Biology subjects

Hamed, M.

Publications and source records attributed to Hamed, M..

11 recordsLinked to original sources

bioRxiv2026

Single-cell profiling reveals tumor grade-dependent immune remodeling in BRAFV600E-driven glioneuronal tumors

BRAFV600E is the key driver variant in epilepsy-associated glioneuronal tumors (GNTs). These tumors often share MAPK/PI3K hyperactivation, a generally benign biological course and rare occurrence of malignant variants. We aimed to characterize the poorly defined immune cell milieu of GNTs with distinct biological behavior. We mapped cellular heterogeneity of the tumor microenvironment (TME) using single-cell transcriptomics on murine models of low-grade (LG-GNT; BRAFV600E/AKTA) and high-grade (HG-GNT; BRAFV600E/AKTA/Trp53KO) tumors, generated via intraventricular in utero electroporation (IUE). Furthermore, ex vivo functional assays with CSF1R-mediated myeloid depletion were utilized to assess the role of identified signaling molecules on tumor viability. We observed a striking, grade-dependent immunological dichotomy: LG-GNT exhibited a permissive niche with prominent surveillance by T cells and pro-inflammatory microglia. In contrast, HG-GNT TME was characterized by a restricted T cell infiltration, massively dominated by myeloid cell infiltrates. Differential gene expression analysis identified Spp1 (osteopontin) as a key mediator of this immunosuppressive HG-GNT TME, exclusively expressed in microglia and border-associated macrophages (BAMs). Crucially, ex vivo functional assays demonstrated that recombinant SPP1 enhances tumor viability through a paracrine mechanism. These findings suggest fundamentally distinct immune activation (a) stimulated by aberrant MAPK/PI3K signaling in LG-GNT, versus (b) malignant tumor feature-driven, e.g. through necrosis in HG-GNT. In the latter, SPP1 signaling creates the immunosuppressive niche. Consequently, while modulating the pro-inflammatory niche may mitigate tumor-related epileptogenicity in LG-GNTs, targeting the SPP1-myeloid axis may restore anti-tumor immunity in HG-GNTs.

neuroscience↗

bioRxiv2026

Loss of nuclear-cytoplasmic compartmentalization is a hallmark of aging in podocytes

The nuclear envelope, with its nuclear pore complexes, establishes a selective barrier that maintains the spatial organization of the cellular proteome. Whether this barrier remains intact in long-lived postmitotic cells during aging is largely unknown. Here, we show that aging podocytes experience a progressive loss of nuclear-cytoplasmic compartmentalization. Using subcellular proteomics and quantitative imaging, we identify a global redistribution of proteins between the nucleus and cytoplasm, affecting key regulators of RNA processing, chromatin organization, and cellular metabolism. Loss of compartmentalization is accompanied by mitochondrial dysfunction, increased reactive oxygen species production, and aberrant nuclear accumulation of YAP1, linking nuclear barrier failure to metabolic and transcriptional dysregulation. These findings identify nuclear envelope dysfunction as a driver of proteome disorganization and cellular decline in aging podocytes and suggest that loss of nuclear-cytoplasmic compartmentalization represents a general mechanism contributing to dysfunction in long-lived cells.

cell biology↗

bioRxiv2025

TWEAK Signaling Drives the Transition from Psoriasis to Atopic Dermatitis-like Inflammation in Paradoxical Skin Reactions

Targeted biologics have significantly advanced the treatment of inflammatory skin diseases such as psoriasis; however, some patients paradoxically develop eczematous skin reactions during or after anti-TNF, IL-17, or IL-23 therapy. Although these paradoxical reactions resemble atopic dermatitis clinically and histologically, the molecular mechanisms that drive their development are not fully understood. Here, we generated high-resolution cellular and spatial maps of healthy skin, psoriasis, atopic dermatitis, and paradoxical reactions using single-cell RNA sequencing, spatial transcriptomics, immunohistochemistry, and in vitro assays. In paradoxical reactions, we identified a distinct transcriptional landscape characterized by myeloid and T-cell expansion and an altered keratinocyte phenotype shaped by TWEAK signaling. Mechanistically, we showed that TWEAK synergizes with IL-13 to drive the Th2/type I interferon-polarized epithelial program. Notably, anti-TNF therapy induced TWEAK gene expression in myeloid cells, suggesting a compensatory inflammatory circuit. Together, these findings identify the TWEAK-IL-13 axis as a central driver of paradoxical skin reactions and provide a mechanistic framework for how cytokine blockade may rewire cutaneous immune responses. One Sentence Summary The TWEAK-IL-13 signaling axis is a key driver of immune reprogramming underlying paradoxical skin reactions.

