Biology subjects

Fernandez, A.

Publications and source records attributed to Fernandez, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Free Amino Acids Accelerate the Time-Dependent Inactivation of Rat Liver Nucleotide Pyrophosphatase / Phosphodiesterase Enpp3 elicited by EDTA

Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn2+-metallohydrolases of nucleoside-5-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (ki(ap)). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased ki(ap). The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2,3-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2,3-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2,3-cGAMP, and that Enpp1 and Enpp3 account for all the 2,3-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2,3-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3.

biochemistry↗

bioRxiv2024

AI-assisted decoding of molecular signatures essential for condensation and function of NON-PHOTOTROPIC HYPOCOTYL3

The plasma membrane-associated protein NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of plant phototropism. Blue light induces the release of NPH3 into the cytosol, where it dynamically transitions into membrane-less biomolecular condensates. Here, we combine experimental data with the power of AI-based protein structure prediction to uncover a bipartite C-terminal motif that enables self-interaction of NPH3. We further demonstrate its importance for both the association of NPH3 with the plasma membrane and the assembly of condensates in the cytosol, with a different part of the bipartite motif playing the key role in each case. However, the formation of cytosolic condensates additionally requires the co-operative action of an N-terminal NPH3 signature. We propose that NPH3 forms a crosslinked 3D network in the cytosol based on distinct molecular determinants that simultaneously self-associate. NPH3 variants incapable of condensation are non-functional, suggesting a fundamental role of NPH3 phase separation for phototropism. This structural snapshot could have direct implications for future analyses of the plant-specific NPH3/RPT2-Like protein family.

plant biology↗

bioRxiv2024

Directed evolution for cell separation in budding yeast

Natural isolates of the yeast Saccharomyces cerevisiae were evolved under a transfer protocol that selected for cell separation and against clumpy growth. Whole-genome sequencing of haploid populations revealed strong selection to deactivate AMN1, a known regulator of post-mitotic cell separation, as well as multiple instances of loss-of-function mutations on the Rim101 pathway, pointing to a previously unknown role of the Rim101 pathway in regulating cell separation. In diploid populations, we observed repeated large partial deletions of chromosome III caused by fusions of the mating type loci MAT and HMR (Hawthornes deletion) or MAT and HML (Stratherns circle). We measured the spontaneous rate of Hawthornes deletion and found that it is within an order of magnitude of previously measured rates of whole-chromosome aneuploidy. A diploid population in which neither large deletion was detected instead fixed a heterozygous nonsynonymous mutation to the calcium channel CCH1, also pointing to a novel role for this gene in relation to cell separation.

evolutionary biology↗

bioRxiv2024

A single-cell atlas of the murine pancreatic ductal tree identifies novel cell populations with potential implications in pancreas regeneration and exocrine pathogenesis.

Background and aimsPancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells, terminal, intercalated, intracalated ducts, and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. MethodsWe used scRNA-seq to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. ResultsWe have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential in vitro, including Wnt-responsive-population, ciliated-population and FLRT3+ cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples, highlighting a putative role of WNT-responsive, IFN-responsive and EMT-populations in pancreatic exocrine pathogenesis as their expression increases in chronic pancreatitis and PanIN lesions. ConclusionsIn light of our discovery of previously unidentified ductal populations, we unmask the potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis.

cell biology↗

bioRxiv2023

Adaptation and exogenous attention interact in the early visual cortex: A TMS study

Transcranial magnetic stimulation (TMS) to early visual cortex modulates the effect of adaptation and eliminates the effect of exogenous (involuntary) attention on contrast sensitivity. Here we investigated whether adaptation modulates exogenous attention under TMS to V1/V2. Observers performed an orientation discrimination task while attending to one of two stimuli, with or without adaptation. Following an attentional cue, two stimuli were presented in the stimulated region and its contralateral symmetric region. A response cue indicated the stimulus whose orientation observers had to discriminate. Without adaptation, in the distractor-stimulated condition, contrast sensitivity increased at the attended location and decreased at the unattended location via response gain-but these effects were eliminated in the target-stimulated condition. Critically, after adaptation, exogenous attention altered performance similarly in both distractor-stimulated and target-stimulated conditions. These results reveal that (1) adaptation and attention interact in the early visual cortex, and (2) adaptation shields exogenous attention from TMS effects.

neuroscience↗

bioRxiv2023

A slc38a8 mouse model of FHONDA syndrome faithfully recapitulates the visual deficits of albinism without pigmentation defects

