Biology subjects

Martinez, M.

Publications and source records attributed to Martinez, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Microengineered transplantation of human solid tumors for in vitro studies of CAR T immunotherapy

Treatment of solid malignancies using chimeric antigen receptor (CAR) T cells remains a significant challenge, but current efforts to advance this therapy are challenged by our limited capacity to probe and understand cancer-immune interactions in human solid tumors. Here, we present a microengineered platform for in vitro modeling of malignant solid tumors during CAR T therapy. This system makes it possible to vascularize human tumor explants and perfuse them with blood-borne immune cells in a controlled manner. We first present a microphysiological model of human lung adenocarcinomas infused with CAR-T cells and show how this system can be used to simulate, visualize, and interrogate tumor-directed trafficking and effector function of CAR T cells. We then demonstrate the proof-of-principle of testing a chemokine-directed CAR T cell engineering strategy in a model of malignant pleural mesothelioma and validating our in vitro assessment using a matching in vivo mouse model. Finally, we describe a potential therapeutic target discovered by single-cell RNA sequencing that can be pharmacologically modulated to increase the efficacy of CAR T cells for lung adenocarcinoma, for which we also present specific biomarkers identified by global metabolomics analysis. We believe that the bioengineering principle demonstrated here will make important contributions to developing new capabilities for preclinical studies of adoptive cell therapies for cancer and other complex diseases.

bioengineering↗

bioRxiv2025

Aviadenovirus structure: a highly thermostable capsid in the absence of stabilizing proteins

High-resolution structural studies have mainly focused on two out of the six adenovirus genera: mastadenoviruses and atadenoviruses. Here we report the high-resolution structure of an aviadenovirus, the poultry pathogen fowl adenovirus serotype 4 (FAdV-C4). FAdV-C4 virions are highly thermostable, despite lacking minor coat and core proteins shown to stabilize the mast- and atadenovirus particles, and having no genus-specific cementing proteins. Unique structural features of the FAdV-C4 hexon include a large insertion at the trimer equatorial region, and a long N-terminal tail. Protein IIIa conformation is closer to atadenoviruses than to mastadenoviruses, while protein VIII diverges from all previously reported structures. We interpret these differences in light of adenovirus evolution. Finally, we discuss the possible role of core composition in determining capsid stability properties. These results enlarge our view on the structural diversity of adenoviruses, and provide useful information to counteract fowl pathogens or use non-human adenoviruses as vectors.

molecular biology↗

bioRxiv2025

Enhancement of Therapeutic Transgene Insertion for Murine Phenylketonuria

Low in vivo transgene integration frequency limits the therapeutic efficacy of homology-directed repair (HDR)-mediated gene insertion as a treatment for Mendelian disorders. This study demonstrates improved efficacy of HDR-mediated gene insertion for the treatment of murine phenylalanine hydroxylase (PAH) deficiency, a model of human phenylketonuria (PKU), through pharmacologic inhibition of competing DNA repair pathways. Targeted integration of a Pah-expressing transgene into the hepatocytes of neonatal mice was enhanced with vanillin, a potent inhibitor of non-homologous end joining (NHEJ). This was further improved following combination of vanillin and novobiocin, an inhibitor of microhomology-mediated end joining (MMEJ). Combined NHEJ and MMEJ inhibition yielded PAH-expressing transgene insertions in approximately 10% of targeted alleles and was associated with a 70.6% decrease in serum phenylalanine. Demonstrating that pharmacologic inhibition of DNA repair pathways that compete with HDR can significantly enhance HDR-mediated transgene insertion in vivo.

