Biology subjects

Hidalgo, S.

Publications and source records attributed to Hidalgo, S..

12 recordsLinked to original sources

bioRxiv2026

The differentiation and functional landscape of tumor-resident CD8⁺ T cells shapes immunity and clinical outcomes in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a common and lethal kidney cancer in which CD8+ T cell-targeted immunotherapies are standard of care. However, tumor CD8+ T cell infiltration correlates with divergent clinical outcomes, reflecting functional heterogeneity. We comprehensively characterized tumor-specific memory CD8+ T cells in ccRCC using phenotypic, transcriptional and functional analyses. Three major subsets were identified based on tissue-residency markers: circulating (Tcirc), tissue-resident (Trm) and CD69+CD103- Trm-like cells, all recognizing autologous RCC cells in an HLA class I-dependent manner. Tcirc cells exhibited greater stemness and cytotoxic potential, whereas Trm populations displayed tissue-residency programs, enhanced tumor reactivity and exhaustion features. Single-cell transcriptomics analyses uncovered substantial heterogeneity within Trm cells, including progenitor, activated, transitional, type-17, interferon-responsive, regulatory and terminally exhausted states. Trajectory and TCR analyses indicated progressive differentiation from central-memory Tcirc cells toward tissue residency while losing circulation, cytotoxicity and stemness potentials and then progress toward terminal exhaustion. Enrichment of progenitor Trm cells associated with improved survival and favorable immunotherapy response, whereas regulatory or exhausted Trm cells correlated with poor outcomes. These findings define the differentiation and functional landscape of tumor-specific CD8+ T cells that shapes immunity and clinical outcomes in ccRCC.

immunology↗

bioRxiv2025

Splicing of a core clock gene regulates seasonal adaptations by a winter gating mechanism

Seasons bring changes to the environment. Many organisms adjust their physiology and behavior in response to seasonal changes in order to survive. Although the molecular mechanisms mediating the integration of seasonal cues are still unclear, the working model indicates the involvement of the circadian clock. Notably, the circadian neuropeptide Pigment Dispersing Factor (PDF), an output of the circadian clock, has been shown to alter its expression and activity in response to seasonal changes to facilitate seasonal adaptations in insects. Here, we show that the alternative splicing (AS) of a circadian clock gene, timeless (tim), regulates the seasonal responses of PDF signaling in Drosophila melanogaster. We first showed that tim-sc, the predominant isoform in winter, is regulated by photoperiod in addition to temperature, while the expression of the canonical tim-l isoform is primarily sensitive to temperature. We then demonstrated that tim-sc maintains physiology and behavior in a "winter lock" state by modulating PDF. At the cellular and molecular level, TIM-SC behavior differs from the canonical TIM-L. Interestingly, flies expressing tim-sc did not fully phenocopy wild-type flies reared in winter conditions, suggesting that other mechanisms are at play in regulating seasonal adaptations, despite the importance of tim AS.

neuroscience↗

bioRxiv2025

Global changes in alternative splicing are associated with insecticide response and resistance

Alternative splicing (AS) promotes phenotypic plasticity to adverse conditions by altering transcripts involved in stress adaptation. However, whether changes in AS occur transcriptome-wide or in only a few key genes is unclear. Agricultural insect pests are regularly exposed to xenobiotic stress from insecticide applications and thus develop resistance. We show that the pest Drosophila suzukii, resistant to multiple insecticides, exhibits increased global AS events compared to susceptible flies. Alternatively spliced genes are enriched in multiple processes including stress response and insecticide resistance, suggesting AS is a mechanism underlying insecticide resistance development. Furthermore, sublethal insecticide exposure promotes AS events even in the absence of substantial differential gene expression. This study provides insights into the role of AS in enabling insects to diversify genome function to survive acute insecticide treatment and to develop xenobiotic resistance.

genomics↗

bioRxiv2025

The Innexin 7 gap junction protein contributes to synchronized activity in the Drosophila antennal lobe and regulates olfactory function

