Targeting host O-linked glycan biosynthesis affects Ebola virus replication efficiency and reveals differential GalNAc-T acceptor site preferences on the Ebola virus glycoprotein.

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.

Crimean-Congo haemorrhagic fever virus uses LDLR to bind and enter host cells.

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.

Unraveling Negative Expectations and Nocebo-Related Effects in Musculoskeletal Pain.

This Perspective adapts the ViolEx Model, a framework validated in several clinical conditions, to better understand the role of expectations in the recovery and/or maintenance of musculoskeletal (MSK) pain. Here, particular attention is given to the condition in which dysfunctional expectations are maintained despite no longer being supported by confirmatory evidence (i.e., belief-lifting the arm leads to permanent tendon damage; evidence-after the patient lifts the arm no tendon damage occurs). While the ViolEx Model suggests that cognitive immunization strategies are responsible for the maintenance of dysfunctional expectations, we suggest that such phenomenon can also be understood from a Bayesian Brain perspective, according to which the level of precision of the priors (i.e., expectations) is the determinant factor accounting for the extent of priors' updating (i.e., we merge the two frameworks, suggesting that highly precise prior can lead to cognitive immunization responses). Importantly, this Perspective translates the theory behind these two frameworks into clinical suggestions. Precisely, it is argued that different strategies should be implemented when treating MSK pain patients, depending on the nature of their expectations (i.e., positive or negative and the level of their precision).

Online teaching in physiotherapy education during COVID-19 pandemic in Italy: a retrospective case-control study on students' satisfaction and performance.

BACKGROUND: During COVID-19 pandemic, physiotherapy lecturers faced the challenge of rapidly shifting from face-to-face to online education. This retrospective case-control study aims to compare students' satisfaction and performances shown in an online course to a control group of students who underwent the same course delivered face-to-face in the previous five years. METHODS: Between March and April 2020, a class (n = 46) of entry-level physiotherapy students (University of Verona - Italy), trained by an experienced physiotherapist, had 24-hours online lessons. Students exposed to the same course in the previous five academic years (n = 112), delivered with face-to-face conventional lessons, served as a historical control. The course was organized in 3 sequential phases: (1) PowerPoint presentations were uploaded to the University online platform, (2) asynchronous video recorded lectures were provided on the same platform, and (3) between online lectures, the lecturer and students could communicate through an email chat to promote understanding, dispel any doubts and collect requests for supplementary material (e.g., scientific articles, videos, webinars, podcasts). Outcomes were: (1) satisfaction as routinely measured by University with a national instrument and populated in a database; (2) performance as measured with an oral examination. RESULTS: We compared satisfaction with the course, expressed on a 5-point Likert scale, resulting in no differences between online and face-to-face teaching (Kruskal-Wallis 2 = 0.24, df = 1, p = 0.62). We weighted up students' results by comparing their mean performances with the mean performances of the same course delivered face-to-face in the previous five years, founding a statistical significance in favour of online teaching (Wilcoxon rank sum test W = 1665, p < 0.001). CONCLUSIONS: Online teaching in entry-level Physiotherapy seems to be a feasible option to face COVID-19 pandemic, as satisfies students as well as face-to-face courses and leading to a similar performance. Entry-level Bachelors in Physiotherapy may consider moving to eLearning to facilitate access to higher education. Universities will have to train lecturers to help them develop appropriate pedagogical skills, and supply suitable support in terms of economic, organizational, and technological issues, aimed at guaranteeing a high level of education to their students. TRIAL REGISTRATION: Retrospectively registered.

Cranberry (Vaccinium macrocarpon) Extract Impairs Nairovirus Infection by Inhibiting the Attachment to Target Cells.

