Human adenovirus 7d is a respiratory pathogen capable of causing acute respiratory disease of variable severity. Phylogenetic analysis of whole-genome sequences of 15 strains isolated from cases of influenza-like-illness during 2017–2019 demonstrated the circulation of 2 distinct clades of genomic variant 7d in colleges in New York, USA. We characterized 15 HAdV-7 strains isolated from influenza virus–negative respiratory specimens collected from students with influenza-like illness at colleges in Tompkins, Albany, and Clinton counties in New York during the 2016–17, 2017–18, and 2018–19 influenza seasons. We used next-generation whole-genome sequencing and phylogenetic analysis to investigate possible epidemiologic connections among the New York college outbreaks and to monitor the dispersion of this reemerging variant within the United States.https://www.illumina.com), as previously described (https://www.geneious.com). We constructed a maximum-likelihood tree using MEGA6 . Phylogenetic analysis of whole-genome sequencing demonstrated the co-circulation of 2 distinct clades of HAdV-7d strains in New York, even within the same county, in the sampled time period (Initial digestion of viral DNA with endonucleases e period . The fir>2 introductions of genomic variant 7d into New York state. Spatiotemporal analysis of a larger genomic sequence dataset with better representation of strains isolated in other US states, as well as in other countries, over an extended period is necessary to more accurately track the introduction of lineages and follow their dispersion.Our phylogenetic analysis provides strong evidence of As in our previous studies and those of others (All HAdV-7d strains examined in this study were isolated from persons with influenza-like illness. Numerous recent publications report severe and even fatal cases of ARD in association with this genomic variant. All HAdV-7d strains sequenced thus far are indistinguishable by restriction enzyme analysis but not identical when examined at the whole-genome level. Characterization of more strains of diverse origins and associated disease and thorough mining of sequence data is needed to identify candidate determinants of virulence for HAdV-7. Host-related and other environmental risk factors are likely to contribute to the level of susceptibility, clinical presentation, and outcome of the associated disease.


Fusobacterium nucleatum could aggravate chemoresistance of colorectal cancer by decreasing the expression of miR-18a* and miR-4802. What’s more, miRNAs can shape the gut microbiota composition, ultimately affecting the host's physiology and disease. miR-515-5p and miR-1226-5p could promote the growth of Fusobacterium nucleatum (Fn) and Escherichia coli (E.coli), which have been reported to drive colorectal cancer. Here, we will review current findings of the interactions between the gut microbiota and microRNAs and discuss how the gut microbiota–microRNA interactions affect host pathophysiology including intestinal, neurological, cardiovascular, and immune health and diseases.The gut microbiota regulates the biological processes of organisms acting like ‘another’ genome, affecting the health and disease of the host. MicroRNAs, as important physiological regulators, have been found to be involved in health and disease. Recently, the gut microbiota has been reported to affect host health by regulating host miRNAs. For example,  They both stimulated the innate immune and neuroinflammatory pathways by activating nuclear factor kappa-B (NF-κB) and inducing microRNA-146a. B. fragilis toxins and B. fragilis LPS induced up-regulation of NF-κB signaling and the expression of miR-146a in primitive cultured cells derived from human central nervous system (CNS) tissue. At the same time, HSV-1 infection induced the expression of miR-146a through regulating NF-κB and AD-type inflammatory gene signaling in human neuronal-glial (HNG) cells in primary culture (Zhao and Lukiw It has been shown that the gut microbiota dysbiosis affected the formation and development of Alzheimer’s disease (AD), mainly through microbial metabolites, such as amyloids, Lipopolysaccharide (LPS), and short-chain fatty acids (SCFAs) -(2RS)-propyl]-CysSer-Lys4), TLR4 (LPS), TLR5 (E. coli Flagellin FliC), TLR9 (CpG oligodeoxynucleotide), and NOD2 (muramyl dipeptide)) interaction via the MyD88-dependent pathway. This regulation may help to relieve intestinal inflammation and maintain intestinal homeostasis by targeting IL-12/IL23p40, which played a key role in the innate immune response to commensal bacteria  could trigger an excessive mucosal immune response associated with Crohn's disease (CD) in wild-type or IL-10 knockout mice by inhibiting the expression of let-7b and thereby caused overexpression of TLR4  mice co-housed with miR-146a knock-out (KO) mice was higher than that of WT mice raised alone (Du et al. P. falciparum infection and reduced microbiota, which may be related to the regulation of several immune effector genes with the target genes of miR-305 (Dennison et al. In addition, the ability to fight pathogen infection is essential for all living species. The gut microbiota–miRNA interactions are involved in the host's immune response to pathogen infection (Sesto et al. Growing evidence shows us that homeostasis of the gut microbiota is an essential part of a healthy body. Recently, a new drug named GV-971 treating Alzheimer's disease by targeting the gut microbiota has been launched in China, which opens up a new therapeutic perspective for the treatment of Alzheimer's disease. The medical value of the gut microbiota attracts our attention. Although the above-mentioned reports identified that the gut microbiota–miRNA interactions influence the host pathophysiology, the specific mechanisms by which the gut microbiota–miRNA interactions work in the host’s physiological process are unclear. For example, which genes and pathways does miR-21 regulate to affect the gut microbiota? Furthermore, which signal molecules derived from the gut microbiota participate in the inhibition of miR-10a and regulation of IBD? Which signaling pathways does the gut microbiota impact to increase intestinal inflammation? In addition, which specific bacteria are regulated by miR-275 and how miR-275 specifically affects the intestinal flora of mosquitoes? What’s more, whether there are other miRNAs involved in the regulation of the gut microbiota which improves host cognitive impairment? Does lack of miR-275 also affect intestinal function in humans and mice? More in vivo experiments and studies of molecular mechanisms need to be carried out to confirm the effects of the gut–microbiota–miRNA interactions on host physiological pathology. Overall, deciphering the signals of the gut–microbiota–miRNA interactions will provide new insights into the crosstalk between the gut microbiota and host, and offer a new direction for the study of mechanisms that affect health and disease.In this brief review, we summarize the interactions between the gut microbiota and miRNAs, and these roles of the gut microbiota–miRNAs interaction are involved in the pathophysiological process of the host. The study about these interactions may provide new directions for clinical treatment of intestinal, neurological, cardiovascular and immune diseases.


Electron-event representation saves the spatial and temporal coordinates of every electron with ultimate accuracy. Through maximum signal extraction, it will improve the performance of electron cryo-microscopy. Up until a decade ago, photographic film provided the best performance for the recording of cryo-electron microscopy (cryo-EM) images. Digital cameras using a phosphor scintillator have also been available since the late 1990s, but despite being more convenient to use their signal-to-noise characteristics were significantly inferior to those of film. Irrespective of their differences, both recording methods were far from the theoretically achievable performance and limited the typical resolution of cryo-EM reconstructions to a ‘blobology’ (~10 Å) level. The inadequacy of these legacy image capture methods was resolved around 2012 with the first commercial introduction of direct electron detectors (DEDs). Together with substantial advances in image processing algorithms, these new cameras played a fundamental role in the great leap in cryo-EM performance.et al., 2016As the name suggests, DEDs detect electrons through their direct interaction with the active silicon layer of the camera chip  space (Fig. 1In their paper in this issue of ce Fig. 1. ConsequEER is another significant step in the development of cryo-EM detector technology. It removes experimental restrictions and improves detector performance while also freeing researchers from mandatory data reduction decisions that had to be made with previous recording methods.


Objective: Primary family caregiver (CG), other family members (FM), and medical professionals (MP) play important roles in medical decision-making for older adults with dementia, who often have lost the capacity to make decisions on their own. Power dynamics within the CG-FM-MP triad relationship determine the process and outcome of the decision-making. Guided by Rahl’s relational power model, this study is among the first to understand the experiences of advance care planning among Chinese. Method: This study includes a total of 25 primary CGs or FMs and 5 MPs from 3 neurology departments. Hybrid grounded theory method was used to analyze the preliminary data we had so far. Based on the dimensions of power, we analyzed the power base, means, and scope of each agent in each interview to determine the power comparability. Results: Three types of triadic power relations were categorized: 1) shared-power with shared-decision, in which three agents shared the power of decision-making and CG as the lawful decision-maker makes the final decisions; 2) balanced-power with reversed-patriarchal decisions, in which FM’s power is over both CG and MP and become the actual decision-maker; and 3) unbalanced power with conflicting decisions, in which neither CG and FM has absolute power over each other and MP becomes the actual decision-maker implicitly. Conclusion: The study provides a framework for researchers and practitioners to understand the ACP process for Chinese older adults, which helps develop intervention strategies to improve surrogates’ ACP knowledge and reduce potential conflicts during the stressful process for the population.


