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Signal propagation in photosensory proteins is a complex and multidimensional event. Unraveling such mechanisms site-specifically in real time is an eligible but a challenging goal. Here, we elucidate the site-specific events in a red-light sensing phytochrome using the unnatural amino acid azidophenylalanine, vibrationally distinguishable from all other protein signals. In canonical phytochromes, signal transduction starts with isomerization of an excited bilin chromophore, initiating a multitude of processes in the photosensory unit of the protein, which eventually control the biochemical activity of the output domain, nanometers away from the chromophore. By implementing the label in prime protein locations and running two-color step-scan FTIR spectroscopy on the Deinococcus radiodurans bacteriophytochrome, we track the signal propagation at three specific sites in the photosensory unit. We show that a structurally switchable hairpin extension, a so-called tongue region, responds to the photoconversion already in microseconds and finalizes its structural changes concomitant with the chromophore, in milliseconds. In contrast, kinetics from the other two label positions indicate that the site-specific changes deviate from the chromophore actions, even though the labels locate in the chromophore vicinity. Several other sites for labeling resulted in impaired photoswitching, low structural stability, or no changes in the difference spectrum, which provides additional information on the inner dynamics of the photosensory unit. Our work enlightens the multidimensionality of the structural changes of proteins under action. The study also shows that the signaling mechanism of phytochromes is accessible in a time-resolved and site-specific approach by azido probes and demonstrates challenges in using these labels.

Background: Due to unmet need for bone augmentation, our aim was to promote osteogenic differentiation of human adipose stem cells (hASCs) encapsulated in gellan gum (GG) or collagen type I (COL) hydrogels with bioactive glass (experimental glass 2-06 of composition [wt-%]: Na 2 O 12.1, K 2 O 14.0, CaO 19.8, P 2 O5 2.5, B 2 O 3 1.6, SiO 2 50.0) extract based osteogenic medium (BaG OM) for bone construct development. GG hydrogels were crosslinked with spermidine (GG-SPD) or BaG extract (GG-BaG)., Methods: Mechanical properties of cell-free GG-SPD, GG-BaG, and COL hydrogels were tested in osteogenic medium (OM) or BaG OM at 0, 14, and 21 d. Hydrogel embedded hASCs were cultured in OM or BaG OM for 3, 14, and 21 d, and analyzed for viability, cell number, osteogenic gene expression, osteocalcin production, and mineralization. Hydroxyapatite-stained GG-SPD samples were imaged with Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy (SPIM) in OM and BaG OM at 21 d. Furthermore, Raman spectroscopy was used to study the calcium phosphate (CaP) content of hASC-secreted ECM in GG-SPD, GG-BaG, and COL at 21 d in BaG OM., Results: The results showed viable rounded cells in GG whereas hASCs were elongated in COL. Importantly, BaG OM induced significantly higher cell number and higher osteogenic gene expression in COL. In both hydrogels, BaG OM induced strong mineralization confirmed as CaP by Raman spectroscopy and significantly improved mechanical properties. GG-BaG hydrogels rescued hASC mineralization in OM. OPT and SPIM showed homogeneous 3D cell distribution with strong mineralization in BaG OM. Also, strong osteocalcin production was visible in COL., Conclusions: Overall, we showed efficacious osteogenesis of hASCs in 3D hydrogels with BaG OM with potential for bone-like grafts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Phytochrome proteins regulate many photoresponses of plants and microorganisms. Light absorption causes isomerization of the biliverdin chromophore, which triggers a series of structural changes to activate the signaling domains of the protein. However, the structural changes are elusive, and therefore the molecular mechanism of signal transduction remains poorly understood. Here, we apply two-color step-scan infrared spectroscopy to the bacteriophytochrome from Deinococcus radiodurans. We show by recordings in H 2 O and D 2 O that the hydrogen bonds to the biliverdin D-ring carbonyl become disordered in the first intermediate (Lumi-R) forming a dynamic microenvironment, then completely detach in the second intermediate (Meta-R), and finally reform in the signaling state (Pfr). The spectra reveal via isotope labeling that the refolding of the conserved "PHY-tongue" region occurs with the last transition between Meta-R and Pfr. Additional changes in the protein backbone are detected already within microseconds in Lumi-R. Aided by molecular dynamics simulations, we find that a strictly conserved salt bridge between an arginine of the PHY tongue and an aspartate of the chromophore binding domains is broken in Lumi-R and the arginine is recruited to the D-ring C═O. This rationalizes how isomerization of the chromophore is linked to the global structural rearrangement in the sensory receptor. Our findings advance the structural understanding of phytochrome photoactivation.