Hazara virus (HAZV) belongs to the Nairoviridae family and is included in the same serogroup of the Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is the most widespread tick-borne arbovirus. It is responsible for a serious hemorrhagic disease, for which specific and effective treatment and preventive systems are missing. Bioactive compounds derived from several natural products may provide a natural source of broad-spectrum antiviral agents, characterized by good tolerability and minimal side effects. Previous in vitro studies have shown that a cranberry (Vaccinium macrocarpon Ait.) extract containing a high content of A-type proanthocyanidins (PAC-A) inhibits the replication of herpes simplex and influenza viruses by hampering their attachment to target cells. Given the broad-spectrum antimicrobial activity of polyphenols and the urgency to develop therapies for the treatment of CCHF, we investigated the antiviral activity of cranberry extract against HAZV, a surrogate nairovirus model of CCHFV that can be handled in Level 2 Biosafety Laboratories (BSL-2). The results indicate that the cranberry extract exerts an antiviral activity against HAZV by targeting early stages of the viral replication cycle, including the initial adsorption to target cells. Although the details of the molecular mechanism of action remain to be clarified, the cranberry extract exerts a virucidal effect through a direct interaction with HAZV particles that leads to the subsequent impairment of virus attachment to cell-surface receptors. Finally, the antiviral activity of the cranberry extract was also confirmed for CCHFV. As a whole, the evidence obtained suggests that cranberry extract is a valuable candidate to be considered for the development of therapeutic strategies for CCHFV infections.

Percutaneous Saphenous Vein Arterialization.

PURPOSE: In patients with no-option critical limb-threatening ischemia, venous foot arterialization can be considered the last chance before major amputation. Up until now, a really significant limitation of endovascular arterialization compared with surgery was the possibility to obtain arterial flow into the foot only through the deep plantar network. TECHNIQUE: Two 5-mm snares are placed: one in the proximal tibial artery and the other in the great saphenous vein. After passing through these snares with a needle and a guidewire and closing the snares, the guidewire is pulled through the proximal arterial sheath and the distal venous sheath. Thus, the arterial-venous connection is created. A covered stent is, then, placed between the artery and the vein to avoid leakage. CONCLUSIONS: Based on our knowledge, this is the first described totally percutaneous arterialization of the superficial dorsal venous foot system, through reverse flow in the great saphenous vein.

Hypothiocyanite and Hypothiocyanite/Lactoferrin Mixture Exhibit Virucidal Activity In Vitro against SARS-CoV-2.

SARS-CoV-2 replicates efficiently in the upper airways during the prodromal stage, resulting in environmental viral shedding from patients with active COVID-19 as well as from asymptomatic individuals. There is a need to find pharmacological interventions to mitigate the spread of COVID-19. Hypothiocyanite and lactoferrin are molecules of the innate immune system with a large spectrum cidal activity. The Food and Drug Administration and the European Medicines Agency designated the hypothiocyanite and lactoferrin combination as an orphan drug. We report an in vitro study showing that micromolar concentrations of hypothiocyanite exhibit dose- and time-dependent virucidal activity against SARS-CoV-2 and that the latter is slightly enhanced by the simultaneous presence of lactoferrin.

What Physiotherapists Specialized in Orthopedic Manual Therapy Know About Nocebo-Related Effects and Contextual Factors: Findings From a National Survey.

OBJECTIVE: The aim of this study was to investigate the knowledge of orthopedic manual therapists (OMTs) regarding context factors (CFs) capable of triggering nocebo effects during the treatment and how this knowledge is related to their socio-demographic features. DESIGN: A cross-sectional online survey. SETTING: National. MAIN OUTCOME MEASURES: A 20 items questionnaire composed by open-ended and closed single-choice questions was administered to explore: (a) socio-demographic variables (10 questions); (b) the relation between different CFs and nocebo-related effects (2 questions); and (c) the knowledge of participants about nocebo-related effects and how they managed them in the clinical practice (8 questions). PARTICIPANTS: 1288 OMTs were recruited from the database of the Master in Rehabilitation of Musculoskeletal Disorders (MRDM) of the University of Genova from March to May 2019. Inclusion criteria were: (a) to possess a valid email account; (b) to understand and use as a native language the Italian; (c) to be graduated as OMTs; and (d) to be employed as physiotherapists specialized-OMTs during the survey. RESULTS: 791 responses were received (61.4%); 473 of them were male (59.8%), with an average age of 31.0 ± 7.1 years. OMTs defined nocebo-related effects as the psychosocial context effects around therapy and patient with specific biological bases (72.2%). OMTs know that their clinical practice is pervaded by nocebo-related effects (42.5%), triggered by CFs. Participants communicated nocebo-related effects balancing the positive features of the therapy with the negative ones (50.9%), during the decision of the therapeutic plan (42.7%). They reported associative learning as the main mechanism involved in nocebo-related effects (28.8%). OMTs taught and trained patient's strategies to manage nocebo-related effects (39.6%) through an evaluation and correction of patient's anxieties, doubts and expectations (37.7%). OMTs most frequently considered themselves to have a "medium" education about nocebo-related effects (48.2%) and that their management should be taught during bachelor (78.6%). CONCLUSION: OMTs believed that nocebo-related effects were present in their clinical practice and that they can be triggered by CFs.

