles was a intravenously injected, followed by the infusion of MSCs, which were labeled by CM-Dil. Myocardium was harvested 48 h later and the distribution of MSCs was observed by laser scanning confocal microscope after frozen sectioning. Echocardiography, histological examination, RT-PCR, and western blotting were performed four weeks after the cell transplantation.

Results

Ultrasound combined with 1:70 NO micro-bubbles had no significant impact on the proliferation or apoptosis of MSCs. Transwell chamber findings demonstrated that MSCs migrated more efficiently in group that underwent ultrasound combined with 1:70 NO micro-bubbles. The Real-time PCR results indicated that the expression of CXCR4 was much higher in the group undergoing ultrasound combined with 1:70 NO micro-bubbles. The normalized fluorescence intensity greatly increased in the group of US+NO micro-bubbles and the cardiac function was also markedly improved. Immunohistochemical staining showed that the capillary density was much greater in the group of US+NO micro-bubbles as compared to that of the other groups. RT-PCR and western blotting also revealed a higher SDF-1 and VEGF expression in the group of US+NO micro-bubbles.

Conclusions

NO micro-bubbles could be used in the cell transplantation, which efficiently promoted the MSC homing into the infarcted myocardium.     

Root iron plaque formation and characteristics under N2 flushing and its effects on translocation of Zn and Cd in paddy rice seedlings (Oryza sativa)

Background and Aims

Anoxic conditions are seldom considered in root iron plaque induction of wetland plants in hydroponic experiments, but such conditions are essential for root iron plaque formation in the field. Although ferrous ion availability and root radial oxygen loss capacity are generally taken into account, neglect of anoxic conditions in root iron plaque formation might lead to an under- or over-estimate of their functional effects, such as blocking toxic metal uptake. This study hypothesized that anoxic conditions would influence root iron plaque formation characteristics and translocation of Zn and Cd by rice seedlings.

Methods

A hydroponic culture was used to grow rice seedlings and a non-disruptive approach for blocking air exchange between the atmosphere and the induction solution matrix was applied for root iron plaque formation, namely flushing the headspace of the induction solution with N₂ during root iron plaque induction. Zn and Cd were spiked into the solution after root iron plaque formation, and translocation of both metals was determined.

Key Results

Blocking air exchange between the atmosphere and the nutrient solution by N₂ flushing increased root plaque Fe content by between 11 and 77 % (average 31 %). The N₂ flushing treatment generated root iron plaques with a smoother surface than the non-N₂ flushing treatment, as observed by scanning electron microscopy, but Fe oxyhydroxides coating the rice seedling roots were amorphous. The root iron plaques sequestrated Zn and Cd and the N₂ flushing enhanced this effect by approx. 17 % for Zn and 71 % for Cd, calculated by both single and combined additions of Zn and Cd.

Conclusions

Blocking of oxygen intrusion into the nutrient solution via N₂ flushing enhanced root iron plaque formation and increased Cd and Zn sequestration in the iron plaques of rice seedlings. This study suggests that hydroponic studies that do not consider redox potential in the induction matrices might lead to an under-estimate of metal sequestration by root iron plaques of wetland plants.     

Trehalose Maintains Bioactivity and Promotes Sustained Release of BMP-2 from Lyophilized CDHA Scaffolds for Enhanced Osteogenesis In Vitro and In Vivo

Calcium phosphate (Ca-P) scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2) loaded calcium-deficient hydroxyapatite (CDHA) scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP) activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2) promoted osteogenic differentiation of bone marrow stromal cells (bMSCs) significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ±5.32%) and area (40.71% ±7.14%) as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ±0.44%, calcein: 6.08% ±1.37%) and mineral apposition rate (4.13±0.62 µm/day) in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17±15.02 Mpa) and load of fracture (144.67±16.13 N). These results lay a potential framework for future study by using trehalose to preserve growth factor bioactivity and optimize release profile of Ca-P based delivery system for enhanced bone regeneration.     

Mud-puddling in the yellow-spined bamboo locust, Ceracris kiangsu (Oedipodidae: Orthoptera): Does it detect and prefer salts or nitrogenous compounds from human urine?

