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Biology subjects

Romano, G.

Publications and source records attributed to Romano, G..

17 recordsLinked to original sources

PSGL-1 blockade delays relapse to BRAF/MEK inhibition in cutaneous melanoma

Advanced BRAF-mutant cutaneous melanoma can be treated with targeted therapy when immune checkpoint inhibitors (ICIs) fail or are not a feasible option. Nevertheless, most patients do not achieve a durable response, highlighting the critical need for therapeutic partners that enhance the long-term efficacy of targeted therapy. Transcriptomic analysis of a BRAF-mutant melanoma model of acquired resistance identified P-selectin glycoprotein ligand-1 (PSGL-1) as a top-upregulated immune mediator upon resistance acquisition. PSGL-1 is a key regulator of CD8+ T cell exhaustion and differentiation, and its inhibition has been shown to enhance T cell function across multiple disease models. Based on these observations, we hypothesized that combined targeting of BRAF/MEK and PSGL-1 would improve anti-tumor responses. Here, we demonstrate that dual inhibition of BRAF/MEK and PSGL-1 elicits durable tumor control in a preclinical model of PD-1-refractory cutaneous melanoma. Single-cell RNA sequencing of the tumor microenvironment reveals robust reprogramming of intratumoral CD8+ T cells toward a less terminally differentiated, memory-like phenotype following combined BRAF/MEK and PSGL-1 targeting. Consistent with these findings, CD8+ T cells in the tumor-draining lymph nodes of PSGL-1-/- mice exhibit enhanced functionality and a less differentiated state of exhaustion when compared with wild-type mice. To extend these observations to a translationally relevant setting, we further show that antibody-mediated blockade of PSGL-1, in combination with BRAF/MEK inhibition, yields superior anti-tumor activity compared with either monotherapy. Collectively, these findings identify PSGL-1 as a promising therapeutic target to enhance the durability of targeted therapy and provide a strong rationale for future clinical evaluation.

immunology↗

A multi-omics approach to identify the impact of miR-411ed on NSCLC TKI resistance

Tyrosine Kinase inhibitors (TKIs) are widely used as effective chemotherapeutic agents for treating patients with EGFR-mutated NSCLC. Unfortunately, after treatment, patients eventually develop resistance to TKI therapy. The most common resistance mechanism for the TKI Osimertinib is the overexpression of the MET Proto-Oncogene, Receptor Tyrosine Kinase (MET). We previously demonstrated that miR-411-5p A-to-I edited at position 5 (miR-411ed) can directly target MET in A549 and H1299 cells. MiR-411ed in combination with Osimertinib reduced cell proliferation in two TKI resistant EGFR-mutated cell lines: HCC827R and PC9R. MiR-411ed did not downregulate MET expression in HCC827R, suggesting an alternative mechanism for TKI response. In this study, we aim to identify the mechanism of miR-411ed TKI response using a multi-omics approach of RNAseq and protein mass spectrometry. In our cellular model, we identified miR-411ed affected genes independent of MET activity, resulting in 211 genes (RNAseq) and 36 proteins (proteomics). Pathway analysis identified an increase in interferon signaling for RNAseq and combined omics, and a decrease in ERK/MAPK signaling in proteomics. Using the IsoTar target prediction tool, we identified STAT3 as a key regulator and confirmed STAT3 protein downregulation upon transfection with miR-411ed. We further investigated the effect of miR-411ed in vivo, observing a reduction in tumor size with miR-411ed in combination with Osimertinib but not with miR-411ed or Osimertinib treatment alone, confirming the effectiveness of miR-411ed in TKI response.

cancer biology↗

Quantitative evaluation of growth rate and survival of planktonic yeast under dual-parameter photodynamic treatment