Treatment of bypass failure in patients with chronic limb threatening ischemia - open surgery vs. percutaneous mechanical thrombectomy.

Background: Lower limb bypass occlusion in patients with chronic limb threating ischemia remains a challenge. We can choose between different treatment options: open surgery, local thrombolysis, thrombectomy/atherectomy devices. In this pilot study, we compare clinical outcomes and treatment costs between open surgery (OS) and percutaneous mechanical thrombectomy (pMTH). Patients and methods: This pilot study represents a retrospective analysis of hospital data of 48 occluded bypasses admitted from 2013 to 2018. Only patients presenting with severe ischemia and recrudescence of symptoms (Rutherford 4-6) were included in the current analysis. Two cohorts of patients were analysed: patients who underwent OS and patients that underwent pMTH. Primary clinical outcomes were one-year cumulative patency and limb salvage rates. Total cost was calculated as a sum of intra- and post-operative costs. To weigh clinical benefits against the economic consequences of OS versus pMTH a cost-effectiveness framework was adopted. Results: We analysed a series of 48 occluded bypasses 17 treated with open surgery and 31 with pMTH. Procedural success was 100% in both groups. When comparing one-year death rates (p-value = .22) and re-occlusion rates (p-value = .43), no statistically significant differences were observed between the two cohorts. Mean patency duration in the surgery cohort was significantly shorter (p-value < .05). Primary patency (OS 41.2% vs. pMTH 48.4%) and limb salvage rate (OS 88.2% vs. pMTH 90.3%) at one year are similar in both groups. The total cost of surgery was substantially higher (OS 10,159€ vs. pMTH 8,401€) Conclusions: This pilot study, although limited to 48 occluded bypasses, demonstrates that endovascular treatment with pMTH is less invasive, less time consuming and less expensive, and produces greater health benefits than traditional OS.

An Italian experience with mechanochemical ablation of the saphenous vein since 2012.

OBJECTIVE: Mechanochemical endovenous ablation is a nonthermal method to ablate superficial incompetent veins. The aim of this paper was to assess short-term complications and 5-year follow-up outcomes. METHODS: This is a retrospective single-center study of data collected prospectively. We treated, in an outpatient setting, 395 primary, symptomatic, unilateral, incompetent varicose saphenous veins. No patients were treated bilaterally in the same session. The majority were great saphenous veins (92.3%), and the others were small saphenous veins. Procedures were performed with a mechanochemical endovenous occlusion catheter (ClariVein endovenous occlusion catheter; Merit Medical, South Jordan, Utah) and polidocanol 2% in liquid form. RESULTS: Follow-up was available for 329 patients treated between September 2012 and September 2017 with a mean follow-up time of 20 ± 18 months (range, 6-60 months). Technical success was achieved in 99.5%; in two patients, we were unable to complete the procedures because of vein spasm leading to catheter damage and inability to infuse the sclerosant. Follow-up was performed with clinical evaluation and duplex ultrasound scan at 1 week, 1 month, 6 months, and 1 year and then once every year. The overall survival rate free from recanalization was 92.4%. Anatomic success is 94% at 1 year, 91% at 2 years, 88% at 3 years, 88% at 4 years, and 84% at 5 years. The follow-up at 5 years includes 23 patients, 5 of whom presented with recanalization; moreover, in this series, in 36% of cases, the veins completely disappeared on duplex ultrasound scan. CONCLUSIONS: The ClariVein catheter is associated with a good occlusion rate, comparable with other techniques including thermal techniques, without major complications.

The Knowledge of Contextual Factors as Triggers of Placebo and Nocebo Effects in Patients With Musculoskeletal Pain: Findings From a National Survey.

