Biology subjects

Garcia, P.

Publications and source records attributed to Garcia, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Setdb1 safeguards genome integrity in muscle stem cells to allow for regenerative myogenesis and inflammation

Modulations in chromatin structure orchestrate gene expression and direct stem cell fate. More specifically, the histone 3 lysine 9 Methyltransferase Setdb1 controls transcriptional repression to regulate pluripotency and self-renewal. While Setdb1 functions have been extensively studied in embryonic stem cells and in cancer cells, less is known on its role in adult stem cells in vivo. Here, we show that Setdb1 expression by adult muscle stem cells (MuSCs) is required for muscle tissue regeneration following acute injury. We find that SETDB1 represses the expression of the endogenous retroviruses (ERVs) family of transposable elements in MuSCs. ERV de-expression in Setdb1-null MuSCs prevents their amplification following exit from quiescence and promotes cell death. Multi-omics profiling further shows that the absence of SETDB1 in MuSCs leads to the activation of the DNA-sensing cGAS-STING pathway, entailing increased cytokine expression. In vivo, conditional disruption of Setdb1 in MuSCs provokes aberrant infiltration of inflammatory cells including the appearance of a pathological macrophage lineage. The ensuing histiocytosis is accompanied by necrosis of the newly formed muscle fibers which, in addition with the progressive loss of MuSCs, completely abolish skeletal muscle tissue repair. In contrast, disruption of Setdb1 gene in another muscle-resident cell type, the fibro-adipogenic progenitors (FAPs), does not impact regenerative inflammation. In conclusion, the control of genome stability by SETDB1 in an adult somatic stem cell is necessary for both its regenerative potential and an adequate inflammation regulating tissue repair.

cell biology↗

bioRxiv2023

Heavy Metal Pollution From a Major Earthquake and Tsunami in Chile Is Associated With Geographic Divergence of Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Latin America

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

microbiology↗

bioRxiv2023

Dynamics of the MRSA Population in A Chilean Hospital: A Phylogenomic Analysis (2000-2016)

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in the last years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary healthcare center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000-2016 in a tertiary healthcare center in Chile. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r=0.8748, p<0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016. The temporal trend analysis revealed that in the period 2000-2003 most of the isolates (94.2%; n=98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013-2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage in Chile. However, this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. ImportanceMethicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or classical typing techniques with several limitations to depict an accurate description of their genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000-2016 in Chile to provide the largest and most detailed study of clonal dynamics of MRSA carried out in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing their frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

microbiology↗

bioRxiv2022

Dietary fibre deprivation and bacterial curli exposure shift gut microbiome and exacerbate Parkinson's disease-like pathologies in an alpha-synuclein-overexpressing mouse

The microbiome-gut-brain axis has been proposed as a pathogenic path in Parkinsons disease (PD). Dietary driven dysbiosis and reduced gut barrier function could facilitate the interaction of toxic external or internal factors with the enteric nervous system, where PD could start. Amyloid bacterial protein such as curli can act as seed to corrupt enteric -synuclein and lead to its aggregation. Misfolded -synuclein can propagate to and throughout the brain. Here, we aimed at understanding if fibre deprivation and amyloidogenic protein curli could, individually or together, exacerbate the phenotype in both enteric and central nervous systems of a transgenic mouse overexpressing wild-type human -synuclein. We analysed the gut microbiome, motor behaviour, gastrointestinal and brain pathologies in these mice. Our findings show that external interventions, akin to unhealthy life habits in humans, can exacerbate PD-like pathologies in mice. We believe that our results shed light on how lifestyle affects PD progression.

neuroscience↗

bioRxiv2022

Discordant population structure inferred from male- and female-type mtDNAs from Limecola balthica, a bivalve species characterized by doubly uniparental inheritance of mitochondria

