Enterococcus spp. is still lacking. In this study, we found the first plasmid-medieted fosB in Enterococcus faecalis from pigs, and all the fosfomycin resistant Enterococcus spp. (FRE) isolates were multi-drug resistant. S1-PFGE, Southern blot and conjugation experiments indicated that the fosB gene located on ~54.7 kb transferable plasmids. Relative competition assay confirmed that the fosB-carrying plasmid impaired fitness in recipient E. faecalis JH2-2. Illumina and the MinION sequencing data revealed that both E. faecalis ES-1 and ES-2 isolates belonged to novel ST (ST964), and had 71 SNPs difference. WGS showed that the genetic environments of fosB were diverse among different species, and the linezolid resistance gene optrA was found in the fosB-carrying strains. To summarize, for the first time, we reported plasmid-mediated fosB in E. faecalis from pigs. And, the co-occurrence of fosB and optrA pose a serious threat to public health.Plasmid-mediated fosfomycin determinants is a global public health concern due to the increasing dissemination of fosfomycin resistance and limited clinical treatment options. Information about the fosfomycin resistant and molecular genetic among  Enterococcus spp. are natural inhabitants of the environment and essential components of the intestinal microbiota of healthy humans and animals [Enterococcus presently contains over 50 species, among which Enterococcusfaecalis  and Enterococcusfaecium (E. faecium) are the most predominant isolated species, accounting for more than 80% of isolates [Enterococcus spp. (FRE), together with the limited availability of novel antimicrobial agents, has significantly restricted effective therapeutic options [ animals ,2. The gisolates . It has isolates . However options . Therefo options .Enterococcus spp. [fosB-plasmid mediated fosfomycin resistance in Enterococcus faecium are available [fosB in E. faecalis is still unkown. Therefore, further research on the phenotype and genetic molecule of FRE and fosB-positive E. faecalis is urgently required. The purpose of this study is to investigate the antimicrobial resistance and molecular genetic of FRE from pigs.Several mechanisms have been proposed to be related to fosfomycin resistance ,9,10. Thcus spp. ,15. Repovailable . HoweverfosA, fosB, fosC and fosX encoding fosfomycin-enzymes were detected by PCR and followed by Sanger sequencing as described previously [365 health pig anal swab samples were collected in one commercial pig farms for one time in Jiangsu province, China, 2017. All the samples were screened on the mEI agar plate  supplemented with 128 mg/L fosfomycin. The identification of bacterial species was performed using MALDI-TOF MS , and then confirmed by 16S rRNA sequence analysis as described previously [eviously . Primershttp://www.eucast.org) (6 Decemeber 2020) guidelines.The MICs of FRE to penicillin, ampicillin, vancomycin, erythromycin, tetracycline, marbofloxacin, florfenicol, rifampin, linezolid, valnemulin, streptomycin, gentamycin and fosfomycin were determined using the broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (document VET01-A4). The MIC results were interpreted according to CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) . XbaI restricted DNA of S. enterica serovar Braenderup H9812 was used as DNA marker. This method allowed the detection and estimation of the size of large bacterial plasmids in the presence of genomic DNA using pulsed-filed gel electrophoresis (PFGE).S1 nuclease-PFGE and Southern blotting were performed to locate escribed . In briefosB was examined using wild-type strain as donor cell and E. faecalis JH2-2 as recipient cell. Transconjugants were confirmed by PCR targeting the fosB gene and cooperated with the results of MICs. The transfer frequency was calculated as the number of transconjugants per recipient. The conjugation assay was described in detail in our previous study . BrieflyfosB-carrying plasmid was investigated by in vitro competition assays [fosB-bearing transconjugant JH-ES-1 and JH-ES-2 against E. faecalis JH2-2 (the tester strain). The overnight cultures of competitor and the tester were mixed at a rate of 1:1 at 0 h and diluted in LB broth. Subsequently, the mixture was incubated for 24 h, and diluted in 1000-fold into LB broth at 37 ℃ for 24 h. The mixtures at both startpoint (0 h) and endpoint (24 h) were plated on LB plates without or with 128 mg/L fosfomycin and incubated at 37 ℃ for 12 h. The relative competitive fitness W was calculated using the formula W = (ln(Rf/Ri)) / (ln(Sf/Si)). Ri and Si indicate transconjugant and recipient cells at 0 h, while Rf and Sf indicate transconjugant and recipient cells at 24 h. Statistical analysis was carried out using GraphPad Prism 5.0.The biological cost of the acquisition of n assays . CompetiE. faecalis was performed in silico by multilocus sequence typing (MLST) analysis using the published database [Genomic DNA of the isolates were extracted using the Magen DNA Purification Kit , following the manufacturer’s instructions. The samples were sequenced with two different platforms, comprising Illumina and the Oxford Nanoproe Technologies (ONT) MinION platform. Paired-end libraries were constructed and sequenced using the Illumina HiSeq 2500 System . Sequencing reads were assembled de novo with the SPAdes 3.5 tool. A Rapid Barcoding Sequencing Kit was used to construct the libraries sequenced in a MinION device as previously reported. Guppy basecalling software (v2.2) was used to generate fast5 files harboring the 1D DNA sequence from fast5 files with only raw data in the tmp folder. High-quality complete plasmids were constructed by hybrid de novo assembly of Illumina short reads and nanopore long reads data using the Unicycler v0.3 tool. Reference sequences of antibiotic resistance genes were from database ARG-ANNOT . The seqdatabase .E. faecalis ES-1 determined in this study have been deposited in GenBank under the BioProject number of PRJNA609523. The genome sequences of E. faecalis isolates and the others were E. faecium isolates. The isolation rates of fosfomycin resistant E. faecalis and E. faecium from pigs were 7.67% and 7.12%, respectively, which were higher than 0.3% in E. faecalis and 4.9% in E. faecium, respectively, from a China teaching hospital [Enterococcus spp. in animals maybe more serious than that in human in China [In total, 54 FRE isolates were obtained from 365 feces samples, of which 28 