Biology subjects

Ortiz, S.

Publications and source records attributed to Ortiz, S..

11 recordsLinked to original sources

bioRxiv2026

Adaptation of Iterative Ultrastructure Expansion Microscopy (iU-ExM) enables high-resolution imaging of the cytoskeleton and subcellular architecture of Trypanosoma cruzi

Kinetoplastid parasites, including Trypanosoma cruzi, possess a highly organized subpellicular microtubule network essential for maintaining cell shape, organelle positioning, and dividing. Here we describe results obtained using immunofluorescence microscopy of cells processed using conventional ultrastructure expansion microscopy (U-ExM), that improved visualization of the cytoskeletal structures, with an ~4.5-fold expansion, as well as the recently described and iterative ultrastructure expansion microscopy (iU-ExM) with about 6.2-fold and 18-fold expansion of epimastigotes of T. cruzi. The optimized protocol involves the formation of an initial hydrogel crosslinked with N,N'(1,2-dihydroxyethylene) bisacrylamide (DHEBA), a temperature- and alkaline pH-sensitive cleavable crosslinker, followed by thermal denaturation at 85 degrees Celsius, intermediate immunostaining, neutral gel re-embedding, and the formation of a second hydrogel crosslinked with N,N' methylenebisacrylamide (BIS). Subsequent dissolution of the first hydrogel under alkaline conditions enables a second round of expansion. This approach achieved approximately 6.2-fold expansion during the first round and a final linear expansion of approximately 18-fold. Using conventional confocal microscopy, iU-ExM enabled high-resolution visualization of individual subpellicular microtubules and specialized internal microtubule arrangements, including those associated with the basal body, flagellar pocket, Golgi complex, and cytopharynx. Furthermore, the enhanced spatial resolution facilitated the examination of cytoskeletal reorganization at different stages of the cell cycle, revealing changes in microtubule organization associated with mitotic spindle formation and cytokinesis. Together, these findings establish iU-ExM as an accessible high-resolution imaging approach for investigating the complex subcellular architecture of T. cruzi, providing new opportunities to explore cytoskeletal organization and remodeling during parasite cell division using conventional confocal microscopy.

cell biology↗

bioRxiv2025

Activity-driven proprioceptive synaptic refinement in the developing spinal cord by complement signaling mechanisms

Proprioceptive group Ia afferents detect muscle stretch to guide effortless and purposeful movement and make monosynaptic connections with spinal a-motor neurons to mediate reflexes, such as the stretch reflex. It is thought that proprioceptive Ia afferents target motor neurons of the same spinal segment; yet, how this specificity, if any, is established during early development is unknown. Using ex vivo spinal cord electrophysiology preparations from neonatal mice of both sexes, we identified a developmental period during which proprioceptive la afferents evoke both segmental and intersegmental responses at monosynaptic latencies. We provide anatomical evidence that motor neurons in the lumbar segment 4 (L4) receive direct input from proprioceptive Ia afferents in L5 during early postnatal development. Intersegmental responses (L4/L5) were prominent at postnatal days (P) 4-7 but were virtually absent by P11-13. To test the role of proprioceptor activity on segmental specification, we analyzed NaV1.6 conditional knockout mice (NaV1.6cKO), in which proprioceptor signaling is impaired, and found that intersegmental responses persist up to P11-13 but were absent in age-matched floxed controls. We predict this is due to impaired activation of complement signaling pathways, as NaV1.6cKO mice showed reduced C1qA expression in the ventral spinal cord at P9. Consistent with this, C1qA knockout mice also retain intersegmental responses at P11-13. Collectively, these findings identify an important postnatal window during which segmental specificity of proprioceptive circuits emerges and suggest that proprioceptor activity induces C1qA-mediated elimination of excessive intersegmental connectivity. Key points summaryO_LIDuring the first ten days of postnatal development, the spinal monosynaptic reflex arc possesses intersegmental proprioceptive afferent projections onto motor neurons of an adjacent segment. C_LIO_LIThese exuberant Ia-motoneuron projections are accompanied by evocable intersegmental reflex responses. In normal conditions, the intersegmental response is lost by postnatal day 11. Conversely, in a mouse model of impaired proprioceptive signaling, the intersegmental response persists. C_LIO_LIWe find that impaired proprioceptive signaling through the monosynaptic reflex arc leads to low expression levels of the complement signaling protein, C1qA, near ChAT-positive motor neurons, whereas microglia recruitment is unaffected. This was consistent with the persistence of robust intersegmental responses in C1qA knockout mice. C_LIO_LICollectively, our results show that proprioceptor activity induces complement cascade signaling to prune exuberant intersegmental synapses in the spinal cord formed during early development, resulting in segmental restriction of the monosynaptic reflex arc by the end of the second postnatal week. C_LI

