Biology subjects

Javed, A.

Publications and source records attributed to Javed, A..

18 recordsLinked to original sources

bioRxiv2026

Azacitidine Response in Myelodysplastic Syndromes is Marked by NK-like CD8 T-Cell Expansion and CXCL12+ Reticular Cell Remodeling

Myelodysplastic syndromes (MDS) are driven by somatic mutations in hematopoietic stem and progenitor cells (HSPCs), leading to clonal expansion and ineffective hematopoiesis. Hypomethylating agents (HMAs; azacitidine or decitabine) are the standard of care for higher-risk MDS. However, their effects on the bone marrow (BM) microenvironment, and the extent to which these changes correlate with clinical response, remain poorly understood. We performed longitudinal analyses of BM aspirates, trephine biopsies, and peripheral blood samples from MDS patients treated with azacitidine in a clinical trial (NCT03493646), integrating CyTOF, 5' single-cell RNA and TCR sequencing, plasma proteomics, and multiplex immunofluorescence microscopy to characterize changes associated with azacitidine response. Clinical responders showed expansion of GzmBCD56CD8 T cells together with increased type I and type II interferon signaling within the T-cell compartment. Responders also exhibited marked alterations in circulating platelet- and myeloid-derived factors with the potential to remodel the BM niche. Spatial analyses revealed expansion of "neighborhoods" enriched for CXCL12-abundant reticular cells and CD8 T cells in responders, whereas HSPC-enriched neighborhoods were largely unchanged. In contrast, several HSPC-enriched neighborhoods expanded in non-responders. These microenvironmental changes were accompanied by evidence of enhanced myelopoiesis in clinical responders. Our findings support a model in which azacitidine response extends beyond direct effects on malignant hematopoietic cells to involve coordinated remodeling of the BM microenvironment which may be reinforced by platelet- and myeloid-derived signals that establish a feed-forward circuit promoting productive hematopoiesis. One Sentence SummaryLongitudinal bone marrow profiling reveals stromal and immune correlates of azacitidine response in myelodysplastic syndrome.

cancer biology↗

bioRxiv2026

Pandemic Coronavirus Genome Packaging Relies on Multiple Dispersed Packaging Signals

Packaging of viral genomes into progeny virions is a critical step in the viral life cycle. Coronaviruses such as SARS-CoV-2 possess unusually large RNA genomes ([~]30 kb), yet the mechanism by which these genomes are selectively condensed and incorporated into virions remains poorly understood. Here, we demonstrate that the SARS-CoV-2 genome contains multiple dispersed RNA structures, termed packaging signals (PSs), which cooperate with a dominant central PS to direct genomic RNA incorporation into infectious particles. We identify the dominant PS within the coding region of the nsp15 gene, downstream of a packaging signal previously described in Embecoviruses. Using virus-like particles (VLPs), we investigate its role in virion assembly and selective genomic RNA packaging, and show that it promotes the formation of ribonucleoprotein (RNP) complexes with the viral nucleocapsid protein (N), as revealed by mass photometry. Notably, we uncover a unique N-induced conformational rearrangement of the PS RNA, from an extended structure to a double stem-loop architecture. This dominant PS acts together with a nearby stem-loop element to assemble a higher-order RNP complex containing 12 N-protein dimers. Using a SARS-CoV-2 replicon system, we further demonstrate functional cooperativity between the dominant PS, its proximal partner stem-loop, and additional packaging elements located approximately 10 kb upstream. Collectively, our findings support a highly dynamic and cooperative mechanism of SARS-CoV-2 genome packaging that relies on multiple dispersed packaging signals organized around a dominant central PS. These insights provide a mechanistic framework for understanding coronavirus genome packaging and reveal new opportunities for antiviral intervention through disruption of this process. They also have important implications for vaccine development and may enable the design of membrane-based vector systems capable of efficiently delivering large nucleic acid cargoes, expanding the potential of bionanotechnology and genetic medicine.