were hospital . Althougin China .E. faecalis and E. faecium were resistant to erythromycin, tetracycline and fosfomycin, and all strains were susceptible to vancomycin and valnemulin  and ampicillin (3.57%), while fosfomycin resistant E. faecium showed a high resistance rate . Inversely, fosfomycin resistant E. faecalis were highly resistant to linezolid (76.92%), but fosfomycin resistant E. faecium presented a low resistance rate   A,B. Acco, 7.69%) C,D. MorefosA, fosB, fosC and fosX). Results showed that two E. faecalis isolates, named ES-1 and ES-2, were positive for fosB, being confirmed by Sanger sequence. Of note, it was found that most fosfomycin resistance Enterococcus isolates were negative for the known fos genes, indicated that novel fosfomycin resistance determinants or murA mutations may exist among these isolates [To explore the plasmid-mediated determinants in those strains for fosfomycin resistance, PCR was used to detect the plasmid encoding genes , and presented resistance to fosfomycin, gentamicin and erythromycin  of 0.78 and 0.73, respectively, when compared with its parental strain JH2-2  analysis was performed. WGS analysis showed that the two isolates both belonged to novel ST type (ST964), and had 71 SNPs. This indicated that clonal dissemination with novel ST type emerged in fosB-carrying E. faecalis strains. Additionally, one complete chromosome and two plasmids sequences were obtained from each one strain, and fosB gene was located on a 53.121-kb plasmid in both strains, which were correspondence with the S1-PFGE results. The fosB-carrying plasmid pES-fosB had 57% query cover and 97.68% identity with E. faecalis plasmid pKUB3007-3 (accession no. AP018546.1). Both the two plasmids carried fosB, macrolide resistance gene erm(B) and toxin-antitoxin (TA) systems  and IS members of ISEfm, IS1216E and ΔIS1216 (To investigate the molecular genetic characteristic of the two  systems C. Additi ΔIS1216 C.fosB-carrying contigs from NCBI showed that fosB in E. faecium was flanked by tnpA. And plasmid pEMA120 (genbank: KX853854) possessed a Tn1546-like element, with tnpA and fosB genes inserted in the vanRS-vanH intergenic region (1546 (GenBank M97297). fosB gene also emerged in Staphylococcus haemolyticus and Staphylococcus aureus in small plasmids, and flanked by rep gene at right end  and aac(6’)-aph(2’’)  . Those aerm(B), chloramphenicol resistance gene cat, tetracycline resistance genes tet(L) and tet(M), TA systems, IS1216E and ΔIS1297, copper ATPases (CopA and CopB), and copper transport repressor (CopY) (The second plasmid in ES-1 and ES-2 named pES-erm(B), which has a size of 85.096-kb, and carried r (CopY) .optrA gene, which conferred resistance to oxazolidinones and phenicols, was in chromosome. Macrolide resistance gene erm(A) and florfenicol resistance gene fexA were flanked on the left of optrA, and the 11.739 kb (tnpB-erm(A)-fexA-optrA-hp) segment has 100% query cover and 99.81% identity with E. faecalis 743142 (accession no. MF443377.1), which located on plasmid. Partial segment containing fexA-optrA in E. faecalis ES1 also emerged in homo E. faecalis plasmid from China and Ireland with accession no. MH018572.1 and MN831417.1, respectively, as well as homo E. faecalis chromosome (accession no. MH225421.1) (aph(3’)-III, lincosamide and streptogramin A resistance gene lsa(A), sulphonamide resistance gene dfrG and macrolide-lincosamide-streptogram B resistance gene erm(B). The emergence of multidrug-resistant genes will render antibiotic treatment ineffective.Further analysis of WGS showed that one copy 25421.1) . AdditioEnterococcus. spp from health pigs in Jiangsu Province, China, was evaluated. As previously reported, fosfomycin has good activity for the treatment of complicated Enterococcus. spp infection [Enterococcus. spp infection at least in some commercial pig farms, and it is necessary to monitor the antimicrobial resistance rate in FRE. Additionally, our results showed that all the FRE in this study were susceptible to vancomycin and valnemulin, which means that these two drugs could inhibit FRE growth at a low concentration, therefore, they have the potential to be used as specific medicines for infections caused by FRE strains.In this study, fosfomycin resistance in clinical nfection . Howevernfection . AccordifosB-carrying E. faecalis was obtained by WGS. The two strains shared the same novel ST964 with 71 SNPs difference. It was noting that clonal dissemination emerged in fosB-carrying E. faecalis strains. Additionally, competitive assay showed that fosB-carrying plasmid brought a fitness cost to the recipient strain. This finding partially explained why fosB is unfrequently detected in E. faecalis. The comparison of pES-fosB and related E. faecalis strains (genebank: AP018546.1) revealed that their plasmid backbones were relatively conserved. The sequence surrounding the fosB gene was diverse among different species. For example, the rep genes were adjacent to fosB in staphylococcal plasmid, while a tnpA gene existed in the downstream of fosB and was included in an ISL3-like transposon in E. faecium plasmid, [hp genes located up- and downstream of fosB in E. faecalis strains, and we speculated that recombination of the fosB gene may have occurred when it was transferred to E. faecalis. Previous study demonstrated that fosB and vanRS co-located on the same plasmid [fosB, erm(B) and aac(6’)-aph(2’’) emerged on one single plasmid, this may be the results of cross-use antimicrobial agents. Since the fosB-carrying plasmid pKUB3007-3 hasn’t been reported until now, this is the first report of transferable fosB-carrying plasmids in E. faecalis.The complete sequences of two plasmid, . However plasmid . And in erm(B) on pES-erm(B) plasmid was detected, and one copy erm(B) located on pES-fosB plasmid. The complicated existence of erm(B) could explain why ES-1 and ES-2 had high MIC values of erythromycin. Additionally, the coexistence of fosB and optrA in one single E. faecalis strain was firstly found, which was consistent with the bacterial drug resistance phenotype. As we know, essential trace elements such as chromium (Cr), zinc (Zn), and copper (Cu) participate in a number of biological processes and are important constituents of several key enzymes being involved in several oxidation-reduction reactions [cadA, copper transport related protein CopZ/B/Y were incorporated into plasmid pES-erm(B) and pES-fosB, which hinted