Photodynamic Antimicrobial Chemotherapy (PACT) relies on the concomitant use of light and a photosensitizer molecule for microbial disinfection. Adding the dimension of light to the system increases the difficulty and variables to consider, yielding time and material-consuming experiments, with their reproducibility depending greatly on the disclosure of the light irradiation conditions. In the present work we analyse the effect of increasing irradiant exposures and rose bengal (RB) concentrations against Candida albicans ATCC 10231, in a checkerboard fashion. For this, we propose the use of the exponential phase coefficients, growth rate and initial number of cells, to assess the efficiency of the treatment. The coefficients are able to accurately describe the decrease of starting number of cells as consequence of the PACT effect, while also showing the metabolic burden of the presence of RB at high concentrations, with further hints that the incorrect combination of RB and radiant exposure decreases the growth rate without affecting the survivability. Our proposed methodology can then be used as a high-throughput screening method for new photosensitizers and new formulations, exploring their needs of radiant exposure for an efficient microbial disinfection.

microbiology↗

AAV2-mediated intravitreal delivery of exon-specific U1 snRNA rescues optic neuropathy in a mouse model of familial dysautonomia.

Familial dysautonomia (FD) is a rare autosomal recessive neurodegenerative disorder caused by a splicing mutation in the ELP1 gene. It predominantly affects the sensory and autonomic nervous systems, with progressive vision loss due to optic neuropathy being a universal and debilitating symptom. Retinal pathology in FD involves progressive thinning of the retinal nerve fiber layer (RNFL), resulting from the degeneration of retinal ganglion cells (RGCs). Notably, FD-associated vision loss has a postnatal onset, offering a critical window for therapeutic intervention before severe visual impairment develops in adolescence. Currently, no approved treatments exist to prevent or reverse vision loss in FD. In this study, we present a novel RNA-based therapeutic approach targeting ELP1 pre-mRNA splicing in the retina. We engineered exon-specific U1 small nuclear RNAs (ExSpeU1s) to enhance inclusion of exon 20 in the mutant ELP1 transcripts in the retina, thereby restoring full-length ELP1 expression. Delivery of ExSpeU1 via adeno-associated virus serotype 2 (AAV2) to the retina improved ELP1 splicing, rescued RGC loss, and visual function in an FD mouse model. These findings highlight ExSpeU1-mediated splicing correction as a promising therapeutic approach for treating optic neuropathy in FD, offering potential to preserve vision and improve quality of life for patients.

molecular biology↗

Disabling PSGL-1 abrogates immune suppression and resistance to PD-1 blockade in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer for which there is a critical need to identify novel therapeutic targets. Herein we define PSGL-1 as a checkpoint inhibitor using a syngeneic orthotopic model of PDAC. As with PDAC patients, CD8+ T cells within murine PDAC tumors expressed high levels of PSGL-1. PSGL-1-/- mice displayed striking T cell-dependent control of primary tumors and lung metastases. Extensive spatial remodeling within PDAC tumors occurred in PSGL-1-/- mice with a dramatic loss of proliferating tumor cells and an increase in CD8+ T cell engagement of antigen-presenting cells. The prominent CD8+ T cell infiltrates included subsets of pre-exhausted T cells retaining hallmarks of stemness and multifunctional effector capacity. These changes enabled a near complete response of PDAC to therapeutic PD-1 blockade. Our findings identify PSGL-1 as a key regulator of anti-tumor immunity in PDAC, highlighting its potential as a therapeutic target to limit CD8+ T cell exhaustion and enhance immunotherapy response. SummaryHope et al describe a pivotal function of PSGL-1 in CD8+ T cell responses to pancreatic ductal adenocarcinoma. Genetic deletion of PSGL-1 elicits tumor control by increasing T cell infiltration and maintaining functional subsets, thereby promoting sensitivity to PD-1 blockade.

immunology↗

Population analysis and immunologic landscape of melanoma in people living with HIV

