On-line high performance liquid chromatography measurements of extracellular metabolites in an aerobic batch yeast (<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>) culture

We constructed a bioprocess environment enabling automatic sampling from a bioreactor combined with a compact on-line high performance liquid chromatography (HPLC) unit. This setup allowed us to measure extracellular glucose, ethanol, glycerol, and acetate concentrations automatically at 5 min intervals during the cultivation. This environment also provides mechanical measurement of the optical density (OD) of cells and enables us to collect and store (−35°C) samples for further off-line analyses. Among the available devices, the performance of the sampling-analysis unit is by far the best with regard to speed and number of analytes. Both the sampling and analysis phases are easily controlled by software; thus, providing a unique environment to perform various bioprocess activity tasks, whether they would be cell line screening or optimisation of conditions for growth and productivity. Complex research set-ups can be created and continuous automated measurements empower long-term cultivations with a time series. We provide evidence for the applicability of this environment by performing three comparable batch cultivations with Saccharomyces cerevisiae yeast and show that both the on-line sampling and analysis modes produce reliable data for further use in the monitoring and controlling of bioprocesses. On-line data provided new insight into the dynamics of the diauxic shift during aerobic glucose batch cultivation. When cell growth and carbon dioxide production ceased for the first time during the diauxic shift, acetate accumulation and consumption of the remaining glucose below 0.15 g/L continued to occur for 1 h. At the same time, glycerol and ethanol began to be consumed. Samples were also collected during cultivation for later analysis of intracellular metabolites and to collect more valuable information about metabolism.     

Attenuated expression of tenascin-c in ovalbumin-challenged STAT4-/- mice

Background

Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity.

Methods

Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro.

Results

OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings.

Conclusions

Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.     

Adenosine A(2A) receptors measured with [C]TMSX PET in the striata of Parkinson's disease patients

Adenosine A(2A) receptors (A2ARs) are thought to interact negatively with the dopamine D(2) receptor (D2R), so selective A2AR antagonists have attracted attention as novel treatments for Parkinson's disease (PD). However, no information about the receptor in living patients with PD is available. The purpose of this study was to investigate the relationship between A2ARs and the dopaminergic system in the striata of drug-na?ve PD patients and PD patients with dyskinesia, and alteration of these receptors after antiparkinsonian therapy. We measured binding ability of striatal A2ARs using positron emission tomography (PET) with [7-methyl-(11)C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([(11)C]TMSX) in nine drug-na?ve patients with PD, seven PD patients with mild dyskinesia and six elderly control subjects using PET. The patients and eight normal control subjects were also examined for binding ability of dopamine transporters and D2Rs. Seven of the drug-na?ve patients underwent a second series of PET scans following therapy. We found that the distribution volume ratio of A2ARs in the putamen were larger in the dyskinesic patients than in the control subjects (p<0.05, Tukey-Kramer post hoc test). In the drug-na?ve patients, the binding ability of the A2ARs in the putamen, but not in the head of caudate nucleus, was significantly lower on the more affected side than on the less affected side (p<0.05, paired t-test). In addition, the A2ARs were significantly increased after antiparkinsonian therapy in the bilateral putamen of the drug-na?ve patients (p<0.05, paired t-test) but not in the bilateral head of caudate nucleus. Our study demonstrated that the A2ARs in the putamen were increased in the PD patients with dyskinesia, and also suggest that the A2ARs in the putamen compensate for the asymmetrical decrease of dopamine in drug-na?ve PD patients and that antiparkinsonian therapy increases the A2ARs in the putamen. The A2ARs may play an important role in regulation of parkinsonism in PD.     

