Biology subjects

Villanueva, P.

Publications and source records attributed to Villanueva, P..

5 recordsLinked to original sources

bioRxiv2026

Adeno-Associated Virus (AAV) Synthetic Inverted Terminal Repeats Enhance Tissue-Specific Transduction and Alter the Vector Induced Stress Response

While adeno-associated virus (AAV) vectors have shown therapeutic benefit in clinical applications, noted challenges include low transduction efficiencies, poor cellular targeting, and vector related adverse events. Recently, it was demonstrated that a rationally designed synthetic inverted terminal repeat (SynITR) altered the AAV vector-induced DNA damage response and abrogated apoptosis in human embryonic stem cells. To explore the utility of AAV-SynITR for diverse gene therapy applications, vector production, transduction, and the cellular response were evaluated in various contexts. Regarding production, SynITR preparations exhibited comparable titers to wtITR in a serotype/transgene-independent manner. Despite slightly decreased transduction efficiency in various cell lines, intravenous administration of AAV8 vectors showed SynITR enhanced transduction in a tissue-specific manner in liver (>7-fold) and kidney and pancreas (>2-fold) at equivalent vector copy numbers; however, no differences were observed in muscle/heart/spleen tissues. Interestingly, persistent {gamma}H2AX, a marker of aging/chronic inflammation, was abundant in the liver and spleen following wtITR (but not SynITR) transduction. In human corneas, SynITR enhanced transduction up to 16-fold over wtITRs. These data demonstrate that SynITRs elicit tissue-specific transduction enhancement and alter the cellular stress response. Importantly, the SynITRs offer an alternative context to elucidate wtITR biology for targeted, enhanced, and potentially safer human gene therapy.

molecular biology↗

bioRxiv2025

Profiling of Vaginal Microbial Communities in Chilean Women Via Self-sampling and Full-length 16S rRNA Sequencing

The vaginal microbiota is a dynamic ecosystem that plays a crucial role in womens health, with Lactobacillus species predominating in healthy individuals. Disruptions in this microbiota can lead to dysbiosis, increasing susceptibility to infections such as bacterial vaginosis and other reproductive health complications. While most studies on the vaginal microbiome have been conducted in North American and European populations, data from Latin America, particularly Chile, remain scarce. This study presents a comprehensive analysis of the vaginal microbiota in Chilean women using full-length 16S rRNA gene sequencing with Oxford Nanopore Technology (ONT). We implemented a self-sampling methodology and a bioinformatics pipeline to achieve species-level resolution of microbial communities. Taxonomic profiling using Emu revealed that Lactobacillus was the dominant genus in most samples, while others exhibited microbial compositions consistent with Community State Type (CST) IV, characterized by higher diversity and lower Lactobacillus abundance. Alpha and beta diversity analyses further demonstrated distinct microbial clustering patterns across CSTs, with species-level resolution providing enhanced classification accuracy. Our findings highlight the potential of long-read sequencing and self-sampling for improving vaginal microbiome studies, particularly in underrepresented populations. This research contributes valuable insights into the composition and diversity of the vaginal microbiota in Chilean women, paving the way for more precise diagnostics and targeted interventions in reproductive health.

microbiology↗

bioRxiv2022

Genome-wide screening reveals metabolic regulation of translational fidelity

Translational quality control is critical for maintaining the accuracy of protein synthesis in all domains of life. Mutations in aminoacyl-tRNA synthetases and the ribosome are known to affect translational fidelity and alter fitness, viability, stress responses, neuron function, and life span. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified 30 nonessential genes that are critical for maintaining the fidelity of stop-codon readthrough. Most of these identified genes have not been shown to affect translational fidelity previously. Intriguingly, we show that several genes controlling metabolism, including cyaA and guaA, unexpectedly enhance stop-codon readthrough. CyaA and GuaA catalyze the synthesis of cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (GMP), respectively. Both CyaA and GuaA increase the expression of ribosomes and tRNAs, allowing aminoacyl-tRNAs to compete with release factors and suppress stop codons. In addition, the effect of guaA deletion on stop-codon readthrough is abolished by deleting prfC, which encodes release factor 3 (RF3). Our results suggest that nucleotide and carbon metabolism is tightly coupled with translational fidelity.

genetics↗

bioRxiv2022

TENT2, TUT4, and TUT7 selectively regulate miRNA sequence and abundance

TENTs generate miRNA isoforms by 3 tailing. However, little is known about how tailing regulates miRNA function. Here, we generated isogenic HEK293T cell lines in which TENT2, TUT4 and TUT7 were knocked out individually or in combination. Together with rescue experiments, we characterized TENT-specific effects by deep sequencing, Northern blot and in vitro assays. We found that 3 tailing is not random but highly specific. In addition to its known adenylation, TENT2 contributes to guanylation and uridylation on mature miRNAs. TUT4 uridylates most miRNAs whereas TUT7 is dispensable. Removing adenylation has a marginal impact on miRNA levels. By contrast, abolishing uridylation leads to dysregulation of a set of miRNAs. Besides let-7, miR-181b and miR-222 are negatively regulated by TUT4/7 via distinct mechanisms while the miR-888 cluster is upregulated specifically by TUT7. Our results uncover the selective actions of TENTs in generating 3 isomiRs and pave the way to investigate their functions.

molecular biology↗

bioRxiv2020

MetaFunPrimer: primer design for targeting genes observed in metagenomes

High throughput primer design is needed to simultaneously design primers for multiple genes of interest, such as a group of functional genes. We have developed MetaFunPrimer, a bioinformatic pipeline to design primer targets for genes of interests, with a prioritization based on ranking the presence of gene targets in references, such as metagenomes. MetaFunPrimer takes inputs of protein and nucleotide sequences for gene targets of interest accompanied by a set of reference metagenomes or genomes for determining genes of interest. Its output is a set of primers that may be used to amplify genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 HT-qPCR primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 soil metagenomes. We demonstrate that these primers can significantly improve targeting of amoA-AOB genes in soil metagenomes compared to previously published primers. IMPORTANCEAmplification-based gene characterization allows for sensitive and specific quantification of functional genes. Often, there is a large diversity of genes represented for a function of interest, and multiple primers may be necessary to target associated genes. Current primer design tools are limited to designing primers for only a few genes of interest. MetaFunPrimer allows for high throughput primer design for functional genes of interest and also allows for ranking gene targets by their presence and abundance in environmental datasets. This tool enables high throughput qPCR approaches for characterizing functional genes.

bioinformatics↗