Adaptive responses of the ileum of NOD mice to low-dose fluoride: A proteomic exploratory study.

Fluoride (F) has been employed worldwide to control dental caries. More recently, it has been suggested that the consumption of low doses of F in the drinking water may reduce blood glucose levels, introducing a new perspective for the use of F for the management of blood glucose. However, the exact mechanism by which F affects blood glucose levels remains largely unexplored. Given that the small gut plays a pivotal role in glucose homeostasis, the aim of this study was to investigate the proteomic changes induced by low doses of F in the ileum of female nonobese-diabetic (NOD) mice. Forty-two female NOD mice were divided into two groups based on the F concentration in their drinking water for 14 weeks: 0 (control) or 10 mgF/L. At the end of the experimental period, the ileum was collected for proteomic and Western blot analyses. Proteomic analysis indicated an increase in isoforms of actin, gastrotropin, several H2B histones, and enzymes involved in antioxidant processes, as well as a decrease in enzymes essential for energy metabolism. In summary, our data indicates an adaptive response of organism to preserve protein synthesis in the ileum, despite significant alterations in energy metabolism typically induced by F, therefore highlighting the safety of controlled fluoridation in water supplies.

Think positive! Resolving human motion ambiguity in the presence of disease threat.

Recently, approach-avoidance tendencies and visual perception biases have been increasingly studied using bistable point-light walkers (PLWs). Prior studies have found a facing-the-viewer bias when one is primed with general threat stimuli (e.g. angry faces), explained by the "error management theory", as failing to detect a threat as approaching is riskier than the opposite. Importantly, no study has explored how disease threat - linked to the behavioural immune system - might affect this bias. This study aimed to explore whether disease-signalling cues can alter how we perceive the motion direction of ambiguous PLWs. Throughout 3 experiments, participants indicated the motion direction of a bistable PLW previously primed with a control or disease-signalling stimuli - that is, face with a surgical mask (Experiment 1), sickness sound (Experiment 2), or face with a disease cue (Experiment 3). Results showed that sickness cues do not significantly modulate the perception of approach-avoidance behaviours. However, a pattern emerged in Experiments 2 and 3, suggesting that sickness stimuli led to more facing away percepts. Unlike other types of threat, this implies that disease-related threat stimuli might trigger a distinct perceptual bias, indicating a preference to avoid a possible infection source. Nonetheless, this finding warrants future investigations.

The impact of COVID-19 concerns on perceiving others' faces.

Unlike most infectious diseases, COVID-19 is characterised by an absence of facial disease-signalling cues. Yet, it is still unclear whether it has influenced face perception. Understanding this may help clarify if and how our motivation toward social interactions is conditional on situational pathogen threats. The present study investigated if priming disease concerns about COVID-19 would change people's perception of neutral faces on perceived disease, social discomfort and arousal elicited by such faces; this condition was compared with other infectious/non-infectious diseases and a non-disease priming condition. One-hundred sixty-six participants recruited nationally performed the online task. When compared with the non-disease condition, participants primed for COVID-19 perceived faces as sicker and tended to view them as eliciting more social discomfort; no difference occurred in arousal. No other difference was found between conditions. These findings suggest that the pandemic context can shape how we perceive others' apparent sickness. Overall, these might reflect adaptations intertwined with the behavioural immune system's defence mechanisms.

Effect of chitosan solutions with or without fluoride on the protection against dentin erosion in vitro.

The aim of this study was to assess the protective effect of experimental solutions containing chitosan at different viscosities with or without fluoride (TiF4 /NaF) on dentin loss in vitro. Bovine dentin samples (n = 15) were prepared and allocated to one of the following treatments: (i) 0.5% chitosan (500 mPas); (ii) 0.5% chitosan (2,000 mPas); (iii) 0.042% NaF and 0.049% TiF4 ; (iv) as (iii) with addition of 0.5% chitosan (500 mPas); (v) as (iii) with addition of 0.5% chitosan (2,000 mPas); (vi) commercial solution with SnCl2 /AmF/NaF (positive control); or (vii) deionized water (negative control). The samples were submitted to pH cycling for 7 d (0.1% citric acid, 4 × 90 s d-1 ). The treatment was applied once a day for 30 s. The dentin loss was quantified using a contact profilometer. Three samples per group were evaluated using scanning electron microscopy. The dentin loss (µm) was submitted to anova and Tukey's test for differences between treatments. Among the treatments tested, only chitosan 500 mPas was able to statistically significantly reduce the dentin loss compared to the negative control, being similar to the positive control. TiF4 /NaF, whether with or without chitosan, had no protective effect. Chitosan 500 mPas and SnCl2 /AmF/NaF solutions have comparable protective effect against dentin erosion in vitro.

