Gait dysfunction and fall risk have been well documented in people with Alzheimer's Disease (AD) and individuals with mild cognitive impairment (MCI). Normal locomotor adaptation may be an important prerequisite for normal and safe community walking function, especially in older adults with age-related neural, musculoskeletal, or cardiovascular changes and cognitive impairments. The split-belt walking task is a well-studied and robust method to evaluate locomotor adaptation . Here, we capitalized on the split-belt adaptation task to test our hypothesis that a decreased capacity for locomotor adaptation may be an important contributing factor and indicator of increased fall risk and cognitive decline in older individuals with MCI and AD. The objectives of this study were to (1) compare locomotor adaptation capacity in MCI and AD compared to healthy older adults (HOA) during split-belt treadmill walking, and (2) evaluate associations between locomotor adaptation and cognitive impairments. Our results demonstrated a significant decrease in split-belt locomotor adaptation magnitude in older individuals with MCI and AD compared to HOA. In addition, we found significant correlations between the magnitude of early adaptation and de-adaptation vs. cognitive test scores, demonstrating that individuals with greater cognitive impairment also display a reduced capacity to adapt their walking in response to the split-belt perturbation. Our study takes an important step toward understanding mechanisms underlying locomotor dysfunction in older individuals with cognitive impairment. The ability to walk without the risk of falling is a defining feature of independent community function for elderly individuals. Individuals with cognitive impairment, such as Alzheimer's Disease (AD), are reported to experience falls and loss of independence twice as often as age-matched healthy older adults (HOA) , 2. Manyvia sensorimotor adaptation—a process through which sensorimotor mappings update in response to errors caused by environmental perturbations or demands. Over the course of multiple exposures to such environmental perturbations, adaptation processes can aid the formation of new motor memories, contributing to flexible and robust motor behaviors  than the other. When exposed to this 2:1 split-belt treadmill condition, the participant initially “limps” , and within 10–15 min of split-belt walking, gait symmetry is restored  and age-matched healthy controls. We also evaluated the hypothesis that locomotor adaptation capacity would be associated with cognitive function. To our knowledge, this is the first analysis of locomotor adaptation and its relationships with cognition in MCI and AD individuals.All study procedures were approved by the Emory Institutional Review Board, and all participants provided informed written consent.All subjects were recruited from the Emory Alzheimer's Disease Research Center Registry. These subjects had undergone standard evaluations including measures that comprise the Uniform Data Set of the National Alzheimer's Coordinating Center. HOA subjects had received a diagnosis of normal cognition within 6 months before completing the study, while MCI and AD subjects received a diagnosis of MCI or AD, respectively, within 6 months before completing the study protocol. The MCI and AD subjects were grouped together as MCI/AD for data analysis. All subjects had no history of psychiatric (Axis I) disorders, alcohol/substance-related abuse, and neurologic conditions such as stroke or Parkinson's disease. Additionally, the subjects had no current significant alcohol use, were not taking hypoglycemic agents, no newly diagnosed neurologic conditions, and no orthopedic problems in the lower limbs or spine that limit walking.A 7-camera motion capture system  and an instrumented split-belt treadmill  were used to collect marker and ground reaction force data during the walking assessment. Retro-reflective markers were attached to the subjects' upper back, pelvis, bilateral hip, knee, and ankle joints with adhesive skin tape, as detailed in our previous publications . The splThe walking assessment consisted of three phases: a baseline phase in which the belts operated at the same speed (Pre-tied), a phase in which the belts operated at different speeds (Split-belt), and a final phase in which the belts operated at the same speed (Post-tied) . At the After assessing the subject's self-selected speed, data were collected during the pre-tied phase, with the subject walking on the treadmill with belt speeds tied for 1 min at the fast speed, followed by 1 min at the slow speed.Following the pre-tied phase, the belt underneath the subject's dominant leg was increased to the fast speed, while the belt underneath the non-dominant leg remained at the slow speed. Thus, the treadmill belt speeds were split to a 2:1 speed ratio. This change in speed induced an initial asymmetry or limp in the subject's gait pattern. The subject continued to walk with this split-belt adaptation condition for 15 min. Gait data collected during this period were used to evaluate each individual's locomotor adaptation capacity by assessing the difference in inter-limb step symmetry that the split-belt induced, and the number of steps required to reach a plateau in step symmetry.After the conclusion of the split-belt phase, during the post-adaptation period, the belt moving at the fast speed was returned to the slow speed. The subject walked at this tied-belt slow speed for 2 min. Then, both belts increased to the fast speed, and the subject walked for an additional 2 min. After 2 min of fast walking, both belts slowed to a stop. Gait data from this phase were used to evaluate aftereffects or the locomotor system's ability to de-adapt following the split-belt adaptation.Following the treadmill assessment, the experimenters administered the Montreal Cognitive Assessment (MoCA) and the n-back subtests of the NIH EXAMINER .Marker data were labeled using Vicon Nexus software and then transferred to Visual 3D software  for further processing. Bilateral step lengths were calculated as the antero-posterior distance between the heel markers of the leading foot and the trailing foot at heel strike. Step length was defined with reference to the leading leg . To compare the fast and slow steps, step length symmetry was calculated for each step as follows :Using this formula, a step symmetry of zero would correspond to equal step lengths for both the fast and slow steps.Step symmetry data for the split-belt and post-tied periods were normalized for each individual by subtracting with respect to the average of the last 5 steps of the pre-tied period. Therefore, a step symmetry equal to zero for each individual corresponds to that individual's baseline step symmetry.Four periods were primarily used to assess the magnitude of adaptation and de-adaptation :Early adaptation: mean of first five steps of the split-belt period.Late adaptation: mean of last five steps of the split-belt period.Early aftereffects: mean of first five steps of the post-tied period.Late aftereffects: mean of last five steps of the post-tied period.magnitude of adaptation since it is the initial magnitude of change induced at the beginning of the split-belt adaptation period.The early adaptation step symmetry is also referred to as the rate of adaptation was defined as the number of steps taken after the split-belt period begins for the subject to reach the adaptation plateau, defined as the average step symmetry of the last 30 steps of the split-belt period. A custom MatLab  program was used to compare the average step symmetry of every five steps with the step symmetry in the plateau window, defined as the adaptation plateau ± the standard deviation of the step symmetry of the last 30 steps. The plateau was considered to be reached when five consecutive 5-step averages were within the plateau window. The rate of adaptation was then defined as the step number of the first of those five consecutive 5-step averages.The rate of de-adaptation was calculated in the same manner, with the exception that the plateau was calculated as the mean of the last five steps (instead of the last 30 steps) because the de-adaptation period did not contain as many steps as the split-belt period.The step symmetry, magnitude of adaptation, and rate of adaptation. A 2-way ANOVA was used to evaluate the effect of group  and time  on step symmetry. A 1-way ANOVA was used to evaluate the effect of group  on the magnitude of adaptation  and the rate of adaptation. Post-hoc t-tests were used for specific comparisons that showed differences after completing the ANOVAs. Secondary variables included MOCA and n-back scores. T-tests were performed to evaluate the difference in MOCA scores and n-back scores between the HOA and MCI/AD groups. Pearson correlations were computed to detect correlations between the primary (locomotor) and secondary (cognitive) variables. SPSS version 24 (IBM) was used for all statistical analyses. We also similarly included analysis on belt speeds to evaluate whether group differences in belt speeds influence adaptation. Alpha level was set as 0.05.The primary dependent variables for analysis were Participant demographics are listed in A two-way ANOVA found no significant difference in belt speeds (slow or fast) between HOA and MCI/AD groups , 2. A PeThe average baseline or pre-tied step symmetry for HOA (0.001 ± 0.080) and MCI/AD (−0.022 ± 0.049) revealed no difference between groups , Table 2The one-way ANOVA revealed a larger magnitude of adaptation for HOA (−0.267 ± 0.102) compared to MCI/AD (−0.140 ± 0.048) . The ANOThe one-way ANOVA revealed no significant difference in magnitude of de-adaptation for HOA compared to MCI/AD . SimilarThe 2-way ANOVA evaluating the effect of group  and time  on step symmetry revealed a significant main effect of group and time. There was no interaction effect , Table 2Planned, pairwise comparisons revealed a significant difference between each time point pooled across groups . PlannedPlanned, pairwise comparisons between groups revealed a significant difference between HOA and MCI/AD at early adaptation , Table 2A significant difference was observed in the MOCA scores between HOA (28.8 ± 1.6) and MCI/AD (18.9 ± 5.2), as well as the n-back scores between HOA (0.84 ± 0.07) and MCI/AD (0.63 ± 0.16) . For botPearson correlation analyses revealed a significant relationship between MOCA score and early adaptation magnitude, with a higher MOCA score (better cognitive status) correlating to a greater magnitude of adaptation , Table 2We found a significant difference in split-belt locomotor adaptation between healthy older adults (HOA) and older individuals with mild cognitive impairments and Alzheimer's disease (MCI/AD). Individuals with MCI/AD showed a significantly reduced magnitude of locomotor adaptation . We found no between-group differences in baseline  step length symmetry or in the magnitudes and rate of de-adaptation. While our small sample preliminary study did not reveal differences in the rate of adaptation between HOA and MCI/AD, we observed much higher inter-individual variability in the time course and patterns of adaptation in individuals with MCI/AD. Furthermore, our correlation analyses revealed that individuals who showed smaller magnitudes of adaptation also demonstrated greater cognitive impairment . Interestingly, although there were no between-group differences during the de-adaptation phase of split-belt walking, we also found significant correlations between the magnitude of de-adaptation and cognitive impairment. Our study takes the first step toward our long-term goal of elucidating mechanisms underlying locomotor adaptation dysfunction and fall risk in older individuals with cognitive impairment.Revealing a significant effect of group , our results depicted a significantly reduced adaptation ability in cognitively impaired participants during early adaptation compared to healthy age-matched controls. We did not find significant differences during de-adaptation. Additionally, based on lack of significant differences in and correlations with belt speed, we infer that belt speed was not a major contributing factor for our observed effects on adaptation. Step-symmetry differences observed in HOA during early adaptation are somewhat consistent with previous literature on changes in gait with aging . Bruijn Walking in the real-world environment places high demands on the interplay between cognitive  and motor functions to adapt walking to rapidly evolving situations, terrains, and weather conditions. A well-functioning ability to sustain, shift, and divide attention between environmental and body function factors is essential for safe ambulation in everyday life. Unfortunately, cognitive dysfunction, the hallmark of MCI and AD, directly impacts the cognitive-motor neural resources available to carry out such activities of daily living , 2. TherSplit-belt walking, a unique adaptation task that induces complex asymmetries in the spatial and temporal coordination of walking patterns, has been used to investigate locomotor adaptation in various populations , 30–32. Despite finding a significant difference in magnitude of adaptation, we observed no significant difference between HOA and MCI/AD for the magnitude of de-adaptation nor the rates of adaptation and de-adaptation. The lack of significant difference for the magnitude of de-adaptation may be due to a small sample size. Due to the small sample, MCI and AD were grouped together to represent cognitively impaired individuals; however, our individual subject data suggested that AD participants showed greater average de-adaptation magnitude compared to those with MCI, both of which were greater than HOA. Both MOCA and n-back scores showed significant relationships with the magnitude of adaptation, suggesting that individuals with greater cognitive impairment also demonstrate a reduced capacity to adapt their walking in response to the split-belt perturbation. These relationships suggest that cognitive status may be an important contributor to walking function and the risk of falls in older individuals with cognitive impairments. Furthermore, given the absence of between-group differences in de-adaptation, we were surprised to find significant associations between de-adaptation magnitude and cognition, such that individuals who showed a larger magnitude of de-adaptation also showed greater cognitive impairment. Notably, we found considerable inter-individual variability in the time course of adaptation and de-adaptation in people with greater cognitive impairment in our cohort. Additionally, individual participant data revealed several examples wherein a small magnitude of early adaptation was accompanied by a relatively large magnitude of de-adaptation . The mecvia a “risky” adaptation , as seen in dementia patients, or a “secure” adaptation , common in HOA  to TK, NIH NICHD Grant (K12HD055931) to JN, and NIH NINDS (T32NS096050) to TP. This work was also funded by a pilot project grant awarded to TK and JN from the Emory Alzheimer's Disease Research Center (ADRC).