A Flagellin-Derived Toll-Like Receptor 5 Agonist Stimulates Cytotoxic Lymphocyte-Mediated Tumor Immunity

Toll-like receptor (TLR) mediated recognition of pathogen associated molecular patterns allows the immune system to rapidly respond to a pathogenic insult. The “danger context” elicited by TLR agonists allows an initially non-immunogenic antigen to become immunogenic. This ability to alter environment is highly relevant in tumor immunity, since it is inherently difficult for the immune system to recognize host-derived tumors as immunogenic. However, immune cells may have encountered certain TLR ligands associated with tumor development, yet the endogenous stimulation is typically not sufficient to induce spontaneous tumor rejection. Of special interest are TLR5 agonists, because there are no endogenous ligands that bind TLR5. CBLB502 is a pharmacologically optimized TLR5 agonist derived from Salmonella enterica flagellin. We examined the effect of CBLB502 on tumor immunity using two syngeneic lymphoma models, both of which do not express TLR5, and thus do not directly respond to CBLB502. Upon challenge with the T-cell lymphoma RMAS, CBLB502 treatment after tumor inoculation protects C57BL/6 mice from death caused by tumor growth. This protective effect is both natural killer (NK) cell- and perforin-dependent. In addition, CBLB502 stimulates clearance of the B-cell lymphoma A20 in BALB/c mice in a CD8⁺ T cell-dependent fashion. Analysis on the cellular level via ImageStream flow cytometry reveals that CD11b⁺ and CD11c⁺ cells, but neither NK nor T cells, directly respond to CBLB502 as determined by NFκB nuclear translocation. Our findings demonstrate that CBLB502 stimulates a robust antitumor response by directly activating TLR5-expressing accessory immune cells, which in turn activate cytotoxic lymphocytes.     

Decreasing the Effective Thermal Conductivity in Glass Supported Thermoelectric Layers

As thermoelectric devices begin to make their way into commercial applications, the emphasis is put on decreasing the thermal conductivity. In this purely theoretical study, finite element analysis is used to determine the effect of a supporting material on the thermal conductivity of a thermoelectric module. The simulations illustrate the heat transfer along a sample, consisting from Cu, Cu₂O and PbTe thermoelectric layers on a 1 mm thick Pyrex glass substrate. The influence of two different types of heating, at a constant temperature and at a constant heat flux, is also investigated. It is revealed that the presence of a supporting material plays an important role on lowering the effective thermal conductivity of the layer-substrate ensemble. By using thinner thermoelectric layers the effective thermal conductivity is further reduced, almost down to the value of the glass substrate. As a result, the temperature gradient becomes steeper for a fixed heating temperature, which allows the production of devices with improved performance under certain conditions. Based on the simulation results, we also propose a model for a robust thin film thermoelectric device. With this suggestion, we invite the thermoelectric community to prove the applicability of the presented concept for practical purposes.     

Amide Proton Solvent Protection in Amylin Fibrils Probed by Quenched Hydrogen Exchange NMR

Amylin is an endocrine hormone that accumulates in amyloid plaques in patients with advanced type 2 diabetes. The amyloid plaques have been implicated in the destruction of pancreatic β-cells, which synthesize amylin and insulin. To better characterize the secondary structure of amylin in amyloid fibrils we assigned the NMR spectrum of the unfolded state in 95% DMSO and used a quenched hydrogen-deuterium exchange technique to look at amide proton solvent protection in the fibrils. In this technique, partially exchanged fibrils are dissolved in 95% DMSO and information about amide proton occupancy in the fibrils is determined from DMSO-denatured monomers. Hydrogen exchange lifetimes at pH 7.6 and 37°C vary between ∼5 h for the unstructured N-terminus to 600 h for amide protons in the two β-strands that form inter-molecular hydrogen bonds between amylin monomers along the length of the fibril. Based on the protection data we conclude that residues A8-H18 and I26-Y37 comprise the two β-strands in amylin fibrils. There is variation in protection within the β-strands, particularly for strand β1 where only residues F15-H18 are strongly protected. Differences in protection appear to be due to restrictions on backbone dynamics imposed by the packing of two-layers of C2-symmetry-related β-hairpins in the protofilament structure, with strand β1 positioned on the surface and β2 in the interior.     

Palaeoecological evidence from buried topsoils and colluvial layers at the Bronze Age fortification Corneşti-Iarcuri,SW Romania: results from palynological,sedimentological, chronostratigraphical and plant macrofossil analyses

Vegetation History and Archaeobotany - Located in the Romanian Banat region, the Late Bronze Age (LBA) fortification Corneşti-Iarcuri is the largest known prehistoric settlement in Europe....     

