Biology subjects

Mendez, L.

Publications and source records attributed to Mendez, L..

5 recordsLinked to original sources

bioRxiv2025

Origins of cin: Lateral Gene Transfer of Cytoplasmic Incompatibility Nuclease Operon to Orientia tsutsugamushi

CinB nucleases are Wolbachia proteins that induce cytoplasmic incompatibility (CI) through tandem nuclease domains nuc1 and nuc2 1. CI is a form of reproductive parasitism (RP) whereby males are conditionally sterilized 2. The system behaves as a toxin-antidote (TA) system 1-8 where operon gene cinA encodes an antidote and gene cinB encodes a toxin 1,5. Cin operons are purportedly the cause of gene-drive induced by wolbachiae infecting Drosophila simulans 1,9-12. An unanswered research question is whether lateral transfer of CI operons to bacteria outside wolbachiae would transfer RP phenotype and activate gene-drive. We demonstrate that a cin operon, capable of gene-drive, has jumped into the genome of Orientia tsutsugamushi, a human pathogen and causative agent of lethal scrub typhus. When expressed in transgenic Drosophila melanogaster, the wildtype cinBoTsu was capable of inducing CI independent of its partner antidote cinAoTsu. In addition, cinAoTsu rescued the phenotype in accordance with strict TA functionality. To understand the diverging roles of the tandem nuclease domains we mutated the domains and tested all permutations of active/inactive forms. Finally, we isolated IS5 transposon variants flanking the operon in O. tsutsugamushi and re-activated them to test their mobility. We demonstrate that these transposons can transfer genes and initiate lateral gene transfers into E. coli. These data demonstrate that active bacterial transposons can mobilize and transfer CI factors (cifs) to diverse bacteria. Overall, our data contribute mechanistic understanding in support of the TA model of CI and illuminate biochemical mechanisms that mobilize cifs across genomes from phylogenetically diverse taxa. Significance StatementCI operons are foundational genes that directly contribute to the success of Wolbachia-based bio-control strategies. Two applications of RP-inducing Wolbachia strains are insect population replacement and the incompatible insect technique. Both these techniques do not work if cifs do not function. Thus, a mechanistic understanding of cif function contributes to worldwide bio-control implementations. Furthermore, certain wolbachiae have long been studied as broad-spectrum inducers of diverse RP phenotypes, including CI, parthenogenesis, male-killing, and feminization. How these diverse phenotypes evolve and if they are all induced by cif genes is a long-standing field question. For example, Orientia tsutsugamushi, the causative agent of a deadly scrub typhus, induces parthenogenesis in Leptotrombidium mites, which putatively have reproductive advantages over uninfected mites. How Orientia induces RP is an important epidemiological question in vector biology. In our report, we show that an active cin operon is capable of CI and jumped into Orientia genomes via an IS5 transposon. We reconstructed this transposon and engineered it as a biotechnological tool. In toto, our study leads to the proposal of a new hypothesis whereby CI and parthenogenesis phenotypes might both be connected to the same cif expressed under divergent host genetic contexts.

molecular biology↗

bioRxiv2024

Extracellular vesicles from preeclampsia disrupt the blood-brain barrier via reduced claudin-5: potential role of vascular endothelial growth factor

BackgroundPhysiopathology of life-treating cerebrovascular complications in preeclampsia are yet unknown. We investigated whether disruption of the blood-brain barrier (BBB), generated using circulating small extracellular vesicles (sEVs) from women with preeclampsia or placentae cultured under hypoxic conditions, impairs the expression of tight junction proteins, such as claudin 5 (CLDN5), mediated by VEGF and activation of VEGF receptor 2 (KDR). MethodssEVs were isolated from plasma (normal pregnancy, sEVs-NP, n=9); preeclampsia, sEVs-PE, n=9) or placental explants from normotensive pregnancies, cultured in normoxia (sEVs-Nor, n=10) or hypoxia (sEVs-Hyp, n=10). The integrity of the BBB was evaluated using in vitro (human and mice brain endothelial cell lines) and in vivo (non-pregnant C57BL/6 mice (4 to 5 months old, (n=10) were injected with sEVs-Hyp), models. ResultssEVs-PE and sEVs-Hyp reduced CLND5 levels (p<0.05) in the endothelial cell membrane without affecting other tight junction proteins. These results were negated with sEVs-PE sonication. sEVs-Hyp injected into non-pregnant mice generated neurological deficits and BBB disruption, specifically in the posterior area of the brain, associated with reduction in CLND5 levels in the brain cortex. Furthermore, sEVs-PE and sEVs-sHyp had higher VEGF levels than sEVs-NP and sEVs-Nor, respectively. Human brain endothelial cells exposed to sEVs-PE or sEVs-sHyp exhibited a reduction in the activation of KDR. ConclusionsEVs from hypoxic placentae and plasma from women with preeclampsia disrupt the BBB, via reduction of CLDN5, a phenomenon that may involve VEGF contained within these vesicles. These findings will improve the elucidation of cerebrovascular alterations in women with preeclampsia.