There is no national representative and conclusive data regarding parent-young communication. Therefore, this study aimed to estimate the pooled prevalence of parent-young communication on sexual and reproductive health issues and its association with sex and perceptions of young people about its importance in Ethiopia, 2020.2 and Egger test statistics were used to test heterogeneity and publication bias respectively.The Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) was used during systematic review and meta-analysis. The study included both published and gray literatures which were searched using appropriate key terms. The articles were searched from different databases: PubMed, Cochrane Library, PsycINFO, CINAHL, Global Health, HINARI and Google scholar. Data were extracted in a Microsoft Excel sheet and STATA/SE 14 was used for meta-analysis. ITwenty-nine articles were included in this systematic review and meta-analysis. The pooled prevalence of parent-young communication on sexual and reproductive health issues in Ethiopia was 42.96% [95% CI: ]. Positive perception of young people towards parent-young communication  and female sex  were significantly associated with parent-young communication on sexual and reproductive health issues.The prevalence of parent-young communication on sexual and reproductive health issues in Ethiopia was low as compared to other literatures conducted outside Ethiopia. Both perceptions of young people towards parent-young communication and sex were significantly associated with parent-young communication on sexual and reproductive health issues. Interventions targeting males and young who had negative perceptions regarding parent-young communication should be the primary focus of the government and their parents.CRD42020161252.The protocol of this systematic review and meta-analysis was registered in the PROSPERO International prospective of systematic reviews with a specific registration number:  According to the World Health Organization (WHO), young were populations aged from 10 to 24 years. Globally, the numbers of the young population were estimated to be 1.8 billion, of which 90% of them were lived in developing countries , 4. The The problem in parent-adolescent communication has been associate with an increase in a wide range of adolescent risk behaviors , 12–16. Parent-young communication was associated with socio-demographic characteristics of young  , 15, 22,CRD42020161252.The study was designed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA-2009 Guidelines) . It was ❖ Population: This systematic review included studies conducted among young and/or parents having young (aged 10 to 24 years) in Ethiopia.❖ Exposure: Female young and young who had positive perception towards parent-young communication.❖ Comparison: Male young and young who had negative perception towards parent-young communication.❖ Outcome: Studies conducted parent-young communication as primary outcome.❖ Study design: All observational studies were included❖ Time frame: All studies irrespective of data collection and publication year until the end of November 1, 2020❖ Publication: Either published in peer-reviewed journals or unpublished studies.❖ Language: studies published only in English language were included in this systematic review and meta-analysis.❖ Studies in which the outcome did not clearly reported❖ Studies in which the full texts were not available after 2 times authors request were excluded from systematic review and meta-analysis.Parent-young communication on sexual and reproductive health issues was measured in two ways. Thirteen articles used as having talked about at least one , 41–46 aThose articles identified in all databases were exported to Endnote X8 and duplicate files were excluded. The remaining articles and abstracts were independently screened by two groups (YD and BK) for inclusion in the full-text appraisal. It was assessed using Joanna Brigg’s Institute (JBI) critical appraisal checklist according to the study design of each article , 56. TwoData were extracted using Microsoft excel 2010 sheet and the sheet contained the following list of variables for the first objective: authors name followed by initials, year of study, year of publication, study setting, study design, sample size, response rate, quality score, sex of participants, region, study finding. For the second objective, in addition to the above mentioned, studies reported at least one of the above mentioned factors as predictors were identified and extracted. Two authors (MY and YW) extract the data for both objectives and any disagreements between the two authors during extractions were solved through discussion and consensus.P-value less than 0.05 . The pooled prevalence of parent-young communication was in line to a study conducted in Nigeria (46.7%)  and ZambIn addition, the highest prevalence of parent-young communication was observed in studies conducted among young as compared to studies conducted among parents. This may be due to the fact that parents had less exposure to education and information than young. Moreover, the parents might think it as a positive reinforcing factor for early sexual initiation.The pooled odds of parent-young communication on sexual and reproductive health issues among young who had positive perception regarding parent-young communication were statistically high as compared to counterparts. The finding of this study was in agreement with a study conducted in Bangladesh . It is aThe pooled odds of parent-young communication on sexual and reproductive health issues among females were high as compared to males. The finding was congruent to a study conducted in Nigeria . The finDespite its strength, the study was not without limitation. The review was restricted to articles published in the English language and it may not be representative for articles published in other languages. Not only this, but also all the studies included in meta-analysis were cross-sectional and the pooled estimate may be influenced by study design.The prevalence of parent-young communication on sexual and reproductive health issue in Ethiopia was low as compared to other literatures. Both positive perceptions of young people towards parent-young communication and female sex were significantly associated with parent-young communication on sexual and reproductive health issues. So, the government should focus on interventions that could change the perception of young on parent-young discussion. Interventions targeting male and young who had negative perception regarding parent-young communication should be the primary focus of the parents.


Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and both ceftazidime and/or imipenem-resistant Acinetobacter spp. (ACB), and ceftazidime and/or imipenem-resistant Pseudomonas spp. (PSA)).In 2015, antimicrobial resistance was identified as a public health priority for the South-Western Indian Ocean (SWIO) , Reunion Island (France), and Seychelles). However, in 2020, colonization rates of antimicrobial-resistant bacteria (ARB) in human populations on most islands in SWIO were still not known and neither hospital nor community colonization rates had been estimated. The aim of this study was to estimate the prevalence of colonization of six ARB groups in hospitalized patients residing in the SWIO territories. The six groups comprise extended-spectrum betalactamase producing Enterobacteriaceae (ESBL-E), carbapenem-resistant Enterobacteriaceae (CRE), methicillin-resistant Based on comprehensive hospital laboratory ARB screening data, we provide the first estimation of ARB colonization rates in hospitalized patients residing in SWIO (2015–2017). Using ARB colonization rates in Reunion Island (France) as the reference for estimating odds ratio, we identified at risk patients based on their territory of residence.The survey pointed to significantly higher overall ARB colonization rates in patients from Comoros, Madagascar, Mayotte, and Seychelles compared to Reunion Island as the reference. Extended-spectrum betalactamase producing Enterobacteriaceae was found to be the most common ARB group colonizing patients from SWIO territories. The highest MRSA colonization rates were observed in patients from Mayotte and Seychelles. Colonization by carbapenem-resistant Enterobacteriaceae (CRE) was highest in patients from Mauritius.These results identify high ARB colonization rates in hospitalized patients from SWIO territories that require further investigation, particularly CRE in Mauritius and MRSA in Seychelles and Mayotte. This study is the first step toward the implementation of a broader regional ARB surveillance system. Staphylococcus aureus (MRSA) [An increase in the prevalence of antimicrobial-resistant bacteria (ARB) was observed in Reunion Island (France) between 1997 and 2007 [The South-Western Indian Ocean (SWIO) region contains the islands of the Union of Comoros, Madagascar, Mauritius, Mayotte (France), Reunion Island (France), and Seychelles. Based on a review of the literature, ESBL-E and carbapenem-resistant Enterobacteriaceae (CRE) were identified as a main public and veterinary health issue for SWIO . AntimicFelix-Guyon University hospital in Reunion Island is well suited for medical evacuations and receives most patients evacuated from other islands of SWIO. Since 2015, an ARB screening strategy has been in place for all patients residing abroad who arrive via medical evacuation, or who visited a foreign country in the three preceding months, and/or were hospitalized abroad in the past year. All patients admitted to the intensive care patients in Felix-Guyon hospital are screened to avoid introducing ARB in the unit.Acinetobacter spp. (ACB), and ceftazidime and/or imipenem-resistant Pseudomonas spp. (PSA)) of hospitalized patients residing in SWIO. This is the first estimation of the ARB colonization rates in patients from SWIO territories using standard indicators.Based on comprehensive hospital laboratory ARB screening data, we estimated the prevalence of colonization by ARB . For Reunion Island, only patients admitted to the intensive care unit  were used as the reference to estimate odds ratio.Bacterial species were routinely identified for all isolates using MALDI-TOF mass spectrometry . Antimicrobial susceptibility testing was performed using the disc diffusion method according to guidelines published by the Committee on Antimicrobial Susceptibility Testing of the French Society of Microbiology in 2015 .(i)Staphylococcus aureus resistant to oxacillin was designated as methicillin-resistant Staphylococcus aureus (MRSA) according to the French national multidrug-resistant bacteria surveillance network [ network ;(ii)Enterobacteriaceae resistant to cefotaxime and/or ceftazidime and/or cefepime were designated as ESBL-E if a synergy between third-generation cephalosporins and clavulanic acid was confirmed by the disc diffusion method according to French recommendations . ESBL-E (iii)Enterobacteriaceae resistant to imipenem and/or ertapenem were confirmed for the presence of relevant resistance genes by PCR  and designated as carbapenem-resistant Enterobacteriaceae (CRE).(iv)Enterococcus faecium resistant to vancomycin and/or teicoplanin and confirmed for the presence of relevant resistance genes by PCR  were designated by vancomycin-resistant enterococci (VRE);(v)Acinetobacter spp. (ACB) and (vi) Pseudomonas spp. (PSA) included in our survey were ceftazidime and/or imipenem resistant.both The ARB groups included in the survey were:A patient was considered ARB positive if one or more ARB group was isolated from that patient.p-values < 0.05. Confidence intervals of ARB prevalence were estimated using the Wilson method. All statistical analyses were performed using R software version 3.4.2 [ARB colonization rates in patients were compared based on their territory of residence using a logistic regression analysis with ARB colonization as the dependent variable and the patient’s territory of residence as the explanatory variable. Odds ratio were calculated using patients residing in Reunion Island as the reference and the statistical significance threshold was set at on 3.4.2 , the pacon 3.4.2 , binom [on 3.4.2 , and theon 3.4.2 .The study was approved by the French national commission on data protection and liberties .From January 1st 2015 to December 31st 2017, a total of 4135 hospitalized patients from the SWIO territories were surveyed. A total of 978 out of 4135 (23.7%) patients tested positive for ARB colonization.The number of hospitalized patients varied between the SWIO territories of residence ranging from 13 patients residing in Seychelles to 2184 patients residing in Reunion Island. The highest ARB colonization rates were observed for patients residing in Seychelles (61.5%) and Madagascar (41.3%) ; ESBL-E colonization rates followed the same trend Table . MRSA cop < 0.001). These results are the first step toward a regional hospital-based ARB surveillance system as planned by the Indian Ocean Commission in 2015 [This study pointed to higher ARB colonization rates in hospitalized patients from Comoros, Madagascar, Mayotte, and Seychelles compared to patients who reside in Reunion Island. ESBL-E colonization was common in all SWIO territories whereas specific epidemiological trends were observed for MRSA and CRE. Higher MRSA colonization rates were reported in patients from Mayotte and Seychelles and a high CRE colonization rate was identified in patients residing in Mauritius  the patient’s territory of residence is a known risk factor for ARB infection and carriage –14 and tESBL-E occurrence in SWIO has already been highlighted as a public health issue both in the community and in hospitals  for whicOur study provides the first estimate of ARB colonization rates in hospitalized patients in the SWIO. Overall high ESBL-E colonization rates were identified in patients from SWIO territories; MRSA and CRE colonization rates were high in certain territories. These results raise awareness on the circulation of specific ARB groups in local SWIO hospitals. This is the first step toward a targeted action plan for the prevention and control of ARB in the region. The results of this study will serve as a basis for the implementation of broader regional surveillance systems.