PurposeTo dissect the clinical and immunological features of people living with HIV (PLWH) diagnosed with melanoma, who have consistently shown worse outcomes than HIV-negative individuals (PLw/oH) with the same cancer. Experimental DesignWe analyzed electronic health records from 1,087 PLWH and 394,437 PLw/oH with melanoma. Demographic and clinical characteristics were compared. Spatial immune transcriptomics (72 immune-related genes) was performed on melanoma tumor samples (n=11), with downstream validation using multiplex immunofluorescence (n=15 PLWH, n=14 PLw/oH). ResultsPLWH were diagnosed at a younger age, had greater representation of Hispanic and Black individuals, and showed reduced survival. They also had a markedly increased risk of brain metastases. PLWH experienced significant delays in initiating immune checkpoint inhibitor (ICI) therapy and had worse post-ICI survival, even after balancing covariates. Spatial transcriptomics revealed a more immunosuppressive tumor microenvironment in PLWH, with increased transcription of immune checkpoints (PD1, LAG3) and reduced antigen-presentation markers (HLA-DRB, B2M), with distinct spatial distributions in tumors and surrounding microenvironments. Multiplex immunofluorescence demonstrated features of an exhausted CD8 T cell compartment, including enrichment of PD1intLAG3- and PD1intLAG3 subpopulations, and a significant accumulation of myeloid-derived suppressor cells (CD11b HLA-DR- CD33). ConclusionsMelanoma in PLWH is associated with distinct clinical and immunological features, including delayed ICI treatment, reduced survival, and an immunosuppressive microenvironment with exhausted CD8 T cells and expanded myeloid-derived suppressor cells. These findings suggest that chronic HIV infection may impair antitumor immunity in melanoma. Targeting the pathways identified here may improve therapeutic responses and outcomes in this population. Statement of translational relevanceThis study reveals critical barriers to effective melanoma treatment in people living with HIV (PLWH). Despite receiving immune checkpoint inhibitors (ICIs), PLWH face delayed therapy initiation, a greater likelihood of brain metastases, and significantly higher long-term mortality, even after adjusting for demographic covariates. Transcriptional immune profiling further uncovers a tumor microenvironment enriched in immunosuppressive myeloid-derived suppressor cells and CD8 T cell populations with features of exhaustion. These findings suggest that poorer outcomes in PLWH stem not only from delayed care, but also from distinct targetable mechanisms of immune dysfunction. For example, strategies to reverse MDSC accumulation in the tumor or tailored ICI regimens could enhance immune responsiveness and improve treatment efficiency. By defining the clinical and immunological features of this population, this work highlights opportunities for precision immunotherapy tailored to PLWH with melanoma, with direct implications for improving survival and reducing disparities.

cancer biology↗

Experimental and theoretical approaches reveal that antimicrobial blue light killing efficiency decreases with biofilm growth in a Pseudomonas aeruginosa model.

Recently, the use of antimicrobial blue light (aBL) has gained interest across various applications. However, a comprehensive framework that addresses the key factors driving bacterial photoinhibition remains lacking--particularly concerning biofilms, the predominant bacterial lifestyle. The goal of this work was to evaluate the potential of photokilling in this wide-spread microbial adherent community type, and to decipher the specific mechanisms at stake. To investigate aBL killing efficiency, we conducted experiments in a Pseudomonas aeruginosa biofilm model using a well-defined millifluidic device that allows real-time microscopy and quantitative analysis of a living biofilm under local irradiation at a defined light dose. In addition, we developed a theoretical model for light-biofilm interaction that accounts for the threedimensional structure of the bacterial biofilm. To inform our model, we examined the light doseresponse in isolated cells and found a profile indicative of a multi-target mechanism of lethality. By comparing the experimental and theoretical results, we identified a loss in killing efficiency as the biofilm grows, due in part to the increase in thickness of the living material inherent to this mode of development. Our findings also highlight a reduction in the intrinsic bacterial sensitivity to blue light as biofilm development progresses, which we attribute to the low oxygen levels typical of densely populated bacterial environments. These findings reveal new features of the photokilling mechanism and redefine the approach to designing effective antimicrobial photoinactivation strategies by integrating the key physical characteristics of bacterial biofilms. Significance StatementAwareness of the bacterial worlds global importance is steadily growing in both science and society. Among the critical challenges, the continuing increase in multidrug resistance to antibiotics represents a major public health concern reinforcing the urgency of alternative antimicrobial therapies with photoinactivation as a promising approach. However, its full potential can only be achieved through a better understanding of the involved mechanisms in relevant environments. In this study, we combined experimental and theoretical approaches to investigate the photoinactivation of bacteria within a developing biofilm, the dominant bacterial lifestyle. Our comprehensive analysis sheds light on the mechanisms and limitations of photoinactivation in the fight against microbes, which is essential for designing novel antibacterial phototherapies.

