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Objective: The present study investigated how pupil size and heart rate variability (HRV) can contribute to the prediction of operator performance. We illustrate how focusing on mental effort as the conceptual link between physiological measures and task performance can align relevant empirical findings across research domains., Background: Physiological measures are often treated as indicators of operators' mental state. Thereby, they could enable a continuous and unobtrusive assessment of operators' current ability to perform the task., Method: Fifty participants performed a process monitoring task consisting of ten 9-minute task blocks. Blocks alternated between low and high task demands, and the last two blocks introduced a task reward manipulation. We measured response times as primary performance indicator, pupil size and HRV as physiological measures, and mental fatigue, task engagement, and perceived effort as subjective ratings., Results: Both increased pupil size and increased HRV significantly predicted better task performance. However, the underlying associations between physiological measures and performance were influenced by task demands and time on task. Pupil size, but not HRV, results were consistent with subjective ratings., Conclusion: The empirical findings suggest that, by capturing variance in operators' mental effort, physiological measures, specifically pupil size, can contribute to the prediction of task performance. Their predictive value is limited by confounding effects that alter the amount of effort required to achieve a given level of performance., Application: The outlined conceptual approach and empirical results can guide study designs and performance prediction models that examine physiological measures as the basis for dynamic operator assistance., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Thioflavin T (ThT) is a typical dye used to visualize the aggregation and formation of fibrillar structures, e.g., amyloid fibrils and peptide nanofibrils. ThT has been considered to produce stable fluorescence when interacting with aggregated proteins. For single-molecule localization microscopy (SMLM)-based optical super-resolution imaging, a photoswitching/blinking fluorescence property is required. Here we demonstrate that, in contrast to previous reports, ThT exhibits intrinsic stochastic blinking properties, without the need for blinking imaging buffer, in stable binding conditions. The blinking properties (photon number, blinking time, and on-off duty cycle) of ThT at the single-molecule level (for ultralow concentrations) were investigated under different conditions. As a proof of concept, we performed SMLM imaging of ThT-labeled α-synuclein fibrils measured in air and PBS buffer.

Physiological measures are often used to assess the mental state of human operators in supervisory process control tasks. However, the diversity of research approaches creates a heterogeneous landscape of empirical evidence. To map existing evidence and provide guidance to researchers and practitioners, this paper systematically reviews 109 empirical studies that report relationships between peripheral nervous system measures and mental state dimensions (e.g. mental workload, mental fatigue, stress, and vigilance) of interest. Ocular and electrocardiac measures were the most prominent measures across application fields. Most studies sought to validate such measures for reliable assessments of cognitive task demands and time on task, with measures of pupil size receiving the most empirical support. In comparison, less research examined the utility of physiological measures in predicting human task performance. This approach is discussed as an opportunity to focus on operators' individual response to cognitive task demands and to advance the state of research.

Formation of liquid condensates plays a critical role in biology via localization of different components or via altered hydrodynamic transport, yet the hydrogen-bonding environment within condensates, pivotal for solvation, has remained elusive. We explore the hydrogen-bond dynamics within condensates formed by the low-complexity domain of the fused in sarcoma protein. Probing the hydrogen-bond dynamics sensed by condensate proteins using two-dimensional infrared spectroscopy of the protein amide I vibrations, we find that frequency-frequency correlations of the amide I vibration decay on a picosecond time scale. Interestingly, these dynamics are markedly slower for proteins in the condensate than in a homogeneous protein solution, indicative of different hydration dynamics. All-atom molecular dynamics simulations confirm that lifetimes of hydrogen-bonds between water and the protein are longer in the condensates than in the protein in solution. Altered hydrogen-bonding dynamics may contribute to unique solvation and reaction dynamics in such condensates.

