Biology subjects

Aguilar, E.

Publications and source records attributed to Aguilar, E..

11 recordsLinked to original sources

bioRxiv2025

FLASH Radiotherapy Mitigates Radiation-Induced Lymphopenia and Prevents Immunosuppression via Chk1-STAT3 Axis Modulation in a Preclinical Thoracic Irradiation Model

Background and AimsRadiation-induced lymphopenia (RIL) is a frequent side effect of conventional radiation therapy (CONV RT), due to the high radiosensitivity of circulating lymphocytes. Ultra-high dose rate "FLASH" RT may preferentially spare normal tissue while maintaining tumor control. This study evaluates the impact of single-fraction and multi-fraction thoracic FLASH RT on lymphocyte preservation, apoptosis, and immunosuppressive signaling in mice. MethodsWe compared the immunological impact of thoracic FLASH RT and CONV RT in C57BL/6 mice using single-fraction (17 Gy) and multi-fraction (2 Gy x 5) regimens using the Mobetron (IntraOp). Longitudinal blood sampling was performed at multiple time-points post-irradiation through facial vein bleed with flow cytometry analysis for CD4+, CD8+, CD19+, and NK cells to assess lymphocyte counts, apoptotic lymphocytes through Annexin V staining, and immune suppression by examining regulatory T cells (Tregs) and PD-1/PD-L1 expression. Mechanistic studies included immunofluorescence and Western blot analyses of splenic tissues to evaluate Chk1 and STAT3 signaling pathways. ResultsIn single-fraction RT, FLASH significantly reduced lung and heart fibrosis (p < 0.0001) at 28 weeks post-RT. The FLASH effect was also seen acutely on circulating immune cells, with significantly reduced lymphocyte apoptosis and accelerated recovery of CD4, CD8, CD3, NK, and B cell populations compared to CONV RT in both single-fraction and multi-fraction regimens. Conversely, CONV RT induced long-lasting increases in Tregs and sustained PD-1 and PD-L1 expression on T- and B-cells at 2- and 5-months post-irradiation in both fractionation regimens. Within the spleen, we also found CONV RT induced sustained activation of the Chk1-STAT3 pathway in CD45+ immune cells, which correlates with increased PD-1/PD-L1 expression. ConclusionFLASH RT mitigates RIL, reduces lymphocyte apoptosis, and prevents long-term immunosuppression by reduced activation of the Chk1-STAT3 pathway. These findings suggest FLASH RT may confer immunological advantages over CONV RT to enhance therapeutic efficacy.

cancer biology↗

bioRxiv2025

Erucamide regulates retinal neurovascular crosstalk

Neurovasculoglial crosstalk is critical in establishing and maintaining a functional neurovascular unit. Breakdown in the unit is central to many neurodegenerative disorders of the CNS of which the retina is a component. A growing literature indicated that primary fatty acid amides (PFAMs) can regulate this crosstalk between vasculature and neuronal tissues. In this study we describe a central role for erucamide, a 22:1 mono-unsaturated omega-9 fatty acid amide, in degenerating retinal tissues. Using high-resolution global mass spectrometry-based metabolomics, we cataloged metabolites in murine models of retinal degeneration and show that while PFAMs, in general, are highly dysregulated, erucamide is the one most significantly diminished during photoreceptor atrophy. Using rodent models of retinal degeneration and novel organosilane-modified porous silicon nanoparticles (pSiNPs) for the in vivo delivery of erucamide, we demonstrate that erucamide activates CD11b+ myeloid cells, leading to the upregulation of angiogenic and neurotrophic cytokines that stabilize retinal degeneration. We identified TMEM19 as a novel binding protein for erucamide that is crucial for human iPSC-derived macrophage precursor cells activation and subsequent neurotrophic and angiogenic factor production. These findings reveal a previously unknown PFAM pathway that is modulated during retinal degenerative diseases, demonstrating that erucamide or functional analogues and their action through TMEM19 may be useful as a therapeutic alternative to neuroprotective and stem cell-based approaches for the treatment of retinal degenerative diseases.

