Biology subjects

Mamede, J. I.

Publications and source records attributed to Mamede, J. I..

5 recordsLinked to original sources

bioRxiv2025

An HIV-1 Reference Epitranscriptome

Post-transcriptional modifications to RNA, which comprise the epitranscriptome, play important roles in RNA metabolism, gene regulation, and human disease, including viral pathogenesis. Modifications to the RNA viral genome and transcripts of human immunodeficiency virus 1 (HIV-1) have been reported and investigated in the context of virus and host biology. However, the diversity of experimental approaches used has made clear correlations across studies, as well as the significance of the HIV-1 epitranscriptome in biology and disease, difficult to assess. Therefore, we established a reference HIV-1 epitranscriptome. We sequenced the model NL4-3 HIV-1 genome from infected primary CD4+ T cells and the Jurkat cell line using the latest nanopore chemistry, optimized RNA preparation methods, and the most current and readily available base-calling algorithms. A highly reproducible sense and a preliminary antisense HIV-1 epitranscriptome were created, where N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (psi), inosine, and 2-O-methyl (Nm) modifications could be identified by rapid multiplexed base-calling. We observed that sequence and neighboring modification contexts induced modification miscalling, which could be corrected with synthetic HIV-1 RNA fragments. We validated m6A modification sites with STM2457, a small molecule inhibitor of methyltransferase-like 3 (METTL3). We find that modifications are quite stable under combination antiretroviral therapy (cART) treatment, in primary CD4+ T cells, and in HIV-1 virions. Sequencing samples from people living with HIV (PLWH) revealed conservation of m6A modifications. However, analysis of spliced transcript variants suggests transcript-dependent modification levels. Our approach and reference data offer a straightforward benchmark that can be adopted to help advance rigor, reproducibility, and uniformity across HIV-1 epitranscriptomics studies. They also provide a roadmap for the creation of reference epitranscriptomes for many other viruses or pathogens.

genetics↗

bioRxiv2024

Structural Repetition Detector: multi-scale quantitative mapping of molecular complexes through microscopy

From molecules to organelles, cells exhibit recurring structural motifs across multiple scales. Understanding these structures provides insights into their functional roles. While super-resolution microscopy can visualise such patterns, manual detection in large datasets is challenging and biased. We present the Structural Repetition Detector (SReD), an unsupervised computational framework that identifies repetitive biological structures by exploiting local texture repetition. SReD formulates structure detection as a similarity-matching problem between local image regions. It detects recurring patterns without prior knowledge or constraints on the imaging modality. We demonstrate SReDs capabilities on various fluorescence microscopy images. Quantitative analyses of three datasets highlight SReDs utility: estimating the periodicity of spectrin rings in neurons, detecting HIV-1 viral assembly, and evaluating microtubule dynamics modulated by EB3. Our open-source ImageJ and Fiji plugin enables unbiased analysis of repetitive structures across imaging modalities in diverse biological contexts.

cell biology↗

bioRxiv2024

HIV-1 infection promotes neuroinflammation and neuron pathogenesis in novel microglia-containing cerebral organoids.

Cerebral organoids (COs) are a valuable tool to study the intricate interplay between glial cells and neurons in brain development and disease, including HIV-associated neuroinflammation. We developed a novel approach to generate microglia containing COs (CO-iMs) by co-culturing hematopoietic progenitors and induced pluripotent stem cells. This approach allowed for the differentiation of microglia within the organoids concomitantly to the neuronal progenitors. CO- iMs exhibited higher efficiency in generation of CD45+/CD11b+/Iba-1+ microglia cells compared to conventional COs with physiologically relevant proportion of microglia ([~]7%). CO-iMs exhibited substantially higher expression of microglial homeostatic and sensome markers as well as markers for the complement cascade. CO-iMs showed susceptibility to HIV infection resulting in a significant increase in several pro-inflammatory cytokines/chemokines and compromised neuronal function, which were abrogated by addition of antiretrovirals. Thus, CO-iM is a robust model to decipher neuropathogenesis, neurological disorders, and viral infections of brain cells in a 3D culture system.

microbiology↗

bioRxiv2019

Correlated cryogenic fluorescence microscopy and electron cryotomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Members of the TRIM protein family have been shown to gather into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy (cryo-CLEM) to intact mammalian cells expressing YFP-rhTRIM5 and found hexagonal nets were present whose arm-lengths were similar to those of the hexagonal nets formed by purified TRIM5 in-vitro. We also observed YFP-rhTRIM5 within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within a double-membraned vesicle (autophagosomes), and sequestosomes within multi-vesicular autophagic vacuoles (autolysosomes or amphisomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data (i) support recent reports that TRIM5 can form both well-organized signaling complexes and non-signaling aggregates, (ii) offer the first images of the macroautophagy pathway in a near-native state, and (iii) reveal that vaults arrive early in macroautophagy.

cell biology↗

bioRxiv2017

Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells

Cryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

cell biology↗