Phytochrome proteins translate light into biochemical signals in plants, fungi and microorganisms. Light cues are absorbed by a bilin chromophore, leading to an isomerization and a rotation of the D-ring. This relays the signal to the protein matrix. A set of amino acids, which is conserved across the phytochrome superfamily, holds the chromophore in the binding pocket. However, the functional role of many of these amino acids is not yet understood. Here, we investigate the hydrogen bonding network which surrounds the D-ring of the chromophore in the resting (Pr) state. We use UV/vis spectroscopy, infrared absorption spectroscopy and X-ray crystallography to compare the photosensory domains from Deinococcus radiodurans, the phytochrome 1 from Stigmatella aurantiaca, and a D. radiodurans H290T mutant. In the latter two, an otherwise conserved histidine next to the D-ring is replaced by a threonine. Our infrared absorption data indicate that the carbonyl of the D-ring is more strongly coordinated by hydrogen bonds when the histidine is missing. This is in apparent contrast with the crystal structure of the PAS-GAF domain of phytochrome 1 from S. aurantiaca (pdb code 4RPW), which did not resolve any obvious binding partners for the D-ring carbonyl. We present a new crystal structure of the H290T mutant of the PAS-GAF from D. radiodurans phytochrome. The 1.4 Å-resolution structure reveals additional water molecules, which fill the void created by the mutation. Two of the waters are significantly disordered, suggesting that flexibility might be important for the photoconversion. Finally, we report a spectral analysis which quantitatively explains why the histidine-less phytochromes do not reach equal Pfr-type absorption in the photoequilibrium compared to the Deinococcus radiodurans wild-type protein. The study highlights the importance of water molecules and the hydrogen bonding network around the chromophore for controlling the isomerization reaction and spectral properties of phytochromes.

Phytochromes sense red light in plants and various microorganism. Light absorption causes structural changes within the protein, which alter its biochemical activity. Bacterial phytochromes are dimeric proteins, but the functional relevance of this arrangement remains unclear. Here, we use time-resolved X-ray scattering to reveal the solution structural change of a monomeric variant of the photosensory core module of the phytochrome from Deinococcus radiodurans. The data reveal two motions, a bend and a twist of the PHY domain with respect to the chromophore-binding domains. Infrared spectroscopy shows the refolding of the PHY tongue. We conclude that a monomer of the phytochrome photosensory core is sufficient to perform the light-induced structural changes. This implies that allosteric cooperation with the other monomer is not needed for structural activation. The dimeric arrangement may instead be intrinsic to the biochemical output domains of bacterial phytochromes.

Three new epithiodiketopiperazine natural products [outovirin A (1), outovirin B (2), and outovirin C (3)] resembling the antifungal natural product gliovirin have been identified in extracts of Penicillium raciborskii, an endophytic fungus isolated from Rhododendron tomentosum. The compounds are unusual for their class in that they possess sulfide bridges between α- and β-carbons rather than the typical α-α bridging. To our knowledge, outovirin A represents the first reported naturally produced epimonothiodiketopiperazine, and antifungal outovirin C is the first reported trisulfide gliovirin-like compound. This report describes the identification and structural elucidation of the compounds by LC-MS/MS and NMR.

