Prospective Multicenter Validation of a Simple Blood Test for the Diagnosis of Glut1 Deficiency Syndrome.

BACKGROUND AND OBJECTIVE: GLUT1 deficiency syndrome (Glut1DS) is a treatable neurometabolic disease that causes a wide range of neurologic symptoms in children and adults. However, its diagnosis relies on an invasive test, that is, a lumbar puncture (LP) to measure glycorrhachia, and sometimes complex molecular analyses of the SLC2A1 gene. This procedure limits the number of patients able to receive the standard of care. We wished to validate the diagnostic performance of METAglut1, a simple blood test that quantifies GLUT1 on the erythrocyte surface. METHODS: We performed a multicenter validation study in France, involving 33 centers. We studied 2 patient cohorts: a prospective cohort consisting of patients with a clinical suspicion of Glut1DS explored through the reference strategy, that is, LP and analyses of the SLC2A1 gene, and a retrospective cohort that included patients previously diagnosed with Glut1DS. All patients were blind-tested with METAglut1. RESULTS: We analyzed 428 patients in the prospective cohort, including 15 patients newly diagnosed with Glut1DS, and 67 patients in the retrospective cohort. METAglut1 was 80% sensitive and >99% specific for the diagnosis of Glut1DS. Concordance analyses showed a substantial agreement between METAglut1 and glycorrhachia. In the prospective cohort, the positive predictive value of METAglut1 was slightly higher than that of glycorrhachia. METAglut1 succeeded to identify patients with Glut1DS with SCL2A1 mosaicism and variants of unknown significance. DISCUSSION: METAglut1 is an easily performed, robust, and noninvasive diagnostic test for the diagnosis of Glut1DS, which allows wide screening of children and adults, including those with atypical forms of this treatable condition. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that a positive METAglut1 test accurately distinguishes patients with suspected GLUT1 deficiency syndrome from other neurologic syndromes as compared with invasive and genetic testing.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

CRISPR knockout (KO) screens have identified host factors regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Here, we conducted a meta-analysis of these screens, which showed a high level of cell-type specificity of the identified hits, highlighting the necessity of additional models to uncover the full landscape of host factors. Thus, we performed genome-wide KO and activation screens in Calu-3 lung cells and KO screens in Caco-2 colorectal cells, followed by secondary screens in four human cell lines. This revealed host-dependency factors, including AP1G1 adaptin and ATP8B1 flippase, as well as inhibitors, including mucins. Interestingly, some of the identified genes also modulate Middle East respiratory syndrome coronavirus (MERS-CoV) and seasonal human coronavirus (HCoV) (HCoV-NL63 and HCoV-229E) replication. Moreover, most genes had an impact on viral entry, with AP1G1 likely regulating TMPRSS2 activity at the plasma membrane. These results demonstrate the value of multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential targets for therapeutic interventions.

Identification of Copper Transporter 1 as a Receptor for Feline Endogenous Retrovirus ERV-DC14.

Vertebrates harbor hundreds of endogenous retroviral (ERV) sequences in their genomes, which are considered signs of past infections that occurred during evolution. On rare occasions, ERV genes like env are maintained and coopted by hosts for physiological functions, but they also participate in recombination events with exogenous retroviruses to generate rearranged viruses with novel tropisms. In domestic cats, feline leukemia virus type D (FeLV-D) has been described as a recombinant virus between the infectious FeLV-A and likely the ERV-DC14 env gene that resulted in an extended tropism due to the usage of a new uncharacterized retroviral receptor. Here, we report the identification of SLC31A1 encoding the copper transporter 1 (CTR1) as a susceptibility gene for ERV-DC14 infection. Expression of human CTR1 into nonpermissive cells was sufficient to confer sensitivity to ERV-DC14 pseudotype infection and to increase the binding of an ERV-DC14 Env ligand. Moreover, inactivation of CTR1 by genome editing or cell surface downmodulation of CTR1 by a high dose of copper dramatically decreased ERV-DC14 infection and binding, while magnesium treatment had no effect. We also investigated the role of CTR1 in the nonpermissivity of feline and hamster cells. While feline CTR1 was fully functional for ERV-DC14, we found that binding was strongly reduced upon treatment with conditioned medium of feline cells, suggesting that the observed resistance to infection was a consequence of CTR1 saturation. In contrast, hamster CTR1 was inactive due to the presence of a N-linked glycosylation site at position 27, which is absent in the human ortholog. These results provide evidence that CTR1 is a receptor for ERV-DC14. Along with chimpanzee endogenous retrovirus type 2, ERV-DC14 is the second family of endogenous retrovirus known to have used CTR1 during past infections of vertebrates. IMPORTANCE Receptor usage is an important determinant of diseases induced by pathogenic retroviruses. In the case of feline leukemia viruses, three subgroups (A, B, and C) based on their ability to recognize different cell host receptors, respectively, the thiamine transporter THTR1, the phosphate transporter PiT1, and the heme exporter FLVCR1, are associated with distinct feline diseases. FeLV-A is horizontally transmitted and found in all naturally infected cats, while FeLV-B and FeLV-C have emerged from FeLV-A, respectively, by recombination with endogenous retroviral env sequences or by mutations in the FeLV-A env gene, both leading to a switch in receptor usage and in subsequent in vivo tropism. Here, we set up a genetic screen to identify the retroviral receptor of ERV-DC14, a feline endogenous provirus whose env gene has been captured by infectious FeLV-A to give rise to FeLV-D in a process similar to FeLV-B. Our results reveal that the copper transporter CTR1 was such a receptor and provide new insights into the acquisition of an expanded tropism by FeLV-D.