microbiology↗

bioRxiv2025

Early lineage divergence segregates sensory and non-sensory thalamic circuits

The thalamus integrates sensory, motor, and associative functions via distinct nuclei, yet the developmental principles driving their specification remains unclear. Here, we generate a spatiotemporal single-cell multiomic and spatial transcriptomic atlas of the embryonic mouse thalamus, combined with barcoding-based lineage tracing. We identify two major glutamatergic lineages--sensory and motor/associative--each governed by different gene regulatory programs and temporal dynamics. These lineages arise from spatially and molecularly isolated progenitors, revealing an early segregation of functional identities. Within the sensory lineage, we resolve two discrete trajectories leading to first-order visual and somatosensory neurons, both emerging from a shared higher-order default state. Using in silico predictions and in vivo perturbations, we identify Sp9 as a key regulator of visual thalamic fate. Together, our findings define the molecular architecture and developmental trajectories that underpin thalamic modality specification, providing a framework to understand how functional circuits emerge in the mammalian brain.

neuroscience↗

bioRxiv2025

Developmental gene expression patterns driving species-specific cortical features

The cerebral cortex shows species-specific variations in size and organization, likely accounting for distinct behavioral abilities. These structural differences may reflect evolutionary changes in the developmental expression of shared genes. To investigate this possibility, we compared cell-type-specific gene expression across species in the developing mouse and human neocortex, and human cortical organoids, by generating a shared transcriptional reference framework. This identified genes with conserved/divergent expression patterns, providing a molecular foundation to interrogate species-specific cellular properties. Using this resource, we discovered that the transcription factor JUNB is expressed in human but not mouse progenitors. Through cell-type-specific gain- and loss-of-function experiments in mice and human organoids, we demonstrate that JUNB bidirectionally controls human cortical features, including progenitor proliferation rates, neuronal production timing, and total neuronal output. This reveals how cell-type-specific regulation of shared genes during development can drive species-specific cortical features, providing a framework for understanding the molecular basis of cortical evolution.

neuroscience↗

bioRxiv2024

Piezo1 balances membrane tension and cortical contractility to stabilize intercellular junctions and maintain epithelial barrier integrity

Formation of a bi-directional skin barrier is essential for organismal survival and maintenance of tissue homeostasis. Barrier formation requires positioning of functional tight junctions (TJ) to the most suprabasal viable layer of the epidermis through a mechanical circuit that is driven by generation of high tension at adherens junctions. However, what allows the sensing of tension build-up at these adhesions and how this tension is balanced to match the requirements of tissue mechanical properties is unclear. Here we show that the mechanosensitive ion channel Piezo1 is essential for the maturation of intercellular junctions into functional, continuous adhesions. Deletion of Piezo1 results in an imbalance of cell contractility and membrane tension, leading to a delay in adhesion maturation. Consequently, the requirement for Piezo1 activity can be bypassed by lowering contractility or elevating membrane tension. In vivo, Piezo1 function in adhesion integrity becomes essential only in aged mice where alterations in tissue mechanics lead to impaired TJs and barrier dysfunction. Collectively these studies reveal an essential function of Piezo1 in the timely establishment and maintenance of cell-cell junctions in the context of a mechanically tensed epidermis.

cell biology↗

bioRxiv2024

GRASP55 Regulates Sorting and Maturation of the Lysosomal Enzyme β-Hexosaminidase A