PurposeWe aimed to generate and phenotype a mouse model of FHONDA (Foveal Hypoplasia, Optic Nerve Decussation Defects, and Anterior Segment Dysgenesis), a rare disease associated with mutations in SLC38A8 that causes severe visual alterations similar to albinism without affecting pigmentation. MethodsThe FHONDA mouse model was generated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology using an RNA guide targeting the Scl38a8 murine locus. The resulting mice were backcrossed to C57BL/6J. Melanin content was measured using spectrophotometry. Retinal cell architecture was analyzed through light and electron microscopy. Retinal projections to the brain were evaluated with anterograde labelling in embryos and adults. Visual function was assessed by electroretinography (ERG) and the optomotor test (OT). ResultsFrom numerous Slc38a8 mouse mutant alleles generated, we selected one that encodes a truncated protein (p.196Pro*, equivalent to p.199Pro* in the human protein) closely resembling a mutant allele described in patients (p.200Gln*). Slc38a8 mutant mice exhibit wild-type eye and coat pigmentation with comparable melanin contents. Subcellular abnormalities were observed in retinal pigment epithelium cells of Slc38a8 mutant mice. Anterograde labelling experiments of retinal projections in embryos and adults showed a reduction of ipsilateral fibers. Functional visual analyses revealed a decreased ERG response in scotopic conditions and a reduction of visual acuity in mutant mice measured by OT. ConclusionsSlc38a8 mutant mice recapitulate the phenotype of FHONDA patients concerning their normal pigmentation and their abnormal visual system, as observed in all types of albinism. These mice will be helpful in better understanding the pathophysiology of this genetic condition.

neuroscience↗

bioRxiv2023

Vitamin D deficiency induces erectile dysfunction: role of superoxide

Epidemiological studies suggest a relationship between vitamin D deficiency and erectile dysfunction (ED) but its causal relationship and the mechanism involved are unclear. Here we demonstrate that isolated corpora cavernosa (CC) from human donors with low vitamin D levels show reduced NO-dependent erectile function. This ED is also reproduced in vitamin D deficient rats and vitamin D receptor knockout mice in vivo and ex vivo and is associated with penile fibrosis. Vitamin D deficiency also blunts the response of CC to the phosphodiesterase 5 inhibitor sildenafil. CC from deficient rats show increased superoxide and their impaired erectile function is restored by superoxide scavengers. These results suggest that vitamin D deficiency induces ED via increased superoxide production.

pharmacology and toxicology↗

bioRxiv2023

Intrinsic control of DRG sensory neuron diversification by Pten

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by deficits in social interactions, repetitive behaviors, and hyper- or hyposensitivity to sensory stimuli. The mechanisms underlying the emergence of sensory features in ASD are not fully understood, but recent studies in rodent models highlight that these may result from differences in primary sensory neurons themselves. We examined sensory behaviors in a Pten haploinsufficient mouse model (PtenHet) for syndromic ASD and identified elevated responses to mechanical stimuli and a higher threshold to thermal responses. Transcriptomic and in vivo anatomical analysis identified alterations in subtype-specific markers of primary somatosensory neurons in PtenHet dorsal root ganglia (DRG). These defects emerge early during DRG development and involve dysregulation of multiple signaling pathways downstream of Pten. Finally, we show that mice harboring an ASD-associated mutation (PtenY69H) also show altered expression of somatosensory neuron subtype-specific markers. Together, these results show that precise levels of Pten are required for proper somatosensory development and provide insight into the molecular and cellular basis of sensory abnormalities in a model for syndromic ASD.

neuroscience↗

bioRxiv2023

De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8

The RGD (Arg-Gly-Asp)-binding integrins v{beta}6 and v{beta}8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of v{beta}6 and v{beta}8 with high selectivity. The v{beta}6 and v{beta}8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7[A] root-mean-square deviation (RMSD) to the computational design models; the designed v{beta}6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-v{beta}6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the v{beta}8 inhibitor maintains the constitutively fixed extended-closed v{beta}8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the v{beta}6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

biochemistry↗

bioRxiv2023

Modulation of functional phosphorylation sites by basic residues in the Unique domain of c-Src

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulate the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The preexisting positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or coupling various phosphosites into a functional unit. In this paper we use pH dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight on the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationship observed between the mutation induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH induced chemical shifts of the NH groups of these residues provides a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

biophysics↗

bioRxiv2023

Featural representation and internal noise underlie the eccentricity effect in contrast sensitivity

