Biology subjects

Rahmani, A.

Publications and source records attributed to Rahmani, A..

11 recordsLinked to original sources

bioRxiv2026

Synaptic proteomics identifies cathepsin B as a regulator of synapse remodelling during learning

Learning requires dynamic changes in synaptic protein composition. Most synaptic proteomic studies capture endpoint snapshots following training, overlooking molecular changes occurring during learning itself. We previously established TurboID proximity proteomics as an approach to capture protein-level changes during learning in the nervous system of Caenorhabditis elegans. Here, we apply this strategy to synapses. Trained synapses exhibited a distinct proteome compared with mock-trained controls, with pathways enriched for neurotransmission, synaptic reorganisation, protein trafficking, and autophagy. Enrichment of autophagy proteins led us to investigate the cathepsin B orthologue CPR-4, a previously uncharacterised candidate for learning identified exclusively in trained synapses. Functional validation identified CPR-4 as a novel regulator of memory: CPR-4 is required for learning, and the absence of CPR-4 results in trained synaptic proteomes that are more like those of mock-trained synapses. Our findings support a model through which CPR-4 promotes synaptic remodelling required for learning through targeted protein clearance.

neuroscience↗

bioRxiv2026

Enhanced Pathogenicity and Contact Transmissibility of Human-origin Avian Influenza H5N1 Clade 2.3.4.4b Genotype B3.13 Compared to D1.1 in Ferrets

Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness. SignificanceInfluenza H5N1 viruses continue to diversify genetically while expanding into mammalian hosts, increasing opportunities for viral adaptation and zoonotic transmission, including humans. However, whether the predominant clade 2.3.4.4b genotype differs in its capacity to infect, transmit, and evolve in mammals remains poorly understood. Using the ferret model of influenza infection and transmission, we demonstrated that the currently circulating B3.13 and D1.1 genotypes exhibit distinct pathogenic and transmission characteristics despite retaining similar receptor-binding characteristics, NA functions, and antigenic profiles. While B3.13 readily infects and transmits in ferrets and does not develop further adaptive mutations associated with increased replication and transmission, D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential. These findings show that genotype-specific biological properties can influence zoonotic risk independently of antigenic similarity and emphasize the importance of integrating phenotypic characterization with genomic surveillance to improve pandemic preparedness and guide public health risk assessment.

microbiology↗

bioRxiv2026

A One-Shot Multivalent Live-Attenuated Candidate Influenza Vaccine against Divergent Zoonotic H5N1 Clades

The continued emergence of genetically diverse high pathogenicity avian influenza (HPAI) H5N1 viruses with zoonotic potential highlights the urgent need for developing vaccines capable of providing broad protection against multiple circulating clades. Here, we developed a one-shot, multivalent, live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive (ts), cold-adapted (ca), and attenuated (att) influenza A/Ann Arbor/6/1960 master donor virus (MDV) that incorporates the hemagglutinin (HA) and neuraminidase (NA) glycoproteins from representative clades 2.3.4.4b (A/Louisiana/12/2024), 2.3.2.1a (A/Victoria/149/2024), and 2.3.2.1e (A/Cambodia/2302009/2023) H5N1 viruses. A single intranasal (IN) immunization of C57BL/6 mice with the multivalent LAIV elicited robust humoral immune responses, with immune sera exhibiting broad cross-reactivity against antigens from all three H5N1 clades included in the vaccine. Following homologous viral challenge, vaccinated C57BL/6 mice were completely protected from disease, demonstrating the immunogenicity and protective efficacy of the multivalent LAIV. By simultaneously targeting antigenically distinct H5N1 lineages with pandemic potential, this strategy expands antigenic coverage within a single LAIV to confirm pan-H5N1 protection. Together, these findings support the development and implementation of this multivalent LAIV as a broadly protective pan-H5N1 LAIV for pandemic preparedness. SignificanceThe increasing genetic diversity of zoonotic H5N1 viruses complicates vaccine design. We developed a multivalent live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive, cold-adapted, and attenuated (ts, ca, att) master donor virus (MDV) influenza A/Ann Arbor/6/1960 backbone that expresses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins of H5N1 clades 2.3.4.4b, 2.3.2.1a, and 2.3.2.1e. A single intranasal (IN) immunization with the multivalent LAIV induced broadly cross-reactive neutralizing antibody (NAb) responses and protected experimental vaccinated animals against homologous lethal viral challenge, demonstrating the feasibility of the multivalent LAIV to protect against H5N1 clades of highest concern to humans. These findings demonstrate the feasibility of developing and implementing this multivalent LAIV as a broad protective pan-H5N1 LAIV against divergent H5N1 viruses for human use.

microbiology↗

bioRxiv2026

Antarctic fish cell cultures show adaptation of organelle morphology and dynamics to extreme cold