Bioactive glasses are known for their ability to induce osteogenic differentiation of stem cells. To elucidate the mechanism of the osteoinductivity in more detail, we studied whether ionic extracts prepared from a commercial glass S53P4 and from three experimental glasses (2-06, 1-06 and 3-06) are alone sufficient to induce osteogenic differentiation of human adipose stem cells. Cells were cultured using basic medium or osteogenic medium as extract basis. Our results indicate that cells stay viable in all the glass extracts for the whole culturing period, 14 days. At 14 days the mineralization in osteogenic medium extracts was excessive compared to the control. Parallel to the increased mineralization we observed a decrease in the cell amount. Raman and Laser Induced Breakdown Spectroscopy analyses confirmed that the mineral consisted of calcium phosphates. Consistently, the osteogenic medium extracts also increased osteocalcin production and collagen Type-I accumulation in the extracellular matrix at 13 days. Of the four osteogenic medium extracts, 2-06 and 3-06 induced the best responses of osteogenesis. However, regardless of the enhanced mineral formation, alkaline phosphatase activity was not promoted by the extracts. The osteogenic medium extracts could potentially provide a fast and effective way to differentiate human adipose stem cells in vitro., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: In recent decades, Raman spectroscopy has entered the biological and medical fields. It enables nondestructive analysis of structural details at the molecular level and has been used to study viruses and their constituents. Here, we used Raman spectroscopy to study echovirus 1 (EV1), a small, nonenveloped human pathogen, in two different uncoating states induced by heat treatments. Raman signals of capsid proteins and RNA genome were observed from the intact virus, the uncoating intermediate, and disrupted virions. Transmission electron microscopy data revealed general structural changes between the studied particles. Compared to spectral characteristics of proteins in the intact virion, those of the proteins of the heat-treated particles indicated reduced α-helix content with respect to β-sheets and coil structures. Changes observed in tryptophan and tyrosine signals suggest an increasingly hydrophilic environment around these residues. RNA signals revealed a change in the environment of the genome and in its conformation. The ionized-carbonyl vibrations showed small changes between the intact virion and the uncoating intermediate, which points to cleavage of salt bridges in the protein structure during the uncoating process. In conclusion, our data reveal distinguishable Raman signatures of the intact, intermediate, and disrupted EV1 particles. These changes indicate structural, chemical, and solute-solvent alterations in the genome and in the capsid proteins and lay the essential groundwork for investigating the uncoating of EV1 and related viruses in real time., Importance: In order to combat virus infection, we need to know the details of virus uncoating. We present here the novel Raman signatures for opened and intact echovirus 1. This gives hope that the signatures may be used in the near future to evaluate the ambient conditions in endosomes leading to virus uncoating using, e.g., coherent anti-Stokes Raman spectroscopy (CARS) imaging. These studies will complement structural studies on virus uncoating. In addition, Raman/CARS imaging offers the possibility of making dynamic live measurements in vitro and in cells which are impossible to measure by, for example, cryo-electron tomography. Furthermore, as viral Raman spectra can be overwhelmed with various contaminants, our study is highly relevant in demonstrating the importance of sample preparation for Raman spectroscopy in the field of virology., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Preventive interventions early in life are likely to lower the risk of intergenerational transmission of criminal behavior. We investigated if psychopathy among homicidal offenders is associated with criminal offending among the offenders' offspring. The basic sample consisted of consecutive Finnish homicide offenders (during 1995-2004) who had been subjected to a forensic psychiatric examination and rated for a file-based PCL-R, and their offspring. Criminal behavior among both genders of the offspring was more common than in the general population. In general, the offspring's crimes against others (e.g., threat, intimidation, deprivation of freedom, breach of domicile) were associated with their parent's psychopathy. A grandfather's major mental disorder was associated with a high rate of crime committed by the offspring. Especially, the sons of male psychopathic homicidal offenders had the highest rate of committing crimes, which was often expressed as vandalism. However, both genders of offspring seem to require special preventive programs to ameliorate these problems.

Background: Filicide, the killing of one's child, is an extraordinary form of homicide. It has commonly been associated with suicide and parental psychiatric illness. In the research on filicide, nationwide studies with comparison groups, specific perpetrator subgroups, and assessment of possible risk factors have been called for. The purpose of the current study was to provide all that., Methods: In this nationwide register-based case-control study all filicide offenders who were in a forensic psychiatric examination in Finland 1995-2004 were examined and compared with an age- and gender matched control group of homicide offenders. The assessed variables were psychosocial history, index offence, and psychiatric variables as well as psychopathy using the PCL-R., Results: Filicide offenders were not significantly more often diagnosed with psychotic disorders than the controls but they had attempted suicide at the crime scene significantly more often. Filicide offenders had alcohol abuse/dependence and antisocial personality less often than the controls. Filicide offenders scored significantly lower on psychopathy than the controls. Within the group of filicide offenders, the psychopathy items with relatively higher scores were lack of remorse or guilt, shallow affect, callous/lack of empathy, poor behavioral controls, and failure to accept responsibility., Conclusion: Since filicide offenders did not seem significantly more mentally disordered than the other homicide offenders, psychiatry alone cannot be held responsible for the prevention of filicide. Extensive international studies are needed to replicate our findings and provide more specific knowledge in order to enhance prevention.

