Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging.

Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound-coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

Reduction in the colonization of Staphylococcus aureus on the skin surface under calcium-/magnesium-depleted conditions.

Excessive expansion of Staphylococcus aureus is associated with several skin diseases, including atopic dermatitis (AD). Recently, we have demonstrated that washing skins with ultra-pure soft water containing little bivalent metal ions improved skin conditions of atopic subjects. In this study, we investigated the roles of calcium or magnesium on the proliferation of S. aureus both in vitro and in vivo. Depletion of calcium and magnesium in the culture medium significantly suppressed the expansion of S. aureus growth. When S. aureus, diluted with water containing calcium/magnesium at the concentration of medium-hard water (83·0 mg l-1 as CaCO3 ) or the one that contains little calcium/magnesium, was applied onto the tape-stripped skin of Hos:HR-1 mice, growth of S. aureus in water without those minerals on the skin was suppressed. These results suggest that depletion of both calcium and magnesium abrogate the proliferation of S. aureus not only in the culture system but also on the skin surface of mice. Since colonization of S. aureus on the skin is well-known to exacerbate AD symptoms, usage of ultra-pure soft water containing less calcium and magnesium may improve the skin condition through the suppression of S. aureus growth on the skin of patients with skin problems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the importance of calcium and magnesium for the colonization and growth of Staphylococcus aureus by using both in vitro culture systems and in vivo experiments on the murine skin. Our results indicate that the removal of these metal ions is probably beneficial for protecting the skin from S. aureus. Thus, using ultra-pure soft water without metal ions may improve the skin condition of patients with skin problems through the protection from S. aureus colonization.

FOXO3 is essential for CD44 expression in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal types of cancer and the 5-year survival rate is only 5%. Several studies have suggested that cancer stem cells (CSCs) are thought to be involved in recurrence and metastasis and so it is essential to establish an approach targeting CSCs. Here we have demonstrated that cyclic guanosine monophosphate (cGMP) suppressed CD44 expression and the properties of CSCs in PDAC. Microarray analysis suggested that cGMP inhibited Forkhead box O3 (FOXO3), which is known as a tumor suppressor. Surprisingly, our data demonstrated that FOXO3 is essential for CD44 expression and the properties of CSCs. Our data also indicated that patients with high FOXO3 activation signatures had poor prognoses. This evidence suggested that cGMP induction and FOXO3 inhibition could be ideal candidates for pancreatic CSC.

Linoleic acid salt with ultrapure soft water as an antibacterial combination against dermato-pathogenic Staphylococcus spp.

AIMS: Skin colonization of Staphylococcus spp. critically affects the severity of dermatitis in humans and animals. We examined different types of fatty acid salts for their antibacterial activity against Staphylococcus spp. when used in ultrapure soft water (UPSW). We also evaluated their therapeutic effect on a spontaneous canine model of dermatitis. METHODS AND RESULTS: UPSW, in which Ca(++) and Mg(++) were replaced with Na(+) , was generated using a water softener with cation-exchange resin. Staphylococcus aureus (Staph. aureus), Staphylococcus intermedius (Staph. intermedius), and Staphylococcus pseudintermedius (Staph. pseudintermedius) were incubated with various fatty acid salts in distilled water (DW) or UPSW and the number of bacteria was counted. Among the fatty acids, oleic acid salt and linoleic acid (LA) salt reduced the number of these bacteria. Also, UPSW enhanced the antibacterial effect of LA on Staph. spp. In spontaneously developed itchy dermatitis in companion dogs, shampoo treatment with liquid soap containing 10% LA in UPSW improved skin conditions. CONCLUSIONS: LA salt showed antibacterial activity against Staph. spp. Treatment with soap containing LA with UPSW reduced clinical conditions in dogs with dermatitis. SIGNIFICANCE AND IMPACT OF THE STUDY: Because colonization of Staph. spp. on the skin exacerbates dermatitis, the use of LA-containing soap in UPSW may reduce unpleasant clinical symptoms of the skin.