Adenosine A2A Receptors Measured with [11C]TMSX PET in the Striata of Parkinson's Disease Patients

Adenosine A_2A receptors (A2ARs) are thought to interact negatively with the dopamine D₂ receptor (D2R), so selective A2AR antagonists have attracted attention as novel treatments for Parkinson''s disease (PD). However, no information about the receptor in living patients with PD is available. The purpose of this study was to investigate the relationship between A2ARs and the dopaminergic system in the striata of drug-naïve PD patients and PD patients with dyskinesia, and alteration of these receptors after antiparkinsonian therapy. We measured binding ability of striatal A2ARs using positron emission tomography (PET) with [7-methyl-¹¹C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([¹¹C]TMSX) in nine drug-naïve patients with PD, seven PD patients with mild dyskinesia and six elderly control subjects using PET. The patients and eight normal control subjects were also examined for binding ability of dopamine transporters and D2Rs. Seven of the drug-naïve patients underwent a second series of PET scans following therapy. We found that the distribution volume ratio of A2ARs in the putamen were larger in the dyskinesic patients than in the control subjects (p<0.05, Tukey-Kramer post hoc test). In the drug-naïve patients, the binding ability of the A2ARs in the putamen, but not in the head of caudate nucleus, was significantly lower on the more affected side than on the less affected side (p<0.05, paired t-test). In addition, the A2ARs were significantly increased after antiparkinsonian therapy in the bilateral putamen of the drug-naïve patients (p<0.05, paired t-test) but not in the bilateral head of caudate nucleus. Our study demonstrated that the A2ARs in the putamen were increased in the PD patients with dyskinesia, and also suggest that the A2ARs in the putamen compensate for the asymmetrical decrease of dopamine in drug-naïve PD patients and that antiparkinsonian therapy increases the A2ARs in the putamen. The A2ARs may play an important role in regulation of parkinsonism in PD.     

Indigenous utilization of termite mounds and their sustainability in a rice growing village of the central plain of Laos

Background

This study was conducted to identify medicinal plants and spices used for medicine by the community of Beni-Sueif, Upper Egypt.

Methods

Ethnobotanical data from local people was collected using direct interviews and a semi-structured questionnaire.

Results

Forty-eight plant species belonging to twenty-seven families and forty-seven genera were encountered during the study. Their botanical and vernacular names, plant parts used and medicinal uses are given. Results of the study were analyzed using two quantitative tools. The factor informant consensus indicated the agreement in the use of plants and the fidelity level indicated the ratio between the number of informants who independently suggested the use of a species for the same major purpose and the total number of informants who mentioned the plant for any use. The results of the factor informant consensus showed that the cardiovascular category has the greatest agreement, followed by the immunological, gastrointestinal and respiratory categories. The most important species according to their fidelity are: Hibiscus sabdariffa L. for the cardiovascular category; Trigonella foenum-graecum L. for the immunological category; Mentha piperita L. for the gastrointestinal category and Pimpinella anisum L. for the respiratory category.

Conclusions

Medicinal plants are still used for treatment in Beni-Sueif community despite the availability of prescribed medications. Documentation of this ethnomedicinal knowledge is important. Evaluation of pharmacological activity for the promising medicinal plants is suggested.     

Three-Dimensional cryoEM Reconstruction of Native LDL Particles to 16? Resolution at Physiological Body Temperature

Background

Low-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. The most prominent structural components in LDL are the core-forming cholesteryl esters (CE) and the particle-encircling single copy of a huge, non-exchangeable protein, the apolipoprotein B-100 (apoB-100). The shape of native LDL particles and the conformation of native apoB-100 on the particles remain incompletely characterized at the physiological human body temperature (37°C).

Methodology/Principal Findings

To study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles vitrified at 6°C and 37°C resulted in reconstructions at ∼16 Å resolution at both temperatures. 3D variance map analysis revealed rigid and flexible domains of lipids and apoB-100 at both temperatures. The reconstructions showed less variability at 6°C than at 37°C, which reflected increased order of the core CE molecules, rather than decreased mobility of the apoB-100. Compact molecular packing of the core and order in a lipid-binding domain of apoB-100 were observed at 6°C, but not at 37°C. At 37°C we were able to highlight features in the LDL particles that are not clearly separable in 3D maps at 6°C. Segmentation of apoB-100 density, fitting of lipovitellin X-ray structure, and antibody mapping, jointly revealed the approximate locations of the individual domains of apoB-100 on the surface of native LDL particles.

