Outer Cell Surface Research Articles

Engineering Saccharomyces cerevisiae for the production of natural osmolyte glucosyl glycerol from sucrose and glycerol through Ccw12-based surface display of sucrose phosphorylase

BackgroundYeast Saccharomyces cerevisiae is widely recognised as a versatile chassis for constructing microbial cell factories. However, producing chemicals from toxic, highly concentrated, or cell-impermeable substrates, or chemicals dependent on enzymatic reactions incompatible with the yeast’s intracellular environment, remains challenging. One such chemical is 2-O-(α-D-glucopyranosyl)-sn-glycerol (glucosyl glycerol, αGG), a natural osmolyte used in the cosmetics and healthcare industries. This compound can be synthesised in a one-enzyme reaction from sucrose and glycerol by Leuconostoc mesenteroides sucrose phosphorylase (SucP), an enzyme which, in a low-water, glycerol-rich, phosphate-free environment, transfers the glucosyl moiety from sucrose to glycerol.ResultsIn this study, we engineered a yeast microbial cell factory for αGG production. For this purpose, we first focused on the abundant yeast GPI-anchored cell wall protein Ccw12 and used our insights to develop a miniature Ccw12-tag, which adds only 1.1 kDa to the enzyme of interest while enabling its covalent attachment to the cell wall. Next, we Ccw12-tagged SucP and expressed it in an invertase-negative strain of yeast S. cerevisiae from the PHO5 promoter, i.e., promoter strongly induced under phosphate-free conditions. Such SucP isoform, covalently C-terminally anchored to the outer cell surface, produced extracellularly 37.3 g l− 1 (146 mM) of αGG in five days, while the underlying chassis metabolised reaction by-products, thereby simplifying downstream processing.ConclusionsThe here-described S. cerevisiae strain, displaying C-terminally anchored sucrose phosphorylase on its cell surface, is the first eukaryotic microbial cell factory capable of a one-step αGG production from the readily available substrates sucrose and glycerol.

Methods for faster estimation of the entropy profile of a lithium-ion battery: A comparison of accelerated potentiometry and the estimation of entropy through thermal signatures

The operating temperature of a lithium-ion battery (LIB) has strong effects on its electrochemical performance, safety, and lifetime. Reliable predictions of cell temperature and heat generation rate are required for the design and thermal management of battery systems. The entropy variation of a LIB has a significant influence on the heat generation and the cell temperature variation, especially at a lower C-rate. Since the entropy is a function of State-of-Charge (SoC), and may change during ageing, the entropy profile may as well be used as an indicator for battery State-of-Health diagnostics. The present experimental methods, such as potentiometric or calorimetric methods, for determining the entropy variation throughout a battery's full SoC window are time-consuming and require specialized equipment with good thermal insulation and instrumentation. Hence, faster and more convenient methods are needed. In this paper, we compare an accelerated potentiometric method with a new entropy characterization method where the entropy profile is extracted from the thermal signature of a LIB during charge and discharge at low C-rates. The accelerated potentiometric method aims to obtain the entropy from thermal cycling while the cell is still not completely relaxed to its stable OCV. The thermal signature method monitors the variations of a cell's outer surface temperature during a low constant current charge or discharge, from which the continuous variation of entropy with SoC can be extracted. The obtained entropy profile was validated through a modeling approach that combines a P2D-electrochemical model for battery operation and a 3D-finite-element model for the thermal behavior during battery operation. Good agreement between the model predictions and experiments was achieved on the temporal variation of the cell's outer surface temperature.