Backgrounds: Contextual factors (CFs) have been recently proposed as triggers of placebo and nocebo effects in musculoskeletal pain. CFs encompass the features of the clinician (e.g. uniform), patient (e.g. expectations), patient-clinician relationship (e.g. verbal communication), treatment (e.g. overt therapy), and healthcare setting (e.g. design). To date, the researchers' understanding of Italian patients' knowledge about the role of CFs in musculoskeletal pain is lacking. Objectives: The aim of this study was to investigate attitudes and beliefs of Italian patients with musculoskeletal pain about the use of CFs in clinical practice. Methods: A national sample of Italian patients with musculoskeletal pain was recruited from 12 outpatient private clinics in Italy. An invitation to participate in an online survey was sent to patients: a) exhibiting musculoskeletal pain; b) aged 18-75; c) with a valid e-mail account; and d) understanding Italian language. Survey Monkey software was used to deliver the survey. The questionnaire was self-reported and included 17 questions and 2 clinical vignettes on the patients' behavior, beliefs, and attitudes towards the adoption of CFs in clinical practice. Descriptive statistics and frequencies described the actual number of respondents to each question. Results: One thousand one hundred twelve patients participated in the survey. Five hundred seventy-four participants were female (52%). The average age of patients was 41.7 ± 15.2 years. Patients defined CFs as an intervention with an unspecific effect (64.3%), but they believed in their clinical effectiveness. They identified several therapeutic effects of CFs for different health problems. Their use was considered ethically acceptable when it exerts beneficial psychological effects (60.4%), but it was banned if considered deceptive (51.1%). During clinical practice, patients wanted to be informed about the use of CFs (46.0%) that are accepted as an addition to other interventions to optimize clinical responses (39.3%). Moreover, patients explained the power of CFs through body-mind connections (37.1%). Conclusion: Patients with musculoskeletal pain had positive attitudes towards the use and effectiveness of CFs when associated with evidence-based therapy. They mostly perceived the adoption of CFs in clinical practice as ethical.

Neurocognitive changes after eversion carotid endarterectomy under local anesthesia.

BACKGROUND: The effects of carotid endarterectomy (CEA) on cognitive function have yet to be fully elucidated. The aim of our study was to administer a battery of cognitive tests to identify any cognitive changes occurring in a consecutive series of patients who underwent CEA using the eversion technique under local anesthesia. METHODS: This prospective study was designed to analyze a consecutive series of patients undergoing eversion CEA under local anesthesia for significant carotid stenosis at our vascular surgery unit over a period of 6 months. Patients underwent tests to rule-out those who already had cognitive impairments or states of depression/anxiety capable of interfering with cognitive testing outcomes. Patients then completed a battery of 10 neurocognitive tests preoperatively and again 30 days and 4 months after surgery to assess the functions of both cerebral hemispheres as thoroughly as possible. RESULTS: Of the 48 patients initially considered for our study, 39 completed the follow-up. They were 71.4 ± 8.2 (mean ± SD) years of age; 30 were men and 9 were women. Six were symptomatic for carotid stenosis and 33 were asymptomatic. All patients were examined by a neurologist and underwent pre- and postoperative nuclear MRI or CT scan of the brain to identify any cerebral ischemia potentially correlated with the surgical procedure. In all cases, the cognitive test findings tended to improve postoperatively; this improvement was statistically significant in 7 tests. Post-hoc analysis confirmed an improvement between the pre- and postoperative test results. Among the different variables considered, only age <75 years seems to have influenced cognitive improvement. CONCLUSIONS: The effects on cognitive function of carotid stenosis, particularly CEA, is still a much debated issue. The data reported in the literature vary considerably, preventing any final conclusions from being drawn. The mechanisms capable of inducing changes in cognitive status after CEA have yet to be precisely clarified. In our study, a suitable battery of tests were used to analyze the trend of cognitive function correlating with eversion CEA under local anesthesia. Our results demonstrate substantially improved cognitive function after CEA, which was statistically significant in 7 of 10 tests. We surmise that the CEA procedure, per se, can help to protect patients against cognitive deterioration, especially in those <75 years of age.