Comparison between micro-computed tomography and transverse microradiography of sound dentine treated with fluorides and demineralized by microcosm biofilm.

The study aimed to apply micro-computed tomography (micro-CT) and transverse microradiography (TMR) to measure dentine demineralization and to test the preventive effect of titanium tetrafluoride (TiF4 ) under microcosm biofilm. Sound dentine specimens from bovine root were treated for 6 h with: (i) 4.0% titanium tetrafluoride (TiF4 ) varnish [pH 1.0, 2.45% fluoride (F-); (ii) 5.42% sodium fluoride (NaF) varnish (pH 5.0, 2.45% F); (iii) 2% chlorhexidine (CHX) gel (pH 7.0); (iv) placebo varnish (pH 5.0); or (v) no agent (untreated). Dentine specimens were then exposed to human saliva mixed with McBain saliva for 8 h. Thereafter, McBain saliva containing 0.2% sucrose was applied daily, for 5 d, onto dentine specimens to stimulate formation of microcosm biofilm. Although a high correlation was found between the results of both methods regarding integrated mineral loss, the results of the methods did not show good agreement in Bland-Altman plots, with significant biases in calculations of lesion depth. Fluoride varnishes were able to reduce dentine demineralization (P < 0.05), while CHX failed to do so. Fluorides are still the best option to reduce dentine demineralization. Micro-CT may be used to measure dentine mineral loss, but not the lesion depth, for which TMR is superior.

Protein Profile of the Acquired Enamel Pellicle after Rinsing with Whole Milk, Fat-Free Milk, and Water: An in vivo Study.

This study detected changes in the protein profile of the acquired enamel pellicle (AEP) formed in vivo after rinsing with whole milk, fat-free milk, or water. Nine subjects in good oral condition took part in the study. The acquired pellicle was formed in the morning, for 120 min, after prophylaxis with pumice. Following this, the volunteers rinsed with 10 mL of whole milk, fat-free milk, or deionized water for 30 s, following a blinded crossover protocol. After 60 min, the pellicle was collected with filter paper soaked in 3% citric acid and processed for analysis by liquid chromatography-electrospray ionization tandem mass spectrometry. The obtained tandem mass spectrometry spectra were searched against a human protein database (Swiss-Prot). The proteomic data related to protein quantification were analysed using the PLGS software. A total of 260 proteins were successfully identified in the AEP samples collected from all groups. Forty-nine were common to all 3 groups, while 72, 62, and 49 were specific to the groups rinsing with whole milk, fat-free milk, and water, respectively. Some were typical components of the AEP, such as cystatin-B, cystatin-SN, isoforms of α-amylase, IgA and IgG, lysozyme C, protein S100 A78, histatin-1, proline-rich protein 27, statherin, and lactotransferrin. Other proteins are not commonly described as part of the AEP but could act in defence of the organism against pathogens. Distinct proteomic profiles were found in the AEP after rinsing with whole or fat-free milk, which could have an impact on bacterial adhesion and tooth dissolution. The use of fat-free milk could favourably modulate the adhesion of bacteria to the AEP as well as biofilm formation when compared with whole milk.

Hepatitis C virus NS3-4A inhibits the peroxisomal MAVS-dependent antiviral signalling response.

