Search

Vijay Bhooshan Kumar,1 Michal Natan,2 Gila Jacobi,2 Ze’ev Porat,3,4 Ehud Banin,2 Aharon Gedanken1,5 1Department of Chemistry, 2Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan Institute for Nanotechnology and Advanced Materials Bar-Ilan University, Ramat Gan, 3Division of Chemistry, Nuclear Research Center-Negev, 4Institutes of Applied Research, Ben-Gurion University of the Negev, Be’er-Sheva, Israel; 5National Cheng Kung University, Department of Materials Science and Engineering, Tainan, Taiwan Abstract: The opportunistic pathogen Pseudomonas aeruginosa causes infections that are difficult to treat by antibiotic therapy. This research article reports on the synthesis of gallium (Ga) doped in carbon (C)-dots (Ga@C-dots) and their antimicrobial activity against free-living P. aeruginosa bacteria. The synthesis of Ga@C-dots was carried out by sonicating molten Ga (for 2.5 h) in polyethylene glycol-400, which acts as both a medium and carbon source. The resultant Ga@C-dots, having an average diameter of 9±2 nm, showed remarkably enhanced antibacterial activity compared with undoped C-dots. This was reflected by the much lower concentration of Ga doped within Ga@C-dots which was required for full inhibition of the bacterial growth. These results highlight the possibility of using Ga@C-dots as potential antimicrobial agents. Keywords: C-dots, Ga@C-dots, sonochemistry, gallium, antibacterial, Pseudomonas aeruginosa

Paz Elia,1 Raya Zach,1 Sharon Hazan,2 Sofiya Kolusheva,2 Ze’ev Porat,1,3 Yehuda Zeiri1,3 1Department of Biomedical Engineering, 2Ilse Katz Institute of Nanotechnology, Ben-Gurion University of the Negev, 3Division of Chemistry, Negev Nuclear Research Center Beersheba, Israel Abstract: Gold nanoparticles (GNPs) were prepared using four different plant extracts as reducing and stabilizing agents. The extracts were obtained from the following plants: Salvia officinalis, Lippia citriodora, Pelargonium graveolens and Punica granatum. The size distributions of the GNPs were measured using three different methods: dynamic light scattering, nanoparticle-tracking analysis and analysis of scanning electron microscopy images. The three methods yielded similar size distributions. Biocompatibility was examined by correlation of L-cell growth in the presence of different amounts of GNPs. All GNPs showed good biocompatibility and good stability for over 3 weeks. Therefore, they can be used for imaging and drug-delivery applications in the human body. High-resolution transmission electron microscopy was used to view the shapes of the larger GNPs, while infrared spectroscopy was employed to characterize the various functional groups in the organic layer that stabilize the particles. Finally, active ingredients in the plant extract that might be involved in the formation of GNPs are proposed, based on experiments with pure antioxidants that are known to exist in that plant. Keywords: gold nanoparticles, Lippia citriodora, Salvia officinalis, Pelargonium graveolens, Punica granatum, antioxidants, size distribution, zeta potential

YES-associated protein (YAP) is a central transcription coactivator that functions as an oncogene in a number of experimental systems. However, under DNA damage, YAP activates pro-apoptotic genes in conjunction with p73. This program switching is mediated by c-Abl (Abelson murine leukemia viral oncogene) via phosphorylation of YAP at the Y357 residue (pY357). YAP as an oncogene coactivates the TEAD (transcriptional enhancer activator domain) family transcription factors. Here we asked whether c-Abl regulates the YAP-TEAD functional module. We found that DNA damage, through c-Abl activation, specifically depressed YAP-TEAD-induced transcription. Remarkably, c-Abl counteracts YAP-induced transformation by interfering with the YAP-TEAD transcriptional program. c-Abl induced TEAD1 phosphorylation, but the YAP-TEAD complex remained unaffected. In contrast, TEAD coactivation was compromised by phosphomimetic YAP Y357E mutation but not Y357F, as demonstrated at the level of reporter genes and endogenous TEAD target genes. Furthermore, YAP Y357E also severely compromised the role of YAP in cell transformation, migration, anchorage-independent growth, and epithelial-to-mesenchymal transition (EMT) in human mammary MCF10A cells. These results suggest that YAP pY357 lost TEAD transcription activation function. Our results demonstrate that YAP pY357 inactivates YAP oncogenic function and establish a role for YAP Y357 phosphorylation in cell-fate decision. [ABSTRACT FROM AUTHOR]