that these genes may be spread to divergent bacteria by horizontal transfer [The presence of two copies of eactions ,25. Howetransfer . AdditiofosB in E. faecalis strains with novel ST964 from pigs, and also studied the antimicrobial resistance and molecular genetic of fosfomycin resistant Enterococcus spp.. The fosB-carrying plasmid could transfer between different strains, which lead to decreased sensitivity to multiple antibiotics in the recipient bacteria. fosB-carrying plasmid brought fitness cost in recipient E. faecalis JH2-2, which partially explained the low detection rate of fosB in E. faecalis. The genetic environments of fosB were diverse in different species, and fosB could coexist with optrA, which has a potential threat to public health. Given that fosfomycin and linezolid are the last-resort antibiotic for treating infections caused by VRE, the co-occurrence of fosB and optrA in one single E. faecalis may seriously compromise the effectiveness of clinical therapy. This finding, taking a “One Health” perspective, provided an importance theory to the increasing fosfomycin resistance. Continuous monitoring will be necessary to prevent further dissemination of these resistant elements in Enterococcus spp. isolates from humans and food producing animals.In conclusion, we firstly reported the emergence of plasmid-mediated fosfomycin resistant gene


This review aims to present and assess available and new methodologies to increase the clinical evidence of proton therapy data for patients with head and neck cancer. Despite the increasing number of scientific reports showing the feasibility and effectiveness of proton therapy in head and neck cancer, clinical evidence on the potential benefits of its use remains low for several reasons. In this article, the pros and cons of consolidated and new methodologies in this setting such as randomized clinical trials, the model-based approach, and the use of prospective multicentric registries will be detailed. Radiation therapy (RT) has an important role in the treatment of patients with head and neck cancer, both in the definitive and the adjuvant settings . In the Lancet Oncology Commission in 2011 [In the past decade, PT registered an exponential rise in both the number of patients treated and in the centers currently in operation worldwide, which exceeded 100 in July 2020 . The uni in 2011 , becauseAmong professionals, the debate is intense regarding the ethical acceptability of conducting RCTs comparing protons with photons because of the superior radiation dose distribution of protons .Another issue to consider when comparing cancer treatments is the cost of cancer care. The already-mentioned advances in RT technology, in addition to having increased the precision and tolerability of the treatments, have also inevitably increased the costs of cancer care. In this context, recent studies , 16 haveIn this article, we aim to present and discuss consolidated and novel approaches to adequate evidence generation for the use of PT in HN cancer: (1) randomized trials, (2) model-based methodology, and (3) registries .Randomized controlled trials represent the “gold standard” method for establishing evidence of new medical therapies. However, as mentioned in the “Introduction”, several issues exist concerning their applicability in assessing evidence of technologic advances, such as PT in comparison with conventional radiotherapy. Ethically, quoting Sullivan et al , “randomIn contrast, RCTs have several well-known, intrinsic weaknesses: generalizability, costs, and duration . The extFor the HN cancer population, it is informative and helpful to look back to the history of the development of another RT technologic advance such as IMRT: a clinical effect of the dosimetric advantage  in parotid sparing using IMRT versus conformal RT was demonstrated on a large scale in the Parotid-Sparing Intensity Modulated versus Conventional Radiotherapy in Head and Neck Cancer (PARSPORT) multicentric randomized trial , which wOther methodologies, apart from RCTs, should be applied when evaluating the development of new medical devices. In the context of RT, in silico planning comparative studies have been extensively used to analyze and report the different dose distribution of investigated techniques –39. The As mentioned, because of the continuous technologic improvements and for ethical reasons, a “traditional” clinical trial may be impractical or excessively challenging to conduct for radiotherapy technologies and, in particular, for PT. As proposed by US Food and Drug Administration guidance , analyseObservational studies, such as registries, have several advantages over RCTs, such as costs, generalizability, long-term outcomes, and relevant outcomes, rather than intermediate outcomes, among others. In particular, long-term follow-up of effectiveness and safety outcomes is relevant when RCTs  or model-based approaches (mainly focused on toxicities) are combined with registries. Moreover, as demonstrated by Concato et al , althougHowever, registries and model-based approaches are prone to selection bias because numerous factors may influence the enrolment of patients in a registry and may be difficult to identify in advance and to prevent.Registries should contain all the information relating to the technology used account for its evolution, costs, and clinical outcomes. As underscored by Porter et al , there iTo ensure the reliability of the RWD, registries should have relevant time windows for data element collection . An aid in the search for standardization of outcome measures could come from the International Consortium for Health Outcomes Measurement (ICHOM)  and the One of the main issues with observational data is that it is difficult to reliably compare 2 treatments because of “confounding by indication,” meaning patients receive a certain treatment  for a reason. Therefore, more-sick individuals may receive protons, whereas healthier individuals receive photons. If so, a direct comparison could show a benefit for photons, but that effect could, in part, be attributed those being treated with photons being healthier. Therefore, confounding by indications needs to be assessed. Obviously, the best way to do that is by randomization, as explained in the “Randomized Trials.” The model-based approach also accounts for this difficulty by comparing between expected toxicity and observed toxicity in the same patient. Regarding