<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells

Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 g dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (_m) and the plasma membrane (_p) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K⁺:Na⁺:Ca²⁺ of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.Abbreviations RP-HPLC Reversed-phase high-performance liquid chromatography - BLM Black lipid membrane - DAD Diode-array detector - _m Mitochondrial membrane potential - _p Plasma membrane potential - JC-1 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenz-imidazolo carbocyanine iodide - AM Calcein acetoxymethyl ester - PI Propidium iodide - MALDI-TOF-MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry - ESI-IT-MS Electrospray ionization ion trap mass spectrometry - EC₅₀ Endpoint concentration which caused 50% change in the viability parameters - FCCP Carbonyl cyanide 4-trifluoromethoxyphenylhydrazone     

Occurrence of Double Strand DNA Breaks and Pairing of Homologous Chromosomes are Preconditions of Crossing Over in Drosophila Melanogaster, and Its Timing

Brood pattern analysis of the effect of a heat shock (35°C 24 h) given to the parental females in presence and absence of In (2L + 2R) Cy in heterozygous condition in the second chromosome on recombination and interference in the cv − v − f region of the X chromosome showed the following: (i) In the absence of the inversion recombination frequencies in both gene intervals increased in the daily broods, which represent oocytes in which premeiotic DNA synthesis was occurring during the heat shock treatment. (ii) In the presence of the inversion recombination frequencies did not increase in any of the daily broods. (iii) The frequencies of double crossovers did not increase in any broods either in the presence or in the absence of inversion. (iv) The coefficient of coincidence changed in virtually all broods both in the presence and absence of the inversion, indicating that the heat shock affects the distribution of single crossovers. (v) The results showed that the effect of the heat shock on the coefficient of coincidence was similar during premeiotic DNA synthesis both in the absence and presence of the inversion, but different in the broods representing oocytes which were undergoing the first meiotic division, suggesting that crossing over occurs during this division, probably during pachytene. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Occurrence of Double Strand DNA Breaks is not the Sole Condition for Meiotic Crossing over in Drosophila Melanogaster

Analysis of the interchromosomal effects of In(2L+2R)Cy, In(3L+3R)LVMand their joint effect on the frequencies of single and double crossovers in the cv-v-fregion of the X chromosome as well as interference showed that both inversions, occurring separately, increased the frequency of single as well as double crossovers and the coefficient of coincidence. However, when the inversions occurred together the frequencies of single crossovers no longer increased, but the frequency of double crossovers, as well as the coefficient of coincidence did increase. These results indicate firstly that the interchromosomal effects influence some precondition of exchange, but that this precondition is not an occurrence of double strand DNA breaks. Thus, the occurrence of double strand DNA breaks is not the sole condition for crossing over in Drosophila melanogaster.     

Physiology of resistant Deinococcus geothermalis bacterium aerobically cultivated in low-manganese medium

This dynamic proteome study describes the physiology of growth and survival of Deinococcus geothermalis, in conditions simulating paper machine waters being aerobic, warm, and low in carbon and manganese. The industrial environment of this species differs from its natural habitats, geothermal springs and deep ocean subsurfaces, by being highly exposed to oxygen. Quantitative proteome analysis using two-dimensional gel electrophoresis and bioinformatic tools showed expression change for 165 proteins, from which 47 were assigned to a function. We propose that D. geothermalis grew and survived in aerobic conditions by channeling central carbon metabolism to pathways where mainly NADPH rather than NADH was retrieved from the carbon source. A major part of the carbon substrate was converted into succinate, which was not a fermentation product but likely served combating reactive oxygen species (ROS). Transition from growth to nongrowth resulted in downregulation of the oxidative phosphorylation observed as reduced expression of V-type ATPase responsible for ATP synthesis in D. geothermalis. The battle against oxidative stress was seen as upregulation of superoxide dismutase (Mn dependent) and catalase, as well as several protein repair enzymes, including FeS cluster assembly proteins of the iron-sulfur cluster assembly protein system, peptidylprolyl isomerase, and chaperones. Addition of soluble Mn reinitiated respiration and proliferation with concomitant acidification, indicating that aerobic metabolism was restricted by access to manganese. We conclude that D. geothermalis prefers to combat ROS using manganese-dependent enzymes, but when manganese is not available central carbon metabolism is used to produce ROS neutralizing metabolites at the expense of high utilization of carbon substrate.     

Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system

The enzyme controlled substrate delivery cultivation technology EnBase(?) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.     