This work aimed at the development of a stable albumin-perfluorocarbon (o/w) emulsion as an artificial oxygen carrier suitable for clinical application. So far, albumin-perfluorocarbon-(o/w) emulsions have been successfully applied in preclinical trials. Cross-linking a variety of different physical and chemical methods for the characterization of an albumin-perfluorocarbon (PFC)-(o/w) emulsion was necessary to gain a deep understanding of its specific emulsification processes during high-pressure homogenization. High-pressure homogenization is simple but incorporates complex physical reactions, with many factors influencing the formation of PFC droplets and their coating. This work describes and interprets the impact of albumin concentration, homogenization pressure, and repeated microfluidizer passages on PFC-droplet formation; its influence on storage stability; and the overcoming of obstacles in preparing stable nanoemulsions. The applied methods comprise dynamic light scattering, static light scattering, cryo- and non-cryo-scanning and transmission electron microscopies, nuclear magnetic resonance spectroscopy, light microscopy, amperometric oxygen measurements, and biochemical methods. The use of this wide range of methods provided a sufficiently comprehensive picture of this polydisperse emulsion. Optimization of PFC-droplet formation by means of temperature and pressure gradients results in an emulsion with improved storage stability (tested up to 5 months) that possibly qualifies for clinical applications. Adaptations in the manufacturing process strikingly changed the physical properties of the emulsion but did not affect its oxygen capacity.

Although the objective assessment of mental workload has been a focus of human factors research, few studies have investigated stakeholders' attitudes towards its implementation in real workplaces. The present study addresses this research gap by surveying N = 702 managers in three European countries (Germany, United Kingdom, Spain) about their expectations and concerns regarding sensor-based monitoring of employee mental workload. The data confirm the relevance of expectations regarding improvements of workplace design and employee well-being, as well as concerns about restrictions of employees' privacy and sovereignty, for the implementation of workload monitoring. Furthermore, Bayesian regression models show that the examined expectations have a substantial positive association with managers' willingness to support workload monitoring in their company. Privacy concerns are identified as a significant barrier to the acceptance of workload monitoring, both in terms of their prevalence among managers and their strong negative relationship with monitoring support., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1-/- compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1-/- than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure., (© 2021 Pütz et al.)

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The present study investigated the affective modulation of conflict adaptation. In a first step, we conducted a direct replication of a previous study (Schuch & Pütz, 2018). Positive vs. negative mood state was induced by a success-failure manipulation (between-groups, N = 40 per group). In a subsequent task-switching experiment, the congruency sequence effect was assessed in task repetitions and task switches, measuring conflict adaptation within tasks and between tasks, respectively. We found conflict adaptation (averaged across task repetitions and task switches) to be enhanced in negative mood. We did not replicate our previous finding of enhanced conflict adaptation in task switches in positive mood. In a second step, we combined the replication data with the original data set, yielding a larger database with N = 80 per mood group. Using diffusion modeling, we explored the affective modulation of conflict adaptation in task repetitions. Conflict adaptation was reflected in drift rate, consistent with the idea that response conflict triggers an increase in processing selectivity, thereby attenuating the influence of the irrelevant stimulus dimension. Conflict adaptation was also reflected in boundary separation, suggesting that response conflict on the previous trial triggered an increase in response caution. The mood manipulation did not seem to affect processing selectivity (as captured by drift rate) but affected the setting of response caution (as captured by the boundary separation parameter), with faster and more error-prone responding in the negative than positive mood group. We discuss theoretical implications of these findings, and also briefly consider the affective modulations of other cognitive control measures.

The novel coronavirus caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached more than 160 countries and has been declared a pandemic. SARS-CoV-2 infects host cells by binding to the angiotensin-converting enzyme 2 (ACE-2) surface receptor via the spike (S) receptor-binding protein (RBD) on the virus envelope. Global data on a similar infectious disease spread by SARS-CoV-1 in 2002 indicated improved stability of the virus at lower temperatures facilitating its high transmission in the community during colder months (December-February). Seasonal viral transmissions are strongly modulated by temperatures, which can impact viral trafficking into host cells; however, an experimental study of temperature-dependent activity of SARS-CoV-2 is still lacking. We mimicked SARS-CoV-2 with polymer beads coated with the SARS-CoV-2 S protein to study the effect of seasonal temperatures on the binding of virus-mimicking nanospheres to lung epithelia. The presence of the S protein RBD on nanosphere surfaces led to binding by Calu-3 airway epithelial cells via the ACE-2 receptor. Calu-3 and control fibroblast cells with S-RBD-coated nanospheres were incubated at 33 and 37 °C to mimic temperature fluctuations in the host respiratory tract, and we found no temperature dependence in contrast to nonspecific binding of bovine serum ablumin-coated nanospheres. Moreover, the ambient temperature changes from 4 to 40 °C had no effect on S-RBD-ACE-2 ligand-receptor binding and minimal effect on the S-RBD protein structure (up to 40 °C), though protein denaturing occurred at 51 °C. Our results suggest that ambient temperatures from 4 to 40 °C have little effect on the SARS-CoV-2-ACE-2 interaction in agreement with the infection data currently reported.