neuroscience↗

bioRxiv2025

FLASH radiotherapy spares lymphocytes in tumor-draining lymph nodes and increases infiltration of immune cells in tumors

Radiotherapy (RT) delivered at conventional dose rates (CONV) can both stimulate antitumor immune responses and inhibit these immune responses by depleting circulating lymphocytes. Given the observed normal tissue sparing associated with ultra-high dose rate (FLASH) RT, we hypothesized that FLASH RT may protect lymphocytes while increasing the immunogenicity of cancer cells. We irradiated cancer cell lines in vitro with FLASH RT or CONV RT and assessed immunogenic mRNA and protein expression. Both HPV-positive cell lines MEER and TC-1 showed upregulation of Calr, Hmgb1, and cGAS-STING family members after FLASH RT but not after CONV RT in vitro. To assess changes in lymphocyte populations, we irradiated murine mEER tumors in syngeneic C57BL/6 mice with 27 Gy in 3 fractions of FLASH RT or CONV RT. In mice bearing FLASH irradiated tumors, tumor-draining lymph nodes contained greater numbers of CD8+ T cells (FLASH 1.7x104 vs 0.8x104 CONV; P<0.001) and CD4+ T cells (FLASH 2.3x104 vs CONV 1.2x104; P<0.001) after irradiation. FLASH RT was associated with increased numbers of activated CD44+CD62LloCD8+ and CD4+ lymphocytes. In irradiated tumors, FLASH RT was associated with increased CD8+ tumor-infiltrating lymphocytes, increased PD1 expression on these lymphocytes and increased PDL1 expression on macrophages. Compared with CONV RT, FLASH RT spared activated T cells in tumor-draining lymph nodes and in tumors but increased checkpoint inhibitor expression in tumors. These results suggest that FLASH RT may enhance antitumor immune responses by maintaining the immunogenic effects of RT while preserving lymphocyte numbers, which may be augmented with immune checkpoint blockade. SignificanceRadiation-induced lymphopenia is associated with poorer survival outcomes. New treatment approaches, like FLASH radiation therapy (FLASH RT), which reduce lymphopenia and enhance the antitumor response, could potentially lead to better outcomes for cancer patients.

cancer biology↗

bioRxiv2025

Mitochondrial Responses to Conventional and Ultra-high Dose Rate (FLASH) Radiation

PurposeUltra-high dose rate (>40 Gy/s, FLASH) radiation therapy (RT) provides equivalent tumor control while reducing normal tissue toxicity relative to conventional dose rate (CONV) RT. However, the mechanisms underlying the observed FLASH effect are unknown. We hypothesized that the preservation of mitochondrial integrity in nontumorigenic cells by FLASH RT could be a key factor in reducing normal tissue toxicity and improving overall treatment outcomes. MethodsWe examined mitochondrial health and function after CONV and FLASH in vitro, ex vivo, and in vivo through assays of metabolic flux, mitochondrial membrane potential, mitochondrial reactive oxygen species (ROS), mitochondrial DNA damage and copy number, mitochondrial morphology, and tumor growth and survival. ResultsIn in vitro assays, murine pancreatic cancer (PDAC) cells showed evidence of equal mitochondrial damage in response to CONV and FLASH, but nontumorigenic pancreatic cells were spared by FLASH. These results were recapitulated ex vivo, and mice treated with FLASH showed higher response rates and longer survival time than mice treated with CONV in an in vivo tumor model. ConclusionsCollectively, these results suggest that FLASH spares mitochondrial function in nontumorigenic cells, but not in PDAC cells, relative to CONV. The preservation of mitochondrial integrity in nontumorigenic cells may be a key mechanism underlying the reduced normal tissue toxicity observed with FLASH RT.

cancer biology↗

bioRxiv2024

Modernizing histopathological analysis: a fully automated workflow for the digital image analysis of the intestinal microcolony survival assay