physiology↗

bioRxiv2023

Genomic signatures of past megafrugivore-mediated dispersal in Malagasy palms

O_LISeed dispersal is a key process in the generation and maintenance of genetic diversity and genetic differentiation of plant populations in tropical ecosystems. During the Last Quaternary, most seed-dispersing megafauna was lost globally, but whether this has caused dispersal limitation, loss of genetic diversity, and increased genetic differentiation between plant populations with large, megafaunal fruits (i.e., > 4 cm - megafruits) remains unclear. C_LIO_LIHere, we assessed whether megafrugivore extinctions in Madagascar (e.g., giant lemurs, elephant birds) have affected the genetic diversity and genetic differentiation of four animal-dispersed Malagasy palm (Arecaceae) species with large (Borassus madagascariensis), medium-sized (Hyphaene coriacea, Bismarckia nobilis), and small (Chrysalidocarpus madagascariensis) fruits. We integrated double-digest restriction-site-associated DNA sequencing (ddRAD) of 167 individuals from 25 populations with (past) distribution ranges for extinct and extant seed-dispersing animal species, climate and human impact data, and applied linear mixed-effects models to explore the drivers of variation in genetic diversity and genetic differentiation across Malagasy palm populations. C_LIO_LIWe detected higher genetic diversity in species with megafruits than in the species with small fruits, and genetic differentiation was lowest for the human-used medium-sized megafruit species. Furthermore, we found that a higher number of shared extinct megafrugivore species between palm population pairs was associated with less genetic differentiation, indicating higher gene flow, whereas no relationship with extant frugivores - that are not able to swallow and disperse the seeds - was found. Finally, genetic diversity decreased with road density, whereas genetic differentiation decreased with increasing human population density, but only for populations with megafruits. C_LIO_LIOur results suggest that the legacy of megafrugivores regularly achieving long dispersal distances is still reflected in the genetic diversity and genetic differentiation of palms that were formerly dispersed by such large animals. Furthermore, high genetic diversity and low genetic differentiation were possibly maintained after the megafauna extinctions through human-mediated dispersal, long generation times, and long lifespans of these palms. Our study illustrates how integrating genetics with ecological data on species interactions, climate, and human impact, provides novel insights into the consequences of megafauna extinctions for plants with megafruits. C_LI

evolutionary biology↗

bioRxiv2020

CRISPR/Cas9 technology abolishes the BCR/ABL1 oncogene effect in chronic myeloid leukemia and restores normal hematopoiesis

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease produced by a unique oncogenic event involving the constitutively active tyrosine kinase (TK) BCR/ABL1. TK activity explains most features of CML, such as tumor development and maintenance. TK-inhibitory (TKI) drugs have changed its prognosis and natural history. Unfortunately, the ABL1 gene persists unaffected by TKIs, leukemic stem cells (LSCs) remains, resistant mutations arise and adverse effects may occur during treatment. To address this problem, we have designed a potential therapeutic alternative with CRISPR/Cas9 genome editing nucleases that target LSCs. The strategy was successfully developed in murine and human cell lines and finally was evaluated in primary LSCs isolated from CML transgenic mice and from CML patients. Mouse CML-LSCs edited were orthotopic transplanted in immunodeficient NSG niches where restored the normal hematopoiesis. Importantly, patient-derived xenografts with CD34+-LSCs edited, repopulated and restored the normal hematopoiesis in immunodeficient NSG niches. We show, for the first time, how CRISPR technology efficiently interrupts the BCR/ABL1 oncogene in murine and human LSCs to provide a significant therapeutic benefit. We propose human CML as a potential candidate for CRISPR therapy, providing proof-of-principle for genome editing in CML patients, and open new avenues for the application of this technique in other fusion genes. Key pointsCRISPR system destroys BCR/ABL oncogene and induces a therapeutic benefit in a CML mouse model and CML patient derived xenografts.

cancer biology↗

bioRxiv2019

Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

CRISPR/Cas9 enables the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.

molecular biology↗