Halofilins as emerging bactofilin families of archaeal cell shape plasticity orchestrators

Bactofilins are rigid, nonpolar bacterial cytoskeletal filaments that link cellular processes to specific curvatures of the cytoplasmic membrane. Although homologs of bactofilins have been identified in archaea and eukaryotes, functional studies have remained confined to bacterial systems. Here, we characterize representatives of two families of archaeal bactofilins from the pleomorphic archaeon Haloferax volcanii, halofilin A (HalA) and halofilin B (HalB). HalA and HalB polymerize in vitro, assembling into straight bundles. HalA polymers are highly dynamic and accumulate at positive membrane curvatures in vivo, whereas HalB forms more static foci that localize in areas of local negative curvatures on the outer cell surface. Gene deletions and live-cell imaging show that halofilins are critical in maintaining morphological integrity during shape transition from disk (sessile) to rod (motile). Morphological defects in ΔhalA result in accumulation of highly positive curvatures in rods but not in disks. Conversely, disk-shaped cells are exclusively affected by halB deletion, resulting in flatter cells. Furthermore, while ΔhalA and ΔhalB cells imprecisely determine the future division plane, defects arise predominantly during the disk-to-rod shape remodeling. The deletion of halA in the haloarchaeon Halobacterium salinarum, whose cells are consistently rod-shaped, impacted morphogenesis but not cell division. Increased levels of halofilins enforced drastic deformations in cells devoid of the S-layer, suggesting that HalB polymers are more stable at defective S-layer lattice regions. Our results suggest that halofilins might play a significant mechanical scaffolding role in addition to possibly directing envelope synthesis.

A New Bacterial Chassis for Enhanced Surface Display of Recombinant Proteins.

Bacterial surface display is a valuable biotechnology technique for presenting proteins and molecules on the outer surface of bacterial cells. However, it has limitations, including potential toxicity to host bacteria and variability in display efficiency. To address these issues, we investigated the removal of abundant non-essential outer membrane proteins (OMPs) in E. coli as a new strategy to improve the surface display of recombinant proteins. We targeted OmpA, a highly prevalent OMP in E. coli, using the lambda red method. We successfully knocked out ompA in two E. coli strains, K-12 MG1655 and E. coli BL-21, which have broad research and therapeutic applications. We then combined ompA knockout strains and two OMPs with three therapeutic proteins including an anti-toxin enzyme (ClbS), interleukin 18 (IL-18) for activating cytotoxic T cells and an anti- CTLA4 nanobody (αCTLA4) for immune checkpoint blockade. A total of six different display constructs were tested for their display levels by flow cytometry, showing that the ompA knockout strains increased the percentage as well as the levels of display in bacteria compared to those of isogenic wild-type strains. By removing non-essential, highly abundant surface proteins, we develop an efficient platform for displaying enzymes and antibodies, with potential industrial and therapeutic applications. Additionally, the enhanced therapeutic efficacy opens possibilities for live bacteria-based therapeutics, expanding the technology's relevance in the field. The online version contains supplementary material available at 10.1007/s12195-024-00819-w.

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell‐Cell Click Chemistry

AbstractDirect interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR‐1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell‐cell click chemistry strategy. By introducing alkyne‐ or azide‐modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C‐type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell‐cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

Expression of filaments of the Geobacter extracellular cytochrome OmcS in Shewanella oneidensis.

The physiological role of Geobacter sulfurreducens extracellular cytochrome filaments is a matter of debate and the development of proposed electronic device applications of cytochrome filaments awaits methods for large-scale cytochrome nanowire production. Functional studies in G. sulfurreducens are stymied by the broad diversity of redox-active proteins on the outer cell surface and the redundancy and plasticity of extracellular electron transport routes. G. sulfurreducens is a poor chassis for producing cytochrome nanowires for electronics because of its slow, low-yield, anaerobic growth. Here we report that filaments of the G. sulfurreducens cytochrome OmcS can be heterologously expressed in Shewanella oneidensis. Multiple lines of evidence demonstrated that a strain of S. oneidensis, expressing the G. sulfurreducens OmcS gene on a plasmid, localized OmcS on the outer cell surface. Atomic force microscopy revealed filaments with the unique morphology of OmcS filaments emanating from cells. Electron transfer to OmcS appeared to require a functional outer-membrane porin-cytochrome conduit. The results suggest that S. oneidensis, which grows rapidly to high culture densities under aerobic conditions, may be suitable for the development of a chassis for producing cytochrome nanowires for electronics applications and may also be a good model microbe for elucidating cytochrome filament function in anaerobic extracellular electron transfer.