Our approach presents a one-step method that permits regulation of an Au(I) precursor to form either visible-absorbing gold nanospheres or near-infrared-window (NIRW)-absorbing anisotropic AuNPs. A collection of shapes is obtained for the NIR-absorbing AuNPs herein, giving rise to spontaneously formed nanomosaic  without a dominant geometry for the tesserae elements that comprise the mosaic. Nonetheless, NIRAuNM exhibited high stability; one test sample remains stable with the same SPR absorption profile 7 years post-synthesis thus far. These NIRAuNM are generated within thermoresponsive poly(N-isopropylacrylamide) (PNIPAm) microgels, without the addition of any growth-assisting surfactants or reducing agents. Our directed-selection methodology is based on the photochemical reduction of a light-, heat-, and water-sensitive Au(I) precursor via a disproportionation mechanism. The NIRAuNM stabilized within the thermoresponsive microgels demonstrates a light-activated size decrease of the microgels. On irradiation with a NIR lamp source, the percent decrease in the size of the microgels loaded with NIRAuNM is at least five times greater compared to the control microgels. The concept of photothermal shrinkage of hybrid microgels is further demonstrated by the release of a model luminescent dye, as a drug release model. The absorbance and emission of the model dye released from the hybrid microgels are over an order of magnitude higher compared to the absorbance and emission of the dye released from the unloaded-control microgels.This work demonstrates the dynamic potential for tailoring the surface plasmon resonance (SPR), size, and shapes of gold nanoparticles (AuNPs) starting from an Au(I) precursor, chloro(dimethyl sulfide)gold (I) (Au(Me N-isopropylacrylamide) (PNIPAm) is often used because of its thermoresponsiveness results in “smart gel” materials. PNIPAm, which has a lower critical solution temperature (LCST)–32 °C, can be tuned by varying its composition and crosslinking density ·2H2O)  from hybrid microgels upon NIR irradiation  based on photothermally driven “shrinking” of PNIPAm-s 32 °C, . Based oadiation . Retentiadiation . UV/VIS/adiation  with simadiation A,B. Initadiation C. In theadiation  that potadiation D. There 2S)Cl complex, a commercially available well-known starting material for Au(I) complexes, has proven to be an effective precursor for making both spherical and anisotropic AuNPs by tuning experimental conditions. The instantaneous reaction of gold(I) sulfide in aqueous media is shown to be responsible for the reduction process without exogenous reducing agents. The flexibility of the reduction of Au(Me2S)Cl to form AuNPs within a wide variety of media is demonstrated. Even with a mixture of shapes to compose nanotesserae, a direct correlation is noticed between the size of anisotropic AuNPs on the one hand (longest dimension for representative tesserae of each sample) and the NIR SPR peak maximum on the other hand. Additionally, a strong direct correlation is observed between the precursor concentration and average longitudinal length and NIR SPR of the anisotropic structures. The higher concentration of Au(I) resulted in large size particles with red-shifted NIR absorption. The demonstration of photothermally driven shrinkage in size followed by the release of a dye from the hybrid microgels exhibits a proof-of-concept result toward potential drug release studies in the future, but for this, higher LCST slightly above body temperature is required. The proof-of-concept result, nonetheless, led to validation studies of the photothermal transduction property of NIRAuNM produced by our distinct one-step photochemical methodology. Based on our ongoing cytotoxicity tests and because these particles do not rely on CTAB or additional chemicals, they are expected to be far less toxic compared to CTAB-containing NIRAuNPs. Of consequence is our development of this method that uses conditions comparatively milder than the literature standard for the formation of different sizes and shapes of AuNPs. In this regard, we suggest our method will add to the existing “green” synthesis methods, and thus will have an impact on reducing the environmental footprint of these technologies. Finally, the preparation of specific drug delivery systems with a variety of model drugs is underway, taking advantage of the approach herein.The present investigation demonstrates two important discoveries: (1) development and use of an unexplored methodology wherein an Au(I) complex serve as a gold precursor to generate AuNPs of different sizes and shapes (nanomosaics) in various stabilizing media and (2) the first-ever direct formation of NIR-absorbing AuNPs within thermoresponsive PNIPAm microgels. The facile wet photochemical method resulted in NIR-absorbing Au nanomosaic (NIRAuNM) of diverse anisotropic sizes and shapes and a corresponding wide absorption cross-section. No exogenous reducing agents are needed to reduce the gold precursor. The Au(Me


For each trial, the risk of bias was assessed applying Cochrane Collaboration methodology. The findings indicate that vitamin K does not consistently prevent progression of calcification, atherosclerosis or arterial stiffness. There may be some benefit in people with calcification at study entry. Studies were heterogenous, with relatively short follow-up and outcome measures were varied. While vitamin K supplementation clearly improves the carboxylation of dephosphoylated MGP, its role in mitigating vascular calcification is uncertain, based on current evidence.Matrix gla protein (MGP) is an important vitamin K-dependent inhibitor of vascular calcification. High levels of uncarboxylated, dephosphorylated MGP have been associated with vascular calcification and are responsive to vitamin K treatment. In this systematic review, we summarize the available evidence examining whether vitamin K supplementation improves surrogate measures of cardiovascular disease including artery and valve calcification, atherosclerosis and artery stiffening. Data from controlled trials of adults were obtained by searching Ovid MEDLINE, Embase, the Cochrane Central Register of Controlled Trials and the Web of Science Core Collection. We identified nine randomized controlled trials for review, including trials of vitamin K Cardiovascular disease (CVD) is the leading cause of morbidity and mortality globally. Calcification of arteries and valves increases the risk for CVD and death in the aging population. Calcification occurs frequently in people with chronic kidney disease (CKD) and diabetes mellitus, where it is an independent risk factor for cardiovascular mortality . Though Matrix Gla protein (MGP) is a vitamin K-dependent protein (VKDP) that counteracts vascular mineralization. MGP is highly expressed in calcifying arteries where it is thought to inhibit extra-osseous calcification by binding to growing hydroxyapatite crystals and inhibiting BMP-2 . VitaminThis systematic review was conducted according to the guidelines for Preferred Reporting Items for Systematic Reviews and Meta-Analyses . The revThe following inclusion criteria were applied to obtain the selected studies: parallel randomized controlled trials of adult participants of any ethnicity that involved vitamin K supplementation with a control group (placebo or no-treatment control group) for a period of >12 weeks and measured a surrogate outcome related to cardiovascular disease at baseline and study end. Trials published up until April, 2020 were considered.1  or vitamin K2 —compared with a non-supplemented group were considered for inclusion. Co-interventions were permissible if they were received by both groups.Studies were restricted to >12 weeks in duration. All studies in which participants received a vitamin K supplement—either vitamin KFor the assessment of the impact of vitamin K treatment on CVD, data from any clinical trial that included a surrogate measure of CVD was considered. Surrogate measures of CVD included an image-based quantification of calcification burden or ultrasound assessment of atherosclerosis or arterial stiffness. Calcification was measured by computed tomography (CT) or positron emission tomography (PET) and was assessed in coronary arteries, aorta, femoral arteries and cardiac valves. Atherosclerosis was assessed by carotid intima media thickness (CIMT). Arterial stiffness was assessed by ultrasound-based measurement of pulse wave velocity (PWV) between the carotid and femoral arteries (cfPWV), as well as the carotid and radial arteries (crPWV). We excluded studies reporting only laboratory parameters. The primary outcome was the change in the measurement of the surrogate marker from baseline to study end. Secondary outcomes assessed within each of the identified trials included any additional surrogate measures of CVD that were assessed in addition to the primary outcome, as well as changes in MGP carboxylation and adverse effects.A three-step search strategy was utilized to locate published studies for this review. An initial limited search of Ovid MEDLINE was executed, followed by an analysis of eligible studies to identify relevant text words contained in the title, abstract and author provided keywords, and the subject headings assigned to the article. A second search using all relevant text words and subject headings was then executed in Ovid MEDLINE and subsequently adapted for the other databases . The folTwo authors  independently screened all abstracts identified from the literature search. Those that were clearly irrelevant were excluded. Full texts of remaining studies were obtained, screened and excluded if irrelevant. Disagreements were resolved by discussion or with a third reviewer (RH).The following data was extracted by two independent reviewers : study design, inclusion and exclusion criteria for patients, method for assigning patients to different treatment groups, details of the intervention and control arms, number of patients randomized, lost to follow-up and analyzed, demographic and clinical information for patients including age and sex and important co-morbidities, and types of surrogate measures of cardiovascular disease. No formal assessment of agreement between raters was obtained. In addition, we obtained the first author’s name, year of publication, funding source and source of vitamin K. We extracted the net change in the surrogate measure of CVD before and after vitamin K treatment as the main outcome measure.Data was synthesized in a narrative form