Biology subjects

Rodriguez, J. E.

Publications and source records attributed to Rodriguez, J. E..

8 recordsLinked to original sources

bioRxiv2025

High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB

Genome editing has revolutionized plant biology research. However, efficient and straightforward delivery of editing reagents remains a major challenge. Viral delivery systems can address these issues, but CRISPR-Cas nucleases are often too large for viral vectors. Recently, smaller editors like TnpBs have been identified, but wild-type TnpBs are significantly less active than commonly used Cas9 nucleases. Here, we optimized a tobacco rattle virus (TRV)-based system to deliver newly discovered, highly active engineered ISDra2 TnpB variants. Our results demonstrate that the eTnpBc variant delivered via TRV enables effective somatic editing in systemic leaves and achieves up to 90% editing efficiency at target loci, significantly higher than that of wild-type ISDra2 TnpB. Additionally, up to 89% of offspring exhibit a mutant phenotype, with editing efficiencies reaching 100%. The design principles outlined here are expected to accelerate broader adoption of eTnpBc for transformation- and transgene-free genome editing in plants.

plant biology↗

bioRxiv2025

Discovery of widespread activating mutations in a compact RNA-guided endonuclease

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the proteins mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

molecular biology↗

bioRxiv2022

Interspecies isobaric labeling-based quantitative proteomics reveals protein changes in the ovary of Aedes aegypti co-infected with ZIKV and Wolbachia

Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by Ae. aegypti. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barre syndrome. One of the actual most promising strategies to mitigate arbovirus transmission involves releasing Ae. aegypti mosquitoes carrying the maternally inherited endosymbiont bacteria Wolbachia pipientis. The presence of Wolbachia is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits as fecundity and fertility. However, the mechanisms by which Wolbachia influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of co-infections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of Wolbachia wMel and ZIKV infection in Ae. aegypti ovaries. To the best of our knowledge, this is the most complete proteome of Ae. aegypti ovaries reported so far, with a total of 3,913 proteins identified, also,were able to quantify a total of 1,044 Wolbachia proteins in complex sample tissue of Ae. aegypti ovary.. Furthermore, we discuss proteins and pathways altered in Ae. aegypti during ZIKV infections, Wolbachia infections, co-infection Wolbachia/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of Ae. aegypti. The modified aspects were mostly related to the immune priming enhancement by Wolbachia presence and the modulation of the Juvenile Hormone pathway caused by both microorganisms infection. HighlightsO_LIProteome changes in Ae. aegypti, Wolbachia, and ZIKV interactions C_LIO_LIA great diversity of Wolbachia proteins were quantified in Ae. aegypti ovary C_LIO_LIJuvenile Hormone pathway is modulated by both infections C_LIO_LIWolbachia enhances Ae. aegypti immune priming mechanism C_LIO_LIZIKV unsettles host immune response by reducing antimicrobial peptides production C_LIO_LICoinfection triggers oxidative stress and a lack of vitellogenin precursors C_LI

biochemistry↗

bioRxiv2021

Single Cell Chemical Proteomics (SCCP) Interrogates the Timing and Heterogeneity of Cancer Cell Commitment to Death

Chemical proteomics studies the effects of drugs upon cellular proteome. Due to the complexity and diversity of tumors, the response of cancer cells to drugs is also heterogeneous, and thus proteome analysis at single cell level is needed. Here we demonstrate that single cell proteomics techniques have become quantitative enough to tackle the drug effects on the target proteins, enabling single cell chemical proteomics (SCCP). Using SCCP, we studied here the time-resolved response of individual adenocarcinoma A549 cells to anticancer drugs methotrexate, camptothecin and tomudex, revealing the early emergence of cellular subpopulations committed and uncommitted to death. As a novel and useful approach to exploring the heterogeneous response to drugs of cancer cells, SCCP may prove to be a "killer application" for single cell proteomics.

cancer biology↗

bioRxiv2021

Implications of central carbon metabolism in SARS-CoV-2 replication and disease severity

Viruses hijack host metabolic pathways for their replicative advantage. Several observational trans-omics analyses associated carbon and amino acid metabolism in coronavirus disease 2019 (COVID-19) severity in patients but lacked mechanistic insights. In this study, using patient- derived multi-omics data and in vitro infection assays, we aimed to understand i) role of key metabolic pathways in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) reproduction and ii) its association with disease severity. Our data suggests that monocytes are key to the altered immune response during COVID-19. COVID-19 infection was associated with increased plasma glutamate levels, while glucose and mannose levels were determinants of the disease severity. Monocytes showed altered expression pattern of carbohydrate and amino acid transporters, GLUT1 and xCT respectively in severe COVID-19. Furthermore, lung epithelial cells (Calu-3) showed a strong acute metabolic adaptation following infection in vitro by modulating central carbon metabolism. We found that glycolysis and glutaminolysis are essential for virus replication and blocking these metabolic pathways caused significant reduction in virus production. Taken together, our study highlights that the virus utilizes and re-wires pathways governing central carbon metabolism leading to metabolic toxicity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.