The Golgi apparatus plays a crucial role in the delivery of lysosomal enzymes. Golgi Reassembly Stacking Proteins, GRASP55 and GRASP65, are vital for maintaining Golgi structure and function. GRASP55 depletion results in the missorting and secretion of the lysosomal enzyme Cathepsin D (Xiang et al., 2013), though the mechanisms remain unclear. In this study, we conducted secretomic analyses of GRASP55 knockout (KO) cells and found a significant increase in lysosome-associated proteins in the extracellular medium. Using the lysosomal beta-hexosaminidase subunit alpha (HEXA) as a model, we found that GRASP55 depletion disrupted normal trafficking and processing of HEXA, resulting in increased secretion of the immature (pro-form) HEXA into the extracellular milieu, along with decreased levels of the mature form and enzymatic activity within the cell. GRASP55 depletion significantly reduced the complex formation between HEXA and mannose-6-phosphate (M6P) receptors (MPR), despite no overall change in MPR expression. And finally, we found there was a notable reduction in the expression of GNPTAB, leading to a reduction in M6P modification of HEXA, hindering its efficient targeting to lysosomes. These findings reveal the role of GRASP55 in regulating lysosomal enzyme dynamics, emphasizing its role in the sorting and trafficking of lysosomal proteins.

cell biology↗

bioRxiv2023

Dual lineage origins of neocortical astrocytes

Astrocytes represent one of the most abundant cell types in the central nervous system, and play an essential role in nearly all aspects of brain functions1. Recent studies have challenged the notion that cortical astrocytes are a uniform population, and have highlighted their diverse characteristics at the morphological, molecular, and functional levels2-5. However, how this diversity originates and establishes during cortical development, remains largely unknown. Using single-cell RNA sequencing, we identified five distinct astrocyte subtypes displaying unique spatial patterns in the mouse neocortex, and discovered essential regulators for their formation. Furthermore, we used TrackerSeq6, a method that integrates heritable DNA barcodes into the genome of electroporated progenitors, to track clonally related astrocytes, and identified two distinct lineages that give rise to the five astrocyte subtypes. The first lineage derives from Emx1+ multipotent progenitors that first generate neurons and then switch to produce cortical astrocytes. The second lineage stems from a fate-restricted progenitor population that exclusively gives rise to a specific subset of cortical astrocytes, marked by Olig2. The knockout of this gene in cortical progenitors is sufficient to promote a fate switch between the two lineages. These findings offer novel insights into the cellular mechanisms underlying astrocyte diversity, highlighting the presence of multiple progenitor subtypes, responsible for generating distinct subtypes of astrocytes.

neuroscience↗

bioRxiv2023

Regional Differences in Progenitor Consumption Dynamics Shape Brain Growth during Development

Developing mammalian brains are characterized by disproportionate growth of the forebrain compared to other regions. How this localized expansion occurs is, however, largely unknown. To address this, we identified region-specific neurogenic patterns by creating a single-cell-resolution birthdate atlas of the mouse brain (https://www.neurobirth.org). We report that in forebrain regions, neurogenesis is sustained compared to the hindbrain, where neurogenesis is transient and limited to early brain development. Sustained forebrain neurogenesis reflects lengthened cell cycle and reduced consumptive divisions of ventricular zone progenitors, resulting in a preserved germinal cell pool. Using single-cell RNA sequencing, we identify functional molecular programs of ventricular zone progenitors that spatially and temporally regulate progenitor cycling properties, including through loss-of-function of the forebrain-enriched mitochondrial membrane protein Fam210b. These results reveal a parsimonious mechanism to locally regulate neuronal production, in which the time window during which progenitors generate cells is a critical determinant of region-specific brain expansion.

neuroscience↗

bioRxiv2023

Exonuclease assisted mapping of protein-RNA interactions (ePRINT)

RNA processing is a fundamental mode of gene regulation that is perturbed in a variety of diseases including cancer and neurodegenerative disorders. RNA-binding proteins (RBPs) regulate key aspects of RNA processing including alternative splicing, mRNA degradation and localization by physically binding RNA molecules. Current methods to map these interactions, such as CLIP, rely on purifying single proteins at a time. We have developed a new method (ePRINT) to map RBP-RNA interaction networks on a global scale without purifying individual RBPs. ePRINT allows precise mapping of the 5 end of the RBP binding site, and can uncover direct and indirect targets of an RBP of interest. Importantly, ePRINT can also uncover RBPs that are differentially activated between cell fate transitions, for instance, as neural progenitors differentiate into neurons. Given its versatility, ePRINT has vast application potential as an investigative tool for RNA regulation in development, health and disease.