BackgroundManual analysis of histopathological images is often not only time-consuming and painstaking but also prone to error from subjective evaluation criteria and human error. To address these issues, we created a fully automated workflow to enumerate jejunal crypts in a microcolony survival assay to quantify gastrointestinal damage from radiation. Methods and MaterialsAfter abdominal irradiation of mice, jejuna were obtained and prepared on histopathologic slides, and crypts were counted manually by trained individuals. The automated workflow (AW) involved obtaining images of jejunal slices from the irradiated mice, followed by cropping and normalizing the individual slice images for resolution and color; using deep learning-based semantic image segmentation to detect crypts on each slice; using a tailored algorithm to enumerate the crypts; and tabulating and saving the results. A graphical user interface (GUI) was developed to allow users to review and correct the automated results. ResultsCrypts counted manually exhibited a mean absolute percent deviation of (34 {+/-} 26)% between individuals vs the group mean across counters, which was reduced to (11 {+/-} 6)% across the 3 most-experienced counters. The AW processed a sample image dataset from 60 mice in a few hours and required only a few minutes of active user effort. AW counts deviated from experts mean counts by (10 {+/-} 8)%. The AW thereby allowed rapid, automated evaluation of the microcolony survival assay with accuracy comparable to that of trained experts and without subjective inter-observer variation. HighlightsO_LIWe fully automated the digital image analysis of a microcolony survival assay C_LIO_LIAnalyzing 540 images takes a few hours with only minutes of active user effort C_LIO_LIThe automated workflow (AW) is just as accurate as trained experts C_LIO_LIThe AW eliminates subjective inter-observer variation and human error C_LIO_LIHuman review possible with built-in graphical user interface C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/627578v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@4d178eorg.highwire.dtl.DTLVardef@1457d39org.highwire.dtl.DTLVardef@120644eorg.highwire.dtl.DTLVardef@1996992_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Neuro-immunology in a mouse model of anti-NMDAR encephalitis and assessment of treatment approaches

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a disorder mediated by autoantibodies against the GluN1 subunit of NMDAR. It occurs with severe neuropsychiatric symptoms that often improve with immunotherapy. Clinical studies and animal models based on patients antibody transfer or NMDAR immunization suggest that the autoantibodies play a major pathogenic role. Yet, there is an important need of models offering an all-inclusive neuro-immunobiology of the disease together with a clinical course long enough to facilitate the assessment of potential new treatments. Toward this end, eight-week-old female mice (C57BL/6J) were immunized (days 1 and 28) with GluN1356-385 peptide or saline with AddaVax adjuvant and pertussis toxin. After symptom development ([~]day 35), subsets of mice were treated with an anti-CD20 (day 35), a positive allosteric modulator (PAM) of NMDAR (NMDAR-PAM, SGE-301) from days 45 to 71, or both. GluN1-antibody synthesis, epitope spreading, effects of antibodies on density and function of NMDAR, brain immunological infiltrates, microglial activation and NMDAR phagocytosis, and antibody synthesis in cultured inguinal and deep cervical lymph nodes (DCLN) were assessed with techniques including immunohistochemistry, calcium imaging, confocal and super-resolution microscopy, electrophysiology, or flow cytometry. Changes of memory and behaviour were assessed with a panel of behavioural tests, and clinical/subclinical seizures with brain-implanted electrodes. Immunized mice, but not controls, developed serum and CSF NMDAR-antibodies (IgG1 predominant) against the immunizing peptide and other GluN1 regions (epitope spreading) resulting in a decrease of synaptic and extrasynaptic NMDAR clusters and reduction of hippocampal plasticity. These findings were associated with brain inflammatory infiltrates, mainly B- and plasma cells, microglial activation, colocalization of NMDAR-IgG complexes with microglia, and presence of these complexes within microglial endosomes. Cultures of DCLC showed GluN1-antibody production. These findings were associated with psychotic-like behaviour (predominant at disease onset), memory deficit, depressive-like behaviour, abnormal movements (15% of mice), and lower threshold for developing pentylenetetrazole-induced seizures (hypoactivity, myoclonic jerks, continuous tonic-clonic) which correlated with regional cFOS expression. Most symptoms and neurobiological alterations were reversed by the anti-CD20 and PAM, alone or combined. Initial repopulation of B cells, by the end of the study, was associated with re-emergence of clinical-neurobiological alterations, which were abrogated by PAM. Overall, this model offers an all-inclusive neuro-immunobiology of the disease, allowing testing novel treatments, supporting the potential therapeutic role of NMDAR-PAM, and suggesting an immunological paradigm of systemic antigen presentation and brain NMDAR epitope spreading, which along the DCLN might contribute to fine-tune the polyclonal immune response.

