New glucose isomerase - fit for biorefinery challenge

Biofuel is not the bio-product with the highest value-addition, especially with the currently low oil price in mind. Modern concepts of biorefineries therefore tend to include production of more valuable products than ethanol, such as bioplastics. Hydroxymethyl furfural (HMF) is considered to be a central platform chemical for biomaterials production. HMF can be produced from hexose sugars, however the conversion is much more efficient ketoses ketoses such fructose than with aldehydes (glucose, mannose and so on). Glucose is the only sufficiently abandoned sugar monomer to potentially become a row material for commodity chemicals manufacturing, wood being the most likely source of glucose. Glucose can be then enzymatically converted to its ketose isomer – fructose using xylose (glucose) isomerase. The glucose isomerases are widely commercially available: they are one of the largest in volume in the industrial enzyme market for their production of widely-used High Fructose Syrups (HFS) for food applications. However, the currently available commercial enzymes are highly sensitive to the substrate sugar purity, which is well acceptable in food industry application. Typically, even sugar produced from starch requires activated carbon filtration, ion exchange chromatography and degasification before it can proceed to isomerization reaction. Sugars produced in 2nd generation biorefinery (especially from wood) have much more impurities than starch derived sugar, including lignin, extractives, etc, and required level of purity is not justified for the technical sugar. Taking this challenge, we set to develop and industrial glucose isomerase that can work directly in lignocellulosic biomass hydrolysate. To address the choice of enzyme prototypes covering most structurally diverse groups, we obtained a custom made 3dm database (ordered from Bio-Prodict BV, Netherlands). The database contained around 25 000 protein sequences from public databases aligned and uniformly numbered based on structural alignment or strong homology. Database, where annotation of proteins was not taken in account while building it, eventually contained xylose isomerases L-rhamnose isomerases, hydropiruvate isomerase, innosose isomerases, D-tagatose epimerases, L-ribulose-phosphate epimerases, mannonate dehydratases and endonucleases. This tool gave a general view on structural diversity of known characterized and just annotated xylose isomerase, and helped to find a representative pool of prototypes to test for our special requirements. Among over 20 tested candidates, we found a new extremely robust enzyme, which outperformed every reference enzyme in glucose isomerization in crude lignocellulosic hydrolysate. The broad scope of proteins represented in 3dm database allowed unprecedented opportunity to analyze the proteins sharing the same fold in terms of what makes them functionally distinct. Focusing our attention on xylose isomerases, we were able to identify several positions, which make a protein with such fold a xylose isomerase. Some of those positions have never been mentioned in the literature as mutational hot spots or as residues essential for the function. We constructed focused libraries with variation in these positions and were able to find enzyme variants with strongly altered substrate preferences between glucose and fructose and enabled doubling the efficiency of glucose isomerization by the new enzyme. The enzyme production in E.coli was scaled up to industrial scale. Thus using bioinformatics approach combined with protein engineering, we developed an industrial enzyme that enables sugar valorization and platform chemicals production in biorefinery streamline. This work was supported by EU via Horizon 2020 projects RETAPP, and BIOFOREVER

Crime and income trajectories preceding lethal and non-lethal violence

Purpose: This study analyzes the pathways leading to violent offending. We examine whether the lethality of a violent crime could be predicted based on individuals' prior history of violent crime and income, and whether the trajectories of lethal offenders are distinguishable from the pathways of non-lethal offenders. Methods: We use a sample of police-reported violent crimes committed in Finland in 2010-2011 (N = 26,303) and contrast the pathways to homicide with the trajectories leading to petty assault, assault, aggravated assault, and attempted homicide. Group-based trajectory modeling is applied for identifying individuals with similar trajectories, and multilevel modeling is used for estimating the mean trajectories for offenders of differing severity. Results: Results indicate that homicide offenders display a clear escalation in the frequency of violent offending and a slight decrease in income prior to the offense, but the pathways to homicide largely resemble the pathways to aggravated assault and attempted homicide. Conclusions: The lethality of violent offending cannot be predicted from the offender's crime and income. The greatest divide in the violence severity continuum is between offenders of assaults and offenders of aggravated assaults, with the latter group largely resembling offenders of completed and attempted homicides.Peer reviewe

The Effects of Intensive Weight Reduction on Body Composition and Serum Hormones in Female Fitness Competitors

Worries about the potential negative consequences of popular fat loss regimens for aesthetic purposes in normal weight females have been surfacing in the media. However, longitudinal studies investigating these kinds of diets are lacking. The purpose of the present study was to investigate the effects of a 4-month fat-loss diet in normal weight females competing in fitness-sport. In total 50 participants finished the study with 27 females (27.2 +/- 4.1 years) dieting for a competition and 23 (27.7 +/- 3.7 years) acting as weight-stable controls. The energy deficit of the diet group was achieved by reducing carbohydrate intake and increasing aerobic exercise while maintaining a high level of protein intake and resistance training in addition to moderate fat intake. The diet led to a similar to 12% decrease in body weight (P <0.001) and a similar to 3550% decrease in fat mass (DXA, bioimpedance, skinfolds, P <0.001) whereas the control group maintained their body and fat mass (diet x group interaction P <0.001). A small decrease in lean mass (bioimpedance and skinfolds) and in vastus lateralis muscle cross-sectional area (ultrasound) were observed in diet (P <0.05), whereas other results were unaltered (DXA: lean mass, ultrasound: triceps brachii thickness). The hormonal system was altered during the diet with decreased serum concentrations of leptin, triiodothyronine (T3), testosterone (P <0.001), and estradiol (P <0.01) coinciding with an increased incidence of menstrual irregularities (P <0.05). Body weight and all hormones except T3 and testosterone returned to baseline during a 34 month recovery period including increased energy intake and decreased levels aerobic exercise. This study shows for the first time that most of the hormonal changes after a 3550% decrease in body fat in previously normal-weight females can recover within 34 months of increased energy intake.Peer reviewe

British perceptions of the Balkan Slavs: Professional and popular categorizations before 1914

The purpose of this thesis is to deepen our understanding about the ways in which the Balkan Slavs were perceived and represented in Britain between 1856 and 1914 by concentrating on religious, military, commercial and satirical discourses. These specific areas have not received enough detailed analysis in the existing secondary literature. This thesis has three aims: first, to investigate the ways and the extent to which British domestic and imperial concerns connected to and were explained through Balkan questions; second, to examine the effects of perceptions and prejudices on decision-making; and third, to analyze the levels of accuracy of perceptions and the concrete consequences of possible misconceptions. In terms of source material, previous studies have mostly relied on travel-writing and literature. This study widens the scope by examining a variety of textual and visual materials, ranging from diplomatic and military writing to religious treatises and cartoons. The main conclusions include that British domestic and imperial concerns coincided and were discussed through Balkan questions on a very concrete level, of which the Anglican-Orthodox reunion debate, the Bulgarian atrocities agitation campaign, issues related to domestic and imperial defence, social problems as well as representations of commercial potential of the Balkans, were among the most visible ones. The attention that the region attracted in Britain was also more nuanced than has been argued in the current literature. Preconceptions and prejudices had an effect on military and business decision-making more often than on religious positions which remained largely unaltered. In many cases, representations of the Balkans were at least partly accurate, although grave misunderstandings and misinterpretations also occurred. These views had concrete relevance most visibly in connection with newly-established British firms, whose misconceptions about the commercial potential of the Balkans partly contributed to their business failures in the region