immunology↗

bioRxiv2023

Palmitoylethanolamide shows limited efficacy in controlling cerebral cryptococcosis in vivo

Cryptococcus neoformans (Cn) is an encapsulated neurotropic fungal pathogen and the causative agent of cryptococcal meningoencephalitis (CME) in humans. Recommended treatment for CME is Amphotericin B (AmpB) and 5-fluorocytosine (5-FC). Though effective, AmpB has displayed numerous adverse side effects due to its potency and nephrotoxicity, prompting investigation into alternative treatments. Palmitoylethanolamide (PEA) is an immunomodulatory compound capable of promoting neuroprotection and reducing inflammation. To investigate the efficacy of PEA as a therapeutic alternative for CME, we intracerebrally infected mice with Cn and treated them with PEA or AmpB alone or in combination. Our results demonstrate that PEA alone does not significantly prolong survival nor reduce fungal burden, but when combined with AmpB, PEA exerts an additive effect and promotes both survivability and fungal clearance. However, we compared this combination to traditional AmpB and 5-FC treatment in a survivability study and observed lower efficacy. Overall, our study revealed that PEA alone is not effective as an antifungal agent in the treatment of CME. Importantly, we describe the therapeutic capability of PEA in the context of Cn infection and show that its immunomodulatory properties may confer limited protection when combined with an effective fungicidal agent.

microbiology↗

bioRxiv2022

Clinical and pathological characterization of Central Nervous System cryptococcosis in an experimental mouse model of stereotaxic intracerebral infection

Infection of the Central Nervous System (CNS) by the encapsulated fungus Cryptococcus neoformans can lead to high mortality meningitis, most commonly in immunocompromised patients. While the mechanisms by which the fungus crosses the blood-brain barrier to initiate infection in the CNS are well recognized, there are still substantial unanswered questions about the disease progression once the fungus is established in the brain. C. neoformans is characterized by a glucuronoxylomannan (GXM)-rich polysaccharide capsule which has been implicated in immune evasion, but its role during the host CNS infection needs further elucidation. Therefore, the present study aims to examine these key questions about the mechanisms underlying cryptococcal meningitis progression and the impact of fungal GXM release by using an intracerebral rodent infection model via stereotaxic surgery. After developing brain infection, we analyzed distinct brain regions and found that while fungal load and brain weight were comparable one-week post-infection, there were region-specific histopathological (with and without brain parenchyma involvement) and disease manifestations. Moreover, we also observed a region-specific correlation between GXM accumulation and glial cell recruitment. Further, mortality was associated with the presence of subarachnoid hemorrhaging and GXM deposition in the meningeal blood vessels and meninges in all regions infected. Our results show that using the present infection model can facilitate clinical and neuropathological observations during the progression of neurocryptococcosis. Importantly, this mouse model can be used to further investigate disease progression as it develops in humans. IMPORTANCECryptococcus neoformans is the causative agent of cryptococcal meningoencephalitis (CME), a severe infection of the central nervous system (CNS) in humans. The high mortality and morbidity rates of this disease in immunosuppressed and HIV+/AIDS-infected individuals necessitate a reliable model for understanding the mechanisms and manifestations of cryptococcosis infection in the CNS. This study presents a mouse model of infection that utilizes a stereotaxic surgical setup to provide a comparative analysis of C. neoformans infection in the brain. Though not typical of infection in humans, this method provides a controlled experimental system to observe the progression of C. neoformans infection in specific regions of the brain. Additionally, this study characterizes murine manifestations of the disease, including GXM deposition in brain tissue and blood vessels, and documents the role of microglia, the resident immune cells of the CNS, in engulfing fungal cells. Depending on the region of the brain infected, distinct disease pathology can be observed with this model, expanding our understanding of cryptococcosis progression in the CNS. The importance of this work extends to the clinical setting, where many questions regarding cerebral cryptococcosis remain unresolved. Utilization of this model to further understand the mechanisms of this pathogen will provide means to develop new therapies to combat infection in humans.

microbiology↗

bioRxiv2021

Semi-quantitative detection for L-asparaginase producing fungi and the impact of carbon and nitrogen sources on enzyme activity

