Biology subjects

Kamel, M.

Publications and source records attributed to Kamel, M..

14 recordsLinked to original sources

bioRxiv2026

A dual sensor regulates P-glycoproteins structural plasticity

P-glycoprotein is an efflux pump with an exceptionally broad substrate profile which drives its profound clinical impact. Despite its biological importance, it remains obscure how P-glycoprotein achieves its polyspecificity and how substrate binding and the lipid environment stimulate its activity. Structural data highlight the importance of transmembrane helices 4 and 10, which surround the binding pocket, and identify them as key players in substrate recognition. Here, we used cryo-EM to study P-glycoprotein in detergent and nanodiscs to strategically leverage environment- and substrate-dependent phenotypes. This approach allowed us to decipher unexpected and distinct roles of transmembrane helices 4 and 10, which structurally explain differences in ATPase activity. Our data highlights helix 4 as an environment sensor and helix 10 as the key player in substrate recognition constituting a dual regulation mechanism for the functional plasticity of P-glycoprotein, and visualizes the intricate interplay between a membrane protein and its environment.

molecular biology↗

bioRxiv2025

Detection of five viruses commonly implicated with Bovine Respiratory Disease using loop-mediated isothermal amplification

Herein, we present novel quantitative loop-mediated isothermal amplification (qLAMP) and reverse-transcription qLAMP (RT-qLAMP) assays for the detection of five viruses commonly implicated with the onset and progression of bovine respiratory disease (BRD): Bovine Alphaherpesvirus Type 1 (BHV-1), Bovine Adenovirus Type 3 (BAV-3), Bovine Respiratory Syncytial Virus (BRSV), Bovine Viral Diarrhea Virus Type 1 (BVDV-1), and Bovine Parainfluenza Virus Type 3 (BPIV-3). Using contrived samples spiked with whole viruses, we found that our extraction-free assays have limits of detection between 30 and 1,057 copies per reaction (1.8% final sample concentration) with minimal sample processing. Using dual-tipped swabs and 1.4 mL resuspension volumes, these limits of detection are on the order of 2 x 105 copies per swab for BAV-3 and BHV-1 and between 6.31 x 106 to 8.22 x 106 copies per swab in the case of BPIV-3, BRSV, and BVDV-1. Analytical sensitivities ranged from 73 - 100% and analytical specificities ranged from 90 - 100%. Additionally, we introduced a streamlined pipeline to minimize the experimental workload to design, screen, select, and characterize LAMP performance for developing assays. The assays targeting these BRD viruses can be utilized to develop colorimetric LAMP assays that enable the sensitive and specific detection of these viruses chute side to aid in diagnosing and treating BRD. The associated development pipeline enables more rapid development of LAMP-based diagnostic tools targeting emerging pathogens.

bioengineering↗

bioRxiv2025

Development and Evaluation of Novel Quantitative PCR (qPCR) and Loop-Mediated Isothermal Amplification (LAMP) Assays for Bovine Adenovirus Type 7

Herein, we report the development of molecular assays for the specific detection of bovine adenovirus type 7 (BAV-7), a prevalent pathogen associated with bovine respiratory disease (BRD). To overcome the limitations of the current diagnostic methods, we developed and optimized a TaqMan quantitative polymerase chain reaction (qPCR) assay with a limit of detection (LOD) of 50 copies per reaction (10 copies/{micro}L) and 100% analytical specificity, showing no cross-reactivity with nine other viral pathogens commonly linked to BRD. Additionally, we designed a fluorescent quantitative loop-mediated isothermal amplification (qLAMP) assay demonstrating equivalent LOD and specificity. Both assays were evaluated using 24 bovine nasal swabs from BRD animals, revealing a 92% agreement between qPCR and qLAMP results. These assays provide powerful tools for advancing our understanding of BAV-7 epidemiology and improving disease management strategies. The qLAMP assay, with its rapid and isothermal amplification, presents potential for future development as a field-deployable diagnostic tool to enable timely detection of BAV-7 in cattle.

bioengineering↗

bioRxiv2025

Substrate-induced assembly and functional mechanism of the bacterial membrane protein insertase SecYEG-YidC