The demand to adapt the work processes to hygiene- and contact reduction requirements in order to curtail the spread of the COVID-19 pandemic in Germany has led to short-term and sometimes radical changes in companies and organizations. Based on 52 expert interviews with company and organization representatives, this research project analyzed which pandemic-related changes in the context of work occur on a supraregional and cross-sectoral level, how they are to be evaluated, and which new ways of working might also prove beneficial to companies after the pandemic. In line with existing studies, an increasing flexibilization of work location and working hours, an acceleration of the digitalization of work processes, and effects on leadership culture were identified. Other key experiences relate to the adaptation of internal and external communication processes and the operational handling of crisis-related challenges. Based on the results, good practices were derived in form of normative-narrative use-case based scenarios. Practical Relevance : Dealing with the COVID-19 pandemic has been the central challenge for many companies over the past year and a half. The presented results show measures that have been successfully implemented in practice to overcome the challenges posed by the crisis, as well as approaches for the future development of forms of work. The measures are contextualized by outlining necessary framework conditions and success factors, thus promoting transfer to other companies and sectors of the economy., (© The Author(s) 2021.)

Fibronectin is a large multidomain protein of the extracellular matrix that harbors two heparin binding sites, Hep-I and Hep-II, which support the heparin-dependent adhesion of melanoma and neuroblastoma cells [Barkalow, F. J. B., and Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818; McCarthy, J. B., et al. (1988) Biochemistry 27, 1380-1388; Drake, S. L., et al. (1993) J. Biol. Chem. 268, 15859-15867]. The stronger heparin/HS binding site on fibronectin, Hep-II, spans fibronectin type III domains 12-14. Previous site-directed mutagenesis, nuclear magnetic resonance (NMR) chemical shift perturbation, and crystallographic structural studies all agree that the main heparin binding site is located on the surface of fibronectin type III domain 13 [Ingham, K. C., et al. (1993) Biochemistry 32, 12548-12553; Sharma, A., et al. (1999) EMBO J. 18, 1468-1479; Sachchidanand, L. O., et al. (2002) J. Biol. Chem. 277, 50629-50635]. However, the "synergy site" for heparin binding located on fibronectin type III domain 14 remained elusive because the actual binding sites could not be identified. Using NMR spectroscopy and isothermal titration calorimetry, we show here that heparin is able to bind to a cationic 'cradle' of fibronectin type III domain 14 formed by the PRARI sequence, which is involved in the integrin α 4 β 1 interaction [Mould, A. P., and Humphries, M. J. (1991) EMBO J. 10, 4089-4095], and to the flexible loop comprising residues KNNQKSE between the last two β-strands, D and E, of FN14. Our data reveal that the individual FN14 domain binds to the sulfated sugars Dp8 and Reviparin with affinities similar to those of the individual domain FN13 [Breddin, H. K. (2002) Expert Opin. Pharmacother. 3, 173-182]. It is noteworthy that by introduction of the last β-strand of FN13 and the linker region between FN type III domains 13 and 14, the perturbation of NMR chemical shifts by heparin is significantly reduced, especially at the PRARI site. This indicates that the Hep-II binding site of fibronectin is mainly located on FN13 and the synergistic binding site on FN14 involves only the KNNQKSE sequence.

Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, represents a large multi-domain non-transmembrane scaffolding protein that contains five consecutive PDZ domains. Here, we report the solution structures of the extended murine PTPN13 PDZ3 domain in its apo form and in complex with its physiological ligand, the carboxy-terminus of protein kinase C-related kinase-2 (PRK2), determined by multidimensional NMR spectroscopy. Both in its ligand-free state and when complexed to PRK2, PDZ3 of PTPN13 adopts the classical compact, globular D/E fold. PDZ3 of PTPN13 binds five carboxy-terminal amino acids of PRK2 via a groove located between the EB-strand and the DB-helix. The PRK2 peptide resides in the canonical PDZ3 binding cleft in an elongated manner and the amino acid side chains in position P0 and P-2, cysteine and aspartate, of the ligand face the groove between EB-strand and DB-helix, whereas the PRK2 side chains of tryptophan and alanine located in position P-1 and P-3 point away from the binding cleft. These structures are rare examples of selective class III ligand recognition by a PDZ domain and now provide a basis for the detailed structural investigation of the promiscuous interaction between the PDZ domains of PTPN13 and their ligands. They will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTPN13 and could ultimately contribute to low molecular weight antagonists that might even act on the PRK2 signaling pathway to modulate rearrangements of the actin cytoskeleton., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The present study investigated the influence of mood state (positive vs. negative) on the cognitive control process of conflict adaptation. A task-switching paradigm was applied, allowing to assess conflict adaptation both within tasks and across tasks. A success-failure manipulation was applied for mood induction. Within-task conflict adaptation tended to be larger in negative mood than in positive mood, in line with previous findings in the literature. Across-task conflict adaptation was also observed, but only in the positive mood group: Participants in positive mood showed larger conflict adaptation than participants in negative mood. We suggest that different cognitive mechanisms underlie this double dissociation: Within-task conflict adaptation is enhanced in negative mood because response conflict is aversive in nature, and is therefore congruent to negative mood states. Between-task conflict adaptation is only present in positive mood because of less distinct task representations under positive mood, leading conflict adaptation to transfer to the other task. The data show that influences of mood state on cognitive control are multifaceted, with some control processes being enhanced and others attenuated in negative relative to positive mood. (PsycINFO Database Record, ((c) 2018 APA, all rights reserved).)

Collagen is the predominant protein in animal connective tissues and is widely used in tissue regeneration and other industrial applications. Marine organisms have gained interest as alternative, nonmammalian collagen sources for biomaterial applications because of potential medical and economic advantages. In this work, we present physicochemical and biofunctionality studies of acid solubilized collagen (ASC) from jellyfish Catostylus mosaicus (JASC), harvested from the Persian Gulf, compared with ASC from rat tail tendon (RASC), the industry-standard collagen used for biomedical research. From the protein subunit (alpha chain) pattern of JASC, we identified it as a type I collagen, and extensive molecular spectroscopic analyses showed similar triple helical molecular signatures for JASC and RASC. Atomic force microscopy of fibrillized JASC showed clear fibril reassembly upon pH neutralization though with different temperature and concentration dependence compared with RASC. Molecular (natively folded, nonfibrillized) JASC was shown to functionalize rigid substrates and promote MC3T3 preosteoblast cell attachment and proliferation better than RASC over 6 days. On blended collagen-agarose scaffolds, both RASC and JASC fibrils supported cell attachment and proliferation, and scaffolds with RASC fibrils showed more cell growth after 6 days compared with those scaffolds with JASC fibrils. These results demonstrate the potential for this new type I collagen as a possible alternative to mammalian type I collagen for biomaterial applications.

Cell-penetrating peptides (CPPs) are short peptide sequences that can translocate across cellular plasma membranes and are thus potential delivery vectors for diagnostic and therapeutic applications. Many CPPs exhibit some sort of structural polymorphism, where the secondary structure of the peptide is altered strongly by its local environment, which is believed to facilitate membrane translocation and uptake. However, much less is known about the fate and structure of CPPs within cells largely due to measurement difficulty. Here we employ isotopic labeling combined with hyperspectral, quantitative coherent Raman microscopy to localize a model CPP-penetratin-and determine its secondary structure in different cellular compartments. Our results show that penetratin is mostly α-helical in the cytosol and acquires a more β-sheet and random coil character in the nucleus. The increased helicity in the cytosol is similar to that seen in previous studies with model lipid membranes, suggesting that the peptide is associated with membranes in, e.g., endosomes (or lysosomes) in the cytosol. The ability to both localize and determine the secondary structure of a CPP within cells is critical for clarifying the mechanism of peptide-mediated translocation and delivery of cargo molecules to specific cellular destinations.