C. kiangsu adults were observed visiting human urine, especially on hot summer days. The main chemicals in fresh human urine include inorganic salts and CO(NH₂)_2. When human urine was incubated, NH₄HCO₃ became the richest nitrogenous compound. The phagostimulants, repellents and attractants in urine were identified here. On the filter papers treated with fresh or incubated urine samples, the 5th instar nymphs and the adults started and continued gnawing around the edges, in contrast to the 3rd and the 4th instar nymphs. The consumed areas were dramatically greater on the filters treated with the urine samples incubated for 3-6 days. The feedings of both male and female adults were also stimulated by several urine-borne components such as NaCl, NaH₂PO₄, Na₂SO₄, KCl, NH₄Cl and NH₄HCO₃ but not by CO(NH₂)₂. Among them NaCl was the most powerful phagostimulant. The repelling, or attractive/arresting effects of CO(NH₂)₂ and NH₄HCO₃ were also evaluated by a two-choice test. When exposed to water- and CO(NH₂)₂ solution-immersed filters simultaneously, the adults prefer to stay on water-immersed filter. In contrast, when provided water- and NH₄HCO₃ solution-treated filters, the adults prefer to stay on NH₄HCO₃ solution-treated filter. This demonstrated that CO(NH₂)₂ acted as a repellent and NH₄HCO₃ as an attractant/arrestant. In the bamboo forest, similar feeding behavior was also elicited by NaCl, NH₄HCO₃ but not by CO(NH₂)₂. Comparing to NaCl solution, a mixed solution of NaCl and CO(NH₂)₂ (1:1) significantly decreased the consumed area of the treated filters whereas a mixed solution of NaCl and NH₄HCO₃ (1:1) dramatically increased the consumed area. These results demonstrated that the phagostimulatory effect by NaCl was reduced by CO(NH₂)₂ in fresh urine and was enhanced by NH₄HCO₃ in incubated urine.     

利用纤维床反应器固定化发酵生产丙酸

构建了一种纤维床反应器(FBB), 并将其应用于丙酸的生产。将棉纤维绕成桶状, 固定于反应器中, 即可用于丙酸固定化发酵。以40 g/L的葡萄糖为碳源, 与游离细胞相比, 利用FBB生产丙酸, 丙酸产量由14.58 g/L提高至20.41 g/L, 发酵时间由120 h缩短至60 h。研究了不同糖浓度条件下FBB生产丙酸情况, 并将补料策略应用于丙酸发酵中。结果表明: 补料发酵能够有效改善Propionibacterium freudenreichii CCTCC M207015在高糖条件下丙酸对葡萄糖转化率较低、副产物较多的问题。经补料发酵280 h, 丙酸产量达45.91 g/L, 丙酸质量约占有机酸总质量比例为72.31%。     

Research on hepatitis B virus genotypes and subgenotypes among Bai nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B C) (P=0.077/P=0.663). In Dali, genotypes B, B C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B C) in patients.     

LC-MS/MS determination of helicid in human plasma and its application in pharmacokinetic studies

Helicid is a traditional Chinese medicine used to treat headache and insomnia with definite effects. To facilitate pharmacokinetic studies of helicid in man, a sensitive and specific LC-MS/MS method for the quantitative detection of helicid in human plasma was developed and validated. The method involved the addition of bergeninum as the internal standard (IS), protein precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor→product ion transitions were monitored at m/z 282.8→120.9 for helicid and m/z 326.9→192.2 for the IS, respectively. The lower limit of quantification (LLOQ) was 0.2 μg/L. The calibration curves for helicid was linear over a concentration range of 0.2-20 μg/L. The intra- and inter-batch analyses of QC samples at 0.4, 2, 20 μg/L indicated good precision (%R.S.D. between 2.69 and 5.47%) and accuracy (between 96.15 and 105.05%). The helicid was stable in human plasma stored at room temperature for at least 24h, 4°C for at least 24h, -20°C for at least 1 month, and for routine three freeze-thaw cycles. This accurate and specific assay provides a useful method for evaluating the pharmacokinetic profile of helicid in humans.