registries, the issue can  be corrected in the statistical analyses.To minimize bias, all the data included in the registries “should be accurate, as complete as possible, and have an appropriate scope to address the question at hand ” . Being sFor a proper analysis of RWD, some tools are essential, such as (1) a predefined common set of data elements, (2) a common definitional framework for common understanding of information, and (3) the choice of predefined time intervals for data collection and research analyses. We have 2 conflicting needs: registries cannot be too general or synthetic because of the risk of being uninformative and incurring bias; however, they must not be too complex and detailed to be introduced into clinical practice and in different health contexts. It is important not only to identify a new working data set but also to plan for integration and use in current systems.The use of PT for patients with HN cancer could improve the QoL of survivors by reducing treatment toxicity. The process of increasing the level of evidence for its use is complex because (1) being a technologic advancement, the use of RCTs could be limited and prone to ethical and practical issues as described; (2) because of the high costs of PT, a high level of evidence is required by national health systems and insurance companies before investing in its full implementation; and (3) although increasing, the number of patients with HN treated by any single PT center is low, thus compromising the possibility of evidence generation by traditional, hierarchic methodologies.As described previously, every solution proposed has pros and cons and is prone to criticisms and risks of bias . Integra


Human male infertility has a notable genetic component, including well-established diagnoses such as Klinefelter syndrome, Y-chromosome microdeletions and monogenic causes. Approximately 4% of all infertile men are now diagnosed with a genetic cause, but a majority (60–70%) remain without a clear diagnosis and are classified as unexplained. This is likely in large part due to a delay in the field adopting next-generation sequencing (NGS) technologies, and the absence of clear statements from field leaders as to what constitutes a validated cause of human male infertility (the current paper aims to address this). Fortunately, there has been a significant increase in the number of male infertility NGS studies. These have revealed a considerable number of novel gene–disease relationships (GDRs), which each require stringent assessment to validate the strength of genotype–phenotype associations. To definitively assess which of these GDRs are clinically relevant, the International Male Infertility Genomics Consortium (IMIGC) has identified the need for a systematic review and a comprehensive overview of known male infertility genes and an assessment of the evidence for reported GDRs.In 2019, the first standardised clinical validity assessment of monogenic causes of male infertility was published. Here, we provide a comprehensive update of the subsequent 1.5 years, employing the joint expertise of the IMIGC to systematically evaluate all available evidence (as of 1 July 2020) for monogenic causes of isolated or syndromic male infertility, endocrine disorders or reproductive system abnormalities affecting the male sex organs. In addition, we systematically assessed the evidence for all previously reported possible monogenic causes of male infertility, using a framework designed for a more appropriate clinical interpretation of disease genes.in-vitro cell and in-vivo animal model phenotypes. A final score was used to determine the clinical validity of each GDR, across the following five categories: no evidence, limited, moderate, strong or definitive. Variants were also reclassified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines and were recorded in spreadsheets for each GDR, which are available at imigc.org.We performed a literature search according to the PRISMA guidelines up until 1 July 2020 for publications in English, using search terms related to ‘male infertility’ in combination with the word ‘genetics’ in PubMed. Next, the quality and the extent of all evidence supporting selected genes were assessed using an established and standardised scoring method. We assessed the experimental quality, patient phenotype assessment and functional evidence based on gene expression, mutant The primary outcome of this review was an overview of all known GDRs for monogenic causes of human male infertility and their clinical validity. We identified a total of 120 genes that were moderately, strongly or definitively linked to 104 infertility phenotypes.Our systematic review curates all currently available evidence to reveal the strength of GDRs in male infertility. The existing guidelines for genetic testing in male infertility cases are based on studies published 25 years ago, and an update is far overdue. The identification of 104 high-probability ‘human male infertility genes’ is a 33% increase from the number identified in 2019. The insights generated in the current review will provide the impetus for an update of existing guidelines, will inform novel evidence-based genetic testing strategies used in clinics, and will identify gaps in our knowledge of male infertility genetics. We discuss the relevant international guidelines regarding research related to gene discovery and provide specific recommendations to the field of male infertility. Based on our findings, the IMIGC consortium recommend several updates to the genetic testing standards currently employed in the field of human male infertility, most important being the adoption of exome sequencing, or at least sequencing of the genes validated in this study, and expanding the patient groups for which genetic testing is recommended. Male infertility is a common condition, affecting at least 7% of men worldwide, and is often predicted to be largely genetic in origin . A majorAdvances in next-generation sequencing (NGS) have greatly facilitated the unbiased exome-wide (whole-exome sequencing (WES)) and genome-wide (whole-genome sequencing (WGS)) detection of any genetic variants that may play a role in male infertility  as PROSPERO 2021: CRD42021229164.A literature search was performed as described in https://conradlab.shinyapps.io/HISTA). To avoid bias in gene–disease evaluation and any conflicts of interest, reviewers were