Hepatitis C virus (HCV) is the cause of one of the most prevalent viral infections worldwide. Upon infection, the HCV genome activates the RIG-I-MAVS signalling pathway leading to the production of direct antiviral effectors which prevent important steps in viral propagation. MAVS localizes at peroxisomes and mitochondria and coordinate the activation of an effective antiviral response: peroxisomal MAVS is responsible for a rapid but short-termed antiviral response, while the mitochondrial MAVS is associated with the activation of a stable response with delayed kinetics. The HCV NS3-4A protease was shown to specifically cleave the mitochondrial MAVS, inhibiting the downstream response. In this study, we have analysed whether HCV NS3-4A is also able to cleave the peroxisomal MAVS and whether this would have any effect on the cellular antiviral response. We show that NS3-4A is indeed able to specifically cleave this protein and release it into the cytosol, a mechanism that seems to occur at a similar kinetic rate as the cleavage of the mitochondrial MAVS. Under these conditions, RIG-I-like receptor (RLR) signalling from peroxisomes is blocked and antiviral gene expression is inhibited. Our results also show that NS3-4A is able to localize at peroxisomes in the absence of MAVS. However, mutation studies have shown that this localization pattern is preferred in the presence of a fully cleavable MAVS. These findings present evidence of a viral evasion strategy that disrupts RLR signalling on peroxisomes and provide an excellent example of how a single viral evasion strategy can block innate immune signalling from different organelles.

Elimination of the vesicular acetylcholine transporter in the forebrain causes hyperactivity and deficits in spatial memory and long-term potentiation.

Basal forebrain cholinergic neurons, which innervate the hippocampus and cortex, have been implicated in many forms of cognitive function. Immunolesion-based methods in animal models have been widely used to study the role of acetylcholine (ACh) neurotransmission in these processes, with variable results. Cholinergic neurons have been shown to release both glutamate and ACh, making it difficult to deduce the specific contribution of each neurotransmitter on cognition when neurons are eliminated. Understanding the precise roles of ACh in learning and memory is critical because drugs that preserve ACh are used as treatment for cognitive deficits. It is therefore important to define which cholinergic-dependent behaviors could be improved pharmacologically. Here we investigate the contributions of forebrain ACh on hippocampal synaptic plasticity and cognitive behavior by selective elimination of the vesicular ACh transporter, which interferes with synaptic storage and release of ACh. We show that elimination of vesicular ACh transporter in the hippocampus results in deficits in long-term potentiation and causes selective deficits in spatial memory. Moreover, decreased cholinergic tone in the forebrain is linked to hyperactivity, without changes in anxiety or depression-related behavior. These data uncover the specific contribution of forebrain cholinergic tone for synaptic plasticity and behavior. Moreover, these experiments define specific cognitive functions that could be targeted by cholinergic replacement therapy.

Control of white spot lesion adjacent to orthodontic bracket with use of fluoride varnish or chlorhexidine gel.

The aims of this study were to compare the effectiveness of fluoride varnish and chlorhexidine gel in controlling white spot lesions (WSLs) adjacent to orthodontic brackets and to compare the ability of Quantitative Light-Induced Fluorescence (QLF) to measure mineral uptake with that of transverse microradiography (TMR). Thirty premolars with artificially induced WSLs were randomly assigned to three groups: (1) two applications of 5% NaF-varnish (F), with one-week interval, (2) two applications of 2% chlorhexidine gel (CHX), with one-week interval, and (3) control (CO), no treatment. QLF was used to measure changes in fluorescence before and after caries induction, 1 week after each application and 1, 2, and 3 months after the last application of F or CHX. TMR was performed to quantify lesion depth and mineral content after caries induction to evaluate the effects of F, CHX, and CO 3 months after the last application of agents. The data were analyzed by repeated measures ANOVA and Tukey's test. All treatments increased the mineral content during the experimental period; however, F induced faster remineralization than CHX. The correlation between QLF and TMR was significantly moderate. Two applications of fluoride varnish or 2% chlorhexidine gel at one-week intervals were effective in controlling WSLs.

ChAT-ChR2-EYFP mice have enhanced motor endurance but show deficits in attention and several additional cognitive domains.

