Tissue Dynamics (Israel)

The differences in the phospholipid profile between bacterial and mammalian membranes present a promising direction for designing antimicrobial peptides (AMPs). A key challenge, however, is engineering AMPs with a high affinity for bacterial anionic phospholipid (phosphatidylglycerol, PG) while minimizing off-target interactions with mammalian phosphatidylserine (PS). Herein, we report a multiarmed radially amphiphilic AMP (maRAP) design that enhances PG recognition, exhibiting potent antibacterial activity and exceptional antibacterial selectivity. Compared to the single-arm RAP (1aRAP), maRAPs exhibited superior affinity for PG, yet they exhibited negligible binding to PS. They also demonstrated significantly enhanced bactericidal efficacy and selectivity over 1aRAP in complex physiological environments. In the murine bladder-infection model, maRAPs more effectively reduced the bacterial load and attenuated infection-induced tissue damage, as demonstrated by an intact urothelial lining and minimal lamina propria edema at the infection site. This work presents the multiarm strategy as an effective approach for advanced AMP development.

The evaluation of research in higher education emerges as a means of assessing its performance, promoting its quality, and detecting its niches of excellence. The aim of this paper is to set out a general conceptual framework that can be used to instrument research performance assessment in higher education. Indeed, the performance of scientific research remains complex and difficult to circumvent, essentially due to its contingent, relative, multidimensional, all-encompassing, and integrating aspects. Our methodology is based on an exploratory approach, based essentially on an in-depth reading of a rich theoretical corpus questioning the performance of scientific research and its various facets, which has enabled us to create anastomoses between a number of academic writings. We tried to remove the ambiguity surrounding the meaning of search performance under its multidimensional character. As a result, ten facets of research performance are put forward namely, Research outputs quantity, Quality and satisfaction of academics, Sustainability and Social impact, Economic and financial facet, Technological facet, Organizational facet, Ethical facet, Internationalization, Partnership and effectiveness of the doctoral cycle.

Background: Pediatric oncological diagnoses and treatments pose complex biopsychosocial challenges for both patients and their families. These experiences can significantly disrupt daily life, evoke intense emotional responses, and raise concerns about the future, often leading to long-term psychological implications. Objective: This study aimed to assess the emotional functioning of children undergoing cancer treatment and to explore the lasting psychological effects reported by young adult survivors of pediatric cancer. Methods: A mixed-methods approach was employed. In total, 52 minors and their caregivers were recruited from two pediatric oncology units in Italy (Turin and Bari), while 18 young adults diagnosed during childhood were recruited from clinical and community settings in Ancona and Bari. Standardized instruments were used to evaluate emotional symptoms and broader psychological functioning in the pediatric sample, while self-report measures assessed psychological well-being and quality of life in the young adult group. To complement these data, semi-structured interviews were conducted with the young adult survivors to investigate the long-term psychosocial and psychological impacts of pediatric cancer. Results: The quantitative results indicate generally low levels of psychological distress in both groups. However, the qualitative findings reveal the complexity of post-cancer identity, highlighting experiences of resilience, emotional ambivalence, and redefinition of self. Conclusions: These results underscore the importance of addressing the psychological well-being of pediatric oncology patients and young adult survivors beyond the medical treatment phase, with a focus on long-term psychosocial support and individualized care.

Abstract Peripheral Myelin Protein 22 (PMP22) is a tetraspan membrane protein whose altered dosage causes the most common hereditary neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). Despite its clinical significance, the physiological functions of PMP22 and the mechanism behind its tightly controlled gene dosage sensitivity remain unknown since over 30 years, in part due to limited knowledge of its protein-protein interactions (PPIs). In fact, integral membrane proteins such as PMP22 are significantly underrepresented in known cellular interactomes, likely due to limited suitability or technical challenges specific to these hydrophobic molecules in the major PPI discovery approaches. Here, we applied a rigorously optimized co-immunoprecipitation and mass spectrometry workflow using the mild detergent DDM and the high affinity ALFA-tag/anti-ALFA nanobody interaction to identify cellular PMP22-associated proteins. In a cross-context approach, we ran our standardized pipeline across multiple cell types including HEK293T, MDCKII epithelial cells, the Schwann cell line MSC80, and primary rat Schwann cells. We confirm known interactors, and uncover distinct, cell type-specific enrichment patterns following functional annotation analysis. Adhesion-related PPIs dominated in MDCKII cells (e.g., CD47, CLDN1, ATP1B1), while in Schwann cells myelin-associated PPIs were enriched. Importantly, we identified novel PPI candidates that may be highly relevant for PMP22 function including enzymes of the de novo sphingolipid biosynthesis pathway.