Physical interactions between two immune cells or between immune and cancer cells play a major role in shaping the immune response in the tumor microenvironment, making them prime therapeutic targets for bispecific engagers. Here, we present a protocol for assessing murine cell doublet engagement and subsequent effects using flow cytometry and imaging flow cytometry. We describe steps for identifying bispecific cell engager antibodies at the cell-cell interface, doublet quantification, and characterizing cellular protein morphology and processes within the doublet. For complete details on the use and execution of this protocol, please refer to Shapir Itai et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Parasitic diseases remain a major global health problem for humans. Parasites employ a variety of strategies to invade and survive within their hosts and to manipulate host defense mechanisms, always in the pathogen's favor. Extracellular vesicles (EVs), membrane-bound nanospheres carrying a variety of bioactive compounds, were shown to be released by the parasites during all stages of the infection, enabling growth and expansion within the host and adaptation to frequently changing environmental stressors. In this review, we discuss how the use of existing nanotechnologies and high-resolution imaging tools assisted in revealing the role of EVs during parasitic infections, enabling the quantitation, visualization, and detailed characterization of EVs. We discuss here the cases of malaria, Chagas disease and leishmaniasis as examples of parasitic neglected tropical diseases (NTDs). Unraveling the EVs' role in the NTD pathogenesis may enormously contribute to their early and reliable diagnostic, effective treatment, and prevention., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

This "Best Practices in User Consultation" article is the result of a 2022 International Society for the Advancement of Cytometry (ISAC) membership survey that collected valuable insights from the shared research laboratory (SRL) community and of a group discussion at the CYTO 2022 workshop of the same name. One key takeaway is the importance of initiating a consultation at the outset of a flow cytometry project, particularly for trainees. This approach enables the improvement and standardization of every step, from planning experiments to interpreting data. This proactive approach effectively mitigates experimental bias and avoids superfluous trial and error, thereby conserving valuable time and resources. In addition to guidelines, the optimal approaches for user consultation specify communication channels, methods, and critical information, thereby establishing a structure for productive correspondence between SRL and users. This framework functions as an exemplar for establishing robust and autonomous collaborative relationships. User consultation adds value by providing researchers with the necessary information to conduct reproducible flow cytometry experiments that adhere to scientific rigor. By following the steps, instructions, and strategies outlined in these best practices, an SRL can readily tailor them to its own setting, establishing a personalized workflow and formalizing user consultation services. This article provides a pragmatic guide for improving the caliber and efficacy of flow cytometry research and aggregates the flow cytometry SRL community's collective knowledge regarding user consultation., (© 2024 The Author(s). Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Organisms evolve mechanisms that regulate the properties of biogenic crystals to support a wide range of functions, from vision and camouflage to communication and thermal regulation. Yet, the mechanism underlying the formation of diverse intracellular crystals remains enigmatic. Here we unravel the biochemical control over crystal morphogenesis in zebrafish iridophores. We show that the chemical composition of the crystals determines their shape, particularly through the ratio between the nucleobases guanine and hypoxanthine. We reveal that these variations in composition are genetically controlled through tissue-specific expression of specialized paralogs, which exhibit remarkable substrate selectivity. This orchestrated combination grants the organism with the capacity to generate a broad spectrum of crystal morphologies. Overall, our findings suggest a mechanism for the morphological and functional diversity of biogenic crystals and may, thus, inspire the development of genetically designed biomaterials and medical therapeutics., (© 2024. The Author(s).)

