Blastocyst formation, embryo transfer and breed comparison in the first reported large scale cloning of camels.

Cloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.

Insulin secretion and sensitivity before and after surgical treatment for aldosterone-producing adenoma.

AIM: Primary aldosteronism, which is usually caused by an aldosterone-producing tumour, affects glucose metabolism. The effects of this condition on insulin secretion and insulin sensitivity have remained unclear, however. To gain insight into the influence of primary aldosteronism on glucose tolerance, various parameters related to insulin secretion or insulin sensitivity in patients with an aldosterone-producing tumour were comprehensively analyzed. METHODS: To assess 14 patients with an aldosterone-producing tumour, hyperglycaemic and hyperinsulinaemic-euglycaemic clamp tests as well as oral glucose tolerance tests (OGTTs) were performed before and after tumour excision. Time between presurgical analysis and surgery was 27-559 (194±132) days, and 14-142 (51±38) days between surgery and postsurgical analysis. Various parameters related to insulin secretion or sensitivity as determined by OGTT as well as hyperglycaemic and hyperinsulinaemic-euglycaemic clamp analyses were evaluated. RESULTS: Surgical treatment of tumours ameliorated hypokalaemia and reduced plasma aldosterone levels. First and second phases of insulin secretion during the hyperglycaemic clamp, as well as the insulinogenic index and total insulin secretion measured during OGTT, were also improved after surgery. In addition, the insulin sensitivity index determined during the hyperinsulinaemic-euglycaemic clamp was reduced after surgery. CONCLUSION: Primary aldosteronism impairs insulin secretion.

Astaxanthin improves the developmental competence of in vitro-grown oocytes and modifies the steroidogenesis of granulosa cells derived from bovine early antral follicles.

In this study we investigated the effect of astaxanthin (Ax), which exhibits strong antioxidant activity, during invitro growth (IVG) on the developmental competence of oocytes and steroidogenesis of granulosa cells derived from early antral follicles. Bovine oocyte-cumulus-granulosa complexes collected from early antral follicles were cultured for 12 days in the presence or absence (control) of 500µM Ax. The viability of oocytes and antrum formation in the granulosa cell layer during IVG culture were greater in the presence than absence of Ax (P<0.05). Regardless of Ax treatment, 17ß-oestradiol production increased during IVG culture; however, progesterone production was significantly lower in the presence than absence of Ax (P<0.05). Reactive oxygen species levels were lower in Ax-treated oocytes than in controls after IVG (P<0.05). Although nuclear maturation and cleavage rates did not differ between the Ax-treated and control groups, Ax treatment led to weaker cathepsin B activity in oocytes and better blastocyst rates than in controls (P<0.05). Accordingly, Ax treatment during IVG increased the total number of cells in blastocysts (P<0.05). These results indicate that Ax supplementation of IVG medium improves the quality of bovine oocytes due to its antioxidative effects on growing oocytes and its suppression of the luteinisation of granulosa cells.

Vascularized Tissue-Engineered Model for Studying Drug Resistance in Neuroblastoma.

Neuroblastoma is a vascularized pediatric tumor derived from neural crest stem cells that displays vasculogenic mimicry and can express a number of stemness markers, such as SOX2 and NANOG. Tumor relapse is the major cause of succumbing to this disease, and properties attributed to cancer stem-like cells (CSLC), such as drug-resistance and cell plasticity, seem to be the key mechanisms. However, the lack of controllable models that recapitulate the features of human neuroblastoma limits our understanding of the process and impedes the development of new therapies. In response to these limitations, we engineered a perfusable, vascularized in vitro model of three-dimensional human neuroblastoma to study the effects of retinoid therapy on tumor vasculature and drug-resistance. METHODS: The in vitro model of neuroblastoma was generated using cell-sheet engineering and cultured in a perfusion bioreactor. Firstly, we stacked three cell sheets containing SKNBE(2) neuroblastoma cells and HUVEC. Then, a vascular bed made of fibrin, collagen I and HUVEC cells was placed onto a collagen-gel base with 8 microchannels. After gelling, the stacked cell sheets were placed on the vascular bed and cultured in the perfusion bioreactor (perfusion rate: 0.5 mL/min) for 4 days. Neuroblastoma models were treated with 10µM isotretionin in single daily doses for 5 days. RESULTS: The bioengineered model recapitulated vasculogenic mimicry (vessel-like structure formation and tumor-derived endothelial cells-TECs), and contained CSLC expressing SOX2 and NANOG. Treatment with Isotretinoin destabilized vascular networks but failed to target vasculogenic mimicry and augmented populations of CSLCs expressing high levels of SOX2. Our results suggest that CSLCs can transdifferentiate into drug resistant CD31+-TECs, and reveal the presence of an intermediate state STEC (stem tumor-derived endothelial cell) expressing both SOX2 and CD31. CONCLUSION: Our results reveal some roles of SOX2 in drug resistance and tumor relapse, and suggest that SOX2 could be a therapeutic target in neuroblastoma.

