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Biology subjects

Rivera, M.

Publications and source records attributed to Rivera, M..

14 recordsLinked to original sources

TorPause: Efficient Static Organ Preservation for Transplants

Transplantation is limited by how long organs survive outside the body. Existing preservation methods slow metabolism or sustain physiological support, but neither eliminates ischemia-reperfusion injury (IRI), which shortens storage and drives organ discard. Embryonic diapause and hibernation show that mammalian cells retain a latent capacity for reversible metabolic arrest. Screening the PI3K-mTOR axis, we identified a dual inhibitor that pauses embryonic stem cells reversibly at ambient oxygen and holds intact mouse embryos in arrest for 11 days. Combined with a mitochondria-targeted hydrogen sulfide donor, this yields TorPause: a two-component flush pairing metabolic pausing with mitochondrial protection, compatible with existing preservation protocols. In a rat liver transplantation model, TorPause reduced structural injury and improved recipient survival after severe warm ischemic storage, potentially expanding the usable donor pool.

developmental biology↗

A shared transcriptional network in the nucleus accumbens supports resilience to chronic stress across sex.

Although chronic stress increases the risk for depression, only a subset of exposed individuals develop psychiatric illness. The biological mechanisms that protect against depression remain incompletely understood, particularly at the molecular level. Here, we identify a transcriptional network in the nucleus accumbens (NAc), a central brain reward region, that supports stress resilience in both sexes and demonstrate the causal contribution of key hub genes. Using chronic social defeat stress, RNA-seq, and co-expression network analysis, we find sex-specific but overlapping gene modules linked to resilience, anchored by shared hub genes embedded within a common network architecture. Overexpression of these hub genes in stress-naive mice confers stress protection and induces a transcriptional state that is discrete from both susceptible and resilient profiles. These findings position resilience as a structured and targetable molecular phenotype and provide a basis for investigating sex-informed mechanisms of stress adaptation.

neuroscience↗

Co-expression-based models improve eQTL predictions and highlightnovel transcriptome-wide genes associated with schizophrenia

Non-coding genetic variants statistically associated with complex heritability phenotypes are thought to act primarily through transcriptome regulatory mechanisms. Predictions of gene expression in tissue like the human brain traditionally rely primarily on cis-eQTLs. Here, we introduce INGENE and MODULE, trans-eQTLs models designed to enhance the prediction of gene expression by capturing the collective impact of candidate trans-eQTLs acting within co-expression networks. Exploiting RNA-seq data in six post-mortem brain regions (amygdala, caudate nucleus, dorsal/subgenual anterior cingulate cortex, dorsolateral prefrontal cortex, and hippocampus), we validate our models on two testing datasets, demonstrating increased gene predictability compared to both an original cis-based model and to EpiXcan, the leading benchmark in cis-model performance. Integration of cis- and trans-predictions significantly improves gene-level expression imputation (MLE = 0.05) for 18,744 genes across the six brain regions considered. Applying cis and trans models to PGC wave 3 genotypes identifies 766 SCZ-associated genes across brain regions (pFDR < .01), emphasizing the complementary nature of cis and trans predictions in trait association discovery. Of these genes, 641 represent novel transcriptome-wide associations with schizophrenia, highlighting the role of trans-heritability and genetic interactions underlying risk for this disorder, in addition to further supporting 125 previous candidates.