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vaccination is a critical intervention in the fight against the coronavirus disease 2019 (COVID-19) pandemic. Various levels of COVID-19 vaccination acceptance have been observed around the world. However, a high percentage of the general population and healthcare professionals (HCPs), refuse the COVID-19 vaccination. This study aims to examine the factors influencing COVID-19 vaccine uptake among HCPs and the general population in Cyprus.An online cross-sectional study was conducted, using a self-administered questionnaire to collect information covering various potential determinants including sociodemographic and health-related characteristics, trust in the healthcare system, satisfaction with it, utilization of preventive healthcare services, COVID-19 vaccination information, and general vaccination knowledge.A total of 2582 participants completed the survey, with 53.5% of individuals in the general population, and 70.0% of the HCPs received the COVID-19 vaccination. We found that as the age increases by one year among the general population, the odds of being vaccinated against COVID-19 increase by 1.02 units , whilst those with increased trust in national healthcare authorities’ guidelines  and increased vaccination knowledge scores  were significantly more likely to be vaccinated. Furthermore, male HCPs , and those who reported increased trust in national healthcare authorities’ guidelines  were significantly more likely to be vaccinated.Public health policymakers can use national campaigns and long-term planning to build public trust in national healthcare authorities and educate and raise awareness about the benefits of vaccination. Such strategies could pave the way for adequate vaccine uptake and prepare the public for unfavorable scenarios, such as future pandemics.• Our results revealed the importance of vaccination knowledge and trust in healthcare system in respect to COVID-19 vaccination uptake.• The urgent need for national campaigns and long-term planning to build public trust in national healthcare authorities.


The currently preferred minimally invasive approaches have substantially improved outcomes of infected walled-off pancreatic necrosis (iWON). However, iWON with deep extension (iWONde) still poses a tricky challenge for sufficient necrosis evacuation by one stand-alone approach, often requiring repeated interventions. The aim of this study was to assess the effectiveness and safety of a minimal-access video-assisted retroperitoneal and/or transperitoneal debridement  in the management of iWONde.a ≥ 75% reduction in size of necrotic collection (in any axis) on CT and clinical resolution of sepsis or organ dysfunction. The primary safety endpoint was a composite of major complications or death during follow-up. Six-month postdischarge follow-up was available.Patients who had developed an iWONde were recruited to receive the VARTD in this prospective single-arm study. The primary efficacy endpoint was clinical improvement up to day 28 after the VARTD, defined as Between July 18, 2018, and November 12, 2020, we screened 95 patients with necrotizing pancreatitis; of these, 21 iWONde patients  were finally enrolled. The primary efficacy endpoint was achieved by most participants . No participants required repeated interventions. The primary safety endpoint occurred in six patients (29%). Except one in-hospital death attributable to repeated intra-abdominal hemorrhage, others were discharged without any major complication.The VARTD approach appears to have a reasonable efficacy with acceptable complication rates and thus might be an option for improving clinical management of iWONde.This study is registered with Chinese Clinical Trial Registry .The online version contains supplementary material available at 10.1186/s40001-023-01030-9. Acute pancreatitis is one of the leading causes of gastrointestinal-related admission to hospital , 2. ApprThe invasive approaches for managing WON have evolved over the past decade –3. HistoNevertheless, in the case of iWON with deep extension , it is something that still represents a challenging scenario for those minimal-access approaches , 3. In tIn this study, we attempted to solve the dilemma that no stand-alone minimal-access approach existed is suited for management of the infected necrosum with deep extension after failure of the “step-up” approach. Here, we introduced a minimal-access video-assisted retroperitoneal and/or transperitoneal debridement  that applies multi-mini-incision access for providing a practicable avenue to achieve sufficient clearance of the large avascular necrosis as much as possible, along with a continuous postoperative lavage by way of the flow that flushes from upper cavity towards lower zone, which may allow easy to irrigation and drainage of residual necrotic debris. The aim of this prospective single-arm study was to assess the effectiveness and safety of the VARTD for the iWONde.Patients admitted in our high-volume pancreas center with a diagnosis of necrotizing pancreatitis were screened for enrollment Fig. . The incThis single-center prospective single-arm trial was conducted at Daping Hospital, Army Medical University, China. This trial is registered with the Chinese Clinical Trial Registry, number ChiCTR1800016950. Ethical approval was obtained from the Ethics Board of Daping Hospital, Army Medical University, China (reference number: 2018-17). Signed informed consent was obtained from all participants or their legal representatives before enrollment.To ensure that all the participants undergo a more standardized and uniform VARTD approach to all the procedures and avoid biases caused by differences in surgical technical skill, allowing for evidence-based recommendations for its future use, the VARTD was performed by a team consisting of two experienced pancreatic surgeons (Y Tang and H Liu).After induction of general anesthesia, the patient was placed in the left/right lateral decubitus position. The decision about where to perform the skin incision depends on prior confirmation of the location of the necrosis on the CECT image, allowing the closest access route to the necrotic collection. First, a longitudinal/oblique mini-incision (approximately 3–5 cm) was made in the midaxillary line between the costal margin and the iliac crest Fig. A, B. TheTo assess the efficacy of the combined VARTD and continuous postoperative lavage in the treatment of iWONde, we prespecified the primary efficacy endpoint as clinical improvement up to day 28 after the VARTD, which defined as a reduction of 75% or greater in size of necrotic collection (in any axis) on CT and clinical resolution of sepsis or organ dysfunction within the first 4 postoperative weeks. Baseline CECT-derived parameters were measured before the VARTD. Repeat CECT scan was performed routinely for a period of 7–14 days, or when less than 50 mL per day fluid drained was observed, or in case of clinical suspicion of enteral/pancreatic fistula or intra-abdominal bleeding. The secondary efficacy endpoint was reintervention on for an additional debridement/necrosectomy. The primary safety endpoint was a composite of major complications comprising enterocutaneous or pancreatic fistula, visceral perforation, and intra-abdominal hemorrhage that require intervention; new-onset organ failure; in-hospital death, and death within 6 months after discharge. The secondary safety endpoints included individual primary endpoint components, biliary strictures, incisional hernia, wound infections, pancreatic endocrine and exocrine insufficiency, and intensive care unit (ICU) and hospital length of stay after the VARTD. Definitions of the safety endpoints were consistent with previous reports [The sample size of this study was estimated using software PASS version 15 . Based on published data, incidence rates of major complications/death are 40–69.5% for minimal-access surgical management in patients with necrotizing pancreatitis . On the Since this was an exploratory study, we used descriptive statistics to summarize the findings. All analyses were followed an intention-to-treat principle. Continuous variables were described using mean ± standard deviation (SD), median and interquartile range (IQR), and range. Categorical variables were reported as numbers and proportions. The statistical analyses were carried out using SPSS software version 20 .Between July 18, 2018, and November 12, 2020, a total of 95 NP patients admitted to our high-volume pancreatic center were screened, 66 of whom developed WON; of these, except for 1 patient who was eligible but declined participation, 21 iWONde patients  were finally enrolled and underwent the VARTD  who enrolled in the present study and underwent the VARTD approach (Table 1, 67% whResults for the primary and secondary safety endpoints are summarized in Table Currently, a step-up approach applying PCD and other minimal-access interventions has been proven to have more favorable outcomes for the management of WON , 14–18. Although several therapeutic schemes have emerged and significant progress towards reducing mortality and the risk of medical complications has been made in the treatment for NP during the last decade, it is clear that sufficient drainage and/or debridement remain the most important component in the management of WON, a delayed but life-threatening NP complication –3, 5, 9.The VARTD technique allows for good efficacy while evacuating the necrotic collections that located both centrally and diffusely throughout the abdomen. The continuous postoperative lavage also provides advantage in constant evacuations of necrotic debris, inflammatory exudate, vasoactive and toxic products, active enzymes, and bacteria and the toxins thereof , 29. In Results reported here also indicate an acceptable safety profile of the VARTD approach. The rates of postoperative complication-morbidity and mortality of the VARTD were substantially lower when compared with those of open surgical debridement as previously reported , 17, 25.Percutaneous catheter drainage or endoscopically transluminal drainage for necrotic collection is preferably postponed, usually > 4 weeks after the disease onset, waiting until necrosis has been encapsulated . FurtherThis was a proof-of-concept study and has some limitations. The small sample size and lack of the risk factor-matched control groups do not allow us to make conclusions about the real safety of the VARTD. Such may also lead to an overestimation of effectiveness of this technique in the face of improving patient outcomes, thus its efficacy should be interpreted in mind. Furthermore, a potential discrepancy between the participants initially treated at primary hospitals and at tertiary referral centers regarding patterns of clinical management prior to receiving the VARTD was not resolved, advertising the potential for an indispensable selection bias. In addition, there remain limited knowledge regarding infected WON with deep extension, and there is no consensus on standard definition for its clinical improvement, thus our a priori definition may result in design bias. This may lead to a reduction in the reintervention rate after the VARTD as compared with other minimal-access techniques as reported previously.In this study, the VARTD was performed as safely as the currently preferred minimal-access surgical approaches for the treatment of infected WON; the VARTD combined with continuous postoperative lavage showed a good efficacy for evacuating necrotic debris while avoiding additional interventions; thus, this approach may be an option for selected patients especially the ones developed the iWONde after failure of the “step-up” approach. To test these findings further, a large-scale, randomized trial involving multiple institutions and comparing the effects of the VARTD with other minimal-access techniques on outcomes of patients who had developed iWONde is warranted.Additional file 1. The following are available as supplementary data in the Additional files: Table S1 Individual participant data for demographics and baseline characteristics. Table S2 Measurements of CT-derived variables of necrosis at an individual level. Table S3 Intraoperative data. Table S4 Efficacy endpoints at an individual level. Table S5 Safety endpoints at an individual level.