Human visual performance for basic visual dimensions (e.g., contrast sensitivity and acuity) peaks at the fovea and decreases with eccentricity. The eccentricity effect is related to the larger surface area of the visual cortex corresponding to the fovea, but it is unknown if differential feature tuning contributes to this eccentricity effect. Here, we investigated two system-level computations underlying the eccentricity effect: featural representation (tuning) and internal noise. Observers (both sexes) detected a Gabor embedded in filtered white noise which appeared at the fovea or one of four perifoveal locations. We used psychophysical reverse correlation to estimate the weights assigned by the visual system to a range of orientations and spatial frequencies (SFs) in noisy stimuli, which are conventionally interpreted as perceptual sensitivity to the corresponding features. We found higher sensitivity to task-relevant orientations and SFs at the fovea than the perifovea, and no difference in selectivity for either orientation or SF. Concurrently, we measured response consistency using a double-pass method, which allowed us to infer the level of internal noise by implementing a noisy observer model. We found lower internal noise at the fovea than perifovea. Finally, individual variability in contrast sensitivity correlated with sensitivity to and selectivity for task-relevant features as well as with internal noise. Moreover, the behavioral eccentricity effect mainly reflects the foveal advantage in orientation sensitivity compared to other computations. These findings suggest that the eccentricity effect stems from a better representation of task-relevant features and lower internal noise at the fovea than at the perifovea. SignificancePerformance in many visual tasks worsens with eccentricity. Many studies attribute this eccentricity effect to retinal and cortical factors, like higher cone density and a larger cortical surface area representing the foveal than peripheral locations. We investigated whether system-level computations for task-relevant visual features also underlie this eccentricity effect. Measuring contrast sensitivity in visual noise, we showed that the fovea better represents task-relevant orientation and spatial frequency and has lower internal noise than the perifovea, and that individual variability in these two computations correlates with that in performance. These findings reveal that both representations of these basic visual features and internal noise underlie the difference in performance with eccentricity.

neuroscience↗

bioRxiv2023

BQsupports: systematic assessment of the support and novelty of new biomedical associations

Living a Big Data era in Biomedicine, there is an unmet need to systematically assess experimental observations in the context of available information. This assessment would offer a means for an unbiased validation of the results and provide an initial estimate of the potential novelty of the findings. Here we present BQsupports, a web-based tool built upon the Bioteque biomedical descriptors that systematically analyzes and quantifies the current support to a given set of observations. The tool relies on over 1,000 distinct types of biomedical descriptors, covering over 11 different biological and chemical entities, including genes, cell lines, diseases and small molecules. By exploring hundreds of descriptors, BQsupports provide support scores for each observation across a wide variety of biomedical contexts. These scores are then aggregated to summarize the biomedical support of the assessed dataset as a whole. Finally, the BQsupports also suggests predictive features of the given dataset, which can be exploited in downstream machine learning applications. Availability and implementationThe web application and underlying data are available online (https://bqsuppports.irbbarcelona.org). Contactpatrick.aloy@irbbarcelona.org

bioinformatics↗

bioRxiv2023

Dissociable roles of human frontal eye fields and early visual cortex in presaccadic attention -- evidence from TMS