In the mammalian olfactory bulb (OB), gap junctions coordinate synchronous activity among mitral and tufted cells to process olfactory information. In insects, gap junctions are also present in the Antennal Lobe (AL), a structure homologous to the mammalian OB. The invertebrate gap junction protein ShakB contributes to electrical synapses between AL Projections Neurons (PNs) in Drosophila. Other gap junction proteins, including Innexin 7 (Inx7), are also expressed in the Drosophila AL, but little is known about their contribution to intercellular communication during olfactory information processing. Here we report spontaneous calcium transients in PNs grown in cell culture that are highly synchronous when these neurons are physically connected. RNAi-mediated knock down of Inx7 in cultured PNs blocks calcium transient neuronal synchronization. In vivo, downregulation of Inx7 in the AL impairs both vinegar-induced electrophysiological calcium responses and behavioral responses to this appetitive stimulus. These results demonstrate that Inx7-encoded gap junctions functionally coordinate PN activity and modulate olfactory information processing in the adult Drosophila AL.

neuroscience↗

bioRxiv2024

Transcriptome analysis of Drosophila suzukii reveals molecular mechanisms conferring pyrethroid and spinosad resistance

Drosophila suzukii lay eggs in soft-skinned, ripening fruits, making this insect a serious threat to berry production. Since its 2008 introduction into North America, growers have used insecticides, such as pyrethroids and spinosads, as the primary approach for D. suzukii management, resulting in detections of insecticide resistance in this pest. This study sought to identify the molecular mechanisms conferring insecticide resistance in these populations. We sequenced the transcriptomes of two pyrethroid- and two spinosad-resistant isofemale lines. In both pyrethroid-resistant lines and one spinosad-resistant line, we identified overexpression of metabolic genes that are implicated in resistance in other insect pests. In the other spinosad-resistant line, we observed an overexpression of cuticular genes that have been linked to resistance. Our findings enabled the development of molecular diagnostics that we used to confirm persistence of insecticide resistance in California, U.S.A. To validate these findings, we leveraged D. melanogaster mutants with reduced expression of metabolic or cuticular genes that were found to be upregulated in resistant D. suzukii to demonstrate that these genes are involved in promoting resistance. This study is the first to characterize the molecular mechanisms of insecticide resistance in D. suzukii and provides insights into how current management practices can be optimized.

genomics↗

bioRxiv2024

Long-term efficacy of adoptive cell therapy is determined by host CD8+ T cells and undermined by lymphodepleting preconditioning

Adoptive T cell therapy (ACT) has demonstrated remarkable efficacy in treating hematological cancers. However, its efficacy against solid tumors remains limited and the emergence of cancer cells that lose expression of targeted antigens often promotes resistance to ACT. Importantly, the mechanisms underlying effective and durable ACT-mediated tumor control are incompletely understood. Here, we show that adoptive transfer of TCR-transgenic CD8+ T cells eliminates established murine melanoma tumors, with concomitant accumulation of tumor-infiltrating CD8+ T cells exhibiting both progenitor-exhausted and terminally-differentiated phenotypes. Interestingly, host CD8+ T cells contributed to ACT-mediated elimination of primary tumors and rejected ACT-resistant melanoma cells lacking the targeted antigen. Mechanistically, ACT induced TNF-- and cross-presenting dendritic cell-dependent tumor accumulation of endogenous CD8+ T cells and effective tumor elimination. Importantly, although lymphodepleting preconditioning enhanced ACT-mediated tumor elimination, it abrogated host antitumor immunity and protection against ACT-resistant melanoma cells. Enrichment of transcriptional signatures associated with TNF- signaling, cross-presenting dendritic cells and tumor-specific CD8+ T cells in human melanoma tumors correlated with favorable responses to ACT and increased survival. Our findings reveal that long-term efficacy of ACT is determined by the interplay between transferred and endogenous CD8+ T cells and is undermined by lymphodepleting preconditioning, which ultimately favors ACT resistance.

