Biology subjects

Martinez, A.

Publications and source records attributed to Martinez, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Mobile microscopy and telemedicine platform assisted by deep learning for quantification of Trichuris trichiura infection.

Soil-transmitted helminths (STH) are the most prevalent pathogens among the group of neglected tropical diseases (NTDs). Kato-Katz technique is the diagnosis method recommended by WHO and although is generally more sensitive than other microscopic methods in high transmission settings, it often presents a decreased sensitivity in low transmission settings and it is labour intensive. Digitizing the samples could provide a solution which allows to store the samples in a digital database and perform remote analysis. Artificial intelligence methods based on digitized samples can support diagnostics efforts by support diagnostics efforts by performing an automatic and objective quantification of disease infection. In this work, we propose an end-to-end pipeline for microscopy image digitization and automatic analysis of digitized images of soil-transmitted helminths. Our solution includes (1) a digitalization system based on a mobile app that digitizes the microscope samples using a low-cost 3D-printed microscope adapter, (2) a telemedicine platform for remote analysis and labelling and (3) novel deep learning algorithms for automatic assessment and quantification of parasitological infection of STH. This work has been evaluated by comparing the STH quantification using both a manual remote analysis based on the digitized images and the AI-assisted quantification against the reference method based on conventional microscopy. The deep learning algorithm has been trained and tested on 41 slides of stool samples containing 949 eggs from 6 different subjects using a cross-validation strategy obtaining a mean precision of 98,44% and mean recall of 80,94%. The results also proved the potential of generalization capability of the method at identifying different types of helminth eggs. In conclusion, this work has presented a comprehensive pipeline using smartphone-based microscopy integrated with a telemedicine platform for automatic image analysis and quantification of STH infection using artificial intelligence models.

microbiology↗

bioRxiv2020

PKA drives paracrine crisis and WNT4-dependent testis tumor in Carney complex

Large Cell Calcifying Sertoli Cell Tumors (LCCSCTs) are among the most frequent lesions occurring in Carney complex (CNC) male patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A leading to unrepressed PKA activity, the LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the CNC testis lesion induction. We demonstrate that Prkar1a mutation is required in both stromal and Sertoli cells for the occurrence of LCCSCT. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to understand the human LCCSCT pathogenesis and demonstrated unprecedented PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in mouse model and human CNC testes.

pathology↗

bioRxiv2020

Identification of NPC1 as a novel SARS-CoV-2 intracellular target

Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol trafficking, which is crucial in the Ebola virus (EBOV) cycle. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cell by binding of the viral spike (S) protein to the ACE2 receptor. This requires S-protein processing either by the surface transmembrane serine protease TMPRSS2 for plasma membrane fusion or cathepsin L for endosomal entry. Additional host factors are required for viral fusion at endosomes. Here, we report a novel interaction of the SARS-CoV-2 nucleoprotein (N) with the cholesterol transporter NPC1. Moreover, small molecules interfering with NPC1 that inhibit EBOV entry, also inhibited human coronavirus. Our findings suggest an important role for NPC1 in SARS-CoV-2 infection, a common strategy shared with EBOV, and a potential therapeutic target to fight against COVID-19.

microbiology↗

bioRxiv2020

Macrocybin, a mushroom natural triglyceride, reduces tumor growth in vitro and in vivo through caveolin-mediated interference with the actin cytoskeleton

Mushrooms have been used for millennia as cancer remedies. Our goal was to screen several species from the rain forest of Costa Rica looking for new antitumor molecules. Mushroom extracts were screened using two human cell lines: A549 (lung adenocarcinoma) and NL20 (immortalized normal lung epithelium). Extracts able to kill tumor cells while preserving nontumor cells were considered "anticancer". The mushroom with better properties was Macrocybe titans. Positive extracts were fractionated further and tested for biological activity on the cell lines. The chemical structure of the active compound was partially elucidated through nuclear magnetic resonance, mass spectrometry, and other ancillary techniques. Chemical analysis showed that the active molecule was a triglyceride containing oleic acid, palmitic acid, and a more complex fatty acid with 2 double bonds. Synthesis of all possible triglycerides and biological testing identified the natural compound, which was named Macrocybin. A xenograft study showed that Macrocybin significantly reduces A549 tumor growth. In addition, Macrocybin treatment resulted in the upregulation of Caveolin-1 expression and the disassembly of the actin cytoskeleton in tumor cells (but not in normal cells). In conclusion, we have shown that Macrocybin constitutes a new biologically active compound that may be taken into consideration for cancer treatment.