neuroscience↗

bioRxiv2024

Redefining FLASH RT: the impact of mean dose rate and dose per pulse in the gastrointestinal tract

BackgroundThe understanding of how varying radiation beam parameter settings affect the induction and magnitude of the FLASH effect remains limited. PurposeWe sought to evaluate how the magnitude of radiation-induced gastrointestinal (GI) toxicity (RIGIT) depends on the interplay between mean dose rate (MDR) and dose per pulse (DPP). MethodsC57BL/6J mice were subjected to total abdominal irradiation (11-14 Gy single fraction) under conventional irradiation (low DPP and low MDR, CONV) and various combinations of DPP and MDR up to ultra-high-dose-rate (UHDR) beam conditions. The effects of DPP were evaluated for DPPs of 1-6 Gy while the total dose and MDR were kept constant; the effects of MDR were evaluated for the range 0.3- 1440 Gy/s while the total dose and DPP were kept constant. RIGIT was quantified in non-tumor-bearing mice through the regenerating crypt assay and survival assessment. Tumor response was evaluated through tumor growth delay. ResultsWithin each tested total dose using a constant MDR (>100 Gy/s), increasing DPP led to better sparing of regenerating crypts, with a more prominent effect seen at 12 and 14 Gy TAI. However, at fixed DPPs >4 Gy, similar sparing of crypts was demonstrated irrespective of MDR (from 0.3 to 1440 Gy/s). At a fixed high DPP of 4.7 Gy, survival was equivalently improved relative to CONV for all MDRs from 0.3 Gy/s to 104 Gy/s, but at a lower DPP of 0.93 Gy, increasing MDR produced a greater survival effect. We also confirmed that high DPP, regardless of MDR, produced the same magnitude of tumor growth delay relative to CONV using a clinically relevant melanoma mouse model. ConclusionsThis study demonstrates the strong influence that the beam parameter settings have on the magnitude of the FLASH effect. Both high DPP and UHDR appeared independently sufficient to produce FLASH sparing of GI toxicity, while isoeffective tumor response was maintained across all conditions.

cancer biology↗

bioRxiv2022

A plant virus causes symptoms through the deployment of a host-mimicking protein domain to attract the insect vector

Viruses are obligate intracellular parasites with limited proteomes that heavily rely on the cell molecular machinery for their multiplication and spread. Plant viruses frequently cause symptoms through interference with host developmental programs. Despite the agricultural relevance of symptom development in virus-infected crops, the molecular mechanisms underlying these viral effects remain elusive. Here, we show that the symptoms triggered by tomato yellow leaf curl virus (TYLCV) depend on the physical interaction between the host-mimicking domain of a virus-encoded protein, C4, and a plant-specific family of RCC1-like domain-containing (RLD) proteins. C4 outcompetes endogenous interactors of RLDs, disrupting RLD function in the regulation of endomembrane trafficking and polar auxin transport, ultimately leading to the developmental alterations recognized as symptoms of the viral infection. Importantly, symptoms do not have a detectable effect on the performance of the virus in the plant host, but they serve as attractants for the viral insect vector, the whitefly Bemisia tabaci, hence promoting pathogen spread. Our work uncovers the molecular underpinnings of the viral manipulation that leads to symptom development in the TYLCV-tomato pathosystem, and suggests that symptoms have evolved as a strategy to promote viral transmission by the insect vector. Given that most plant viruses are insect-transmitted, the principles described here might have broad applicability to crop-virus interactions.

