Functional conformational changes of endo-1,4-xylanase II from Trichoderma reesei: A molecular dynamics study

Recent crystallographic studies have revealed a range of structural changes in the three-dimensional structure of endo-1,4-xylanase (XYNII) from Trichoderma reesei. The observed conformational changes can be described as snapshots of an open-close movement of the active site of XYNII. These structures were further analyzed in this study. In addition, a total of four 1 ns molecular dynamics (MD) simulations were performed representing different states of the enzyme. A comparison of the global and local changes found in the X-ray structures and the MD runs suggested that the simulations reproduced a similar kind of active site opening and closing as predicted by the crystal structures. The open-close movement was characterized by the use of distance difference matrixes and the Hingefind program (Wriggers and Schulten, Proteins 29:1–14, 1997) to be a ‘hinge-bending’ motion involving two large rigidly-moving regions and an extended hinge. This conformational feature is probably inherent to this molecular architecture and probably plays a role in the function of XYNII. Proteins 31:434–444, 1998. © 1998 Wiley-Liss, Inc.     

Objective working hour characteristics and work–life conflict among hospital employees in the Finnish public sector study

This epidemiological cohort study, based on Finnish public sector data, investigated the associations between objective working hour characteristics and work–life conflict in day and shift work. The comprehensive data of hospital workers (n = 8 931, 92% women, average age 45 years), consisted of survey responses from 2012, linked with the payroll data of working hour characteristics from 91 days preceding the survey. Logistic regression analysis was used to investigate the associations between working hour characteristics and experiencing work–life conflict often/very often. The analyses were adjusted for age (< 39, 40–49 and >50 years), sex, level of education, marital status, number of small (0–6 years) and school-aged (7–18 years) children, and the overall stressfulness of the life situation. We also conducted stratified analyses of age and sex on the basis of significant interactions. Difficulties in combining work and life were more often associated with shift work without night shifts and shift work with night shifts than with day work (41% and 34 versus 27%; OR for shift work with night shifts 1.78, 95% CI 1.59–2.00, OR for shift work without night shifts 1.42, 95% CI 1.26–1.60). A high proportion (> 25%) of long (> 40h, (OR 1.26, 95% 1.14–1.39) and very long (> 48h, OR 1.31, 95% CI 1.15–1.49) weekly working hours were associated with work–life conflict, and in the stratified analysis, the latter was also true among women (OR 1.54, 95% CI 1.25–1.89). Of the unsocial working hour characteristics, a relatively large amount (> 10% of all shifts) of evening (OR 1.56, 95% CI 1.41–1.72) and night shifts (OR 1.46, 95%CI 1.32–1.61), a high proportion (> 25% of all shifts) of quick returns (< 11h) (OR 1.46, 95% CI 1.31–1.63), and weekend work (OR 1.44, 95% CI 1.31–1.58) were associated with work–life conflict. A large amount of single days off (> 25% of all days off) was associated with work–life conflict among men (OR 1.90, 95% CI 1.11–3.25), but not in the whole sample. When the two types of shift work were analyzed separately, shift work without night shifts and very long work weeks had higher odds (OR 1.47, 95% CI 1.20–1.80) of work–life conflict than shift work with night shifts. Conversely, weekend work and evening shifts had higher odds of work–life conflict among shift workers with night shifts (OR 1.74, 95% 1.55–1.96; (OR 1.57, 95% CI 1.40–1.77) than among those without night shifts. To conclude, this study shows that shift workers with and without night shifts more often have difficulties combining work and life than day workers. Several unsocial working hour characteristics, including long work weeks, evening and night shifts, weekend work, and quick returns, are associated with work–life conflict.     

Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

A combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature survey was (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4, IC(50) of 610+/-15microM). In the virtual screening 46,400 compounds were docked and subjected to a pharmacophore search. Of these compounds, 29 were purchased and tested in a biological assay, allowing three novel inhibitors containing an aromatic scaffold to be identified. Based on one of the hits from the virtual screening a small library of 15 analogues was synthesized producing four compounds that inhibited glutamine synthetase.     

Biomass allocation and nutrient use in fast-growing woody and herbaceous perennials used for phytoremediation

This study assessed the suitability of two deciduous woody perennials (Salix spp. and Populus spp.) and two summer green herbaceous perennials (Phragmites australis and Urtica dioica) for purification of nutrient enriched wastewater. The main hypothesis tested was that species with a particular trait combination of high relative growth rate (RGR), low nutrient productivity (A) and high mean residence time (MRT) of nutrients would be most effective in accumulating nutrients. The nitrogen and phosphorus use efficiency at the whole plant level was analysed. Four treatments comprising two possible phytoremediation substrates (municipal wastewater and landfill leachate) and two control plant nutrition situations (balanced nutrient solution and pure water) were applied in four replications to the four plant species. Generally, all four species studied showed a high RGR and a low P productivity in the balanced nutrient solution treatment, while the opposite (low RGR and high P productivity) was seen in the phytoremediation substrate and pure water treatments. The general conclusion is that if P is present in marginal proportions in the wastewater, a vegetation filter with Phragmites would have an advantage since biomass and nutrient accumulation in Phragmites does not decrease as much during phytoremediation as that in deciduous woody perennials.     

Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts

c-Myc is known to induce or potentiate apoptotic processes predominantly by triggering or enhancing the activity of caspases, but the activation mechanisms of caspases by c-Myc remain still poorly understood. Here we found that in MycER™ rat fibroblasts the activation of c-Myc led to an early activation and cleavage of the initiator caspase-8, and concurrent processing and activation of the effector caspases 3 and 7. Interestingly, the expression of cellular FLICE inhibitory protein (c-FLIP) mRNA and the encoded protein, c-FLIP_L, a catalytically inactive homologue of caspase-8, were down-regulated prior to or coincidently with the activation of caspase-8. Of the other known initiators, caspase-9, involved in the mitochondrial pathway, was activated/processed surprisingly late, only after the effector caspases 3/7. Further, we studied the potential involvement of the Fas- and tumor necrosis factor receptor (TNFR)-mediated signaling in the activation of caspase-8 by c-Myc. Blocking of the function of these death receptors by neutralizing antibodies against Fas ligand and TNF-α did not prevent the processing of caspase-8 or cell death. c-Myc was neither found to induce any changes in the expression of TNF-related apoptosis inducing ligand (TRAIL) or its receptor. These data suggest that caspase-8 does not become activated through an extrinsic but an “intrinsic/intracellular” apoptotic pathway unleashed by the down-regulation of c-FLIP by c-Myc. Moreover, ectopic expression of c-FLIP_L inhibited the c-Myc-induced apoptosis.     

Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low‐cost purification by surfactant‐based aqueous two‐phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY‐2) suspension cell platform and the establishment of pilot‐scale propagation and downstream processing including first‐step purification by ATPS. Green fluorescent protein‐hydrophobin fusion (GFP‐HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY‐2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP‐HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large‐scale hydrophobin‐assisted production of recombinant proteins in tobacco BY‐2 cell suspensions.     

Permanent night workers´ sleep and psychosocial factors in hospital work. A comparison to day and shift work

ABSTRACT

We aimed to study whether permanent night workers sleep and psychosocial factors differ from day workers and shift workers. The participants (n = 9 312, 92% females, average age 45 years, most commonly nurses and departmental secretaries) were day workers (DW, n = 2 672), shift workers (SW, n = 6 486) and permanent night workers (PNW, n = 154). The Finnish Public Sector survey responses from six hospital districts from 2012 were combined to payroll data from 91 days preceding the survey. The data were analyzed using Pearson χ²-test, one-way ANOVA and multinomial logistic regression analysis. The PNWs reported slightly longer average sleep length than the SWs or the DWs (7:27 vs. 7:13 and 7:10 h, p < 0.001). The PNWs reported least often difficulties in maintaining sleep (p < 0.001) compared to the SWs and the DWs. The PNWs reported most often difficulties to fall asleep and fatigue during free-time (p-values <0.001). The DWs and PNWs experienced less often work-life conflict than the SWs (25 and 26 vs. 38%, p < 0.001). The PNWs were more often satisfied with autonomy at work and appreciation and fair treatment by colleagues than the DWs or the SWs (p < 0.001). The SWs and PNWs reported remarkably higher occurrence of verbal (p < 0.001, OR 3.71, 95% CI 3.23–4.27 and OR 7.67, 95% CI 5.35–10.99, respectively) and physical workplace violence (p < 0.001, OR 9.24, 95% CI 7.17–11.90 and OR 28.34, 95% CI 16.64–43.06, respectively) compared to DWs. Conclusively, PNWs reported contradictory differences in sleep quality compared to DWs and SWs. PNWs are more often satisfied with their colleagues and autonomy at work than DWs or SWs but face workplace violence remarkably more often.     

New method for toxicity assessment in marine and brackish environments using the macroalga Gracilaria tenuistipitata (Gracilariales,Rhodophyta)

A growth inhibition test method was developed using the macroalga Gracilaria tenuistipitata as the test organism. This alga was chosen because of its high laboratory growth rates, commonly 30–40% d^–1, which are reached in salinities between 5 and 40, and its epiphyte resistance. The toxicity of a number of substances, including heavy metals, herbicides and complex wastewaters towards the alga was assayed. Anti-fouling paints were tested with a modification of the method. EC50 values for heavy metals varied between 0.05 and 17 mg l^–1 and for herbicides between 0.002 and 0.02 mg l^–l. The sensitivity to the toxicant was generally higher at low salinity. Omitting nitrogen and phosphorus additions to the test medium increased the sensitivity and a semi-static performance was possible with maintained or increased sensitivity. Preliminary tests done with a computerised photosynthesis inhibition method produced promising results.In conclusion, this is a simple, sensitive and reproducible test method for assessing the toxicity of substances, wastewaters and anti-fouling paints in brackish and marine environments.     

Variation and repeatability of courtship song characters among wild-caught and laboratory-rearedDrosophila montana andD. littoralis males (Diptera: Drosophilidae)

InDrosophila montana andD. littoralis (species of theD. virilis group), females use male courtship song in their mate choice in wild preferring males which produce short and dense sound pulses (Aspi and Hoikkala, 1995). In the present study these song characters were found to be repeatable among overwintered males. Male progenies of wild-caught flies reared in the laboratory, and inD. montana also the males collected in wild before overwintering, exhibited very little variation between males in these characters. Contrary to pulse characters, pulse train characters measured forD. montana song varied significantly between laboratory-reared males. Our findings suggest that inD. montana andD. littoralis song characters playing a part in sexual selection in the wild are more condition dependent than song characters which are not the direct targets of female choice.