and then sub-grouped based on the different surrogate outcome measures of CVD  and arterial stiffness (PWV)). Summary tables and text provide study descriptions and clinical characteristics. The over-arching goal was to summarize the direction of any observed effects of vitamin K across studies. Meta-analysis was not performed.Two review authors independently assessed the risk of bias for each individual study using the Cochrane Risk of Bias tool  . DisagreAll trials reported continuous measures at baseline and study end with tests of statistical significance to determine treatment effect. For the purposes of summarizing results, we categorized the trials according to the primary or secondary outcome measures reported. For example, some trials reported the impact of vitamin K treatment on more than one surrogate measure of CVD  score and PWV).The results of the bibliographic search are presented in n = 9) in alphabetical order by the first author’s surname. The included studies involved 1589 participants. Seven trials were randomized and controlled whereas two were open-label. Only one trial was conducted outside of Europe and the majority of trials were conducted in a single-center. The intervention received in three trials was VK1 and in the remaining six trials was VK2. The dose of VK1 ranged from 500 mcg to 2 mg daily and the dose of VK2 ranged from 90 μg to 2000 μg daily. Follow-up ranged from 6 months to 3 years. The primary outcome was a measure of calcification in six studies and a measure of atherosclerosis in three studies. The timing of outcome measures was at baseline and study end.All nine studies selected for review were randomized trials published in English. Quality measures of the randomized trials are presented in n = 40) that enrolled only patients with pre-existing CAC found a trend toward improved CAC scores with MK-7 supplementation [p = 0.06) when those with very high baseline CAC scores (>1000 AU) were excluded from the analysis . A longitudinal study examining changes in CAC over 18 months in patients on HD with non-valvular atrial fibrillation (AF) using different anticoagulation strategies  found no significant difference between its three arms [n = 1), aortic valve (n = 2), thoracic aorta (n = 1), abdominal aorta (n = 1) and femoral arteries (n = 1). An open label trial that enrolled patients without CKD and with minimal aortic valve calcification (AVC) at baseline found that AVC progressed by 10% in patients taking 2 mg vitamin K1, compared to 22% for those taking placebo (p = 0.04) [18F-NaF PET scan, which reportedly measures active calcification [18F-NaF PET-CT scan showed increased calcification activity in the MK-7 group compared to placebo . Overall, the role of vitamin K supplementation in modifying vascular calcification is unclear, especially considering the contrasting results obtained when using different imaging modalities.Six of the nine included trials examined the effect of vitamin K supplementation on the calcification of major vessels and cardiac valves . Three sentation . This firee arms . Five st = 0.04) . However = 0.04) . The Oikfication . Interes1 per day study showed no benefit in attenuating CIMT progression over time. In a small study of participants with CKD, MK-7 and vitamin D resulted in an attenuated increase in CIMT over 9 months but this difference was not significant. Overall, the results of these trials do not support a role for vitamin K in modifying atherosclerosis when measured by CIMT.Change over time in CIMT was evaluated in three of the nine trials but was not the primary end-point for any of the studies ,15,17 T. In totaOf the nine vitamin K treatment trials, three examined changes in PWV, and in one trial this was the primary end-point . Fulton Of the nine clinical trials included in this review, the response of dp-ucMGP to either vitamin K1 or MK-7 treatment was examined in eight of the studies and the change in total MGP was reported in two studies . A signiOf the nine clinical trials included in this review, five reported the frequency and types of adverse events experienced by trial participants . In threWe performed a systematic review of trials that have been conducted to examine whether vitamin K supplementation mitigates cardiovascular disease progression by examining correlates of CVD including measured vascular calcification, intima-media thickness, and stiffness. While vitamin K supplementation improves vitamin K status, based on the carboxylation status of dpMGP, its role in mitigating vascular calcification is unclear, based on current evidence. Vitamin K supplementation does not appear to significantly affect atherosclerosis and the results derived from studies examining arterial stiffness are not consistent.The mechanisms by which vascular calcification occur are multifactorial and becoming progressively clearer . Local i18F-NaF PET scanning of arteries can detect microcalcifications and may be more sensitive for newly formed calcium deposits [Total vascular calcification is typically quantified using the Agatston score, which is a weighted calculation of macroscopic calcification densities observed on CT imaging . While tdeposits , whereasdeposits .p = 0.04), including when the data were indexed to BSA [Of the six trials that measured changes in calcification of major vessels or valves, five concluded that there was no significant benefit of vitamin K. Sub-analyses of two of these studies suggest there might be lowered CAC progression in patients with significant calcification at baseline (Agatston score ≥10) ,19. One d to BSA . However18F-NaF PET scanning, after 6 months of MK-7 supplementation [18F-NaF PET scan, they saw that the target-to-background ratio (TBR) tended to increase in the MK-7 group compared with placebo, suggesting that there may be more ongoing active vascular calcification in the MK-7 treatment group. Resolution limits may therefore bias the assessment of arterial calcification scores via conventional imaging modalities such as CT scan. Taken together, in this review, we did not find conclusive evidence that vitamin K supplementation impedes vascular calcification.When using the CT scan-based Agatston score, CAC progression should be assessed over a minimum 1-year follow-up period in order to detect macroscropic changes in calcification ,30. A 20Intimal calcification occurs in conjunction with atherosclerotic plaque formation and is associated with dyslipidemia, inflammation and other traditional Framingham risk factors. Ultrasound measurement of the carotid intima media thickness (CIMT) is one established surrogate marker of atherosclerosis burden . CIMT isDephosphorylated uncarboxylated MGP has been associated with CIMT in healthy post-menopausal women  but has 1 per day in attenuating CIMT progression over time [Our assessment of the three trials does not support a role for vitamin K in modifying atherosclerosis in humans when measured by CIMT. In the study of older people with established CVD, 100 mcg of MK-7 daily for 6 months had no impact on CIMT compared to placebo . Howeverver time . The appver time . In contver time . HoweverMedial calcification is characterized by hydroxyapatite deposition in the medial layer of the artery. This type of calcification has classically been associated with vascular smooth muscle cell conversion to osteoblast-like phenotype. It can occur in the absence of atherosclerosis and may involve the arterial tree diffusely. Clinical sequelae of vascular stiffening include hypertension and left ventricular hypertrophy. Measurement of pulse wave velocity (PWV) is a surrogate measure of vascular stiffness. The most validated method to assess vascular stiffness is based on the measurement of the velocity of pulse waves as they travel along the arterial tree between two sites most typically between the carotid and femoral artery. PWV increases with age and with hypertension and is linked to inflammation ,40. HighThe level of ucMGP has been consistently associated with higher PWV in studies of the general population, as reviewed by Roumeliotis et al. , whereasOf the nine vitamin K treatment trials, three examined changes in PWV, with one as the primary end-point for the trial. MK-7 was not beneficial in either elderly people or participants with non-dialysis dependent CKD over 6 to 12 months . De VrieThe level of dp-ucMGP has been associated with several adverse cardiovascular risk factors, including obesity and hypertension, and predicts cardiovascular events in the general population ,50. CrosOne pharmaceutical company, Nattopharma, was the source of vitamin K for five of the nine clinical trials. Three trials were conducted with the support of federal or national-based health society funds, three trials were conducted with industry funding, two trials were conducted using university-level funding and one trial received no funding. We did not observe any clear patterns related to source of funding or vitamin K with regards to study outcomes and conclusions.The studies included in this review used varying doses of vitamin K. We therefore cannot rule out that a beneficial effect might be observed if higher doses were given, particularly in high risk groups. Two trials currently being conducted in hemodialysis patients use substantially higher doses of vitamin K1 than have been previously studied ,53. AlthThere is presently a lack of randomized trial evidence to support a beneficial role for vitamin K in preventing the worsening of surrogate measures of CVD. The trials conducted to date are primarily small, single-center trials and are heterogeneous with regard to type of vitamin K administered, dose of vitamin K, population studied and outcome measures. However, an alternative explanation may be that abnormalities in dp-ucMGP, which are sensitive to vitamin K supplementation, may be a consequence of CVD. Our assessment is that an improvement in surrogate measures of CVD with vitamin K supplementation has not been consistently demonstrated in the clinical trials to date and no clinical trial has examined important clinical events including mortality. In those trials that demonstrate a vitamin K treatment effect on a surrogate measure of CVD, the causal pathway linking the benefit to an improvement in dp-ucMGP has not been demonstrated. At present, it is not clear from human clinical trials that a causal pathway exists between vitamin K and reduced cardiovascular end-points that operates through enhanced MGP carboxylation and is responsive to vitamin K supplementation.