Regenerative medicine has emerged as a promising field to address tissue damage and organ failure, with porous microspheres playing a crucial role as cell carriers in tissue engineering applications. However, conventional fabrication methods often result in heterogeneous size distributions and poorly controlled pore structures, limiting their effectiveness. This study leverages microfluidic technology to overcome these challenges and develop uniform poly (L-lactic acid) (PLLA) porous microspheres. We comprehensively evaluate their physical and biological properties, including morphology, size distribution, pore structure, and biocompatibility. Through long-term culture experiments, we investigated the microspheres' capacity to support cell growth and proliferation, demonstrating their effectiveness as cell scaffolds. Our research provides valuable insights into the potential of microfluidic-produced PLLA porous microspheres and its further translational medicine.

Abstract Antibiotic resistance is a growing threat, underscoring the need to understand the underlying mechanisms. Aminoglycosides kill bacteria by disrupting translation fidelity, leading to the synthesis of aberrant proteins. Surprisingly, mutations in fusA, a gene encoding translation elongation factor G (EF-G), frequently confer resistance, even though EF-G neither participates in mRNA decoding nor blocks aminoglycoside binding. Here, we show that EF-G resistance variants selectively slow ribosome movement along mRNA when aminoglycosides are bound. This delay increases the chance that the drug dissociates before misreading occurs. Over several elongation cycles, this selective silencing of drug-bound ribosomes prevents error cluster formation, preserving proteome and membrane integrity. As a result, fusA mutations confer resistance early in treatment by preventing self-promoted aminoglycoside uptake. Translation on drug-free ribosomes remains sufficiently rapid to sustain near-normal bacterial growth. This previously unrecognized resistance mechanism— selective silencing of corrupted targets—reveals a novel antibiotic resistance strategy with potential therapeutic implications.

Abstract Encoding of several sensory modalities into neural signals is mediated by ribbon synapses. The synaptic ribbon tethers synaptic vesicles at the presynaptic active zone (AZ) and may act as a super-scaffold organizing AZ topography. Here we employed a synthetic biology approach to reconstitute structures mimicking ribbon-type AZs in HEK293 cells for probing minimal molecular requirements and studying presynaptic Ca2+ channel clustering. Co-expressing a membrane-targeted version of the AZ-protein Bassoon and the ribbon core protein RIBEYE, we observed structures recapitulating basic aspects of ribbon-type AZs, which we call synthetic ribbons or SyRibbons. Super-resolution STED microscopy and cryo-correlative electron tomography revealed SyRibbons were similar to native ribbons at AZs of cochlear inner hair cells in shape and size. SyRibbons with Ca2+ channel clusters formed upon additional expression of CaV1.3 Ca2+ channels and RIM-binding protein 2 (RBP2). CaV1.3 Ca2+ channel clusters associated with SyRibbons were larger than ribbonless CaV1.3 Ca2+ channels clusters and functional analysis by Ca2+-imaging in combination with patch clamp showed partial confinement of the Ca2+ signal at SyRibbons. In summary, we identify Ca2+ channels, RBP, membrane-anchored Bassoon, and RIBEYE as minimal components for reconstituting a basic ribbon-type AZ. SyRibbons might complement animal studies on molecular interactions of AZ proteins.