genomics↗

bioRxiv2024

Modulation of leg trajectory by transcranial magnetic stimulation during walking

The primary motor cortex is involved in initiation and adaptive control of locomotion. However, the role of the motor cortex in controlling gait trajectories remains unclear. In animals, cortical neuromodulation allows for precise control of step height. We hypothesized that a similar control framework applies to humans, whereby cortical stimulation would primarily increase foot elevation. Transcranial magnetic stimulation (TMS) was applied over the motor cortex to assess the involvement of the corticospinal tract over the limb trajectory during human walking. Eight healthy adults (aged 20-32 years) participated in treadmill walking at 1.5 km/h. TMS was applied over the left motor cortex at an intensity of 120% of the threshold to elicit a dorsiflexion of the right ankle during the swing phase of gait. Electromyographic (EMG) measurements and three-dimensional (3D) lower limb kinematics were collected. When delivered during the early swing phase, TMS led to a significant increase in the maximum height of the right toe by a mean of 40.7% {+/-} 14.9% (25.6mm {+/-} 9.4 mm, p = 0.0352) and knee height by 57.8%{+/-} 16.8%; (32mm {+/-} 9.3 mm; p = 0.008) across participants. These findings indicate that TMS can influence limb trajectory during walking, highlighting its potential as a tool for studying cortical control of locomotion.

neuroscience↗

bioRxiv2024

The small MAF transcription factor MAFG co-opts MITF to promote melanoma progression

Transcription factor deregulation potently drives melanoma progression by dynamically and reversibly controlling gene expression programs. We previously identified the small MAF family transcription factor MAFG as a putative driver of melanoma progression, prompting an in-depth evaluation of its role in melanoma. MAFG expression increases with human melanoma stages and ectopic MAFG expression enhances the malignant behavior of human melanoma cells in vitro, xenograft models, and genetic mouse models of spontaneous melanoma. Moreover, MAFG induces a melanoma phenotype switch from a melanocytic state to a more dedifferentiated state. Mechanistically, MAFG interacts with the lineage transcription factor MITF which is required for the pro-tumorigenic effects of MAFG. MAFG and MITF co-occupy numerous genomic sites and MAFG overexpression influences the expression of genes harboring binding sites for the MAFG[~]MITF complex. These results establish MAFG as a potent driver of melanomagenesis through dimerization with MITF and uncover an unappreciated mechanism of MITF regulation. Significance statementMITF is critically involved in melanoma progression and phenotype switching. We discovered that MAFG interacts with MITF to influence expression of MITF target genes and facilitate a shift toward a dedifferentiated melanoma cell state. This study demonstrates that MAFG promotes melanomagenesis by influencing MITF activity, an unappreciated mechanism of MITF regulation.

cancer biology↗

bioRxiv2024

Prenatal Dexamethasone Programs Autonomic Dysregulation in Female Rats

Autonomic dysfunction is associated with cardiovascular and neurological disease, including hypertension, heart failure, anxiety, and stress-related disorders. Prior studies demonstrated that late gestation exposure to dexamethasone (DEX) resulted in female-biased increases in stress-responsive mean arterial pressure (MAP) and heart rate (HR), suggesting a role for glucocorticoid-mediated programming of autonomic dysfunction. The present study investigated the influence of sympathetic (SYM) or parasympathetic (PS) blockade on cardiovascular function in male and female rat offspring of mothers injected with DEX in utero (gestation days [GD]18-21). At 11-12-weeks of age, MAP, HR, and heart rate variability (HRV) were evaluated at baseline and in response to SYM antagonists (1-adrenoceptor + {beta}1-adrenoceptor), a PS (muscarinic) antagonist, or saline (SAL). To assess stress-responsive function, rats were exposed to acute restraint. Tyrosine hydroxylase was measured in adrenals and left ventricle, and gene expression for the {beta}1 adrenergic receptor was measured in left ventricle. Maternal DEX injection reduced basal HRV in male and female offspring. SYM blockade attenuated increases in stress-responsive HR and MAP. PS blockade elevated stress-responsive HR and MAP to a greater extent in Vehicle females. SYM and PS blockade produced equivalent effects on HR and MAP responses in male offspring, regardless of maternal treatment. Based on these findings, we suggest that maternal DEX injection disrupted autonomic regulation of cardiovascular function in females, resulting in a shift toward greater SYM input and less input from PS. Future studies will investigate whether changes in autonomic function are mediated by changes in central autonomic circuitry. New and NoteworthyThese studies use pharmacological antagonists to characterize the nature of the autonomic dysregulation induced in female offspring exposed to the synthetic glucocorticoid, dexamethasone, in utero. The female offspring of dams injected with dexamethasone in late gestation show a reduction in vulnerability to parasympathetic blockade and an increase in responses to acute restraint stress even in the presence of sympathetic blockade. This suggests that late gestation dexamethasone disrupts the normal development of the autonomic function in females leading to a shift in the sympathovagal balance.