Understanding the proportion of patients with COVID-19 who have respiratory bacterial co-infections and the responsible pathogens is important for managing COVID-19 effectively while ensuring responsible antibiotic use.To estimate the frequency of bacterial co-infection in COVID-19 hospitalized patients and of antibiotic prescribing during the early pandemic period and to appraise the use of antibiotic stewardship criteria.Systematic review and meta-analysis was performed using major databases up to May 5, 2021. We included studies that reported proportion/prevalence of bacterial co-infection in hospitalized COVID-19 patients and use of antibiotics. Where available, data on duration and type of antibiotics, adverse events, and any information about antibiotic stewardship policies were also collected.We retrieved 6,798 studies and included 85 studies with data from more than 30,000 patients. The overall prevalence of bacterial co-infection was 11% . When only confirmed bacterial co-infections were included the prevalence was 4% . Overall antibiotic use was 60% . Empirical antibiotic use rate was 62% . Few studies described criteria for stopping antibiotics.There is currently insufficient evidence to support widespread empirical use of antibiotics in most hospitalised patients with COVID-19, as the overall proportion of bacterial co-infection is low. Furthermore, as the use of antibiotics during the study period appears to have been largely empirical, clinical guidelines to promote and support more targeted administration of antibiotics in patients admitted to hospital with COVID-19 are required.The online version contains supplementary material available at 10.1186/s12879-022-07942-x. The COVID-19 pandemic has impacted health systems worldwide, with SARS-CoV-2 infection being implicated in more than 6 million deaths to date , 4.Distinguishing between viral pneumonia and bacterial co-infection at presentation and during the course of COVID-19 disease can be challenging due to various similarities, including characteristically high inflammatory markers and the frequent presence of pulmonary infiltrates on chest X-ray or computed tomography (CT) imaging . There iWe performed a systematic review to estimate the frequency of confirmed bacterial co-infection in patients admitted to hospital with COVID-19 pneumonitis, the frequency of empirical antibiotic use in this patient group, and to identify any antibiotic stewardship criteria that have been used during the COVID-19 pandemic to date.We registered the review protocol at the PROSPERO international prospective register of systematic reviews CRD 42020181215). We followed the method for the elaboration of systematic reviews recommended by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . Althoug81215. WeWe included studies with patients admitted to a hospital setting with suspected lower respiratory tract infection (LRTI) and with SARS-CoV-2 infection confirmed by PCR. Due to the high number of publications, we only included original studies with at least 10 participants and which provided enough information to appraise the methods used. Randomised and non-randomised studies that presented at least one of the following outcomes of interest were included: (a) prevalence of bacterial co-infection in patients with confirmed SARS-CoV-2 infection; (b) the proportion of patients with confirmed SARS-CoV-2 infection that were commenced on empirical antibiotic treatment. Where available, we collected information on the duration and type of antibiotics and on any related adverse events. In cases receiving specific treatment for COVID-19 as part of a clinical trial, we only included standard-of-care comparator arms. We excluded antibiotic use for indications other than bacterial LRTI . In order for our findings to be readily generalisable, we excluded pregnant women and patients with chronic immunosuppressive conditions, these being specific populations with different and increased infection risk profiles. We also excluded studies that mentioned bacterial co-infection rates but did not provide clinical details . Given that many authors provided only limited descriptions of antibiotic use, we performed two sub-analyses: one of studies clearly stating bacterial co-infection confirmed by cultures taken less than 48 h from point of admission, and another including only studies that clearly stated the empirical use of antibiotics. In the latter, we also describe any antibiotic stewardship strategies.We also performed sub-group analyses of any available data on critically ill patients, defined as those patients identified by study authors as requiring admission to high-dependency or intensive care. Definitions of bacterial co-infection provided by study authors were accepted.We searched the following databases up to May 5, 2021: Pubmed, LILACS, Embase, Web of Science and Cochrane Library. Our search strategy is given in Additional file We used a two-stage screening process to identify publications that would be eligible for inclusion: title and abstract, followed by full text review. Any original manuscripts referenced by systematic reviews but not identified by the initial search were also included if they were eligible. All publications were then screened in duplicate and independently by reviewers working in pairs ; any disagreements in screening were resolved by a third, independent reviewer (EH or BP). Data from eligible papers were extracted by two independent reviewers into separate, piloted and standardised Microsoft Excel spreadsheets; the third reviewer was then asked to resolve any discrepancies and a single consensus dataset was produced after discussion.2 statistic as a measure of the overall variation in the proportion that was attributable to between-study heterogeneity. STATA 17.0 was used for all analyses.We present the results of all included studies according to the selected outcomes of interest. We analysed our data using a proportion meta-analysis. We applied an arc-sine transformation to stabilise the variance of proportions (Freeman-Tukey variant of the arc-sine square-root of transformed proportions method), where y = arcsine[√(r/(n + 1))] + arcsine[√(r/(n + 1)/(n + 1)], with a variance of 1/(n + 1), with n being the population size. The pooled proportion was calculated as the back-transformation of the weighted mean of the transformed proportions, using inverse arcsine variance weights for the fixed and random effects models. Where heterogeneity between studies was found we applied DerSimonian-Laird weights for the random effects model. We calculated the ITo describe the quality of the prevalence data extracted from the included studies, we used The Joanna Briggs Institute (JBI) Critical Appraisal Checklist for cross-sectional/prevalence data . This isQuality of included studies were assessed independently by two investigators; any disagreements were resolved by a third senior investigator.Database searches identified 6798 studies. After removing duplicates and reviewing the secondary reference lists from included papers we screened a total of 4,132 studies for title and abstract. Of these, 162 (3.9%) went to full text review and 85 (2.1%) were selected for data extraction  and USA . The main characteristics of the included studies are shown in Table 2 99.2%)   Table . Meta-an2 94.2%) in the overall population  in critically ill patients.  gave a clear definition of bacterial co-infection, stating that this was diagnosed within 48 h from admission. All of them included cultures, urinary antigen and PCR for definitions of bacterial co-infection. We performed a meta-analysis of this subgroup that showed a prevalence of 4%    studies were included in the analysis of antibiotic use Table . Meta-an8%) Fig. A; sub-gr2%) Fig. B.Table 32 95.1%)   clearly described empirical antibiotic use. A sub-analysis of these papers found that overall empirical antibiotic use was 62%  in the overall population. Interestingly, we observed that bacterial co-infection was lower when including only studies with clear definitions of bacterial co-infection , critically ill patients 12% (95% CI 4 to 22%)). Our results are similar to that found by other authors who have evaluated co-infections in patients with COVID-19. For example, Rawson et al.  conducteIt is important to acknowledge that our estimates of the prevalence of bacterial co-infection prevalence were derived from a number of different definitions, as provided by the authors of the source papers. This is relevant, as although microbiological cultures are the gold standard for diagnosis, these are neither quick nor universally available tools on which to base prescribing decisions, particularly in patients with severe disease.Our study also finds that, as expected, the overall use of antibiotics in patients with COVID-19 is high compared to the estimated prevalence of bacterial co-infection. We identified a prevalence of empirical antibiotic use of 62% (95%CI 55 to 29%). These estimates are similar to those of Langford et al. , who fouThe final aim of our study was to identify to what degree decisions to stop empirically prescribed antibiotics were being made according to any defined criteria. This aspect has not been addressed previously in published systematic reviews and meta-analyses. Despite terms related to stewardship being specifically included in our search strategy and despite meticulously reading all included citations in full, including discussion sections, we found very little information on stewardship measures. We found this absence of information particularly notable given that antimicrobial resistance is widely acknowledged as being one of the most serious public health challenges of our times –25. WhilOur study has some important limitations. The most important being that the COVID-19 pandemic has given rise to an unprecedented situation in the scientific world in terms of a seemingly exponential increase in the volume of related publications over a very short time. Thus, at the time of writing there are likely to be additional studies that would have qualified for inclusion. This rapidity of publication would necessitate updating searches and analysis on as much as a weekly basis, which we suggest would be unrealistic for a piece of peer-reviewed work such as this. It is reassuring to know, however, that other groups pursuing similar research questions , 22, 28 Another important limitation is one that is inherent to this type of analysis. There is a consensus that the methodology for systematic reviews of prevalence data is not well developed, with a notable lack of methodological and reporting guidance for systematic reviews of prevalence data , 30. ThuIn this study we have reported bacterial co-infection and antibiotic use during the first 18 months of the SARS-CoV-2 pandemic. This work can help clinicians to reflect on and understand the initial response to a global pandemic of a novel respiratory virus. Our results show that there is currently insufficient evidence to support the use of empirical use of antibiotics in most hospitalised patients with COVID-19, as the overall proportion of bacterial co-infection in these patients is low. Furthermore, as the use of antibiotics in COVID-19 appears to have been largely empirical, it is necessary to identify clinical and laboratory markers and to formulate guidelines to promote more targeted administration of antibiotics in patients admitted to hospital with COVID-19.Additional file 1. Material S1. PRISMA check list. Material S2. Search Strategy. Material S3. Quality Assessment of included studies. Material S4. Reference list of included studies.