immunology↗

bioRxiv2024

Alternative splicing of clock transcript mediates the response of circadian clocks to temperature changes

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In Drosophila, seasonal adaptations and temperature compensation are regulated by temperature-sensitive alternative splicing (AS) of period (per) and timeless (tim) genes that encode key transcriptional repressors of clock gene expression. Although clock (clk) gene encodes the critical activator of clock gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. We therefore sought to investigate whether clk exhibits AS in response to temperature and the functional changes of the differentially spliced transcripts. We observed that clk transcripts indeed undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternative clk transcript, hereinafter termed clk-cold, which encodes a CLK isoform with an in-frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1-dependent phosphorylation site, is among the four amino acids deleted in CLK-cold protein. Using a combination of transgenic fly, tissue culture, and in vitro experiments, we demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing CLK occupancy at clock gene promoters. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature, which can be explained by the higher amounts of CLK-cold isoforms that lack S13 residue. This study provides new insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock.

neuroscience↗

bioRxiv2022

The circadian clock conveys thermal and photoperiodic cues to modulate EYES ABSENT via the neuropeptide PDF to regulate seasonal physiology

Organisms adapt to seasonal changes in photoperiod and temperature to survive; however, the mechanisms by which these signals are integrated in the brain are poorly understood. We previously reported that EYES ABSENT (EYA) in Drosophila shows higher levels in cold temperature or short photoperiod, and genetic ablation of eya in the fly brain inhibits reproductive dormancy, suggesting that EYA promotes winter physiology. Nevertheless, the mechanisms by which EYA senses seasonal cues are unclear. Pigment-Dispersing Factor (PDF) is a neuropeptide important for photoentrainment and regulation of circadian output rhythms. Interestingly, PDF also regulates reproductive dormancy, suggesting that it may mediate the function of the circadian clock in modulating seasonal physiology. In this study, we investigated the role of PDF signaling in mediating the impact of EYA on seasonal biology. First, we subjected flies to different photoperiodic and temperature regimes and observed that PDF abundance is lower in cold and short days, compared to warm and long days. Interestingly, the response of PDF to seasonal cues is opposite of what was observed for EYA. We then determined the potential for PDF to convey seasonal cues and modulate EYA function in seasonality by assessing coexpression of EYA and PDF receptor. Our results indicated that PDF receptor (PDFR) is indeed coexpressed with EYA in the fly brain, including in the circadian clock neuronal network and neurons in the pars intercerebralis. We then manipulated PDF signaling in eya+ cells to show that PDF modulates seasonal adaptations in daily activity rhythm and ovary development via EYA-dependent and independent mechanisms. At the molecular level, manipulating PDF signaling impacted EYA protein abundance. Specifically, we showed that protein kinase A (PKA), an effector of PDF signaling, phosphorylates EYA and promotes its degradation. This explains the opposite responses of PDF and EYA abundance to changes in seasonal cues. In summary, our results support a model in which PDF signaling negatively modulates EYA levels to regulate seasonal physiology, linking the circadian clock to the modulation of seasonal adaptations.

neuroscience↗

bioRxiv2022

CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters

Circadian clock and chromatin remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin remodeling complex promotes the repression of circadian gene expression in Drosophila. In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to the period (per) promoter. We observed reduced BRM binding to the DNA in clk null flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to the per promoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. This conclusion is further supported by elevated BRM binding to the per promoter in flies subjected to constant light. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin remodeling complex. Author SummaryCircadian clocks are endogenous time-keeping mechanisms that allow organisms to anticipate and adapt to daily changes in their external environment. These clocks are driven by a molecular oscillator that generates rhythms in the expression of many genes, termed clock-controlled genes. The genomic DNA containing these clock-controlled genes are also modified in a rhythmic manner throughout the day. DNA are more tightly packaged with histone proteins when transcription of clock-controlled genes is repressed while the interaction between DNA and histone proteins are more relaxed during transcriptional activation. We found that two key clock proteins, CLOCK and TIMELESS, regulate daily rhythmicity in the binding of BRAHMA, a chromatin remodeler, to DNA spanning clock-controlled genes to facilitate their rhythmic gene expression cycles. Moreover, because TIMELESS is sensitive to light, our study provides new insights into how light can affect DNA structure and gene expression.