ObjectiveTo semi-quantitively screen filamentous fungi isolated from different habitats for L-asparaginase production by three indicators; phenol red, cresol red and bromothymol blue and to examine the impact of different carbon and nitrogen sources on the enzyme production using different fungal isolates. Materials and methodsFifty-five fungal isolates were tested for L-asparaginase production by plate assay using Modified Czapek-Dox (MCD) medium. The enzyme activity was estimated using the Nessler method which measures the concentration of ammonia formed owing to the enzyme action on the substrate. The impact of nitrogen and carbon sources on the enzyme production was done by using the best three L-asparaginase producers from the semi-quantitative screening. Results and conclusionsA total of 53/55 (96.36%) fungal isolates were L-asparaginase producing strains, of them, Cladosporium tenuissimum, Penicillium camembertii and Aspergillus carneus showed high enzyme production. Production of L-asparaginase was higher with the glucose and urea as carbon and nitrogen sources, respectively. The highest enzyme level (5,558 U/ml) was produced by C. tenuissimum in a glucose-containing medium. This study shows that P. camemberti, A. carneus, and C. tenuissimum are good L-asparaginase producers and thus could be used for L-asparaginase production

microbiology↗

bioRxiv2020

Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity

Cytotoxic CD8+ T lymphocytes (CTL) eliminate infected cells or transformed tumour cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+-influx through store operated Ca2+ channels, formed by STIM-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. Here, we isolated total RNA from natural killer (NK) cells, non-stimulated CD8+ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+ dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, this strategy led to the identification of KCNN4, RCAN3, CCR5 and BCL2 as potential candidates to regulate the efficiency of Ca2+-dependent target cell killing.

immunology↗

bioRxiv2020

The possible anti-inflammatory activity of macrolide antibiotics in some animal models

Recently, there has been increasing evidence on the use of macrolide antibiotics in treatment of chronic inflammatory diseases through mechanisms distinct from their antibacterial activity. The key desired effect lies somewhere between these two therapeutic potentials and has not been identified yet. The aim of the present study was to evaluate the anti-inflammatory activity of the macrolide antibiotic azithromycin in formaldehyde induced arthritis and carrageenan induced air pouch in albino rats in comparison to the anti-rheumatic drug meloxicam. Results of Formaldehyde induced arthritis revealed that pretreatment of animals with a single daily dose of either azithromycin (10, 20 and 40 mg/kg) or meloxicam (4 mg/kg) for 15 days produced a significant reduction in inflammation size. Histopathological study showed that Formaldehyde produced marked inflammatory cell infiltration, congestion of blood vessels and soft tissue edema which were attenuated by azithromycin in dose dependent manner. The radiological study revealed that azithromycin attenuates soft tissue edema, periarticular bone resorption, narrowing of joint spaces and joint deformities induced by formaldehyde. This effect was marked with 40mg/kg azithromycin pre-treatment. In carrageenan, induced air pouch, results demonstrate that group of animals pretreated with azithromycin (10, 20 and 40 mg/kg) or meloxicam (4 mg/kg) for 6 days significantly attenuated the mean increase in total leukocyte count in air pouch exudate. In conclusion, the present work showed that azithromycin has antiinflammatory activity in the models tested and suggests that it can exert therapeutics effects independent of its anti-bacterial activity. Also, the anti-inflammatory effect of azithromycin was potent and even comparable to that of meloxicam.

pharmacology and toxicology↗

bioRxiv2019

Unraveling the heterogeneous mutational signature of spontaneously developing tumors in MLH1-/- mice