With an estimated global prevalence of chronic kidney disease (CKD) of 11–13%, non-invasive biomarkers of renal pathology are desperately required to enhance early diagnosis, guide prognosis and monitor response to treatment [ex vivo perfusion systems. The stable detection of miRNAs in many of these samples is enhanced by their binding to proteins (e.g. Argonaute and lipoproteins) and/or their encapsulation in extracellular vesicles—both of which limit their degradation by RNases. In addition to their role as biomarkers, their ability to negatively regulate key networks of genes by post-transcriptional repression of mRNAs has resulted in critical mechanistic roles being identified in development, homeostasis and disease in multiple organs including the kidney [The ideal biomarker should be non-invasive, stable and sensitive, and should dynamically and specifically reflect disease pathology so as to guide diagnosis, treatment response and prognosis. Since their discovery in 1993, the small (20–22 nucleotides) non-coding RNAs microRNAs (miRNAs) have been shown to have many of these qualities. MiRNAs in multiple renal diseases have now been explored in renal tissue and biofluids including blood, urine , serum, plasma and perfusate from renal The current classical pipeline for miRNA biomarker development is firstly high-throughput measurement of miRNAs by small-RNA sequencing or microarray in a discovery cohort containing the disease and sample of interest, followed by validation of key miRNAs in large and ideally prospective cohort studies by quantitative real-time PCR (qRT-PCR). Although miRNAs can be readily detected from a limited RNA quantity by qRT-PCR, the output from high-throughput sequencing often requires a greater RNA quantity that can often only be reliably obtained from renal tissue. However, the expression of many miRNAs initially profiled in renal tissue is often not faithfully reflected within contemporaneous biofluids. A further technical challenge in biofluid miRNA quantification is the normalization of miRNA expression levels to an exogenous and/or endogenous control. This is particularly important when quantifying miRNAs in biofluids such as urine that may drastically vary in concentration. At present, there is little consensus in the field on the optimal normalization approach and this may contribute to incongruous results between studies .et al. conducted a large international study and demonstrated that serum miR-148b and miR-let-7b could discriminate patients with IgA nephropathy from both controls and patients with other forms of glomerulonephritis [With their ability to regulate and reflect disease-specific molecular pathways, a potential advantage of miRNAs is that they may non-invasively offer diagnostic discrimination over generic biomarkers of renal injury. For example, microRNA-148b (miR-148b) and miR-let-7b have been shown experimentally to regulate the O-glycosylation process of immunoglobulin A1 (IgA1) in the peripheral blood mononuclear cells of patients with IgA nephropathy [ephritis . While aAnother potential clinical utility of miRNAs is as predictive markers. Within acute kidney injury (AKI), a key unmet need is to develop a biomarker that predicts the risk of progression to severe AKI. MiR-21 remains one of the most extensively studied miRNAs, in the context of ischaemic AKI upregulation of urinary and to a lesser extent plasma miR-21 predicted AKI progression in patients undergoing cardiac surgery . In tranet al., allowing them to conclude that miR-126 and the miR-770 family in urine and blood were the most promising markers to predict progression of diabetic nephropathy [In the context of CKD, another large study examined miR-126 and miR-223 in the serum of 601 CKD patients (Stages 1–5) with follow-up for 6 years  and founhropathy .An attractive use of miRNAs would be as treatment modifiable biomarkers to assess response to treatment. In patients with hypertensive nephropathy (HN), miR-103a-3p was upregulated as compared with healthy controls in urine and serum. Mechanistically, the authors demonstrated that miR-103a-3p contributed to angiotensin II-induced HN via the SNRK/nuclear factor-κB/p65 regulatory axis [A number of promising non-invasive miRNA biomarkers have now been identified in renal disease with the potential to augment diagnosis, guide prognosis and monitor response to treatment—just a few are highlighted here . Large pThe authors are supported by funding from the Medical Research Council (MR/S001743/1) and Kidney Research UK .L.D. is an awardee of PhD studentships co-funded by Regulus Therapeutics and GSK.


How HetR controls heterocyst-specific expression from DIF1 motif promoters remains to be answered. This study presents evidence that the expression from DIF1 motif promoters of hetR, patS and patX is more directly dependent on hetZ, a gene regulated by HetR via a HetR-recognition site. The HetR-binding site upstream of hetR is not required for the autoregulation of hetR. PatU3 (3′ portion of PatU) that interacts with HetZ may modulate the expression of hetR, hetZ and patS. These findings contribute to understanding of the mutual regulation of hetR, hetZ-patU and patS/patX in a large group of multicellular cyanobacteria.HetR and PatS/PatX-derived peptides are the activator and diffusible inhibitor for cell differentiation and patterning in heterocyst-forming cyanobacteria. HetR regulates target genes via HetR-recognition sites. However, some genes (such as  With the rise of atmospheric oxygen, certain filamentous species developed the capability to form specialized N2-fixing cells, called heterocysts, to protect nitrogenase from inactivation by oxygen  but not in differentiating cells of 7120hetZdel4-201 carrying the same plasmid; substitutions at TCCGGA abolished the expression of gfp in the wild-type strain (patS in (pro)heterocysts largely depends on HetZ and the DIF1-motif promoter. Similarly, expression from the DIF1-motif promoter of patX is also dependent on the function of hetZ  of hetR and the same DNA fragment without the HetR-binding site or the DIF1 motif. Expression from the promoter of hetR was upregulated in (pro)heterocysts of Anabaena 7120, and the upregulated expression was abolished by substitutions at the DIF1 motif but not at the HetR-binding site  upstream of gfp (pHB6821) and introduced the plasmid into Anabaena 7120 and the hetZ mutant. As shown in Anabaena 7120 [pHB6821] but barely expressed in differentiating cells of the hetZ mutant. The copy numbers of zeta-based plasmids showed some changes in different strains but were still comparable with each other. Apparently, the upregulated expression of hetR in (pro)heterocysts is also mediated by HetZ via the DIF1 motif promoter.To confirm the role of the DIF1 motif in heterocyst-specific expression of Anabaena 7120, PhetR-DIF1-, with the DIF1 motif substituted with GATATC in the chromosomal DNA. Compared to the wild type, the PhetR-DIF1- strain showed delayed heterocyst differentiation and lowered heterocyst frequency  is not involved in heterocyst differentiation [ntiation . EmployihetR and patS in the wild type and the 7120patU3::C.K4 strain at 6 h after nitrogen stepdown heterocysts. Substitutions at the DIF1 motif greatly reduced the transcription activity of PpatS; a mutant of Anabaena 7120 with the DIF1 motif of hetR substituted in the genome showed no transcription from the tsp -272 heterocysts in the wild type, and the expression was greatly weakened in the 7120hetZdel4-201 strain. These results indicated that HetZ directly or indirectly regulates the expression of these genes via DIF1 motif promoters.First, we showed that DIF1-motif promoters are responsible for the upregulation of ild type ). UpstrehetR in a hetR-minus background? Because HetR and the global nitrogen regulator NtcA are dependent on each other for upregulated expression during heterocyst differentiation [ntcA from being upregulated. For this question, we think that NtcA and HetR do not directly regulate each other’s encoding gene. In at least one substrain of Anabaena 7120, NrrA mediates the regulation of hetR by NtcA [hetR mutant with PntcA-hetZ-hetP implied that genes regulated by NtcA were properly expressed in developing cells. Presumptively, the expression of hetZ and hetP from PntcA allowed sufficient expression of NtcA in those developing cells (the relationship between hetZ/hetP and ntcA awaits investigation), and NtcA in turn enhanced the expression of PntcA-hetZ-hetP and indirectly upregulated PhetR. (2) How to explain the differentiating cells in the 7120delhetZ4-201 mutant? In the hetZ mutant generated with Anabaena 7120 in our laboratory (substrain IHB), we found that the mRNA level of hetR was increased after nitrogen stepdown as in the wild type (hetR could have initiated cell differentiation that ceased at the very early stage in less regular pattern (consistent with the low expression of patS). This is a difference between the hetZ mutant generated in this study and that reported by Videau et al [For the results we presented, two points need to be addressed in particular. (1) How to explain the upregulation of Pntiation , lack of by NtcA , 50. Actild type , therefoau et al .hetZ is involved in initiation of heterocyst differentiation and regulation of patS/patX and hetR. patX is not required for de novo heterocyst patterning in Anabaena 7120 , but its counterparts in most other heterocyst-forming species may play a role in heterocyst patterning. Therefore, HetR, HetZ and PatS/PatX form the core regulatory circuit in most heterocyst-forming cyanobacteria. This conclusion is important, because HetZ may provide an additional site for modulation of the expression of patS/patX, the sources of diffusible inhibitors for de novo pattern formation. PatU3 is a candidate for the modulator. It interacts with HetZ and somehow modulates the expression of hetR, hetZ and patS Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Sirs:LMNA giving rise for lamin A and C by a splice variant. Mutations lead to several diseases from progeria to skeletal muscular dystrophy. In the heart, mutations in the LMNA gene may cause cardiomyopathy, conduction disease, atrial and ventricular arrhythmias as well as sudden cardiac death (SCD) [LMNA mutations at an early stage of atrial remodeling [Lamins are the nuclear intermediary filaments that stabilize the nucleus in eukaryotic cells. They are encoded by the gene th (SCD) . Patientth (SCD)  and as ith (SCD) . Little modeling .In general, Laminopathy in the heart is associated with a poor prognosis, related to heart failure due to dilated cardiomyopathy and sudden cardiac death, caused by ventricular arrhythmia or conduction block. First phenotypic changes include conduction abnormalities like AV block type I or supraventricular arrhythmias , 6. BradHere, we report on a 37-year-old female patient presented at our center in 2017 for family screening of numerous cases of SCD. On presentation, she was asymptomatic and her 12-lead resting ECG revealed an atrioventricular block (AVB) grade I and low atrial amplitude suggestive for a laminopathy  were documented in the holter of the ICD (AHRE)  and in 1We report a case of successful treatment of symptomatic AF with a two-step approach of PVI in a patient with lamin A/C mutation. In this young patient treatment with PVI did not only reduce AF episodes but most importantly improved quality of life during the short-term follow-up. Despite the fact that mid- to long-term effects of PVI is still missing in our patient, it is nevertheless important to communicate the need for further case studies and randomized-trials in specialized centers to assess the time course of atrial remodeling and the long-term effects of AF ablation in this highly arrhythmogenic patient collective. Numerous patients have undergone PVI procedures as in recent ablation registries and studies –29 and i


SARS-CoV-2, a betacoronavirus is the infectious viral agent causing the current pandemic of COVID-19. The disease causes acute respiratory distress syndrome, an inflammatory response which is the major cause of mortality of this disease. The current pandemic has been projected to cause hundreds of thousands of deaths worldwide . In the in vitro activity against SARS-CoV-2 [Several pharmacological approaches are being evaluated as therapeutics to reduce disease burden until effective vaccines are developed. The primary focus has been to repurpose approved therapeutics to target COVID-19 by using anti-inflammatories and antivirals. The aim of the first approach is to reduce the overactive inflammatory response to SARS-CoV-2 which has been suggested as a mechanism of increased disease severity , while tRS-CoV-2 .In this context, we would like to discuss an interesting link between druggable epigenetic modifications and the innate immune response mediated by Type I interferons (IFNs). The Type I IFN pathway is a key antiviral response induced early on in infection via host sensing of pathogen-associated molecular patterns which are typically aberrant nucleic acid structures not normally present in the host. The pathway ultimately leads to the transcriptional activation of interferon stimulated genes which are potent antiviral effectors.A recent study showed that pathogenic influenza A viruses and other betacoronaviruses (MERS-CoV and SARS-CoV) inhibit the activation of IFN-dependent genes in infected cells  which inPharmacologic inhibitors of PRC2 are currently in advanced clinical trials for several malignancies  and coulTherefore, inhibitors targeting the PRC2 complex could be used to counteract viral repression of interferon stimulated genes and enhance the adaptive immune response in the case of SARS-CoV-2 infection. When testing the antiviral properties of PRC2 inhibitors, it will be essential to evaluate the optimal timing and duration of treatment and its use in combination with other therapies. In addition, most of the data on PRC2 inhibitors have been collected from cancer models so it will be important to study the role of these and other epigenetic drugs in the specific context of SARS-CoV-2 infection in relevant models. However, we think that the current situation requires every scientist to contribute new and possibly unconventional ideas, in the hope that some will soon produce effective treatments.