plant biology↗

bioRxiv2021

Combinatorial interactions between viral proteins expand the functional landscape of the viral proteome

As intracellular parasites, viruses need to manipulate the molecular machinery of their host cells in order to enable their own replication and spread. This manipulation is based on the activity of virus-encoded proteins. The reduced size of viral genomes imposes restrictions in coding capacity; how the action of the limited number of viral proteins results in the massive cell reprogramming observed during the viral infection is a long-standing conundrum in virology. In this work, we explore the hypothesis that combinatorial interactions expand the multifunctionality of viral proteins, which may exert different activities individually and when in combination, physical or functional. We show that the proteins encoded by a plant-infecting DNA virus physically associate with one another in an intricate network. Our results further demonstrate that these interactions can modify the subcellular localization of the viral proteins involved, and that co-expressed interacting viral proteins can exert novel biological functions in planta that go beyond the sum of their individual functions. Based on this, we propose a model in which combinatorial physical and functional interactions between viral proteins enlarge the functional landscape of the viral proteome, which underscores the importance of studying the role of viral proteins in the context of the infection.

plant biology↗

bioRxiv2019

cfrB,cfrC, and a potential new cfr-like gene in Clostridium difficile strains recovered across Latin America

Cfr is a radical S-adenosyl-L-methionine (SAM) enzyme that confers cross-resistance to all antibiotics targeting the large ribosomal subunit through hypermethylation of nucleotide A2503 of 23S rRNA. Of the four known cfr genes known to date, cfr(B) and cfr(C) have been sporadically found in C. difficile, yet functional characterization of cfr(C) is still lacking. We identified genes for putative Cfr-like enzymes among clinical C. difficile strains from Mexico, Honduras, Costa Rica, and Chile. To confirm their identity and activity, we obtained minimum inhibitory concentrations for ribosome-targeting antibiotics, annotated whole genome sequences, and performed a functional characterization of Cfr(C). The seven representative isolates analyzed displayed different levels of resistance to PhLOPSA antibiotics in the absence of the ribosome protection factor OptrA, and mutations in genes for 23S rRNAs or the ribosomal proteins L3 and L4. cfr(B) was detected in four isolates as part of a Tn6218-like transposon or an un-described mobile genetic element. In turn, cfr(C) was found integrated into an ICE-element. One isolate harbored a putative cfr-like gene that shows only 51-58% of sequence identity to Cfr and known Cfr-like enzymes. Moreover, our in vitro assays confirmed that Cfr(C) methylates E. coli and C. difficile 23S rRNA fragments. These results indicate selection of cfr-like genes in C. difficile from Latin America, suggest that the diversity of cfr-like resistance genes is larger than anticipated, and provide the first assessment of the methylation activity of Cfr(C).

microbiology↗

bioRxiv2019

LINE-1 retrotransposition impacts the genome of human pre implantation embryos and extraembryonic tissues

Long Interspersed Element 1 (LINE-1/L1) is an abundant retrotransposon that has greatly impacted human genome evolution. LINE-1s are responsible for the generation of millions of insertions in the current human population. The characterization of sporadic cases of mosaic individuals carrying pathogenic L1-insertions, suggest that heritable insertions occurs during early embryogenesis. However, the timing and potential genomic impact of LINE-1 mobilization during early embryogenesis is unknown. Here, we demonstrate that inner cell mass of human pre-implantation embryos support the expression and retrotransposition of LINE -1s. Additionally, we show that LINE-1s are expressed in trophectoderm cells of embryos, and identify placenta-restricted endogenous LINE-1 insertions in newborns. Using human embryonic stem cells as a model of post-implantation epiblast cells, we demonstrate ongoing LINE-1 retrotransposition, which can impact expression of targeted genes. Our data demonstrate that LINE-1 retrotransposition starts very shortly after fertilization and may represent a previously underappreciated factor in human biology and disease.

genomics↗