genomics↗

Rapid generation of ventral A9-like dopaminergic neurons from patterned iPSCs

In vitro modelling of highly vulnerable nigral dopaminergic (DA) neuronal subtypes in Parkinsons disease (PD), is necessary for studying disease mechanisms. Here, we optimized a new approach by expressing the pioneer neurogenic transcription factor, Achaete-scute-like 1 (Ascl1), implicated in determining dopaminergic fate. Sequential small-molecule patterning of iPSCs into early floor plate mesencephalic progenitors, followed by inducible Ascl1 expression, rapidly differentiates midbrain DA neurons. Immunocytochemistry and transcriptomic analysis of these patterned Ascl1-driven DA neurons (PA-DANs) confirmed midbrain-lineage specificity. Importantly, we found an enrichment of DA subpopulations that corresponded to the adult human ventral SOX6-positive A9 DA subtypes vulnerable in PD. Furthermore, we combined these ventral A9-like PA-DANs with human iPSC-derived midbrain astrocytes and microglia in defined ratios to generate mature 3D A9-like assembled organoids that display characteristic spontaneous neuronal activity and electrical propagation along the axon. Our method efficiently generates a mature and functional A9-like DA neuronal platform to study PD. HighlightsO_LISequential midbrain patterning and Ascl1 expression accelerates DA differentiation C_LIO_LIPA-DANs resemble human adult ventral A9-like DA subtypes vulnerable in PD C_LIO_LI3D assembled organoids show mature identity of PA-DANs, iAstrocytes and iMicroglia C_LIO_LIPA-DANs matured in 3D organoids show neuronal network activity within weeks C_LI eTOC blurbIn this study, Ullian and colleagues have developed a rapid method to differentiate dopaminergic neurons, using small molecules to generate early floor plate mesencephalic progenitors from human iPSCs and sequentially expressing a pioneer transcription factor, Ascl1, that accelerates uniform dopaminergic neurogenesis. Patterned Ascl1-driven dopaminergic neurons (PA-DANs) in 2D and 3D assembled organoids serve as a platform to study Parkinsons disease O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/685897v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1d83eb3org.highwire.dtl.DTLVardef@1fc7fa5org.highwire.dtl.DTLVardef@2033e3org.highwire.dtl.DTLVardef@2e8fa3_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Efficient in vivo pharmacological inhibition of deltaFOSB, an AP1 transcription factor, in brain

{Delta}FOSB, an unusually stable member of the AP1 family of transcription factors, mediates long-term maladaptations that play a key role in the pathogenesis of drug addiction, cognitive decline, dyskinesias, and several other chronic neurological and psychiatric conditions. We have recently identified that 2-phenoxybenzenesulfonic acid-containing compounds disrupt the binding of {Delta}FOSB to DNA in vitro in cell-based assays, and one such compound, JPC0661, disrupts {Delta}FOSB binding to genomic DNA in vivo in mouse brain with partial efficiency. JPC0661 binds to a groove outside of the DNA-binding cleft of the {Delta}FOSB/JUND bZIP heterodimer in the co-crystal structure. Here, we generated a panel of analogs of JPC0661 with the goal of establishing structure-activity relationships and improving its in vivo efficacy by replacing the amino-pyrazolone cap moiety with various substituents. We show that one such analog, YL0441, disrupts the binding of {Delta}FOSB to DNA in vitro and in vivo, and suppresses {Delta}FOSB-function in cell-based assays. Importantly, infusion of YL0441 into the hippocampus of APP mice (a mouse model for Alzheimers disease) leads to virtually complete loss of {Delta}FOSB bound to genomic DNA by CUT&RUN sequencing. Our findings corroborate that DNA binding/release of AP1 transcription factors can be controlled via small molecules, even by analogs of a compound that binds to a groove outside of the DNA-binding cleft, and that our lead can be optimized via medicinal chemistry to yield a highly efficacious inhibitor of {Delta}FOSB function in vivo. These findings define a strategy to design small-molecule inhibitors for other AP1- and AP1-related transcription factors. IN BRIEFWe demonstrate the creation of a highly effective inhibitor, YL0441, of {Delta}FOSB, an AP1 transcription factor, which decreases the number of {Delta}FOSB-bound sites to genomic DNA by [~]94% upon in vivo infusion to the hippocampus of APP mice, a mouse model for Alzheimers disease. This work generates a highly novel probe compound to assess the therapeutic value of {Delta}FOSB in vivo, a transcription factor with a critical role in mediating long-term changes in gene expression in several neuropsychiatric disorders in addition to Alzheimers disease, including drug addiction, seizure-related cognitive decline, and dyskinesias.