Early detection of marginal bone loss is vital for treatment planning and prognosis of teeth and implant. This study was conducted to assess diagnostic accuracy of CBCT compared to intra-oral (IO) radiography for detection, classification, and measurement of peri-implant bone defects in an animal model.p value of < 0.05 was considered as statistically significant.Fifty-four mandible blocks with implants were harvested from nine male health adult beagle dogs with acquisition of IO, CBCT and micro-CT images from all samples. Peri-implant bone defects from 16 samples were diagnosed using micro-CT and classified into 3 defect categories: dehiscence (n = 5), infrabony defect (n = 3) and crater-like defect (n = 8). Following training and calibration of the observers, they asked to detect location  and shape of the defect  utilizing both IO and CBCT images. Both observers assessed defect depth and width on IO, CBCT and micro-CT images at each side of peri-implant bone defect via CT-analyzer software. Data were analyzed using SPSS software and a Overall, there was a high diagnostic accuracy for detection of bone defects with CBCT images (sensitivity: 100%/100%), while IO images showed a reduction in accuracy (sensitivity: 69%/63%). Similarly, diagnostic accuracy for defect classification was significantly higher for CBCT, whereas IO images were unable to correctly identify vestibular dehiscence, with incorrect assessment of half of the infrabony defects. For accuracy of measuring defect depth and width, a higher correlation was observed between CBCT and gold standard micro-CT , whereas a lower correlation was seen for IO images .The diagnostic accuracy and reliability of CBCT was found to be superior to IO imaging for the detection, classification, and measurement of peri-implant bone defects. The application of CBCT adds substantial information related to the peri-implant bone defect diagnosis and decision-making which cannot be achieved with conventional IO imaging. Peri-implantitis is a pathological inflammatory reaction leading to progressive loss of the supporting bone which exceeds the physiological bone remodeling around an implant , 2. To dTwo-dimensional (2D) imaging modalities such as intraoral (IO) and panoramic radiography are the most commonly utilized radiographic methods for defect detection in a clinical practice based on their low radiation exposure and cost-effectiveness –13. HoweFurthermore, microscopic computed tomography (micro-CT), one of the most versatile non-invasive investigative techniques has been regarded as a standard tool for quantifying the density and architecture of bone in preclinical investigations. Micro-CT functions by illuminating a rotated object with x-rays and collects the magnified projection images via planar x-ray detector. Thereafter, multiple angular images are obtained and stacked together to form the 3D image. Henceforth, micro-CT not only has the ability of imaging the internal biological structures without the need for sample preparation but also provides with 3D representation of the anatomical structures. Previous studies have widely reported on the feasibility and reliability of using micro-CT to evaluate morphologic characteristics of cortical and trabecular bone in both animal and human models –28.Therefore, the current study was conducted to assess the diagnostic accuracy of CBCT compared to IO radiography using the micro-CT as the standard for the detection, classification, and measurement of peri-implant bone defects in an animal model.Following ethical approval from the Bioethics Committee of Sichuan University (Reference No: WCCSIRB-D-2014-010), nine health male adult beagle dogs  with completely sound oral condition were recruited by following the ARRIVE guidelines  for precFollowing diagnosis, both observers measured the defect depth and width on IO, CBCT and micro-CT images at each side of peri-implant bone defect via CT-analyzer software . The slices in 3D images were standardized to that of the 2D image. A mesio-distal and bucco-lingual slice was selected from the sagittal and coronal view individually on the 3D images and were oriented parallel to the long axis of implant. The width and depth of the defects were measured as shown in Fig. p value of < 0.05 was considered as statistically significant.Data were analyzed using SPSS software . The diagnostic accuracy of IO and CBCT images for the defect detection and classification was assessed by calculating the sensitivity and intra- and inter‐rater reliability (Cohen’s and Fleiss’s Kappa) of each method and observer. The interpretation of Kappa values was carried out as suggested by Landis and Koch . Intra-cOverall, a high agreement  and reliability  was observed for the detection and classification of the defect on IO and CBCT images. Intra- and inter-rater agreement for the diagnosis with CBCT was found to be almost perfect for each observer. The reliability of CBCT images was also considerably high when compared with the micro-CT for the detection and classification of the bone defect . Additionally, the intra-rater and inter-rater agreement between both observers was higher with CBCT compared to IO images . A higher correlation was observed when CBCT was compared with micro-CT , whereas slightly lower correlation was seen with IO images .p = 0.003) and buccal side (p = 0.002) showed a significantly larger depth in crater-like defects. Similarly, a larger defect width was observed for crater-like defects in both IO and CBCT images, except mesial side on CBCT images which showed a larger width in infrabony defect. Whereas, IO images showed a significantly larger width on the mesial side of the crater-like defect.The relationship between the bone defect morphology and size is demonstrated in Fig. Evidence suggests multiple studies underlining the importance of the accurate detection and classification of bone defect which can influence the treatment planning and prognosis of dental implant –36. TherOur findings suggested a higher reliability and diagnostic accuracy for the detection and classification of peri-implant defects with CBCT compared to IO images. Based on the 2D nature of IO radiographs, it only allowed accurate diagnosis of the horizontal and 1-wall vertical bone defects in a mesiodistal direction . This coIn relation to the mesiodistal depth and width measurements of the bone defect, a high correlation was found for both IO (r = 0.82) and CBCT (r = 0.92) images when compared with the micro-CT. The slightly lower correlation value with IO could have resulted due to the overlapping effect and blurring of the peri-implant region. We found the size of defect to be significantly larger in crater-like defect compared to infrabony defect. A previous study observed peri-implant bone defects to be larger on buccal side compared to other walls in patients with peri-implantitis . In contOur study had certain limitations. Firstly, the sample size was not sufficient for drawing a meaningful conclusion related to the relationship between the bone defect type and size. Secondly, the setup for the high resolution CBCT was obviously diverting from that of a normal patient acquisition. We realise that this may cause some deviation when transferring the present imaging data and outcome to the clinic situation. However, the high-dose allowed accurate investigation of infra-bony defects which might get impeded due to the presence of implant-related artefacts if a lower dose is applied. Further studies are required to assess the diagnostic accuracy of low-dose CBCT protocols in patients for detecting and classifying peri-implant bone defects. In the midst of these limitations, we provided evidence related to the detection of bone defects which might not be visible on IO radiographs, thereby leading to an inaccurate diagnosis and treatment planning. Although IO radiography is considered as a clinical standard for assessing peri-implant bone defects , the impThe diagnostic accuracy and reliability of CBCT was found to be superior to IO imaging for the detection, classification, and measurement of peri-implant bone defects. The application of CBCT adds substantial information related to the peri-implant bone defect diagnosis and decision-making which cannot be achieved with conventional IO imaging. However, benefit-risk ratio should be kept in mind and CBCT should be acquired for cases where a peri-implant bone defect might influence the implant survival rate.


We describe and evaluate a novel white-box fuzzer for C programs named FuSeBMC, which combines fuzzing and symbolic execution, and applies Bounded Model Checking (BMC) to find security vulnerabilities in C programs. FuSeBMC explores and analyzes C programs (1) to find execution paths that lead to property violations and (2) to incrementally inject labels to guide the fuzzer and the BMC engine to produce test-cases for code coverage. FuSeBMC successfully participates in Test-Comp’21 and achieves first place in the Cover-Error category and second place in the Overall category.


