Biology subjects

Pinto, A. M.

Publications and source records attributed to Pinto, A. M..

5 recordsLinked to original sources

bioRxiv2025

Validation of the NovaSeq6000 Platform and automated library preparation for CE-IVD equivalence

The implementation of next-generation sequencing (NGS) technologies in clinical diagnostics requires rigorous validation of sequencing platforms and analytical workflows. In this study, we validated the performance of the Illumina NovaSeq6000 Research Use Only (RUO) platform, combined with automated library preparation using the Hamilton Microlab STAR system, by comparison to the CE-IVD certified NovaSeq6000Dx platform, which currently relies on manual library preparation. A total of 96 clinical samples underwent whole-exome sequencing (WES) on both platforms. Variant detection performance was assessed for single nucleotide variants (SNVs) and copy number variants (CNVs). The RUO platform demonstrated 100% concordance with the CE-IVD system for clinically relevant SNVs, with full agreement across positive, negative, and overall percent agreement metrics. For CNVs larger than 150 kb, the positive percent agreement was 79%, rising to 91.7% for CNVs >900 kb. Coverage uniformity and autosomal callability were consistently high across platforms. These results confirm the analytical equivalence of the NovaSeq6000 RUO configuration with automated library preparation for clinical-grade WES. This validation framework supports the adoption of scalable, cost-effective workflows that can achieve diagnostic performance comparable to CE-IVD certified systems and may facilitate routine implementation of exome sequencing in clinical laboratories. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/671033v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1735683org.highwire.dtl.DTLVardef@134d50dorg.highwire.dtl.DTLVardef@18f6e07org.highwire.dtl.DTLVardef@180a3e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Maturational Differences in Affective Behaviors Involves Changes in Frontal Cortical-Hippocampal Functional Connectivity and Metabolomic Profiles

The differential expression of emotional reactivity from early to late adulthood may involve maturation of prefrontal cortical responses to negative valence stimuli. In mice, age-related changes in affective behaviors have been reported, but the functional neural circuitry warrants further investigation. We assessed age variations in affective behaviors and functional connectivity in male and female C57BL6/J mice. Mice aged 10, 30 and 60 weeks (wo) were tested over 8 weeks for open field activity, sucrose preference, social interactions, fear conditioning, and functional neuroimaging. Prefrontal cortical and hippocampal tissues were excised for metabolomics. Our results indicate that young and old mice differ significantly in affective behavioral, functional connectome and prefrontal cortical-hippocampal metabolome. Young mice show a greater responsivity to novel environmental and social stimuli compared to older mice. Conversely, late middle-aged mice (60wo group) display variable patterns of fear conditioning and with re-testing with a modified context. Functional connectivity between a temporal cortical/auditory cortex network and subregions of the anterior cingulate cortex and ventral hippocampus, and a greater network modularity and assortative mixing of nodes was stronger in young versus older adult mice. Metabolome analyses identified differences in several essential amino acids between 10wo mice and the other age groups. The results support differential expression of emotionality across distinct stages of the mouse lifespan involving greater prefrontal-hippocampal connectivity and neurochemistry.

neuroscience↗

bioRxiv2023

Inflammation causes insulin resistance via interferon regulatory factor 3 (IRF3)-mediated reduction in FAHFA levels

Obesity-induced inflammation causes metabolic dysfunction, but the mechanisms remain elusive. Here we show that the innate immune transcription factor interferon regulatory factor (IRF3) adversely affects glucose homeostasis through induction of the endogenous FAHFA hydrolase androgen induced gene 1 (AIG1) in adipocytes. Adipocyte-specific knockout of IRF3 protects mice against high-fat diet-induced insulin resistance, whereas overexpression of IRF3 or AIG1 in adipocytes promotes insulin resistance on a high-fat diet. Furthermore, pharmacological inhibition of AIG1 reversed obesity-induced insulin resistance and restored glucose homeostasis in the setting of adipocyte IRF3 overexpression. We, therefore, identify the adipocyte IRF3/AIG1 axis as a crucial link between obesity-induced inflammation and insulin resistance and suggest an approach for limiting the metabolic dysfunction accompanying obesity.

physiology↗

bioRxiv2022

Dynamics in vertical transmission of viruses in naturally selected and traditionally managed honey bee colonies across Europe

The suppressed in-ovo virus infection trait (SOV) was the first trait applied in honey bee breeding programs aimed to increase resilience to virus infections, a major threat for colony survival. By screening drone eggs for viruses, the SOV trait scores the antiviral resistance of queens and its implications for vertical transmission. In this study, queens from both naturally surviving and traditionally managed colonies from across Europe were screened using a two-fold improved SOV phenotyping protocol. First, a gel-based RT-PCR was replaced by a RT-qPCR. This not only allowed quantification of the infection load but also increased the test sensitivity. Second, a genotype specific primer set was replaced by a primer set that covered all known deformed wing virus (DWV) genotypes, which resulted in higher virus loads and fewer false negative results. It was demonstrated that incidences of vertical transmission of DWV were more frequent in naturally surviving populations than in traditionally managed colonies, although the virus load in the eggs remained the same. Dynamics in vertical transmission were further emphasized when comparing virus infections with queen age. Interestingly, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies when compared with younger queens. Seasonal variation in vertical transmission was found with lower infection frequencies in spring compared to summer for DWV and black queen cell virus. Together, these patterns in vertical transmission suggest an adaptive antiviral response of queens aimed at reducing vertical transmission over time.

ecology↗

bioRxiv2019

U1 snRNP regulates cancer cell migration and invasion

Stimulated cells and cancer cells have widespread shortening of mRNA 3-utranslated regions (3UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in the last exon and in introns. U1 snRNA (U1), vertebrates most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread mRNA shortening. Here we show that U1 AMO also modulates cancer cells phenotype, dose-dependently increasing migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected link between U1 regulation and oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.

molecular biology↗