PERSISTENT CATATONIA IN A PREGNANT NIGERIAN WOMAN: A CASE REPORT.

BACKGROUND: The syndrome of catatonia appears to exist with many conditions, yet goes undetected by the skillful eyes of clinicians. This case which is rarely reported in literatures shows the effectiveness of antipsychotic augmenting in a persistent catatonic schizophrenia disorder. METHOD: This is a case narration of persistent catatonia in a 24-years old pregnant Nigerian woman with schizophrenia disorder. RESULTS: First line management with benzodiazepines and electroconvulsive therapy (ECT) failed to resolve the syndrome which later responded to Electroconvulsive Therapy with low dose antipsychotic augmentation. CONCLUSIONS: Cautious augmenting of electroconvulsive therapy with neuroleptics may be a quick and relatively safe procedure in the relief of schizophrenia with catatonia in pregnancy.

Antifungal effects of palmitic acid salt and ultrapure soft water on Scedosporium apiospermum.

AIMS: Scedosporium apiospermum sometimes causes serious infectious diseases on the skin of immunodeficient subjects. Antifungal effects of fatty acid salts in soap against S. apiospermum were investigated under different water conditions. METHODS AND RESULTS: Ultrapure soft water (UPSW) was generated by the water softener with cation-exchange resin. The calcium and magnesium ions were replaced with sodium ions in UPSW. Scedosporium apiospermum was incubated with different fatty acid salts that constituted soap in distilled water (DW), tap water (TW) and UPSW. After incubation, the number of fungi was counted. Among the fatty acids, palmitic acid salt (C16) reduced the number of S. apiospermum. UPSW enhanced the antifungal effect of C16 on S. apiospermum. The absence of both calcium and magnesium ions and the existence of sodium chloride in UPSW were responsible for its antifungal effect. In addition, repeated short-term treatment with UPSW and C16 decreased the number of S. apiospermum. CONCLUSIONS: Antifungal effects of C16 on S. apiospermum were demonstrated. Moreover, the use of UPSW promoted the antifungal effect of C16. SIGNIFICANCE AND IMPACT OF STUDY: This study provides the preventive method for diseases associated with S. apiospermum infection using novel palmitic acid soap in UPSW.

Effect of plasma uric acid on pharmacokinetics of cyclosporine A in living-related renal transplant recipients and pharmacokinetic study in rats with experimental hyperuricaemia.

OBJECTIVES: Renal transplant recipients are thought to have an increased risk of hyperuricaemia (HU); therefore, the effects of plasma uric acid (UA) on the pharmacokinetics (PK) of cyclosporine A (CyA), an immunosuppressant, in renal transplant recipients and experimental animals were investigated. METHODS: An open-label, non-randomized, retrospective study was performed in renal transplant recipients. Data from 76 subjects who received a renal transplantation with CyA medication were included. We compared the PK of CyA of recipients showing a high UA level with the other recipients. In addition, PK studies were performed using hyperuricaemic-model rats (HU rats) prepared by subcutaneous injection of the uricase inhibitor, potassium oxonate and intraperitoneal injection of UA. RESULTS: The area under the blood concentration vs. time curve (AUC) up to 9 h, the blood level at 2 h after dose and peak level in high UA recipients (UA > 7.0 mg/dL) was significantly lower (about 10-16%) than that in the other recipients, although there were no differences in dose, and the trough blood level. On the contrary, there were no differences in PK parameters after intravenous administration of CyA between HU and control rats; however, AUC, peak level and bioavailability in HU rats (2.01 ± 0.56 µg h/mL, 0.47 ± 0.26 µg/mL and 0.186 ± 0.05, respectively) after oral administration were significantly lower than in the control animals (6.13 ± 0.97 µg h/mL, 0.82 ± 0.17 µg/mL and 0.458 ± 0.07 µg/mL, respectively). In addition, the absorptions of CyA and midazolam, an ideal probe for CYP3A, from the intestinal loop in HU rats were significantly less (about 50% and 37%, respectively) than in the controls. CONCLUSIONS: The absorption of CyA was affected by plasma UA in transplant recipients and experimental rats. The contribution of intestinal metabolism by CYP3A to decreasing CyA absorption in HU rats was significant. These results suggest that transplant recipients with high UA may have poor absorption of CyA.