Doubly Uniparental Inheritance (DUI) of mitochondria is a remarkable exception to the Strictly Maternal Inheritance (SMI) in metazoans. In species characterized by DUI --almost exclusively gonochoric bivalve mollusks--females (F) transmit mitochondria to offspring of both sexes, while males (M) pass on their mitochondria exclusively to their sons. Under DUI, males are heteroplasmic, somatic tissues containing F-transmitted mtDNA and gametic cells containing M-transmitted mtDNAs. The aforementioned transmission routes make M- and F-transmitted mtDNA interesting as sex-specific markers which can differ in their effective population sizes, mutation rates, and selective constraints. For these reasons, looking at both markers can provide significant insights into the genetic structure of populations and investigate its determinants. In this study, we document differences in genetic diversity, divergence, inter-populational differentiation and biogeographic structure between M- and F-type cox1 mt genes in the Baltic tellin (Macoma balthica) to test whether cox1m and cox1f genes bear the marks of similar phylogeographic histories. These markers were sequenced for 302 male individuals sampled from the North Sea to the Gironde Estuary (Southern France) encompassing the intra-subspecific M. b. rubra hybrid zone in the Gulf of Saint Malo. Both genes supported a scenario of cladogenesis of M.b. rubra clades prior to the last glacial maximum. Nucleotide diversity and net divergence were over twice higher in cox1m compared to cox1f. Genetic differentiation between northern and southern populations was nearly 3 times higher at cox1m compared to cox1f (global {Phi}ST = 0.345 and 0.126 respectively) and the geographic localization of the strongest genetic break significantly differed between the markers (Finistere Peninsula at cox1f; Cotentin Peninsula at cox1m, [~]250 km apart). A higher mutation rate, relaxed negative selection and differences in effective population sizes (depending on locations) at cox1m could explain differences in population genetic structure. As both F- and M-type mtDNAs interact with nuclear genes for oxidative phosphorylation and ATP production, geographical discordances in genetic clines in a context of secondary contact could be linked to mito-nuclear genetic incompatibilities.

evolutionary biology↗

bioRxiv2022

Monomodular Pseudomonas aeruginosa phage JG004 lysozyme (Pae87) contains a bacterial surface-active antimicrobial peptide-like region and a possible substrate-binding subdomain

Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy for introducing a more precise approach in the way we apply antimicrobial therapy. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had been previously identified as an antimicrobial agent candidate, able to interact with the Gram-negative surface and disrupt it. Further evidence is hereby provided on this matter, based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18 [A] away from the catalytic site and located at the opposite side of the lysin molecule. This substrate binding region was conserved among phylogenetically related lysins lacking an additional cell wall binding domain, but not among those containing such a module. Two glutamic acids were identified as relevant for the peptidoglycan degradation activity, although Pae87 antimicrobial activity was seemingly unrelated to it. In contrast, an antimicrobial peptide-like region within Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and have antibacterial activity by itself. Therefore, we propose an antimicrobial mechanism for Pae87 in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell wall function, either with or without the participation of Pae87 catalytic activity. SynopsisThe structure of the monomodular Pseudomonas aeruginosa bacteriophage JG004 lysin Pae87 is presented and investigated in relationship with its function repurposed as an antimicrobial agent. The structure with its peptidoglycan ligand revealed a possible cell wall binding region. A C-terminal antimicrobial peptide-like region is shown to be important for disrupting the bacterial cell wall.

molecular biology↗

bioRxiv2021

Non-coding RNAs Associated with Prader-Willi Syndrome Regulate Transcription of Neurodevelopmental Genes in Human Induced Pluripotent Stem Cells

Mutations and aberrant gene expression during cellular differentiation lead to neurodevelopmental disorders such as Prader-Willi syndrome (PWS) which results from the deletion of an imprinted locus on chromosome 15. We analysed chromatin-associated RNA in human induced pluripotent cells (iPSCs) upon depletion of hybrid small nucleolar long non-coding RNAs (sno-lncRNAs) and 5 snoRNA capped and polyadenylated long non-coding RNAs (SPA-lncRNAs) transcribed from the locus deleted in PWS. We found that rapid ablation of these lncRNAs affects transcription of specific gene classes. Downregulated genes contribute to neurodevelopment and neuronal maintenance while genes that are upregulated are predominantly involved in the negative regulation of cellular metabolism and apoptotic processes. Our data revealed the importance of SPA-lncRNAs and sno-lncRNAs in controlling gene expression in iPSCs and provided a platform for synthetic experimental approaches in PWS studies. We conclude that ncRNAs transcribed from the PWS locus are critical regulators of a transcriptional signature important for neuronal differentiation and development.

molecular biology↗

bioRxiv2021

Sequence-function relationships in phage-encoded bacterial cell wall lytic enzymes and their implications for phage-derived products design

Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a landscape view of lysins structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G-) (higher net charge, amphipathic helices) are responsible for an improved interaction with G- envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecules design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G- surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages host bacteria and sequence-predicted physicochemical properties. Based on such classifiers, an investigation on the differential appearance of certain features was conducted. Such analyses revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. Particularly, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment and aliphatic index) were associated to G- phage lysins, appearing specifically at their C-terminal end. Evidences on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts have been provided, specifically supporting the nowadays common hypothesis that lysins from G- usually contain AMP-like regions. IMPORTANCEPhage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The lysins potential as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance, but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysins diversity landscape by examining a set of phage lysin genes. This way, we have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures, and, thus, also the reach of their specialization regarding cell wall structures. These results provide clarity and evidences to sustain some of the common hypothesis in current literature, as well as make available an updated and characterized database of lysins sequences for further developments.

microbiology↗

bioRxiv2021

Crosstalk between AML and stromal cells triggers acetate secretion through the metabolic rewiring of stromal cells

Acute myeloid leukaemia (AML) cells interact and modulate components of their surrounding microenvironment into their own benefit. Stromal cells have been shown to support AML survival and progression through various mechanisms. Nonetheless, it is unclear whether AML cells could establish beneficial metabolic interactions with stromal cells. Here, we identify a novel metabolic crosstalk between AML and stromal cells where AML cells prompt stromal cells to secrete acetate for their own consumption to feed the tricarboxylic acid cycle (TCA). By performing transcriptome analysis, tracer-based metabolic NMR analysis and ROS measurements, we observe that stromal cells present a higher rate of glycolysis. We also find that acetate in stromal cells is derived from pyruvate via chemical conversion under the influence of reactive oxygen species (ROS) following ROS transfer from AML to stromal cells via gap junctions. Overall, we present a unique metabolic communication between AML and stromal cells that could potentially be exploited for adjuvant therapy.

cell biology↗

bioRxiv2020

Strain- and age-dependent features of the nigro-striatal circuit in three common laboratory mouse strains, C57BL/6J, A/J, and DBA/2J - Implications for Parkinson's disease modeling

Mouse models have been instrumental in understanding genetic determinants of aging and its crucial role in neurodegenerative diseases. However, few studies have analyzed the evolution of the mouse brain over time at baseline. Furthermore, mouse brain studies are commonly conducted on the C57BL/6 strain, limiting the analysis to a specific genetic background. In Parkinsons disease, the gradual demise of nigral dopaminergic neurons mainly contributes to the motor symptoms. Interestingly, a decline of the dopaminergic neuron function and integrity is also a characteristic of physiological aging in some species. Age-related nigro-striatal features have never been studied in mice of different genetic backgrounds. In this study, we analyze the morphological features in the striatum of three common mouse strains, C57BL/6J, A/J, and DBA/2J at 3-, 9- and 15 months of age. By measuring dopaminergic markers, we uncover age-related changes that differ between strains and evolve dynamically over time. Overall, our results highlight the importance of considering background strain and age when studying the murine nigro-striatal circuit in health and disease. HighlightsO_LIStudy of the integrity of the nigro-striatal circuit in C57BL/6J, A/J, and DBA/2J at different ages C_LIO_LIAge related evolution of essential features of nigral dopaminergic neurons differ between strains C_LIO_LIConsider background strain and age is crutial to study the nigrostriatal circuit in health and disease C_LI

neuroscience↗

bioRxiv2020

Neurodegeneration and neuroinflammation are linked, but independent of alpha-synuclein inclusions, in a seeding/spreading mouse model of Parkinson disease