Conclusions/Significance

Our study provides molecular background for further understanding of the link between structure and function of native LDL particles at physiological body temperature.     

Unfolding of the amyloid β-peptide central helix: mechanistic insights from molecular dynamics simulations

Alzheimer's disease (AD) pathogenesis is associated with formation of amyloid fibrils caused by polymerization of the amyloid β-peptide (Aβ), which is a process that requires unfolding of the native helical structure of Aβ. According to recent experimental studies, stabilization of the Aβ central helix is effective in preventing Aβ polymerization into toxic assemblies. To uncover the fundamental mechanism of unfolding of the Aβ central helix, we performed molecular dynamics simulations for wild-type (WT), V18A/F19A/F20A mutant (MA), and V18L/F19L/F20L mutant (ML) models of the Aβ central helix. It was quantitatively demonstrated that the stability of the α-helical conformation of both MA and ML is higher than that of WT, indicating that the α-helical propensity of the three nonpolar residues (18, 19, and 20) is the main factor for the stability of the whole Aβ central helix and that their hydrophobicity plays a secondary role. WT was found to completely unfold by a three-step mechanism: 1) loss of α-helical backbone hydrogen bonds, 2) strong interactions between nonpolar sidechains, and 3) strong interactions between polar sidechains. WT did not completely unfold in cases when any of the three steps was omitted. MA and ML did not completely unfold mainly due to the lack of the first step. This suggests that disturbances in any of the three steps would be effective in inhibiting the unfolding of the Aβ central helix. Our findings would pave the way for design of new drugs to prevent or retard AD.     

Measuring Eco-efficiency of Production with Data Envelopment Analysis

Aggregation of environmental pressures into a single environmental damage index is a major challenge of eco-efficiency measurement. This article examines how the data envelopment analysis (DEA) method can be adapted for this purpose. DEA accounts for substitution possibilities between different natural resources and emissions and does not require subjective judgment about the weights. Although DEA does not require subjective or normative judgment, soft weight restrictions can be incorporated into the framework. The proposed approach is illustrated by an application to assessing ecoefficiency of road transportation in the three largest towns of eastern Finland.     

Variation in metal concentrations in the brown alga Undaria pinnatifida in Osaka Bay, Japan

In order to evaluate the usefulness of a biomonitoring system using seaweeds for assessing the geographic distribution of metal ions in coastal seawaters, the metal concentrations in the sporophytes of an annual kelp, Undaria pinnatifida (Harvey) Suringar, were collected at 15 localities in Osaka Bay, Japan and compared. About 160 cm² of the blade was cut out from the central part of clean sporophytes, rinsed in filtered seawater using an ultrasonic cleaning bath, and freeze‐dried. After digestion with 12% HNO₃ in a microwave apparatus, metal concentrations in the samples were determined by inductively coupled plasma mass spectrometry. The concentrations (dry weight basis) of most examined elements were in the parts per million range; Cr, 0.48–3.18; Ni, 0.77–5.94; Cu, 3.20–43.8; Zn, 11.3–86.8; Pb, 0.14–3.53. Comparisons of metal compositions of the U. pinnatifida samples from the northeastern area of the bay, which has a large urban population and highly developed industries inland, showed high concentrations of Cd, Pb and Cu compared with the samples from the southwestern area of the bay where the population and industries are much smaller. This suggests that U. pinnatifida metal loads can be used as a marker to track the geographic distributions of the metal concentrations in coastal seawaters, reflecting inland human activities such as shipbuilding and repairing in port areas, and can be used as a useful biomonitoring system of coastal environments for long‐term trend.