cancer biology↗

bioRxiv2020

Host-directed FDA-approved drugs with antiviral activity against SARS-CoV-2 identified by hierarchical in silico/in vitro screening methods

The unprecedent situation generated by the COVID-19 global emergency prompted us to actively work to fight against this pandemic by searching for repurposable agents among FDA approved drugs to shed light into immediate opportunities for the treatment of COVID-19 patients. In the attempt to proceed toward a proper rationalization of the search for new antivirals among approved drugs, we carried out a hierarchical in silico/in vitro protocol which successfully combines virtual and biological screening to speed up the identification of host-directed therapies against COVID-19 in an effective way. To this end a multi-target virtual screening approach focused on host-based targets related to viral entry followed by the experimental evaluation of the antiviral activity of selected compounds has been carried out. As a result, five different potentially repurposable drugs interfering with viral entry, cepharantine, clofazimine, metergoline, imatinib and efloxate, have been identified.

microbiology↗

bioRxiv2020

Evolutionary analysis of SARS-CoV-2 spike protein for its different clades

ObjectiveThe spike protein of SARS-CoV-2 has become the main target for antiviral and vaccine development. Despite its relevance, there is scarce information about its evolutionary traces. The aim of this study was to investigate the diversification patterns of the spike for each clade of SARS-CoV-2 through different approaches. MethodsTwo thousand and one hundred sequences representing the seven clades of the SARS-CoV-2 were included. Patterns of genetic diversifications and nucleotide evolutionary rate were estimated for the spike genomic region. ResultsThe haplotype networks showed a star shape, where multiple haplotypes with few nucleotide differences diverge from a common ancestor. Four hundred seventy nine different haplotypes were defined in the seven analyzed clades. The main haplotype, named Hap-1, was the most frequent for clades G (54%), GH (54%), and GR (56%) and a different haplotype (named Hap-252) was the most important for clades L (63.3%), O (39.7%), S (51.7%), and V (70%). The evolutionary rate for the spike protein was estimated as 1.08 x 10-3 nucleotide substitutions/site/year. Moreover, the nucleotide evolutionary rate after nine months of pandemic was similar for each clade. ConclusionsIn conclusion, the present evolutionary analysis is relevant since the spike protein of SARS-CoV-2 is the target for most therapeutic candidates; besides, changes in this protein could have consequences on viral transmission, response to antivirals and efficacy of vaccines. Moreover, the evolutionary characterization of clades improves knowledge of SARS-CoV-2 and deserves to be assessed in more detail since re-infection by different phylogenetic clades has been reported.

evolutionary biology↗

bioRxiv2020

Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the unfolded protein response (UPR) in models of neurodegeneration

Alteration to endoplasmic reticulum (ER) proteostasis is observed on a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR-target genes. Here, we designed an ATF6f-XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has stronger an effect in reducing the abnormal aggregation of mutant huntingtin and alpha-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinsons and Huntingtons disease. These results support the concept where directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

neuroscience↗

bioRxiv2020

Dynamics of central remyelination and treatment evolution in a model of Multiple Sclerosis with Optic Coherence Tomography

The need for remyelinating drugs is essential for healing important diseases such as multiple sclerosis (MS). One of the reasons for the lack of this class of therapies is the impossibility to follow remyelination in vivo, which is of utmost importance to perform good clinical trials. Here, we show how the optical coherence tomography (OCT), a cheap and non-invasive technique commonly used in ophthalmology, may be used to follow remyelination in vivo in MS patients. Our pioneer study validates the study of myelin/remyelination in the optic nerve using OCT and reflects what is occurring in non accessible CNS structures, like the spinal cord. For this study we used the oral bioavailable small molecule VP3.15, confirming its therapeutical potential as neuroprotective, antinflammatory and remyelinating drug for MS. Altogether, our present results confirm the usefulness of OCT to monitor the effectivity of remyelinating therapies in vivo and underscore the relevance of VP3.15 as potential disease modifying drug for MS therapy.

neuroscience↗

bioRxiv2020

Habitat differences filter functional diversity of low dispersive microscopic animals

