Inside Cover: Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs (J. Biophotonics 5/2018)

Optical tissue clearing is a method allowing post‐mortem deep imaging of organs in three dimensions. By optimizing the CUBIC clearing protocol, the authors provide rapid and simple approach to clear the entire adult rat organism within as little as four days, which is accompanied by the variety of its staining and imaging techniques. The image was captured with polarizers and demonstrates transparent rodent heart with thread‐like crystals of clearing reagent. Further details can be found in the article by Pawe? Matryba et al. ( e201700248 ).

     

Stabilization of a prokaryotic LAT transporter by random mutagenesis

The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis l-serine/l-threonine exchanger is the best-known prokaryotic paradigm of the mammalian l–amino acid transporter (LAT) family. Unfortunately, SteT’s lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.     

Structural and Functional Analyses Reveal Insights into the Molecular Properties of the Escherichia coli Z Ring Stabilizing Protein,ZapC

In Escherichia coli cell division is driven by the tubulin-like GTPase, FtsZ, which forms the cytokinetic Z-ring. The Z-ring serves as a dynamic platform for the assembly of the multiprotein divisome, which catalyzes membrane cleavage to create equal daughter cells. Several proteins effect FtsZ assembly, thereby providing spatiotemporal control over cell division. One important class of FtsZ interacting/regulatory proteins is the Z-ring-associated proteins, Zaps, which typically modulate Z-ring formation by increasing lateral interactions between FtsZ protofilaments. Strikingly, these Zap proteins show no discernable sequence similarity, suggesting that they likely harbor distinct structures and mechanisms. The 19.8-kDa ZapC in particular shows no homology to any known protein. To gain insight into ZapC function, we determined its structure to 2.15 Å and performed genetic and biochemical studies. ZapC is a monomer composed of two domains, an N-terminal α/β region and a C-terminal twisted β barrel-like domain. The structure contains two pockets, one on each domain. The N-domain pocket is lined with residues previously implicated to be important for ZapC function as an FtsZ bundler. The adjacent C-domain pocket contains a hydrophobic center surrounded by conserved basic residues. Mutagenesis analyses indicate that this pocket is critical for FtsZ binding. An extensive FtsZ binding surface is consistent with the fact that, unlike many FtsZ regulators, ZapC binds the large FtsZ globular core rather than C-terminal tail, and the presence of two adjacent pockets suggests possible mechanisms for ZapC-mediated FtsZ bundling.     

Prevalence and species distribution of the low-complexity,amyloid-like,reversible, kinked segment structural motif in amyloid-like fibrils

Membraneless organelles (MLOs) are vital and dynamic reaction centers in cells that compartmentalize the cytoplasm in the absence of a membrane. Multivalent interactions between protein low-complexity domains contribute to MLO organization. Previously, we used computational methods to identify structural motifs termed low-complexity amyloid-like reversible kinked segments (LARKS) that promote phase transition to form hydrogels and that are common in human proteins that participate in MLOs. Here, we searched for LARKS in the proteomes of six model organisms: Homo sapiens, Drosophila melanogaster, Plasmodium falciparum, Saccharomyces cerevisiae, Mycobacterium tuberculosis, and Escherichia coli to gain an understanding of the distribution of LARKS in the proteomes of various species. We found that LARKS are abundant in M. tuberculosis, D. melanogaster, and H. sapiens but not in S. cerevisiae or P. falciparum. LARKS have high glycine content, which enables kinks to form as exemplified by the known LARKS-rich amyloidogenic structures of TDP43, FUS, and hnRNPA2, three proteins that are known to participate in MLOs. These results support the idea of LARKS as an evolved structural motif. Based on these results, we also established the LARKSdb Web server, which permits users to search for LARKS in their protein sequences of interest.     

Removal of naturally grown human biofilm with an atmospheric pressure plasma jet: An in‐vitro study

The removal of biofilm is a prerequisite for a successful treatment of biofilm‐associated diseases. In this study, we compared the feasibility of an atmospheric pressure plasma device with a sonic powered brush to remove naturally grown supragingival biofilm from extracted teeth. Twenty‐four periodontally hopeless teeth were extracted. Argon jet plasma with an oxygen admixture of 1 vol% and a sonically driven brush were used to remove biofilm with application times of 60 s, 180 s and 300 s. The treatment efficiency was assessed with light microscopy, scanning electron microscopy (SEM) and X‐ray photoelectron spectroscopy (XPS). The highest biofilm removal rate was observed after an application time of 180 s/300 s with the sonic brush (80.4%/86.2%), plasma (75.5%/89.0%). These observations were confirmed by SEM. According to XPS analysis, plasma treatment decreased the amount of carbon and nitrogen, indicative of an extensive removal of proteins. Plasma treatment of naturally grown biofilm resulted in an effective cleaning of the tooth surface and was comparable to mechanical treatment. Treatment time had a significant influence on plaque reduction. These results showed that plasma could be a useful adjuvant treatment modality in cases where biofilm removal or reduction plays a decisive role, such as periodontitis and peri‐implantitis.

Plasma‐treated biofilm on an extracted tooth.     

Photoactive modified chitosan

A water-soluble polymeric photosensitizer that contains naphthyl chromophores and absorbs light in the near UV region was obtained by modification of chitosan. The excitation energy can be used to induce photochemical reactions via energy and electron transfer.     

Never born proteins as a test case for ab initio protein structures prediction

The number of natural proteins although large is significantly smaller than the theoretical number of proteins that can be obtained combining the 20 natural amino acids, the so-called “never born proteins” (NBPs). The study of the structure and properties of these proteins allows to investigate the sources of the natural proteins being of unique characteristics or special properties. However the structural study of NPBs can also been intended as an ideal test for evaluating the efficiency of software packages for the ab initio protein structure prediction. In this research, 10.000 three-dimensional structures of proteins of completely random sequence generated according to ROSETTA and FOD model were compared. The results show the limits of these software packages, but at the same time indicate that in many cases there is a significant agreement between the prediction obtained.     

The dynamics of health in wild field vole populations: a haematological perspective

1. Pathogens have been proposed as potentially important drivers of population dynamics, but while a few studies have investigated the impact of specific pathogens, the wealth of information provided by general indices of health has hardly been exploited. By evaluating haematological parameters in wild populations, our knowledge of the dynamics of health and infection may be better understood. 2. Here, haematological dynamics in natural populations of field voles are investigated to determine environmental and host factors associated with indicators of inflammatory response (counts of monocytes and neutrophils) and of condition: measures of immunological investment (lymphocyte counts) and aerobic capacity (red blood cell counts). 3. Individuals from three field vole populations were sampled monthly for 2 years. Comparisons with individuals kept under controlled conditions facilitated interpretation of field data. Mixed effects models were developed for each cell type to evaluate separately the effects of various factors on post-juvenile voles and mature breeding females. 4. There were three well-characterized 'physiological' seasons. The immunological investment appeared lowest in winter (lowest lymphocyte counts), but red blood cells were at their highest levels and indices of inflammatory response at their lowest. Spring was characterized by a fall in red blood cell counts and peaks in indicators of inflammatory response. During the course of summer-autumn, red blood cell counts recovered, the immunological investment increased and the indicators of inflammatory response decreased. 5. Poor body condition appeared to affect the inflammatory response (lower neutrophil and monocyte peaks) and the immunological investment (lower lymphocyte counts), providing evidence that the capacity to fight infection is dependent upon host condition. 6. Breeding early in the year was most likely in females in better condition (high lymphocyte and red blood cell counts). 7. All the haematological parameters were affected adversely by high population densities.