Background: The contribution of women to violent offending, including homicide, may be increasing as society changes., Aims: The aim of this paper was to test for trends in homicide by women in Finland., Methods: A retrospective register-based study was conducted by comparing two national cohorts: one from 1982 to 1992 and the other from 1993 to 2005., Results: There was a small increase in the proportion of homicides committed by women over time, but the most striking difference between the cohorts was in the significantly higher frequency of alcohol abuse/dependence in the later cohort and of being under the influence of alcohol during the crime. Fewer perpetrators were regarded as lacking or being of diminished responsibility in the later cohort. The victims of the earlier cohort were emotionally closer to the offender than those of the later one., Conclusions: In Finland, there have been changes in characteristics of women who commit homicide and their crimes over time, with the apparent development of a subgroup of women who kill who are much more like men who kill than women in the 1980s and early 1990s. Preventing substance abuse and marginalization are likely to be important ways of preventing homicide by both female and male perpetrators., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Approximately 9% of the homicides in Finland are committed by adolescents under 20 years of age. The purpose of this study was to investigate the offence and offender characteristics in homicidal adolescents. Forensic psychiatric evaluation statements of adolescent offenders accused of a homicide during 1990-2001 were reviewed retrospectively (n=57). In 38% of the cases, there were multiple offenders. In 58% of the cases, the victim was an acquaintance, in 25% a stranger, in 12% a family member and in 5% of the cases an (ex)intimate partner. Sixty-nine percent of the offenders were intoxicated and 21% under the influence of drugs at the time of the killing. The most frequent motives were an argument (25%) and a robbery (25%). Sixty-four percent of the offenders had developmental problems and 42% had a crime history. Approximately half were diagnosed as having a conduct or a personality disorder, but 32% of the offenders were considered not to suffer from a mental illness or substance abuse. For 63%, the level of intellectual functioning was average or above average. There were signs of more than one form of violence in 54% of the cases and 28% of the cases contained excessive violence. The use of multiple and excessive violence was significantly related to the offender age, multiple offenders, offender-victim relationship and substance abuse, but not related to having developmental problems, crime history or mental illness.

Background: It is not currently known how psychotic symptoms are associated with the nature of violence among homicide offenders with schizophrenia, or, more specifically, whether different psychotic symptoms are differentially linked with excessive violence., Aim: To identify factors associated with the use of excessive violence among homicide offenders with schizophrenia., Methods: Forensic psychiatric examination statements and Criminal Index File data of 125 consecutive Finnish homicide offenders with a diagnosis of schizophrenia were analysed., Results: Nearly one-third of the cases in this sample involved extreme violence, including features such as sadism, mutilation, sexual components or multiple stabbings. Excessive violence was a feature of acts when the offender was not the sole perpetrator or when there was a previous homicidal history. Positive psychotic symptoms, including delusions, were not associated with the use of excessive violence., Conclusions: These results highlight the importance of variables other than clinical state when examining qualitative aspects of homicidal acts, such as the degree and nature of violence, by offenders with schizophrenia. Further study is needed with a more specific focus on the qualities of the violence among different subgroups of offenders, but inclusive of those with psychosis., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

In Finland, homicide by ligature strangulation is a rare event. The purpose of this study was to investigate recent homicide cases by ligature strangulation and to analyse offence and offender characteristics. All forensic psychiatric evaluation statements of offenders accused of ligature strangulation homicides during the 7-year period 1996--2002 (n=19) were reviewed retrospectively. Thirty-five percent of the victims were females. In four cases, the victim was the offender's intimate partner; in one case, it was the stepson; in one case, a father and in the rest of the cases (n=13), an acquaintance. None of the cases involved planning, sadism or sexual elements. In 89% of the cases, the offender was either intoxicated or on drugs at the time of the offence and in 94% of the cases, the victim was found at the scene of the killing. Of the offenders, 63% had crime history, and approximately half of the offenders had experienced parental alcohol abuse, domestic violence or previous trauma. Most of the offenders were unemployed at the time of the offence. In all, 89% were diagnosed as having personality disorder and two were schizophrenic.