A key process of neurodegeneration in Parkinson disease (PD) is the transneuronal spreading of alpha-synuclein. Alpha-synuclein is a presynaptic protein that is implicated in the pathogenesis of PD and other synucleinopathies, where it forms, upon intracellular aggregation, pathological inclusions. Other hallmarks of PD include neurodegeneration and microgliosis in susceptible brain regions. Whether it is primarily transneuronal spreading of alpha-synuclein particles, inclusion formation, or other mechanisms, such as inflammation, that cause neurodegeneration in PD is unclear. We used spreading/aggregation of alpha-synuclein induced by intracerebral injection of alpha-synuclein preformed fibrils into the mouse brain to address this question. We performed quantitative histological analysis for alpha-synuclein inclusions, neurodegeneration, and microgliosis in different brain regions, and a gene expression profiling of the ventral midbrain, at two different timepoints after disease induction. We observed significant neurodegeneration and microgliosis in brain regions not only with, but also without alpha-synuclein inclusions. We also observed prominent microgliosis in injured brain regions that did not correlate with neurodegeneration nor with inclusion load. In longitudinal gene expression profiling experiments, we observed early and unique alterations linked to microglial mediated inflammation that preceded neurodegeneration, indicating an active role of microglia in inducing neurodegeneration. Our observations indicate that alpha-synuclein inclusion formation is not the major driver in the early phases of PD-like neurodegeneration, but that diffusible, oligomeric alpha-synuclein species, which induce unusual microglial reactivity, play a key role in this process. Our findings uncover new features of alpha-synuclein induced pathologies, in particular microgliosis, and point to the necessity of a broader view of the process of prion-like spreading of that protein.

neuroscience↗

bioRxiv2020

Quantitative trait locus mapping identifies Col4a6 as a novel regulator of striatal dopamine level and axonal branching in mice.

The features of dopaminergic neurons (DAns) of nigrostriatal circuitry are orchestrated by a multitude of yet unknown factors, many of them genetic. Genetic variation between individuals at baseline can lead to differential susceptibility to and severity of diseases. As decline of DAns, a characteristic of Parkinsons disease, heralds a significant decrease in dopamine level, measuring dopamine can reflect the integrity of DAns. To identify novel genetic regulators of the integrity of DAns, we used the Collaborative Cross (CC) mouse strains as model system to search for quantitative trait loci (QTLs) related to dopamine levels in the dorsal striatum. The dopamine levels in dorsal striatum varied greatly in the eight CC founder strains, and the differences were inheritable in 32 derived CC strains. QTL mapping in these CC strains identified a QTL associated with dopamine level on chromosome X containing 393 genes. RNA-seq analysis of the ventral midbrain of two of the founder strains with large striatal dopamine difference (C57BL/6J and A/J) revealed 24 differentially expressed genes within the QTL. The protein-coding gene with the highest expression difference was Col4a6, which exhibited a 9-fold reduction in A/J compared to C57BL/6J, consistent with decreased dopamine levels in A/J. Publicly available single cell RNA-seq data from developing human midbrain suggests that Col4a6 is highly expressed in radial glia-like cells and neuronal progenitors, indicating possible involvement in neurogenesis. Interestingly, the lowered dopamine levels were accompanied by reduced striatal axonal branching of striatal DAns in A/J compared to C57BL/6J. Because Col4a6 is known to control axogenesis in non-mammal model organisms, we hypothesize that different dopamine levels in mouse dorsal striatum are due to differences in axogenesis induced by varying COL4A6 levels during neural development.

neuroscience↗

bioRxiv2020

Single nuclei chromatin profiles of ventral midbrain reveal cell identity transcription factors and cell type-specific gene regulatory variation

Cell types in ventral midbrain are involved in diseases with variable genetic susceptibility such as Parkinsons disease and schizophrenia. Many genetic variants affect regulatory regions and alter gene expression. We report 20 658 single nuclei chromatin accessibility profiles of ventral midbrain from two genetically and phenotypically distinct mouse strains. We distinguish ten cell types based on chromatin profiles and analysis of accessible regions controlling cell identity genes highlights cell type-specific key transcription factors. Regulatory variation segregating the mouse strains manifests more on transcriptome than chromatin level. However, cell type-level data reveals changes not captured at tissue level. To discover the scope and cell-type specificity of cis-acting variation in midbrain gene expression, we identify putative regulatory variants and show them to be enriched at differentially expressed loci. Finally, we find TCF7L2 to mediate trans-acting variation selectively in midbrain neurons. Our dataset provides an extensive resource to study gene regulation in mesencephalon.

genomics↗

bioRxiv2020

An ATM-MYBL2-CDC7 axis regulates replication initiation and prevents replication stress in pluripotent stem cells

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR-dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be substantially different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed between hundred to thousand-fold more highly in ESCs compared to somatic cells. Here we show that MYBL2 functions to activate ATM and suppress replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs, results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, this study proposes that in addition to ATR, a MYBL2-MRN-ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

cell biology↗

bioRxiv2020

Ablation of MYB-dependent leukaemia phenotype in MLL-driven AML correlates with increased expression of MAFB.