Abstract Study question What does a secreted oocyte-specific protein, ZP2, do inside mouse embryos during cleavage? Summary answer Contrary to prevalent notion, ZP2 protein synthesis is not silenced after ovulation, but goes on in preimplantation embryos to support the morula-blastocyst transition What is known already A tenet of reproductive biology is that the expression of oocyte-specific genes must remain silent in embryos. Among such genes, those of the ZP encode glycoproteins that are secreted until end of oogenesis and tasked with extracellular roles during folliculogenesis and fertilization. No further synthesis of ZP proteins is thought to occur after ovulation. However, already since the mouse gene knockout was reported in 2001 it was noticed that ZP2 null oocytes failed to develop to term even when fertilized correctly (PMID 11245577). Likewise, rabbit oocytes in which ZP4 was mutated at pronuclear stage featured defective embryogenesis (PMID 31635692). Study design, size, duration This is a treatment vs control study conducted in wild-type mice. For profiling of ZP2 protein expression,zygotes were produced in vivo and cultured 4 days in the presence of natural vs isotopic amino acids, and compared to embryos grown in vivo. For analysis of ZP2 function, zygotic knock-down of ZP2 was compared with that of a protein not present in wild-type embryos (green fluorescent protein, GFP), followed by transfer of both groups to uterus. Participants/materials, setting, methods B6C3F1 female mice were stimulated with gonadotropins and mated to CD1 males to produce zygotes. These were labeled continuously in vitro or in vivo using isotopic Arginine + Lysine, and sampled at consecutive stages for monoclonal immunofluorescence and mass spectrometry. To achieve knockdown of ZP2 during embryogenesis, zygotes were microinjected with Trim-Away reagents (PMID 29153837) and/or morpholino oligonucleotide anti-ZP2, followed by scoring of cleavage rates, blastocyst quality (germ layers) and transcriptome and proteome compositions. Main results and the role of chance The most abundant protein of the mouse zona pellucida, ZP2, not only was still present inside embryos up to blastocyst, but also became significantly more abundant in the 4-8 cell stage, as determined by direct immunofluorescence with monoclonal antibody (+25%, p < 0.05, Wilcoxon test). Mass spectrometry after isotopic labeling of protein synthesis during embryo culture confirmed that ZP2 incorporated the label, meaning: de novo protein synthesis of an oocyte-specific gene product in embryos. Zygotic knockdown via morpholino oligonucleotides and Trim-Away resulted in 50% reduction of ZP2 abundance at the 8-cell stage (p < 0.05, Wilcoxon test), accompanied by growth retardation at the 8-cell and morula stage. Embryos with depletion of ZP2 formed blastocysts with significantly smaller trophectoderm and primitive endoderm compartments. These effects were not observed in controls subjected to mock knockdown (GFP). Consistent with these morphometric data, transcriptome and proteome analyses returned ‘embryonic lethality’ and ‘embryonic growth retardation’ among the over-represented gene ontology terms of ZP2 (but not GFP) knockdown embryos. Thus, intra-embryonic ZP2 appears necessary to keep up the pace of cleavage progression and secure blastocyst fitness. This discovery can explain the historical conundrum of why ZP2 null oocytes formed blastocysts and yet these could not develop to birth (PMID 11245577). Limitations, reasons for caution Although there is sufficient evidence for expanding on the tasks of ZP proteins in mouse development, human oocytes contain ZP4 in addition to ZP1-3. While the novel tasks of ZP2 in mice hinge on de novo protein synthesis, it is unclear if this occurs also in humans. Wider implications of the findings Our study provides novel insight into the pathogenic mechanism of ZP gene mutations, adding a novel intracellular route to the longstanding extracellular roles. If ZP proteins function inside human oocytes and embryos, then using ICSI to overcome problems of thick or thin zona pellucida remains ineffective. Trial registration number No

Abstract Nuclear transport receptors (NTRs) carry cargo across the permeability barrier of nuclear pore complexes (NPCs) - an FG phase condensed from disordered but cohesive FG repeats. This phase repels ‘normal’ macromolecules but allows NTR passage. When HIV infects non-dividing cells, its capsid is transported into nuclei not like cargo but crosses NPCs like NTRs. We now uncovered the molecular determinants of the capsid’s NTR behavior: The FG-binding pocket is insufficient. Hexameric and pentameric capsomers contribute. The highly exposed outer capsid surface is key. It lacks ‘FG-repulsive’ charged residues (K,D,E) that are very abundant on ‘normal’ protein surfaces. ‘FG-attractive’ residues dominate the capsid surface. Introducing FG-repulsive ones impedes FG phase-partitioning, NPC-targeting and NPC-passage of assembled capsids. Capsids are thus made FG phase-soluble by myriads of transient FG-attractive interactions originating from individual surface sidechains. We discuss that CPSF6 releases the capsid from NPCs by switching its surface from FG-attractive to FG-repulsive.