physiology↗

bioRxiv2024

Evolutionary plasticity and functional repurposing of the essential metabolic enzyme MoeA

MoeA, or gephyrin in higher eukaryotes, is crucial for molybdenum cofactor biosynthesis required in redox reactions. Gephyrin is a moonlighting protein also involved in postsynaptic receptor clustering, a feature thought to be a recent evolutionary trait. We showed previously that a repurposed copy of MoeA (Glp) is involved in bacterial cell division. To investigate how MoeA acquired multifunctionality, we used phylogenetic inference and protein structure analyses to understand the diversity and evolutionary history of MoeA. Glp-expressing Bacteria have at least two copies of the gene, and our analysis suggests that Glp has lost its enzymatic role. In Archaea we identified an ancestral duplication where one of the paralogs might bind tungsten instead of molybdenum. In eukaryotes, the acquisition of the moonlighting activity of gephyrin comprised three major events: first, MoeA was obtained from Bacteria by early eukaryotes, second, MogA was fused to the N-terminus of MoeA in the ancestor of opisthokonts, and finally, it acquired the function of anchoring GlyR receptors in neurons. Our results support the functional versatility and adaptive nature of the MoeA scaffold, which has been repurposed independently both in eukaryotes and bacteria to carry out analogous functions in network organization at the cell membrane.

evolutionary biology↗

bioRxiv2024

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ER) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ER ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ER breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29s activities in breast cancer cells harboring Y537S ER. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

cancer biology↗

bioRxiv2024

BETA-HYDROXYBUTYRATE COUNTERACTS THE DELETERIOUS EFFECTS OF A SATURATED HIGH-FAT DIET ON SYNAPTIC AMPA RECEPTORS AND COGNITIVE PERFORMANCE

The ketogenic diet, characterized by high fat and low carbohydrates, has gained popularity not only as a strategy for managing body weight but also for its efficacy in delaying cognitive decline associated with neurodegenerative diseases and the aging process. Since this dietary approach stimulates the livers production of ketone bodies, primarily {beta}-hydroxybutyrate (BHB), which serves as an alternative energy source for neurons, we investigated whether BHB could mitigate impaired AMPA receptor trafficking, synaptic dysfunction, and cognitive decline induced by metabolic challenges such as saturated fatty acids. Here, we observe that, in cultured primary cortical neurons, exposure to palmitic acid (200M) decreased surface levels of glutamate GluA1-containing AMPA receptors, whereas unsaturated fatty acids, such as oleic acid and {omega}-3 docosahexaenoic acid (200M), and BHB (5mM) increased them. Furthermore, BHB countered the adverse effects of palmitic acid on synaptic GluA1 levels in hippocampal neurons, as well as excitability and plasticity in hippocampal slices. Additionally, daily intragastric administration of BHB (100 mg/kg/day) for two months reversed cognitive impairment induced by a saturated high-fat diet (49% of calories from fat) in a mouse experimental model of obesity. In summary, our findings underscore the significant impact of fatty acids and ketone bodies on AMPA receptors abundance, synaptic function and neuroplasticity, shedding light on the potential use of BHB to delay cognitive impairments associated with metabolic diseases.

neuroscience↗

bioRxiv2024

Feedforward circuits enable modality-specific control of cortical inhibition during behavioral state transitions.

