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Castillo, J.

Publications and source records attributed to Castillo, J..

14 recordsLinked to original sources

Deep Microbial Colonization in 2-Billion-Year-Old Ultramafic Rock from the Bushveld Complex

Archean cratons may provide stable microbial habitats in the deep subsurface, as evidenced by the discovery of billion-year-old crustal fluids1,2. However, the long-term habitability of these cratonic environments is uncertain, as polymetamorphic evolution in most cratons typically destroys microbial habitats through mineral reactions and porosity loss3,4. Preservation of deep microbial habitats is more likely where mantle-derived magma intruded the craton after metamorphic overprinting4. Here we report the discovery of dense microbial colonization at 814 m depth within the 2.05-billion-year-old, unmetamorphosed Bushveld Igneous Complex intrusion, South Africa5. Using advanced contamination-control protocols6,7 and synchrotron-based X-ray spectroscopy, we identified indigenous microbial cells localized at the rims of phlogopite, a hydrous phyllosilicate mineral. Our study reveals that microbial colonization is associated with Fe(III) derived from the structure of phlogopite, where the dehydrogenation likely oxidizes Fe(II) to Fe(III) coupled to H2 generation8. Despite the absence of fracture-driven fluid ingress in the unfractured rock matrix, aqueous alteration evidenced at the rims by potassium removal indicates a self-sustaining habitat driven by an internal redox gradient9. These findings demonstrate that aqueous alteration of ultramafic rocks can sustain isolated microbial life over geological timescales, significantly expanding the potential for long-term habitability on both Earth and Mars4,10.

microbiology↗

Irradiation and nitrogen metabolism: differential responses in high yield indica and japonica rice commercial cultivars.

Photosynthesis accounts for most of the final grain yield in rice, making improvements in radiation use efficiency (RUE) a key strategy for enhancing productivity. Agronomically, RUE is defined as the biomass produced per unit of total solar radiation or photosynthetically active radiation intercepted by the canopy. However, the interaction between carbon and nitrogen metabolism plays a critical role in determining plant growth and grain yield. Assimilated nitrogen is required for the synthesis of photosynthetic pigments and enzymes, while the reduction of nitrate (NOLL) and nitrite (NOLL), as well as the assimilation of ammonium (NHLL), depend on the reducing power and carbon skeletons generated by photosynthesis. In this study, two high-yielding rice (Oryza sativa) cultivars--an indica-type (El Paso 144) and a japonica-type (INIA Parao) were subjected to two nitrogen treatments (3 mM and 9 mM NOLL/NHLL) and two light intensities (850 and 1500 mol mL{superscript 2} sL{superscript 1}). A strong interaction between light intensity and nitrogen metabolism was observed, with contrasting responses between subspecies. These differences reflect a coordinated regulation of carbon assimilation and primary nitrogen metabolism. The results provide new insights into the metabolic strategies underlying nitrogen compound accumulation under variable irradiance. Such knowledge is essential for improving nitrogen fertilizer use efficiency and yield performance in elite rice genotypes cultivated under commercial field conditions.

plant biology↗

A Secreted Subunit SARS-CoV-2 RBD-CpE Yeast Oral Vaccine Induces an Adaptive Immune Response in BALB/c Mice

Despite their differences, all the current COVID-19 vaccines need to be refrigerated and administered intramuscularly by a health care worker. They are also relatively expensive. These characteristics often make COVID-19 vaccine storage, distribution, and administration relatively complex, especially in low- and middle-income countries where refrigeration and healthcare workers are limited. To address these challenges, we are developing a COVID-19 oral vaccine utilizing the yeast Saccharomyces boulardii as a delivery platform. We have successfully engineered recombinant strains of Saccharomyces boulardii to express and secrete the Receptor Binding Domain (RBD) of the Spike protein from the original Wuhan-Hu-1 strain of the SARS- CoV-2 virus, fused to the C-terminal fragment of Clostridium perfringens enterotoxin (CpE). Animal trials suggest that our candidate secreted oral yeast vaccine can elicit detectable RBD- specific IgG and IgA, as well as an IFN-{gamma} but not an IL-4 response, in BALB/c mice. As such, our data suggest that Saccharomyces boulardii remains a promising and novel vaccine delivery platform not only for COVID-19 but also for other infectious diseases.

immunology↗

Loss of tight junction protein claudin 18 uncovers alveolar epithelial stem cell plasticity and emergence of non-fibrogenic transitional progenitors