neuroscience↗

bioRxiv2025

CloneCoordinate: Open-source software for collaborative DNA construction

Custom DNA constructs have never been more common or important in the life sciences. Many researchers therefore devote substantial time and effort to molecular cloning, aided by abundant computer-aided design tools. However, support for managing and documenting the construction process, and for effectively handling and reducing the frequency of setbacks, is lacking. To address this need, we developed CloneCoordinate, a free, open-source electronic laboratory notebook specifically designed for cloning and fully implemented in Google Sheets. By maintaining a real-time, automatically prioritized task list, a uniform physical sample inventory, and standardized data structures, CloneCoordinate enables productive, collaborative cloning for individuals or teams. We demonstrate how the information captured by CloneCoordinate can be leveraged to troubleshoot assembly problems and provide data-driven insights into cloning efficiency. CloneCoordinate offers a new and uniquely accessible model for how to carry out, and iteratively improve on, real-world DNA assembly.

synthetic biology↗

bioRxiv2025

Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1+Tcf1+ stem-like reservoir and overcomes resistance to PD-1 axis blockade

PD-1+Tcf1+ stem-like cells are critical mediators of endogenous T cell responses to PD-1/PD-L1 blockade and are maintained by MHC-dependent interactions with professional antigen-presenting cells (APCs). Unlike conventional T cells, CAR-T cells are activated by intact antigen expressed on tumors, not by peptide/MHC expressed on APCs, restricting their activation to the hostile tumor microenvironment (TME) that may impair preservation of this critical stem-like subset. Indeed, in an autochthonous model of ROR1+ lung cancer that we developed, CAR-T cells targeting the tumor-associated antigen ROR1 were uniformly Tcf1+ prior to infusion but rapidly downregulated Tcf1 in vivo and became terminally exhausted, similar to observations in patients, resulting in faster attrition and no enhancement in response to PD-L1 blockade. We hypothesized that overexpression of AP-1 family transcription factors, which can regulate T cell exhaustion, could enable CAR-Ts to maintain this critical PD-1+Tcf1+ subset within tumors independently of APCs and sensitize them to PD-1/PD-L1 blockade. Overexpression of the AP-1 TF c-Jun, but not BATF, improved preservation of PD-1+Tcf1+ CAR-T cells within tumors in a cell-intrinsic manner that correlated with increased persistence deeper within tumors. Notably, c-Jun overexpression alone was insufficient to prevent CAR-T exhaustion in the lung TME, in contrast to prior work in xenograft models, with progressive CAR-T dysfunction correlated with PD-1-dependent downregulation of c-Jun. However, c-Jun overexpression dramatically sensitized CAR-Ts to PD-L1 blockade, which restored c-Jun levels in CAR-Ts, drove log-fold expansion of CAR-Ts within tumors, and induced nearly complete eradication of ROR1+ tumor in highly aggressive models of lung cancer. Altogether, our data show that combination with PD-L1 blockade is necessary to unleash the full potential of c-Jun-overexpressing CAR-T cells in aggressive solid tumors like lung cancer and suggest that strategies to enhance formation of intratumoral PD-1+Tcf1+ reservoirs can overcome CAR-T resistance to PD-1 blockade.

immunology↗

bioRxiv2024

Differential encoding of mammalian proprioception by voltage-gated sodium channels