MLH1 knock out mice represent a preclinical model that resembles features of the human counterpart. As these mice develop mismatch repair deficient (MMR-D) neoplasias in a sequential twin-peaked manner (first lymphomas, then gastrointestinal tumors) we aimed at identification of the underlying molecular mechanisms. Using whole-exome sequencing, we focused on (I) shared and (II) mutually exclusive mutations and described the processes of ongoing mutational events in tumor-derived cultures.\n\nA heterogeneous genetic landscape was found, with few mutations shared among different neoplasias (ARID1A and IDH2). Mutations in tumor suppressor genes SMAD4 and POLE were mutually exclusive in lymphomas, most likely contributing to a more aggressive in vivo phenotype. Comparing the mutational profile of selected primary tumors and their corresponding cell line upon in vitro culture revealed continuous increased numbers of somatic gene mutations. The same was true for coding microsatellite mutations in selected MMR-D target genes, showing a gradual increase during in vitro passage. With respect to this latter type of mutations, partial overlap was detectable, yet recognizing shared vaccination antigens. The two most promising candidates are AKT3, a RAC-gamma serine/threonine-protein kinase with relevance in maintenance of cellular homeostasis and the endonuclease ERCC5 (Excision Repair 5), involved in DNA excision repair.\n\nNovel results of a comparison between spontaneously developing lymphomas and gastrointestinal tumors as models for MMR-D driven tumorigenesis are reported. In addition to identification of ARID1A as a potentially causative mutation hotspot, our comprehensive characterization of the mutational signature is a starting point for immune-based approaches to therapy.\n\nAuthor SummaryThis study describes the mutational spectrum of MLH1-/--associated tumors, spontaneously developing in mice. While these tumors arise at the bottom of the same germline mutation, the clinical presentations as well as resulting molecular alterations are heterogeneous, and thus likely being directly linked. Highly aggressive lymphomas, developing early in life are ultra-hypermutated and harbor mutations in tumor suppressor genes SMAD4 and POLE. Gastrointestinal tumors develop later in life and show different mutations. By performing in-depth whole exome sequencing analysis, we here identified for the first time a common mutational hotspot. ARID1A constitutes a potentially causative mutation, shared among different MLH1-/--associated tumors and thus irrespective of the origin. Additional interesting and identified candidate genes include AKT3, a RAC-gamma serine/threonine-protein kinase and the endonuclease ERCC5. Both genes are bona fide tumor suppressors with significant relevance in DNA excision repair and maintenance of cellular homeostasis. This finding is of particular relevance for subsequent therapeutic and - even more important - prophylactic vaccination approaches aiming at entity-overlapping treatment of MLH1-/--related tumors.

cancer biology↗

bioRxiv2018

Pathway maps enable straightforward yet customized and semi-automated yet insightful analyses of omics data

To explore the molecular processes underlying some biological theme of interest based on public data, gene lists are used herein as input for the construction of annotated pathway maps, employing Cytoscape apps, and then high-throughput (\"omics\") gene expression data are overlaid onto these maps. Seeded with a published set of marker genes of the senescence-associated secretory phenotype and the genes of the cellular senescence KEGG pathway, a gene/protein interaction network and annotated clusters (a \"pathway map\") of cellular senescence are derived. The map can be amended, by adding some application-specific genes, and overlaid with gene expression data describing cellular senescence of fibroblasts and with disease-related gene expression data associated with prostate and pancreatic cancer, and with ischemic stroke, allowing insights into the role of cellular senescence in disease. Some gene expression data are derived from the \"Biomarker Benchmark repository\". The pathway map approach can be followed in principle for any biological theme of interest, fostering much-needed independence from the investigator-biased expert networks usually used for overlaying gene expression data.

genomics↗

bioRxiv2018

Healthspan pathway maps in C. elegans and humans highlight transcription, prolifera-tion/biosynthesis and lipids

The molecular basis of aging and of aging-associated diseases is being unraveled at an increasing pace. An extended healthspan, and not merely an extension of lifespan, has become the aim of medical practice. However, a precise definition of health and healthspan is not straightforward, and the causal molecular basis of health \"per se\" is largely unknown. Here, we define health based on the absence of diseases and dysfunctions. Based on an extensive review of the literature, in particular for humans and C. elegans, we compile a list of features of health and of the genes associated with them. Clusters of these genes based on molecular interaction data give rise to maps of healthspan pathways for humans, featuring the themes transcription initiation, proliferation and cholesterol/lipid processing, and for C. elegans, featuring the themes immune response, mitochondrion and biosynthesis based on genetic and compound intervention data, and lipids, biosynthesis and transcription based on WormBase compound intervention data. Overlaying healthspan-related gene expression data (describing effects of metabolic intervention associated with improvements in health) onto the aforementioned healthspan pathway maps, we observe the downregulation of Notch signalling in humans and of proliferation/cell-cycle in C. elegans. The former reflects the proinflammatory role of the Notch pathway. We identify transcription, proliferation/biosynthesis and lipids as a common theme on the annotation level, and proliferation-related kinases on the gene/protein level. Our literature-based data corpus, including visualization, is available as a reference for future investigations, at http://www.h2020awe.eu/index.php/pathways/.

bioinformatics↗