Exploration of factors related to hara-kiri as a method of suicide and suicidal behavior.

OBJECTIVE: The objective of this study was to explore factors associated with hara-kiri as a method of suicide and suicidal behavior in contemporary Japan. METHODS: A retrospective study was conducted on medical records of 421 patients (174 male; 247 female) who were considered suicidal and treated at the Kitasato University Hospital Emergency Medical Center in Japan between January 2006 and March 2008. We compared hara-kiri and all other methods regarding sociodemographics and clinical features of all suicidal patients. RESULTS: Instances of hara-kiri suicide attempt had the highest proportion of males (63%) among all suicide and suicidal behavior. One-way analysis of variance revealed significant differences between hara-kiri and other suicide attempt methods in the age of the suicidal patients. Result of multiple logistic regression analysis indicated that those who attempted hara-kiri suicide were likely to be male, be diagnosed with schizophrenia, survive, and be married. CONCLUSION: Our findings indicate that hara-kiri as a method of suicide and suicidal behavior remains prevalent in Japan, and the study findings also suggest that both clinical and cultural factors might play a role in hara-kiri as a method of suicide and suicidal behavior.

The experience of burnout among home caregivers of patients with dementia: relations to depression and quality of life.

Caregivers of patients with dementia often experience chronic and emotional strains, which may lead to emotional exhaustion and burnout. The objective of this study was to examine the relationships between burnout, depression, and quality of life in home caregivers of patients with dementia. The participants were 84 home caregivers (39 male; 45 female), and they were asked to complete three questionnaires: the Beck Depression Inventory Second Edition (BDI-II), the WHO Quality of Life 26 (WHO-QOL26), and the Pines Burnout Measure (BM). Results from a bivariate correlation showed that BM scores significantly correlated with the scores on BDI-II and WHO-QOL26. Results from ANOVA comparing the level of burnout with the scores on BDI-II and WHO-QOL26 revealed that there were differences between the burnout groups and the scores on BDI-II and WHO-QOL26. The higher levels of burnout were found to correspond to higher levels of depressive symptoms and lower QOL. These findings suggest that caregiver intervention to reduce burnout might benefit from strategies to reduce depression and increase QOL.

Experimental concept for examination of biological effects of magnetic field concealed by gravity.

Space is not only a place to study biological effects of gravity, but also provides unique opportunities to examine other environmental factors, where the biological actions are masked by gravity on the ground. Even the earth's magnetic field is steadily acting on living systems, and is known to influence many biological processes. A systematic survey and assessment of its action are difficult to conduct in the presence of dominant factors, such as gravity. Investigation of responses of biological systems against the combined environment of zero-gravity and zero-magnetic field might establish the baseline for the analysis of biological effects of magnetic factors. We propose, in this paper, an experimental concept in this context, together with a practical approach of the experiments, both in orbit and on the ground, with a thin magnetic shielding film. Plant epicotyl growth was taken as an exemplar index to evaluate technical and scientific feasibility of the proposed system concept.

Abutment region dosimetry for the monoisocentric three-beam split field technique in the head and neck region using asymmetrical collimators.

