This paper contributes to the public and academic debate on the appropriateness of young Westerners' participation in projects of volunteer tourism conducted in developing countries. Ethnographic ...research was carried out in the context of an Australian program that organizes short-term group placements for university students in countries like Vietnam, Mexico and Fiji. The results illustrate that such projects can produce similar benefits to other educational initiatives of international volunteering and service (IVS) in terms of global engagement, career development, intercultural competence and psychological support. However, for these projects to avoid public critiques and negative outcomes, they need to harmonize personal and institutional expectations with real volunteer capacities. Thus, until IVS programs in the university context distance themselves from a development aid discourse, they will potentially fall under the umbrella of "neo-colonialism". The research provides a model of impact analysis and raises challenging questions for universities or similar organizations involved with short-term group placements of volunteer tourism.
Finding new enzyme variants with the desired substrate scope requires screening through a large number of potential variants. In a typical in silico enzyme engineering workflow, it is possible to ...scan a few thousands of variants, and gather several candidates for further screening or experimental verification. In this work, we show that a Graph Convolutional Neural Network (GCN) can be trained to predict the binding energy of combinatorial libraries of enzyme complexes using only sequence information. The GCN model uses a stack of message-passing and graph pooling layers to extract information from the protein input graph and yield a prediction. The GCN model is agnostic to the identity of the ligand, which is kept constant within the mutant libraries. Using a miniscule subset of the total combinatorial space (204–208 mutants) as training data, the proposed GCN model achieves a high accuracy in predicting the binding energy of unseen variants. The network’s accuracy was further improved by injecting feature embeddings obtained from a language module pretrained on 10 million protein sequences. Since no structural information is needed to evaluate new variants, the deep learning algorithm is capable of scoring an enzyme variant in under 1 ms, allowing the search of billions of candidates on a single GPU.
Abstract
The detailed understanding of the binding of small molecules to proteins is the key for the development of novel drugs or to increase the acceptance of substrates by enzymes. Nowadays, ...computer-aided design of protein–ligand binding is an important tool to accomplish this task. Current approaches typically rely on high-throughput docking essays or computationally expensive atomistic molecular dynamics simulations. Here, we present an approach to use the recently re-parametrized coarse-grained Martini model to perform unbiased millisecond sampling of protein–ligand interactions of small drug-like molecules. Remarkably, we achieve high accuracy without the need of any a priori knowledge of binding pockets or pathways. Our approach is applied to a range of systems from the well-characterized T4 lysozyme over members of the GPCR family and nuclear receptors to a variety of enzymes. The presented results open the way to high-throughput screening of ligand libraries or protein mutations using the coarse-grained Martini model.
The present study evaluates the influence of continuous light on phenotypic sex ratios in Chirostoma estor, a temperature sex determination animal model. Relative gene expression levels of 5 day old ...larvae were performed on two early gonad differentiation genes (sox9 and foxl2), two stress axis activation genes (gcr1 and crf) and four reactive oxygen species (ROS) antagonist effector genes (sod2, ucp2, gsr and cat). Two light treatments were applied from fertilization; control (12L:12D) simulated natural photoperiod and a continuous illumination photoperiod. By the end of the trial (12 weeks after hatching), differentiated and normal gonads were clearly identifiable in both treatments by histological observations. Regarding sex ratio, 73% of phenotypic males were found in continuous illumination compared with 40% in controls. Consistently, the sox9 gene (involved in early testis differentiation) showed an over expression in 64% of the individual larvae analysed compared with foxl2 (ovarian differentiation) suggesting a masculinization tendency in continuous illumination. On the other hand, only 36% of individuals showed the same tendency in the control treatment consistent with phenotypic sex ratios found under normal culture conditions. Relative gene expression results did not show significant difference in sod2, ucp2 and gcr1 levels, but cat, gsr and crf showed significantly higher expression levels in the continuous illumination treatment suggesting that both, the stress axis and ROS response mechanisms were activated at this time. This study suggests, a link between continuous light, oxidative stress and environmental sex determination in vertebrates. However, further research is necessary to describe this possible upstream mechanism that may drive some aspects of sexual plasticity in vertebrates.
The co-localization of Cluster-of-Differentiation-44 protein (CD44) and cytoplasmic adaptors in specific membrane environments is crucial for cell adhesion and migration. The process is controlled by ...two different pathways: On the one hand palmitoylation keeps CD44 in lipid raft domains and disables the linking to the cytoplasmic adaptor, whereas on the other hand, the presence of phosphatidylinositol-4,5-biphosphate (PIP2) lipids accelerates the formation of the CD44-adaptor complex. The molecular mechanism explaining how CD44 is migrating into and out of the lipid raft domains and its dependence on both palmitoylations and the presence of PIP2 remains, however, elusive. In this study, we performed extensive molecular dynamics simulations to study the raft affinity and translocation of CD44 in phase separated model membranes as well as more realistic plasma membrane environments. We observe a delicate balance between the influence of the palmitoylations and the presence of PIP2 lipids: whereas the palmitoylations of CD44 increases the affinity for raft domains, PIP2 lipids have the opposite effect. Additionally, we studied the association between CD44 and the membrane adaptor FERM in dependence of these factors. We find that the presence of PIP2 lipids allows CD44 and FERM to associate in an experimentally observed binding mode whereas the highly palmitoylated species shows no binding affinity. Together, our results shed light on the sophisticated mechanism on how membrane translocation and peripheral protein association can be controlled by both protein modifications and membrane composition.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BACKGROUNDRecently, serum soluble urokinase receptor (suPAR) has been proposed as a cause of two thirds of cases of focal segmental glomerulosclerosis (FSGS). It was noted to be uniquely elevated in ...cases of primary FSGS, with higher levels noted in cases that recurred after transplantation. It is also suggested as a possible target and marker of therapy.