genomics↗

bioRxiv2023

O-GlcNAc modification forces the formation of an α-Synuclein amyloid-strain with notably diminished seeding activity and pathology

The process of amyloid fibril formation remains one of the primary targets for developing diagnostics and treatments for several neurodegenerative diseases (NDDs). Amyloid-forming proteins such -Synuclein and Tau, which are implicated in the pathogenesis of Alzheimers and Parkinsons disease, can form different types of fibril structure, or strains, that exhibit distinct structures, toxic properties, seeding activities, and pathology spreading patterns in the brain. Therefore, understanding the molecular and structural determinants contributing to the formation of different amyloid strains or their distinct features could open new avenues for developing disease-specific diagnostics and therapies. In this work, we report that O-GlcNAc modification of -Synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by Cryo-EM, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinsons disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the -Synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that post-translational modifications, such as O-GlcNAc modification, of -Synuclein are key determinants of -Synuclein amyloid strains and pathogenicity. These findings have significant implications for how we investigate and target amyloids in the brain and could possibly explain the lack of correlation between amyloid burden and neurodegeneration or cognitive decline in some subtypes of NDDs.

biochemistry↗

bioRxiv2023

A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthase (FPGS), which normally promotes intracellular accumulation and activity of both natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. In this study, we characterize a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase (DHFR) and downstream one-carbon metabolism. Contrary to methotrexate, however, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folate concentrations for interaction with DHFR. Indeed, we show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1-induced growth inhibition, while FPGS-high CRC organoids are more sensitive to methotrexate. Our results thus argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, employing a vulnerability created by polyglutamylation deficiency.

biochemistry↗

bioRxiv2022

Pou3f1 orchestrates a gene regulatory network controlling contralateral retinogeniculate projections

The balance of contralateral and ipsilateral retinogeniculate projections is critical for the establishment of binocular vision, but the transcriptional programs regulating this process remain ill-defined. In this study, we show that the Pou class homeobox protein POU3F1 is selectively expressed in developing mouse contralateral retinal ganglion cells (cRGCs). Inactivation of Pou3f1 increases the number of ipsilateral RGCs produced, leading to abnormal ipsilateral to contralateral projection ratio at the optic chiasm, whereas expression of Pou3f1 in retinal progenitors increases the production of cRGCs. Using Cut&Run and RNA sequencing in wildtype and Pou3f1 mouse knockout retinas, we demonstrate that Pou3f1 is necessary for the establishment of a cRGC gene regulatory network through activation and repression of several known contralateral and ipsilateral determinants, respectively. Finally, we report that POU3F1 is sufficient to induce production of RGC-like cells sending projections to the optic nerve, even in late-stage retinal progenitors not normally competent to generate RGCs. This work uncovers POU3F1 as a master regulator of the cRGC transcriptional program and opens new possibilities for optic nerve regenerative therapies.

developmental biology↗

bioRxiv2022

Development of neonatal brain functional centrality and alterations associated with preterm birth

Formation of the functional connectome in early life underpins future learning and behaviour. However, our understanding of how the functional organisation of brain regions into interconnected hubs (centrality) matures in the early postnatal period is limited, especially in response to factors associated with adverse neurodevelopmental outcomes such as preterm birth. We characterised voxel-wise functional centrality (weighted degree) in 366 neonates from the Developing Human Connectome Project. We tested the hypothesis that functional centrality matures with age at scan in term-born babies and is disrupted by preterm birth. Finally, we asked whether neonatal functional centrality predicts general neurodevelopmental outcomes at 18 months. We report an age-related increase in functional centrality predominantly within visual regions and decrease within motor and auditory regions in term-born infants. Preterm-born infants scanned at term equivalent age had higher functional centrality predominantly within visual regions and lower measures in motor regions. Functional centrality was not related to outcome at 18 months old. Thus, preterm birth appears to affect functional centrality in regions undergoing substantial development during the perinatal period. Our work raises the question of whether these alterations are adaptive or disruptive, and whether they predict neurodevelopmental characteristics that are more subtle or emerge later in life.