Abstract 3189: A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity

Abstract Transmembrane 4 super family member 4(TM4SF4) protein has been shown to be involved in EMT-associated stemness through autocrine of insulin-like growth factor 1(IGF1) and osteopontin(OPN) in non-small cell lung cancer(NSCLC) cells. However, its potential as a therapeutic target has not been evaluated. In this study, anti-human TM4SF4 monoclonal antibodies(anti-hTM4SF4 mAbs) were prepared in mouse as part of a strategy for developing anticancer drug using humanized antibody. A synthetic peptide(15 mer, 126-140th AA) derived from the extracellular loop(ECL) 2 of human TM4SF4 exposed on outer cell surface was used as an immunization antigen. Among the selected anti-hTM4SF4 mAbs, ECL-2B7 and ECL-12A8 showed the best affinity for the antigen(Kd), approximately 2.66 nM and 8.34 nM, as determined by surface plasmon resonance analysis. Using mouse xenograft of NSCLC cell line, it was confirmed that ECL-2B7 was superior to ECL-12A8 in producing humanized antibody. ECL-2B7 inhibited EMT-associated stemness of TM4SF4 positive cancer cells by blocking cellular signaling events such as IGF1Rβ and CD44 and their downstream signals, PI3K/AKT/GSK3β/NF-κB and JAK2/STAT3 pathways. ECL-2B7 also enhanced antitumor activity by downregulating cellular and exosomal level of PD-L1 and B7-H4, immune-checkpoint ligands that elicit immunosuppression of T cell, and by mediating antibody-dependent cellular cytotoxicity. Treatment with OPN- and IGF1-neutralizing antibodies suppressed intracellular PD-L1 and B7-H4 level. It means that the regulation of PD-L1 and B7-H4 levels by TM4SF4 is closely related to the autocrine action of OPN and IGF1 induced by TM4SF4. Using patient-derived xenograft model, it was confirmed that the ECL-2B7 has excellent antitumor activity that effectively inhibits tumor progress. These results strongly suggest that it is necessary to construct humanized antibody based on ECL-2B7 to confirm its potential as a novel anticancer drug. Citation Format: Rae Kwon Kim, Chang-Kyu Heo, Yeon-jee Kahm, Uhee Jung, Byung-Chul Shin, Won-Yong Kim, Bum-Jin Kim, Sung-Chul Kim, Chun-jeih Ryu, Eun-Wie Cho, In-Gyu Kim. A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3189.

Blockade of CD300A enhances the ability of human NK cells to lyse hematologic malignancies.

The human cluster of differentiation (CD)300A, a type-I transmembrane protein with immunoreceptor tyrosine-based inhibitory motifs, was investigated as a potential immune checkpoint for human natural killer (NK) cells targeting hematologic malignancies (HMs). We implemented a stimulation system involving the CD300A ligand, phosphatidylserine (PS), exposed to the outer surface of malignant cells. Additionally, we utilized CD300A overexpression, a CD300A blocking system, and a xenotransplantation model to evaluate the impact of CD300A on NK cell efficacy against HMs in in vitro and in vivo settings. Furthermore, we explored the association between CD300A and HM progression in patients. Our findings indicated that PS hampers the function of NK cells. Increased CD300A expression inhibited HM lysis by NK cells. CD300A overexpression shortened the survival of HM-xenografted mice by impairing transplanted NK cells. Blocking PS-CD300A signals with antibodies significantly amplified the expression of lysis function-related proteins and effector cytokines in NK cells, thereby augmenting the ability to lyse HMs. Clinically, heightened CD300A expression correlated with shorter survival and an "exhausted" phenotype of intratumoral NK cells in patients with HMs or solid tumors. These results propose CD300A as a potential target for invigorating NK cell-based treatments against HMs.