microbiology↗

bioRxiv2021

Type-I interferon signatures in SARS-CoV-2 infected Huh7 cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has caused a global health emergency. A key feature of COVID-19 is dysregulated interferon-response. Type-I interferon (IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in the pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identified that SARS-CoV-2 infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to inhibit RIG-I mediated IFN-{beta} production. Our results also confirm the recent findings that IFN-I pretreatment is able to reduce susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Moreover, senescent Huh7 cells, in spite of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection, and the virus effectively inhibited IFIT1 in these cells. Finally, proteomic comparison between SARS-CoV-2, SARS-CoV and MERS-CoV revealed a distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Investigation of different interferon stimulated genes and their role in inhibition of SARS-CoV-2 pathogenesis may direct novel antiviral strategies.

microbiology↗

bioRxiv2020

Metabolic reprogramming and host-immune response against Crimean-Congo Hemorrhagic Fever Viruses

The pathogenesis and host-viral interactions of the Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) are convoluted and not well evaluated. Application of the multi-omics system biology approaches including biological network analysis in elucidating the complex host-viral response, allow for interrogating the viral pathogenesis. The present study aimed to fingerprint the system-level alterations during acute CCHFV-infection and the cellular immune responses during productive CCHFV-replication in vitro. We used system-wide network-based system biology analysis of peripheral blood mononuclear cells (PBMCs) from a longitudinal cohort of CCHF patients during the acute phase of infection and after one year of recovery (convalescent phase) and untargeted quantitative proteomics analysis of the most permissive CCHFV-infected Huh7 and SW13 cells. In the RNAseq analysis of the PBMCs, comparing the acute and convalescent-phase, we observed system-level hosts metabolic reprogramming towards central carbon and energy metabolism (CCEM) with distinct upregulation of oxidative phosphorylation (OXPHOS) during CCHFV-infection. Upon application of network-based system biology methods, negative coordination of the biological signaling systems like FOXO/Notch axis and Akt/mTOR/HIF-1 signaling with metabolic pathways during CCHFV-infection were observed. The temporal quantitative proteomics in Huh7 showed a dynamic change in the CCEM over time and was in agreement with the cross-sectional proteomics in SW13 cells. By blocking the two key CCEM pathways, glycolysis and glutaminolysis, viral replication was inhibited in vitro. Activation of key interferon stimulating genes during infection suggested the role of type I and II interferon-mediated antiviral mechanisms both at system-level and during progressive replication. Significance StatementA combination of multi-modal systems-wide host-immune response and in vitro temporal analysis identified molecular re-arrangement in CCEM and fingerprinting the interferon-mediated antiviral mechanism during CCHFV-infection. Using the newly gained insights, we then modulated the key pathways of CCEM by drugs and inhibited the productive CCHFV-replication in in vitro infection models. Our study thus provides a comprehensive, system-level picture of the regulation of cellular and metabolic pathways during productive CCHFV-infection for the first time that aids in identifying novel therapeutic targets and treatment strategies.

microbiology↗

bioRxiv2019

Exome Sequencing Reveals a High Prevalence of BRCA1 and BRCA2 Founder Variants in a Diverse Population-Based Biobank

Pathogenic variants in BRCA1 and BRCA2 (BRCA1/2) lead to increased risk of breast, ovarian, and other cancers, but most variant positive individuals in the general population are unaware of their risk, and little is known about the prevalence of pathogenic BRCA1/2 variants in non-European populations. We investigated BRCA1/2 prevalence and impact using exome sequencing and electronic health record (EHR) data from 30,223 adult participants of the BioMe Biobank in New York City. There were 218 (0.7%) individuals harboring expected pathogenic variants, resulting in an overall prevalence of 1 in 139. In sub-populations defined by genetic ancestry, the highest prevalence was in individuals of Ashkenazi Jewish (AJ; 1 in 49), Filipino and Southeast Asian (1 in 81), and Non-AJ European (1 in 103) descent. Among 218 variant positive individuals, 112 (51.4%) harbored known founder variants: 80 had AJ founder variants (BRCA1 c.5266dupC and c.68_69delAG, and BRCA2 c.5946delT), 7 had a Puerto Rican founder variant (BRCA2 c.3922G>T), and 25 had one of 19 other founder variants. Non-European populations were more likely to harbor BRCA1/2 variants that were not classified in ClinVar, or that had uncertain or conflicting evidence for pathogenicity. Within mixed ancestry populations, such as Hispanic/Latinos with genetic ancestry from Africa, Europe, and the Americas, there was a strong correlation between the proportion African genetic ancestry and the likelihood of harboring a BRCA1/2 variant with uncertain or conflicting evidence for pathogenicity. Based on EHR and participant questionnaire data, [~]28% of variant positive individuals had a personal history, and [~]45% a personal or family history of BRCA1/2-associated cancers. Approximately 27% of variant positive individuals had evidence of prior clinical genetic testing for BRCA1/2. However, individuals with AJ founder variants were twice as likely to have had a clinical test (38%) than those with other pathogenic variants (19%). These findings deepen our knowledge about BRCA1/2 variants and associated cancer risk in diverse populations, indicate a gap in knowledge about potential cancer-related variants in non-European populations, and suggest that genomic screening in diverse patient populations may be an effective tool to identify at-risk individuals.

genomics↗