O_LIWhereas the study of patterns of distribution of microscopic animals has long been dominated by the ubiquity paradigm, we are starting to appreciate that microscopic animals are not as widespread as previously thought and that habitat preferences may have a strong role in structuring their patterns of occurrence. However, we still ignore to what extent and through which mechanisms the environment selects for specific communities or traits in microscopic animals. This gap is partly due to the lack of data on the relevant traits of many species, and partly because measuring environmental variables at an appropriate resolution may be problematic. C_LIO_LIWe here overcome both issues by analysing the functional space of marine mite communities living in a sea-grass (Posidonia oceanica) meadow across two habitats: the leaves and the matte. The strictly benthic lifestyle and the conserved morphology of mites allow for unambiguous characterization of their functional traits, while the discrete nature of the two habitats alleviates the uncertainty in their ecological characterization. C_LIO_LIOur results show that habitat filters the distribution of certain traits favouring a higher diversity, dispersion, and evenness of functional traits in the matte than in the leaves. We further observed temporal variations in the functional diversity of communities, potentially following the seasonal renovation and decay of seagrass leaves. However, in spite of the stark ecological differences between the two habitats and across seasons, the filtering effect is partial and affects mostly relative species abundances. C_LIO_LIWe conclude that in other microscopic organisms, habitat filtering might appear even more subtle especially if they are capable of long distance dispersal or occur in ecological systems where environmental variables vary continuously or fluctuate through time. Our study therefore emphasises the need of moving from a merely taxonomical toward a functional view of ecological studies of microscopic organisms if we want to achieve a mechanistic understanding of their habitat and distribution patterns. C_LI Data availability statementRaw data and R script to generate the analyses will be deposited in a public repository upon acceptance.

ecology↗

bioRxiv2020

Specialized terminology limits the reach of new scientific knowledge

Words are the building blocks of science. As our understanding of the world progresses, scientific disciplines naturally enrich their specialized vocabulary (jargon). However, in the era of interdisciplinarity, the use of jargon may hinder effective communication amongst scientists that do not share a common linguistic background. The question of how jargon limits the transmission of scientific knowledge has long been debated, but rarely addressed quantitatively. We explored the relationship between the use of jargon and citations using 21,486 articles focusing on cave research, a multidisciplinary field particularly prone to terminological specialization and where linguistic disagreement among peers is frequent. We demonstrate that the use of jargon in the title and abstract significantly reduces the number of citations a paper receives. Given that these elements are the hook to readers, we urge scientists to restrict jargon to sections of the paper where its use is unavoidable.

scientific communication and education↗

bioRxiv2020

A novel USP30 inhibitor recapitulates genetic loss of USP30 and sets the trigger for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinsons Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

cell biology↗

bioRxiv2020

Scientometric correlates of high-quality reference lists in ecological papers

It is said that the quality of a scientific publication is as good as the science it cites, but the properties of high-quality reference lists have never been numerically quantified. We examined seven numerical characteristics of reference lists of 50,878 primary research articles published in 17 ecological journals between 1997 and 2017. Over this 20-years period, there have been significant changes in reference lists’ properties. On average, more recent ecological papers have longer reference lists, cite more high Impact Factor papers, and fewer non-journal publications. Furthermore, we show that highly cited papers across the ecology literature have longer reference lists, cite more recent and impactful papers, and account for more self-citations. Conversely, the proportion of ‘classic’ papers and non-journal publications cited, as well as the temporal range of the reference list, have no significant influence on articles’ citations. From this analysis, we distill a recipe for crafting impactful reference lists.View Full Text

scientific communication and education↗

bioRxiv2020

Phylogeny and distribution of Y-chromosomal haplotypes in domestic, ancient and wild goats

The male-specific part of the Y-chromosome is in mammalian and many other species the longest haplotype that is inherited without recombination. By its paternal transmission it has a small effective population size in species with dominant males. In several species, Y-chromosomal haplotypes are sensitive markers of population history and introgression. Previous studies have identified in domestic goats four major Y-chromosomal haplotypes Y1A, Y1B, Y2A and Y2B with a marked geographic differentiation and several regional variants. In this study we used published whole-genome sequences of 70 male goats from 16 modern breeds, 11 ancient-DNA samples and 29 samples from seven wild goat species. We identified single-copy male-specific SNPs in four scaffolds, containing SRY, ZFY, DBY with SSX3Y and UTY, and USP9Y with UMN2001, respectively. Phylogenetic analyses indicated haplogroups corresponding to the haplotypes Y1B, Y2A and Y2B, respectively, but Y1A was split into Y1AA and Y1AB. All haplogroups were detected in ancient DNA samples from southeast Europe and, with the exception of Y1AB, in the bezoar goat, which is the wild ancestor of the domestic goats. Combining these data with those of previous studies and with genotypes obtained by Sanger sequencing or the KASP assay yielded haplogroup distributions for 132 domestic breeds or populations. The phylogeographic differentiation indicated paternal population bottlenecks on all three continents. This possibly occurred during the Neolithic introductions of domestic goats to those continents with a particularly strong influence in Europe along the Danubian route. This study illustrates the power of the Y-chromosomal haplotype for the reconstructing the history of mammalian species with a wide geographic range.

