Biology subjects

Fernandez-Gonzalez, A.

Publications and source records attributed to Fernandez-Gonzalez, A..

5 recordsLinked to original sources

bioRxiv2025

Development of metagenomic methods for non-invasive health monitoring of endangered species: Unveiling hidden microbial threats in fecal samples

Metagenomic analysis of feces is emerging as a powerful tool for improving the monitoring of endangered species. A critical aspect in assessing the extinction risk of a species is the analysis of the burden of parasites and pathogens that can negatively affect the health of individuals. However, the identification of pathogens in non-model species using metagenomics is a major challenge due to the lack of reference genome sequences or data limited to distantly related species. In this study, we developed a pipeline for detecting potentially pathogenic bacteria from metagenomic sequences by mapping unassembled reads to available reference genomes. The approach uses the breadth of genome coverage rather than the number of mapped reads for species identification, thereby minimizing false positives due to conserved or repetitive genomic regions. We applied this method to fresh fecal samples of the Iberian desman (Galemys pyrenaicus), a critically endangered semi-aquatic mammal. Our analysis revealed the presence of 19 potentially pathogenic bacterial species, with prevalences ranging from a single individual to 30% of the samples. We also detected some desmans with elevated or altered pathogen loads, suggesting variations in individual health status or different environmental exposures. This work represents a novel application of metagenomic methods for species-level pathogen detection in wildlife using fecal samples. Application of this method across populations and over time for endangered species may provide essential health and epidemiological information to improve conservation strategies.

genomics↗

bioRxiv2024

CCQM-P199: Interlaboratory comparability study of HIV-1 RNA copy number quantification

Infection with human immunodeficiency virus (HIV)-1 leads to acquired immunodeficiency syndrome (AIDS) if left untreated. According to UN figures, approximately 39 million people globally were living with HIV in 2022, with 76% of those individuals accessing antiretroviral therapy. Measurement of plasma viral RNA load using calibrated nucleic acid amplification tests (like reverse transcription quantitative PCR, RT-qPCR) is routinely performed to monitor response to treatment and ultimately prevent viral transmission. RNA quantities measured by commercial tests can vary over many orders of magnitude, from trace single copy levels to, in cases, over 109 /mL of plasma, presenting an analytical challenge for calibrating across a broad measurement range. Interlaboratory study CCQM-P199 "HIV-1 RNA copy number quantification" (April to September 2019) was conducted under the auspices of the Consultative Committee for Amount of Substance (CCQM) Nucleic Acid analysis Working Group (NAWG), with the aims of supporting national metrology institutes (NMIs) and designated institutes (DIs) development of the capacity and evaluating candidate reference measurement procedures for applied viral nucleic acid measurements. Thirteen laboratories participated in CCQM-P199 and were requested to report the RNA copy number concentration, expressed in copies per microliter, of the HIV-1 group specific antigen (gag) gene of in vitro transcribed RNA molecules at low ({approx} 103 /L) and high concentration ({approx} 109 /L) (Study Materials 1 and 2, linked by gravimetric dilution) and purified genomic RNA from cultured virus (Study Material 3). Study Materials 1 and 3 were measured by participants using one-step reverse transcription digital PCR (RT-dPCR) (Bio-Rad reagents) and/or two-step RT-dPCR with alternative cDNA synthesis reagents. Study Material 2 was measured by both RT-dPCR (one-step) (n = 4) and orthogonal methods: single molecule flow cytometric counting (n = 2), high performance liquid chromatograph (HPLC) (n = 1) and isotype dilution-mass spectrometry (ID-MS) (n = 1). Interlaboratory reproducibilities (expressed as %CV) were 21.4 %, 15.3 % and 22.0 % for Study Materials 1, 2 and 3 respectively. Analysis of overdispersion showed that the interlaboratory variation for all three Study Materials was not accounted for in their reported uncertainties, indicating uncharacterized sources of variation remain. Although the mean values of RT-dPCR and orthogonal method results were not statistically significantly different (p = 0.46), the extrapolated mean Study Material 2 results were higher than mean Study Material 1 results (1196 vs. 808 /L; p < 0.05). Follow-up analysis of Study Material 2 purity by ultra-performance liquid chromatography (UPLC) indicated higher molecular weight (MW) impurities constituted 16.6 % of the molecules, which are hypothesised to be the cause of the HPLC and ID-MS results being higher than the majority of Study Material 1 and 2 results. This study demonstrates that reproducible measurement of RNA templates was achieved by metrology laboratories, illustrating the potential of RT-dPCR combined with complimentary orthogonal approaches to support traceability and precision of contemporary methods for RNA quantification. This study also highlighted that detailed characterization of RNA materials and sources of bias affecting measurements such as RT efficiency is needed to further establish RT-dPCR as a primary reference measurement procedure for RNA copy number quantification.

molecular biology↗

bioRxiv2024

CCQM-P199b: Interlaboratory comparability study of SARS-CoV-2 RNA copy number quantification