Significance: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed.Aim: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging.Approach: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology.Results: Our imaging system enabled macroscopic FLIM of a Conclusions: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation. We describe the overall architecture of our device and characterize the system in regard to future intraoperative use. Finally, we validate our method by imaging human brain tumor samples and compare the results to conventional PpIX fluorescence imaging as well as histology.2Based on the feedback from neurosurgeons, we opted for a minimum WD of 200 mm. We modified the assistant’s surgeon port of the surgical microscope  for integration of the FLIM system and designed a compact scanner block, where we integrated the fiber port for the excitation laser as well as the beam steering and collimation optics. The object-sided numerical aperture (NA) computed using Code V was 0.013, implying a theoretical diffraction-limited resolution of In brief, the FLIM system is based upon laser scanning an amplitude-modulated 405-nm laser  for excitation and performing a nondescanned homodyne detection using a sensitive photomultiplier (PMT) as detector . As shown in For comparative wide-field imaging, a consumer grade color camera  was attached to the other camera port. Two camera settings were used. First, an image under white-light illumination was acquired. Subsequently, the white-light illumination was switched off and the 405-nm laser was scanned over the sample repeatedly. During the laser scan, the camera was set to a long exposure (10 s). The pixel dwell time was set such that the laser was scanned over the full FOV four times during the exposure time of the camera. A 430-nm longpass filter  was used to block the excitation laser and detect the fluorescence only. The integrated color fluorescence image was then overlaid on the white-light image in postprocessing.The fluorescence lifetime acquisition was controlled through an open-source microscopy suite . This is in good agreement with the theoretically expected resolution of In the first step, we characterized the lifetime accuracy for integration times of the lock-in amplifier ranging from ex vivo on human tumor samples within an ongoing study approved by the ethics committee of the Medical University of Vienna (EK419/2008—Amendment 04/2018). We show two representative samples from two patients undergoing tumor resection of suspected low-grade gliomas after obtaining informed and written consent. A detailed study design can be found in our previous publication.Imaging with the FLIM system was performed In the second patient, the surgeon excised a focal fluorescent area, which was then further characterized using FLIM. Although the fluorescence was clearly visible in the camera image  and exhi,Our first results seem promising for enhanced tumor detection. Both specimen imaged with our system showed areas with lifetimes At present, the acquisition time of around a minute limits the potential for real-time surgical guidance. Future studies should focus on increasing the imaging speed. Due to the stereoscopic design and the long WD, the NA of commercial surgical microscopes is currently limited to 4We presented the characterization and validation of a modified surgical microscope with integrated FLIM capability. While surgical microscopes have been equipped with alternative modalities such as spectroscopic imaging,Click here for additional data file.Click here for additional data file.


We conducted a prospective, dual-site, crossover-randomised study to determine the ability of a 16-item smartphone-based assessment (the index test) to predict subitems from the Movement Disorder Society-Unified Parkinson’s Disease Rating Scale part III (MDS-UPDRS III) as assessed by three blinded clinical raters (the reference-standard). We analysed data from 60 subjects . Subject-level predictive performance was quantified as the leave-one-subject-out cross-validation (LOSO-CV) accuracy. A pre-specified analysis classified 70.3% (SEM 5.9%) of subjects into a similar category to any of three blinded clinical raters and was better than random  classification. Post hoc optimisation of classifier and feature selection improved performance further , although individual subtests were variable (range 53.2–97.0%). Smartphone-based measures of motor severity have predictive value at the subject level. Future studies should similarly mitigate against subjective and feature selection biases and assess performance across a range of motor features as part of a broader strategy to avoid overly optimistic performance estimates.Digital assessments of motor severity could improve the sensitivity of clinical trials and personalise treatment in Parkinson’s disease (PD) but have yet to be widely adopted. Their ability to capture  But it is time-consuming for clinicians and its poor calibration and sensitivity3 may have played a part in the gross failure of novel therapies to translate into clinical practice5.Precise and sensitive measures of the motor severity of Parkinson’s disease (PD) remain elusive but critical if new therapies are to be fairly and quickly evaluated. The internationally validated, familiar and easily interpretable Movement Disorder Society-Unified Parkinson’s Disease Rating Scale Part III (MDS-UPDRS III)repeated monitoring of motor severity that are likely to improve the sensitivity of clinical trials in which they are used as end-points. Digital assessments show promise8 but have yet to be widely adopted partly due to the lack of transparency and harmonisation of the analysis methods used and the lack of subsequent high-quality subject-level evidence comparing them to current reference-standard measures9.Mobile devices, such as wearables and smartphones, enable low-cost objective 8 and subcomponents of it10 but the equivalence of digital and clinical measures at the individual level remains unclear. Cross-validated subject-level prediction of disease category and total MDS-UPDRS III has been demonstrated in ten patients7, but when scaled to larger numbers, a model discriminating disease category—the simpler task—was only accurate at the cost of unstable features11.Feasibility studies have reported that digital assessments correlate with total MDS-UPDRS IIIsubjective bias within them, rather than removing it. Second, approaches that consist of only 5–7 digital subtests may be too blunt to capture the individual heterogeneity12 evident within the 33-item clinical MDS-UPDRS III, and third, previous studies are at high risk of providing over-optimistic results due to feature selection bias if a large number of post hoc candidate digital features or machine-learning algorithms are tested within a limited size study.This failure to generalise may be due to three fundamental reasons. First, digital models trained with single human scores incorporate 6 or pre-registered to reduce post hoc feature selection bias.We designed the CloudUPDRS Smartphone Software in Parkinson’s (CUSSP) study to address these concerns. We assessed the degree to which subject-level smartphone-based measures predicted subject-level MDS-UPDRS III subitems. A randomised crossover design and blinded assessment by three clinical raters mitigated concerns about subjective bias. We used a larger 16-item smartphone-based assessment to increase the capacity to capture individual heterogeneity, and methods were pre-publishedOverall, 62 participants were recruited with a minority (5) from the second site , which started recruitment later. Network faults during data capture and storage errors of video-recordings, or incorrect task performance (detected on review of video-recordings) resulted in loss of data for two subjects, 18/1008 remaining smartphone tests and 6/882 remaining clinical MDS-UPDRS III items. Only three participants opted for an OFF/ON recording, the treatment status of the remaining participants being considered intermediate. Subsequently, the final analysis included 60 patients, 63 sessions, 990 smartphone subtests, 876 MDS-UPDRS III item videos and 2628 human score ratings. Twenty participants were female, eight participants were left-handed. Demographic details are shown in Table median MDS-UPDRS III subscores overall  but this was improved with optimised classifier and feature selection  classification accuracy of the smartphone-based prediction of the MDS-UPDRS III subscores. A fully pre-specified analysis classified 70.3% SEM 5.9%) of subjects into a similar category to a clinical rater see Fig. . This wa.9% of suItem-specific LOSO-CV accuracy for each of the 16 smartphone tests is shown in Fig. 10. Here, we mitigate against this bias by training on the median of three blinded human ratings with mostly moderate-to-substantial inter-rater agreement  was as expected less accurate overall  but still better than a random baseline .New measures of motor severity in PD must be robustly evaluated at the subject level prior to widespread use. Although digital assessments such as finger tapping speed are objective and more likely to be reproducible, they can only be meaningfully interpreted as severity scales either when matched to prima facie valid subject-level outcomes such as degree of functional dependence or, more commonly, when matched to well-established and familiar but subjective scales such as the MDS-UPDRS III. Subsequently, most digital tools trained on single human classifications or ratings incorporate the subjective bias of that particular rater within them10. Our selection criteria successfully captured those with mild to moderate disease who may have mild/moderate depression or mild cognitive impairment (MoCA > 20), but not of a severity that would impair understanding of the simple motor tasks required. The sample is, therefore, broader and more transparent than previous reports and more representative of a typical secondary care population, recruiting from two hospital sites and not excluding patients based on poor compliance with home monitoring, or phone availability for example. In spite of this, some clinical features (e.g. leg tremor) did not have enough sample variation to train a useful predictive model, and generally models were more sensitive to changes in mild to moderate (rather than severe) disease. This highlights an important limitation to most current digital studies that focus on mild/moderately affected individuals—if classifiers are not trained on data from more severely affected patients (or patients assess in the ‘OFF’ state), they will not generalise well to these populations. In our study, only three subjects opted to be assessed after overnight withdrawal from dopaminergic medication. Although the effect of treatment is not the question we are addressing here, this may have limited the number of high MDS-UPDRS III scores in our cohort. Similarly to other digital tools, therefore, our smartphone software may be of most benefit to populations with mild/moderate disease undergoing interventions to modify disease progression or treat motor fluctuations. Future work should aim to address this by collecting more data or changing inclusion criteria to incorporate more severely affected patients. Because all assessments were performed under clinical supervision and reviewed by video, we can also have confidence that the smartphone tasks were performed adequately without undue influence from idiosyncratic factors . Although mild cognitive impairment and depression may correlate with disease severity and therefore slower movements, this is likely to equally affect the clinical MDS-UPDRS III and smartphone subtests and therefore is unlikely to be a significant confound to the primary outcome. Finally, the reproducibility of these findings also depends on the number of blinded clinical raters and their inter-rater agreement. Our three blinded raters were of similar clinical experience and so population-level and subject-level inter-rater agreement was reasonable. Future work may look at the effect of a larger and more varied pool of trained blinded raters.Any clinical study is limited by how well the study cohort represents the clinical population in question. We analysed data from 60 subjects which cannot represent the entire population but compares favourably with other studies comparing supervised smartphone measures to MDS-UPDRS III scores12. Most current digital assessments that focus on only 5–7 pre-selected subtests17 are likely to have reduced sensitivity as compared to the 33-item MDS-UPDRS III. We rely on 16 independent smartphone subtests to evaluate individual heterogeneity across body parts and report results for individual tests in addition to a combined score . This is an important consideration for current smartphone assessments that typically focus on finger-tapping only16. Future analyses should determine whether a combination of subtests can be used to predict non-tested items (such as limb rigidity) or approximate total MDS-UPDRS III score but this is beyond the scope of the pre-specified analysis plan that we report here. Until this is established, we suggest ongoing assessment