The Sec translocon and the YidC/Oxa1-type insertases universally mediate biogenesis of -helical membrane proteins, but the molecular basis of their cooperation has remained disputed. Recent discoveries of multi-subunit insertases assembled at the back of the translocon in fungi and higher eukaryotes have raised the question about the architecture and mechanism of the putative bacterial ortholog SecYEG-YidC. Here, we combine cryogenic electron microscopy with cell-free protein synthesis to visualize biogenesis of SecYEG/YidC-dependent multipass membrane protein NuoK. We demonstrate that the nascent chain of NuoK does not enter the lateral gate of SecYEG, but crosses over the translocon towards its back side, whereto YidC is recruited in the nascent chain-dependent manner. The SecY-YidC interface promotes folding of the transmembrane helices prior their insertion, in agreement with thermodynamic principles of membrane protein folding. YidC forms extensive contacts with the nascent chain, suggesting its key role in the insertion event. Our data provide detailed insights on the insertase machinery, suggest the evolutionary conservation of the gate-independent insertion route, and offer an expanded view on membrane protein biogenesis.

biochemistry↗

bioRxiv2024

ENACT: End-to-End Analysis of Visium High Definition (HD) Data

MotivationSpatial transcriptomics (ST) enables the study of gene expression within its spatial context in histopathology samples. To date, a limiting factor has been the resolution of sequencing based ST products. The introduction of the Visium High Definition (HD) technology opens the door to cell resolution ST studies. However, challenges remain in the ability to accurately map transcripts to cells and in assigning cell types based on the transcript data. ResultsWe developed ENACT, the first tissue-agnostic pipeline that integrates advanced cell segmentation with Visium HD transcriptomics data to infer cell types across whole tissue sections. Our pipeline incorporates novel bin-to-cell assignment methods, enhancing the accuracy of single-cell transcript estimates. Validated on diverse synthetic and real datasets, our approach is both scalable and effective offering a robust solution for spatially resolved transcriptomics analysis. Availability and implementationENACT source code is available at https://github.com/Sanofi-Public/enact-pipeline. Experimental data is available at https://zenodo.org/records/13887921. Supplementary information: Supplementary data are available at BiorXiv online.

bioinformatics↗

bioRxiv2024

A Paper-based Loop-Mediated Isothermal Amplification (LAMP) Assay for Highly Pathogenic Avian Influenza

Avian influenza outbreaks have had significant economic and public health consequences worldwide. Therefore, prompt, reliable, and cost-effective diagnostic devices are crucial for scrutinizing and confining highly pathogenic avian influenza viruses (HPAIVs). Our study introduced and evaluated a novel paper-based loop-mediated isothermal amplification (LAMP) test for diagnosing the H5 subtype of the avian influenza virus (AIV). We meticulously designed and screened LAMP primers targeting the H5-haemagglutinin (H5-HA) gene of AIV and fine-tuned the paper-based detection assay for best performance. The paper-based LAMP assay demonstrated a detection limit of 500 copies per reaction (25 copies/{micro}L). This inexpensive, user-friendly point-of-need diagnostic tool holds great promise, especially in resource-limited settings. It only requires a water bath for incubation and enables visual detection of results without special equipment. Overall, the paper-based LAMP assay provides a promising method for rapidly and reliably detecting the H5 subtype of AIV, contributing to improved surveillance and early intervention strategies.

bioengineering↗

bioRxiv2024

Polyamines regulate cell fate by altering the activity of histone-modifying enzymes

Polyamines are polycationic alkyl-amines abundant in proliferating stem and cancer cells. How these metabolites influence numerous cellular processes remains unclear. Here we show that polyamine levels decrease during differentiation and that inhibiting polyamine synthesis leads to a differentiated-like cell state. Polyamines are enriched in the nucleus, where their loss drives changes in chromatin accessibility and histone post-translational modifications. Polyamines interact electrostatically with DNA on the nucleosome core, freeing histone tails to conformations accessible to chromatin-modifying enzymes. Consistent with their role in increasing histone-tail accessibility, polyamines are able to replace MYCs role in reprogramming to pluripotency. These data reveal a mechanism by which an abundant metabolite influences chromatin structure and function in a direct but sequence independent manner, facilitating chromatin remodeling during reprogramming and limiting it during fate commitment.

cell biology↗

bioRxiv2024

The ABC transporter MsbA in a dozen environments

High-resolution structure determination of membrane proteins typically requires isolation from the native lipid bilayer and reconstitution into artificial membrane mimics. For this purpose, numerous detergents, amphipols, polymers and membrane scaffold proteins are available. The choice of the specific membrane substitute can strongly affect the proteins specific activity, stability and conformational spectrum, potentially leading to errors or misinterpretation during analysis. The bacterial ATP-binding cassette transporter MsbA is a prominent example of such environment-specific bias, resulting in apparent conformational and activity responses. Here, we present a systematic analysis of the conformational spectrum of MsbA, stabilized in a dozen environments, using cryo-EM. Our data show pronounced structural feedback of the ABC transporter to the respective membrane mimetics. Detergents generally favour a conformation with wide separation of the nucleotide-binding domains, while nanodiscs induce the narrow conformation. Notably, only three of the dozen tested environments allow MsbA to sample the functional conformational spectrum, enabling full movement of the nucleotide-binding domains between narrow and wide inward-facing conformations. We expect this study to serve as a blueprint for other membrane proteins, even where the structural reaction to the hydrophobic environment is not directly visible but still critical for the proteins function.