The Cocoon patent foramen ovale (PFO) Occluder is a new generation nitinol alloy double-disk device coated with nanoplatinum, likely useful in patients with nickel hypersensitivity. Early results and mid-term outcomes of this device in percutaneous PFO closure are missing.To assess the preliminary efficacy and safety profile of PFO closure with Cocoon device in an Italian multi-center registry.This is a prospective registry of 189 consecutive adult patients treated with the Cocoon PFO Occluder at 15 Italian centers from May 2017 till May 2020. Patients were followed up for 2 years.Closure of the PFO with Cocoon Occluder was carried out successfully in all patients, with complete closure without residual shunt in 94.7% of the patients and minimal shunt in 5.3%. Except from a case of paroxysmal supraventricular tachycardia and a major vascular bleeding, no procedural and in-hospital device-related complications occurred. No patient developed cardiac erosions, allergic reactions to nickel, or any other major complications during the follow-up. During the follow-up period, 2 cases of new-onset atrial fibrillation occurred within thirty-day.Percutaneous closure of PFO with Cocoon Occluder provided satisfactory procedural and mid-term clinical follow-up results in a real-world registry. The presence of patent foramen ovale (PFO) can be detected in about 25% of the adult population, with implication in the pathogenesis of different medical conditions as cryptogenic stroke, decompression illness, platypnea-orthodeoxia syndrome and, although still controversial, migraine with aura –3. When Nowadays, different systems are available for percutaneous PFO closure, including a suture-based system , 14. PerThe Cocoon PFO Occluder  was recently released as a novel nitinol alloy double-disk device, with a similar design to Amplatzer Occluder device . The Cocoon PFO Occluder features a specific nanoplatinum coating that should abolish the issue of nickel hypersensitivity (Ni-Hy) and smooth the microscopic geometry of the disks to minimize the risk of erosion. Initial evaluation of Cocoon septal Occluder for closure of atrial septal defects in adults and pediatric patients was recently presented, whereas procedural and follow-up data of the Cocoon PFO Occluder are missing .We herein feature a nation-wide registry concerning the acute procedural data and the mid-term clinical follow-up of adult patients with PFO treated with the Cocoon Occluder.In this multi-center, observational registry, adult patients ≥18 years old, with a clinical indication for PFO closure according to the current guidelines, treated with the Cocoon PFO Occluder were consecutively included –6. All tPatients were eligible for the procedure if they presented a documented history of cryptogenic stroke  or transient ischemic attack (TIA) in the previous 12 months, intractable migraine or need for PFO closure for professional reasons. Exclusion criteria were subjects with previous stroke or TIA with known etiology or with congenital or pre-existing neurological disorders  or intra-cranial disease; subjects with atrial fibrillation/flutter or other known emboligenic heart diseases; subjects with carotid, vertebral or basilar artery stenosis >50%; subjects with previous endocarditis or at risk for endocarditis; subjects with contraindications to aspirin, clopidogrel or anticoagulant therapy; subjects under tutorship, curatorship or unable to take the prescribed medical therapy.Baseline and procedural variables were collected from medical records through a web-based electronic case report form from each participating center. Follow-up data were then obtained by means of telephone calls and/or outpatients clinical visits.For the purpose of our analysis, we considered as efficacy outcome the occurrence of effective PFO closure with a residual minimal RLS or lower in acute and at the follow-up. As regards safety outcome, we considered the occurrence of peri-procedural major adverse events (MAEs) including death, acute neurological disorders, new-onset arrhythmia, major bleeding, cardiac tamponade or complications related to the vascular access, device malposition and embolization. The secondary safety outcome was a composite of MAEs detected during the follow-up including arrhythmia, late neurological disorders, endocarditis and device-related adverse events .Before the procedure, all the patients underwent contrast-enhanced transcranial Doppler ultrasound or transesophageal echocardiography with bubble test, in order to confirm the presence of the PFO and the severity of RLS. The grading of RLS through the PFO was semi-quantitative in accordance to the number of micro-bubbles detected in left atrium at rest and after Valsalva maneuver and defined as severe (≥20 bubbles or opacification), moderate (6–19 bubbles), minimal (1–5 bubbles) or no-shunt (0 bubbles) .All the patients signed an informed written consent.The registry was approved by the local ethical committees of each participating hospital and the study complied with the Declaration of Helsinki.The Cocoon PFO Occluder is an implantable self-expandable double-disk device, structurally similar to the Amplatzer Occluder device , 19. TheAll patients underwent the procedure under general anesthesia or conscious sedation, and were simultaneously studied by transesophageal echocardiography or intra-cardiac echocardiography, at discretion of each participating center. The procedure was performed as for standard for percutaneous PFO closure, with wire crossing through the PFO into the left upper pulmonary vein and delivery sheath advancing over the stiff wire, with final release of the intended device; an illustrative case is displayed in The patients were discharged with aspirin and/or clopidogrel and/or oral anticoagulant in accordance to their clinical history and at discretion of the physicians. In order to evaluate residual RLS, contrast-enhanced transthoracic echocardiography and transcranial Doppler follow-up were done within 3 months post procedure.Clinical FU was evaluated and recorded up to 2 years.th to 75th interquartile range–IQR), as appropriate according to their distribution. Continuous variables were analyzed for normal distribution using the Shapiro-Wilk test. The data were analyzed with SPSS statistics software .Categorical and dichotomous variables were expressed as absolute numbers and percentages whereas continuous variables were expressed as mean±standard deviation (SD) or median . Coagulation disorders were present in just 7.4% of patients. The main indications for PFO closure were cryptogenic stroke (42.3%) and TIA (32.3%). The basal shunt was severe in 65 patients (34.4%) and almost half of the patients presented atrial septum aneurysm (48.1%).*18 mm (73.0%). During the procedure, except a single case of paroxysmal supraventricular tachycardia and a major vascular bleeding (patient was on clopidogrel), no other significant adverse events occurred. Device embolization and recapturing or procedural cardiac erosions neither occurred. Post-procedural residual RLS was absent in most of the patients (94.7%), whereas a minimal RLS was documented in 5.3%.The procedure was performed in conscious sedation in 67.2% of patients . TransesNo adverse events occurred during the hospital stay. Most of the patients were discharged with indication to dual antiplatelet therapy with aspirin and clopidogrel (97.9%), for 1–12 months . Four paLife-long aspirin was prescribed in 27.0% of the cases, whereas life-long clopidogrel in 2.1%. Patients that were in treatment with anticoagulants before the procedure continued their standard treatment with vitamin K antagonists or direct oral anticoagulants.The choice of the antiplatelet/anticoagulant regimen was left to the attending physician in order to mimic the real world, in the absence of clear-cut evidence on the most appropriate therapy after PFO closure.Within 2 years post procedure, 2 cases (1.1%) of new-onset paroxysmal atrial fibrillation occurred, both within 3 weeks from the index procedure. No other major adverse events were reported. In all cases, contrast-enhanced echocardiographic and/or transcranial Doppler follow-up within 3 months after the index procedure showed absence of any severe RLS in all patients.Percutaneous PFO closure is a well-established therapy for paradoxical left thromboembolism , 20. MulThe main findings of this prospective nation-wide registry can be summarized as follows:- The novel Cocoon PFO Occluder showed good results in terms of efficacy  and safety.- The effective closure of the PFO was confirmed in all cases by means of appropriate imaging.- No cardiac erosion, allergic reactions or thrombotic events occurred both in the early phase and during the 2 year FU.- 2 cases (1.1%) of early new-onset paroxysmal atrial fibrillation occurred.The effectiveness of the Cocoon PFO Occluder in permanently closing the PFO seems to be consistent with those observed with the large RCTs concerning the Amplatzer Occluder and Gore Occluder  –9. IndeeThe nanoplatinum coating of the Cocoon PFO Occluder is supposed to soften the structure of the device, and thus minimize the risk of cardiac erosion, that is a rare but dramatic complication, more often observed with devices for the treatment of ASD , 21. WheThe specific design of Cocoon device may supposedly address the demands for device PFO closure in patients with Ni-Hy: the nanoplatinum coating theoretically prevent nickel ions release into the bloodstream. The Ni-Hy represents the classic presentation of a T cell-mediated, delayed-type hypersensitivity response to exogenous agents. The initial step is hapten binding to a skin carrier protein. The complex ultimately produces the sensitization of T cells. Sensitized T cells encountering the antigen at any time later will then lead to the release of cytokines, which in turn leads to macrophage activation and produces the immune response .In literature, the incidence and real magnitude of the device-related Ni-Hy “syndrome” is still a matter of debate, and skin reactions must be distinguished from the systemic reactions which, in extreme situations, may lead to surgical explant: this critical situation always occur weeks to months after the device implant. Preliminary studies have shown that, during the first period of endothelization after Cocoon device implantation, there was no nickel release into the bloodstream –26.A way to evaluate the presence of Ni-Hy might be the use of patches before the PFO closure, although it still unclear their role on top of clinical and physical examination .Contact allergy secondary to the Amplatzer , PFO-Star , and Gore Helex devices have been described previously –32.https://www.sciencedirect.com/topics/medicine-and-dentistry/patent-foramen-ovale PFO closure published in 2017, which included more than 2,000 patients, only 1 device-related allergic reaction was reported among the adverse events  of patients undergoing percutaneous closure had device removal . AllergyFor people who present Ni-Hy, the use of suture-mediated system may be proposed, but this technology has an 18–20% risk of significant residual RLS , 34, thuOn the other hand, whether the use of a Cocoon is the appropriate solution has to be confirmed beyond the theoretical basis.The polypropylene filling of the Coccon occluder may be associated with a reduced device-related thrombotic risk that, according to the literature, has been estimated with other devices between 1 and 2% , 17, 19.Of note, despite 7.4% of patients in our registry presented coagulation disorders, no device-related thrombotic events were recorded in the peri-procedural setting, and up to 2 years.In a recent publication, thrombus formation on the device was the justification for surgical excision in 4 (0.03%) of the 13,736 device implants. Of the 4 devices explanted with thrombus: 3 were CardioSEAL devices (0.15% of such devices) and 1 was an Amplatzer device (0.01%) .A clear understanding of the risk of thrombosis related to different PFO closure devices is quite challenging as only a large scale direct comparison with other devices, with a comparable thrombotic risk and medical therapy might be reliable.*18 mm. Due to the paroxysmal course of the arrhythmia, both cases were treated with antiarrhythmic drugs and life-long anticoagulant therapy. The incidence of AF after procedure seems to be lower than previous reports, likewise the incidence of supraventricular arrhythmias  received dual antiplatelet therapy for a period varying from 1 to 12 months, although the vast majority of them were on dual antiplatelet therapy for 1 to 3 months, and only 4 patients received a short-term single antiplatelet therapy, whereas 5 patients received anticoagulant therapy in association. This variability confirms the lack of a consensus about the pharmacological post-implantation approach, and highlights the need for more evidences –40.This is a prospective single arm registry and the endpoints were reported by the participating centers. The sample size of this study is relatively small, but, to the best of our knowledge, this is the largest experience so far concerning the real-world preliminary efficacy and safety profile of this new technology. The latter seems at least comparable with available and more extensively evaluated devices; the theoretical benefit in terms of increased safety has to be demonstrated.The Cocoon Occluder device showed a good performance in terms of shunt resolution and recurrence of neurological ischemic events. The low rate of peri-procedural complications supports the use of the Cocoon PFO Occluder in patients with indication to PFO closure. Larger scale and comparative studies with long-term follow-up are needed in order to confirm whether the technical features of this new technology actually translate into an improved safety/efficacy profile.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by each participating center EC. The patients/participants provided their written informed consent to participate in this study.LT and AP drafted and revised the manuscript. AP conducted the statistical analysis. LT and FB made substantial expert contributions. All authors contributed to the data collection. All authors contributed to the article and approved the submitted version.