[Nomophobia and the connection anxiety dimensions: a cross-sectional study among students and nurses.] / Nomofobia e le dimensioni dell'ansia da connessione: uno studio cross-sectional tra studenti e infermieri.

INTRODUCTION: Nomophobia, a form of behavioral addiction, is uncontrolled, obsessive fear of being disconnected from the mobile phone network. Excessive use of smartphone during care, a source of errors and distractions, is among the top ten risks related to the use of technologies in healthcare. The study aims to investigate the presence of nomophobia among nurses and students of the Degree in Nursing and any differences based on gender, age group and seniority. METHODS: A cross-sectional quantitative descriptive study conducted at Università Politecnica delle Marche and Azienda Ospedali Riuniti Marche Nord departments; the NMP-Q questionnaire (Nomophobia Questionnaire) was administered to a non-probabilistic sample of nurses and nursing students. The data were analyzed using descriptive and inferential non-parametric statistical measurements. RESULTS: A total of 280 questionnaires were returned, 141 for students and 139 for nurses. The mean total score for students and nurses was moderate and overlapping (M=79.9 vs. 79.3, p>0.05), with no difference by gender and course year (p>0.05); in the sample of nurses score is higher in men (M=89.8 vs. 76.0, p=0.037), under 30 (M=72.0) and over 50 years (M=83.1, p=0.021). The comparison between the two groups shows higher percentages of moderate nomophobic grade among students (+17pp), mild (+8.2 pp) and severe (+7.4 pp) among nurses, without significant gender differences. CONCLUSIONS: In the perspective of proper risk management, the level of nomophobia found in both groups should not be underestimated. This study highlights the importance of monitoring the phenomenon, adopting information and awareness-raising policies aimed at healthcare personnel as early as university training: distracting factors associated with the over-use of smartphones in the workplace make nurses, particularly newly graduates and with less expertise, more vulnerable and more exposed to the risk of error.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses.

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

[Intentional roundings in general medical practice: the experience of the Azienda Ospedali Riuniti Marche Nord (Italy).] / Intentional rounding in medicina generale: l'esperienza dell'Azienda Ospedali Riuniti Marche Nord.

INTRODUCTION: The "intentional roundings" are planned rounds, conducted at regular intervals by nursing staff to anticipate care, comfort, hospitality and psychological needs of hospitalized users. These purposes are achieved with a structured way to make observations and carry out activities for well-being and patient safety, documenting what was done with a structured ad hoc form. In the United Kingdom, as well as in the USA, intentional rounding is an established model of care that improve the safety of provided care, to reduce the occurrence of preventable events, address proactively basic caring needs, and that increase users and staff satisfaction. OBJECTIVE: Implementing in a medical pilot unit the care model named "intentional rounding". METHODS: The care team carried intentional rounds every two hours, in a systematic and documented manner. All patients received admission informations about the organizational method and were invited to participate by completing a satisfaction questionnaire at discharge. At the end of the experimental period organizational impact have been investigated, specifically users and hospital staff satisfaction. RESULTS: About privacy, courtesy, nurse support and quality of care provided, level of satisfaction of patients and caregivers reached high percentage of approval (90-99%). Nurses and other healthcare personnel have perceived they delivered either a safer and most satisfying healthcare (90-94%) as well as inter- and intra-professional dynamics communications (95%). Value of roundings have been less appreciated concerning the optimization about the worktime management (79%). Calls to the bell have had a notable change of their reasons unlikely about the quantity. CONCLUSIONS: Intentional roundings affects very positively to users satisfaction level and to the perception of the quality of care provided; the care team, though express the need to make some changes for a real implementation, recognize the proactivity of intentional roundings as an added value.

XPR1 mutations are a rare cause of primary familial brain calcification.