not allowed to score any GDRs they had published. Scoring was separated into five categories: no evidence (<3 points), limited (3–8 points), moderate (9–12 points), strong (13–15 points) or definitive (>15 points), with a maximum of 17 points. After independent scoring, the individual scores (for each GDR) for both reviewers were compared and any inconsistencies in scoring  were settled by the two assigned reviewers. Where this was not possible, the scoring was discussed with all non-conflicted reviewers. GDRs with a moderate or higher classification were deemed as confidently linked to human male infertility and combined scoring sheets are available at http://www.imigc.org. All results in the study are collated in The clinical validity of each identified GDR was scored using a system published by We performed a literature search using terms related to ‘male infertility’ in combination with keywords related to the word ‘genetics’ in MEDLINE-PubMed and used the same inclusion and exclusion criteria as described previously . The totA total of 1523 publications met all inclusion criteria and were used in this systematic review. Of these, 186 publications were published in the period from 2019 to mid-2020 and were thus not included in the previous clinical validity assessment. Overall, 657 individual GDRs described in these 1523 publications were investigated, of which 136 were novel and 521 were re-evaluated with our updated assessment criteria in order to incorporate any additional supporting evidence .in-vitro and in-vivo loss-of-function animal/cell models. Variants were also reclassified according to the widely accepted the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) standards and then recorded in spreadsheets for each GDR.The quality and the extent of all evidence for the GDRs were assessed by two independent reviewers, using a standardised scoring method. This assessment was performed for GDRs that were new (n = 136), existing with newly published evidence (n = 145) or previously confidently linked  to male infertility with no newly published evidence (n = 19). This score was used to assign a clinical validity of each GDR as: no evidence, limited, moderate, strong or definitive. We assessed the experimental quality, patient phenotype information, functional evidence based on gene expression and the study of vice versa). As detailed above, GDRs were listed as ‘unable to classify’ when the predicted inheritance pattern or genotypes were not reported. In comparison to the previous assessment, 456 scores did not change because no (or insufficient) new evidence was published during the search period  and 43 were classified as having limited evidence. After classification of the existing GDRs that had new evidence published and scoring of GDRs that were previously confidently linked to male infertility with our updated scoring criteria, 45 increased in score since 2019, 11 decreased in score and 9 changed from unable to be classified to no evidence , strong (n = 27) or definitive (n = 48) evidence  and II. As outlined above, in recent years, the identification of genes confidently linked to a male infertility phenotype has risen by 150% from a stable average of approximately four per year in the period of 2000–2016 to 10 per year in the period of 2017–2019 . AdditioThe majority of confident GDRs were endocrine disorders or reproductive system syndromes (n = 67) and isolated infertility phenotypes (n = 36), while a minority were linked to syndromic infertility (n = 17), including primary ciliary dyskinesia PCD; . The sysThe aim of this study was to provide an updated assessment of all genetic variants reported as causative of human male infertility phenotypes. We also highlight male infertility phenotypes that have received significant attention in the last few years and identify a group of promising new candidate genes. These genes are currently classified as limited evidence, requiring additional replication studies and/or functional evidence in order to be classified as strongly associated with male infertility. Based on our findings, the authors/the IMIGC consortium recommend several updates to the genetic testing standards currently employed in the field of human male infertility, most important being the adoption of exome sequencing as the default sequencing approach.Our clinical validity assessment revealed that as of 1 July 2020, a total of 104 genes were linked to a total of 120 male infertility or abnormal genitourinary development phenotypes. This is a significant increase from the 2019 report wherein 78 genes (33% increase to 104) were associated with 92 phenotypes (30% increase to 120) at a moderate or higher level of supporting evidence. As this previous assessment included all published reports from 1958 to 2018, this significant increase in 2 years further emphasises the healthy uptake of studies to elucidate genetic causes of male infertility, specifically those using NGS.The diagnostic rate of genetic tests for all types of isolated male infertility combined currently sits between 4% and 9.2% . The bigARMC2, CFAP43, CFAP44, CFAP65, CFAP69, CFAP91, CFAP251, DNAH1, DNAH17, FSIP2, QRICH2, SEPTIN12, SPEF2 and TTC29) are confidently linked to MMAF and another 9  are listed as candidate genes, i.e. they have a limited classification. As a reflection of collaborative research efforts, MMAF-associated genes also comprise 42% of all confident genes that function during the spermiogenesis period.The phenotype where most progress has been made in recent years is MMAF. Of the 31 newly discovered genes confidently linked to male infertility, 9 were linked to MMAF (29%). When just considering genes likely associated with primary male infertility (i.e. spermatogenesis genes), this constitutes 47% of new, confidently classified genes (9 of 19). Currently, a total of 14 genes  can assist in refining possibly relevant genes containing variants, based on any known biological function(s). However, very little so far has been done outside of meiotic and spermatogonial arrest. Various genes have also been linked to multiple phenotypic outcomes; thus, the establishment of a clear genotype–phenotype correlation is not necessarily straightforward. One such example is the PROK2 gene, for which autosomal dominant, recessive and oligogenicity have been proposed to be causative /Kallmann syndrome. In addition to Mendelian causes, incomplete penetrance and