Bacterial gasdermins reveal an ancient mechanism of cell death

Ancient origin of cell death Gasdermins are cell death proteins in mammals that form membrane pores in response to pathogen infection. Johnson et al . report that diverse bacteria encode structural and functional homologs of mammalian gasdermins. Like their mammalian counterparts, bacterial gasdermins are activated by caspase-like proteases, oligomerize into large membrane pores, and defend against pathogen—in this case, bacteriophage—infection. Proteolytic activation occurs through the release of a short inhibitory peptide, and many bacterial gasdermins are lipidated to facilitate membrane pore formation. Pyroptotic cell death, a central component of mammalian innate immunity, thus has a shared origin with an ancient antibacteriophage defense system. —SMH

Leakage of Endotoxins through the Endotoxin Retentive Filter: An in vitro Study

<b><i>Introduction:</i></b> Ultrapurification of dialysis fluid has enabled highly efficient dialysis treatments. Online hemodiafiltration is one such treatment that uses a purified dialysis fluid as a supplemental fluid. In this method, an endotoxin retentive filter (ETRF) is used in the final step of dialysis fluid purification, with the aim of preventing leakage of endotoxins. Sodium hypochlorite and peracetic acid are used as disinfecting agents for the dialysis fluid pipes containing the ETRF; however, the effects of these agents on ETRF membrane pores have not been fully clarified. <b><i>Methods:</i></b> Water permeability (flux) and endotoxin permeability were assessed in 3 types of ETRFs made with different membrane materials: polyester polymer alloy (PEPA), polyether sulfone (PES), and polysulfone (PS). High-concentration sodium hypochlorite and 2 types of peracetic acid were used as disinfecting agents, and the changes in flux and the endotoxin sieving coefficient (SC) were measured. <b><i>Results:</i></b> After repeated use of high concentrations of sodium hypochlorite and peracetic acid, the PEPA and PES ETRFs did not permit passage of endotoxins, regardless of their flux. However, in the PS ETRF, the flux and endotoxin SC increased with the number of cleaning cycles. No differences were observed according to the concentration of peracetic acid disinfecting agents. <b><i>Conclusion:</i></b> PEPA and PES ETRFs completely prevent endotoxin leakage and can be disinfected at concentrations higher than the conventionally recommended concentration without affecting pore expansion. Even new PS ETRFs have low levels of endotoxin leakage, which increase after disinfection cycles using sodium hypochlorite and peracetic acid.

Membrane adsorber with hierarchically porous HKUST-1 immobilized in membrane pores by flowing synthesis

A membrane adsorber with hierarchically porous HKUST-1 (HP-HKUST-1) immobilized in membrane pores has been fabricated by flowing synthesis. The XRD characteristics indicated that the structure integrity of the HP-HKUST-1 immobilized in the membrane pores can be kept after template agent removed. Other characteristics presented by XPS, FTIR, SEM, TEM and BET proved the effective immobilization of HP-HKUST-1 in membrane pores. Compared with hydrothermal HKUST-1 powder, the adsorption capacity for Congo red and Methylene blue adsorption can be increased several times by hydrothermal HP-HKUST-1 powder, owing to the mesopores with rich active sites for adsorption. When the solution was flowed through the membrane adsorber, the adsorption rate for these adsorbates increased significantly, owing to the enhanced mass transfer in the confined space of the membrane pores with micro or nano scale. After going through seven adsorption-desorption experiments, the membrane adsorber with HP-HKUST-1 immobilized in membrane pores shows a remarkable repeatability.

Single-Entity Electrocatalysis at Electrode Ensembles Prepared by Template Synthesis

Nanoelectrode ensembles (NEEs), prepared by Au template synthesis, are presented as a proof-of-concept sample platform to study individual electrodeposited materials by scanning electrochemical cell microscopy (SECCM). With this platform, the non-conductive membrane support does not contribute to the electrocatalytic activity recorded at each electrode. Use of low-density template membranes results in electrodes that are isolated because initial membrane pores are typically separated by significant (microscale) distances. Electrodeposition of catalytic nanoparticles onto the electrodes of the array and observation of electrocatalytic activity are demonstrated to be suitable for correlative SECCM voltammetric mapping and electron microscopy. Suitability of NEEs for studies of surface Au oxidation, hydrazine oxidation, and hydrogen evolution (hydrogen evolution reaction, HER), and at Pt particles on NEEs (Pt-NEEs) for HER is demonstrated.

Influence of Transglutaminase Crosslinking on Casein Protein Fractionation during Low Temperature Microfiltration

Low temperature microfiltration (MF) is applied in dairy processing to achieve higher protein and microbiological quality ingredients and to support ingredient innovation; however, low temperature reduces hydrophobic interactions between casein proteins and increases the solubility of colloidal calcium phosphate, promoting reversible dissociation of micellar β-casein into the serum phase, and thus into permeate, during MF. Crosslinking of casein proteins using transglutaminase was studied as an approach to reduce the permeation of casein monomers, which typically results in reduced yield of protein in the retentate fraction. Two treatments (a) 5 °C/24 h (TA) and (b) 40 °C/90 min (TB), were applied to the feed before filtration at 5 °C, with a 0.1 µm membrane. Flux was high for TA treatment possibly due to the stabilising effect of transglutaminase on casein micelles. It is likely that formation of isopeptide bonds within and on the surface of micelles results in the micelles being less readily available for protein-protein and protein–membrane interactions, resulting in less resistance to membrane pores and flow passage, thereby conferring higher permeate flux. The results also showed that permeation of casein monomers into the permeate was significantly reduced after both enzymatic treatments as compared to control feed due to the reduced molecular mobility of soluble casein, mainly β-casein, caused by transglutaminase crosslinking.

Fracture healing is delayed in the absence of gasdermin signaling

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1β (IL-1β) and IL-18. Excessive GSDM-mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1β and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

Nanobody-Mediated Inhibition of P2X7 on Microglia Improves Stroke Outcome

BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.

Mitigating Silica Fouling and Improving PPCP Removal by Modified NF90 Using In Situ Radical Graft Polymerization

This study in-situ modified a commercial nanofiltration membrane, NF90, through the concentration-polymerization-enhanced radical graft polarization method by applying two agents of 3-sulfopropyl methacrylate potassium salt (SPM) and 2-hydroxyethyl methacrylate (HEMA) with different dosages. Surface characterization revealed that the modified membranes became rougher and more hydrophilic compared with the pristine membrane. The modified membranes exhibited considerably enhanced separation performance with 5.8–19.6% higher NaCl rejection and 17.2–19.9% higher pharmaceuticals and personal care products (PPCPs) rejection than the pristine membrane. When treating the feedwater with high silica concentration, the modified membranes exhibited relatively less flux decline with high percentage of reversible fouling, especially the ones modified using a lower monomer concentration (0.01 M SPM and 0.01 M HEMA). Moreover, membrane modification enhanced the PPCP rejection (1.3–5.4%) after silica fouling by mitigating foulant deposition on the membrane surface. The fouling mechanism was confirmed to be intermediate blocking of membrane pores. Therefore, the in-situ modification technique with a low monomer concentration proved to be effective for mitigating silica fouling and improving PPCP rejection, which can be easily performed and cost-effective in practical application.