Animals that require purposeful movement for survival are endowed with mechanosensory neurons called proprioceptors that provide essential sensory feedback from muscles and joints to spinal cord circuits, which modulates motor output. Despite the essential nature of proprioceptive signaling in daily life, the mechanisms governing proprioceptor activity are poorly understood. Here, we have identified distinct and nonredundant roles for two voltage-gated sodium channels (NaVs), NaV1.1 and NaV1.6, in mammalian proprioception. Deletion of NaV1.6 in somatosensory neurons (NaV1.6cKO mice) causes severe motor deficits accompanied by complete loss of proprioceptive transmission, which contrasts with our previous findings using similar mouse models to target NaV1.1 (NaV1.1cKO). In NaV1.6cKO animals, loss of proprioceptive feedback caused non-cell- autonomous impairments in proprioceptor end-organs and skeletal muscle that were absent in NaV1.1cKO mice. We attribute the differential contribution of NaV1.1 and NaV1.6 in proprioceptor function to distinct cellular localization patterns. Collectively, these data provide the first evidence that NaV subtypes uniquely shape neurotransmission within a somatosensory modality. TeaserVoltage gated sodium channels differentially encode mammalian proprioception via distinct cellular localization patterns.

neuroscience↗

bioRxiv2023

Temperature influences the excretion time and parasitic load of Trypanosoma cruzi in the Triatoma infestans vector

Trypanosoma cruzi is a protozoan parasite transmitted by triatomine insect vectors, which expel their infectious dejections when they feed, causing Chagas disease in humans. The transmission and incidence of this vector-borne disease depend on the vital traits of its vectors, including Triatoma infestans, the main vector in Southern South America. Being an ectothermic species, its metabolism and its vital traits respond to temperature fluctuations. Here, we evaluated if changes in the average and variability of temperature expected with climate change modify: (i) the extrinsic incubation period (EIP) of T. cruzi within the vector T. infestans, (ii) its parasitic load, and (iii) the probability that its dejections were T. cruzi-positive. We acclimated triatomines infected with Dm28c T. cruzi strain to two constant and two variable temperature treatments and measured T. cruzi in their dejections by qPCR over a 42-day period. We observed that individuals in warm-temperature treatments showed lower EIP and higher parasitic load than cold-temperature treatments. Also, temperature variability can increase the parasitic load peak in cold-temperature treatments. Consequently, in a climate change scenario, there might be an increase in the vector capacity of T. infestans and probably a change in the risk of vectorial transmission of T. cruzi.