In the Antarctic Southern Ocean, cold-blooded animals have evolved to live at stable temperatures of 0{+/-}2 {degrees}C. This extremely low temperature affects their biology at every scale, from protein folding to development. However, how animal (sub)cellular organisation and dynamics are adapted to near-0 {degrees}C temperatures has not been studied. We therefore established methods to culture and fluorescently label cells from the Antarctic plunderfish Harpagifer antarcticus and a temperate comparator species, the shanny Lipophrys pholis. By imaging these cultures live at physiological temperatures, we found that subcellular organisation is broadly conserved in H. antarcticus, featuring known membranous organelles and biomolecular condensates that remain dynamic, with mitochondria in H. antarcticus and L. pholis moving at similar speeds. However, we also identified differences in organelle properties between H. antarcticus and L. pholis, including lysosomal enlargement and mitochondrial morphology changes. These differences may be functionally linked to protein misfolding and slow embryonic development in Antarctic species.

cell biology↗

bioRxiv2025

Single-molecule flow cytometry

Flow cytometry (FC) is a powerful tool for high-throughput characterisation of cell populations, yet its ensemble fluorescence detection and the autofluorescence of cells impose a sensitivity limit of [~]100-1,000 fluorophores per cell. This precludes detection of low-abundance proteins and weak signalling events with biological importance and therapeutic potential. We introduce single-molecule Flow Cytometry (smFC), integrating high-numerical aperture oblique plane microscopy (OPM) with microfluidics to achieve optical sectioning and photon collection efficiencies suitable for single-molecule detection on flowing cells. Using super-bright, large Stokes shift fluorophores, smFC can detect labelled membrane proteins on cells with digital precision down to [~]2 molecules per cell, compared to an unlabelled control. This represents an improvement in the detection limit of 10- to 80-fold depending on the probe. We apply smFC to quantify the distribution of the membrane receptor c-kit in triple-negative breast cancer cells with unprecedented sensitivity. This revealed a previously unidentified distribution of 60% c-kit negative cells and 40% with low-abundance but heterogenous (1-200 mol/cell) surface distribution that is undetectable by conventional FC. These results establish smFC as a robust platform for high-throughput, quantitative single-molecule analysis in cell populations, opening new avenues for detecting rare biomarkers and quantifying the presence of low-abundance membrane proteins and targetable surface molecules with digital precision.

biophysics↗

bioRxiv2024

Active Remote Focus Stabilization in Oblique Plane Microscopy

Light-sheet fluorescence microscopy (LSFM) has demonstrated great potential in the life sciences owing to its efficient volumetric imaging capabilities. For long term imaging, the light-sheet typically needs to be stabilized to the detection focal plane for the best imaging results. Current light-sheet stabilization methods rely on fluorescence emission from the sample, which may interrupt the scientific imaging and add to sample photobleaching. Here we show that for oblique plane microscopes (OPM), a subset of LSFM where a single primary objective is used for illumination and detection, light-sheet stabilization can be achieved without expending sample fluorescence. Our method achieves ~43nm axial precision and maintains the light-sheet well within the depth of focus of the detection system for hour-long acquisition runs in a lab environment that would otherwise detune the system. We demonstrate subcellular imaging of the actin skeleton in melanoma cancer cells with a stabilized OPM.

biophysics↗

bioRxiv2024

Identifying regulators of associative learning using a protein-labelling approach in C. elegans

The ability to learn and form memories is critical for animals to make choices that promote their survival. The biological processes underlying learning and memory are mediated by a variety of genes in the nervous system, acting at specific times during memory encoding, consolidation, and retrieval. Many studies have utilised candidate gene approaches or random mutagenesis screens in model animals to explore the key molecular drivers for learning and memory. We propose a complementary approach to identify this network of learning regulators: the proximity-labelling tool TurboID, which promiscuously biotinylates neighbouring proteins, to snapshot the proteomic profile of neurons during learning. To do this, we expressed the TurboID enzyme in the entire nervous system of C. elegans and exposed animals to biotin only during the training step of an appetitive gustatory learning paradigm. Our approach revealed hundreds of proteins specific to trained worms, including components of molecular pathways previously implicated in memory in multiple species such as insulin signalling, G protein-coupled receptor signalling, and MAP kinase signalling. Most (87-95%) of the proteins identified are neuronal, with relatively high representation for neuron classes involved in locomotion and learning. We validated several novel regulators of learning, including cholinergic receptors (ACC-1, ACC-3, LGC-46) and putative arginine kinase F46H5.3. These previously uncharacterised learning regulators all showed a clear impact on appetitive gustatory learning, with F46H5.3 showing an additional effect on aversive gustatory memory. Overall, we show that proximity labelling can be used in the brain of a small animal as a feasible and effective method to advance our knowledge on the biology of learning.

neuroscience↗

bioRxiv2024

Astigmatism-based focus stabilisation with universal objective lens compatibility, extended operating range and nanometre precision

