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Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients., (© 2024. The Author(s).)

The bacterial cytoplasmic membrane separates the cell from its environment and acts as a selective permeability barrier. In addition, it functions in energy conservation, transport, signaling, and biosynthesis processes. Antimicrobial agents disrupting these functions may lead to pleiotropic effects, including leakage of low molecular weight compounds such as ions, amino acids, and ATP and subsequent membrane depolarization. This updated chapter describes two techniques to assess antibiotic-induced membrane impairment in vivo., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel., Methods: Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1β release after incubation with the P2X7-specific nbs., Results: Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1β release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood-brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size., Conclusion: Mechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage., (© 2022. The Author(s).)

The P2X7 ion channel is a key sensor for extracellular ATP and a key trigger of sterile inflammation. Intravenous injection of nanobodies that block P2X7 has shown to be beneficial in mouse models of systemic inflammation. P2X7 has also emerged as an attractive therapeutic target for inflammatory brain diseases. However, little is known about the ability of nanobodies to cross the BBB. Here we evaluated the ability of P2X7-specific nanobodies to reach and to block P2X7 on microglia following intravenous or intracerebral administration. For this study, we reformatted and sequence-optimized P2X7 nanobodies for higher stability and elevated isoelectric point. Following injection of nanobodies or nanobody-encoding adeno-associated viral vectors (AAV), we monitored the occupancy and blockade of microglial P2X7 in vivo using ex vivo flow cytometry. Our results show that P2X7 on microglia was within minutes completely occupied and blocked by intracerebroventricularly injected nanobodies, even at low doses. In contrast, very high doses were required to achieve similar effects when injected intravenously. The endogenous production of P2X7-antagonistic nanobodies following intracerebral or intramuscular injection of nanobody-encoding AAVs resulted in a long-term occupancy and blockade of P2X7 on microglia. Our results provide new insights into the conditions for the delivery of nanobodies to microglial P2X7 and point to AAV-mediated delivery of P2X7 nanobodies as a promising strategy for the treatment of sterile brain inflammation., Competing Interests: FK-N and CS are co-inventors of a patent covering P2X7-antagonistic nanobodies. The remaining authors declare(s) that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pinto-Espinoza, Guillou, Rissiek, Wilmes, Javidi, Schwarz, Junge, Haag, Liaukouskaya, Wanner, Nicke, Stortelers, Tan, Adriouch, Magnus and Koch-Nolte.)

The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the two-component systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (Moskowitz, S. M.; Antimicrob. Agents Chemother. 2012, 56, 1019-1030; Cheng, H. Y.; J. Biomed. Sci. 2010, 17, 60). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica . The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs.

Magnetic particle imaging (MPI) is an innovative radiation-free tomographic imaging method providing excellent temporal resolution, contrast, sensitivity, and safety. Mobile human MPI prototypes suitable for continuous bedside monitoring of whole-brain perfusion have been developed. However, for the clinical translation of MPI, a crucial gap in knowledge still remains: while MPI can visualize the reduction in blood flow and tissue perfusion in cerebral ischemia, it is unclear whether MPI works in intracranial hemorrhage. Our objective was to investigate the capability of MPI to detect intracranial hemorrhage in a murine model. Intracranial hemorrhage was induced through the injection of collagenase into the striatum of C57BL/6 mice. After the intravenous infusion of a long-circulating MPI-tailored tracer consisting of superparamagnetic iron oxides, we detected the intracranial hemorrhage in less than 3 min and could monitor hematoma expansion in real time. Multicontrast MPI can distinguish tracers based on their physical characteristics, core size, temperature, and viscosity. By employing in vivo multicontrast MPI, we were able to differentiate areas of liquid and coagulated blood within the hematoma, which could provide valuable information in surgical decision making. Multicontrast MPI also enabled simultaneous imaging of hemorrhage and cerebral perfusion, which is essential in the care of critically ill patients with increased intracranial pressure. We conclude that MPI can be used for real-time diagnosis of intracranial hemorrhage. This work is an essential step toward achieving the clinical translation of MPI for point-of-care monitoring of different stroke subtypes.

