Automated cell isolation from photodegradable hydrogel based on fluorescence image analysis

We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO₂ concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.     

Depression and Anxiety Correlate Differently with Salivary Free Cortisol in the Morning in Patients with Functional Somatic Syndrome

Patients presenting with functional somatic syndrome (FSS) are common, and the symptoms are persistent and difficult to treat for doctors and costly for society. The aim of this study was to clarify the common pathophysiology of FSS, especially the relationship between hypothalamic-pituitary-adrenal (HPA) axis function and psychological characteristics of patients with FSS. The subjects were 45 patients with FSS and 29 healthy controls. Salivary free cortisol was measured in the morning, and psychological tests examining depression, anxiety and quality of life (QOL) were performed on the same day. In patients with FSS, depressive scores showed a significant negative correlation with salivary free cortisol in the morning, although in healthy controls, cortisol showed a significant positive correlation with depressive scores. In addition, the correlation between other psychological test scores and cortisol secretion in patients with FSS contrasted with that of controls. The relationship between cortisol and depression, anxiety or QOL, suggests that the HPA axis of patients with FSS is dysfunctional and does not function properly when patients with FSS are under stress. This dysfunction may explain the pathology of medically unexplained persistent symptoms of patients with FSS.     

Organ culture of chicken cecum: Morphologic and physiologic observations after 24 and 48 h of culture

Summary Cecum from chickens, 4 wk old, can best be maintained for 24 h in a serum-free organ culture system using Trowell T8 medium agar sheet at 25°C. As determined by light microscopy as well as scanning and transmission electron microscopy, mucosal architecture involving classic ultrastructure of chicken cecal mucosa was preserved. Protein content of cecal explants did not change up to 48 h of culture. DNA content of cecal explants did not change up to 24 h of culture but decreased significantly to two-thirds of control in 48 h of culture. Based on the morphologic and physiologic findings, it became evident that this organ culture system using Trowell T8 medium at 25°C can be successfully used as an in vitro experimental model for as long as 24 h. The organ culture system could be a useful tool, from the structural integrity of ceca observed in this study, in investigating mucosal function and mucosal response to drugs, carcinogens, trophic factors, and pathogens.     

Comparison of properties of leaf and root glutathione reductases from spinach

Glutathione reductase (GR; EC 1.6.4.2) was purified from spinach roots (rGR) to homogeneity in terms of SDS-PAGE, and its properties were compared with those of the enzyme from spinach leaves (IGR). The two enzymes had similar native molecular (118000) and subunit masses (58000) and immunochemical properties, but different pH optima (ca pH 7.8 for IGR, ca pH 7.2 for rGR) and amino acid compositions. Peptide maps of two GRs showed that they differed from each other. The N-terminal amino acid of the IGR was glycine and that of the rGR was blocked. The partial amino acid sequence of the N-terminal region of the IGR was determined to the 11 th residue and it was found that the sequence of 8 amino acids of the IGR had 100% homology with that of the putative chloroplast GR from Arabidopsis and pea.     

Methoxylation of 3β,7α-dihydroxychol-5-en-24-oic acid,a key intermediate of chenodeoxycholic acid biogenesis,compared with that of its 7β-epimer

The conventional methods of gas liquid chromatography or mass spectrometry failed to be useful for the identification of the biliary 3β, 7α-hydroxychol--en-24-oic acid, a key intermediate of chenodeoxycholic acid biogenesis. It has been preliminarily reported that this acid in human bile was successfully identified by gas chromatography-mass spectrometry, after the methoxylation of its allyl alcohol group. Physical as well as spectral properties of the methoxylation products derived from the acid were reported, compared with those from its 7β-epimer.     

Antifouling Brominated Diterpenoids from Japanese Marine Red Alga Laurencia venusta Yamada

The marine red algal genus Laurencia has abundant halogenated secondary metabolites, which exhibit novel structural types and possess various unique biological potentials, including antifouling activity. In this study, we report the isolation, structure elucidation, and antifouling activities of two novel brominated diterpenoids, aplysin-20 aldehyde ( 1 ), 13-dehydroxyisoaplysin-20 ( 2 ), and its congeners. We screened marine red alga Laurencia venusta Yamada for their antifouling activity against the mussel Mytilus galloprovincialis. Ethyl acetate extracts of L. venusta from Hiroshima and Chiba, Japan, were isolated and purified, and the compound structures were identified using 1D and 2D NMR, HR-APCI-MS, IR, and chemical synthesis. Seven secondary metabolites were identified, and their antifouling activities were evaluated. Compounds 1 , 2 , and aplysin-20 ( 3 ) exhibited strong activities against M. galloprovincialis. Therefore, these compounds can be explored as natural antifouling drugs.     

Beneficial effects of footbaths in controlling spasticity after stroke

Footbaths are considered to provide beneficial thermal therapy for post-stroke patients with spasticity, but their anti-spastic effects have not been investigated comprehensively. The present study aimed to evaluate alterations in motor-neuron excitability using F-wave parameters in post-stroke patients with spastic hemiplegia. Subjects’ legs below the knee joint were immersed in water at 41°C and F-wave recordings were made over the abductor hallucis muscle before, immediately after, and 30 min after thermal treatment. Antidromic stimulation was performed on the tibial nerve at the ankle. Measurements included F-wave amplitude, F-wave/M-response ratio, changes in modified Ashworth scale (MAS), body temperature and surface-skin temperature. The mean values of both F-wave parameters were higher on the affected side before footbath treatment. In post-stroke patients, the mean values of F-wave parameters were significantly reduced after footbath treatment (P < 0.01). The anti-spastic effects of footbath treatment were indicated by decreased F-wave parameters, in parallel with decreases in MAS. Body temperature was significantly increased both immediately after, and 30 min following footbath treatment in both groups, which appeared to play an important role in decreased spasticity. Surface-skin temperature increased immediately after footbath treatment in both groups and returned to baseline 30 min later. These findings demonstrate that the use of footbaths is an effective nonpharmacological anti-spastic treatment that might facilitate stroke rehabilitation.