Deforestation in the tropics is not only responsible for direct carbon emissions but also extends the forest edge wherein trees suffer increased mortality. Here we combine high-resolution (30 m) satellite maps of forest cover with estimates of the edge effect and show that 19% of the remaining area of tropical forests lies within 100 m of a forest edge. The tropics house around 50 million forest fragments and the length of the world's tropical forest edges sums to nearly 50 million km. Edge effects in tropical forests have caused an additional 10.3 Gt (2.1-14.4 Gt) of carbon emissions, which translates into 0.34 Gt per year and represents 31% of the currently estimated annual carbon releases due to tropical deforestation. Fragmentation substantially augments carbon emissions from tropical forests and must be taken into account when analysing the role of vegetation in the global carbon cycle.

Aging causes major alterations of all components of the neurovascular unit and compromises brain blood supply. Here, we tested how aging affects vascular reactivity in basilar arteries from young (<10 weeks; y-BA), old (>22 months; o-BA) and old (>22 months) heterozygous MYPT1-T-696A/+ knock-in mice. In isometrically mounted o-BA, media thickness was increased by ∼10% while the passive length tension relations were not altered. Endothelial denudation or pan-NOS inhibition (100 µmol/L L-NAME) increased the basal tone by 11% in y-BA and 23% in o-BA, while inhibition of nNOS (1 µmol/L L-NPA) induced ∼10% increase in both ages. eNOS expression was ∼2-fold higher in o-BA. In o-BA, U46619-induced force was augmented (pEC 50 ∼6.9 vs. pEC 50 ∼6.5) while responsiveness to DEA-NONOate, electrical field stimulation or nicotine was decreased. Basal phosphorylation of MLC 20 -S19 and MYPT1-T-853 was higher in o-BA and was reversed by apocynin. Furthermore, permeabilized o-BA showed enhanced Ca 2+ -sensitivity. Old T-696A/+ BA displayed a reduced phosphorylation of MYPT1-T696 and MLC 20 , a lower basal tone in response to L-NAME and a reduced eNOS expression. The results indicate that the vascular hypercontractility found in o-BA is mediated by inhibition of MLCP and is partially compensated by an upregulation of endothelial NO release.

The Melanoma Inhibitory Activity (MIA) protein is strongly expressed and secreted by malignant melanoma cells and was shown to promote melanoma development and invasion. The MIA protein was the first extracellular protein shown to adopt an Src homology 3 (SH3) domain-like fold in solution that can bind to fibronectin type III domains. Together with MIA, the homologous proteins OTOR (or FDP), MIA-2, and TANGO (or MIA-3) constitute a protein family of non-cytosolic and - except for fulllength TANGO and TANGO1-like (TALI) - extracellular SH3-domain containing proteins. Members of this protein family modulate collagen maturation and export, cartilage development, cell attachment in the extracellular matrix, and melanoma metastasis. These proteins may thus serve as promising targets for drug development against malignant melanoma. For the last twenty years, NMR spectroscopy has become a powerful technique in medicinal chemistry. While traditional high throughput screenings only report on the activity or affinity of low molecular weight compounds, NMR spectroscopy does not only relate to the structure of those compounds with their activity, but it can also unravel structural information on the ligand binding site on the protein at atomic resolution. Based on the molecular details of the interaction between the ligand and its target protein, the binding affinities of initial fragment hits can be further improved more efficiently in order to generate lead structures that exhibit significant therapeutic effects. The NMR-based approach promises to greatly contribute to the quest for low molecular weight compounds that ultimately could yield drugs to treat skin-related diseases such as malignant melanoma more effectively., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson`s disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Melanoma inhibitory activity (MIA), an extracellular protein highly expressed by malignant melanoma cells, plays an important functional role in melanoma development, progression, and metastasis. After its secretion, MIA directly interacts with extracellular matrix proteins, such as fibronectin (FN). By this mechanism, MIA actively facilitates focal cell detachment from surrounding structures and strongly promotes tumour cell invasion and migration. Hence, the molecular understanding of MIA's function provides a promising target for the development of new strategies in malignant melanoma therapy. Here, we describe for the first time the discovery of small molecules that are able to disrupt the MIA-FN complex by selectively binding to a new druggable pocket, which we could identify on MIA by structural analysis and fragment-based screening. Our findings may inspire novel drug discovery efforts aiming at a therapeutically effective treatment of melanoma by targeting MIA.