neuroscience↗

Astrocytic CREB regulates transcriptional, neuronal, and behavioral responses to cocaine

Drug addiction is characterized by neuronal adaptations that support a shift from goal-directed behaviors to habitual, compulsive drug-seeking with persistent effects on cognition and decision-making. Emerging evidence increasingly indicates that astrocytes are also involved in nervous system disorders, including addiction, but the cocaine-induced astrocyte-specific transcriptome has not yet been investigated. We utilized whole cell sorting of astrocytes, RNA-sequencing, and bioinformatic approaches to characterize the astrocyte transcriptome in the nucleus accumbens (NAc), a key brain region involved in reward-processing, following cocaine self-administration, prolonged abstinence, and "relapse" in male mice. We found that astrocytes exhibit robust and contextually-specific transcriptional signatures that converge strongly with human cocaine use disorder. Bioinformatic analysis revealed CREB as a highly ranked predicted upstream regulator of cocaine-induced transcriptional regulation in NAc astrocytes, and CUT&RUN-sequencing mapped increased CREB binding across the astrocyte genome in response to cocaine. Viral-mediated manipulation of CREB activity selectively in NAc astrocytes, in combination with several measures of addiction-related behaviors including conditioned place preference and self-administration, revealed that astrocytic CREB increases the rewarding and reinforcing properties of cocaine. This effect is sex-specific, with no change in astrocytic CREB activity or CPP found in females. Subsequent experiments identify potential molecular mechanisms of astrocytic CREBs influence through modulating astrocytic Ca2+ signaling in response to cocaine. Finally, we show that astrocytic CREB selectively modulates D1-type medium spiny neurons in NAc to control cocaine-related behaviors. Together, these data demonstrate that the astrocyte transcriptome responds robustly to cocaine and that CREB mediates cocaines effects on gene expression in astrocytes, with consequent effects on neuronal activity and rewarding responses to the drug.

neuroscience↗

Three-dimensional tissue platform co-laid with native collagen fibers and cells for phenotypic screening of stem cell interactions

Phenotypic screening of cell-cell and cell-matrix interactions is critical yet challenging for drug discovery and disease modeling. In this study, a scalable 3D tissue platform was developed by co-laying extracted natural insoluble collagen fibers, mesenchymal stem cells, endothelial cells, and neural progenitor cells for phenotypic screening. Cell growth and interactions were enhanced in the co-laid platform, evident through increased cell proliferation, viability, and vascularization. Dense vascular networks rapidly formed through cell-cell and cell-matrix interactions without adding a traditionally needed growth factor set. Both in vitro and implantation studies confirmed that these blood vessels were of human origin. To evaluate the phenotypic screening of cell-cell and cell-matrix interactions, we propose a phenotype screening prototype for stem cell interactions that utilized multivariate analysis encompassing both cell-cell and cell-matrix interactions and demonstrated its effectiveness to screen vasculature formation and autism spectrum disorder (ASD) models. Using the prototype, we confirmed that collagen crosslinking, ROCK, WNT, and YAP pathways impact vasculogenesis. In addition, ASD donor-derived neural progenitor cells can be distinguished from non-ASD control donor-derived neural progenitor cells.