biophysics↗

An opioid efficacy switch for reversible optical control of peripheral analgesia

The mu-opioid receptor (MOR) is a major target for the treatment of pain. However, opioids are prone to side effects which limit their effectiveness as analgesics and can lead to opioid use disorders or, even, lethal overdose. The systemic administration of opioid agonists makes it both very difficult to decipher their underlying circuit mechanisms of action and to limit drug action to specific receptor subpopulations to isolate therapeutic effects from adverse side effects. Here we design, synthesize, and characterize a reversibly photoswitchable morphinan agonist termed "azo-morphine-3" (AM-3) which interconverts from low to high efficacy in response to different wavelengths of light to enable optical control of MOR signaling. Cryo-EM structures of the low efficacy "trans" and high efficacy "cis" states of AM-3 bound to the MOR reveal distinct binding modes of the photoswitchable azobenzene moiety, each inducing unique structural dynamics, providing insight into the molecular basis of agonist efficacy. In mice, AM-3 drives reversible and repeatable optical control of anti-nociception with a reduced side effect profile owing to its restriction to the periphery and its ability to be locally activated at the site of pain.

pharmacology and toxicology↗

A newly developed whole genome sequencing protocol enables early tracking of Enterovirus D68 molecular evolution.

Background Human enterovirus D68 (EV-D68) has been associated with an increase in mild-to-severe pediatric respiratory diseases worldwide. The rate of circulation of this virus is largely underestimated in the population and genetic evolutionary data are usually available only for partial sequences (e.g VP1 region). To achieve a timely genomic surveillance, a reliable, high-throughput EV-68 sequencing assay is required. Here we report an improved high-throughput EV-D68 whole-genome sequencing (WGS) assay performed directly on clinical samples that is suitable for short-read sequencing platforms. Between June and December 2022, a total 37 (1.9%) respiratory samples were EV-D68 positive and together with 52 additional samples with a median cycle threshold of 28.3, ranging from 18 to 36.8 were included in the validation analyses. Overall, all the primers had good performance and no mismatches were detected in more than 85% of sequences (932 WGS dataset). Using a cut-off of Cq <32 in at least 85.5% of samples a WG or PG was obtained, confirming an acceptable positive sequencing rate for the designed method. A total of 65 WGS were obtained and have a mean coverage of 98.4% across the genome, with a median depth of 6158x (range 2815x-7560x). Based on the obtained data, this method is cost effective resulting in an easy-to-perform protocol helpful for tracing the evolution of EV-D68 in protein different from VP1. EV-D68 could become a significant pathogen for public health in the next future, and thus this protocol for whole genome sequencing could help clinical and molecular virologists to be ready for molecular epidemiology surveillance.