Rab35 controls formation of luminal projections required for bile canalicular morphogenesis

Hepatocytes display a unique biaxial polarity with shared apical luminal connections between adjacent hepatocytes that merge into a network of bile canaliculi. Belicova et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202103003) discovered that hepatocyte apical membranes generate Rab35-dependent extensions that traverse the lumen and are essential for bile canalicular formation and maintenance.

Many crucial functions of the hepatocyte, the predominant liver cell, are dependent on its unique “biaxial” polarity (1)—none more so than the generation and secretion of bile. Hepatocytes receive nutrients from venous blood via sinusoids and secrete bile into bile canaliculi (BC) that merge into larger ducts lined with cholangiocyte epithelial cells with simple “vectorial” polarity (1). Any disturbance in this complicated minute irrigation system can lead to severe and often fatal disease (2). Better understanding of the molecular mechanism of liver parenchymal morphogenesis will uncover therapeutic targets essential for developing effective treatments.Hepatocyte membrane molecular exchange is extremely efficient due to the highly expanded canalicular surface and transporter proteins specifically localized to canalicular or basolateral membranes. Moreover, each hepatocyte generates multiple apices by creating connections with several adjacent hepatocytes. BC-like structures can be mimicked when cells are cultured in 3D; hepatocyte apical connections can become confluent and form continuous BC channels (3). Several pathways have been proposed to play key roles in BC morphogenesis such as mitosis, cytokinesis and endosomal trafficking (4, 5, 6), nevertheless the mechanistic studies are lacking. Belicova and colleagues set out to investigate the basic mechanisms for BC formation (7).The authors generated a cell model of lumen morphogenesis by terminally differentiating primary mouse hepatoblasts isolated from embryonic livers into hepatocytes. The cultured differentiated cells were able to generate BC structures. Using time-lapse microscopy, it was possible to observe how single hepatocytes formed the initial individual lumina by interacting with neighboring cells. From a small point contact, the lumina elongated spanning the whole surface of the cell–cell interaction. Astonishingly, the authors detected in real-time how two lumina fused and thus created an elongated channel (Fig. 1 A). Furthermore, one lumen could branch forming a three-cell contact. At the same time, to their great surprise, they saw stripes being formed in a transverse direction to the lumen elongation. The stripes contained a high concentration of F-actin and were localized as projections into the BC lumen rather than formations outside of it.Open in a separate windowFigure 1.Rab35 impacts the BC formation and stability. (A) WT levels of Rab35 allow the formation of bulkheads in the BC lumen facilitating the hepatocyte biaxial polarity and elongation of BC between neighboring hepatocytes. The formed BC have a radii <6 µm and are not divided into discrete compartments by the bulkheads. (B) Reduction of Rab35 expression by RNA interference leads to bulkheads decreasing in size. This creates luminal bile duct–like structures with radii >6 µm surrounded by 4–5 conical shape hepatocytes. TJ, tight junctions.The authors then used EM and 3D reconstruction of serial sections to establish the nature of these newly discovered intracanalicular projections. The reconstructions showed that each cell participating in a luminal contact generated projections that met in the middle of the lumen and their connection was sealed by tight junctions (TJs) thus forming a structure that completely traversed the BC lumen. They hypothesized that such structure would provide additional tensile strength to the luminal wall just like bulkheads strengthen a boat shell against pressure. Belicova et al. found that the processes they named “bulkheads” had plate-/ridge-like 3D shapes and never completely isolated the BC compartments, thus allowing continuous bile flow along the canaliculus. To test whether bulkheads were present in vivo, they studied EM sections of embryonic day 15.5 (E15.5) and adult mouse livers as well as cultured primary mouse hepatocytes. The 3D reconstruction again showed the presence of bulkheads that originated from the opposing hepatocyte luminal membranes and their contact points sealed by the TJs.To understand whether bulkheads are essential for BC morphogenesis and the molecular mechanisms governing their formation, the authors performed an RNAi screen in their BC cell model. They selected 25 genes of potential interest due to their known involvement in hepatocyte polarity pathways such as apical junction formation, cytoskeleton regulation, and polarized trafficking. One of the identified phenotypes was seen to be of particular interest. The knockdown of small GTPase Rab35 did not affect cell polarity marker localization but changed lumen morphology (Fig. 1 B). Silencing of Rab35 produced elongated tubes with increased radius and large cysts that were still connected to the remaining BCs and their appearance resembled formations of cholangiocytes, although they did not express cholangiocyte marker Sox9. Reintroduction of Rab35 reduced the lumen to the original size.Live-cell time-lapse microscopy was used to investigate the function of Rab35 in BC morphogenesis. Rab35 is typically an endosomal protein, and in the liver cell model it was enriched at the apical surface, lateral plasma membrane, and cytoplasmic vesicles. Moreover, it was also present at the transverse connections within forming lumina. Upon Rab35 depletion, hepatocytes reshaped their apical surface, forming multicellular cysts lined by cells with vectorial polarity. This spherical expansion of the lumen was associated with the absence of actin-rich bulkheads.To test whether the cell model findings stay true in vivo, they targeted E13.5 mouse embryo livers, injecting lipid nanoparticle-coated siRNA against Rab35. 3D tissue reconstruction showed the changes in tissue architecture in siRNA-injected livers in striking detail. As predicted, large tubule formation was seen in contrast to the normal BCs seen in the mock-injected liver parenchyma. The 3D analysis demonstrated that the enlargement of the tubular lumina was not due to pure dilatation but the tissue reorganisation from typical biaxial polarity into a bile duct–like structure with vectorial polarity where multiple (4–5 in number) cells lined the single lumen (Fig. 1 B). Furthermore, the cells changed shape from a typical hepatocyte octagon to a conical profile while they still expressed the usual hepatocyte markers and not those typical of cholangiocytes.Bile canalicular morphogenesis requires anisotropy of surface tension and/or rigidity of the wall to progress from the initial point contact of apical surfaces of the two adjacent hepatocytes toward elongation, branching, and fusion with other canalicular lumina (8). The authors discovered that presence of transverse connections (bulkheads) was associated with maintenance of narrow lumen, which elongates under inner pressure. Thus, similar to their use in boatbuilding, BC bulkheads provide mechanical stability to the elongating BC lumen. It is possible that bulkheads could provide directionality to the bile flow in liver parenchyma. Positional location of the bulkheads may be determined by the sensing mechanisms coupled to the tension and curvature through actin mesh. Primary cilia are assigned a function in sensing directional flow and controlling tissue morphogenesis. Unlike cholangiocytes and many other epithelial cells, hepatocytes lack cilia and the molecular components of the tension sensing mechanism in hepatocytes remain to be determined.Belicova and colleagues showed that Rab35 is required for bulkhead formation and hepatocyte lumen shape. Rab35 was previously found to regulate endosomal recycling and thus it may control intracellular trafficking of proteins required for bulkhead formation, such as the components of TJs. It may also support the fusion of the two projections along the T-shaped TJs. Furthermore, the authors report that their unpublished observations suggest that clusters of Rab35-positive vesicles are seen at the base of the bulkhead, and it may coordinate trafficking and organize actin cytoskeleton or regulate actin remodelling as previously described in neurons (9).Elucidation of the mechanism of hepatocyte canalicular morphogenesis is one of the important research questions highly relevant to understanding the basic processes in both healthy and diseased liver. Liver cirrhosis was one of the top 10 causes of death in low- and middle-income countries in 2019 (10). Cirrhotic changes in the liver architecture include destruction of BC, and the new information provided by Belicova and colleagues may eventually lead to discoveries of effective treatments for devastating liver diseases.     