Autophagy is required for cell survival under L-asparaginase-induced metabolic stress in acute lymphoblastic leukemia cells.

L-asparaginase has been used for more than three decades in acute lymphoblastic leukemia (ALL) patients and remains an essential drug in the treatment of ALL. Poor response to L-asparaginase is associated with increased risk of therapeutic failure in ALL. However, both the metabolic perturbation and molecular context of L-asparaginase-treated ALL cells has not been fully elucidated. Here we identify that treatment with L-asparaginase results in metabolic shutdown via the reduction of both glycolysis and oxidative phosphorylation, accompanied by mitochondrial damage and activation of autophagy. The autophagy is involved in reducing reactive oxygen species (ROS) level by eliminating injured mitochondria. Inhibition of autophagy enhances L-asparaginase-induced cytotoxicity and overcomes the acquired resistance to L-asparaginase in ALL cells. The ROS-p53-positive feedback loop is an essential mechanism of this synergistic cytotoxicity. Thus, our findings provide the rationale for the future development of combined treatment of L-asparaginase and anti-autophagy drug in ALL patients.

Salmonella Enterica Serovar Pomona Infection in Farmed Juvenile American Alligators ( Alligator Mississippiensis).

A fatal epizootic of salmonellosis occurred in farmed juvenile American alligators in Louisiana. Six animals were examined. Gross lesions included severe fibrinonecrotizing enterocolitis, necrotizing splenitis, coelomic effusion, and perivisceral and pulmonary edema. Microscopic examination revealed severe necrotizing enterocolitis and splenitis with intralesional bacteria and pneumocyte necrosis with fibrin thrombi. Salmonella enterica serovar Pomona was isolated from intestine and lung. Clinical salmonellosis is a rare finding in reptiles and salmonellosis caused by S. Pomona is not previously reported in American alligators. Since S. Pomona is a commonly isolated Salmonella serotype from patients with reptile-associated salmonellosis in the United States, and since alligator meat is consumed and the skin is exported to numerous countries, risk of human and animal infection should be considered.

Polymeric immunoglobulin receptor expression and local immunoglobulin A production in bovine sublingual, submandibular and parotid salivary glands.

The submandibular and parotid glands are the main sources of immunoglobulins A (IgAs) in human and rat saliva. These glands express the polymeric immunoglobulin receptor (pIgR), which transports IgAs into saliva. The main source of IgAs in saliva and pIgR expression in salivary glands has not been well documented in cattle. Expressions of pIgR were determined in the major bovine salivary glands (sublingual, submandibular, and parotid) by RT-PCR for mRNA and by Western blot analysis and immunohistochemistry (IHC) using an anti-human pIgR antibody for protein. The protein detected with the antibody was identified by nano-liquid chromatography-quadrupole time of flight mass spectrometry. Additionally, the distribution of Ig-producing plasma cells was analyzed by IHC. RT-PCR showed that pIgR was expressed in the sublingual and submandibular glands, but not in the parotid gland. Higher protein levels were observed in sublingual glands than in submandibular glands by Western blot. By IHC, pIgR was mainly located on the apical side of the cytoplasmic membrane in the sublingual gland, whereas it was observed only on the basal side in the submandibular gland. The highest density of plasma cells expressing IgAs was observed in the sublingual gland. These results suggest that the sublingual gland plays an important role in first-line defence of the oral cavity in cattle in contrast to humans and rats.

