Biology subjects

Leonard, F.

Publications and source records attributed to Leonard, F..

5 recordsLinked to original sources

bioRxiv2026

Single-time-point shotgun metagenomics of four Irish Integrated Constructed Wetlands reveals microbial dynamics in wastewater treatment.

Over the past 20 years, the Integrated Constructed Wetland (ICW) concept has been applied in Ireland for wastewater treatment, offering a nature-based solution for reducing pollutants and antimicrobial resistance genes (ARGs) in effluents and receiving environments. However, wastewater microbial communities remain inadequately characterized and their dynamics across treatment largely unexplored. Here, we present a culture-independent investigation of four Irish ICWs, aiming to advance our understanding of microbial dynamics in wastewater treatment. Shotgun metagenomic sequencing was conducted on influent and effluent samples collected in biological triplicates from four ICWs treating agricultural, companion animal, industrial, and municipal wastewater, alongside eight positive and ten negative controls for filtering and sequencing. End-to-end metagenomic analysis was performed with SqueezeMeta. Taxonomic placement of high-quality metagenome-assembled-genomes (HQ-MAGs) was confirmed with GTDB-Tk. Decontamination and statistical analyses were conducted in R. Coassembled contigs were screened for ARGs, virulence- and plasmid-associated sequences. Results revealed statistically significant shifts in taxonomic and functional profiles across treatment. Effluent populations exhibited generally higher richness than corresponding influents, yet were more similar to one another across locations. Multi-log-scale increases were observed in the relative abundance of environmental genera (Legionella, Methylotenera, Thiothrix), alongside reductions in common faecal/human indicators (Bacteroides, Lactococcus, Prevotella), and up to 80% ARGs removal. Collectively, our findings provide insights into ICW efficacy, showing that they can drive marked shifts in wastewater microbiome and ARG reduction. Our culture-independent approach retrieved 34 HQ-MAGs, including 19 potentially novel taxa, uncovering previously uncharacterized microbial diversity that is potentially unique to the Irish environment and warrants further investigation.

microbiology↗

bioRxiv2026

Intranasal CRISPR- lipid nanoparticles targeting MAPK9 reduce neuroinflammation after traumatic brain injury

Traumatic brain injury (TBI) triggers a sustained neuroinflammatory response driven by activated microglia, which contributes to secondary injury and long-term neurological dysfunction. Therapeutic reprogramming of microglial activation from a pro-inflammatory (M1-like) to a reparative (M2-like) phenotype represents a promising strategy; however, the lack of cell-specific targeting within an injured brain has limited clinical translation. Here, we developed a targeted gene-editing nanotherapy to modulate post-traumatic innate immune responses. Lipid nanoparticles (LNPs) encapsulating CRISPR-Cas12a components were engineered to target mitogen-activated protein kinase-9 (MAPK9), a key regulator of pro-inflammatory signaling, and were conjugated with an Iba-1 antibody (Iba-1-CRISPR-LNPs) to enable selective targeting of microglia. In vitro, MAPK9 editing in primary macrophages inhibited M1 polarization and promoted an M2-like phenotype, leading to reduced production of pro-inflammatory cytokines. In a TBI mouse model, intranasal administration of Iba-1-CRISPR-LNPs achieved efficient delivery to the injured brain, with selective localization in Iba-1+ microglia. MAPK9 CRISPR targeting significantly attenuated microglial activation, reduced central and peripheral inflammatory responses, and decreased pro-inflammatory cytokine levels. Importantly, this approach demonstrated a favorable safety profile, with no detectable toxicity across major organs. Collectively, these findings establish a non-viral, intranasal CRISPR-based strategy for cell-specific modulation of neuroinflammation following TBI. Targeted genome editing of MAPK9 effectively reprograms microglial activation and attenuates acute inflammatory responses, highlighting its potential as a promising and translationally relevant therapeutic platform for TBI and related neuroinflammatory disorders.

neuroscience↗

bioRxiv2025

Rictor CRISPR Gene Editing by Lipid Nanoparticle Delivery Stimulates Anti-Tumor Immunity in Breast Cancer Liver Metastasis Model