microbiology↗

METTL3 alters capping enzyme expression and its activity on ribosomal proteins

The 5 cap, catalyzed by RNA guanylyltransferase and 5-phosphatase (RNGTT), is a vital mRNA modification for the functionality of mRNAs. mRNA capping occurs in the nucleus for the maturation of the functional mRNA and in the cytoplasm for fine-tuning gene expression. Given the fundamental importance of RNGTT in mRNA maturation and expression there is a need to further investigate the regulation of RNGTT. N6-methyladenosine (m6A) is one of the most abundant RNA modifications involved in the regulation of protein translation, mRNA stability, splicing, and export. We sought to investigate whether m6A could regulate the expression and activity of RNGTT. A motif for the m6A writer methyltransferase 3 (METTL3) in the 3UTR of RNGTT mRNA was identified. Knockdown of METTL3 resulted in destabilizing RNGTT mRNA, and reduced protein expression. Sequencing of capped mRNAs identified an underrepresentation of ribosomal protein mRNA overlapping with 5 terminal oligopyrimidine (TOP) mRNAs and genes are dysregulated when cytoplasmic capping is inhibited. Pathway analysis identified disruptions in the mTOR and p70S6K pathways. A reduction in RPS6 mRNA capping, protein expression, and phosphorylation was detected with METTL3 knockdown.

molecular biology↗

Su(var)3-9 mediates age-dependent increase in H3K9 methylation on TDP-43 promoter triggering neurodegeneration

Aging progressively modifies the physiological balance of the organism increasing susceptibility to both genetic and sporadic neurodegenerative diseases. These changes include epigenetic chromatin remodeling events that may modify gene transcription. However, how aging interconnects with disease-causing genes is not well known. Here, we found that Su(var)3-9 causes increased methylation of histone H3K9 in the promoter region of TDP-43, the most frequently altered factor in amyotrophic lateral sclerosis (ALS), affecting the mRNA and protein expression levels of this gene through epigenetic modifications in chromatin organization that appear to be conserved in aged Drosophila brains, mouse and human cells. Remarkably, augmented Su(var)3-9 activity causes a decrease in TDP-43 expression followed by early defects in locomotor activities. In contrast, decreasing Su(var)3-9 action promotes higher levels of TDP-43 expression and reinvigorates motility parameters in old flies, uncovering a novel role of this enzyme in regulating TDP-43 expression and locomotor senescence. The data indicate how conserved epigenetic mechanisms may link aging with neuronal diseases and suggest that Su(var)3-9 may play a role in the pathogenesis of ALS.

molecular biology↗

Microparticle-delivered Cxcl9 delays the relapse of Braf inhibitor-treated melanoma

Patients with BRAF-mutant melanoma show significant responses to combined BRAF and MEK inhibition, but most relapse within 2 years. A major reservoir for drug resistance is minimal residual disease (MRD), comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600E extinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis after BrafV600E extinction revealed that after an initial surge of immune activation, tumors later became immunologically "cold" after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines that may be central players in the process, being strongly upregulated initially and steeply decreasing as the immune response faded. Therefore, we hypothesized that sustaining the chemokine signaling could impair MRD maintenance through increased recruitment of effector T-cells. We show that intratumoral administration of recombinant Cxcl9, either naked or loaded in microparticles, significantly impaired MRD relapse in BRAF-inhibited tumors, including several complete responses after microparticle-delivered rCxcl9 combined with BRAF and MEK-inhibition. Our experiments constitute a proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of frontline treatment methods.

cancer biology↗

TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila brains

Alterations in the function of the RNA-binding protein TDP-43 is largely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease of the human motor system that leads to motoneurons degeneration and reduced life expectancy by molecular mechanisms not well known. Regarding to that, we found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible to convert glutamate to {gamma}-aminobutyric acid (GABA), were downregulated in TBPH-null flies and motoneurons derived from ALS patients carrying mutations in TDP-43 suggesting that defects in the regulation of GAD1 may lead to neurodegeneration by affecting neurotransmitter balance. In this study, we observed that TBPH was required to regulate GAD1 pre-mRNA splicing and GABA levels in Drosophila brains. Interestingly, we discovered that pharmacological treatments aimed to modulate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies revealing novel mechanisms and therapeutic strategies in ALS.