Persistence of senescent alveolar transitional progenitors following lung injury is implicated in the pathogenesis of fibrosis. We identified transitional cells in uninjured Cldn18 knockout (KO) mouse lungs distinct from previously reported damage-associated transitional progenitors (DATPs) with a less fibrogenic transcriptomic profile. Cldn18 KO mice are protected from bleomycin-induced fibrosis, with early restoration of cellular homeostasis. Lineage tracing implicates accelerated differentiation as a mechanism for protection from fibrosis, leading us to name these cells regeneration-associated transitional progenitors (RATPs). Multiome confirms that RATPs and DATPs are epigenetically distinct, with RATPs comprised of RATP2s and RATP1s based on epigenomic proximity to AT2s and AT1s, respectively, and suggests dynamic regulatory remodeling during AT2-to-AT1 differentiation, with NKX2.1 and AP-1 active in early transitions and TEAD factors in later stages. These results reveal an unexpected role for Cldn18 in regulation of AEC plasticity, while identification of RATPs challenges the notion that persistence of transitional alveolar cells is invariably pathologic.

cell biology↗

Notch signaling maintains a progenitor-like subclass of hepatocellular carcinoma

Hepatocellular carcinomas (HCCs) constitute one of the few cancer indications for which mortality rates continue to rise. While Notch signaling dictates a key progenitor lineage choice during development, its role in HCC has remained controversial. Using therapeutic antibodies targeting Notch ligands and receptors to screen over 40 patient-derived xenograft models, we here identify progenitor-like HCCs that crucially depend on a tumor-intrinsic JAG1-NOTCH2 signal. Inhibiting this signal induces tumor regressions by triggering progenitor-to-hepatocyte differentiation, the same cell fate-switch that Notch controls during development. Transcriptomic analysis places the responsive tumors within the well-characterized progenitor subclass, a poor prognostic group of highly proliferative tumors, providing a diagnostic method to enrich for Notch-dependent HCCs. Furthermore, single-cell RNA sequencing uncovers a heterogeneous population of tumor cells and reveals how Notch inhibition shifts cells from a mixed cholangiocyte-hepatocyte lineage to one resembling mature hepatocytes. Analyzing the underlying transcriptional programs brings molecular detail to this process by showing that Notch inhibition de-represses expression of CEBPA, which enables the activity of HNF4, a hepatocyte lineage factor that is otherwise quiescent. We thus describe a compelling and targetable dependency in a poor-prognosis class of HCCs.

cancer biology↗

Cell of origin alters myeloid-mediated immunosuppression in lung adenocarcinoma

Solid carcinomas are often highly heterogenous cancers, arising from multiple epithelial cells of origin. Yet, how the cell of origin influences the response of the tumor microenvironment is poorly understood. Lung adenocarcinoma (LUAD) arises in the distal alveolar epithelium which is populated primarily by alveolar epithelial type I (AT1) and type II (AT2) cells. It has been previously reported that Gramd2+ AT1 cells can give rise to a histologically-defined LUAD that is distinct in pathology and transcriptomic identity from that arising from Sftpc+ AT2 cells1,2. To determine how cells of origin influence the tumor immune microenvironment (TIME) landscape, we comprehensively characterized transcriptomic, molecular, and cellular states within the TIME of Gramd2+ AT1 and Sftpc+ AT2-derived LUAD using KRASG12D oncogenic driver mouse models. Myeloid cells within the Gramd2+AT1-derived LUAD TIME were increased, specifically, immunoreactive monocytes and tumor associated macrophages (TAMs). In contrast, the Sftpc+ AT2 LUAD TIME was enriched for Arginase-1+ myeloid derived suppressor cells (MDSC) and TAMs expressing profiles suggestive of immunosuppressive function. Validation of immune infiltration was performed using flow cytometry, and intercellular interaction analysis between the cells of origin and major myeloid cell populations indicated that cell-type specific markers SFTPD in AT2 cells and CAV1 in AT1 cells mediated unique interactions with myeloid cells of the differential immunosuppressive states within each cell of origin mouse model. Taken together, Gramd2+ AT1-derived LUAD presents with an anti-tumor, immunoreactive TIME, while the TIME of Sftpc+ AT2-derived LUAD has hallmarks of immunosuppression. This study suggests that LUAD cell of origin influences the composition and suppression status of the TIME landscape and may hold critical implications for patient response to immunotherapy.

cancer biology↗

GRAMD2+ alveolar type I cell plasticity facilitates cell state transitions in organoid culture

Alveolar epithelial regeneration is critical for normal lung function and becomes dysregulated in disease. While alveolar type 2 (AT2) and club cells are known distal lung epithelial progenitors, determining if alveolar epithelial type 1 (AT1) cells also contribute to alveolar regeneration has been hampered by lack of highly specific mouse models labeling AT1 cells. To address this, the Gramd2CreERT2 transgenic strain was generated and crossed to RosamTmG mice. Extensive cellular characterization, including distal lung immunofluorescence and cytospin staining, confirmed that GRAMD2+ AT1 cells are highly enriched for green fluorescent protein (GFP). Interestingly, Gramd2CreERT2 GFP+ cells were able to form organoids in organoid co-culture with Mlg fibroblasts. Temporal scRNAseq revealed that Gramd2+ AT1 cells transition through numerous intermediate lung epithelial cell states including basal, secretory and AT2 cell in organoids while acquiring proliferative capacity. Our results indicate that Gramd2+ AT1 cells are highly plastic suggesting they may contribute to alveolar regeneration.