METHODSWe studied serum and urine suPAR from pretransplantation banked samples from 86 well-characterized kidney transplant recipients and 10 healthy controls to determine its prognostic utility. Causes of native kidney disease were primary FSGS, diabetic nephropathy, membranous nephropathy, immunoglobulin A nephropathy, and autosomal dominant polycystic kidney disease. suPAR was measured using a commercially available enzyme-linked immunosorbent assay kit. Urinary suPAR was indexed to creatinine.
RESULTSBoth serum and urine suPAR correlated with proteinuria and albuminuria. Serum suPAR was found to be elevated in all transplant candidates with advanced renal disease compared with healthy controls and could not differentiate disease diagnosis. Urine suPAR was elevated in cases of recurrent FSGS compared with all other causes of end-stage renal disease. Recurrent FSGS cases had substantially higher proteinuria compared with all other cases. However, elevated urinary suPAR showed a trend in providing additional prognostic information beyond proteinuria in the small cohort of recurrent FSGS cases.
CONCLUSIONIn advanced renal disease, elevated serum suPAR is not unique to FSGS cases. Urinary suPAR appears to be higher in cases of FSGS destined for recurrence and merits further evaluation.
ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral ...amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.
In this study, we looked at the population dynamics of a two phages-one host system using phages vB_EcoP_SU10 (SU10) and vB_EcoD_SU57 (SU57) and the bacteria
, strain ECOR57. Phage-specific growth ...curves were observed where infections by SU10 resulted in a moderate production of phages and infections by SU57 resulted in a fast and extensive production of phage progeny. Sequentially adding SU10 followed by SU57 did not produce a significant change in growth rates, whereas adding SU57 followed by SU10 resulted in a decrease in SU10 titer The efficiency of the plating assays showed that ECOR57 exhibited a resistance spectrum after infection by both the single and combined phages. Phage-resistant bacteria exhibited four different morphotypes (i.e., normal, slimy, edgy, and pointy). The normal and edgy morphotypes had a high frequency of developing resistance. Bacterial growth and biofilm assays indicated that the edgy and pointy morphotypes reached a stationary phase faster and produced more biofilm compared to the wild type. These findings suggest that the dynamic structure of phage-bacteria communities dictate resistance evolution and development. Understanding when and how resistances arise and phage(s)-hosts interactions could aid in the design of phage therapy treatments.
ω-Transaminases (ω-TA) are attractive biocatalysts for the production of chiral amines from prochiral ketones via asymmetric synthesis. However, the substrate scope of ω-TAs is usually limited due to ...steric hindrance at the active site pockets. We explored a protein engineering strategy using computational design to expand the substrate scope of an (S)-selective ω-TA from Pseudomonas jessenii (PjTA-R6) toward the production of bulky amines. PjTA-R6 is attractive for use in applied biocatalysis due to its thermostability, tolerance to organic solvents, and acceptance of high concentrations of isopropylamine as amino donor. PjTA-R6 showed no detectable activity for the synthesis of six bicyclic or bulky amines targeted in this study. Six small libraries composed of 7–18 variants each were separately designed via computational methods and tested in the laboratory for ketone to amine conversion. In each library, the vast majority of the variants displayed the desired activity, and of the 40 different designs, 38 produced the target amine in good yield with >99% enantiomeric excess. This shows that the substrate scope and enantioselectivity of PjTA mutants could be predicted in silico with high accuracy. The single mutant W58G showed the best performance in the synthesis of five structurally similar bulky amines containing the indan and tetralin moieties. The best variant for the other bulky amine, 1-phenylbutylamine, was the triple mutant W58M + F86L + R417L, indicating that Trp58 is a key residue in the large binding pocket for PjTA-R6 redesign. Crystal structures of the two best variants confirmed the computationally predicted structures. The results show that computational design can be an efficient approach to rapidly expand the substrate scope of ω-TAs to produce enantiopure bulky amines.
We have developed a continuous wave sub-wavelength terahertz (THz) imaging system that combines two prominent classical optical techniques: solid immersion microscopy and interferometric detection. ...This combination allows for resolution beyond the diffraction limit at 703 GHz. We experimentally demonstrate sub-wavelength spatial resolution working with a relatively low-cost pyroelectric detector and with both high and low contrast samples.
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