Triple-negative breast cancer (TNBC) is a highly aggressive subtype, accounting for 10-15% of breast cancer cases in the United States. Liver metastases, common in advanced TNBC, are linked to especially poor outcomes, with a 5-year survival rate of just 11%. Although immune checkpoint inhibitors (ICIs) targeting PD-1 or PD-L1 show promise, durable responses in TNBC remain uncommon. This is largely due to a profoundly immunosuppressive tumor microenvironment (TME), driven by tumor-associated myeloid cells. Tumor-associated macrophages (TAMs) and neutrophils (TANs) polarize into immunosuppressive M2 and N2 phenotypes, respectively, suppressing T cell activity through cytokines, ROS, and checkpoint ligands such as VISTA. Myeloid-derived suppressor cells (MDSCs) further inhibit immunity by depleting nutrients and inducing regulatory T cells. As a result, despite its immunogenic features, TNBC remains resistant to immunotherapy due to persistent myeloid-mediated suppression. Here, we developed ionizable lipid nanoparticles (iLNPs) engineered to deliver the CRISPR-Cas12a ribonuclease complex targeting Rictor, a critical component of mTORC2, for in vivo reprograming of myeloid cells. The intravenous (IV) injection of CRISPR Rictor-targeting iLNP (CR-Ric-LNP) showed efficient uptake by circulating myeloid cells and accumulation into the breast cancer liver metastases. Notably, Rictor gene editing triggered pro-inflammatory activation of myeloid cells in the TME, enhancing antitumor responses. Single-cell RNA sequencing revealed that Rictor silencing treated samples showed induced rapid remodeling of the TME, with a significant reduction in immunosuppressive macrophages within 24 hours of treatment. Concurrently, cytotoxic T-cell populations exhibited increased interferon-gamma (Ifng) production, driving the emergence of specific myeloid clusters that were responsive to Interferon signaling, particularly in macrophages and neutrophils. A shift from an immunosuppressive to an inflammatory TME was further evidenced by an elevated Cxcl10/Spp1 ratio in myeloid cells. CR-Ric-LNP treatment also enhanced T-cell activation, reducing exhausted T cells and regulatory T cells (Tregs) while expanding natural killer (NK) cells, naive CD4+, and CD8+ T cells. These changes correlated with a decreased proportion of tumor cells and proliferating cells, ultimately leading to a significant survival benefit in a 4T1 breast cancer liver metastasis model. Our findings demonstrate that myeloid-targeted Rictor silencing reprograms the TME, promoting antitumor immunity and improving therapeutic outcomes. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/661471v1_ufig1.gif" ALT="Figure 1"> View larger version (109K): org.highwire.dtl.DTLVardef@10a5e7aorg.highwire.dtl.DTLVardef@1c0c005org.highwire.dtl.DTLVardef@17ec6b5org.highwire.dtl.DTLVardef@27c507_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Combined inhibition of S100A4 and TIGIT suppresses late-stage breast cancer metastasis to the lung by activating T and NK cells

Cancer metastasis is responsible for approximately 90% of cancer-related deaths, but very few treatment options exist currently. While the role of S100A4 in promoting metastasis has been known for decades, this knowledge has not been translated in the clinic. Here, we report that a novel monoclonal antibody against the human S100A4 protein (S1004-11) effectively suppresses breast cancer metastasis in two different mouse models. Importantly, a novel combination of ant-TIGIT and S100A4-11 can suppress lung metastases even in late-stage disease after the lung premetastatic niche (PMN) has already been established similar to a stage when many breast cancer patients are diagnosed. Mechanistically, S1004-11 mAb treatment block the formation of PMN by suppressing neutrophil infiltration and activating natural killer (NK) and T cells in the lung. Single-cell RNA-sequencing and cell:cell communication analyses indicate that TIGIT signaling suppresses NK cells, which is reversed in S100A4-11 treated PMN. In summary, this study provides compelling evidence for a novel mechanism of S100A4 function in PMN formation and the feasibility of using S100A4-11 monoclonal antibody to suppress metastases at different stages of breast cancer progression.

cancer biology↗

bioRxiv2024

Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

Persistent DNA double-strand breaks (DSBs) are enigmatically implicated in neurodegenerative diseases including Huntingtons disease (HD), the inherited late-onset disorder caused by CAG repeat elongations in Huntingtin (HTT). Here we combine biochemistry, computation and molecular cell biology to unveil a mechanism whereby HTT coordinates a Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ) complex. HTT joins TC-NHEJ proteins PNKP, Ku70/80, and XRCC4 with chromatin remodeler Brahma-related Gene 1 (BRG1) to resolve transcription-associated DSBs in brain. HTT recruitment to DSBs in transcriptionally active gene- rich regions is BRG1-dependent while efficient TC-NHEJ protein recruitment is HTT-dependent. Notably, mHTT compromises TC-NHEJ interactions and repair activity, promoting DSB accumulation in HD tissues. Importantly, HTT or PNKP overexpression restores TC-NHEJ in a Drosophila HD model dramatically improving genome integrity, motor defects, and lifespan. Collective results uncover HTT stimulation of DSB repair by organizing a TC-NHEJ complex that is impaired by mHTT thereby implicating dysregulation of transcription-coupled DSB repair in mHTT pathophysiology. Highlights* BRG1 recruits HTT and NHEJ components to transcriptionally active DSBs. * HTT joins BRG1 and PNKP to efficiently repair transcription related DSBs in brain. * Mutant HTT impairs the functional integrity of TC-NHEJ complex for DSB repair. * HTT expression improves DSB repair, genome integrity and phenotypes in HD flies.

neuroscience↗