Introduction: On October 7, 2023, terrorist organizations led by Hamas, launched an extensive attack on Israel. Within days following the initial attack, there was a clear need to provide psychological support to individuals who were exposed to the horrors of the October 7 attacks. Hence, the Tel Aviv Sourasky Medical Center opened the "Ichilov Otef Nefesh" clinic to provide psychological first aid to the first-line victims of the war. As the fighting spread, the services were extended to the entire population. The guiding principle of the intervention was that it is crucial in the acute phase after exposure to significant trauma, and its main purpose is to support the natural course of one's recovery processes. This case series describes three representative cases from among the hundreds of people who applied to the clinic. The intervention provided was guided by an integrated approach that promotes introspection and connectedness to one's environment and community, enhancing cognitive functions, emotional regulation and utilizing available resources to reduce distress. The main themes that emerged shifted between feelings of helplessness and a sense of agency; a breach in basic trust and renewed trust, guilt and self-compassion; anger, abandonment and alienation versus solidarity and connecting to the present and the future, despair versus hope and loss versus acceptance and connectedness. In face of these, the therapists endorsed techniques of validation, restoring self-agency, promoting a sense of self-agency and self-compassion, creating a link between the past and present, and finding connection and hope.

Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1-SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential., (© 2024. The Author(s).)

An essential interaction between sunlight and eukaryotes involves vitamin D production through exposure to ultraviolet (UV) radiation. While extensively studied in vertebrates, the role of vitamin D in non-animal eukaryotes like microalgae remains unclear. Here, we investigate the potential involvement of vitamin D in the UV-triggered response of Emiliania huxleyi , a microalga inhabiting shallow ocean depths that are exposed to UV. Our results show that E. huxleyi produces vitamin D 2 and D 3 in response to UV. We further demonstrate that E. huxleyi responds to external administration of vitamin D at the transcriptional level, regulating protective mechanisms that are also responsive to UV. Our data reveal that vitamin D addition enhances algal photosynthetic performance while reducing harmful reactive oxygen species buildup. This study contributes to understanding the function of vitamin D in E. huxleyi and its role in non-animal eukaryotes, as well as its potential importance in marine ecosystems., Competing Interests: The authors O.E., S.M., and E.S. have a pending patent application under publication number US 20240083843 A1., (© 2024 The Author(s).)

Polymer electrolyte was used as a medium for testing the performance of microband electrodes under conditions of linear diffusion. Cyclic voltammetry (CV) and chronoamperometry (CA) experiments were performed in a highly viscous medium, where diffusion rates are much slower than in fluid solutions. The log i vs. log v (CV) or log i vs. log t (CA) relationships with the current equation confirmed the existence of such conditions, yielding slope values that were lower than the expected 0.5. This could indicate an impure linear diffusion profile, i.e., some contribution from radial diffusion (edge effects). However, the desired value of 0.5 was obtained when performing these tests in monomeric solvents of similar viscosities, such as glycerol or propylene glycol. These results led to the conclusion that the current equations, which are based on Fick's laws, may not be applicable for polymer electrolytes, where various obstructions to free diffusion result in a more complicated process than for monomeric solvents.

Proteolysis of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a crucial role in the immune response to bacterial infections. Here we report the secretion of MMPs associated with proteolytic extracellular vesicles (EVs) released by macrophages in response to Salmonella enterica serovar Typhimurium infection. Specifically, we used global proteomics, in vitro, and in vivo approaches to investigate the composition and function of these proteolytic EVs. Using a model of S. Typhimurium infection in murine macrophages, we isolated and characterized a population of small EVs. Bulk proteomics analysis revealed significant changes in protein cargo of naïve and S. Typhimurium-infected macrophage-derived EVs, including the upregulation of MMP-9. The increased levels of MMP-9 observed in immune cells exposed to S. Typhimurium were found to be regulated by the toll-like receptor 4 (TLR-4)-mediated response to bacterial lipopolysaccharide. Macrophage-derived EV-associated MMP-9 enhanced the macrophage invasion through Matrigel as selective inhibition of MMP-9 reduced macrophage invasion. Systemic administration of fluorescently labeled EVs into immunocompromised mice demonstrated that EV-associated MMP activity facilitated increased accumulation of EVs in spleen and liver tissues. This study suggests that macrophages secrete proteolytic EVs to enhance invasion and ECM remodeling during bacterial infections, shedding light on an essential aspect of the immune response.