Background - Chronic cerebral hypoperfusion (CCH) is an important pathophysiological mechanism of vascular cognitive impairment (VCI). The heterogeneous effects of CCH complicate establishing single target therapies against VCI and its more severe form, vascular dementia (VaD). Intermittent fasting (IF) has multiple targets and is neuroprotective across a range of disease conditions including stroke, but its effects against CCH-induced neurovascular pathologies remain to be elucidated. We therefore assessed the effect of IF against CCH-associated neurovascular pathologies and investigated its underlying mechanisms.Methods - Male C57BL/6NTac mice were subjected to either ad libitum feeding (AL) or IF (16 hours of fasting per day) for 4 months. In both groups, CCH was experimentally induced by the bilateral common carotid artery stenosis (BCAS) method. Sham operated groups were used as controls. Measures of leaky microvessels, blood-brain barrier (BBB) permeability, protein expression of tight junctions, extracellular matrix components and white matter changes were determined to investigate the effect of IF against CCH-induced neurovascular pathologies.Results - IF alleviated CCH-induced neurovascular pathologies by reducing the number of leaky microvessels, BBB breakdown and loss of tight junctional proteins. In addition, IF mitigated the severity of white matter lesions, and maintained myelin basic protein levels, while concurrently reducing hippocampal neuronal cell death. Furthermore, IF reduced the CCH-induced increase in levels of matrix metalloproteinase (MMP)-2 and its upstream activator MT1-MMP, which are involved in the breakdown of the extracellular matrix that is a core component of the BBB. Additionally, we observed that IF reduced CCH-induced increase in the oxidative stress marker malondialdehyde, and increased antioxidant markers glutathione and superoxide dismutase. Overall, our data suggest that IF attenuates neurovascular damage, metalloproteinase and oxidative stress-associated pathways, and cell death in the brain following CCH in a mouse model of VCI.Conclusion - Although IF has yet to be assessed in human patients with VaD, our data suggest that IF may be an effective means of preventing the onset or suppressing the development of neurovascular pathologies in VCI and VaD. Vascular cognitive impairment (VCI) embodies a spectrum of cognitive deficits that range from mild cognitive impairment to vascular dementia (VaD). Due to a considerable increase in the aging population, VCI is becoming a major public health concern worldwide CCH induces a cascade of cellular and molecular mechanisms that contributes to the pathogenesis of VCI - including oxidative stress and inflammation. CCH has been reported to increase the levels of matrix metalloproteinases (MMPs), proinflammatory cytokines such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor (TNF) Intermittent fasting (IF) is defined as an eating pattern that cycles between periods of eating and fasting. IF has been extensively reported to extend both health and lifespan, and decrease the development of age-related disorders including cardiovascular, metabolic and neurodegenerative diseases In this study, we demonstrate for the first time that IF promotes neuroprotective effects in a BCAS model of VaD by maintaining the integrity of the neurovascular structures in the brain. We specifically show that IF attenuated vascular pathology by reducing microvascular leakage and BBB dysfunction, while maintaining the expression of tight junction (TJ) proteins. IF was also effective in decreasing WML formation, hippocampal neuronal cell death and cell death markers, while maintaining myelin basic protein levels. Our data suggest that the effects of IF on the structural integrity of the neurovasculature may be mediated through mechanisms that decrease oxidative stress and matrix metalloproteinase expression. Overall, our findings indicate that prophylactic IF may be a potential therapy in reducing and preventing neurovascular pathology associated with VaD.Figure , at eight weeks of age, the mice were randomly assigned to either the ad libitum (AL) or the intermittent fasting (IF) diet regimen. Mice under the IF regimen underwent 16 hours of fasting every 24-hour period, with food available for the remaining 8 hours from 07:00 to 15:00 for four months . All in vivo experimental procedures were approved by the National University of Singapore, Singapore Animal Care and Use Committee and performed according to the guidelines set forth by the National Advisory Committee for Laboratory Animal Research, Singapore. All efforts were made to minimize any suffering and number of animals used. All sections of the manuscript were performed and reported in accordance to ARRIVE  guidelines. In a separate set of experiments, animals from both AL and IF groups  underwent physiological measurements such as body weight, blood glucose and ketone levels throughout the four months as shown in Figure .Six-week-old wild-type male C57BL/6NTac mice (n=150) were obtained from InVivos, Singapore and housed upon arrival at the National University of Singapore Animal Facility. As shown in At six months of age, AL and IF experimental animals were further divided into three experimental groups: Sham, 15-day bilateral common carotid artery stenosis (BCAS) and 30-day BCAS. C57BL/6NTac mice were subjected to chronic cerebral hypoperfusion injury by bilateral common carotid artery stenosis (BCAS) surgery, as previously described Figure , the brain regions of interest (ROI) between the bregma and the lambda were selected to measure arbitrary units of CBF in the area between the cerebral hemispheres. Briefly, the fur on the head was removed by shaving and the skull was exposed via a midline skin incision. The skull was cleaned gently using a cotton applicator with 1×phosphate-buffered solution (PBS). Throughout the imaging of CBF, the skull was kept moist and in order to improve imaging resolution, a non-toxic silicon oil was applied on the skull. Body temperature was maintained at 37 ± 0.5ºC throughout the measurement periods. In the PeriCam PSI System, a CCD camera (2448×2048 pixels) that can take images in real-time was installed 10cm above the skull (speed 120 frames per second). Images were then acquired and analysed using a dedicated software package .The Laser Speckle Contrast Imager  was used to obtain high-resolution cerebral blood flow (CBF) measurements before BCAS (baseline), immediately after BCAS surgery (post-BCAS) and finally at the end points of BCAS (before sacrifice). As shown in Animals were euthanized by inhaled carbon dioxide and the brains were harvested at their respective time points. Different brain regions  were isolated immediately on ice and frozen in dry ice for future analysis . Detailed immunoblot analysis procedures were performed as previously described th percentile, to best represent the area closest to the bregma.At the end of each timepoint, a subset of animals  were deeply anaesthetized with isoflurane, and perfusion through the heart was performed as described previously At the end of each timepoint, a subset of animals  underwent Evans Blue (EB) Staining analysis as described previously Luxol fast blue (LFB) staining and expression of myelin basic protein (MBP) revealed myelin integrity, while cresyl violet staining and expression of cell death markers were indicative assessments for neuronal loss in the hippocampus and different brain regions. Mouse brain tissues were fixed in 10% neutral buffered formalin and then processed into paraffin wax blocks. Coronal sections (3μm thick) were obtained via microtome sectioning  and collected on slides . Luxol Fast Blue (LFB) staining was performed to detect the severity of white matter lesions (WMLs) in 5 regions  in the brain. Briefly, tissue sections were de-waxed and rehydrated before immersion into the LFB solution  at 37ºC overnight. Excess staining was removed using 95% ethanol treatment followed by washing in deionized water. Gray and white matter differentiation was initiated with the treatment of 0.05% aqueous lithium carbonate  for 20s, followed by 70% ethanol until the nuclei was decolorized. Sections were then immersed in Cresyl Violet solution  for 5 mins and the excess staining was washed in deionized water. The sections were then dehydrated in an ethanol gradient (70-100%), and finally cleared in xylene and mounted onto the slides . Brightfield images were taken under ×60 magnification using an Olympus upright Fluorescence Microscope BX53. A WML severity index was calculated by giving each brain region a grade of either: Grade 0 , Grade 1 (disarrangement of nerve fibres), Grade 2 (formation of marked vacuoles), or Grade 3 (disappearance of myelinated fibres). The severity of the WMLs was scored by three blinded examiners and the number of neurons in CA1, CA2 and CA3 regions of the hippocampus were counted as previously described Experimental data were analysed by GraphPad Prism 8.0 software . All values were expressed as mean ± standard error of the mean. One-way analysis of variance was used, followed by a Bonferroni post-hoc test to determine the significance between experimental groups. A P-value < 0.05 was deemed to be statistically significant. All experimental groups were compared against AL Sham conditions.Fig. 1). At eight weeks of age, the mice were randomly assigned to either the ad libitum (AL) or the intermittent fasting (IF) diet regimen . Male C57BL/6NTac mice were fed a normal chow diet . Mice were randomly assigned to Sham, 15-day BCAS and 30-day BCAS groups at six months of age when surgeries were performed. First, we monitored the physiological effects of IF on these mice. The IF group had a significantly lower body weight, a non-significant reduction in blood glucose level and a significant increase in blood ketone level than AL mice . We examined dynamic changes and recovery of cerebral blood flow (CBF) in the animals  using the Laser Speckle Imaging Technique. The CBF was measured at three time points i.e. baseline, post-surgery and before sacrifice in both AL and IF animals over 15 and 30 days following BCAS. At baseline, animals from all experimental groups had high CBF as indicated by the red perfusion colour. Sham did not show any reduction in CBF, as no microcoils were inserted onto the common carotid arteries. However, expectedly, following BCAS surgery (Post-surgery), AL and IF groups had a significant reduction in CBF compared to their corresponding Sham counterparts. Before sacrifice, at 15 days and 30 days post-surgery, CBF was found to increase and be restored in BCAS animals. Interestingly, for the first time in the VaD model, we show that IF was able to demonstrate a better CBF recovery as compared to AL following hypoperfusive injury.The