Nucleic acid amplification tests including reverse transcription quantitative PCR (RT-qPCR) are used to detect RNA from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Standardized measurements of RNA can facilitate comparable performance of laboratory tests in the absence of existing reference measurement systems early on in a pandemic. Interlaboratory study CCQM P199b "SARS-CoV-2 RNA copy number quantification" was designed to test the fitness-for-purpose of developed candidate reference measurement procedures (RMPs) for SARS-CoV-2 genomic targets in purified RNA materials, and was conducted under the auspices of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) to evaluate the measurement comparability of national metrology institutes (NMIs) and designated institutes (DIs), thereby supporting international standardization. Twenty-one laboratories participated in CCQM P199b and were requested to report the RNA copy number concentration, expressed in number of copies per microliter, of the SARS-CoV-2 nucleocapsid (N) gene partial region (NC_045512.2: 28274-29239) and envelope (E) gene (NC_045512.2: 26245-26472) (optional measurements) in samples consisting of in vitro transcribed RNA or purified RNA from lentiviral constructs. Materials were provided in two categories: lower concentration ({approx} (101-104) /L in aqueous solution containing human RNA background) and high concentration ({approx} 109 /L in aqueous solution without any other RNA background). For the measurement of N gene concentration in the lower concentration study materials, the majority of laboratories (n = 17) used one-step reverse transcription-digital PCR (RT-dPCR), with three laboratories applying two-step RT-dPCR and one laboratory RT-qPCR. Sixteen laboratories submitted results for E gene concentration. Reproducibility (% CV or equivalent) for RT-dPCR ranged from 19 % to 31 %. Measurements of the high concentration study material by orthogonal methods (isotope dilution-mass spectrometry and single molecule flow cytometry) and a gravimetrically linked lower concentration material were in a good agreement, suggesting a lack of overall bias in RT-dPCR measurements. However methodological factors such as primer and probe (assay) sequences, RT-dPCR reagents and dPCR partition volume were found to be potential sources of interlaboratory variation which need to be controlled when applying this technique. This study demonstrates that the accuracy of RT-dPCR is fit-for-purpose as a RMP for viral RNA target quantification in purified RNA materials and highlights where metrological approaches such as the use of in vitro transcribed controls, orthogonal methods and measurement uncertainty evaluation can support standardization of molecular methods.

molecular biology↗

bioRxiv2023

Standardisation of cell-free DNA measurements: An International Study on Comparability of Low Concentration DNA Measurements using cancer variants

For the impact of genomic testing from liquid biopsies to be maximized, mechanisms to ensure reproducible and comparable test performance will be required. This can be established and maintained through reference measurement procedures and materials with property values that are internationally comparable through traceability to a common standard. To achieve this objective, an interlaboratory study was organised to explore digital PCR (dPCR) for standardisation of cell-free DNA (cfDNA) quantification. Blinded samples of wild-type/variant mixtures of two DNA sequences (BRAF p.V600E single nucleotide variant or EGFR exon 19 deletion) were provided to 12 laboratories. Laboratories independently designed and applied dPCR assays to determine absolute and relative quantities, with no guidance provided to harmonise the approach. The mean and coefficient of variation (CV) of copy number concentrations for variant sequences were 18 copies/L (CV 7.2%) (BRAF variant sample) and 9 copies/L (CV 25%) (EGFR variant sample) while the mean variant allele frequencies (vAF) were 8.0% (CV 5.3%) and 0.080% (CV 29%) respectively. This study demonstrated that dPCR was capable of exceptional technical accuracy for variant copy number concentration and vAF, even when different assays and platforms were used. This implies that dPCR offers a unique analytical methodology that can be deployed globally in supporting comparability for cfDNA testing based on the existing framework of the International System of units of measurement.

genetics↗

bioRxiv2023

Immunoregulatory macrophages modify local pulmonary immunity and ameliorate hypoxic-pulmonary hypertension

RationaleMacrophages play a central role in the onset and progression of vascular disease in pulmonary hypertension (PH) and cell-based immunotherapies aimed at treating vascular remodeling are lacking. ObjectiveTo evaluate the effect of pulmonary administration of macrophages modified to have an anti-inflammatory/pro-resolving phenotype in attenuating early pulmonary inflammation and progression of experimentally induced PH. MethodsMouse bone marrow derived macrophages (BMDMs) were polarized in vitro to a regulatory (M2reg) phenotype. M2reg profile and anti-inflammatory capacity were assessed in vitro upon lipopolysaccharide (LPS)/interferon-{gamma} (IFN{gamma}) restimulation, before their administration to 8- to 12-week-old mice. M2reg protective effect was tested at early (2 to 4 days) and late (4 weeks) time points during hypoxia (8.5% O2) exposure. Levels of inflammatory markers were quantified in alveolar macrophages and whole lung, while PH development was ascertained by right ventricular systolic pressure (RSVP) and right ventricular hypertrophy (RVH) measurements. Bronchoalveolar lavage (BAL) from M2reg-transplanted hypoxic mice was collected, and its inflammatory potential tested on naive BMDMs. ResultsM2reg macrophages demonstrated a stable anti-inflammatory phenotype upon a subsequent pro-inflammatory stimulus by maintaining the expression of specific anti-inflammatory markers (Tgf{beta}, Il10 and Cd206) and downregulating the induction of proinflammatory cytokines and surface molecules (Cd86, Il6 and Tnf). A single dose of M2regs attenuated the hypoxic monocytic recruitment and perivascular inflammation. Early hypoxic lung and alveolar macrophage inflammation leading to PH development was significantly reduced and, importantly, M2regs attenuated RVH, RVSP and vascular remodeling at 4 weeks post treatment. ConclusionsAdoptive transfer of M2regs halts the recruitment of monocytes and modifies the hypoxic lung microenvironment, potentially changing the immunoreactivity of recruited macrophages and restoring normal immune functionality of the lung. These findings provide new mechanistic insights on the diverse role of macrophage phenotype on lung vascular homeostasis that can be explored as novel therapeutic targets.

cell biology↗