neuroscience↗

MiREDiBase: a manually curated database of editing events in microRNAs

MicroRNAs (miRNAs) are regulatory small non-coding RNAs that function as translational repressors. MiRNAs are involved in most cellular processes, and their expression and function are presided by several factors. Amongst, miRNA editing is an epitranscriptional modification that alters the original nucleotide sequence of selected miRNAs, possibly influencing their biogenesis and target-binding ability. A-to-I and C-to-U RNA editing are recognized as the canonical types, with the A-to-I type being the predominant one. Albeit some bioinformatics resources have been implemented to collect RNA editing data, it still lacks a comprehensive resource explicitly dedicated to miRNA editing. Here, we present MiREDiBase, a manually curated catalog of editing events in miRNAs. The current version includes 3,059 unique validated and putative editing sites from 626 pre-miRNAs in humans and three primates. Editing events in mature human miRNAs are supplied with miRNA-target predictions and enrichment analysis, while minimum free energy structures are inferred for edited pre-miRNAs. MiREDiBase represents a valuable tool for cell biology and biomedical research and will be continuously updated and expanded at https://ncrnaome.osumc.edu/miredibase.

bioinformatics↗

TDP-43 regulates the expression levels of Disc-large in skeletal muscles to promote the assemble of the neuromuscular synapses in Drosophila

BackgroundAlterations in the intracellular distribution of TDP-43 were observed in the skeletal muscles of patients suffering from ALS. However, it is not clear whether these modifications play an active role in the disease or represent a physiological adaptation to muscles homeostasis.\n\nResultTo answer these questions, we modulated the activity of this protein in Drosophila muscles and observed that TDP-43 was required in these tissues to promote the formation and growth of the neuromuscular synapses. Moreover, we identified that TDP-43 regulates the expression levels of Disc-large (Dlg) and demonstrated that the modulation of Dlg activity, in skeletal muscles or motoneurons, was sufficient to recover the TDP-43-null locomotive and synaptic defects in flies. Additionally, we found that similar mechanisms are conserved in human cell lines and present in tissues derived from ALS patients.\n\nConclusionsOur results uncover the physiological role of TDP-43 in skeletal muscles as well as the mechanisms behind the autonomous and non-autonomous behavior of this protein in the organization of the neuromuscular synapses.

neuroscience↗

Endogenous TDP-43 prevents retrotransposons activation through Dicer-2 activity and the RNA silencing machinery in Drosophila

The aberrant expression of retrotransposable elements (RTEs) was observed in different neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a terminal disorder characterized by functional alterations in the small RNA-binding protein TDP-43, suggesting that these events might be connected. Using genome wide gene expression profiles, we detected RTEs highly upregulated in TDP-43-null Drosophila heads while, the genetic rescue of TDP-43 function reverted these modifications. Furthermore, we found that TDP-43 modulates the small interfering RNA (siRNA) silencing machinery responsible for RTEs repression. Molecularly, we observed that TDP-43 regulates the expression levels of Dicer-2 by direct protein-mRNA interactions in vivo. Accordingly, the genetic or pharmacological recovery of Dicer-2 activity was sufficient to repress retrotransposons activation and revert the neurodegeneration in TDP-43-null Drosophila motoneurons. Our results, describe a novel physiological role of endogenous TDP-43 in the prevention of RTEs-induced neurodegeneration through the modulation of Dicer-2 activity and the siRNA pathway.

cell biology↗

rCASC: reproducible Classification Analysis of Single Cell sequencing data

SummarySingle-cell RNA sequencing has emerged as an essential tool to investigate cellular heterogeneity, and highlighting cell sub-population specific signatures. Nowadays, dedicated and user-friendly bioinformatics workflows are required to exploit the deconvolution of single-cells transcriptome. Furthermore, there is a growing need of bioinformatics workflows granting both functional, i.e. saving information about data and analysis parameters, and computation reproducibility, i.e. storing the real image of the computation environment. Here, we present rCASC a modular RNAseq analysis workflow allowing data analysis from counts generation to cell sub-population signatures identification, granting both functional and computation reproducibility.\n\nAvailability and ImplementationrCASC is part of the reproducible bioinfomatics project. rCASC is a docker based application controlled by a R package available at https://github.com/kendomaniac/rCASC.\n\nSupplementary informationSupplementary data are available at rCASC github

bioinformatics↗