ecology↗

bioRxiv2023

Human induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation

Assisted reproductive technologies (ART) have significantly impacted fertility treatment worldwide through innovations such as in vitro fertilization (IVF) and in vitro maturation (IVM). IVM holds promise as a technology for fertility treatment in women who cannot or do not wish to undergo conventional controlled ovarian stimulation (COS). However, IVM has historically shown highly variable performance in maturing oocytes and generating oocytes with strong developmental capacity. Furthermore, recently reported novel IVM approaches are limited to use in cycles lacking human chorionic gonadotropin (hCG) triggers, which is not standard practice in fertility treatment. We recently reported the development of ovarian support cells (OSCs) generated from human induced pluripotent stem cells (hiPSCs) that recapitulate dynamic ovarian function in vitro. Here we investigate the potential of the se OSCs in an IVM co-culture system to improve the maturation of human cumulus-enclosed immature oocytes retrieved from abbreviated gonadotropin stimulated cycles. We reveal that OSC-IVM significantly improves maturation rates compared to existing IVM systems. Most importantly, we demonstrate that OSC-assisted IVM oocytes are capable of significantly improving euploid blastocyst formation and yielding blastocysts with normal global and germline differential methylation region methylation profiles, a key marker of their clinical utility. Together, these findings demonstrate a novel approach to IVM with broad applicability to modern ART practice. Structured AbstractO_ST_ABSObjectiveC_ST_ABSTo determine if in vitro maturation (IVM) of human oocytes can be improved by co-culture with ovarian support cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs). DesignThree independent experiments were performed in which oocyte donors were recruited to undergo abbreviated gonadotropin stimulation and retrieved cumulus oocyte complexes (COCs) were randomly allocated between the OSC-IVM and control IVM conditions. SubjectsAcross the three experiments, a total of 67 oocyte donors aged 19 to 37 years were recruited for retrieval using informed consent. Anti-mullerian hormone (AMH) value, antral follicle count (AFC), age, BMI, and ovarian pathology were used for inclusion and exclusion criteria. Intervention and ControlThe OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture supplemented with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione and doxycycline. IVM controls comprised commercially-available IVM media without OSCs and contained either the same supplementation as above (media-matched control), or FSH and hCG only (IVM media control). In one experiment, an additional control using fetal ovarian somatic cells (FOSCs) was used with the same cell number and media conditions as in the OSC-IVM. Main Outcome MeasuresPrimary endpoints consisted of metaphase II (MII) formation rate and oocyte morphological quality assessment. A limited cohort of oocytes were utilized for secondary endpoints, consisting of fertilization and blastocyst formation rates with preimplantation genetic testing for aneuploidy (PGT-A) and embryo epigenetic analysis. ResultsOSC-IVM resulted in a statistically significant improvement in MII formation rate compared to the media-matched control, a commercially available IVM media control, and the FOSC-IVM control. Oocyte morphological quality between OSC-IVM and controls did not significantly differ. OSC-IVM displayed a trend towards improved fertilization, cleavage, and blastocyst formation. OSC-IVM showed statistically significant improvement in euploid day 5 or 6 blastocyst formation compared to the commercially available IVM media control. OSC-IVM embryos displayed similar epigenetic global and germline loci profiles compared to conventional stimulation and IVM embryos. ConclusionThe novel OSC-IVM platform is an effective tool for maturation of human oocytes obtained from abbreviated gonadotropin stimulation cycles, supporting/inducing robust euploid blastocyst formation. OSC-IVM shows broad utility with different stimulation regimens, including hCG triggered and untriggered oocyte retrieval cycles, making it a highly useful tool for modern fertility treatment.

developmental biology↗

bioRxiv2022

Nav1.1 in mammalian sensory neurons is required for normal motor behaviors

AO_SCPLOWBSTRACTC_SCPLOWThe voltage-gated sodium channel (NaV), NaV1.1, is well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we identify a new role for NaV1.1 in mammalian proprioception. RNAscope analysis and in vitro patch clamp recordings in genetically identified mouse proprioceptors show ubiquitously channel expression and significant contributions to intrinsic excitability. Notably, genetic deletion of NaV1.1 in sensory neurons caused profound and visible motor coordination deficits in conditional knockout mice of both sexes, similar to conditional Piezo2-knockout animals, suggesting this channel is a major contributor to sensory proprioceptive transmission. Ex vivo muscle afferent recordings conditional knockout mice found that loss of NaV1.1 leads to inconsistent and unreliable proprioceptor firing characterized by action potential failures during static muscle stretch; conversely, afferent responses to dynamic vibrations were unaffected. This suggests that while a combination of Piezo2 and other NaV isoforms are sufficient to elicit activity in response to transient stimuli, NaV1.1 is required for transmission of receptor potentials generated during sustained muscle stretch. Impressively, recordings from afferents of heterozygous conditional knockout animals were similarly impaired, and heterozygous conditional knockout mice also exhibited motor behavioral deficits. Thus, NaV1.1 haploinsufficiency in sensory neurons impairs both proprioceptor function and motor behaviors. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia; therefore, our data suggest sensory neuron dysfunction contributes to the clinical manifestations of neurological disorders in which NaV1.1 function is compromised. Collectively, we present the first evidence that NaV1.1 is essential for mammalian proprioceptive signaling and behaviors.

neuroscience↗

bioRxiv2020

A divergent Articulavirus in an Australian gecko identified using meta-transcriptomics and protein structure comparisons