oligogenic forms are thought to be causative for these disorders  are situated across the USA, Europe and Australia, and are interested in forming additional collaborations for clinical and research purposes. As a less impactful, backup approach, the genetic targets of panels used in targeting sequencing should at least be updated to include the genes validated in this study (those moderately linked and stronger). Additionally, a wider subset of patients/phenotypes should be sequenced. Such approaches will help to achieve more diagnoses, better-personalised treatment, improved risk assessment for the transmission of infertility to offspring and better counselling for potential health risks in the infertile man. It will also help to improve counselling of azoospermic men prior to testicular biopsy, as finding a genetic diagnosis may help in predicting the chances for successful testicular sperm extraction, which is an incredibly invasive procedure.TEX11, which is expressed in the testis and the pancreas according to RNA-seq data . As has been made clear above, this is the case for several cilia genes, where men often present with chronic bronchitis and other lung conditions  for monogenic causes of isolated or syndromic male infertility, endocrine disorders or reproductive system abnormalities affecting the male sex organs. No other published reviews in this field come close to including the ∼600 genes comprehensively evaluated here. We improved the quality of evidence scoring by including a larger number of reviewers from independent research groups with broad expertise in male infertility. As all research groups have been involved in disease gene discovery of various genes described in this study, we prevented bias as much as possible by not allowing scoring by reviewers who have worked on respective genes.Here, we describe an updated clinical validity assessment for a total of 657 male infertility or abnormal genitourinary development GDRs, involving 596 genes. We identified 104 genes linked to 120 phenotypes with sufficient evidence for use in gene panels. These results may help to improve genetic testing in male infertility research and/or diagnostics.Human Reproduction Update online.http://www.crd.york.ac.uk/PROSPERO) as PROSPERO 2021: CRD42021229164. All scoring spreadsheets are accessible via the IMIGC website (www.imigc.org/data-sharing).The present study and the corresponding search protocol were registered with the PROSPERO registry (dmab030_Supplementary_DataClick here for additional data file.


Perceived realism is the result of an evaluation of the virtual world regarding (1) the subjective degree of reality of the depicted environment and (2) its overall plausibility and credibility. Thus, the sense of presence in a VE is conceived as a composite of being there and perceived realism. When in VR, a user will inevitably compare the look of virtual objects to real-world objects and judge the level of congruence . Opticians do not only measure visual acuity but also the visual field, stereoscopic vision, and color vision. In the same vein, presence researchers are encouraged to not only measure the experience of “being there” in the sense of attentional allocation to the virtual environment (VE), but also the Previous papers on presence are based on the assumption that realism enhances presence  to content presented in VR, i.e., it does not require higher-order cognitive processing. A similar concept was proposed by Wirth et al.  virtual objects, sounds and scenes, (2) credibility and plausibility of the story and its characters, and (3) naturalness and ease of the interaction with the VE. This is a new definition which we propose and which will have to be tested and, if necessary, adapted in future research. It has to be pointed out that perceived realism is not a mere function of the technical properties of the VR system but rather results from the interaction between individual expectations and technical properties such as tracking quality and visual appearance of objects. For example, the more natural the way of interacting with the VE , the more realistic it appears, usually. This corresponds with those definitions that include possible actions as a relevant aspect of presence experiences . Empirical findings support this idea, as they show that presence is modulated by individual expectations, personal relevance, and personality traits  and characters  are also evaluated in terms of consistency and plausibility  realism complements the sense of being there and that it needs to be incorporated into a comprehensive definition of presence in VR. In line with Busselle and Bilandzic, we have chosen the term there if equipped with a modern HMD: the entire visual field is taken in by the display, headphones are drowning external noise, and haptic devices emulate the sense of touch . However, we seek for a new, substantially advanced, concept of realism that goes beyond quality assessments of the VE's sensual properties. A judgment about the plausibility of the virtual world is lacking. A graphically unsophisticated world does not necessarily go along with a low presence measure but can very well be experienced as realistic in certain scenarios. The other way around, a visually stunning experience does not necessarily lead to high presence. Presence always depends on the user's individual predispositions and situational aspects, aspects that are not reflected in current presence questionnaires  captures and maintains our attention and (2) is perceived as realistic. Therefore, we suggest dividing presence into the two dimensions Current research on presence does either not consider perceived realism or a distinction between perceived realism and being there is widely missing. This resulted not only in contradictory definitions of presence but also in numerous confusing questionnaire items. Unlike other media, VR creates a highly absorbing experience and evoking a sense of being there is almost automatically achieved by rendering potentially competing external sensory input unavailable. Indeed, creating credible and realistic VEs is crucial for achieving strong feelings of presence in VR. Enhancing the perceived realism of VEs is a main focus of VR development. Yet, it is often neglected in theoretical conceptions or blended in with being there as a form of attentional allocation. Perceived realism is especially relevant for VR experiences, not only because