summarized study design includes the timing of interventions, and blood and tissue collection .We next analysed the effect of IF against hallmark vascular pathologies involving cerebrovascular structural and BBB integrities following CCH. The first vascular pathology examined was the number of microvascular leakages that occurred following CCH injury. Fluorescence imaging of vasculature staining using a carbocyanine dye perfusion indicated a significant increase in the average number of microvascular leaks at 30 days after BCAS in AL animals, while IF significantly reduced the number of microvascular leaks . BBB permeability after CCH was found to be significantly increased following 15 and 30 days of BCAS under AL conditions. In contrast, IF reduced BBB permeability in both sham and BCAS mice . To further analyse the structural integrity of the BBB, we next examined the expression levels of TJ proteins between endothelial cells in the cortex, hippocampus and cerebellar regions . There was evidence of reduced TJ expression following BCAS compared to Sham in AL animals, and IF was able to increase and maintain higher levels of TJ expression in all three brain regions. TJ protein zonula occludens (ZO-1) showed a significant reduction in expression levels following 30-day of BCAS at the cortex and the hippocampus, and a non-significant reduction in the cerebellum. Conversely, IF increased the expression of ZO-1 in the cortex and cerebellum at both 15-day and 30-day time points . Expression of occludin was significantly reduced following 15-day and 30-day BCAS at the cortex and cerebellum regions in AL animals, while IF increased occludin expression significantly at both 15-day and 30-day timepoints in the cortex and cerebellum, but only at the 30-day timepoint in the hippocampus . Claudin-5 levels were maintained following 15-day BCAS, but increased following 30-day BCAS in the cortex and cerebellum. In the hippocampus, Claudin-5 levels showed a significant reduction in AL mice following the 30-day BCAS timepoint. IF increased Claudin-5 levels in the cortex at the 15-day timepoint, and reduced Claudin-5 levels at the cerebellum at the 30-day timepoint. IF maintained Claudin-5 levels following 15-day and 30-day BCAS in the hippocampus . Junctional adhesion molecule (JAM-A) expression was reduced significantly in AL animals following BCAS in the cerebellum and non-significantly in the cortex, but maintained in the hippocampus. IF maintained JAM-A expression levels in the cortex and hippocampus, but increased expression at the 15-day timepoint in the cerebellum . These data thus indicated that IF likely protected against CCH-induced BBB breakdown by maintaining TJ proteins compared to the AL diet.The second vascular pathology examined was the extent of BBB damage following CCH injury. We analysed BBB damage using the Evans Blue technique and additionally examined TJ protein expression levels via immunoblot analysis in the AL and IF mice following BCAS . WM integrity after 15 or 30 days of CCH was assessed in 5 brain regions including the corpus callosum (paramedian), corpus collosum , caudoputamen, internal capsule and optic tract . The severity of WM damage was significantly increased in the corpus callosum (paramedian) at the 30-day timepoint  and caudoputamen at 15-day and 30-day timepoints  in AL mice following BCAS. At the corpus callosum   and internal capsule , AL animals showed a non-significant, but more severe disruption in white matter compared to their IF counterparts. We found no change in white matter disruption at the optic tract . IF was able to generally maintain a lower WM severity score as compared to their AL counterparts at all 5 regions, specifically significant at the internal capsule in Sham and 15-day timepoints. We found a significant decrease in the expression of MBP in the cortex and cerebellum at the 30-day timepoint in AL animals . IF increased MBP expression in the cerebellum in BCAS mice at the 15-day timepoint .We next analysed the effect of IF on neuronal pathologies such as white matter (WM) integrity, neuronal loss and cerebral cell death. The first neuronal pathology we examined was WM integrity and the corresponding WM protein known as myelin basic protein (MBP) . Sham controls in all three regions showed densely packed nuclei, but there were signs of neuronal loss at CA1, CA2 and CA3 regions evident following BCAS in AL animals. In the CA1 region, neuronal count was reduced at the 30-day timepoint in AL animals . In the CA2 region, the neuronal count showed a non-significant reduction in neuronal numbers following BCAS in AL animals . In the CA3 region, there was a reduction in neuronal numbers at the 15-day timepoint following BCAS in AL animals . IF was able to rescue degeneration of neurons significantly at the 15- and 30-day time points as compared to their AL counterparts in all three hippocampal regions .The second neuronal pathology examined was hippocampal neuronal loss. BCAS-induced neuronal loss was evident in hippocampal regions . Our data shows that although total caspase-3 levels remained unchanged in all three brain regions. Cleaved caspase-3 was found to show little change in the cortex , a non-significant increase in the hippocampus , and a significant increase at the 15-day timepoint at the cerebellum  in AL animals, which suggests CCH-mediated brain loss to be partly mediated by activation of the apoptotic caspase pathway. IF animals had reduced expression of cleaved caspase-3 at the sham and 30-day BCAS timepoints at the cortex, and at the 15-day timepoint at the cerebellum.The third and final neuronal pathology examined was cerebral cell death following CCH. Therefore, we analysed the expression levels of caspase-3 in the cortex, hippocampus and cerebellum as a marker for apoptotic cell death . Our data established evidence for significantly increased expression of membrane type-1 matrix metalloproteinase (MT1-MMP) at the cortex, and non-significant increase in MT1-MMP levels in the hippocampus and cerebellum following BCAS . IF was able to significantly reduce MT1-MMP levels at the 15-and 30-day timepoints in the hippocampus and at the 30-day timepoint in the cerebellum . Downstream pro-MMP-2 showed a significant increase following BCAS in the cortex at the 30-day timepoint, and a non-significant increase in the hippocampus and cerebellum in AL animals . IF was able to significantly reduce the CCH-induced increase of pro-MMP-2 levels at the cortex and cerebellum at the 15-day and 30-day time points, and at 30-day timepoint in the hippocampus . Additionally, we found a decrease in pro-MMP-9 levels at the 15-day and 30-day timepoints in the cortex, but no changes in expression of pro-MMP-9 at the hippocampus and cerebellum, although pro-MMP-9 levels were downregulated in the hippocampus in IF BCAS animals at the 30-day timepoint  suggesting that the MMP-9 pathway is not induced during CCH-associated injury.As various cellular and molecular pathways have been implicated in CCH-induced brain injury, we postulated the involvement of matrix metalloproteinases (MMPs) in the breakdown of the extracellular matrix in the brain that could mediate both vascular and neuronal pathologies . Our data showed that the oxidative stress marker, malondialdehyde, was significantly increased following 30 days of BCAS injury in AL animals at the cortex, but non-significantly in the hippocampus and cerebellum . IF reduced malondialdehyde levels at the 30-day timepoint in the cortex and at the 15-day timepoint in the hippocampus and cerebellum . Antioxidant glutathione levels were non-significantly lower in AL animals at the cortex and cerebellum following BCAS, and at the hippocampus, glutathione levels remained unchanged. IF was able to significantly increase expression levels of glutathione at the cortex and cerebellum at the 30-day timepoint, but showed no change at the hippocampus . Antioxidant superoxide dismutase was lower at the 15-day timepoint at the cortex and hippocampus but showed non-significant lower levels at the cerebellum. IF was able to increase superoxide dismutase levels at the cortex and cerebellum at the 30-day and 15-day time points respectively .Another mechanism we postulated to be involved in CCH-induced brain injury was oxidative stress. We, therefore, analysed oxidative stress markers in the cortex, hippocampus and cerebellum . Collectively, these data are the first to demonstrate the effect of IF on vascular and neuronal pathologies following CCH injury, identifying IF as a potential therapeutic intervention for VCI.Our study establishes a consensus of the beneficial effect of IF against age-related diseases. The effect of IF achieved through intermittent metabolic switching which implies recurring periods of a bioenergetic challenge. IF leads to depletion of liver glycogen stores and production of ketones from adipose-cell-derived fatty acids, followed by recovery period. This metabolic switch results in coordinated molecular and cellular adaptations involving various signaling cascades. Current findings indicate such a metabolic switch protects against CCH-induced neurovascular pathology in an animal model of vascular dementia. Prophylactic IF attenuated microvascular leakage, BBB permeability, TJ breakdown, white matter injury, neuronal loss and cell death through a reduction in MMP and oxidative stress levels  at the molecular and cellular levels by which IF alleviates both vascular and neuronal pathologies following CCH. Here, we investigated pathways previously implicated in BBB breakdown, namely the activation of matrix metalloproteinases (MMPs) and oxidative stress. Previously, an increase in matrix metalloproteinases, particularly MMP-2 under CCH has been reported The precise mechanisms through which IF promotes VaD pathological tolerance is not yet completely deciphered. Nevertheless, future work in systematically studying these mechanisms and the molecular pathways are warranted. However, it was previously established that IF protects against age-related diseases such as cardiovascular diseases and neurodegeneration by targeting dysregulated energy mechanisms, oxidative stress, mitochondrial dysfunction, impaired lysosome and proteasome function, inflammation, cellular senescence and promoting the expression of genes by modulating epigenetic mechanisms that are essential for plasticity and regeneration In summary, we have provided substantial evidence that prophylactic IF is effective in reducing vascular and neuronal pathologies following CCH. While our study establishes IF as a potential therapeutic approach for attenuating VCI-related pathologies, further research is needed to test its clinical potential.Supplementary figures.Click here for additional data file.Supplementary data.Click here for additional data file.