Local inhibitory neurons (INs), specifically those involved in disinhibitory circuits like somatostatin (SST) and vasoactive intestinal peptide (VIP)-positive INs, are essential for regulating cortical function during behavior. However, it is unclear the mechanisms by which these INs are recruited during active states and whether their activity is consistent across different sensory cortices. We now reveal that in mice, locomotor activity strongly recruits SST-INs in primary somatosensory (S1) but not visual (V1) cortex. This diverse engagement of SST-INs could not be explained by differences in VIP-IN function but was absent in the presence of visual sensory drive suggesting involvement of feedforward sensory pathways. In agreement, inactivating the somatosensory thalamus, but not decreasing VIP-INs activity, significantly reduces the recruitment of SST-INs in S1 by locomotion. Finally, model simulations suggest differences in SST-INs activity can be explained by varying ratios of VIP-driven inhibition and thalamus-driven cortical excitation. Our work suggests that by integrating feedforward activity with neuromodulation, SST-INs play a central role in adapting sensory processing to behavioral states.

neuroscience↗

bioRxiv2023

Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation

Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria (PKU) by cell transplantation. Hepatocytes from a wildtype donor animal were edited in vitro to create Cypor deficiency and then transplanted into PKU animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from <1% to [~]14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism. We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for PKU with long-term efficacy and a favorable safety profile.

genetics↗

bioRxiv2023

Uncovering and exploiting the return of voluntary motor programs after paralysis using a bi-cortical neuroprosthesis

Rehabilitative and neuroprosthetic approaches after spinal cord injury (SCI) aim to reestablish voluntary control of movement. Promoting recovery requires a mechanistic understanding of the return of volition over action, but the relationship between re-emerging cortical commands and the return of locomotion is not well established. We introduced a neuroprosthesis delivering targeted bi-cortical stimulation in a clinically relevant contusive SCI model. In healthy and SCI cats, we controlled hindlimb locomotor output by tuning stimulation timing, duration, amplitude, and site. In intact cats, we unveiled a large repertoire of motor programs. After SCI, the evoked hindlimb lifts were highly stereotyped, yet effective in modulating gait and alleviating bilateral foot drag. Results suggest that the neural substrate underpinning motor recovery had traded-off selectivity for efficacy. Longitudinal tests revealed that the return of locomotion after SCI was time-locked with recovery of the descending drive, which advocates for rehabilitation interventions directed at the cortical target. HighlightsO_LIA bilateral cortical implant allowed for the delivery of alternate bilateral stimulation coherently with locomotion, which modulated gait trajectories. C_LIO_LIWe analyzed the effects of stimulation parameters - timing, duration, amplitude, and site of stimulation - to maximize the improvement of locomotor output after paralysis. C_LIO_LIA varied repertoire of motor programs evoked in intact cats was reduced to one stereotyped response after spinal cord injury (SCI) consisting in flexion modulation that efficiently alleviated hindlimb dragging. C_LIO_LIAfter SCI, the return of cortical gait control emerged in synchrony with locomotor recovery. C_LI

neuroscience↗

bioRxiv2023

Cochlear dysfunction as an early biomarker for cognitive decline in normal hearing and mild hearing loss

Age-related hearing loss (presbycusis) at moderate levels (>40 dB HL) has been recognized as an important risk factor for cognitive decline. However, whether individuals with mild hearing loss (audiogram thresholds between 25 and 40 dB HL) or even those with normal audiograms (<25 dB HL) have a higher risk of dementia, is still debated. Importantly, these early stages of presbycusis are the most common among the elderly, indicating the need to screen and identify individuals with early presbycusis that have an increased risk of cognitive decline. Unfortunately, in this group of patients, audiogram thresholds are not sufficiently sensitive to detect all the hearing impairments that are related to cognitive decline. Consequently, at the individual level, audiogram thresholds are not good estimators of the dementia risk in the group with mild hearing loss or normal hearing thresholds. Here, we propose to use distortion product otoacoustic emissions (DPOAE), as an objective and sensitive tool to estimate the risk of clinically relevant cognitive decline in elders with normal hearing o mild hearing loss. We assessed neuropsychological, brain magnetic resonance imaging, and auditory analyses on 94 subjects aged >64 years old. In addition, cognitive and functional performance was evaluated with the Clinical Dementia Rating Sum of Boxes (CDR SoB), assessed through structured interviews conducted by neurologists, who were blind to the DPOAE results. We found that cochlear dysfunction, measured by DPOAE -and not by conventional audiometry-, was associated with CDR SoB classification and brain atrophy in the group with mild hearing loss (25 to 40 dB), and normal hearing (<25 dB). Our findings suggest that DPOAE may be a non-invasive tool for detecting neurodegeneration and cognitive decline in the elderly, potentially allowing for early intervention.

