Biology subjects

Golab, J.

Publications and source records attributed to Golab, J..

8 recordsLinked to original sources

bioRxiv2025

Cellohood: multi-granular discovery of cellular neighborhoods with a permutation-invariant set transformer auto-encoder

Discovering cellular neighborhoods and their roles in disease requires computational methods that consider the full breadth of data and offer multi-level granularity and interpretability. Here, we introduce Cellohood, a permutation-invariant set transformer auto-encoder equipped with a clinical association pipeline. Cellohood encodes full readouts for bags of spatially co-localized cells and supports multi-level analyses, providing interpretability by mapping latent dimensions to spatial tissue features. Our model surpasses current methods in accuracy of cellular neighborhood detection for spatial transcriptomics of the human cortex and CODEX spleen data measuring lupus progression. Applied to cancer data across three granularity levels, our method recovers neighborhoods along the expected immune-cold to immune-hot spectrum and further refines them into biologically and clinically meaningful subclasses, revealing spatial patterns linked to prognosis, histology, stage, and tumor mutational burden, and uncovering subgroups that transcend standard classifications. Overall, Cellohood enables in-depth analysis of complex tissues, revealing clinically informative spatial neighborhoods.

bioinformatics↗

bioRxiv2025

PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

PolyA tail regulates mRNA localization, stability, and translation. PolyA length affects the durability and translational activity of both endogenous and exogenously delivered mRNAs. However, long polyA stretches can undergo recombination during amplification in bacterial plasmids, impairing the production of in vitro transcribed (IVT) mRNA with long polyAs. PolyA tail segmentation with heteronucleotide spacers has recently emerged as a solution. Here, we developed segmented polyA patterns that stabilize the sequence during DNA amplification and enhance mRNA translation. We designed 15 novel genetically modified polyA variants, differing in the length, placement, and frequency of spacers, and the overall length (from [~]120 to [~]200 nucleotides). We evaluated their stability in DNA plasmids and homogeneity, translational activity, and durability in cell culture of the resulting mRNAs, comparing them to A90 tail and other known solutions, including those from existing mRNA vaccines. Selected sequences were validated in vivo. Surprisingly, we found that even frequent heteronucleotide insertions produce functional polyA tails. The most notable enhancements in protein production were observed for a segmented tail exceeding 200 nt in length [A30(CA15)11; up to 6-fold compared to mRNA with A90 tails]. Our findings extend the scope of possible polyA modification strategies, offering new possibilities for advancing mRNA therapeutics.

molecular biology↗

bioRxiv2024

Chemical circularization of in vitro transcribed RNA opens new avenues for circular mRNA design

Circularization is at the frontier of therapeutic messenger RNA (mRNA) enhancements. Currently available enzymatic and ribozymatic methods for generating circular RNAs (circRNAs) face several challenges related to sequence limitations, purification, and sub-optimal biological activity. The chemical circularization of synthetic RNA fragments potentially overcomes these limitations but is applicable only to extremely short sequences. Here, we report a novel approach for accessing circular RNAs based on the chemical circularization of in vitro transcribed RNA. We efficiently accessed chemically circularized RNAs (chem-circRNAs) by making in vitro transcribed precursor RNAs modified at the 5' end with an ethylenediamine moiety, which undergoes an intramolecular reaction with the periodate-oxidized RNA 3' end under reductive amination conditions. We demonstrate that this method is modification-compatible and applicable to various sequences. Additionally, we report methods for the effective separation of chem-circRNAs from their linear precursors. Using this approach, we prepared multiple chemically-obtained circular RNAs (chem-circRNAs; 35-1500 nt long) with circularization efficiencies reaching up to 60%. We show that protein-coding chem-circRNAs are translationally active in living cells and exhibit increased durability, similar to enzymatically circularized mRNAs. We also demonstrate that this approach enables unprecedented access to chemically modified circRNAs, such as circ-mRNAs incorporating a functional endocyclic N7-methylguanosine cap or modified with N1-methylpseudouridine within the RNA body. Notably, circRNAs containing an endocyclic cap structure engage in the most efficient, cap-dependent mechanism of translation. Our approach makes chemically-modified circularized full-length protein-coding RNAs easily accessible, thereby opening new avenues for the design, modification, and functionalization of circular mRNAs.