they effectively block external sensory input but also for the better use of the multi-sensory stimulation that is possible with advanced technology. VR experiences have already been enhanced with the sense of touch , a sense of agency , and a sense of body ownership . This concept successfully expands the definition of embodiment, which has been used for explaining the effect of accepting an artificial limb as a part of one's own body  and the researchers' focus on presence (being there). As mentioned, it is not only important to measure the realism associated with each sense being stimulated. It also necessary to keep track of possible interactions between them and ask the user about his integrated sense of perceived realism.A compelling virtual world needs to be believable, authentic, and visually appealing in addition to capturing attention. This is especially true for professional VR applications that rely on maximal comparability to the real world, such as training environments for surgeons, firefighters, or pilots. A clearer separation of being there and perceived realism makes presence more generalizable across media and especially more applicable to VR. In terms of VR, questionnaires need to focus on the perceived realism of the VE. This way, game designers could obtain subjective quality measures of VEs. Researchers could use more detailed presence measures that separate being there and perceived realism. This will lead to stronger and clearer results and strengthen the correlations between presence and different output variables such as enjoyment, therapy outcome, or learning.This opinion paper aims to initiate a discussion about what constitutes presence in VR. We have proposed a plausible definition that is based on existing findings. Nevertheless, in a first step, future research is needed to challenge this definition.An evaluation of the proposed definition could be accomplished by developing a new questionnaire that incorporates relevant aspects in the context of perceived realism, e.g., the perception of virtual objects, sounds and scenes; plausibility of the story; naturalness and ease of interactions. As part of the questionnaire construction, an appropriate item pool would need to be generated to map these aspects  so that attentional resources are bound by the VE. The condition low level of being there is a version of the same game, but in a version that is not involving at all with the result that the attentional resources are not allocated toward the VE; this could for example be an overly simple and unentertaining version of the game, perhaps in combination with a repetitive and unchallenging task. The factor perceived realism could be varied as follows: in the condition high level of perceived realism, the game could be embedded in an overall story  and with more emphasis on a realistic experience in terms of high vividness, refined visual scenes and the involvement of multiple sensory cues . In contrast, the condition low level of perceived realism should not involve any  storyline, characters, or surrounding environment and the environment could look Minecraft-like  the extent to which being there and perceived realism influence possible outcome variables and to assess (2) which dimension explains more variance. For this purpose, it would be promising to conduct an experiment in which being there (high vs. low) and perceived realism (high vs. low) are manipulated in a 2×2 design. This could help to find out more about the relative influence of the two dimensions. Below we outline such an experiment. It is a suggestion that might be useful to find out the influence that the two dimensions can exert. The idea is to transfer a well-known conventional video game, which involves simple graphics yet is fun to play, into VR. A good example could be We are convinced that a more thorough assessment of the VR experience will be beneficial for research and applications alike. VE's are now rapidly gaining importance, and it is by all means necessary to better understand the dimensions that are involved in order to exploit VR's full potential, and also further develop the technology so that it serves society best.SW, DW, and FM contributed to the conception of the manuscript. SW wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Technology-supported rehabilitation can help alleviate the increasing need for cost-effective rehabilitation of neurological conditions, but use in clinical practice remains limited. Agreement on a core set of reliable, valid and accessible outcome measures to assess rehabilitation outcomes is needed to generate strong evidence about effectiveness of rehabilitation approaches, including technologies. This paper collates and synthesizes a core set from multiple sources; combining existing evidence, clinical practice guidelines and expert consensus into European recommendations for Clinical Assessment of Upper Limb In Neurorehabilitation (CAULIN).core set: strong evidence for validity, reliability, responsiveness and clinical utility AND recommended by at least two sources; an extended set: strong evidence OR recommended by at least two sources and a supplementary set: some evidence OR recommended by at least one of the sources.Data from systematic reviews, clinical practice guidelines and expert consensus (Delphi methodology) were systematically extracted and synthesized using strength of evidence rating criteria, in addition to recommendations on assessment procedures. Three sets were defined: a core set recommended for clinical practice and research: Fugl-Meyer Assessment of Upper Extremity (FMA-UE) and Action Research Arm Test (ARAT); the extended set recommended for clinical practice and/or clinical research: kinematic measures, Box and Block Test (BBT), Chedoke Arm Hand Activity Inventory (CAHAI), Wolf Motor Function Test (WMFT), Nine Hole Peg Test (NHPT) and ABILHAND; the supplementary set recommended for research or specific occasions: Motricity Index (MI); Chedoke-McMaster Stroke Assessment (CMSA), Stroke Rehabilitation Assessment Movement (STREAM), Frenchay Arm Test (FAT), Motor Assessment Scale (MAS) and body-worn movement sensors. Assessments should be conducted at pre-defined regular intervals by trained personnel. Global measures should be applied within 24 h of hospital admission and upper limb specific measures within 1 week.In total, 12 measures (with primary focus on stroke) were included, encompassing