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

Cell surface RNAs control neutrophil recruitment

RNAs localizing to the outer cell surface have been recently identified in mammalian cells, including RNAs with glycan modifications known as glycoRNAs. However, the functional significance of cell surface RNAs and their production are poorly known. We report that cell surface RNAs are critical for neutrophil recruitment and that the mammalian homologs of the sid-1 RNA transporter are required for glycoRNA expression. Cell surface RNAs can be readily detected in murine neutrophils, the elimination of which substantially impairs neutrophil recruitment to inflammatory sites invivo and reduces neutrophils' adhesion to and migration through endothelial cells. Neutrophil glycoRNAs are predominantly on cell surface, important for neutrophil-endothelial interactions, and can be recognized by P-selectin (Selp). Knockdown of the murine Sidt genes abolishes neutrophil glycoRNAs and functionally mimics the loss of cell surface RNAs. Our data demonstrate the biological importance of cell surface glycoRNAs and highlight a noncanonical dimension of RNA-mediated cellular functions.

Proteomic and morphological insights into the exposure of Cupriavidus metallidurans CH34 planktonic cells and biofilms to aluminium

Aluminium (Al) is one of the most popular materials for industrial and domestic use. Nevertheless, research has proven that this metal can be toxic to most organisms. This light metal has no known biological function and to date very few aluminium-specific biological pathways have been identified. In addition, information about the impact of this metal on microbial life is scarce. Here, we aimed to study the effect of aluminium on the metal-resistant soil bacterium Cupriavidus metallidurans CH34 in different growth modes, i.e. planktonic cells, adhered cells and mature biofilms. Our results indicated that despite a significant tolerance to aluminium (minimal inhibitory concentration of 6.25 mM Al₂(SO₄)₃.18H₂O), the exposure of C. metallidurans to a sub-inhibitory dose (0.78 mM) caused early oxidative stress and an increase in hydrolytic activity. Changes in the outer membrane surface of planktonic cells were observed, in addition to a rapid disruption of mature biofilms. On protein level, aluminium exposure increased the expression of proteins involved in metabolic activity such as pyruvate kinase, formate dehydrogenase and poly(3-hydroxybutyrate) polymerase, whereas proteins involved in chemotaxis, and the production and transport of iron scavenging siderophores were significantly downregulated.

Nuclear-Targeting Lipid PtIV Prodrug Amphiphile Cooperates with siRNA for Enhanced Cancer Immunochemotherapy by Amplifying Pt-DNA Adducts and Reducing Phosphatidylserine Exposure.

Patients treated with Pt-based anticancer drugs (PtII) often experience severe side effects and are susceptible to cancer recurrence due to the limited bioavailability of PtII and tumor-induced immunosuppression. The exposure of phosphatidylserine on the cell's outer surface induced by PtII results in profound immunosuppression through the binding of phosphatidylserine to its receptors on immune cells. Here, we report a novel approach for enhanced cancer chemoimmunotherapy, where a novel nuclear-targeting lipid PtIV prodrug amphiphile was used to deliver a small interfering RNA (siXkr8) to simultaneously amplify Pt-DNA adducts and reduce the level of exposure of phosphatidylserine. This drug delivery vehicle is engineered by integrating the PtIV prodrug with self-assembly performance and siXkr8 into a lipid nanoparticle, which shows tumor accumulation, cancer cell nucleus targeting, and activatable in a reduced microenvironment. It is demonstrated that nuclear-targeting lipid PtIV prodrug increases the DNA cross-linking, resulting in increased Pt-DNA adduct formation. The synergistic effects of the PtIV prodrug and siXkr8 contribute to the improvement of the tumor immune microenvironment. Consequently, the increased Pt-DNA adducts and immunogenicity effectively inhibit primary tumor growth and prevent tumor recurrence. These results underscore the potential of utilizing the nuclear-targeting lipid PtIV prodrug amphiphile to enhance Pt-DNA adduct formation and employing siXkr8 to alleviate immunosuppression during chemotherapy.

Target Functionalized Carbon Dot Nanozymes with Dual-Model Photoacoustic and Fluorescence Imaging for Visual Therapy in Atherosclerosis.