Tropical forests play an important role in the global carbon cycle, as they store a large amount of carbon (C). Tropical forest deforestation has been identified as a major source of CO2 emissions, though biomass loss due to fragmentation--the creation of additional forest edges--has been largely overlooked as an additional CO2 source. Here, through the combination of remote sensing and knowledge on ecological processes, we present long-term carbon loss estimates due to fragmentation of Neotropical forests: within 10 years the Brazilian Atlantic Forest has lost 69 (±14) Tg C, and the Amazon 599 (±120) Tg C due to fragmentation alone. For all tropical forests, we estimate emissions up to 0.2 Pg C y(-1) or 9 to 24% of the annual global C loss due to deforestation. In conclusion, tropical forest fragmentation increases carbon loss and should be accounted for when attempting to understand the role of vegetation in the global carbon balance.

Landscape simulators are widely applied in landscape ecology for generating landscape patterns. These models can be divided into two categories: pattern-based models that generate spatial patterns irrespective of the processes that shape them, and process-based models that attempt to generate patterns based on the processes that shape them. The latter often tend toward complexity in an attempt to obtain high predictive precision, but are rarely used for generic or theoretical purposes. Here we show that a simple process-based simulator can generate a variety of spatial patterns including realistic ones, typifying landscapes fragmented by anthropogenic activities. The model "G-RaFFe" generates roads and fields to reproduce the processes in which forests are converted into arable lands. For a selected level of habitat cover, three factors dominate its outcomes: the number of roads (accessibility), maximum field size (accounting for land ownership patterns), and maximum field disconnection (which enables field to be detached from roads). We compared the performance of G-RaFFe to three other models: Simmap (neutral model), Qrule (fractal-based) and Dinamica EGO (with 4 model versions differing in complexity). A PCA-based analysis indicated G-RaFFe and Dinamica version 4 (most complex) to perform best in matching realistic spatial patterns, but an alternative analysis which considers model variability identified G-RaFFe and Qrule as performing best. We also found model performance to be affected by habitat cover and the actual land-uses, the latter reflecting on land ownership patterns. We suggest that simple process-based generators such as G-RaFFe can be used to generate spatial patterns as templates for theoretical analyses, as well as for gaining better understanding of the relation between spatial processes and patterns. We suggest caution in applying neutral or fractal-based approaches, since spatial patterns that typify anthropogenic landscapes are often non-fractal in nature.

The antibacterial (bioactive) and antifouling (biopassive) properties of stable, uniform, high surface coverage films of poly(hydroxyethyl methacrylate-co-2-methacryloyloxyethyl phosphorylcholine) (p(HEMA-co-MPC)) with embedded, non-leaching silver nanoparticles (AgNPs) are reported. Based on the experimental findings, a mechanism of action of AgNPs in antibacterial activity in combination with antifouling characteristics is discussed. Long-term antifouling studies of E. coli determine little to no adhesion on p(HEMA-co-MPC)/Ag films at 2.5 × 10 6 CFU mL -1 for 7 d, measured using live/dead staining assays. Agar diffusion tests indicate that there is no leaching of Ag from the films and SEM and EDX analyses of the films before and after incubation with E. coli show no attachment of E. coli and no visible change in film morphology or AgNP dispersal. Antibacterial studies are investigated using E. coli K-12 as a model bacterial strain and are tested in static (CFUs) and dynamic contact assays. Antibacterial efficacy of the films containing extremely low AgNP concentration (3.8 ng cm -2 ) is shown with growth suppression of E. coli in culture medium for 4 h at 1.35 × 10 5 CFU mL -1 and killing greater than 99% of E. coli in only 1 h of exposure to concentrations up to 1 × 10 5 CFU mL -1 . These hybrid films may propose an exciting direction to long-term antibacterial and antifouling films in clinical applications.