biochemistry↗

bioRxiv2023

SARS-CoV-2 and its ORF3a, E and M viroporins activate inflammasome in human macrophages and induce of IL-1α in pulmonary epithelial and endothelial cells

Inflammasome assembly is a potent mechanism responsible for the host protection against pathogens, including viruses. When compromised, it can allow viral replication, while when disrupted, it can perpetuate pathological responses by IL-1 signaling and pyroptotic cell death. SARS-CoV-2 infection was shown to activate inflammasome in the lungs of COVID-19 patients, however, potential mechanisms responsible for this response are not fully elucidated. In this study, we investigated the effects of ORF3a, E and M SARS-CoV-2 viroporins in the inflammasome activation in major populations of alveolar sentinel cells: macrophages, epithelial and endothelial cells. We demonstrated that each viroporin is capable of activation of the inflammasome in macrophages to trigger cell death and IL-1 release from epithelial and endothelial cells. Small molecule NLRP3 inflammasome inhibitors reduced IL-1 release but weakly affected the pyroptosis. Importantly, we discovered that while SARS-CoV-2 could not infect the pulmonary microvascular endothelial cells it induced IL-1 and IL-33 release. Together, these findings highlight the essential role of macrophages as the major inflammasome-activating cell population in the lungs and point to endothelial cell expressed IL-1 as a potential novel component driving the pulmonary immunothromobosis in COVID-19.

immunology↗

bioRxiv2023

Trinucleotide mRNA cap analog N6-benzylated at the site of posttranscriptional m6Am mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo

Eukaryotic mRNAs undergo co-transcriptional 5-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am - a common epitranscriptomic mark unique to the mRNA 5-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analog of the m6Am-cap - m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as an RP-HPLC purification handle, allowing separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic setting as anti-cancer vaccines. The biochemical characterization suggests several phenomena underlying the biological properties of AvantCap: (i) increased competitiveness of the mRNA 5-end for eIF4E protein by reducing its propensity for unspecific interactions, (ii) direct involvement of eIF3 in alternative translation initiation, (iii) subtle differences in mRNA impurity profiles, or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of the m6Am in mRNA.

biochemistry↗

bioRxiv2022

SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response

Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Modernas mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails. We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (m{Psi}), is terminated by a long poly(A) tail (~100 nucleotides) followed by an m{Psi}Cm{Psi}AG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of m{Psi}Cm{Psi}AG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after m{Psi}Cm{Psi}AG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after m{Psi}Cm{Psi}AG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination. Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.

immunology↗

bioRxiv2021

Anti-clotting functions of lymphatics form the natural on-off switch for immune recognition by controlling the antigens and immune cells access to the lymph nodes

The ability of lymph to clot indicates that, like blood vessels, lymphatics must have means to counteract this process. Here, we analyzed lymphatic hemostatic properties, tailoring them for potential therapeutic applications. Inflammatory stimuli induced tissue factor-dependent focal lymph clotting while blocking thrombomodulin leading to widespread but transient occlusion of collecting vessels. Decellularization of lymphatics resulted in tissue factor-independent lymphatic occlusion by widespread and persistent lymph clots. In occluded decellularized ghost vessels, fibrin was eventually reperfused. During the regeneration, ghost lymphatics were filled with granuloma-like clusters of antigen-presenting cells and T cells. Despite that, immune response against allografts placed under non-drained skin did not develop as long lymphatics remained occluded, the effect that could be prolonged by delaying regeneration of the decellularized collectors. When the lymph clotting was blocked, decellularized lymphatics could still drain macromolecules and leukocytes, showing that lymphatic endothelium is not necessary for the classic lymphatic functions. The control of excessive clotting emerges as the essential function of lymphatics that could explain the seeming spandrel presence of lymphatic networks in organs such as the kidney or heart, contribute to microvascular thrombosis during infection, and can be exploited to induce immune ignorance of the subcutaneous endocrine grafts.

physiology↗

bioRxiv2021

Potent but transient immunosuppression of T-cells is a general feature of erythroid progenitor cells

CD71+ erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-induced L-arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-{gamma} production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-{gamma} production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71highCD235adim cells). The suppressive properties disappear during erythroid differentiation as more differentiated CECs and mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of CECs in the immune response regulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/427109v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@136fbd8org.highwire.dtl.DTLVardef@d05d00org.highwire.dtl.DTLVardef@189baceorg.highwire.dtl.DTLVardef@1472b2b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