Multifunctional nanomedicines have been used in atherosclerosis theranostics. Herein, phosphatidylserine-specific peptide CLIKKPF-functionalized carbon-dots nanozymes (pep-CDs) are reported for specific and efficient noninvasive theranostic of atherosclerosis. Surprisingly, pep-CDs are discovered to not only inherit the inherent properties of carbon dots (CDs), including deep-red fluorescence emission, photoacoustic response, and superoxide dismutase-like antioxidant, and anti-inflammatory activities but also possess the ability to target recognition on foam cells and target localization on plaques due to the specific interaction of CLIKKPF with phosphatidylserine on the membrane outer surface of foam cells. Furthermore, the target localization effect of pep-CDs vastly promotes the efficient accumulation of CDs in plaque, thus maximizing AS theranostic of CDs. Interestingly, pep-CDs could be developed to image plaque for monitoring atherosclerosis pathological progression in real-time resulting from the different content of foam cells. This work on the one hand proposes a simple and feasible strategy to construct theranostic nanoplatform employing only a single functional unit (i.e., multifunctional CDs) to simplify the fabrication procedure, on the other hand, highlights the advantages of the active target auxiliary mode for atherosclerosis theranostic applications.

Cell Surface RNAs Control Neutrophil Function

Recently, RNAs localizing to the outer cell surface have been reported in mammalian cells, with such RNAs containing glycan modifications (referred to as GlycoRNAs). However, the function of cell surface RNAs are poorly known. In this study, we investigated whether cell surface RNAs exist in neutrophils, what their functions are, and the mechanism by which they function. We first determined that neutrophils express cell surface GlycoRNAs. We utilized two strategies to assess cell surface GlycoRNAs. First, we utilized a sialic acid homologue to metabolically label glycans inside cells. Labeled glycans can be readily detected in purified total RNAs from primary murine neutrophils. The GlycoRNAs signals were depleted upon RNase digestion but not digestion by proteinase or DNase, supporting the existence of GlycoRNAs in neutrophils. Importantly, treating live neutrophils with RNase extracellularly removed over 90% of GlycoRNA signals, supporting that the majority of GlycoRNAs were located on the surface of neutrophils. Second, we directly visualized cell surface RNAs by labeling cellular RNAs with the nucleoside homologue 5'-bromouridine (BrU) and detecting live cells with an anti-BrU antibody applied extracellularly. These data support the existence of cell surface GlycoRNAs on neutrophils. We next revealed that cell surface GlycoRNAs play important functions in neutrophils to mediate transendothelial migration both in vivo and in vitro. We utilized an acute peritonitis model in which primarily neutrophils with cell surface GlycoRNAs removed by extracellular RNase were injected into circulation in mice that were treated with thioglycolate, and the migration of these neutrophils into the peritoneal cavity was quantified and compared to mock treated cells in vivo. We observed a 9-fold decrease of migration by neutrophils treated with extracellular RNase. To determine the in vitro function of cell surface RNAs, we tested the ability of neutrophils to migrate toward a chemoattractant in a trans-well assay. While neutrophils treated with extracellular RNase were viable and migrated similarly as control neutrophils, we observed a substantial defect in migration by extracellular-RNase-treated neutrophils when an endothelial layer was present on the tanswell membrane. A similar defect was observed when assaying neutrophil attachment to endothelial cells in vitro. This defect can be replicated using control neutrophils but by pre-blocking endothelial cells with purified neutrophil GlycoRNAs or the glycan fraction of GlycoRNAs. The defect in neutrophil endothelial interaction in vivo was observed by intravital confocal microscopy. These data support the function of neutrophil cell surface GlycoRNAs in helping transendothelial migration. Lastly, we found that neutrophil GlycoRNAs are bona fide ligands for P-selectin on endothelial surface. Removal of neutrophil surface RNAs with extracellular RNase treatment did not significantly change cell surface integrin levels or reactivity, but reduced recombinant P-selectin binding. Furthermore, recombinant P-selectin, but not recombinant E-selectin, can detect glycoRNAs in purified neutrophil total RNAs. Additionally, blocking endothelial cells with an antibody against P-selectin led to a significant reduction in GlycoRNA binding. These data support that GlycoRNA-P-selectin interaction, at least in part, mediate neutrophil transendothelial migration. Our data demonstrate a critical role of cell surface RNAs in neutrophils, and reveal a new dimension that regulate the function of hematopoietic cells.