Human prion protein and prion-like protein misfolding are widely recognized as playing a causal role in many neurodegenerative diseases. Based on in vitro and in vivo experimental evidence relating to prion and prion-like disease, we extrapolate from the compelling evidence that the spike glycoprotein of SARS-CoV-2 contains extended amino acid sequences characteristic of a prion-like protein to infer its potential to cause neurodegenerative disease. We propose that vaccine-induced spike protein synthesis can facilitate the accumulation of toxic prion-like fibrils in neurons. We outline various pathways through which these proteins could be expected to distribute throughout the body. We review both cellular pathologies and the expression of disease that could become more frequent in those who have undergone mRNA vaccination. Specifically, we describe the spike protein’s contributions, via its prion-like properties, to neuroinflammation and neurodegenerative diseases; to clotting disorders within the vasculature; to further disease risk due to suppressed prion protein regulation in the context of widely prevalent insulin resistance; and to other health complications. We explain why these prion-like characteristics are more relevant to vaccine-related mRNA-induced spike proteins than natural infection with SARS-CoV-2. We note with an optimism an apparent loss of prion-like properties among the current Omicron variants. We acknowledge that the chain of pathological events described throughout this paper is only hypothetical and not yet verified. We also acknowledge that the evidence we usher in, while grounded in the research literature, is currently largely circumstantial, not direct. Finally, we describe the implications of our findings for the general public, and we briefly discuss public health recommendations we feel need urgent consideration.An earlier version of this article was previously posted to the Authorea preprint server on August 16, 2022. C, whereas the misfolded form is called PrPSC (for "scrapie"). Disease propagation occurs through an autocatalytic process whereby external misfolded prion proteins (PrPSC) act as an infectious agent to facilitate misfolding of the same protein expressed in neurons. It is now generally recognized that an intermediate soluble oligomeric form of the protein is the toxic agent. In contrast, the insoluble plaque may even be protective in that it results in the clearing of the soluble oligomers [Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of rare, consistently fatal brain diseases affecting animals and humans. They are caused by 'proteinaceous infectious particles,' which can facilitate disease spread in the absence of a classical infection by a living organism. They include the familiar mad cow disease , scrapie in sheep, and chronic wasting disease (CWD) in deer. The primary human prion disease is known as Creutzfeldt-Jakob disease (CJD), and it is always fatal. Fatal familial insomnia (FFI) is a rare fatal genetic disease caused by certain mutations in the prion protein. In common nomenclature, the naturally folded form of the prion protein is referred to as PrPligomers .It is iligomers ,3. Both ligomers -6. Furthligomers . Proteinligomers .A remarkable study involved a mutated variant of a bacterial plasmid initiator protein, RepA, that builds intracellular amyloid oligomers triggering a lethal cascade in bacteria, similar to the mitochondrial impairment of human cells in neurodegeneration . In thisThe spike protein has prion-like domainshttp://plaac.wi.mit.edu/) employs HMM technology in order to identify candidate prionogenic domains in proteins of viruses, prokaryotes, and eukaryotes [The COVID-19 mRNA vaccines are based on lipid nanoparticles containing mRNA encoding the SARS-CoV-2 spike glycoprotein. The vaccine has been engineered in several ways to protect the mRNA contents from breakdown and to assure that cells transfected with it produce large quantities of spike protein at a high production rate over a long time period -18.A cokaryotes ,23. The karyotes .Table 1karyotes ,25-28. IA study evaluating the amyloidogenic potential of the spike protein used both theoretical and experimental methods to verify that the SARS-CoV-2 spike protein can cause amyloid-like fibrils to appear after the protein has been subjected to proteolysis. Theoretical predictions identified seven potentially amyloidogenic sequences within the spike protein. In laboratory experiments where the protein was incubated with the protease neutrophil elastase, amyloid-like fibrils appeared during 24 hours of coincubation. A specific segment, spike 194-213 (FKNIDGYFKI), was highly abundant after six hours, and it overlapped almost completely with the most amyloidogenic sequence identified theoretically. Neutrophils responding to immune activation release neutrophil elastase into the medium, where it would have access to the spike protein and be able to break it down into the amyloidogenic segments .The SARProf. Luc Montagnier is a recently deceased Nobel-prize winner for his work on HIV. A recently published paper co-authored by Montagnier describes 26 cases where the patient became severely ill with spontaneous symptoms of CJD shortly after a COVID-19 vaccine. Twenty-three out of the 26 cases developed symptoms within 15 days of their second injection of an mRNA vaccine. The other three cases were associated with the AstraZeneca DNA vector vaccine, and symptoms appeared within the first month. Of the 26, 20 had died at the time of writing the paper, and the remaining six were in critical condition. The mean time to death was under five months after the injection . CJD is Biodistribution of mRNA vaccinesAs early as 1979, it was recognized that exposure of mice to scrapie prion protein, regardless of whether it was via intraperitoneal, IV, or multiple subcutaneous routes, always showed the same pattern of spread of infectivity. Propagation in the spleen consistently showed up well before a noticeable spread to the spinal cord, with infectivity in the brain requiring the longest incubation period. A conclusion was that immune cells carry the infectious proteins into the spleen, and further spread of the infectivity occurs mainly along nerves rather than via the vasculature or the lymphatic system .Unlike PrP, which is highly expressed in the nervous system but expressed at much lower levels in a plethora of other tissues, the amyloid precursor protein (APP) is highly expressed in many tissues apart from the nervous system, including muscles, the liver, the immune system (thymus and spleen) and many other organs ,43.Only a few studies have been conducted to examine the biodistribution of mRNA from vaccines subsequent to injection. A study published in 2017 tracked the distribution of mRNA coding for influenza haemagglutinin proteins following injection into mouse muscle. They quantified the maximum level of mRNA found in various organs and used these data to infer the migration pathway of the mRNA. As expected, by far, the highest concentration remained in the muscle . However, a substantial amount was found in the proximal lymph nodes , with significantly smaller amounts in the distal lymph nodes (177.0 ng/mL). Among the organs, the spleen and liver had by far the highest concentrations (86.9 ng/mL in the spleen and 47.2 ng/mL in the liver). Smaller amounts were found in the plasma (5.47 ng/mL), bone marrow (3.35 ng/mL), ileum (3.54 ng/mL), and testes 2.37 ng/mL, with trace amounts in many other organs, including the brain (0.429 ng/mL) .Another study tracked the biodistribution pathway of a rabies mRNA vaccine administered intramuscularly to rats. They found that the mRNA appeared in the draining lymph nodes within one day and was also found in blood, lungs, spleen, and liver . DevelopWe recognize and acknowledge that these are animal-based biodistribution studies. As such, their relevance to humans is speculative. These studies are conducted, though, on the expectation that they have some relevance to human biodistribution. In the absence of human studies that would clarify this topic, we include the in vivo studies as the best available evidence that is potentially analogous to human biodistribution.Exosomes and microRNAsC) and its misfolded (PrPSC) form. Furthermore, exosomes containing PrPSC are infectious [Exosomes are membranous secreted nanovesicles 30-150 nm in size, generated and released by all cells, often under conditions of stress. These extracellular vesicles are produced in late endosomes by the inward budding of the endosomal membrane. Their cargo is diverse and can include nucleic acids, proteins, lipids, and metabolites. They mediate both near and long-distance intercellular communication through contents that include signaling molecules, nutrients, and toxins. In particular, their lipid membrane can protect internalized RNA molecules from degradation by extracellular ribonucleases. A paper published by Wei H et al. in 2021 provides an excellent review of the complex mechanisms that control sorting proteins, RNAs, and other molecules into exosomes for export and delivery to other cells .It has fectious . Exosomefectious . In a mofectious . Tau profectious . Finallyfectious .One of fectious ,57.Bothfectious . Two miRfectious . Both hafectious .It has been demonstrated experimentally that exosomes play an essential role in cell-cell communication between T cells and B cells during the process of antibody production following antigen presentation in germinal centers. Three specific miRNAs, one of which was miR-155, were identified as being present in these exosomes and were essential for eliciting the appropriate B-cell response. The miRNAs promoted survival, proliferation, and antibody class switching in the B cells, all essential for the antibody production process .We haveStaphylococcus aureus [A review paper by Pogue AI and Lukiw WJ showed that miRNAs are abundant in the brain and participate in the progression of many age-related neurological disorders. miR-146a was singled out as a pro-inflammatory contributory factor in several neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), macular degeneration, multiple sclerosis, temporal lobe epilepsy, and CJD .As was s aureus . FurtherIn a previous review publication, we proposed that a major effect of the mRNA vaccines was to inhibit type I interferon (IFN) signaling, leading to increasing susceptibilities to the activation of latent viruses and cancer . This hyCD16+ monocytes and toll-like receptor 4As many as 30% of patients infected with SARS-CoV-2 continue to experience debilitating symptoms long after the virus has cleared. This condition, referred to colloquially as "long COVID," has also been formally named "post-acute sequelae of COVID" (PASC). Common symptoms include breathlessness, fatigue, brain fog, inflammation, and coagulopathies. A study based on 46 individuals suffering from PASC found that two specific non-classical monocyte types,  and  were significantly elevated in the PASC patients up to 15 months after the acute infection. A statistically significant number of these non-classical monocytes were found to still contain SARS-CoV-2 S1 protein up to 15 months post-infection [A follow-on preprint human study involved individuals who experienced PASC-like symptoms post-vaccination for COVID-19. CD16+ monocytes were isolated from six of these patients, and it was confirmed that they, too, contained both S1 and S2 sequences, as well as several mutant S1 peptides . It was SC's cytotoxicity is dependent upon its ability to facilitate LINE-1 retrotransposition activity [SC, and, by analogy, the spike protein itself, which is also an RNA-binding protein, may facilitate the retrotranscription of spike protein mRNA into DNA, mediated by LINE-1. While LINE-1 is inactive in most cells, neurons, like cancer cells and immune cells, actively express LINE-1, especially in association with neurodegenerative diseases [The Gag polyprotein, present in all retroviruses, is an essential nucleic-acid-binding protein that coordinates many aspects of virion assembly as an important step towards reverse transcription and integration into the host DNA . A paperactivity . This lediseases ,75. The Fibrinogen in the blood can clot into an anomalous amyloid form of fibrin that, similar to other β-rich amyloids and prions, is relatively resistant to proteolysis (fibrinolysis). In a human study where blood and platelet-poor plasma samples (PPP) were collected from healthy volunteers and COVID-19 patients, it was concluded that the SARS-CoV-2 spike protein produces blood hypercoagulation and fibrinogen changes in a direct way, even when it is not taken up by cells . A paperBlood monocytes recognize endotoxins produced by Gram-negative bacteria through a toll-like receptor 4 (TLR4) pathway . The TLRA carefully conducted experiment has demonstrated convincingly that the SARS-CoV-2 spike protein binds to TLR4 and activates it. The spike trimer directly binds to the TLR4 receptor with an affinity of ∼300nM, comparable to the binding strength of many virus-receptor interactions. Furthermore, the spike protein robustly induces the inflammatory agent Il-1β, and this induction is lost when TLR4 inhibitors are added . It is cA research paper by a team in Boulder, CO, focused on the S1 subunit of the spike protein and demonstrated that injection of the S1 segment into the cisterna magna of adult male Sprague-Dawley rats resulted in behavioral deficits, microglial activation, and neuroinflammatory response. They determined that S1 signals via a pathogen-associated molecular pattern (PAMP). In vitro experiments on transgenic TLR4 human embryonic kidney (HEK)-293 cells showed that S1 binds to TLR4 receptors to induce upregulation of TNF-α and other proinflammatory cytokines .There is a growing body of evidence supporting the role of TLR4 in PD . TLR4 exFigure Severe COVID symptoms share many features with sepsis . The CD1The  monocytes have been recognized as exhibiting higher expression of proinflammatory cytokines and higher potency in antigen presentation than other monocytes. As such, they play a crucial role in infection and inflammation . In a stFurthermore, abnormally high levels of  monocytes are also associated with sarcoidosis and complex regional pain syndrome, a condition associated with neurogenic inflammation ,94. SarcWe recognize that the connections we are proposing between these cellular events and the clinical manifestation of inflammation and specific pathologies rely upon an inferential chain that links in vitro and in vivo data to histological findings in human subjects. Nevertheless, we believe it is important to consider these potential connections so they might be more rigorously investigated.Potential interactions between the spike protein and PrPThe AIDS virus, HIV, invades the central nervous system where it causes neuroinflammation leading to cognitive impairments. A study published in 2017 demonstrated that TNF-α expression, induced by HIV, led to the shedding of PrP from astrocytes in the brain. The levels of PrP in the cerebrospinal fluid of AIDS patients suffering from cognitive issues were elevated compared to those of AIDS patients without cognitive issues .The S2 segment of the spike protein is responsible for membrane fusion of viral and cellular membranes. A study of the 3D structural aspects of S2 and the gp41 protein from HIV-1 revealed that these two proteins share the same two α helices and could follow an analogous membrane fusion mechanism .C is induced due to spike protein interactions in murine microglia, and human monocytes and macrophages [C numbers can lead to prion conformation misfolding and generates prion and prion-related diseases [The fact that the spike protein induces sharp TNF-α upregulation and causes cognitive issues implies that it might also, like HIV, upregulate PrP expression in the brain. Recently, our team has shown that the expression of PrProphages . In thisdiseases ,100.While it is unclear what the primary function of the prion protein is, it has been shown that it is protective under neuronal stress conditions. PrP expression is increased in the plasma of stroke patients, and it protects neurons from apoptosis . Also, tThe spike protein has been shown experimentally to induce senescence in transfected cells . FurtherIn vitro molecular study shows that macromolecular crowding can facilitate the conversion of native PrP into the neurotoxic soluble β oligomer configuration [SC molecules, which would be released within exosomes from transfected immune cells in the spleen and elsewhere. The transformation of PrPC to the infectious PrPSC molecule is an extremely slow process in the absence of PrPSC. However, under the influence of intermediate PrPs, PrPSC can induce solid and irreversible amyloid genesis via the template-assistance model. In another model, where PrPSC is present and interacts with PrPC, the progression to amyloid genesis is fast and reversible (nuclear-polymerization model), to establish neurotoxicity [guration . It is ctoxicity ,100.Figure A role of Hsp70 and diabetesMultiple studies in humans have shown that people who suffer from diabetes and/or obesity have an increased risk of severe outcomes from COVID-19 ,109. OneNormally, HSR induces the expression of inducible heat shock protein 70 (Hsp70), also known as Hsp72 and Hspa1a, a molecular chaperone with many complex roles in metabolism and regulatory processes. Heat shock proteins can account for up to 2% of the total protein mass in a cell following activation by HSR . Hsp70/7Stressful stimuli can induce the release of intracellular heat shock proteins into the extracellular milieu and circulation. Extracellular Hsp70/72 plays a facilitative role in the adaptive immune response to antigens . Also, eImpaired insulin signaling leads to a deficient ability to induce HSR and the subsequent resolution of inflammation. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that plays essential role in the molecular pathophysiology of many diseases. Its overexpression is linked to insulin resistance . GSK-3 nSC accumulation. Furthermore, mice lacking the gene for Hsp70 experienced accelerated prion disease progression compared with wild-type mice [One of the most important functions of Hsp70 is to protect from neurodegenerative disease. Many papers in the research literature link Hsp70 to protection from various protein-misfolding neurological diseases through its ability to facilitate proper folding and delay fibril formation -126. EviHuman post-mortem studies from patients who died from COVID-19 have shown that a primary target of the SARS-CoV-2 spike protein is immune cells, particularly macrophages, in the spleen and that many infected cells committed apoptosis . ApoptotExosomes represent a novel and efficient method for prion transmission. Stimulation of exosome release increases the intercellular transfer of prion proteins, and conversely, pharmacological inhibition of exosome release decreases prion transfer efficiency . VaccinaA potential role of G quadruplexesC to PrPSC. Understanding how mRNA structures are involved in the (mis-)folding of PrPC and possibly many other RNA-binding proteins with prion-like properties is of prime importance for the development of better treatments of CJD and related diseases" [A consideration in comparing the vaccine spike protein to the protein synthesized by the virus is related to the "codon optimization" step in specifying the mRNA for the vaccines. This practice takes advantage of the redundant nucleotide codes for most amino acids, and it involves replacing the codons used by the virus with ones that are more efficient in protein assembly. It turns out that the most efficient codons, on average, contain more guanines than other codons.Guanine nucleotides, when they are enriched in the nucleotide sequence, are sometimes able to configure into a special structure called a "G quadruplex" (G4) . G4s haviseases" .Reassessment of the risk/benefit ratio of COVID-19 vaccinationA study published in the Lancet tracked the effectiveness of COVID-19 vaccines over time. It showed that once eight months had elapsed since the second injection of the two-injection series, immune function was lower than that among unvaccinated individuals . While bWe acknowledge that we have relied heavily on in vitro and in vivo studies. However, we link these as closely as possible with the documented human histological and pathological correlates with cellular pathways and events. In light of these considerations, the risk/benefit ratio for the mRNA vaccines needs to be reevaluated. With every vaccine comes a flood of spike protein released into the circulation, further advancing the potential for amyloidogenic effects and increasing the risk of future neurodegenerative disease. A comment by Kenji Yamamoto published in BMC is urging the medical community to keep track of the date of the most recent vaccination of hospital patients in order to be better able to assess what role the vaccine may have played in any manifest disease or condition. He also strongly discourages policy that promotes continued boosting of anyone other than the most at-risk patients to death from COVID-19 . There iWe have examined the extensive literature concerning the prion-like properties of the SARS-CoV-2 spike glycoprotein. Furthermore, we identify pathways through which the mRNA vaccines could be capable of delivering the spike protein to the brain, which we suggest happens via exosomes released from germinal centers in the spleen traveling up the vagus nerve, increasing the risk of neurodegenerative disease. Should this happen, it would be expected that the COVID-19 vaccines would shorten the time period before neurodegenerative disease manifests in susceptible individuals. We speculate that the age of onset of neurodegenerative disease at the population level will decrease in the future in countries where vaccine uptake has been high.Particularly concerning is the evidence that CD16+ monocytes can continuously produce spike protein for months after vaccination, possibly through prolonged cytosolic presence of mRNA or reverse transcription of the mRNA into DNA. It has become clear that the antibodies induced through vaccination wane over time, necessitating frequent boosters to raise the antibody levels for sufficient protection from COVID-19. With each booster comes a compounded risk of future neurodegenerative disease. Fortunately, Omicron variant infection has a greatly reduced prion-like capability, and thus, with the cessation of mass vaccination, the expected increase in prion-like diseases could stabilize over the coming years.