Biology subjects

Darula, Z.

Publications and source records attributed to Darula, Z..

9 recordsLinked to original sources

bioRxiv2026

Novel Synthetic Polymyxin Variants Inspired by Newly Uncovered Natural Sequences Explored as Potential Antibiotics

The incidence of infections caused by multidrug-resistant bacterial agents has increased at an alarming rate worldwide. With the aim of identifying novel antimicrobial peptides (AMPs), we screened bacteria isolated from various environmental samples. Our hypothesis was that the sequence space of natural AMPs belonging to known AMP classes is far from saturation. We used a classical pipeline of bacterial culturing, overlay assays, extraction and fractionation to purify AMPs for identification. Using LC/MS analysis, the structure of AMPs isolated from a strain of Paenibacillus was narrowed down and a new subclass of polymyxins was uncovered. These harbor three aliphatic residues within the cyclic C-terminal, and in certain cases Ser replaces Thr at position A2. Four discrete polymyxins fitting the new class, but not identical to the natural polymyxins were synthesized and tested against 29 human pathogenic bacteria. Each displayed an antibacterial spectrum different from that of colistin, with the best candidate surpassing it in potency against ten bacterial strains, but underperforming against three others. These results demonstrate that current screening of natural bacterial isolates can still permit the design of novel antimicrobial peptide variants without the substantial threat of diminishing returns.

microbiology↗

bioRxiv2026

Direct anti-inflammatory actions of N,N-dimethyltryptamine on microglia are revealed by proteomic profiling and receptor pharmacology

N,N-dimethyltryptamine (DMT) is an endogenous psychedelic tryptamine that has recently emerged as a promising therapeutic candidate for acute ischemic stroke. Although DMT consistently reduces infarct size, attenuates neuroinflammation, and improves functional outcome in experimental stroke, the cellular and receptor mechanisms underlying these effects remain poorly understood. Primary rat microglial cultures were used to examine the direct anti-inflammatory effects of DMT following lipopolysaccharide (LPS)-induced activation. Microglial morphology, phagocytosis, and proteomic alterations were analyzed. Radioligand binding assays determined the affinity of DMT for microglial sigma-1 receptors (Sig-1Rs). Pharmacological inhibition of Sig-1Rs and serotonin (5-HT) receptors was performed to define receptor-specific mechanisms. Translational relevance was evaluated in acute mouse brain slices subjected to mild oxygen-glucose deprivation (mOGD) and anoxic episodes, where microglial activation, spreading depolarizations (SDs), and neuronal injury were assessed. DMT directly suppressed LPS-induced microglial activation, promoted a homeostatic morphology, and reduced phagocytic activity. Proteomic profiling demonstrated that DMT selectively reprogrammed inflammatory pathways by suppressing proteins involved in cytokine and chemokine signaling and oxidative stress while largely preserving arachidonic acid-prostaglandin synthesis. DMT bound microglial Sig-1Rs with micromolar affinity comparable to that reported in whole-brain preparations. Pharmacological inhibition revealed that DMT-induced morphological reprogramming required both Sig-1R and serotonergic signaling, whereas suppression of phagocytosis was largely independent of either receptor pathway. In acute brain slices, DMT attenuated microglial activation, reduced SD propagation and ischemic neuronal injury, and tissue-level neuroprotection depended on serotonergic signaling. DMT directly targets microglia and selectively remodels inflammatory states rather than broadly suppressing microglial activation. The receptor mechanisms underlying its actions are context dependent, with Sig-1R and serotonergic signaling contributing differentially according to the cellular response and experimental model. These findings provide mechanistic insight into the neuroprotective actions of DMT and support its ongoing clinical translation as a potential therapy for ischemic stroke.