Comparison between chest digital tomosynthesis and CT as a screening method to detect artificial pulmonary nodules: a phantom study.

OBJECTIVES: The objective of this study was to evaluate the imaging capabilities of chest digital tomosynthesis (DT) as a screening method for the detection of artificial pulmonary nodules, and to compare its efficiency with that of CT. METHODS: DT and CT were used to detect artificial pulmonary nodules (5 mm and 8 mm in diameter, ground-glass opacities) placed in a chest phantom. Using a three-dimensional filtered back-projection algorithm at acquisition angles of 8°, 20°, 30° and 40°, DT images of the desired layer thicknesses were reconstructed from the image data acquired during a single tomographic scan. Both standard and sharp CT reconstruction kernels were used, and the detectability index (DI) valves computed for both the DT scan acquisition angles and CT reconstruction kernel types were considered. For the observer study, we examined 50 samples of artificial pulmonary nodules using both DT and CT imaging. On the basis of evaluations made by five thoracic radiologists, a jackknife free-response receiver operating characteristic (JAFROC) study was performed to compare and assess the differences in detection accuracy between CT and DT imaging. RESULTS: For each increased acquisition angle, DI obtained by DT imaging was similar to that obtained by CT imaging. The difference in the observer-averaged JAFROC figure of merit for the five readings was 0.0363 (95% confidence interval: -0.18, 0.26; F=0.101; p=0.75). CONCLUSION: With the advantages of a decreased radiation dose and the practical accessibility of examination, DT may be a useful alternative to CT for the detection of artificial pulmonary nodules.

Effect of CT fluoroscopy-guided transpulmonary radiofrequency ablation of liver tumours on the lung.

OBJECTIVE: We retrospectively evaluated the effect of transpulmonary radiofrequency ablation (RFA) of liver tumours on the lung. METHODS: 16 patients (10 males and 6 females; mean age, 65.2 years) with 16 liver tumours (mean diameter 1.5 cm) underwent transpulmonary RFA under CT fluoroscopic guidance. The tumours were either hepatocellular carcinoma (n=14) or liver metastasis (n=12). All 16 liver tumours were undetectable with ultrasonography. The pulmonary function values at 3 months after transpulmonary RFA were compared with baseline (i.e. values before RFA). RESULTS: In 8 of 16 sessions, minor pulmonary complications occurred, including small pneumothorax (n=8) and small pleural effusion (n=1). In two sessions, major pulmonary complications occurred, including pneumothorax requiring a chest tube (n=2). These chest tubes were removed at 4 and 6 days, and these patients were discharged 7 and 10 days after RFA, respectively, without any sequelae. The pulmonary function values we evaluated were forced expiratory volume in 1 s (FEV1.0) and vital capacity (VC). The mean values of FEV1.0 before and 3 months after RFA were 2.55 l and 2.59 l, respectively; the mean values of VC before and 3 months after RFA were 3.20 l and 3.27 l, respectively. These pulmonary values did not show any significant worsening (p=0.393 and 0.255 for FEV1.0 and VC, respectively). CONCLUSION: There was no significant lung injury causing a fatal or intractable complication after transpulmonary RFA of liver tumours.

Water irradiation by high-frequency ultrasonic wave: effects on properties of passive film formed on stainless steel.

In this paper an aqueous solution was irradiated with a 1.63MHz ultrasonic wave. It is shown that if stainless steel can passivate under dynamic polarization in this medium, under static polarization, the latter does not show any repassivation behaviour with time. This is attributed to a diminution of the diffusion layer thickness that is developed at the electrode/electrolyte interface, which is associated with a production of H(2) species by sonolysis and which maintains reductive conditions at the interface. The oxide film formed under ultrasonic irradiation for 1h at a passive potential of+0.2V(SCE) shows an early stage of passivation and an increased disordered state, which implies a considerable decrease in the corrosion resistance behaviour of the sample. The polarization resistance of the stainless steel R(p) is divided by a value of 4.5 under ultrasonic conditions.

Hepatitis C virus NS4 protein impairs the Th1 polarization of immature dendritic cells.