An experimental setup is described for measuring laser-induced plasma emission spectra in the near vacuum UV with a Czerny-Turner spectrograph and intensified charge-coupled device under atmospheric pressure. With a simple gas-purge technique, emission lines down to 130 nm could be recorded. The strongest emission lines of bromine, chlorine, and iodine in the near vacuum UV are easily detected.

A theoretical distinction between instrumental and expressive aggression was used in analyzing offender characteristics and their associations with crime scene actions in Finnish homicides. Twenty-one variables reflecting the offenders' criminal activity, previous relationships with intimates and victims, and general social and psychological adjustment were derived from files of single-offender/single-victim homicides occurring between 1980 and 1994 (n = 502). Additionally, three variables describing post-offense actions and police interview behavior were included. A multidimensional scaling procedure was used to investigate the interrelationships between the variables. A distinction between expressive and instrumental characteristics was observable in the empirical structure, which was divided into three subthemes of Instrumental, Expressive: Blood, and Expressive: Intimate. Associations between the characteristics with five previously identified subthemes of crime scene actions were computed. In addition, the subthemes of crime scene actions were related to post-offense actions and police interview behavior, with Expressive themes being associated with less denial as well as a greater likelihood of surrendering and confession. The practical usefulness for police investigations and theoretical implications of the results are discussed.

Causality factors, the responsibility of the driver and driver fatigue-related factors were studied in fatal two-vehicle accidents where a trailer truck driver was involved during the period of 1991-1997 (n = 337). In addition, 251 long-haul truck drivers were surveyed in order to study their views regarding contributing factors in accidents involving trucks and the development of possible countermeasure against driver fatigue. Trailer truck drivers were principally responsible for 16% of all the accidents. Younger driver age and driving during evening hours were significant predictors of being principally responsible. In addition, the probability of being principally responsible for the accident increased by a factor of over three if the driver had a chronic illness. Prolonged driving preceding the accident, accident history or traffic offence history did not have a significant effect. Only 2% of the drivers were estimated to have fallen asleep while driving just prior to the accident, and altogether 4% of the drivers had been tired prior to the accident. Of the drivers 13% had however, been driving over 10 h preceding the accident (which has been criminally punishably in Finland since 1995 under the EC regulation) but no individual factors had a significant effect in predicting prolonged driving. The surveyed views regarding causes of truck accidents correspond well with the accident analysis. Accidents were viewed as being most often caused by other road users and driver fatigue was viewed to be no more than the fifth (out of eight) common cause of accidents. The probability of viewing fatigue as a more common cause increased significantly if the driver had experienced fatigue-related problems while driving. However, nearly half of the surveyed truck drivers expressed a negative view towards developing a technological countermeasure against driver fatigue. The negative view was not related to personal experiences of fatigue-related problems while driving.

This study examined the effects of task and time-on-task on fatigue symptoms in overnight driving. Four participants drove an instrumented car 1200 km overnight and completed the same trip as passengers on another night. Subjective ratings of drowsiness, eye blink frequency and duration, microsleeps, and steering-wheel inputs were analysed as a function of time-on-task, and for separate samples when meeting oncoming heavy vehicles. Four video cameras were used to monitor the road view and the face of both the driver and passenger. In terms of eye closure duration, the reported microsleeps were shorter while driving (mean = 0.7 s, SD = 0.2 s) than as a passenger (mean = 2.6 s, SD = 2.0 s). Blink frequency increased with time-on-task as expected, indicating tiredness, and decreased when approaching an oncoming heavy vehicle, indicating attentive response to a potential critical situation. No consistent effect of time-on-task on high-frequency steering-wheel inputs when meeting oncoming heavy vehicles was found. The results raise the important question of what makes a driver wake from a microsleep earlier than a passenger and, given proper monitoring of long eyelid closures, what the proper intervention should be.