molecular biology↗

bioRxiv2024

The fungal protein Jps1 facilitates unconventional protein secretion through a direct phosphoinositide interaction

Protein secretion is indispensable for essential cellular processes in eukaryotic cells, contributing significantly to nutrient acquisition, defense or communication. Alternative pathways bypassing the endomembrane system collectively referred to as unconventional secretion are gaining increasing attention. A number of important molecules such as cytokines, fibroblast growth factor or viral proteins are being exported through these mechanistically diverse pathways. In the fungal model Ustilago maydis, cytokinesis-dependent unconventional secretion mediates export of the chitinase Cts1 via the fragmentation zone. This membrane-rich compartment is formed during cytokinesis between mother and daughter cells. Recently, we identified Jps1, a previously uncharacterized protein, as a crucial factor for Cts1 localization and export. Combining biochemical experiments and in vivo studies, we here uncover two pivotal features of Jps1: dimerization and phosphoinositide (PIP) binding. Our findings reveal that a conserved structural core domain mediates homodimerization, while surrounding flexible variable regions suggest potential diversification in different basidiomycete species. Jps1 does not harbor a canonical PIP-binding domain but instead specificity of the interaction with the preferred PIP PI(4,5)P2 is determined by basic residues. Importantly, loss of PI(4,5)P2 binding specificity results in mis-localisation, morphological defects and reduced extracellular Cts1 activity, particularly at low cell densities. This discovery sheds light on previously unknown key features of Jps1, elucidating its role in supporting Cts1 secretion, and representing a crucial step towards understanding the broader implications of unconventional secretion in eukaryotic cells.

biochemistry↗

bioRxiv2023

Emergence of the Novel Infectious bursal disease viruse Variant in Vaccinated Poultry Flocks in Egypt

Infectious bursal disease viruses (IBDVs) have a profound impact on poultry production worldwide, directly causing mortality rates of up to 100%, and indirectly through their immunosuppressive effects. Since the emergence of the antigenically modified very virulent IBDV (vvIBDV) in Egypt in late 1999, the country has experienced recurrent outbreaks with high mortality rates and typical vvIBDV gross lesions. However, a notable shift occurred in 2023, characterized by a substantial increase in reported subclinical IBDV cases exhibiting atrophied bursa and associated immunosuppression. To assess the field situation, we examined samples from 21 farms in 2023 and 18 farms from 2021 and 2022, all of which experienced IBD outbreaks based on clinical diagnosis. These samples were submitted to our laboratory for confirmatory testing and subsequently subjected to VP2-HVR sequencing. Phylogenetic analysis revealed that all samples collected in 2021 and 2022 clustered with classical virulent strains and very virulent IBDV. In 2023, one sample clustered with the Egyptian vvIBDV, while one sample clustered with classic virulent IBDV, and the remaining 2023 samples clustered with the Chinese novel variant IBDV (nVarIBDV). The alignment of deduced amino acid sequences for VP2 revealed that all Egyptian classic virulent strains were similar to the Winterfield or Leukert strains. In contrast, vvIBDV strains exhibited two out of the three typical residues found in Egyptian antigenically atypical vvIBDV, namely Y220F and G254S, but not A321T, and one sample was identical to the European vvIBDV (emerged in 1989). Meanwhile, all variant strains recognized in the present study exhibited typical residues found in variant IBDV, in addition to the three conserved amino acid residues found only in Chinese variant IBDVs. However, all Egyptian variant strains showed a mutation at position 321 (321V), which represents the most exposed part of the capsid and is known to have a massive impact on IBDV antigenicity, with the exception of one sample that had 318G instead. This report highlights the emergence of a new variant IBDV clustered with the Chinese new variant in Egypt, causing bursa atrophy and spreading subclinically in broiler farms over a wide geographic distance, resulting in massive economic losses due to immunosuppression.