Creating non-divergent field edges using asymmetric collimators and a single isocentre can improve matchline dosimetry owing to decreased reliance on operator skills and avoidance of couch movement. However, asymmetic jaws have an associated tolerance that can cause abutment to be misaligned. The matching area dose for monoisocentric three-beam split fields commonly used in head and neck cancer treatments using mismatched and matched collimators is the subject of this work. X-ray verification film was exposed in a solid-water phantom, and the dose at the matching area was evaluated using mismatched and matched collimators. In the case of mismatched (consistently overlapped) collimators, digital displays of an asymmetric collimator position within the tolerance indicated in the manufacturer's specifications were investigated for the three-beam split field technique. The effect of this technique on the junctional dose was also determined using matched collimators. Although the collimators showed a consistent overlap, a perfect dose distribution could be obtained at the matching area. The three-beam split field technique yielded an 8% overdose at the matchline using matched collimators. In conclusion, an awareness of the effects of the abutting technique and digital display tolerance is necessary to achieve good junction uniformity using asymmetric collimators.

Focal segmental glomerulosclerosis associated with acromegaly.

Experimental studies in rats have demonstrated an association between focal segmental glomerulosclerosis (FSGS) and growth hormone, but patients with FSGS complicating acromegaly are very rare. In this report we present a case of FSGS associated with acromegaly. With a long history of soft tissue swelling of hands and feet, elevated plasma growth hormone levels and other biochemical abnormalities, a 53-year old male had suffered from acromegaly for over 15 years. He had moderate proteinuria for 6 years, but never evidenced nephrotic syndrome. A renal biopsy specimen revealed FSGS and glomerular hypertrophy. Trans-sphenoidal surgical removal of the pituitary adenoma resulted in the normalization of elevated growth hormone and insulin-like growth factor I levels, but proteinuria continued. This case suggests that the overproduction of growth hormone may participate, at least in part, in the development of human FSGS. It is possible that once FSGS is present in an acromegalic patient, cessation of GH overproduction may not be enough to reverse it.

Role of Notch-1 intracellular domain in activation of rheumatoid synoviocytes.

OBJECTIVE: Notch family proteins are transmembrane receptors that control cell fate and proliferation. Rheumatoid arthritis (RA) is characterized by activation and abnormal proliferation/differentiation of synoviocytes. We examined the expression of Notch-1 and its role in the activation of RA synoviocytes. METHODS: The expression of Notch-1 protein was detected by a specific antibody raised against the Notch-1 intracellular domain. Notch-1 messenger RNA (mRNA) expression in synoviocytes was analyzed by Northern blotting. Notch-1 protein expression was confirmed by Western blotting with anti-Notch-1 antibody. To analyze the role of Notch-1 in synoviocyte proliferation, we examined the effects of antisense Notch-1 oligonucleotides (ODNs) and MW167, a gamma-secretase inhibitor. RESULTS: Notch-1 protein and mRNA were detected in synovium from all study subjects. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 antibody, whereas there was predominantly cytoplasmic staining of normal and osteoarthritis (OA) synoviocytes. Western blotting showed a distinct approximately 63-kd protein detected by anti-Notch-1 antibody in nuclear extracts from RA synoviocytes, indicating that nuclear staining of RA synovium and synoviocytes is likely to be the result of nuclear localization of Notch-1 intracellular domain (NICD). Furthermore, tumor necrosis factor alpha (TNFalpha) increased NICD nuclear translocation in a dose-dependent manner. Antisense Notch-1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFalpha-induced proliferation in both OA and RA synoviocytes. In addition, gamma-secretase inhibitor, which blocks the production of NICD, also inhibited TNFalpha-induced proliferation of RA synoviocytes. CONCLUSION: Our results demonstrate the expression of Notch-1 in synoviocytes and the presence of Notch-1 fragment in the nuclei of RA synoviocytes and suggest the involvement of Notch-1 signaling in the TNFalpha-induced proliferation of RA synoviocytes.

A new anthropoid from the latest middle Eocene of Pondaung, central Myanmar.