genetics↗

bioRxiv2019

Failure to engage the TPJ-pSTS during naturalistic scene processing in schizophrenia

The ability to search for and detect social cues, such as facial expressions of emotion, is critical to the understanding of complex dynamic social situations. This ability involves the coordinated actions of multiple cognitive domains, including face-emotion processing, mentalization, and visual attention. Individuals with schizophrenia are generally impaired in social cognition, and have been shown to have deficits in all of these domains. However, the whether the neural substrates of these impairments are shared or separate remains unclear. One candidate region for a shared substrate is the right temporoparietal junction/posterior superior temporal sulcus (TPJ-pSTS), which contains areas belonging to all of the cortical networks underlying these domains. Here we use functional MRI to examine differences in cortical activity evoked by a naturalistic movie, and link these results to impaired visual scanning and social cognition. 27 schizophrenia participants and 21 healthy controls watched a 15-minute clip of the movie \"The Good, the Bad, and the Ugly\" while high resolution multiband BOLD-fMRI activity was recorded. Inter-subject correlation was used to measure the evoked activity. BOLD-fMRI activity was also correlated with motion content in the movie, with the average activity in other cortical areas, and with frequency of saccades made during the movie. Visual scanning performance was measured in a separate behavioral experiment, and social cognition measured by The Awareness of Social Inference Test (TASIT). Contrasting the groups revealed that the TPJ-pSTS has the largest engagement deficit in both cortical hemispheres in schizophrenia patients versus healthy controls. Follow-up analyses find that brain activity in this region is less correlated with the motion content of the movie, that this region is abnormally synchronized to the other cortical areas involved in the cognitive domains underlying visual scanning of social scenes, and that activity this region is less correlated with the saccades made during the movie. Lastly, schizophrenia participant visual scanning performance of this clip was impaired compared to healthy controls, and correlated across the two groups with social cognition. These results indicate that the TPJ-pSTS plays less of an integral role in the coordination of face-emotion processing, mentalization, and visual attention in schizophrenia participants versus healthy controls. This functional deficit then impacts the visual scanning of a complex dynamic visual scenes, which in turn affects the comprehension of that scene. These findings indicating that the TPJ-pSTS is potentially the shared substrate for all of these deficits will lead to new treatments targeting this region to improve social cognition in individuals with schizophrenia.

neuroscience↗

bioRxiv2019

Cognitive and non-cognitive outcomes associated with student engagement in a novel brain mapping and connectomics course-based undergraduate research experience

Course-based undergraduate research experiences (CUREs) engage emerging scholars in the authentic process of scientific discovery, and foster their development of content knowledge, motivation, and persistence in the science, technology, engineering, and mathematics (STEM) disciplines. Importantly, authentic research courses simultaneously offer investigators unique access to an extended population of students who receive education and mentoring in conducting scientifically relevant investigations and who are thus able to contribute effort toward big-data projects. While this paradigm benefits fields in neuroscience, such as atlas-based brain mapping of nerve cells at the tissue level, there are few documented cases of such laboratory courses offered in the domain.\n\nHere, we describe a curriculum designed to address this deficit, evaluate the scientific merit of novel student-produced brainatlasmapsofimmunohistochemically-identifiednervecellpopulations for the rat brain, and assess shifts in science identity, attitudes, and science communication skills of students engaged in the introductory-level Brain Mapping and Connectomics (BM&C) CURE. BM&C students reported gains in research and science process skills following participation in the course. Furthermore, BM&C students experienced a greater sense of science identity, including a greater likelihood to discuss course activities with non-class members compared to their non-CURE counterparts. Importantly, evaluation of student-generated brain atlas maps indicated that the course enabled students to produce scientifically valid products and make new discoveries to advance the field of neuroanatomy. Together, these findings support the efficacy of the BM&C course in addressing the relatively esoteric demands of chemoarchitectural brain mapping.

scientific communication and education↗

bioRxiv2019

Early-Career Coordinated Distributed Experiments: Empowerment Through Collaboration