Local thermal conductivity, thermal diffusivity, and volumetric heat capacity of all-inorganic halide perovskite thin films are mapped simultaneously and with highest spatial resolution for the first time. These various thermal properties are detected by a scanning near-field thermal microscope operated at two different frequencies simultaneously. We apply this technique to analyze the thermal properties of halide perovskites on the nanoscale. In addition to an ultralow thermal conductivity of 0.43 ± 0.03 and 0.33 ± 0.02 W/(m·K), a low thermal diffusivity of 0.3 ± 0.1 mm 2 /s and a small heat capacity of 0.29 ± 0.9 and 0.18 ± 0.6 J/(g·K) are obtained for CsPbBr 3 and CsPb 2 Br 5 films, respectively. The findings of our thermal microscopy are of great general importance for the thermal design of thin-film devices based on halide perovskites, while the measurement technique itself is generally applicable for other thin-film optoelectronic materials.

Wolbachia endobacteria are obligate intracellular bacteria with a highly reduced genome infecting many arthropod and filarial species, in which they manipulate arthropod reproduction to increase their transmission and are essential for nematode development and survival. The Wolbachia genome encodes all enzymes required for the synthesis of the cell wall building block lipid II, although a peptidoglycan-like structure has not been detected. Despite the ability to synthesize lipid II, Wolbachia from arthropods and nematodes have only a subset of genes encoding enzymes involved in the periplasmic processing of lipid II and peptidoglycan recycling, with arthropods having two more than nematodes. We functionally analyzed the activity of the putative cell wall hydrolase AmiD from the Wolbachia endosymbiont of Drosophila melanogaster , an enzyme not encoded by the nematode endobacteria. Wolbachia AmiD has Zn 2+ -dependent amidase activity and cleaves intact peptidoglycan, monomeric lipid II and anhydromuropeptides, substrates that are generated during bacterial growth. AmiD may have been maintained in arthropod Wolbachia to avoid host immune recognition by degrading cell wall fragments in the periplasm. This is the first description of a wolbachial lipid II processing enzyme putatively expressed in the periplasm.

The bacterial cell envelope is believed to be a principal target for initiating the staphylocidal pathway of chitosan. The present study was therefore designed to investigate possible changes in cell surface phenotypes related to the in vitro chitosan resistance development in the laboratory strain S. aureus SG511-Berlin. Following a serial passage experiment, a stable chitosan-resistant variant (CRV) was identified, exhibiting >50-fold reduction in its sensitivity towards chitosan. Our analyses of the CRV identified phenotypic and genotypic features that readily distinguished it from its chitosan-susceptible parental strain, including: (i) a lower overall negative cell surface charge; (ii) cross-resistance to a number of antimicrobial agents; (iii) major alterations in cell envelope structure, cellular bioenergetics and metabolism (based on transcriptional profiling); and (iv) a repaired sensor histidine kinase GraS. Our data therefore suggest a close nexus between changes in cell envelope properties with the in vitro chitosan-resistant phenotype in S. aureus SG511-Berlin., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The bacterial cytoplasmic membrane separates the cell from its environment and acts as a selective permeability barrier. In addition, it functions in energy conservation, transport, and biosynthesis processes. Antimicrobial agents disrupting these functions may lead to pleiotropic effects, including leakage of low molecular weight compounds such as ions, amino acids and ATP, and subsequent membrane depolarization. This article describes two techniques to assess antibiotic-induced membrane impairment in vivo.

Cationic and amphiphilic peptides are widely distributed in eukaryotic organisms and constitute a first line of host defense against invading pathogens. Some of these host defense peptides (HDPs) combine specific antibiotic activities with modulation of immune responses. Moreover, they are active against bacteria resistant to conventional antibiotics and show only modest resistance development under in vitro selection pressure. Based on these features, HDPs and particularly defensins are considered a promising source of novel anti-infective agents. This review summarizes the current knowledge about defensins from different kingdoms and discusses their potential for therapeutic application., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