Acetylcholine (ACh) is an important neuromodulator in the nervous system implicated in many forms of cognitive and motor processing. Recent studies have used bacterial artificial chromosome (BAC) transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT-ChR2-EYFP) to dissect cholinergic circuit connectivity and function using optogenetic approaches. We report that a mouse line used for this purpose also carries several copies of the vesicular acetylcholine transporter gene (VAChT), which leads to overexpression of functional VAChT and consequently increased cholinergic tone. We demonstrate that these mice have marked improvement in motor endurance. However, they also present severe cognitive deficits, including attention deficits and dysfunction in working memory and spatial memory. These results suggest that increased VAChT expression may disrupt critical steps in information processing. Our studies demonstrate that ChAT-ChR2-EYFP mice show altered cholinergic tone that fundamentally differentiates them from wild-type mice.

The prion protein ligand, stress-inducible phosphoprotein 1, regulates amyloid-ß oligomer toxicity.

In Alzheimer's disease (AD), soluble amyloid-ß oligomers (AßOs) trigger neurotoxic signaling, at least partially, via the cellular prion protein (PrP(C)). However, it is unknown whether other ligands of PrP(C) can regulate this potentially toxic interaction. Stress-inducible phosphoprotein 1 (STI1), an Hsp90 cochaperone secreted by astrocytes, binds to PrP(C) in the vicinity of the AßO binding site to protect neurons against toxic stimuli. Here, we investigated a potential role of STI1 in AßO toxicity. We confirmed the specific binding of AßOs and STI1 to the PrP and showed that STI1 efficiently inhibited AßO binding to PrP in vitro (IC50 of ∼70 nm) and also decreased AßO binding to cultured mouse primary hippocampal neurons. Treatment with STI1 prevented AßO-induced synaptic loss and neuronal death in mouse cultured neurons and long-term potentiation inhibition in mouse hippocampal slices. Interestingly, STI1-haploinsufficient neurons were more sensitive to AßO-induced cell death and could be rescued by treatment with recombinant STI1. Noteworthy, both AßO binding to PrP(C) and PrP(C)-dependent AßO toxicity were inhibited by TPR2A, the PrP(C)-interacting domain of STI1. Additionally, PrP(C)-STI1 engagement activated α7 nicotinic acetylcholine receptors, which participated in neuroprotection against AßO-induced toxicity. We found an age-dependent upregulation of cortical STI1 in the APPswe/PS1dE9 mouse model of AD and in the brains of AD-affected individuals, suggesting a compensatory response. Our findings reveal a previously unrecognized role of the PrP(C) ligand STI1 in protecting neurons in AD and suggest a novel pathway that may help to offset AßO-induced toxicity.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γ1 chain.

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln γ1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln γ1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln γ1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.

In situ effect of sodium fluoride or titanium tetrafluoride varnish and solution on carious demineralization of enamel.

This study evaluated the effect of titanium tetrafluoride (TiF(4)) formulations on enamel carious demineralization in situ. Thirteen subjects took part in this cross-over, split-mouth, double-blind study performed in three phases of 14 d each. In each subject, two sound and two predemineralized specimens of bovine enamel were worn intra-orally and plaque accumulation was allowed. One sound and one predemineralized specimen in each subject was treated once with sodium fluoride (NaF) varnish or solution (Treatment A); TiF(4) varnish or solution (Treatment B); or placebo varnish or no treatment (Treatment C). The initially sound enamel specimens were exposed to severe cariogenic challenge (20% sucrose, eight times daily for 5 min each time), whereas the predemineralized specimens were not. Eleven subjects were able to finish all experimental phases. The enamel alterations were quantified by surface hardness and transversal microradiography. Demineralization of previously sound enamel was reduced by all test formulations except for the NaF solution, while both TiF(4) formulations were as effective as NaF varnish. For the predemineralized specimens, enamel surface hardness was increased only by TiF(4) formulations, while subsurface mineral remineralization could not be seen in any group. Within the experimental protocol, TiF(4) was able to decrease enamel demineralization to a similar degree as NaF varnish under severe cariogenic challenges, while only TiF(4) formulations remineralized the enamel surface.

Perceived Vulnerability to Disease Questionnaire: psychometric validation with a Portuguese sample.