Mutations in XPR1, a gene encoding an inorganic phosphate exporter, have recently been identified in patients with primary familial brain calcification (PFBC). Using Sanger sequencing, we screened XPR1 in 18 unrelated patients with PFBC and no SLC20A2, PDGFB, or PDGFRB mutation. XPR1 variants were tested in an in vitro physiological complementation assay and patient blood cells were assessed ex vivo for phosphate export. We identified a novel c.260T > C, p.(Leu87Pro) XPR1 variant in a 41-year-old man complaining of micrographia and dysarthria and demonstrating mild parkinsonism, cerebellar ataxia and executive dysfunction. Brain (123)I-Ioflupane scintigraphy showed marked dopaminergic neuron loss. Peripheral blood cells from the patient exhibited decreased phosphate export. XPR1 in which we introduced the mutation was not detectable at the cell surface and did not lead to phosphate export. These results confirm that loss of XPR1-mediated phosphate export function causes PFBC, occurring in less than 8 % of cases negative for the other genes, and may be responsible for parkinsonism.

Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export.

Primary familial brain calcification (PFBC) is a neurological disease characterized by calcium phosphate deposits in the basal ganglia and other brain regions and has thus far been associated with SLC20A2, PDGFB or PDGFRB mutations. We identified in multiple families with PFBC mutations in XPR1, a gene encoding a retroviral receptor with phosphate export function. These mutations alter phosphate export, implicating XPR1 and phosphate homeostasis in PFBC.

Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans.

Inorganic phosphate uptake is a universal function accomplished by transporters that are present across the living world. In contrast, no phosphate exporter has ever been identified in metazoans. Here, we show that depletion of XPR1, a multipass membrane molecule initially identified as the cell-surface receptor for xenotropic and polytropic murine leukemia retroviruses (X- and P-MLV), induced a decrease in phosphate export and that reintroduction of various XPR1 proteins, from fruit fly to human, rescued this defect. Inhibition of phosphate export was also obtained with a soluble ligand generated from the envelope-receptor-binding domain of X-MLV in all human cell lines tested, as well as in diverse stem cells and epithelial cells derived from renal proximal tubules, the main site of phosphate homeostasis regulation. These results provide new insights on phosphate export in metazoans and the role of Xpr1 in this function.

Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa.

During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination.

FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei.

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.

Clonal conditional mutagenesis in malaria parasites.

We describe here an efficient method for conditional gene inactivation in malaria parasites that uses the Flp/FRT site-specific recombination system of yeast. The method, developed in Plasmodium berghei, consists of inserting FRT sites in the chromosomal locus of interest in a parasite clone expressing the Flp recombinase via a developmental stage-specific promoter. Using promoters active in mosquito midgut sporozoites or salivary gland sporozoites to drive expression of Flp or its thermolabile variant, FlpL, we show that excision of the DNA flanked by FRT sites occurs efficiently at the stage of interest and at undetectable levels in prior stages. We applied this technique to conditionally silence MSP1, a gene essential for merozoite invasion of erythrocytes. Silencing MSP1 in sporozoites impaired subsequent merozoite formation in the liver. Therefore, MSP1 plays a dual role in the parasite life cycle, acting both in liver and erythrocytic parasite stages.

Host cell traversal is important for progression of the malaria parasite through the dermis to the liver.

The malaria sporozoite, the parasite stage transmitted by the mosquito, is delivered into the dermis and differentiates in the liver. Motile sporozoites can invade host cells by disrupting their plasma membrane and migrating through them (termed cell traversal), or by forming a parasite-cell junction and settling inside an intracellular vacuole (termed cell infection). Traversal of liver cells, observed for sporozoites in vivo, is thought to activate the sporozoite for infection of a final hepatocyte. Here, using Plasmodium berghei, we show that cell traversal is important in the host dermis for preventing sporozoite destruction by phagocytes and arrest by nonphagocytic cells. We also show that cell infection is a pathway that is masked, rather than activated, by cell traversal. We propose that the cell traversal activity of the sporozoite must be turned on for progression to the liver parenchyma, where it must be switched off for infection of a final hepatocyte.

Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force.

H(+)-F(O)F(1)-ATP synthase couples proton flow through its membrane portion, F(O), to the synthesis of ATP in its headpiece, F(1). Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the epsilon subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the gamma subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced gammaLys23 with the DELSEED region of subunit beta stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit gamma rotation which is necessary for the activation.

Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus.

The proton-pumping and the ATP hydrolysis activities of the ATP synthase of Rhodobacter capsulatus have been compared as a function of the ADP and P(i) concentrations. The proton pumping was measured either with the transmembrane pH difference probe, 9-amino-6-chloro-2-methoxyacridine, or with the transmembrane electric potential difference probe, bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, obtaining consistent results. The comparison indicates that an intrinsic uncoupling of ATP synthase is induced when the concentration of either ligand is decreased. The half-maximal effect was found in the submicromolar range for ADP and at about 70 microM for P(i). It is proposed that a switch from a partially uncoupled state of ATP synthase to the coupled state is induced by the simultaneous binding of ADP and P(i).