bioengineering↗

Astrocytic CREB in nucleus accumbens promotes susceptibility to chronic stress

BackgroundIncreasing evidence implicates astrocytes in stress and depression in both rodent models and human Major Depressive Disorder (MDD). Despite this, little is known about the transcriptional responses to stress of astrocytes within the nucleus accumbens (NAc), a key brain reward region, and their influence on behavioral outcomes. MethodsWe used whole cell sorting, RNA-sequencing, and bioinformatic analyses to investigate the NAc astrocyte transcriptome in male mice in response to chronic social defeat stress (CSDS). Immunohistochemistry was used to determine stress-induced changes in astrocytic CREB within the NAc. Finally, astrocytic regulation of depression-like behavior was investigated using viral-mediated manipulation of CREB in combination with CSDS. ResultsWe found a robust transcriptional response in NAc astrocytes to CSDS in stressed mice, with changes seen in both stress-susceptible and stress-resilient animals. Bioinformatic analysis revealed CREB, a transcription factor widely studied in neurons, as one of the top-predicted upstream regulators of the NAc astrocyte transcriptome, with opposite activation states seen in resilient versus susceptible mice. This bioinformatic result was confirmed at the protein level with immunohistochemistry. Viral overexpression of CREB selectively in NAc astrocytes promoted susceptibility to chronic stress. ConclusionsTogether, our data demonstrate that the astrocyte transcriptome responds robustly to CSDS and, for the first time, that transcriptional regulation in astrocytes contributes to depressive-like behaviors. A better understanding of transcriptional regulation in astrocytes may reveal unknown molecular mechanisms underlying neuropsychiatric disorders.

neuroscience↗

Integrative Spatial Analysis of H&E and IHC Images Identifies Prognostic Immune Subtypes Correlated with Progression-Free Survival in HPV-Related Oropharyngeal Squamous Cell Carcinoma

PurposeDeep learning techniques excel at identifying tumor-infiltrating lymphocytes (TILs) and immune phenotypes in hematoxylin and eosin (H&E)-stained slides. However, their ability to elucidate detailed functional characteristics of diverse cellular phenotypes within tumor immune microenvironment (TME) is limited. We aimed to enhance our understanding of cellular composition and functional characteristics across TME regions and improve patient stratification by integrating H&E with adjacent immunohistochemistry (IHC) images. MethodsA retrospective study was conducted on patients with HPV(+) oropharyngeal squamous cell carcinoma (OPSCC). Using paired H&E and IHC slides for 11 proteins, a DL pipeline was used to quantify tumor, stroma, and TILs in the TME. Patients were classified into immune inflamed (IN), immune excluded (IE), or immune desert (ID) phenotypes. By registering the IHC and H&E slides, we integrated IHC data to capture protein expression in the corresponding tumor regions. We further stratified patients into specific immune subtypes, such as IN, with increased or reduced CD8+ cells, based on the abundance of these proteins. This characterization provided functional insight into the H&E-based subtypes. ResultsAnalysis of 88 primary tumors and 70 involved lymph node tissue images revealed an improved prognosis in patients classified as IN in primary tumors with high CD8 and low CD163 expression (p = 0.007). Multivariate Cox regression analysis confirmed a significantly better prognosis for these subtypes. ConclusionsIntegrating H&E and IHC data enhances the functional characterization of immune phenotypes of the TME with biological interpretability, and improves patient stratification in HPV(+) OPSCC.

cancer biology↗

Optimization of host cell-compatible, antimicrobial peptides effective against biofilms and clinical isolates of drug-resistant bacteria.

Here, we describe the continued synthetic molecular evolution of a lineage of host-compatible antimicrobial peptides (AMP) intended for the treatment of wounds infected with drug-resistant, biofilm-forming bacteria. The peptides tested are variants of an evolved AMP called D-CONGA, which has excellent antimicrobial activities in vitro and in vivo. In this newest generation of rational D-CONGA variants, we tested multiple sequence-structure-function hypotheses that had not been tested in previous generations. Many of the peptide variants have lower antibacterial activity against Gram-positive or Gram-negative pathogens, especially variants that have altered hydrophobicity, secondary structure potential, or spatial distribution of charged and hydrophobic residues. Thus, D-CONGA is generally well tuned for antimicrobial activity. However, we identified a variant, D-CONGA-Q7, with a polar glutamine inserted into the middle of the sequence, that has higher activity against both planktonic and biofilm-forming bacteria as well as lower cytotoxicity against human fibroblasts. Against clinical isolates of K. pneumoniae, innate resistance to D-CONGA was surprisingly common despite a lack of inducible resistance in P. aeruginosa reported previously. Yet, these same isolates were susceptible to D-CONGA-Q7. D-CONGA-Q7 is much less vulnerable to AMP resistance in Gram-negative bacteria than its predecessor. Consistent with the spirit of synthetic molecular evolution, D-CONGA-Q7 achieved a critical gain-of-function and has a significantly better activity profile.