neuroscience↗

bioRxiv2026

Rapid direct neuronal reprogramming of human dental pulp stem cells

Direct neuronal reprogramming offers an alternative to induced pluripotent stem cell-based differentiation by converting somatic cells directly into neurons without passage through pluripotency. However, commonly used fibroblast-based protocols are often slow and inefficient. Here, we evaluated human dental pulp stem cells (DPSCs), which originate from the cranial neural crest and possess intrinsic neurogenic potential, as a developmentally relevant source of induced neurons (iNs). Using an all-in-one lentiviral vector, we converted DPSCs into iNs within 17 days, compared with 28 days for fibroblasts reprogrammed with the same vector, and achieved significantly higher neuronal purity under the respective established protocols. Multi-omic profiling revealed coordinated suppression of mesenchymal and cell-cycle programs and induction of neuronal, synaptic, and metabolic pathways. Single-nucleus RNA sequencing resolved fibroblast-like, transitional, maturing neuronal, GABAergic-like, and alternative fates, while trajectory inference suggested divergent neuronal and non-neuronal conversion paths. Whole-cell recordings showed that a subset of DPSC-iNs developed early neuronal excitability and voltage-gated inward and outward currents. Together, our findings establish DPSCs as an accessible and developmentally relevant source for rapid direct neuronal conversion. This integrated molecular, single-nucleus, and electrophysiological characterization defines the cellular heterogeneity of DPSC-to-neuron reprogramming and provides a framework for protocol refinement and future patient-specific disease modeling.

neuroscience↗

bioRxiv2026

Flightless I and LRRFIP work together to regulate lateral growth of the sarcomeres in Drosophila

Myofibrils are structurally highly conserved elements of the cardiac and striated muscles. The striking commonalities in their basic organization principles emerge during myofibrillogenesis when the initially formed premyofibrils grow in length and width to attain their final dimensions, characteristic of each muscle type. Although the molecular composition of the myofibrils is well known, the mechanisms governing their growth remain poorly understood, particularly those driving peripheral thickening. Here, we show that two cardiac disease associated proteins, Flightless I and LRRFIP, are required for lateral integration of the myofilaments that is essential for circumferential myofibril growth in the Drosophila flight muscle. Genetic and biochemical analysis reveal that these proteins form multimeric complexes enriched at the barbed end of the actin filaments. Furthermore, we found that the Flightless I/LRRFIP complex acts in a formin dependent manner. Together, our findings demonstrate that Flightless I and LRRFIP cooperate to promote the spatially controlled integration of peripheral actin filaments at the Z-disc, uncovering a key step in myofibril maturation.

cell biology↗

bioRxiv2026

Unraveling the Regenerative Proteomic Signature of Helix aspersa's Slime in Human Dermal Fibroblasts by Data-driven Proteomics Approach

Proteins orchestrate essential cellular processes, including metabolism, communication, survival, and regeneration, making proteomic profiling a powerful strategy to elucidate complex biological responses. Snail slime (SnS) has emerged as a bioactive material with documented pro-healing, antioxidant, and anti-inflammatory properties; however, its effects at the proteome level in normal human dermal fibroblasts (NHDFs) remain unexplored. In this study, an LC-MS-based proteomic approach (Data are available via ProteomeXchange with identifier PXD075292) combined with network and Gene Ontology enrichment analyses was employed to investigate SnS-induced molecular reprogramming in NHDFs, followed by functional assays. Results show that SnS is well tolerated for up to 72 h, confirming its cytocompatibility, followed by proteomic analysis revealing enrichment of biological processes related to apoptosis regulation, oxidative stress response, wound healing, cell migration, and anti-aging. Network analysis identified AKT, PI3K, SRC, and KRAS family members as key hub proteins, indicating convergence on central signaling pathways controlling survival, redox balance, and migratory activity. Functional assays demonstrated a time-dependent, controlled modulation of apoptosis consistent with cellular turnover, alongside a hormetic redox response characterized by transient ROS signaling followed by enhanced antioxidant capacity. Importantly, SnS significantly accelerated fibroblast migration, achieving complete wound closure within 24 h. Collectively, these findings demonstrate that SnS induces coordinated proteomic and functional reprogramming that integrates redox modulation, controlled apoptosis, and enhanced migration, providing a mechanistic basis for its pro-healing and anti-aging effects and supporting its potential as a regenerative biomaterial.

cell biology↗

bioRxiv2025

Phosphoproteomic profiling reveals post-translational dysregulation in Huntington's disease patient-derived neurons