Tethered lipid membranes or immobilized lipid vesicles are frequently used as biomimetic systems. In this article, the authors presented a suitable method for efficient immobilization of lipid vesicles onto a broad range of surfaces, enabling analysis by quantitative methods even under rigid, mechanical conditions-bare surfaces such as hydrophilic glass surfaces as well as hydrophobic polymer slides or metal surfaces such as gold. The immobilization of vesicles was based on the electrostatic interaction of zwitterionic or negatively charged lipid vesicles with two types of cationic chemically modified bovine serum albumin (cBSA) blood plasma proteins (cBSA-113 and cBSA-147). Quantitative analysis of protein adsorption was performed as the cBSA coatings were characterized by atomic force microscopy, surface zeta potential measurement, fluorescence microscopy, and surface plasmon spectroscopy, revealing a maximal surface coverage 270-280 ng/cm(2) for 0.02 mg/ml cBSA on gold. Small unilamellar vesicles as well as giant unilamellar vesicles (GUVs) were readily immobilized (∼15 min) on cBSA coated surfaces. GUVs with 5-10 mol% negatively charged 1,2,-dipalmitoyl-sn-glycero-3-phosphoglycerol remained stable in liquid for at least 5 weeks.

Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into beta-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into beta-alanine and histamine by Tan activity. Tan thus provides a key function in the recycling pathway of the neurotransmitter histamine. It is also involved in the process of cuticle formation. There, it cleaves beta-alanyl-dopamine, a major component in cuticle sclerotization. Active Tan enzyme is generated by a self-processing proteolytic cleavage from a pre-protein at a conserved Gly-Cys sequence motif. We confirmed the dependence on the Gly-Cys motif by in vitro mutagenesis. Processing time delays the rise to full Tan activity up to 3 h behind its putative circadian RNA expression in head. To investigate its pleiotropic functions, we have expressed Tan as a His(6) fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His(6)-Tan protein co-migrating in size exclusion chromatography with a molecular weight compatible with homodimer formation. We conclude that dimer formation is preceding pre-protein processing. Drosophila tan(1) null mutant analysis revealed that amino acid Arg(217) is absolutely required for processing. Substitution of Met(256) in tan(5), on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His(6)-Tan(5) retains activity. Kinetic parameters of Tan reveal characteristic differences in K(m) and k(cat) values of carcinine and beta-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.

Background: Mass spectrometry based quantification of peptides can be performed using the iTRAQ reagent in conjunction with mass spectrometry. This technology yields information about the relative abundance of single peptides. A method for the calculation of reliable quantification information is required in order to obtain biologically relevant data at the protein expression level., Results: A method comprising sound error estimation and statistical methods is presented that allows precise abundance analysis plus error calculation at the peptide as well as at the protein level. This yields the relevant information that is required for quantitative proteomics. Comparing the performance of our method named Quant with existing approaches the error estimation is reliable and offers information for precise bioinformatic models. Quant is shown to generate results that are consistent with those produced by ProQuant, thus validating both systems. Moreover, the results are consistent with that of Mascot 2.2. The MATLAB scripts of Quant are freely available via http://www.protein-ms.de and http://sourceforge.net/projects/protms/, each under the GNU Lesser General Public License., Conclusion: The software Quant demonstrates improvements in protein quantification using iTRAQ. Precise quantification data can be obtained at the protein level when using error propagation and adequate visualization. Quant integrates both and additionally provides the possibility to obtain more reliable results by calculation of wise quality measures. Peak area integration has been replaced by sum of intensities, yielding more reliable quantification results. Additionally, Quant allows the combination of quantitative information obtained by iTRAQ with peptide and protein identifications from popular tandem MS identification tools. Hence Quant is a useful tool for the proteomics community and may help improving analysis of proteomic experimental data. In addition, we have shown that a lognormal distribution fits the data of mass spectrometry based relative peptide quantification.