cell biology↗

LINC00261 expression reduces mutation accumulation while conferring resistance to cisplatin and altering the tumor-immune microenvironment in lung adenocarcinoma

Immunotherapy has emerged as a major breakthrough that has the ability to improve survival outcomes for multiple solid tumor types, however effective response requires the combination of blocking inhibitory signals on the tumor surface and antigen presentation to the tumor surface for proper immune recognition. Several commercially available and robust methods exist for identification of tumors displaying immune-inhibitory surface receptors, including PD-L1 and CTLA-4, however it is currently difficult to predict effectiveness of antigen presentation on the cell surface. To address this, we utilized clinical and next-generation sequencing data generated by The Cancer Genome Atlas (TCGA) to identify gene signatures that are correlated to tumor mutational burden (TMB) within cancers of epithelial origins as a surrogate for neoantigen signatures. We identified LINC00261 as a top gene correlated to mutational burden (TMB), whose expression activates DNA damage response pathways in vitro along with resistance to cisplatin. LINC00261 expression was also significantly correlated to MHC Class II genes involved in endogenous neoantigen presentation expression within the TCGA-LUAD cohort. This relationship was confirmed in vitro through ectopic reintroduction of LINC00261 for key MHC Class II presentation genes. Interferon gamma-induced MHC gene activation in vitro was also able to induce endogenous expression of LINC00261. Our results suggest there is a mechanistic relationship between LINC00261 expression and functional MHC Class II neoantigen presentation in LUAD. The silencing of LINC00261 in LUAD results in compromised DNA repair, accumulation of mutations, and reduced antitumor immune response.

cancer biology↗

An intranasal stringent response vaccine targeting dendritic cells as a novel adjunctive therapy against tuberculosis

Lengthy tuberculosis (TB) treatment is required to address the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the RelMtb-mediated stringent response. We developed a novel therapeutic DNA vaccine construct involving fusion of the relMtb gene with the immature dendritic cell-targeting gene encoding chemokine MIP-3/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that the intramuscular MIP-3/relMtb (fusion) vaccine potentiates isoniazid activity more than a similar DNA vaccine expressing relMtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log10 colony-forming units, P=0.0001), inducing pronounced Mtb-protective immune signatures. The intranasal fusion vaccine, an approach combining relMtb fusion to MIP-3 and intranasal delivery, demonstrated the greatest therapeutic effect compared to each approach alone, as evidenced by robust Th1 and Th17 responses systemically and locally and the greatest mycobactericidal activity when combined with isoniazid (absolute reduction of Mtb burden: 1.13 log10, P<0.0001, when compared to the intramuscular vaccine targeting relMtb alone). This DNA vaccination strategy may be a promising adjunctive approach combined with standard therapy to shorten curative TB treatment, and also serve as proof-of-concept for treating other chronic infections.

immunology↗

Not all smokers are alike: The hidden cost of sustained attention during nicotine abstinence

BackgroundNicotine Withdrawal Syndrome (NWS)-associated cognitive deficits are heterogeneous, suggesting underlying endophenotypic subgroups. We identified smoker subgroups based on response accuracy during a cognitively demanding Parametric Flanker Task (PFT) and characterized their distinct neuroimaging endophenotypes using a nicotine state manipulation (sated, abstinent). MethodsForty-five smokers completed the 25-min PFT in two fMRI sessions (nicotine sated, abstinent). Task-evoked NWS-associated errors of omission (EOm), brain activity, underlying functional connectivity (FC), and brain-behavior correlations between subgroups were assessed. ResultsBased on their response accuracy in the high demand PFT condition, smokers split into high (HTP, n=21) and low task performer (LTP, n=24) subgroups. Behaviorally, HTPs showed greater response accuracy independent of nicotine state and greater vulnerability to abstinence-induced EOm. HTPs showed greater BOLD responses in attentional control brain regions for the [correct responses (-) errors of commission] PFT contrast across states. A whole-brain FC analysis with these subgroup-derived regions as seeds revealed two circuits: L Precentral : R Insula and L Insula : R Occipital, with abstinence-induced FC strength increases only in HTPs. Finally, abstinence-induced brain (FC) and behavior (EOm) differences were positively correlated for HTPs in a L Precentral : R Orbitofrontal cortical circuit. ConclusionWe used a cognitive stressor (PFT) to fractionate smokers into two subgroups (HTP/LTP). Only the HTPs demonstrated sustained attention deficits during nicotine abstinence, a stressor in dependent smokers. Unpacking underlying smoker heterogeneity with this dual stressor approach revealed distinct smoker subgroups with differential attention deficit responses to withdrawal that could be novel targets for therapeutic interventions to improve cessation outcomes.