microbiology↗

bioRxiv2023

Adaptive capacity of a DNA polymerase clamp-loader ATPase complex

The ability of mutations to facilitate adaptation is central to evolution. To understand how mutations can lead to functional adaptation in a complex molecular machine, we created a defective version of the T4 clamp-loader complex, which is essential for DNA replication. This variant, which is [~]5000-fold less active than the wildtype, was made by replacing the catalytic domains with those from another phage. A directed-evolution experiment revealed that multiple substitutions to a single negatively-charged residue in the chimeric clamp loader - Asp 86 - restore fitness to within [~]20-fold of wildtype. These mutations remove an adventitious electrostatic repulsive interaction between Asp 86 and the sliding clamp. Deep mutagenesis shows that the reduced fitness of the chimeric clamp loader is compensated for by lysine and arginine substitutions of several DNA-proximal residues in the clamp loader or the sliding clamp. Thus, the fitness decrease of the chimeric clamp loader is caused by a reduction in affinity between the clamp loader and the clamp. Our results demonstrate that there is a latent capacity for increasing affinity of the clamp loader for DNA and the sliding clamp, such that even single point mutations can readily compensate for the loss of function due to suboptimal interactions elsewhere.

biochemistry↗

bioRxiv2021

A single-cell atlas of de novo β-cell regeneration reveals the contribution of hybrid β/δ cells to diabetes recovery in zebrafish

Regeneration-competent species possess the ability to reverse the progression of severe diseases by restoring the function of the damaged tissue. However, the cellular dynamics underlying this capability remain unexplored. Here, we use single-cell transcriptomics to map de novo {beta}-cell regeneration during induction and recovery from diabetes in zebrafish. We show that the zebrafish has evolved two distinct types of somatostatin-producing {delta}-cells, which we term {delta}1- and {delta}2-cells. Moreover, we characterize a small population of glucose-responsive islet cells, which share the hormones and fate-determinants of both {beta}- and {delta}1-cells. The transcriptomic analysis of {beta}-cell regeneration reveals that {beta}/{delta} hybrid cells constitute a prominent source of insulin-expression during diabetes recovery. Using in vivo calcium imaging and cell tracking, we further show that the hybrid cells form de novo and acquire glucose-responsiveness in the course of regeneration. The overexpression of dkk3, a gene enriched in hybrid cells, increases their formation in the absence of {beta}-cell injury. Finally, interspecies comparison shows that plastic {delta}1-cells are partially related to PP-cells in the human pancreas. Our work provides an atlas of {beta}-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish

developmental biology↗

bioRxiv2021

Novel intracellular phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly

Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and multiresistance against antibiotics. This bacterium produces and secretes various toxins with hydrolytic activities including phospholipases A, C and D. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. PaPlaB isolated from detergent-solubilized membranes of E. coli rapidly degraded various GPLs including endogenous GPLs isolated from P. aeruginosa cells. Cellular localization studies suggest that PaPlaB is peripherally bound to the inner and outer membrane of E. coli, yet the active form was predominantly associated with the cytoplasmic membrane. In vitro activity of purified and detergent-stabilized PaPlaB increases at lower protein concentrations. The size distribution profile of PaPlaB oligomers revealed that decreasing protein concentration triggers oligomer dissociation. These results indicate that homooligomerisation regulates PaPlaB activity by a yet unknown mechanism, which might be required for preventing bacteria from self-disrupting the membrane. We demonstrated that PaPlaB is an important determinant of the biofilm lifestyle of P. aeruginosa, as shown by biofilm quantification assay and confocal laser scanning microscopic analysis of biofilm architecture. This novel intracellular phospholipase B with a putative virulence role contributes to our understanding of membrane GPL degrading enzymes and may provide a target for new therapeutics against P. aeruginosa biofilms.

biochemistry↗

bioRxiv2021

Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon

The translocon SecYEG forms the primary protein-conducting channel in the cytoplasmic membrane of bacteria, and the associated ATPase SecA provides the energy for the transport of secretory and cell envelope protein precursors. The translocation requires negative charge at the lipid membrane surface, but its dependence on the properties of the membrane hydrophobic core is not known. Here, we demonstrate that SecA:SecYEG-mediated protein transport is immensely stimulated by unsaturated fatty acids (UFAs). Furthermore, UFA-rich tetraoleoyl-cardiolipin, but not bis(palmitoyloleoyl)-cardiolipin, facilitate the translocation via the monomeric translocon. Biophysical analysis and molecular dynamics simulations show that UFAs determine the loosely packed membrane interface, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced manifold at elevated ionic strength. Tight SecA:lipid interactions convert into the augmented translocation. As bacterial cells actively change their membrane composition in response to their habitat, the modulation of SecA:SecYEG activity via the fatty acids may be crucial for protein secretion over a broad range of environmental conditions.

biophysics↗