Non-Vesicular Release of Alarmin Prothymosin α Complex Associated with Annexin-2 Flop-Out.

Nuclear protein prothymosin α (ProTα) is a unique member of damage-associated molecular patterns (DAMPs)/alarmins. ProTα prevents neuronal necrosis by causing a cell death mode switch in serum-starving or ischemic/reperfusion models in vitro and in vivo. Underlying receptor mechanisms include Toll-like receptor 4 (TLR4) and Gi-coupled receptor. Recent studies have revealed that the mode of the fatal stress-induced extracellular release of nuclear ProTα from cortical neurons in primary cultures, astrocytes and C6 glioma cells has two steps: ATP loss-induced nuclear release and the Ca2+-mediated formation of a multiple protein complex and its extracellular release. Under the serum-starving condition, ProTα is diffused from the nucleus throughout the cell due to the ATP loss-induced impairment of importin α-mediated nuclear transport. Subsequent mechanisms are all Ca2+-dependent. They include the formation of a protein complex with ProTα, S100A13, p40 Syt-1 and Annexin A2 (ANXA2); the fusion of the protein complex to the plasma membrane via p40 Syt-1-Stx-1 interaction; and TMEM16F scramblase-mediated ANXA2 flop-out. Subsequently, the protein complex is extracellularly released, leaving ANXA2 on the outer cell surface. The ANXA2 is then flipped in by a force of ATP8A2 activity, and the non-vesicular release of protein complex is repeated. Thus, the ANXA2 flop-out could play key roles in a new type of non-vesicular and non-classical release for DAMPs/alarmins, which is distinct from the modes conducted via gasdermin D or mixed-lineage kinase domain-like pseudokinase pores.

Long-term transformation of nanoscale zero-valent iron explains its biological effects in anaerobic digestion: From ferroptosis-like death to magnetite-enhanced direct electron transfer networks

Nanoscale zero-valent iron (nZVI) has been extensively used for environmental remediation and wastewater treatment. However, the biological effects of nZVI remain unclear, which is no doubt a result of the complexity of iron species and the dynamic succession of microbial community during nZVI aging. Here, the aging effects of nZVI on methanogenesis in anaerobic digestion (AD) were consecutively investigated, with an emphasis on deciphering the causal relationships between nZVI aging process and its biological effects. The addition of nZVI in AD led to ferroptosis-like death with hallmarks of iron-dependent lipid peroxidation and glutathione (GSH) depletion, which inhibited CH4 production during the first 12 days of exposure. With prolonged exposure time, a gradual recovery (12–21 days) and even better performance (21–27 days) in AD were observed. The recovery performance of AD was mainly attributed to nZVI-enhanced membrane rigidity via forming siderite and vivianite on the outer surface of cells, protecting anaerobes against nZVI-induced toxicity. At the end of 27-days exposure, the significantly increased amount of conductive magnetite simulated direct interspecies electron transfer among syntrophic partners, improving CH4 production. Metagenomic analysis further revealed that microbial cells gradually adapted to the aging of nZVI by upregulating functional genes related to chemotaxis, flagella, conductive pili and riboflavin biosynthesis, in which electron transfer networks likely thrived and the cooperative behaviors between consortium members were promoted. These results unveiled the significance of nZVI aging on its biological effects toward multiple microbial communities and provided fundamental insights into the long-term fates and risks of nZVI for in situ applications.

Plasticity and Stereotypic Rewiring of the Transcriptome Upon Bacterial Evolution of Antibiotic Resistance.