A new genus and species of medium-sized fossil primate, Myanmarpithecus yarshensis, is described from the lastest middle Eocene sediments of Pondaung, central Myanmar (Burma). The specimens consist of right maxillary fragments with P(4)-M(3)and a left mandibular corpus with C-P(3)and M(2-3). To date, three purported anthropoids have been discovered from the Pondaung Formation: Pondaungia and Amphipithecus (Amphipithecidae) and Bahinia (Eosimiidae). Myanmarpithecus differs from these other Pondaung primates in having cingular hypocones on upper molars and in lacking paraconids on M(2-3). Although Myanmarpithecus resembles some utahiin omomyines in superficial aspects of the morphology of M(2-3)(i.e., mesiodistally compressed molar trigonid and enamel crenulation), the morphological analysis of upper molars and lower premolars indicates that it is neither an omomyoid nor an adapoid but is more derived than fossil prosimians (such as adapoids, omomyoids, and tarsiers) and more anthropoid-like. On the other hand, it is more primitive (prosimian-like) than early anthropoids from the late Eocene/early Oligocene of the Fayum, Egypt. Myanmarpithecus is likely to be an early, primitive anthropoid ("protoanthropoid").

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.

Cloning and sequencing of the beta-glucosidase gene from Acetobacter xylinum ATCC 23769.

The beta-glucosidase gene (bglxA) was cloned from the genomic DNA of Acetobacter xylinum ATCC 23769 and its nucleotide sequence (2200 bp) was determined. This bglxA gene was present downstream of the cellulose synthase operon and coded for a polypeptide of molecular mass 79 kDa. The overexpression of the beta-glucosidase in A. xylinum caused a tenfold increase in activity compared to the wild-type strain. In addition, the action pattern of the enzyme was identified as G3ase activity. The deduced amino acid sequence of the bglxA gene showed 72.3%, 49.6%, and 45.1% identity with the beta-glucosidases from A. xylinum subsp. sucrofermentans, Cellvibrio gilvus, and Mycobacterium tuberculosis, respectively. Based on amino acid sequence similarities, the beta-glucosidase (BglxA) was assigned to family 3 of the glycosyl hydrolases.

Effects of combinations of anti-rheumatic drugs on the production of vascular endothelial growth factor and basic fibroblast growth factor in cultured synoviocytes and patients with rheumatoid arthritis.

OBJECTIVE: To examine whether different combinations of disease-modifying anti-rheumatic drugs (DMARDs), including bucillamine (BUC), gold sodium thiomalate (GST), methotrexate (MTX), salazosulphapyridine (SASP) and dexamethasone (DEX; a steroid), act by inhibiting the production of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in cultured synoviocytes, causing a decrease in their serum concentrations in patients with rheumatoid arthritis (RA). METHODS: The VEGF and bFGF concentrations in cultured synoviocytes and peripheral blood from patients with RA were measured by enzyme-linked immunosorbent assay and their serum concentrations were measured at two time points. RESULTS: BUC and GST inhibited VEGF production even when given alone, and a combination of BUC, GST and MTX with DEX also inhibited VEGF production. None of the DMARDs or DEX inhibited bFGF production when given alone, but a combination of SASP and GST inhibited the production of bFGF in cultured synoviocytes. Serum VEGF concentrations were significantly decreased 6 months after the commencement of medication compared with their concentrations before medication. CONCLUSION: Our results show that the effects of a combination of DEX with any two of BUC, GST, SASP and MTX on the production of VEGF and bFGF in cultured synoviocytes and on the serum concentrations of VEGF in patients with RA may be based on synergistic or additive effects of the drugs.

Cloning and sequencing of the levansucrase gene from Acetobacter xylinum NCI 1005.

The levansucrase gene (lsxA) was cloned from the genomic DNA of Acetobacter xylinum NCI 1005, and the nucleotide sequence of the lsxA gene (1,293 bp) was determined. The deduced amino acid sequence of the lsxA gene showed 57.4% and 46.2% identity with the levansucrases from Zymomonas mobilis and Erwinia amylovora, respectively, while only 35.2% identity with that from Acetobacter diazotrophicus. The gene product of lsxA (LsxA) that was overproduced in E. coli coded for a polypeptide of molecular mass 47 kDa. The LsxA released glucose and produced polysaccharide from sucrose, the structure of which was analyzed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2,6)-linked polyfructan.