biochemistry↗

Gestationally-Dependent Immune Organization at the Maternal-Fetal Interface

The immune system and placenta have a dynamic relationship across gestation to accommodate fetal growth and development. High-resolution characterization of this maternal- fetal interface is necessary to better understand the immunology of pregnancy and its complications. We developed a single-cell framework to simultaneously immuno-phenotype circulating, endovascular, and tissue-resident cells at the maternal-fetal interface throughout gestation, discriminating maternal and fetal contributions. Our data reveal distinct immune profiles across the endovascular and tissue compartments with tractable dynamics throughout gestation that respond to a systemic immune challenge in a gestationally-dependent manner. We uncover that mononuclear phagocytes and neutrophils drive the temporal immune composition of the placenta with remarkably diverse populations, including PD-L1-expressing subsets having compartmental and early gestational bias. Our approach and accompanying datasets provide a resource for additional investigations into gestational immunology and evoke a more significant role for the innate immune system in establishing the microenvironment of early pregnancy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/449807v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@6235corg.highwire.dtl.DTLVardef@fba572org.highwire.dtl.DTLVardef@12b0af4org.highwire.dtl.DTLVardef@dc44da_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

Salicylic acid mediated immune response of Citrus Sinensis to varying frequencies of herbivory and pathogen inoculation

Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. A one-time (7 d) inoculation access period with CLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response after a long period of infection without herbivory (270 and 330 days). In contrast, while repeated (monthly) pulses of 7 d psyllid feeding injury stimulated immunity in CLas-infected citrus by increasing [SA] in leaves initially (up to 120 d), long-term (270 and 330 days) repeated herbivory caused [SA] to decrease coincident with upregulation of genes associated with SA metabolism (BMST and DMR6). Similarly, transcriptional responses and metabolite (SA and its analytes) accumulation in citrus exposed to a continuously reproducing population of D. citri exhibited a transitory upregulation of genes associated with SA signaling at 120 days and a posterior downregulation after long-term psyllid (adults and nymphs) feeding (270 and 330 days). Herbivory played an important role in regulation of SA accumulation in mature leaves of C. sinensis, whether or not those trees were coincidentally infected with CLas. Our results indicate that prevention of feeding injury inflicted by D. citri from the tritrophic interaction may allow citrus plants to better cope with the consequences of CLas infection, highlighting the importance of vector suppression as a component of managing this cosmopolitan disease. Author SummaryWe explored tritrophic interactions among an insect vector (Diaphorina citri) - phytopathogen (Candidatus Liberibacter asiaticus) - and cultivated fruit crop [sweet orange, Citrus sinensis (L) Osbeck]. Transcriptional and metabolic responses of plants were analyzed over an extended time-course of disease progression after various frequencies of herbivore feeding and durations of pathogen infection using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the presence of the devastating citrus disease, huanglongbing. We found that in the absence of coincident psyllid feeding damage, citrus trees could mount a defense response against the pathogen by activating the salicylic acid (SA) pathway. Repeated, monthly pulses of herbivory led to pronounced stimulation of SA transcription that was coincident with diminished pathogen titers in plants. Although insect injury initially activated SA-dependent defense responses, continuous and/or long-term ([&ge;] 270 d) herbivory shut down PR-1-dependent defense responses against the pathogen. Our results provide a mechanism explaining how vector suppression contributes to maintaining health of cultivated citrus in areas where huanglongbing is endemic. Our results also point to specific gene targets that may yield novel genotypes expressing tolerance against CLas after appropriate manipulations.

plant biology↗

The need to harmonize insecticide resistance testing: methodology, intensity concentrations and molecular mechanisms evaluated in Aedes aegypti populations in Central America and Hispaniola

