The entire N-terminal half of TatC is involved in twin-arginine precursor binding

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.     

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.     

Basic Analysis of the Cerebrospinal Fluid: An Important Framework for Laboratory Diagnostics of the Impairment of the Central Nervous System

Laboratory analysis of basic cerebrospinal fluid (CSF) parameters is considered as essential for any CSF evaluation. It can provide rapidly very valuable information about the status of the central nervous system (CNS). Our retrospective study evaluated parameters of basic CSF analysis in cases of either infectious or non-infectious CNS involvement. Neutrophils are effector cells of innate immunity. Predominance of neutrophils was found in 98.2% of patients with purulent inflammation in CNS. Lymphocytes are cellular substrate of adaptive immunity. We found their predominance in 94.8% of patients with multiple sclerosis (MS), 66.7% of patients with tick-borne encephalitis (TBE), 92.2% of patients with neuroborreliosis, 83.3% of patients with inflammatory response with oxidative burst of macrophages in CNS and 75.0% of patients with malignant infiltration of meninges (MIM). The simultaneous assessment of aerobic and anaerobic metabolism in CSF using the coefficient of energy balance (KEB) allows us to specify the type of inflammation in CNS. We found predominantly aerobic metabolism (KEB > 28.0) in 100.0% CSF of patients with normal CSF findings and in 92.8% CSF of patients with MS. Predominant faintly anaerobic metabolism (28.0 > KEB > 20.0) in CSF was found in 71.8% patients with TBE and in 64.7% patients with neuroborreliosis. Strong anaerobic metabolism (KEB < 10.0) was found in the CSF of 99.1% patients with purulent inflammation, 100.0% patients with inflammatory response with oxidative burst of macrophages and in 80.6% patients with MIM. Joint evaluation of basic CSF parameters provides sufficient information about the immune response in the CSF compartment for rapid and reliable diagnosis of CNS involvement.     

3D (x-y-t) Raman imaging of tomato fruit cuticle: Microchemistry during development

The cuticle is a protective extracellular matrix that covers the above-ground epidermis of land plants. Here, we studied the cuticle of tomato (Solanum lycopersicum L.) fruits in situ using confocal Raman microscopy. Microsections from cuticles isolated at different developmental stages were scanned to visualize cuticle components with a spatial resolution of 342 nm by univariate and multivariate data analysis. Three main components, cutin, polysaccharides, and aromatics, were identified, with the latter exhibiting the strongest Raman scattering intensity. Phenolic acids and flavonoids were differentiated within the cuticle, and three schematic cuticle models were identified during development. Phenolic acids were found across the entire cuticle at the earliest stage of development, i.e. during the formation of the procuticle layer. Based on a mixture analysis with reference component spectra, the phenolic acids were identified as mainly esterified p-coumaric acid together with free p-hydroxybenzoic acid. During the cell expansion period of growth, phenolic acids accumulated in an outermost layer of the cuticle and in the middle region of the pegs. In these stages of development, cellulose and pectin were detected next to the inner cuticle region, close to the epidermal cell where flavonoid impregnation started during ripening. In the first ripening stage, chalconaringenin was observed, while methoxylated chalcones were chosen by the algorithm to fit the mature cuticle spectra. The colocation of carbohydrates, esterified p-coumaric acid, and methoxylated chalconaringenin suggests that the latter two link polysaccharide and cutin domains. Elucidating the different distribution of aromatics within the cuticle, suggests important functions: (1) overall impregnation conferring mechanical and thermal functions (2) the outermost phenolic acid layer displaying UV-B protection of the plant tissue.

Raman mapping and multivariate data analysis provide insights into the distribution of cutin, carbohydrates, and phenolics along cross sections of green and mature tomato fruit cuticles.     

The breakthrough paradox: How focusing on one form of innovation jeopardizes the advancement of science

Research needs a balance of risk‐taking in “breakthrough projects” and gradual progress. For building a sustainable knowledge base, it is indispensable to provide support for both. Subject Categories: Careers, Economics, Law & Politics, Science Policy & Publishing

Science is about venturing into the unknown to find unexpected insights and establish new knowledge. Increasingly, academic institutions and funding agencies such as the European Research Council (ERC) explicitly encourage and support scientists to foster risky and hopefully ground‐breaking research. Such incentives are important and have been greatly appreciated by the scientific community. However, the success of the ERC has had its downsides, as other actors in the funding ecosystem have adopted the ERC’s focus on “breakthrough science” and respective notions of scientific excellence. We argue that these tendencies are concerning since disruptive breakthrough innovation is not the only form of innovation in research. While continuous, gradual innovation is often taken for granted, it could become endangered in a research and funding ecosystem that places ever higher value on breakthrough science. This is problematic since, paradoxically, breakthrough potential in science builds on gradual innovation. If the value of gradual innovation is not better recognized, the potential for breakthrough innovation may well be stifled.

While continuous, gradual innovation is often taken for granted, it could become endangered in a research and funding ecosystem that places ever higher value on breakthrough science.

Concerns that the hypercompetitive dynamics of the current scientific system may impede rather than spur innovative research have been voiced for many years (Alberts et al, 2014). As performance indicators continue to play a central role for promotions and grants, researchers are under pressure to publish extensively, quickly, and preferably in high‐ranking journals (Burrows, 2012). These dynamics increase the risk of mental health issues among scientists (Jaremka et al, 2020), dis‐incentivise relevant and important work (Benedictus et al, 2016), decrease the quality of scientific papers (Sarewitz, 2016) and induce conservative and short‐term thinking rather than risk‐taking and original thinking required for scientific innovation (Alberts et al, 2014; Fochler et al, 2016). Against this background, strong incentives for fostering innovative and daring research are indispensable.     

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid‐encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β‐barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion‐conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco‐harboring pathogens.