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin (HTT) gene. Although transcriptomic and proteomic changes have been characterized in patient-derived neurons, the contribution of post-translational modifications (PTMs), such as phosphorylation, remains poorly understood. Here, we present the first phosphoproteomic analysis by mass spectrometry (P-MS) of human induced neurons (iNs) directly reprogrammed from HD patient fibroblasts. We identified 177 phosphopeptides with significantly altered abundance in HD-iNs, mapping to phosphoproteins associated with key signaling pathways known to be affected in HD, such as splicing and autophagy. By integrating P-MS data with previously published proteomic and transcriptomic data from the same donors, we identified distinct subsets of ON-OFF phosphopeptides that exhibited a complete loss of phosphorylation in either HD- or Ctrl-iNs, without corresponding changes at the RNA or protein level. An exception was MXRA8, previously described in glial cells as a mediator of blood-brain barrier integrity and astrocyte-mediated neuroinflammation. This protein showed increased protein abundance despite the absence of phosphorylation in HD- iNs, suggesting a compensatory mechanism - a pattern also observed in human post mortem cortical HD tissue. Additionally, MXRA8 showed altered protein-protein interactions with lysosomal and metabolic regulators in HD-iNs, highlighting its potential role in autophagy impairment as well as in neurovascular dysfunction. These findings uncover a distinct layer of post-translational dysregulation in HD, suggesting that phospho-switch proteins such as MXRA8 may be candidate effectors of pathology and thus site-specific phosphorylation loss may contribute to impaired signaling and proteostasis in human HD neurons.

neuroscience↗

bioRxiv2024

Assessing Heterogeneity in the N-Telopeptides of Type I Collagen by Mass Spectrometry

Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.

biochemistry↗

bioRxiv2020

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

Band 3 (anion exchanger 1 - AE1) is the most abundant membrane protein in red blood cells (RBCs), the most abundant cell in the human body. A compelling model, based on indirect evidence, posits that - at high oxygen saturation - the N-term cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during RBC aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of blood storage quality and efficacy of transfusion - a life-saving intervention for millions of recipients worldwide. Here we leverage two murine models carrying genetic ablations of AE1 to provide the first direct mechanistic evidence of its role in metabolic regulation and blood storage quality. Observations in mice phenocopied those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and cross-linking proteomics, we provide the first comprehensive analysis of the RBC interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Finally, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recoveries of stored RBCs from healthy human donors and genetically ablated mice, paving the way for the in vivo metabolic manipulation of RBCs facing oxidant stress - a landmark of many diseases. Key pointsO_LIGenetic ablation of N-term of band 3 results in significant metabolic aberrations and poor post-transfusion recoveries in mice and humans; C_LIO_LIStructural studies on the N-term of band 3 reveal a complex interactome with several enzymes, including GAPDH; C_LI

biochemistry↗

bioRxiv2019

Nectar- and stigma-specific expression of a chitinase could partially protect against fire blight in certain apples

To attract pollinators many angiosperms secrete stigma exudate and nectar in their flowers. As these nutritious fluids are ideal infection points for pathogens, both secretions contain various antimicrobial compounds. Erwinia amylovora, the causing bacterium of the devastating fire blight apple disease, is the model pathogen that multiplies in flower secretions and infects through the nectaries. Although Erwinia resistant apples are not available, certain cultivars are tolerant. It was reported that in stigma infection assay, the Freedom cultivar was Erwinia tolerant while the Jonagold was susceptible. We hypothesized that differences in the nectar protein compositions lead to different susceptibility. Indeed we found that an acidic chitinase III protein (Machi3-1) selectively accumulates in the nectar and stigma of the Freedom cultivar. We demonstrate that MYB binding site containing repeats of the Freedom Machi3-1 promoter are responsible for the strong nectar- and stigma-specific expression. As we found that in vitro the Machi3-1 protein impairs growth and biofilm formation of Erwinia at physiological concentration, we propose that the Machi3-1 contribute to the tolerance by inhibiting Erwinia multiplication in the stigma exudate and in the nectar. We show that the Machi3-1 allele was introgressed from Malus floribunda 821 into different apple cultivars including the Freedom.\n\nHighlightCertain apple cultivars accumulate to high levels in their nectar and stigma an acidic chitinase III protein that can protect against pathogens including fire blight disease causing Erwinia amylovora

plant biology↗