The discovery of highly divergent RNA viruses is compromised by their limited sequence similarity to known viruses. Evolutionary information obtained from protein structural modelling offers a powerful approach to detect distantly related viruses based on the conservation of tertiary structures in key proteins such as the viral RNA-dependent RNA polymerase (RdRp). We utilised a template-based approach for protein structure prediction from amino acid sequences to identify distant evolutionary relationships among viruses detected in meta-transcriptomic sequencing data from Australian wildlife. The best predicted protein structural model was compared with the results of similarity searches against protein databases based on amino acid sequence data. Using this combination of meta-transcriptomics and protein structure prediction we identified the RdRp (PB1) gene segment of a divergent negative-sense RNA virus in a native Australian gecko (Geyra lauta) that was confirmed by PCR and Sanger sequencing. Phylogenetic analysis identified the Gecko articulavirus (GECV) as a newly described genus within the family Amnoonviridae, order Articulavirales, that is most closely related to the fish virus Tilapia tilapinevirus (TiLV). These findings provide important insights into the evolution of negative-sense RNA viruses and structural conservation of the viral replicase among members of the order Articulavirales.

microbiology↗

bioRxiv2020

Membrane-Interactive Compounds from Pistacia lentiscus L. Thwart Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa is capable to deploy a collection of virulence factors that are not only essential for host infection and persistence, but also to escape from the host immune system and to become more resistant to drug therapies. Thus, developing anti-virulence agents that may directly counteract with specific virulence factors or disturb higher regulatory pathways controlling the production of virulence armories are urgently needed. In this regard, this study reports that Pistacia lentiscus L. fruit cyclohexane extract (PLFE1) thwarts P. aeruginosa virulence by targeting mainly the pyocyanin pigment production by interfering with 4-hydroxy-2-alkylquinolines molecules production. Importantly, the anti-virulence activity of PLFE1 appears to be associated with membrane homeostasis alteration through the modulation of SigX, an extracytoplasmic function sigma factor involved in cell wall stress response. A thorough chemical analysis of PLFE1 allowed us to identify the ginkgolic acid (C17:1) and hydroginkgolic acid (C15:0) as the main bioactive membrane-interactive compounds responsible for the observed increased membrane stiffness and anti-virulence activity against P. aeruginosa. This study delivers a promising perspective for the potential future use of PLFE1 or ginkgolic acid molecules as an adjuvant therapy to fight against P. aeruginosa infections.

microbiology↗

bioRxiv2019

Anterior cingulate cortex and ventral hippocampal inputs to the basolateral amygdala selectively control generalized fear

Nearly one third of Americans have been afflicted with an anxiety disorder. A common symptom of anxiety disorders is the over generalization of fear across a broad range of contextual cues. We previously found that the anterior cingulate cortex and ventral hippocampus (vHPC) regulate generalized fear. Here, we investigate the functional projections from the ACC and vHPC to the amygdala and their role in governing generalized fear in a preclinical rodent model. A chemogenetic approach (DREADDs) was used to inhibit glutamatergic projections from the ACC or vHPC that terminate within the basolateral amygdala (BLA) at recent (1 day) or remote (28 days) time points after contextually fear conditioning male mice. Inactivating ACC or vHPC projections to the BLA significantly reduced generalized fear to a novel, nonthreatening context but had no effect on fear to the training context. Further, our data indicate that the ACC-BLA circuit supports generalization in a time-independent manner. We also identified for the first time a strictly time-dependent role of the vHPC-BLA circuit in supporting remote generalized contextual fear. Dysfunctional signaling to the amygdala from the ACC or the hippocampus could underlie over-generalized fear responses that are associated with anxiety disorders. Our findings demonstrate that the ACC and vHPC regulate fear expressed in novel, nonthreatening environments via projections to the BLA but do so as a result of training intensity or time, respectively.\n\nSignificance StatementAnxiety disorders are characterized by a common symptom that promotes overgeneralization of fear in non-threatening environments. Dysregulation of the amygdala, anterior cingulate cortex (ACC), or hippocampus has been hypothesized to contribute to increased fear associated with anxiety disorders. Our findings show that the ACC and HPC projections to the basolateral amygdala regulate generalized fear in non-threatening, environments. However, descending ACC projections control fear generalization independent of time, whereas HPC projections play a strictly time-dependent role in regulating generalized fear. Thus, dysfunctional ACC/HPC signaling to the BLA may be a predominant underlying mechanism of non-specific fear associated with anxiety disorders. Our data have important implications for predictions made by theories about aging memories and interactions between the hippocampus and cortical regions.

neuroscience↗