Zeitgebers and their association with rest-activity patterns

Zeitgebers such as light, eating and physical activity provide input to the circadian clock. Chronic circadian misalignment is associated with significant adverse health effects. An improved understanding of the impact of the timing of zeitgebers on the stability of 24-hour rest-activity rhythm in free-living settings may identify behavioural and environmental intervention targets. A total of 133 healthy adults, aged 21–60 years, wore a wrist actigraph for 7 consecutive days. We applied a non-parametric analysis to activity counts to derive rest-activity patterns. We administered a questionnaire through a smartphone app to collect self-reported timing of light exposure, eating episodes and physical activity. To assess the relationship between timing exposures (first and last exposure to outdoor light, first exposure to indoor light, last eating episode, first eating episode, morning physical activity proportion, evening physical activity proportion) and rest-activity or sleep outcomes (bedtimes, total sleep time, inter-daily stability, intra-daily variability, L5 and M10 midpoint), we first calculated Spearman correlations, using the false discovery rate method to control for multiple comparisons. From those significant associations, we then fit regression models adjusting for age, sex, race, household income, education level, study site, body mass index, as well as physical activity. Finally, we tested for interaction between chronotype and each timing-related exposure and stratified the analysis by morning type. All zeitgebers, except for evening physical activity proportion, were correlated with at least four of the seven sleep and rest-activity outcomes. In adjusted analysis, later timing of first (after 6:30 to 7:45 AM versus earlier) and last exposure to indoor light (after 11:00 PM versus earlier) and first (after 7:45–9:45 AM versus earlier) and last eating episode (after 8:00–09:00 PM versus earlier) were associated with a shift of 0.60–1.39 hours to later bedtimes, M10 and L5 midpoints (i.e. timing of peak activities or inactivities). Later timing of first exposure to outdoor light (after 09:30 AM versus earlier) was also associated with 0.51 (95% CI: 0.19 to 0.83) hours longer total sleep time. Higher morning physical activity proportion (> 33%) was associated with 0.95 (95% CI: ?1.38 to ?0.53) hours earlier in-bed time and 0.69 (95% CI: ?1.14 to ?0.24) hours earlier out-of-bed time, 0.92 (95% CI: ?1.41 to ?0.42) hours earlier M10 and 0.96 (95% CI: ?1.42 to ?0.49) min earlier L5 midpoint. The results did not change substantially with further adjustment for total activity. There was a significant interaction between morning chronotype and first eating episode with rest-activity patterns (p < 0.05), with first eating episode associating with timing of activities only in non-morning type adults. Timing of zeitgebers was associated with sleep and rest-activity patterns, including bedtimes, L5 and M10 midpoint. Future research should evaluate the impact of manipulating zeitgebers on both circadian rhythms and health outcomes.     

The chemical character and burial of phosphorus in shallow coastal sediments in the northeastern Baltic Sea

The chemical composition and vertical distribution of sediment phosphorus (P) in shallow coastal sediments of the northeastern Baltic Sea (BS) were characterized by sequential extraction. Different P forms were related to chemical and physical properties of the sediments and the chemistry of pore water and near-bottom water. Sediment P composition varied among the sampling sites located in the Archipelago Sea (AS) and along the northern coast of the Gulf of Finland (GoF): the organic rich sites were high in organic P (OP), while apatite-P dominated in the area affected by sediment transportation. Although the near-bottom water was oxic, the sediments released P. Release of P was most pronounced at the site with high sediment OP and reduced conditions in the sediment-water interface, indicating that P had its origins in organic sources as well as in reducible iron (Fe) oxyhydroxides. The results suggest that even though these coastal areas are shallow enough to lack salinity stratification typical for the brackish BS, they are vulnerable to seasonal oxygen (O₂) depletion and P release because of their patchy bottom topography, which restricts mixing of water. Furthermore, coastal basins accumulate organic matter (OM) and OP, degradation of which further diminishes O₂ and creates the potential for P release from the sediment. In these conditions, an abundance of labile OP may cause marked efflux of P from sediment reserves in the long-term.     

Liver-Fat Accumulation and Insulin Resistance in Obese Women with Previous Gestational Diabetes

Objective: We determined whether fat accumulation in the liver is associated with features of insulin resistance independent of obesity. Research Methods and Procedures: We recruited 27 obese nondiabetic women in whom liver fat (LFAT) content was determined by proton spectroscopy, intra-abdominal and subcutaneous fat by magnetic resonance imaging, and insulin sensitivity by the euglycemic insulin clamp technique. The women were divided based on their median LFAT content (5%) to groups with low (3.2 ± 0.3%) and high (9.8 ± 1.5%) liver fat. The groups were almost identical with respect to age (36 ± 1 vs. 38 ± 1 years in low vs. high-LFAT), body mass index (32.2 ± 0.6 vs. 32.8 ± 0.5 kg/m²), waist-to-hip ratio, intra-abdominal, subcutaneous, and total fat content. Results: Women with high LFAT had features of insulin resistance including higher fasting serum triglyceride (1.93 ± 0.21 vs. 1.11 ± 0.09 mM, p < 0.01) and insulin (14 ± 3 vs. 10 ± 1 mU/L, p < 0.05) concentrations than women with low LFAT. The group with high LFAT also had higher 24-hour blood pressures, and lower whole-body insulin sensitivity compared with the low-LFAT group. Discussion: In obese women with previous gestational diabetes, LFAT, rather than any measure of body composition, is associated with features of insulin resistance.