neuroscience↗

bioRxiv2023

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. Their elaborate multi-layered cell wall, composed primarily of the mycolyl-arabinogalactan-peptidoglycan complex, and their polar growth mode impose a stringent coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the tropomyosin-like protein Wag31. Here, we report the identification of two new divisome members, a gephyrin-like repurposed molybdotransferase (GLP) and its membrane receptor (GLPR). We show that the interplay between the GLPR/GLP module, FtsZ and Wag31 is crucial for orchestrating cell cycle progression. Our results provide a detailed molecular understanding of the crosstalk between two essential machineries, the divisome and elongasome, and reveal that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis similar to the gephyrin/GlyR system that in higher eukaryotes mediates synaptic signaling through network organization of membrane receptors and the microtubule cytoskeleton.

microbiology↗

bioRxiv2023

Evaluating the solubility of compounds intended for intramammary infusion based upon tests conducted across a range of milk products using cephapirin sodium and benzathine as model drugs

The ability to evaluate drug solubility in milk and milk-related products has relevance both to human and veterinary medicine. Model compounds explored in a previous investigation focused on drug solubility assessments when delivered in milk-associated vehicles for administration to human patients. In the current investigation, we focus on the solubility of drugs intended for delivery via intramammary infusion to cattle. Because there are logistic challenges typically associated with obtaining raw milk samples for these tests, there is a need to determine potential alternative media as a substitute for raw bovine milk. Given the complexity of the milk matrix, aqueous media do not reflect the range of factors that could impact these solubility assessments. This led to the current effort to explore the magnitude of differences that might occur when substituting raw bovine milk with off-the-shelf milk products such as whole milk, skim milk, or reconstituted whole milk powder. We considered conclusions based upon the solubility assessments derived from the use of the model compounds studied in our previous report and compared them to conclusions obtained when testing two drugs with differing physicochemical characteristics that are approved for administration via bovine intramammary infusion: cephapirin benzathine and cephapirin sodium. Based upon these results, we recommend that whole milk or reconstituted whole milk can substitute for bovine raw milk for the solubility assessment of compounds intended for administration via intramammary infusion. However, unlike the human drug situation, these tests should be conducted at 38{degrees}C.

pharmacology and toxicology↗

bioRxiv2022

Bacterial outer-membrane polysaccharide export (OPX) proteins occupy three structural classes with selective β-barrel porin requirements for polymer secretion

Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes that implicate outer-membrane (OM) polysaccharide export (OPX) proteins in polymer translocation to the cell surface. In the social predatory bacterium Myxococcus xanthus, exopolysaccharide (EPS)-pathway WzaX, major spore coat (MASC)-pathway WzaS, and biosurfactant polysaccharide-pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning -helices. Comparative genomics across all bacteria, complemented with cryo-electron tomography cell- envelope analyses, revealed WzaX/S/B architecture to be the most common amongst three defined OPX-protein structural classes independent of periplasmic thickness. Fold-recognition and deep- learning analyses revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded adjacent to WzaX/S/B) to be integral OM {beta}-barrels, with structural homology to the poly-N-acetyl-D- glucosamine synthase-dependent pathway porin PgaA. Such porins were identified in bacteria near numerous genes for all three OPX-protein classes. Interior MXAN_7418/3226/1916 {beta}-barrel electrostatics were found to match known properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 absence shown herein to compromise EPS translocation, these data support a novel secretion paradigm for Wzx/Wzy-dependent pathways in which those containing an OPX component that cannot span the OM instead utilize a {beta}-barrel porin to mediate polysaccharide transport across the OM.

microbiology↗

bioRxiv2022

Withdrawal from repeated nicotine vapor exposure increases somatic signs of physical dependence, anxiety-like behavior, and brain reward thresholds in adult male rats.