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management., (© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Immune checkpoint inhibition treatment using aPD-1 monoclonal antibodies is a promising cancer immunotherapy approach. However, its effect on tumor immunity is narrow, as most patients do not respond to the treatment or suffer from recurrence. We show that the crosstalk between conventional type I dendritic cells (cDC1) and T cells is essential for an effective aPD-1-mediated anti-tumor response. Accordingly, we developed a bispecific DC-T cell engager (BiCE), a reagent that facilitates physical interactions between PD-1 + T cells and cDC1. BiCE treatment promotes the formation of active dendritic/T cell crosstalk in the tumor and tumor-draining lymph nodes. In vivo, single-cell and physical interacting cell analysis demonstrates the distinct and superior immune reprogramming of the tumors and tumor-draining lymph nodes treated with BiCE as compared to conventional aPD-1 treatment. By bridging immune cells, BiCE potentiates cell circuits and communication pathways needed for effective anti-tumor immunity., Competing Interests: Declaration of interests The Weizmann Institute has filed a PCT patent application related to this work, on which Y.S.I., O.B., I.A., and R.D. are inventors. The patent application has been licensed to Merck Healthcare KGaA. I.A. is a member of the Cell advisory board., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The thymus is a primary lymphoid organ that is essential for the establishment of adaptive immunity through generation of immunocompetent T cells. In response to various stress signals, the thymus undergoes acute but reversible involution. However, the mechanisms governing its recovery are incompletely understood. Here, we used a dexamethasone-induced acute thymic involution mouse model to investigate how thymic hematopoietic cells (excluding T cells) contribute to thymic regeneration. scRNA-seq analysis revealed marked transcriptional and cellular changes in various thymic populations and highlighted thymus-resident innate lymphoid cells type 2 (ILC2) as a key cell type involved in the response to damage. We identified that ILC2 are activated by the alarmins IL-25 and IL-33 produced in response to tissue damage by thymic tuft cells and fibroblasts, respectively. Moreover, using mouse models deficient in either tuft cells and/or IL-33, we found that these alarmins are required for effective thymus regeneration after dexamethasone-induced damage. We also demonstrate that upon their damage-dependent activation, thymic ILC2 produce several effector molecules linked to tissue regeneration, such as amphiregulin and IL-13, which in turn promote thymic epithelial cell differentiation. Collectively, our study elucidates a previously undescribed role for thymic tuft cells and fibroblasts in thymus regeneration through activation of the type 2 immune response.

Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite-parasite and parasite-host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite-infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells., Competing Interests: No potential conflicts of interest were reported by the authors., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection 1 .Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells 2-7 . Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA + plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Bloom's syndrome (BLM) protein is a known nuclear helicase that is able to unwind DNA secondary structures such as G-quadruplexes (G4s). However, its role in the regulation of cytoplasmic processes that involve RNA G-quadruplexes (rG4s) has not been previously studied. Here, we demonstrate that BLM is recruited to stress granules (SGs), which are cytoplasmic biomolecular condensates composed of RNAs and RNA-binding proteins. BLM is enriched in SGs upon different stress conditions and in an rG4-dependent manner. Also, we show that BLM unwinds rG4s and acts as a negative regulator of SG formation. Altogether, our data expand the cellular activity of BLM and shed light on the function that helicases play in the dynamics of biomolecular condensates., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)