BACKGROUND: Individual differences in one's perceived vulnerability to infectious diseases are implicated in psychological distress, social and behavioral disease avoidance phenomena. The Perceived Vulnerability to Disease Questionnaire (PVD) is the most extensively used measure when it comes to assessing subjective vulnerability to infectious diseases. However, this measure is not yet accessible to the Portuguese population. The present study aimed to adapt and validate the PVD with 136 Portuguese participants. METHODS: Factorial, convergent and discriminant validity (of both the scale and between each factor), and reliability analysis were assessed. RESULTS: A modified bifactorial model, comprised of Perceived Infectability and Germ Aversion factors, was obtained, with acceptable goodness-of-fit indices, adequate convergent and discriminant validity, and good internal consistencies. CONCLUSIONS: Overall, the 10-items European-Portuguese PVD appears to be a reliable and valid measure of one's perceived vulnerability to disease, with potential relevance for application in both research and clinical practice pertaining to disease-avoidance processes.

InfectionCMA: A Cell MicroArray Approach for Efficient Biomarker Screening in In Vitro Infection Assays.

The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the scientific community to acquire knowledge in real-time, when total lockdowns and the interruption of flights severely limited access to reagents as the global pandemic became established. This unique reality made researchers aware of the importance of designing efficient in vitro set-ups to evaluate infectious kinetics. Here, we propose a histology-based method to evaluate infection kinetics grounded in cell microarray (CMA) construction, immunocytochemistry and in situ hybridization techniques. We demonstrate that the chip-like organization of the InfectionCMA has several advantages, allowing side-by-side comparisons between diverse cell lines, infection time points, and biomarker expression and cytolocalization evaluation in the same slide. In addition, this methodology has the potential to be easily adapted for drug screening.

Sensitivity Treatments for Teeth with Molar Incisor Hypomineralization: Protocol for a Randomized Controlled Trial.

BACKGROUND: The sensitivity of teeth with molar incisor hypomineralization (MIH) can affect children's quality of life and is a challenging problem for dentists. Remineralizing agents such as sodium fluoride varnish seem to reduce the sensitivity of teeth with MIH, but long-term clinical trials with large samples are still needed for more evidence about its effectiveness as a desensitizing agent before its clinical recommendation. OBJECTIVE: This randomized clinical trial aims to compare three treatment interventions for teeth with MIH and hypersensitivity. METHODS: A total of 60 children aged 6-10 years presenting with at least one first permanent molar with sensitivity and no loss of enamel will be randomly assigned to three groups: the control group (sodium fluoride varnish; Duraphat, Colgate); experimental group I (4% titanium tetrafluoride varnish); and experimental group II (a coating resin containing surface prereacted glass-ionomer filler; PRG Barrier Coat, Shofu). The sodium fluoride varnish and 4% titanium tetrafluoride varnish will be applied once per week for 4 consecutive weeks and the PRG Barrier Coat resin will be applied in the first session and the application will be simulated the following 3 weeks to guarantee the blinding of the study. The primary outcome will be sensitivity level measured at different moments (before each material application, immediately after application or simulation, and 1, 2, 4, and 6 months after the last application/simulation) by one examiner using the Wong-Baker FACES Pain Rating Scale, the Schiff Cold Air Sensitivity Scale, and the FLACC (Face, Legs, Activity, Cry, Consolability) scale. As secondary outcomes, parental satisfaction and child self-reported discomfort after the treatment will be measured with a questionnaire prepared by the researcher. The data will undergo statistical analysis and the significance level will be set at 5%. RESULTS: The project was funded in 2018, and enrollment was completed in November 2019. The recruitment of participants is currently underway and the first results are expected to be submitted for publication in 2022. CONCLUSIONS: If found effective in reducing the patient's sensitivity long term, these agents can be considered as a treatment choice, and the findings will contribute to the development of a treatment protocol for teeth with sensitivity due to MIH. TRIAL REGISTRATION: Brazilian Registry of Clinical Trials Universal Trial Number U1111-1237-6720; https://tinyurl.com/mr4x82k9. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/27843.

Searching for SARS-CoV-2 in Cancer Tissues: Results of an Extensive Methodologic Approach based on ACE2 and Furin Expression.