neuroscience↗

bioRxiv2021

Ikaros family proteins regulate developmental windows in the mouse retina through convergent and divergent transcriptional programs

Temporal identity factors regulate the competence of neural progenitors to generate specific cell types in a time-dependent manner, but how they operate remains poorly defined. In the developing mouse retina, the Ikaros zinc finger transcription factor Ikzf1 regulates the production of early-born cell types, except cone photoreceptors. In this study we show that Ikzf4, another Ikaros family protein, cooperates with Ikzf1 to control cone photoreceptor production during early stages of retinal development, whereas at late stages, when Ikzf1 is no longer expressed in progenitors, Ikzf4 is instead required for Muller glia production. Using CUT&RUN sequencing, we find that both Ikzf1 and Ikzf4 generally bind to the same genes involved in cone development and other early-born fates, but at different cis-regulatory elements. In late-stage progenitors, Ikzf4 re-localizes to bind target genes involved in Muller glia development and regulate their expression. Specifically, we show that Ikzf4 maintains Hes1 expression in differentiating cells using two Ikzf GGAA binding sites at the Hes1 promoter, thereby favouring Muller glia fate commitment. These results uncover a combinatorial role for Ikaros family members in nervous system development and provide mechanistic insights on how they temporally regulate cell fate output.

developmental biology↗

bioRxiv2021

Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-care Platform

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of 7 wastewater samples from COVID-19 hotspots. RT{square}LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best surrogate marker for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be used as a surrogate marker for detection of SARS-CoV-2. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips.

microbiology↗

bioRxiv2021

Direct neuronal reprogramming by temporal identity factors

Temporal identity factors are sufficient to reprogram developmental competence of neural progenitors, but whether they could also reprogram the identity of fully differentiated cells is unknown. To address this question, we designed a conditional gene expression system combined with genetic lineage tracing that allows rapid screening of potential reprogramming factors in the mouse retina. Using this assay, we report that co-expression of the early temporal identity transcription factor Ikzf1, together with Ikzf4, another Ikaros family member, is sufficient to directly convert adult Muller glial cells into neuron-like cells in vivo, without inducing a proliferative progenitor state. scRNA-seq analysis shows that the reprogrammed cells share some transcriptional signatures with both cone photoreceptors and bipolar cells. Furthermore, we show that co-expression of Ikzf1 and Ikzf4 can reprogram mouse embryonic fibroblasts to induced neurons by remodeling chromatin and promoting a neuronal gene expression program. This work uncovers general neuronal reprogramming properties for temporal identity factors in differentiated cells, opening new opportunities for cell therapy development.

neuroscience↗

bioRxiv2020

Mechanochemical control of epidermal stem cell divisions by B-plexins

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data uncover a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.

cell biology↗

bioRxiv2019

Visualising nascent chain dynamics at the ribosome exit tunnel by cryo-electron microscopy

Ribosomes maintain a healthy cellular proteome by synthesising proteins. The nascent chain (NC), emerges into the cellular milieu via the ribosomal exit tunnel, which is an active component that regulates the NC passage. How the NC dynamics at the exit tunnel affect NC folding remains to be an important question, the answer on which has strong implications to medicine. Here, we report high-resolution cryo-EM maps of ribosome nascent-chain complexes (RNCs) displaying distinct steps during biosynthesis. These RNC structures reveal a range of pathways adopted by the NC. The most pronounced diversity in the NC trajectories were found in the vestibule region. Rearrangements of several ribosomal components further suggest that these elements may actively monitor the emerging NC during translation. The ribosome-NC contacts within the vestibule define these NC pathways and modulate position of a folded immunoglobulin domain outside the ribosome.

biophysics↗