Interaction of metallothionein-2 with platinum-modified 5'-guanosine monophosphate and DNA

Human metallothioneins (MTs), a family of cysteine- and metal-rich metalloproteins, play an important role in the acquired resistance to platinum drugs. MTs occur in the cytosol and the nucleus of the cells and sequester platinum drugs through interaction with their zinc-thiolate clusters. Herein, we investigate the ability of human Zn 7MT-2 to form DNA-Pt-MT cross-links using the cisplatin- and transplatin-modified plasmid DNA pSP73. Immunochemical analysis of MT-2 showed that the monofunctional platinum-DNA adducts formed DNA- cis/ trans-Pt-MT cross-links and that platinated MT-2 was released from the DNA- trans-Pt-MT cross-links with time. The DNA- cis/ trans-Pt-MT cross-links were also formed in the presence of 2 mM glutathione, a strong S-donor ligand. Independently, we used 5'-guanosine monophosphate (5'-GMP) platinated at the N7 position as a model of monofunctional platinum-DNA adducts. Comparison of reaction kinetics revealed that the formation of ternary complexes between Zn 7MT-2 and cis-Pt-GMP was faster than that of the trans isomer. The analysis of the reaction products with time showed that while the formation of ternary GMP- trans-Pt-MT complex(es) is accompanied by 5'-GMP release, a stable ternary GMP- cis-Pt-MT complex is formed. In the latter complex, a fast initial formation of two Pt-S bonds was followed by a slow formation of an additional Pt-S bond yielding an unusual Pt(II)S 3N coordination with N7-GMP as the only N-donor ligand. The ejection of negligible zinc from the zinc-thiolate clusters implies the initial formation of Zn-(mu-SCys)-Pt bridges involving the terminal thiolate ligands. The biological implications of these studies are discussed.     

Structure of the bacteriophage phi KZ lytic transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.