Shortly before each saccadic eye movement, presaccadic attention improves visual sensitivity at the saccade target1-5 at the expense of lowered sensitivity at non-target locations6-11. Some behavioral and neural correlates of presaccadic attention and covert attention -which likewise enhances sensitivity, but during fixation12-are similar13. This resemblance has led to the debatable13-18 notion that presaccadic and covert attention are functionally equivalent and rely on the same neural circuitry19-21. At a broad scale, oculomotor brain structures (e.g., FEF) are also modulated during covert attention22-24 - yet by distinct neuronal subpopulations25-28. Perceptual benefits of presaccadic attention rely on feedback from oculomotor structures to visual cortices29,30 (Fig. 1a); micro-stimulation of FEF in non-human primates affects activity in visual cortex31-34 and enhances visual sensitivity at the movement field of the stimulated neurons35-37. Similar feedback projections seem to exist in humans: FEF+ activation precedes occipital activation during saccade preparation38,39 and FEF TMS modulates activity in visual cortex40-42 and enhances perceived contrast in the contralateral hemifield40. We investigated presaccadic feedback in humans by applying TMS to frontal or visual areas during saccade preparation. By simultaneously measuring perceptual performance, we show the causal and differential roles of these brain regions in contralateral presaccadic benefits at the saccade target and costs at non-targets: Whereas rFEF+ stimulation reduced presaccadic costs throughout saccade preparation, V1/V2 stimulation reduced benefits only shortly before saccade onset. These effects provide causal evidence that presaccadic attention modulates perception through cortico-cortical feedback and further dissociate presaccadic and covert attention. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/529691v2_fig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1567165org.highwire.dtl.DTLVardef@2ecec9org.highwire.dtl.DTLVardef@10a00adorg.highwire.dtl.DTLVardef@1b29dfd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Background and experimental design. (a) Feedback connections originally established in non-human primates29,30,36,90,104,105 assumed to underly perceptual correlates of human presaccadic attention. FEF: Frontal eye field, SC: Superior Colliculus; (b) Response gain effect of presaccadic attention on the contrast response function: presaccadic attention scales the response by a multiplicative gain factor, resulting in an increase of the asymptotic response dmax (the maximal response achieved at high contrast)9,48. (c) Determining occipital (V1/V2) stimulation sites (Exp.1 & 2a) and stimulus placement for both experiments via phosphene mapping: Observers were stimulated laterally around the occipital pole until they perceived a phosphene (in the contralateral visual field), then drew its outline on the screen. The center of the phosphene drawings (stimulated region) and the symmetric region (not stimulated) were used for stimulus placement in the main experiment, where we applied sub-phosphene-threshold TMS using identical coil positioning. (d) Determining rFEF+ stimulation sites (Exp.2b). rFEF+ (yellow ROI) was localized on each individual observers anatomy (two exemplary observers shown here) via a probabilistic topography atlas76 and verified by anatomical landmarks (junction of the precentral and superior frontal sulcus; light gray areas)64,65,67. (e) Presaccadic orientation discrimination task. After a fixation period, a central direction cue (black line) appeared. Observers were instructed to make a saccade to the indicated target marked by placeholder dots. Note that the saccade was equally likely directed to the stimulated and to the symmetric region / hemifield. 100ms after cue onset, a tilted test Gabor patch was presented at either the saccade target (valid; 50%) or at the opposite location (invalid), randomly intermixed; a vertical Gabor was presented at the other location. Importantly, stimuli were presented during saccade preparation, i.e., while gaze was still at the screen center. After saccade offset, a response cue (white dots on placeholder) indicated the location at which the test Gabor had appeared, and observers reported its orientation. In the neutral condition (separately blocked) the cue pointed to both placeholders and observers kept fixating (supplemental Video S1 demonstrates the trials sequence of each condition). (f) Trial timeline. Observers received double-pulse TMS (50ms inter-pulse interval) locked to stimuli onset (Exp.1) or at various times during saccade preparation (0-200ms relative to cue onset; Exp.2). Whereas grating contrast was varied to measure contrast response functions in Exp.1, contrast was fixed to 46% in Exp.2. (g) Experimental conditions. The test was equally likely presented at the stimulated region (test stimulated) or in the opposite hemifield (not stimulated). Moreover, the test was equally likely presented at the saccade target (valid) or opposite of it (invalid). Note that the fixation condition (neutral) was only tested in Exp.1. C_FIG

neuroscience↗

bioRxiv2023

Immunogenicity and safety in pigs of PHH-1V, a SARS-CoV-2 RBD fusion heterodimer vaccine candidate

The continuing high global incidence of COVID-19 and the undervaccinated status of billions of persons strongly motivate the development of a new generation of efficacious vaccines. We have developed an adjuvanted vaccine candidate, PHH-1V, based on a protein comprising the receptor binding domain (RBD) of the Beta variant of SARS-CoV-2 fused in tandem with the equivalent domain of the Alpha variant, with its immunogenicity, safety and efficacy previously demonstrated in mouse models. In the present study, we immunized pigs with different doses of PHH-1V in a prime-and-boost scheme showing PHH-1V to exhibit an excellent safety profile in pigs and to produce a solid RBD-specific humoral response with neutralising antibodies to 7 distinct SARS-CoV-2 variants of concern, with the induction of a significant IFN{gamma}+ T-cell response. We conclude that PHH-1V is safe and elicits a robust immune response to SARS-CoV-2 in pigs, a large animal preclinical model.