The transcription factor MYB plays a pivotal role in haematopoietic homeostasis and its aberrant expression is involved in the genesis and maintenance of acute myeloid leukaemia (AML). Our previous work has demonstrated that not all AML types display the same dependency on MYB expression and that MYB dependence is dictated by the nature of the driver mutation. However, whether this difference in MYB dependency is a general trend in AML still remains to be further elucidated. In this study, we investigate the importance of MYB in human leukaemia by performing siRNA-mediated knock-down in cell line models of AML with different driver lesions. We show that the characteristic reduction in proliferation and the concomitant induction of myeloid differentiation that is observed in MLL-fusion-driven leukaemia upon MYB suppression is not seen in AML cells with a complex karyotype. By performing transcriptome analysis, we demonstrate that a strong activation of MAFB expression driven by MYB ablation is restricted to MYB-dependent cells. In line with these observations, stratification of publicly available patient data reveals a reciprocal relationship between the expression of MYB and MAFB, highlighting a novel connection between those two factors in AML.

cancer biology↗

bioRxiv2020

Pituitary Tumor Transforming Gene 1 orchestrates gene regulatory variation in mouse ventral midbrain during aging

BackgroundDopaminergic neurons in the midbrain are of particular interest due to their role in diseases such as Parkinsons disease and schizophrenia. Genetic variation between individuals can affect the integrity and function of dopaminergic neurons but the DNA variants and molecular cascades modulating dopaminergic neurons and other cells types of ventral midbrain remain poorly defined. Three genetically diverse inbred mouse strains -- C57BL/6J, A/J, and DBA/2J -- differ significantly in their genomes (~7 million variants), motor and cognitive behavior, and susceptibility to neurotoxins. ResultsTo further dissect the underlying molecular networks responsible for these variable phenotypes, we generated RNA-seq and ChIP-seq data from ventral midbrains of the 3 mouse strains. We defined 1000-1200 transcripts that are differentially expressed among them. These widespread differences may be due to altered activity or expression of upstream transcription factors. Interestingly, transcription factors were significantly underrepresented among the differentially expressed genes, and only one TF, Pttg1, showed significant differences among all strains. The changes in Pttg1 expression were accompanied by consistent alterations in histone H3 lysine 4 trimethylation at Pttg1 transcription start site. The ventral midbrain transcriptome of three-month-old C57BL/6J congenic Pttg1-/- mutants was only modestly altered, but shifted towards that of A/J and DBA/2J in nine-month-old mice. Principle component analysis identified the genes underlying the transcriptome shift and deconvolution of these bulk RNA-seq changes using midbrain single cell RNA-seq data suggested that the changes were occurring in several different cell types, including neurons, oligodendrocytes, and astrocytes. ConclusionTaken together, our results show that Pttg1 contributes to gene regulatory variation between mouse strains and influences mouse midbrain transcriptome during aging.

genomics↗

bioRxiv2020

A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR

The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

molecular biology↗

bioRxiv2020

A new synuclein-transgenic mouse model for early Parkinson's reveals molecular features of preclinical disease

Understanding Parkinsons disease (PD) in particular in its earliest phases is important for diagnosis and treatment. However, human brain samples are collected post-mortem, reflecting mainly end stage disease. Because brain samples of mouse models can be collected at any stage of the disease process, they are useful to investigate PD progression. Here, we compare ventral midbrain transcriptomics profiles from -synuclein transgenic mice with a progressive, early PD-like striatum neurodegeneration across different ages using pathway, gene set and network analysis methods. Our study uncovers statistically significant altered genes across ages and between genotypes with known, suspected or unknown function in PD pathogenesis and key pathways associated with disease progression. Among those are genotype-dependent alterations associated with synaptic plasticity, neurotransmission, as well as mitochondria-related genes and dysregulation of lipid metabolism. Age-dependent changes were among others observed in neuronal and synaptic activity, calcium homeostasis, and membrane receptor signaling pathways, many of which linked to G-protein coupled receptors. Most importantly, most changes occurred before neurodegeneration was detected in this model, which points to a sequence of gene expression events that may be relevant for disease initiation and progression. It is tempting to speculate that molecular changes similar to those changes observed in our model happen in midbrain dopaminergic neurons before they start to degenerate. In other words, we believe we have uncovered molecular changes that accompany the progression from preclinical to early PD.

neuroscience↗