θ-Defensins are cyclic antimicrobial peptides expressed in leukocytes of Old world monkeys. To get insight into their antibacterial mode of action, we studied the activity of RTDs (rhesus macaque θ-defensins) against staphylococci. We found that in contrast to other defensins, RTDs do not interfere with peptidoglycan biosynthesis, but rather induce bacterial lysis in staphylococci by interaction with the bacterial membrane and/or release of cell wall lytic enzymes. Potassium efflux experiments and membrane potential measurements revealed that the membrane impairment by RTDs strongly depends on the energization of the membrane. In addition, RTD treatment caused the release of Atl-derived cell wall lytic enzymes probably by interaction with membrane-bound lipoteichoic acid. Thus, the premature and uncontrolled activity of these enzymes contributes strongly to the overall killing by θ-defensins. Interestingly, a similar mode of action has been described for Pep5, an antimicrobial peptide of bacterial origin.

Defensins are small basic amphiphilic peptides (up to 5 kDa) that have been shown to be important effector molecules of the innate immune system of animals, plants and fungi. In addition to immune modulatory functions, they have potent direct antimicrobial activity against a broad spectrum of bacteria, fungi and/or viruses, which makes them promising lead compounds for the development of next-generation antiinfectives. The mode of antibiotic action of defensins was long thought to result from electrostatic interaction between the positively charged defensins and negatively charged microbial membranes, followed by unspecific membrane permeabilization or pore-formation. Microbial membranes are more negatively charged than human membranes, which may explain to some extent the specificity of defensin action against microbes and associated low toxicity for the host. However, research during the past decade has demonstrated that defensin activities can be much more targeted and that microbe-specific lipid receptors are involved in the killing activity of various defensins. In this respect, human, fungal and invertebrate defensins have been shown to bind to and sequester the bacterial cell wall building block lipid II, thereby specifically inhibiting cell wall biosynthesis. Moreover, plant and insect defensins were found to interact with fungal sphingolipid receptors, resulting in fungal cell death. This review summarizes the current knowledge on the mode of action and structure of defensins from different kingdoms, with specific emphasis on their interaction with microbial lipid receptors.

Background and Aim: Achillodynia is often seen as a sign of repetitive strain of dorsal tissue of the calf. A neurogene component is also considered for this disease pattern. This association haven`t been investigated yet. The major goal of this study was the investigation of the dynamics of the sural nerve by ultrasonography with regard to movements of ankle, knee and hip. This concerns the physiological situation and pathological aspects of achillodynia., Subjects/method: In this cross sectional study without a blinding-assay 29 sural nerves of 15 healthy subjects (group A) and 6 sural nerves of 3 subjects with pain or dysaesthesia at the achilles tendon or the lateral foot (group B) have been investigated by diagnostic ultrasonography., Results: The sural nerve has an average size of 3.7 mm (2) (± 0.05 mm (2)) for healthy subjects and 4.1 mm (2) (± 0.1 mm (2)) for those with achillodynia. The lateral (Mlat = 3.89 mm) and anterio-posterior (Map = 0.56 mm) movement of the sural nerve has a statistic significantly greater extent by the whole range of motion of dorsalflexion and plantarflexion than by flexion and extension of the kneejoint (Mlat: p = 0.008; Map: p = 0.0115) or by abduction and adduction of the hipjoint (Mlat: p = 0.0025; Map: p = 0.002) for group A. Within this study, no statistic significant differences between both groups could be observed regarding the dynamics and concerning the size of the sural nerve., (© Georg Thieme Verlag KG Stuttgart · New York.)

Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and Cg-Defm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-N-acetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon resonance measurements showed that oyster defensins bind almost irreversibly to the lipid II peptidoglycan precursor, thereby inhibiting the cell wall biosynthesis. To our knowledge, this is the first detailed analysis of the mechanism of action of antibacterial defensins produced by invertebrates. Interestingly, the three defensins, which were chosen as representative of the oyster defensin molecular diversity, bound differentially to lipid II. This correlated with their differential antibacterial activities. From our experimental data and the analysis of oyster defensin sequence diversity, we propose that oyster defensin activity results from selective forces that have conserved residues involved in lipid II binding and diversified residues at the surface of oyster defensins that could improve electrostatic interactions with the bacterial membranes.

Human beta-defensin 3 (hBD3) is a highly charged (+11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.