immunology↗

bioRxiv2023

Accuracy of direct genomic values and methylation profile through genotype-by-LowPass sequencing using Nanopore technology

Genotype-by-sequencing has been proposed as an alternative to SNP genotyping arrays in genomic selection to obtain a high density of markers along the genome. It requires a low sequencing depth to be cost effective, which may increase the error at the genotype assigment. Third generation Nanopore sequencing technology offers low cost sequencing and the possibility to detect genome methylation, which provides added value to genotype-by-sequencing. The aim of this study was to evaluate the performance of genotype-by-LowPass Nanopore sequencing for estimating the direct genomic value in dairy cattle, and the possibility to obtain methylation marks simultaneously. Latest Nanopore chemistry (LSK14 and Q20) achieved a modal base calling accuracy of 99.55 %, whereas previous kit (LSK109) achieved slightly lower accuracy (99.1 %). The direct genomic value accuracy from genotype-by-Low Pass sequencing ranged between 0.79 and 0.99, depending on the trait, with a sequencing depth as low as 2x and using the latest chemistry (LSK114). Lower sequencing depth led to biased estimates, yet with high rank correlations. The LSK109 and Q20 achieved lower accuracies (0.57-0.93). More than one million high reliable methylated sites were obtained, even at low sequencing depth, located mainly in distal intergenic (87 %) and promoter (5 %) regions. This study showed that the latest Nanopore technology can be use in a LowPass sequencing framework to estimate direct genomic values with high reliability. It may provided advantages in populations with no available SNP chip, or when a large density of markers with a wide range of allele frequencies is needed. In addition, Low Pass sequencing provided with nucleotide methylation status of >1 million nucleotides at [≥] 10x, which is an added value for epigenetic studies.

genomics↗

bioRxiv2023

Cross-β helical filaments of Tau and TMEM106B in Gray and White Matter of Multiple System Tauopathy with presenile Dementia

BackgroundThe Microtubule-Associated Protein Tau (MAPT) is one of the proteins that are central to neurodegenerative diseases. The nature of intracellular tau aggregates is determined by the cell types whether neuronal or glial, the participating tau isoforms, and the structure of the amyloid filament. The transmembrane protein 106B (TMEM106B) has recently emerged as another significant player in neurodegeneration and aging. In the central nervous system, the composition of the gray and white matter differs considerably. The gray matter consists of nerve cell bodies, dendrites, unmyelinated axons, synaptic terminals, astrocytes, oligodendrocytes (satellite cells) and microglia. The white matter differs from the gray for the presence of axonal tracts as the only neuronal component and for the absence of nerve cell bodies, dendrites and synaptic terminals. Cryogenic electron microscopy (cryo-EM) studies have unveiled the structure of tau and TMEM106B, from the cerebral cortex, in several neurodegenerative diseases; however, whether tau and TMEM106B filaments from the gray and white matter share a common fold requires additional investigation. MethodsWe isolated tau and TMEM106B from the cerebral cortex and white matter of the frontal lobes of two individuals affected by multiple system tauopathy with presenile dementia (MSTD), a disease caused by the MAPT intron 10 mutation +3. We used immunostaining, biochemical, genetics and cryo-EM methods to characterize tau and TMEM106B. ResultsWe determined that tau filaments in the gray and the white matter of MSTD individuals can induce tau aggregation and have identical AGD type 2 folds. TMEM106B amyloid filaments were also found in the gray and white matter of MSTD; the filament folds were identical in the two anatomical regions. ConclusionsOur findings show for the first time that in MSTD two types of amyloid filaments extracted from the gray matter have identical folds to those extracted from the white matter. Whether in this genetic disorder there is a relationship in the pathogenesis of the tau and TMEM106B filaments, remains to be determined. Furthermore, additional studies are needed for other proteins and other neurodegenerative diseases to establish whether filaments extracted from the gray and white matter would have identical folds.

neuroscience↗

bioRxiv2022

Preclinical efficacy, safety, and immunogenicity of PHH-1V, a second-generation COVID-19 vaccine, in non-human primates

SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programmes, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG binding and neutralising antibodies against several SARS-CoV-2 variants, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.

immunology↗

bioRxiv2022

Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering

Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci, and CRISPR associated (Cas) proteins of the prokaryotic CRISPR-Cas systems, form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most used for genome engineering, at least in part due to its catalytic robustness and the requirement of an exceptionally short motif (5-NGG-3 PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are demanded. Here we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5-NGG-3 PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.

molecular biology↗