BackgroundThe Zika AIRS Project, a USAID-funded initiative worked across the Latin America and Caribbean regions from 2016 to 2019, as an emergency to contain the spread of the Zika virus. All entomological records in the target countries showed wide distribution and high abundance of Aedes aegypti populations, however the susceptibility profiles of these insects to insecticides commonly employed by vector control campaigns were in most cases incomplete or inexistent. In close collaboration with the Ministries of Health of individual countries, Zika-AIRS teams conducted insecticide susceptibility testing of an array of insecticides in A. aegypti populations of each country. Procedures applied met the standard international protocols instructed by the World Health Organization and Centers for Disease Control and Prevention. Methodology and main findingsThe insecticides tested were selected under categories such as pyrethroids, organophosphates and carbamate. Results showed A. aegypti populations displaying high and widely distributed resistance to all pyrethroids across countries, tolerance to organophosphates and full susceptibility to a carbamate. Key inconsistencies between testing methods are presented and discussed. Additionally, four kdr mutations were analyzed to detect molecular mechanisms of insecticide resistance. The screening for kdr mutations suggested the widespread nature of V1016I mutation, linked to pyrethroid resistance in A. aegypti populations distributed and sampled in the above mentioned regions. Conclusions and perspectivesThis multi-country study contributes with updated information to the public health decision-makers across Central America and the Caribbean. This study provided training and established technical networks for more effective and sustainable insecticide surveillance programs. Most but not all records of insecticide resistance in A. aegypti were consistent between methodologies, thus inconsistent issues are discussed here to call for further improvement in procedures and convey more practical guidelines for surveillance teams in countries where Aedes-borne diseases are endemic. Author summaryAt the forefront of the fight against arboviruses transmission is the insecticide-based vector control. All countries in the Latin American and Caribbean region invest valuable resources from their limited budget to acquire and implement insecticide-based tools, with non-existent or weak insecticide resistance monitoring programs. Hence, the USAID-funded Zika AIRS Project (ZAP) collaborated with the Ministries of Health of multiple countries to update the profile of susceptibility to insecticides in Aedes aegypti populations. We found widespread resistance to pyrethroid and organophosphate insecticides, which account to almost 100% of the products available to control adult mosquitoes. As we used both of World Health Organization and Centers for Disease Control and Prevention standard methods, we found many similarities and some inconsistencies in the susceptibility profiles obtained for the very same vector populations. Additionally, we obtained insight on potential molecular mechanisms of resistance across the countries, finding the kdr mutation V1016I possibly involved in loss of susceptibility. This study is the biggest cross-country update of insecticide resistance for Aedes aegypti in years, and it should be used as evidence for improving the selection of insecticides in these countries and a call for further support to maintain insecticide resistance monitoring programs.

ecology↗

New de novo assembly of the Atlantic bottlenose dolphin (Tursiops truncatus) improves genome completeness and provides haplotype phasing.

High quality genomes are essential to resolve challenges in breeding, comparative biology, medicine and conservation planning. New library preparation techniques along with better assembly algorithms result in continued improvements in assemblies for non-model organisms, moving them toward reference quality genomes. We report on the latest genome assembly of the Atlantic bottlenose dolphin leveraging Illumina sequencing data coupled with a combination of several library preparation techniques. These include Linked-Reads (Chromium, 10x Genomics), mate pairs, long insert paired ends and standard paired ends. Data were assembled with the commercial DeNovoMAGICTM assembly software resulting in two assemblies, a traditional \"haploid\" assembly (Tur_tru_Illumina_hap_v1) that is a mosaic of the two parental haplotypes and a phased assembly (Tur_tru_Illumina_phased_v1) where each scaffold has sequence from a single homologous chromosome. We show that Tur_tru_Illumina_hap_v1 is more complete and accurate compared to the current best reference based on the amount and composition of sequence, the consistency of the mate pair alignments to the assembled scaffolds, and on the analysis of conserved single-copy mammalian orthologs. The phased de novo assembly Tur_tru_Illumina_phased_v1 is the first publicly available for this species and provides the community with novel and accurate ways to explore the heterozygous nature of the dolphin genome.

genomics↗