Dendritic cells (DCs) in chronic hepatitis C patients display impaired function, although the details remain unclear. To investigate the hepatitis C virus (HCV) protein that has the most impact on DC function, we compared five recombinant proteins and seven HCV protein genes in modulating DC phenotype and function. Immature DCs (iDCs) were established from healthy donor peripheral blood monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4. Lipopolysaccharide was used to establish mature DCs (mDCs). Cells were then pulsed with HCV recombinant proteins or transfected with HCV plasmids and subsequently assayed for cell surface marker expression by flow cytometry. For cytokine and proliferative T-cell response analysis, DCs were cultured with autologous CD4 T cells and tuberculin purified protein derivative (PPD). Mean fluorescent intensity of CD86 was reduced in HCV protein-pulsed iDCs. Proliferative T-cell responses and Th1 cytokine concentrations were reduced with HCV nonstructural proteins (NS), particularly with HCV NS4. HCV nonstructural proteins, particularly NS4, change the iDC phenotype and reduce antigen-specific T-cell stimulatory function with Th1 cytokine reductions.

A sulfoglycolipid beta-sulfoquinovosyldiacylglycerol (betaSQDG) binds to Met1-Arg95 region of murine DNA polymerase lambda (Mmpol lambda) and inhibits its nuclear transit.

Beta-sulfoquinovosyldiacylglycerol (betaSQDG) is a synthetic sulfoglycolipid that shows inhibitory activity of DNA polymerase lambda (pol lambda). Here we identified a betaSQDG binding region within murine pol lambda (Mmpol lambda) using T7 phage display technology. We compared the binding intensity of betaSQDG with recombinant phages (phages lambda1-6) that displayed different segments of Mmpol lambda. The binding assay clearly showed that phage lambda1, which displayed the non-structural Met1-Arg95 region including the nuclear localization signal (NLS) and part of the BRCT domain, bound more strongly to betaSQDG than the other recombinant phages. Binding assays using recombinant proteins gave similar results, showing specific betaSQDG binding to Met1-Arg95 with a K(D) value of 9.9 nM. Furthermore, in a cell-based assay, nuclear localization of EGFP-pollambda was inhibited in the presence of betaSQDG possibly due to binding of betaSQDG to NLS. These experiments clearly show that the binding region of betaSQDG within Mmpol lambda could be successfully identified using T7 phage display technology. We suggest that the strategy we describe here will be of value for identifying the binding site within a protein for small ligands, and will provide information that cannot be obtained using other experimental techniques due to their inherent technical limitations.

Molecular analysis of physiological responses to changes in nitrogen in a marine macroalga, Porphyra yezoensis (Rhodophyta).

The rhodophyte seaweed Porphyra yezoensis, known more commonly world-wide as "nori", is an important commercial crop in Japan. Cultivation of nori in Japan is often affected by outbreaks of "iroochi", a discoloration of the thalli due to a decrease in inorganic nutrients in the culture area that in turn decreases the amount of photosynthetic pigments in the thalli. Treating thalli with inorganic nitrogen can reverse iroochi. In this paper, we report on the characterization of three P. yezoensis genes, a nitrate transporter (PyNRT2) and two urea transporters (PyUT1 and PyUT2), which may be involved in reversing iroochi. The predicted length of the PyNRT2 protein was 479 amino acids (AA), while PyUT1 and PyUT2 were 740 and 680 AA, respectively. PyNRT2 was more similar to NRT2 from a chromophyte than to NRTs from Chlamydomonas and higher plants. The two P. yezoensis UTs had 56% AA identity to each other, and showed the closest relationship to higher plant and yeast DUR3 proteins which formed a subfamily of the sodium-solute symporter protein family. Hydrophobicity plots of the AA sequences showed that the PyNRT2, PyUT1, and PyUT2 included 12, 15, and 16 transmembrane domains, respectively. Southern blot analysis indicated that the P. yezoensis genome has a single NRT2-encoding gene and at least four UT-encoding genes. Expression analysis of PyNRT2 and PyUT genes showed that the messenger RNA level of the PyNRT2 gene reached a maximum after 48 h in the nitrate starvation condition and was then restored to the constitutive level, while expression of the PyUT genes was induced in proportion to treatment times in the nitrate starvation condition. These results suggest that the PyNRT2 and PyUT are responsible for the high-affinity nitrate/urea transport systems that operate under low external nitrate concentrations.