Coordinated distributed experiments (CDEs) enable the study of large-scale ecological patterns in geographically dispersed areas, while simultaneously providing broad academic and personal benefits for the participants. However, the effective involvement of early-career researchers (ECRs) presents major challenges. Here, we analyze the benefits and challenges of the first CDE exclusively led and conducted by ECRs (i.e. ECR-CDE), which sets a baseline for similar CDEs, and we provide recommendations for successful CDE execution. ECR-CDEs achieve most of the outcomes identified in conventional CDEs as well as extensive benefits for the young cohort of researchers, including: (i) receiving scientific credit, (ii) peer-training in new concepts and methods, (iii) developing leadership and communication skills, (iv) promoting a peer network among ECRs, and (v) building on individual engagement and independence. We also discuss the challenges of ECR-CDEs, which are mainly derived from the lack of independence and instability of the participants, and we suggest mechanisms to address them, such as resource re-allocation and communication strategies.

scientific communication and education↗

bioRxiv2019

Molecular determinants of Arc oligomerization and formation of virus-like capsids

Expression of activity-regulated cytoskeleton-associated protein (Arc) is critical for long-term synaptic plasticity, memory formation, and cognitive flexibility. The ability of Arc to self-associate and form virus-like capsid structures implies functionally distinct oligomeric states. However, the molecular mechanism of Arc oligomerization is unknown. Here, we identified a 28-amino-acid region necessary and sufficient for Arc oligomerization. This oligomerization region is located within the second coil of a predicted anti-parallel coiled-coil in the N-terminal domain (NTD). Using alanine scanning mutagenesis, we found a 7-amino-acid motif critical for oligomerization and Arc-mediated transferrin endocytosis in HEK cells. Intermolecular fluorescence lifetime imaging in hippocampal neurons confirmed self-association mediated by the motif. To quantify oligomeric size, we performed a single-molecule photobleaching analysis of purified Arc wild-type and mutant. This analysis revealed a critical role for the NTD motif in the formation of higher-order Arc oligomers (30-170 molecules). Moreover, assembly of higher-order wild-type Arc oligomers was significantly enhanced by addition of GFP RNA. Purified wild-type Arc formed virus-like capsids, as visualized by negative-stain EM, and was estimated by light scattering analysis to contain 40-55 Arc units. In contrast, mutant Arc formed a homogenous dimer population as demonstrated by single-molecule TIRF imaging, size-exclusion chromatography with multi-angle light scattering analysis, small-angle X-ray scattering analysis, and single-particle 3D EM reconstruction. Thus, the dimer appears to be the basic building block for assembly. Herein, we show that the NTD motif is essential for higher-order Arc oligomerization, assembly of virus-like capsid particles, and facilitation of oligomerization by exogenous RNA. SIGNIFICANCEArc protein is rapidly expressed in neurons in response to synaptic activity and plays critical roles in synaptic plasticity, postnatal cortical developmental, and memory. Arc has diverse molecular functions, which may be related to distinct oligomeric states of the protein. Arc has homology to retroviral Gag protein and self-assembles into retrovirus-like capsid structures that are capable of intercellular transfer of RNA. Here, we identified a motif in the N-terminal coiled-coil domain of mammalian Arc that mediates higher-order oligomerization and formation of virus-like capsids. The basic building block is the Arc dimer and exogenous RNA facilitates further assembly. The identified molecular determinants of Arc oligomerization will help to elucidate the functional modalities of Arc in the mammalian brain.

biochemistry↗

bioRxiv2019

The structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin

Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone. Here we present the first structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the pre-catalytic state. Crystal structures, solved at 3.18[A] resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH4. BH4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic FeII. Upon BH4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH4. Importantly, BH4 binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH4 from the pre-catalytic towards the active conformation, a molecular mechanism that was supported by site directed mutagenesis and targeted MD simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations. Significance StatementThe present crystal structure of phenylalanine hydroxylase (PAH) provides the first and long-awaited 3D-structure of the full-length human PAH, both unbound and complexed with the tetrahydrobiopterin (BH4) cofactor. The BH4-bound state is physiologically relevant, keeping PAH stable and in a pre-catalytic state at low L-Phe concentration. Furthermore, a synthetic form of BH4 (Kuvan(R)) is the only drug-based therapy for a subset of phenylketonuria patients. We found two tetramer conformations in the same crystal, depending on the active site occupation by BH4, which aid to understand the stabilization by BH4 and the allosteric mechanisms in PAH. The structure also reveals the increased mobility of human-compared with rat PAH, in line with an increased predisposition to disease-associated mutations in human. Classification: Biological Science (major), biochemistry (minor) The authors declare no conflict of interest. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 6HYC and 6HPO). The EM maps have been deposited in the EMDB with accession s EMD-4605 This article contains supporting information.

biochemistry↗