Focus stabilisation is vital for long-term fluorescence imaging, particularly in the case of high-resolution imaging techniques. Current stabilisation solutions either rely on fiducial markers that can be perturbative, or on beam reflection monitoring that is limited to high-numerical aperture objective lenses, making multimodal and large-scale imaging challenging. We introduce a beam-based method that relies on astigmatism, which offers advantages in terms of precision and the range over which focus stabilisation is effective. This approach is shown to be compatible with a wide range of objective lenses (10x-100x), typically achieving <10 nm precision with >10 m operating range. Notably, our technique is largely unaffected by pointing stability errors, which in combination with implementation through a standalone Raspberry Pi architecture, offers a versatile focus stabilisation unit that can be added onto most existing microscope setups.

biophysics↗

bioRxiv2023

Increased beta2-adrenergic signaling is a targetable stimulus essential for bone healing by promoting callus neovascularization

Traumatic brain injury (TBI) is associated with a hyperadrenergic state and paradoxically causes systemic bone loss while accelerating fracture healing. Here, we identify the beta2-adrenergic receptor (Adrb2) as a central mediator of these skeletal manifestations. While the negative effects of TBI on the unfractured skeleton can be explained by the established impact of Adrb2 signaling on bone formation, Adrb2 promotes neovascularization of the fracture callus under conditions of high sympathetic tone, including TBI and advanced age. Mechanistically, norepinephrine stimulates the expression of Vegfa and Cgrp primarily in periosteal cells via Adrb2, both of which synergistically promote the formation of osteogenic type-H vessels in the fracture callus. Accordingly, the beneficial effect of TBI on bone repair is abolished in mice lacking Adrb2 or Cgrp, and aged Adrb2-deficient mice without TBI develop fracture nonunions despite high bone formation in uninjured bone. Pharmacologically, the Adrb2 antagonist propranolol impairs, and the agonist formoterol promotes fracture healing in aged mice by regulating callus neovascularization. Clinically, intravenous beta-adrenergic sympathomimetics are associated with improved callus formation in trauma patients with long bone fractures. Thus, Adrb2 is a novel target for promoting bone healing, and widely used beta-blockers may cause fracture nonunion under conditions of increased sympathetic tone. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/548550v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@cea57corg.highwire.dtl.DTLVardef@d29642org.highwire.dtl.DTLVardef@186ea5aorg.highwire.dtl.DTLVardef@83eaae_HPS_FORMAT_FIGEXP M_FIG Artwork was created in BioRender. C_FIG

cell biology↗

bioRxiv2022

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

Tpp49Aa1 from Lysinibacillus sphaericus is a Toxin_10 family protein that - in combination with Cry48Aa1, a 3-domain crystal protein - has potent mosquitocidal activity, specifically against Culex quinquefasciatus mosquitoes. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser, allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 1.62 [A] resolution from native nanocrystals. This revealed the packing of Tpp49Aa1 within these nanocrystals, isolated from sporulated bacteria, as a homodimer with a large intermolecular interface, shedding light on natural crystallization. Complementary experiments conducted at varied pH also enabled investigations of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals. Using modelling, we propose a potential interaction between Tpp49Aa1 and Cry48Aa1 that may play a role in their codependency and broaden our understanding of this two-component system. We expand the known target range, demonstrating Tpp49Aa1/Cry48Aa1 susceptibility of larvae from Anopheles stephensi, Aedes albopictus and Culex tarsalis - substantially increasing the potential use of this toxin pair in mosquito control. Further functional insights are gained using Culex cell lines to characterise cellular models for future investigations into Cry48Aa1/Tpp49Aa1 mechanism of action and to demonstrate transient detrimental effects of individual toxin components. Significance StatementThe Tpp49Aa1/Cry48Aa1 protein pair kills mosquito larvae. Innovative use of nano-focused X-ray free electron laser to match the size of natural Tpp49Aa1 nanocrystals and the highest beam intensity available in any XFEL for high-throughput data collection, allowed structural resolution to 1.62 [A]. Tpp proteins show a range of interactions with different partners to elicit toxicity. To gain insight into Tpp49Aa1, its interaction with Cry48Aa1 was modelled. We also establish cell-based assays of Tpp49Aa1/Cry48Aa1 activity. We expand the known target range to include three more mosquito species: Anopheles stephensi, Aedes albopictus and Culex tarsalis. This study will underpin future Tpp mode of action investigations and aid insecticide optimization against mosquito vectors of emerging diseases such as West Nile Virus and malaria.

biochemistry↗

bioRxiv2021

An extension of Wang protein design model using Blosum62 substitution matrix

Humans life depends on the functionality of molecules in the body. One of these essential molecules is the protein that plays a vital role in our life, such that its malfunction can cause severe damages. Such roles make protein structure and its functionality necessary to understand. One of the problems that help us understand the relation between protein structure is the well-known protein design problem which attempts to find an amino acid sequence that can fold into a desired tertiary structure. However, despite having an acceptable accuracy in protein design, this accuracy is an identical percentage of amino acid retrieving. At the same time, it is well-known that amino acids can replace each other in evolution while the function and structure of protein stay the same. Thus the designed sequence does not have the opportunity to be close to the target in the evolutionary aspect. This paper presents an extension to Wangs deep learning model, which uses evolutionary information in the Blosum62 substitution matrix to take amino acid replacement probability into account while designing a sequence.

bioinformatics↗