of a broader range of motor tests to improve the sensitivity of subject-level smartphone-based measures to diverse motor presentations of disease.PD is heterogeneous—the motor signs of tremor and bradykinesia typically vary within a patient across body parts and over timefeature selection bias where predictive accuracy is over-optimistic and relies on chance relations in the data . This problem is magnified when the ratio of features to observations is high and may explain why previous models failed to generalise11. We mitigated against this by pre-registering our design and features6 and using a standard linear classification algorithm. Our approach, based on more restrictive pre-specified analysis and the consideration of individual features only, is conservative compared to the majority of prior studies in the literature. We additionally performed an exploratory graded feature and classifier selection process which maximally led to an improved overall LOSO-CV accuracy of 78.7% (SEM 5.1). The combined results provide conservative and relatively unbiased benchmark accuracies suitable for clinical translation (full pre-specification) together with increasingly optimised results which are at risk of bias, but facilitate comparison with other studies and suggest optimal features and classifiers to be tested by future research.Conversion of a raw digital measurement into a useful digital biomarker typically requires specific data selection and transformation (feature selection) and choice of an optimal machine-learning algorithm. The greater the pool of post hoc features and algorithms from which the selection takes place, the greater the risk of This study demonstrates how digital assessments for PD can be robustly validated within prospective clinical trials—a necessary step prior to widespread adoption. We mitigated against prior concerns of subjective bias and feature selection bias whilst remaining sensitive to the diverse motor presentations of the condition and utilising open-source analysis software. Our evaluation provides a conservative but relatively unbiased benchmark performance of how smartphone-based measures correspond to standard-reference measures at the subject level. Future smartphone-based studies should consider similar precautions and incorporate more severely affected patients to improve the reliability and generalisability of their conclusions.We designed and conducted a prospective, pre-registered (NCT02937324), dual-site crossover-randomised study comparing structured single time-point smartphone-based and blinded clinical rater assessments of motor severity in PD. The primary outcome was the degree to which subject-level smartphone-based measures predicted subject-level MDS-UPDRS III as calculated by three blinded clinical raters. This was quantified as the leave-one-subject-out cross-validation (LOSO-CV) predictive accuracy. This work has received ethical approval from the UK HRA and the local research ethics committee and written informed consent was obtained from all participants.20, (2) age over 18 years old, (3) a score >20/30 on the Montreal Cognitive Assessment (MoCA)21, (4) no anti-parkinsonism medication changes within the last week, (5) capacity to consent, (6) ability to understand English well enough to operate the phone software, follow its instructions and be able to answer the study questions. Exclusion criteria were: (1) concurrent acute medical illness, (2) other co-morbidity that in the opinion of the Investigator may preclude their participation in the study, (3) inability to consent. A target of 60 participants was pre-specified based on feasibility rather than a power calculation.Participants were recruited from both the National Hospital for Neurology and Neurosurgery, London, UK and Homerton University Hospital, London, UK between 8 August 2017 and 1 March 2019. The inclusion criteria were (1) diagnosis of probable idiopathic PD according to Brain Bank criteria1, MoCA21, Beck Depression Inventory22, PDQ-3923 and Hoehn and Yahr24. Participants opted to perform the motor assessments immediately, or at a second morning visit after overnight withdrawal of their anti-parkinsonian medications. Participants attending for a second visit performed the test initially in a practically defined ‘OFF’ state, and then took their usual medication and repeated the testing in the ‘ON’ state. The latter test was carried out after a maximum of 1.5 h from the initial testing and only after both the examiner and participant agreed that the participant had entered the ‘ON’ state. Participants attending for only one visit were labelled as an ‘Intermediate’ state.Patient data were collected over one or two visits, as individually convenient. Screening assessments performed after enrolment included the full MDS-UPDRS1 by a member of the movement disorder clinical team and a 16-item smartphone-based assessment supervised by the clinician. Smartphone assessments were performed on either the participant’s own phone or on a smartphone supplied by the study team depending on availability. The smartphone and clinical assessments were performed one immediately after the other in a crossover design—their order randomised by software within the CloudUPDRS application at the point of enrolment for each participant.Each motor assessment included a standard video-recorded assessment of the 33-item MDS-UPDRS IIIvideoed examination of each MDS-UPDRS III subcomponent included in the study 26. The software guides each participant through 17 subtests that correspond to subcomponents of the MDS-UPDRS III. Each subtest has specific written, visual and audio instructions (available at http://www.updrs.net/help/) and lasts between 60 and 90 s during which time the relevant phone sensor is recorded at the maximum sampling rate the phone allows (minimum of 50 Hz). Raw sensor data are time-stamped, assigned a unique identification number and stored locally. On completion of the test subsets, data are automatically uploaded to a remote secure server via an encrypted link. Full computational details have been published previously26.The CloudUPDRS system is a CE marked software device consisting of the CloudUPDRS application for Android smartphones, a cloud-based scalable data-collection service and a data-mining toolkit that we developed left hand first’, ‘tap as fast as you can for 1 min’). All smartphone examinations were also video-recorded and reviewed to confirm correct task performance. Trials where there were substantial errors, such as unintended use of wrong hand, were removed.Smartphone subtests were designed to resemble the MDS-UPDRS III as far as possible, whilst also considering safety and practicality in the context of concurrent smartphone use. Three types of subtests were performed. Finger tapping required participants to tap one or two consistently spaced targets on the smartphone whilst screen sensors recorded the onset, duration, upwards or downwards movement and the coordinates of each touch. Tremor and proximal limb bradykinesia tests required the participant to hold or repeatedly move the phone in a stereotyped pattern whilst acceleration in 3- or 6-axes was recorded. Walking tests required the participant to place the phone in their pocket and walk 5 m, turn around and walk back . Supplementary Table https://github.com/pdkit/pdkit), an open-source data science toolkit for PD running in a Python 3 environment that has been previously developed by some of the authors of this study. Data were ingested and converted to a standardised time-series data type. There are many ways in which subject-level acceleration time-series data and touch event data can be summarised by single numbers (features). For example, acceleration data can be characterised by the power at its peak frequency or the amplitude over a frequency range. Touch event data can be summarised as a function of the hold-time of a tap, the time between taps or the spatial proximity of the touch to the visual target. PDKit provides robust, transparent and automated extraction of hundreds of distinct features currently in use in the literature and is designed to support harmonisation and comparison of metrics within one fully transparent and non-proprietary platform. We calculated nine features for finger tapping subtests, and 35 features for each other subtest. A persistent archive of feature names and the code used to calculate them is available at: 10.5281/zenodo.3632529.Raw data were stored in a custom flat text files and processed offline using PDkit version 1.2.1 (6) and the best-performing feature for each subtest (see below). Pre-specified features were calculated exactly as previously. That is, for tremor subtests, the magnitude of the scalar sum acceleration in three axes was filtered with a high-pass second-order Butterworth filter at 2 Hz prior to fast Fourier transform. Tremor amplitude was calculated as the sum of the resultant power spectrum between 2 and 10 Hz. For bradykinesia assessments excluding tapping tests, the signal had DC removed prior to applying a low-pass second-order Butterworth filter at 4 Hz and subsequent fast Fourier transform. The amplitude of the remaining signal was calculated as the sum of the amplitude between 0 and 4 Hz. Note that in this context, amplitude relates to the ‘magnitude’ of tremor in the frequency domain rather than the measured distance travelled during movement. For single- or dual-tapping tests we calculated the tapping frequency as the total number of taps divided by the time period of the task (60 s).We separately report analyses of pre-specified features  maximally free from bias but likely to under-fit the data (under-optimistic accuracy). At the exploratory end, we selected both the feature and classifier with maximum out-of-sample predictive accuracy which is moderately likely to over-fit the data (over-optimistic accuracy). Intermediate accuracy values where either the classifier or the feature were selected based on performance are also presented. A full description of the feature and classifier selection process and details of the pre-specified features are presented in Supplementary Note https://pdkit.readthedocs.io/).With many more features available than observations, post hoc selection of one or a few of these features is a commonly used strategy to improve model stability. This can, however, subsequently induce feature selection bias. Post hoc selection of a classifier can induce a similar bias and so we adopted a graded approach to address both issues. At the conservative end, we used single pre-specified features from our previously reported studyThe primary outcome was the overall LOSO-CV classification accuracy of the smartphone-based prediction of selected subitems of the MDS-UPDRS III . For each subject, each item from the MDS-UPDRS III was blindly rated three times and so the median of these clinical ratings was taken forward as the dependent variable for model training. For each of the 16 smartphone subtests, a single  smartphone feature was entered into a classifier using the corresponding median MDS-UPDRS III subscore as the target variable. The target score maximally consisted of five categories reflecting the ordinal 5-point scale of each MDS-UPDRS III item, and often consisted of fewer categories being restricted to measures in the study population sample. To reduce the risk of over-fitting and to understand how classifiers would perform on new unseen subjects, we performed LOSO-CV. The out-of-sample prediction for each subject was therefore made from a model trained only on the remaining data. Correct classification was defined as an individual prediction consistent with any of the three individual clinical raters (any-rater criterion). We additionally performed a more stringent analysis in which correct classification was defined as an individual prediction consistent with the median of three individual clinical raters (median-rater criterion). Higher accuracy can reflect characteristics of the target distribution (the distribution of MDS-UPDRS III item scores) such as class imbalance, rather than the utility of the smartphone data and so we performed two additional checks. First, we determined whether the pre-specified models were predicting a range of categories rather than consistently predicting one category. Secondly, we provide a reference ‘random baseline’ accuracy from a classifier that randomly assigns subjects into each available clinical category with uniform probability across categories. As well as using pre-specified features and the standard multinomial logistic regression model, we tested a number of other feature and classifier combinations using the same LOSO-CV procedure , moderately (2 raters agreed) or disagreed . Inter-rater agreement was calculated in Matlab R2020a  using the Fleiss toolbox (https://www.github.com/dnafinder/Fleiss).To ensure the three blinded clinical raters were consistent with each other, we additionally calculated inter-rater reliability for each MDS-UPDRS III subscore using Fleiss’ Kappa, which is a generalisation of Cohen’s Kappa to more than two ratersFurther information on research design is available in the Supplementary Materialreporting summary