molecular biology↗

bioRxiv2022

Internal needs drive the cephalic phase response in Drosophila melanogaster

Hungry animals consistently show a desire to obtain food. Even a brief sensory detection of food can trigger bursts of physiological and behavioral changes. However, the underlying mechanisms by which the sensation of food triggers the acute behavioral response remain elusive. We have previously shown in Drosophila that hunger drives a preference for low temperature. Because Drosophila is a small ectotherm, a preference for low temperature implies a low body temperature and a low metabolic rate. Here, we show that taste sensing triggers a switch from a low to a high temperature preference in hungry flies. We show that taste stimulation by artificial sweeteners or optogenetics triggers an acute warm preference, but is not sufficient to reach the fed state. Instead, nutrient intake is required to reach the fed state. The data suggest that starvation recovery is controlled by two components: taste-evoked and nutrient-induced warm preferences, and that taste and nutrient quality play distinct roles in starvation recovery. Animals are motivated to eat based on time of day or hunger. We found that clock genes and hunger signals profoundly control the taste-evoked warm preferences. Thus, our data suggest that the taste-evoked response is one of the critical layers of regulatory mechanisms representing internal energy homeostasis and metabolism.

neuroscience↗

bioRxiv2021

Lipophorin receptors regulate mushroom bodies development and participate in learning, memory, and sleep in flies

BackgroundDrosophila melanogaster Lipophorin Receptors (LpRs), LpR1 and LpR2, mediate lipid uptake. The orthologs of these receptors in vertebrates, ApoER2 and VLDL-R, bind Reelin, a glycoprotein not present in flies. These receptors are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain. It is currently unknown whether LpRs play similar roles in the Drosophila brain. ResultsWe report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the Mushroom Bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurites. This effect depends on LpRs and Dab, the Drosophila ortholog of the reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin. ConclusionsThese findings demonstrate that LpRs contribute to MB development and function, supporting the existence of LpR-dependent signaling in Drosophila.

neuroscience↗

bioRxiv2020

Neonicotinoids disrupt memory, circadian behaviour and sleep

Globally, neonicotinoids are still the most used insecticides, despite their well-documented sub-lethal effects on beneficial insects1. Neonicotinoids are agonists at the nicotinic acetylcholine receptors, the main mediator of synaptic transmission in the insect brain2-5, making them highly potent neurotoxins and insecticides6,7. Memory, circadian rhythmicity and sleep are essential for efficient foraging in many pollinating insects, and involve nicotinic acetylcholine receptor signalling2,4,8-10. The effect of field-relevant concentrations of European Union-banned neonicotinoids: imidacloprid, clothianidin and thiamethoxam, as well as the currently unbanned thiacloprid were tested on Drosophila memory, circadian rhythms and sleep. Field-relevant concentrations of imidacloprid, clothianidin and thiamethoxam disrupted learning, behavioural rhythmicity and sleep whilst thiacloprid exposure only affected sleep. Exposure to imidacloprid and clothianidin directly affected neurophysiology, preventing the day/night remodelling and accumulation of pigment dispersing factor neuropeptide in the dorsal terminals of clock neurons. Knockdown of the neonicotinoid susceptible D1 and D{beta}2 nicotinic acetylcholine receptor subunits in the mushroom bodies or clock neurons recapitulated the neonicotinoid like deficits in memory or circadian/sleep behaviour demonstrating that neonicotinoid effects are likely mediated in the mushroom body and clock circuitry. Disruption to learning, circadian rhythmicity and sleep are likely to have far-reaching detrimental effects on beneficial insects in the field.

pharmacology and toxicology↗