Bacterial evolution of antibiotic resistance frequently has deleterious side effects on microbial growth, virulence, and susceptibility to other antimicrobial agents. However, it is unclear how these trade-offs could be utilized for manipulating antibiotic resistance in the clinic, not least because the underlying molecular mechanisms are poorly understood. Using laboratory evolution, we demonstrate that clinically relevant resistance mutations in Escherichia coli constitutively rewire a large fraction of the transcriptome in a repeatable and stereotypic manner. Strikingly, lineages adapted to functionally distinct antibiotics and having no resistance mutations in common show a wide range of parallel gene expression changes that alter oxidative stress response, iron homeostasis, and the composition of the bacterial outer membrane and cell surface. These common physiological alterations are associated with changes in cell morphology and enhanced sensitivity to antimicrobial peptides. Finally, the constitutive transcriptomic changes induced by resistance mutations are largely distinct from those induced by antibiotic stresses in the wild type. This indicates a limited role for genetic assimilation of the induced antibiotic stress response during resistance evolution. Our work suggests that diverse resistance mutations converge on similar global transcriptomic states that shape genetic susceptibility to antimicrobial compounds.

Recent advancements in Cancer Targeting Therapy with the Hyaluronic Acid as a Potential Adjuvant

Introduction. The non-sulfated natural compound, Hyaluronic acid (HA), is a mucopolysaccharide that has an essential role in cellular biology; it is a fundamental element of the living cell. HA plays an essential role in targeted drug delivery; it has recently acquired much attention because of various advantages like biocompatibility, biodegradability, non-immunogenicity, and non-toxicity.
 Methods. This narrative review is based on the literature searched with keywords Hyaluronic acid, Hyaluronic acid in cancer therapy, Hyaluronic acid in cancer targeting, Hyaluronic acid in drug targeting, was done on PubMed and Elsevier database from January to May 2021. Research published in the last five years was considered; however, no such timeline is followed in cross-referencing.
 Results. From the literature, it is found that HA can recognize distinct receptors that are abnormally revealed in large numbers on the outer surface of cancerous tissues or cells; hence can be used for conjugation with anticancer drugs, facilitating their enhanced therapeutic activity over the cancer cells than normal cells. It is also found that HA-based systems also provide increased stability and solubility of anticancer agents in biological surroundings. Based on these findings and advantages, HA is conjugated with various delivery systems like micelles, liposomes, hydrogels, nanoparticles, etc. As per recent research, the HA-based system provides immunotherapy, gene therapy, targeted chemotherapy, and combination therapy with enormous applications in the evolution of highly efficacious and cost-effective therapy for the ministration of cancer.
 Conclusion. In this context, various literatures on HA as adjuvant for drug delivery system for cancer targeting represents the HA as a potential adjuvant for cancer treatment.

Impacts of Climate Change on the Plant Water Interactions

Climate change has an impact on ecosystem structure and function globally by altering the relationships between plants and soil organisms. Despite the fact that water is the most plentiful molecule on Earth's surface, water scarcity is the element that most severely limits global terrestrial plant production. Little is known about the climatic factors that drive phenological responses to climate change, and less attention has been paid to the fact that phenology is also responsive to other climatic. The aim of this study was to assess the impacts of climate change on plant water interactions. This study was guided by the specific objectives, which included examining the relationship between climate change and plant function; finding out the impacts of climate change on plant water interactions; and assessing how plants handle water scarcity. It was found that there was a linkage between climate change and plant function. The evaporation of water molecules from the outer surfaces of the mesophyll cells initiates the upward transpiration pull in the leaves, and respiring starches and sugars are created during photosynthetic processes using sunlight energy. Climate change enhanced the most enormous movement of species that has occurred without direct human intervention. It was also found that precipitation was a key driver of phenological changes in desert ecosystems. It was also found that drought was one of the most significant biotic challenges faced by plants, with considerable genetic variation in water deficit responses. There is a need for research on climate change to ease biodiversity conservation. Keywords: Climate change, Plant water interactions, Plant, Water DOI: 10.7176/JRDM/87-01 Publication date: September 30 th 2022