neuroscience↗

Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 105 TCID50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFN{beta} and TNF, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population. ImportanceMen experience more severe outcomes from COVID-19 than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human clinical isolate of SARS-CoV-2. After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in females than male hamsters. Greater lung pathology during the acute phase combined with reduced antiviral antibody responses during the recovery phase of infection in males than females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection. One Sentence SummaryFollowing SARS-CoV-2 infection, male hamsters experience worse clinical disease and have lower antiviral antibody responses than females.

microbiology↗

Neural representation of abstract task structure during generalization

Cognitive models in psychology and neuroscience widely assume that the human brain maintains an abstract representation of tasks. This assumption is fundamental to theories explaining how we learn quickly, think creatively, and act flexibly. However, neural evidence for a verifiably generative abstract task representation has been lacking. Here, we report an experimental paradigm that requires forming such a representation to act adaptively in novel conditions without feedback. Using functional magnetic resonance imaging, we observed that abstract task structure was represented within left mid-lateral prefrontal cortex, bilateral precuneus and inferior parietal cortex. These results provide support for the neural instantiation of the long-supposed abstract task representation in a setting where we can verify its influence. Such a representation can afford massive expansions of behavioral flexibility without additional experience, a vital characteristic of human cognition.

neuroscience↗

Time varying connectivity across the brain changes as a function of nicotine abstinence state

BackgroundThe Nicotine Withdrawal Syndrome (NWS) includes affective and cognitive disruptions whose incidence and severity vary across time during acute abstinence. However, most network-level neuroimaging employs static measures of resting state functional connectivity (rsFC), assuming time-invariance, and unable to capture dynamic brain-behavior relationships. Recent advances in rsFC signal processing allow characterization of "time varying connectivity" (TVC), which characterizes network communication between sub-networks that reconfigure over the course of data collection. As such, TVC may more fully describe network dysfunction related to the NWS. MethodsTo isolate alterations in the frequency and diversity of communication across network boundaries as a function of acute nicotine abstinence we scanned cigarette smokers in the nicotine sated and abstinent states and applied a previously-validated method to characterize TVC at a network and nodal level within the brain. ResultsDuring abstinence, we found brain wide decreases in the frequency of interactions between network nodes in different modular communities (i.e. temporal flexibility; TF). In addition, within a subset of the networks examined the variability of these interactions across community boundaries (i.e. spatiotemporal diversity; STD) also decreased. Finally, within two of these networks the decrease in STD was significantly related to NWS clinical symptoms. ConclusionsEmploying multiple measures of TVC in a within subjects design, we characterized a novel set of changes in network communication and link these changes to specific behavioral symptoms of the NWS. These reductions in TVC provide a meso-scale network description of the relative inflexibility of specific large-scale brain networks as a result of acute abstinence.

animal behavior and cognition↗

INCLUSION CRITERIA UPDATE OF THE INTRALUMINAL ISCHEMIC MODEL IN RAT FOR PRECLINICAL STUDIES

A proper occlusion of the medial cerebral artery (MCA) determined by laser Doppler during cerebral ischemia in rat models is an important inclusion criteria in experimental studies. However, a successful occlusion of the artery does not always guarantee a reproducible infarct volume which is critical to validate the efficacy of new protective drugs. In this study, we have compared the variability of infarct size in ischemic animals when the artery occlusion is monitored with laser Doppler alone and in combination with MRI during artery occlusion. Infarct volume determined at 24 hours was compared between animals with laser Doppler monitoring alone and in combination with MR angiography (MRA) and diffusion weighted images (DWI). Twenty-eight animals presented a successful occlusion and reperfusion determined by Doppler monitoring with an infarct size at 24 hours of 16.71{+/-}11.58%. However, when artery occlusion and infarct damage were analyzed in these animals by MRA and DWI, 15 animals were excluded and only 13 animals were included based on Doppler and MRI inclusion criteria, with an infarct size of 21.65{+/-}6.15% at 24 hours. These results show that laser Doppler monitoring is needed but not enough to guarantee a reproducible infarct volume in rat ischemic model.\n\nSummary statementLaser Doppler monitoring in combination with DWI and MR angiography represents a reliable inclusion protocol during ischemic surgery for the analysis of protective drugs in the acute phase of stroke.

neuroscience↗