In recent years, there has been a dramatic increase in nicotine vapor consumption via electronic nicotine delivery systems (i.e., e-cigarettes), particularly in adolescents. While recent work has focused on the health effects of nicotine vapor exposure, its effects on the brain and behavior remain unclear. In this study, we assessed the effects that cessation from repeated nicotine vapor exposure had on behavioral and neuronal measures of withdrawal. For Experiment 1, fifty-six adult male rats were tested for plasma cotinine levels, somatic withdrawal signs, and anxiety-like behavior in the elevated plus maze, immediately following precipitated withdrawal from repeated exposure to 12 or 24 mg/mL nicotine vapor. In Experiment 2, twelve adult male rats were tested for intracranial self-stimulation (ICSS) across 14 days of exposure to 24 mg/mL nicotine vapor and across the 14 days immediately following nicotine exposure. Results revealed that plasma cotinine, somatic signs, anxiety-like behavior, and ICSS stimulation thresholds were all observed to be elevated during withdrawal in the 24 mg/mL nicotine group, when compared to vehicle controls (50/50 vegetable glycerin/propylene glycol). The data suggest that cessation from repeated nicotine vapor exposure using our preclinical model leads to nicotine dependence and withdrawal, and demonstrates that the vapor system described in these experiments is a viable pre-clinical model of e-cigarette use in humans. Further characterization of the mechanisms driving nicotine vapor abuse and dependence is needed to improve policies and educational campaigns related to e-cigarette use. HighlightsO_LIA rodent model of nicotine e-cigarette vapor use was utilized to assess effects of cessation from repeated nicotine vapor exposure on behavioral and neuronal measures of drug withdrawal. C_LIO_LICessation of repeated nicotine vapor exposure resulted in increased plasma cotinine levels, somatic withdrawal signs, and anxiety-like behavior. C_LIO_LICessation of repeated nicotine vapor exposure resulted in elevations of ICSS reward threshold. C_LIO_LIElectrode implantations for ICSS were mapped by location and threshold to a standardized reference atlas of the rat brain to facilitate comparisons with the published literature. C_LI

neuroscience↗

bioRxiv2022

In situ structure and priming mechanism of the rhoptry secretion system in Plasmodium revealed by cryo-electron tomography

Apicomplexan parasites secrete the contents of rhoptries into host cells to permit their invasion and establishment of an infectious niche. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is positioned at the cell apex and associates with an enigmatic apical vesicle (AV), which docks one or two rhoptries at the site of exocytosis. The interplay among the rhoptries, the AV, and the parasite plasma membrane for secretion remains unclear. Moreover, it is unknown if a similar machinery exists in the deadly malaria parasite Plasmodium falciparum. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct AV docking the tips of the two rhoptries to the RSA. We also discover two new organizations: one in which the AV is absent with one of the two rhoptry tips docks directly to the RSA, and a second in which the two rhoptries fuse together and the common tip docks directly to the RSA. Interestingly, rhoptries among the three states show no significant difference in luminal volume and density, suggesting that the exocytosis of rhoptry contents has not yet occurred, and that these different organizations likely represent sequential states leading to secretion. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to each state, including the opening of a gate-like density in the rhoptry-fused state. These conformational changes of the RSA uncover structural details of a priming process for major rhoptry secretion, which likely occur after initial interaction with a red blood cell. Our results highlight a previously unknown step in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

microbiology↗