SARS-CoV-2 pandemics have been massively characterized on a global scale by the rapid generation of in-depth genomic information. The main entry gate of SARS-CoV-2 in human cells is the angiotensin-converting enzyme 2 (ACE2) receptor. The expression of this protein has been reported in several human tissues, suggesting a correlation between SARS-CoV-2 organotropism and ACE2 distribution. In this study, we selected (a series of) 90 patients who were submitted to surgery for tumor removal between the beginning of the SARS-CoV-2 pandemic and the closure of operating rooms (by the end of March 2020) in two different countries-Portugal and Brazil. We evaluated the expressions of ACE2 and furin (another important factor for virus internalization) in colon (n = 60), gastric (n = 19), and thyroid (n = 11) carcinomas. In a subseries of cases with PCR results for SARS-CoV-2 detection in the peri-operatory window (n = 18), we performed different methodological approaches for viral detections in patient tumor samples. Our results show that colon and gastric carcinomas display favorable microenvironments to SARS-CoV-2 tropism, presenting high expression levels of ACE2 and furin. From the subseries of 18 cases, 11 tested positive via PCR detection performed in tumor blocks; however, a direct association between the ACE2 expression and SARS-CoV-2 infection was not demonstrated in cancer cells using histology-based techniques, such as immunohistochemistry or in situ hybridization. This study raises the possibility of ACE2-mediated viral tropism in cancer tissues to be clarified in future studies.

Shedding Light on the African Enigma: In Vitro Testing of Homo sapiens-Helicobacter pylori Coevolution.

The continuous characterization of genome-wide diversity in population and case-cohort samples, allied to the development of new algorithms, are shedding light on host ancestry impact and selection events on various infectious diseases. Especially interesting are the long-standing associations between humans and certain bacteria, such as the case of Helicobacter pylori, which could have been strong drivers of adaptation leading to coevolution. Some evidence on admixed gastric cancer cohorts have been suggested as supporting Homo-Helicobacter coevolution, but reliable experimental data that control both the bacterium and the host ancestries are lacking. Here, we conducted the first in vitro coinfection assays with dual human- and bacterium-matched and -mismatched ancestries, in African and European backgrounds, to evaluate the genome wide gene expression host response to H. pylori. Our results showed that: (1) the host response to H. pylori infection was greatly shaped by the human ancestry, with variability on innate immune system and metabolism; (2) African human ancestry showed signs of coevolution with H. pylori while European ancestry appeared to be maladapted; and (3) mismatched ancestry did not seem to be an important differentiator of gene expression at the initial stages of infection as assayed here.

Dynamics of a Dual SARS-CoV-2 Lineage Co-Infection on a Prolonged Viral Shedding COVID-19 Case: Insights into Clinical Severity and Disease Duration.

A few molecularly proven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases of symptomatic reinfection are currently known worldwide, with a resolved first infection followed by a second infection after a 48 to 142-day intervening period. We report a multiple-component study of a clinically severe and prolonged viral shedding coronavirus disease 2019 (COVID-19) case in a 17-year-old Portuguese female. She had two hospitalizations, a total of 19 RT-PCR tests, mostly positive, and criteria for releasing from home isolation at the end of 97 days. The viral genome was sequenced in seven serial samples and in the diagnostic sample from her infected mother. A human genome-wide array (>900 K) was screened on the seven samples, and in vitro culture was conducted on isolates from three late samples. The patient had co-infection by two SARS-CoV-2 lineages, which were affiliated in distinct clades and diverging by six variants. The 20A lineage was absolute at the diagnosis (shared with the patient's mother), but nine days later, the 20B lineage had 3% frequency, and two months later, the 20B lineage had 100% frequency. The 900 K profiles confirmed the identity of the patient in the serial samples, and they allowed us to infer that she had polygenic risk scores for hospitalization and severe respiratory disease within the normal distributions for a Portuguese population cohort. The early-on dynamic co-infection may have contributed to the severity of COVID-19 in this otherwise healthy young patient, and to her prolonged SARS-CoV-2 shedding profile.

Endocytosis of prion protein is required for ERK1/2 signaling induced by stress-inducible protein 1.

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrP(C)) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrP(C) trafficking and tested whether this process controls PrP(C)-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrP(C), induced PrP(C) endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrP(C); however, heterologous expression of PrP(C) reconstituted both PKA and ERK1/2 activation. In contrast, a PrP(C) mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrP(C) endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.