body function and activity level of the International Classification of Functioning and Health. The The CAULIN recommendations for outcome measures and assessment procedures provide a clear, simple, evidence-based three-level structure for upper limb assessment in neurological rehabilitation. Widespread adoption and sustained use will improve quality of clinical practice and facilitate meta-analysis, critical for the advancement of technology-supported neurorehabilitation. Neurological conditions are a leading cause of disability world-wide. Incidence is rising due to an ageing world population and prevalence is increasing due to growth of the world population, better survival rates and improved long-term care . The resRecent advances in neurorehabilitation research have resulted in a better understanding of recovery, giving rise to new promising approaches such as increased intensity of practice, early intervention and use of technology. Of those, the use of technology in rehabilitation may help alleviate the pressure on the healthcare system. Moreover, technologies could enable access to rehabilitation throughout the lifespan and has been advocated by the World Health Organisation (WHO) as an investment in human capital that contributes to health, economic and social development .For a successful transfer of therapeutic interventions using rehabilitation technology into clinical practice, evidence of their effectiveness is essential. This is reflected in national strategies and frameworks emphasising the need for informed decision making in healthcare that is research-led and evidence-based. Yet, several national guidelines cite limited research evidence to justify the use of rehabilitation technologies –5. IndeeAgreement on outcome measures (OM) and corresponding procedures for assessment are critical to advancing the field. For new approaches to be used effectively in clinical practice , clinicians need clear assessment guidelines to enable them to make informed decisions. The use of agreed, uniform OM is not only useful in order to compare the effectiveness of different training approaches, but also to identify which patients benefit most from which training approach and dose.For example, the use of different technologies for task-oriented training of the upper limb was investigated in highly functional chronic stroke patients in two separate clinical trials using a sensor system  or a robIn addition, practical and accurate tools are emerging that can predict recovery, with the potential to significantly improve patient management and reduce costs of health services . EstabliThe European Network on Robotics for Neurorehabilitation  has developed a set of recommendations for upper limb assessment in neurological conditions, to evaluate both conventional and technology-supported therapy. These European recommendations aim to improve the quality of upper limb neurorehabilitation in clinical practice globally, through the adoption of standardised, agreed protocols for assessment in clinical practice and research. The recommendations will directly support clinical research and facilitate larger scale multi-centre studies, allowing meta-analyses, essential for informing and stimulating investigation of prediction for patient-specific training approaches and more generally advancing understanding of recovery. It will also inform and influence the development of new upper limb neurorehabilitation technologies both as therapies and assessment tools, and assist in the translation of useful technologies into clinical practice.The present paper collates and synthesizes the recommendations from multiple sources, combining existing evidence, current clinical practice guidelines and expert consensus, into the recommendations for Clinical Assessment of Upper Limb In Neurorehabilitation (CAULIN). The CAULIN recommendations provide evidence-based recommendations for upper limb assessment of patients with neurological conditions before, during and after therapy , including the recommended time frame of applying structured assessment where available.The CAULIN recommendations were developed within the framework of a European network (COST Action TD1006). This enabled involvement of more than 200 experts and stakeholders from over 24 European countries, with a wide range of backgrounds: physical therapists, occupational therapists, physicians and nurses ; clinical researchers from the same or related professions; engineers, technology developers; neurological patients; and other stakeholders such as neurorehabilitation educators and healthcare insurers.A systematic approach, in correspondence with the Appraisal of Guidelines for Research and Evaluation (AGREE) II methodology , addressAlthough the evidence and information on which the recommendations are based focus primarily on stroke, other neurological conditions are addressed as well, including spinal cord injury (SCI), multiple sclerosis (MS), and traumatic brain injury (TBI). The recommendations are primarily targeted at supporting clinicians during clinical decision making, but they are also applicable to all professionals working in neurorehabilitation, including research, to establish uniform methods for reporting clinical outcomes.A structured approach was applied to generate the recommendations, synthesizing three published sources of evidence: existing scientific literature, clinical practice guidelines and expert consensus Fig. . ScientiA recommendation on specific sets of OM on body functions and structures and activity level,A recommendation on assessment procedures specifying when, how and by whom the assessments should be done.The CAULIN recommendations for upper limb assessment in neurorehabilitation include:The systematic overview of 13 systematic reviews (published between 2004 and 2014) focused on the psychometric properties and clinical utility of upper limb OM in stroke . From 53The evidence from 34 records (published between 2007 and 2017), including existing national clinical guidelines and published practice guidelines, on assessment of upper limb in neurorehabilitation provided input from clinical practice to the CAULIN recommendations . The speA Delphi consensus exercise with six consensus rounds performed between 2011 and 2015 provided evidence from five expert groups, consisting of 208 clinicians and researchers from medical and engineering fields across Europe. In each expert group, votes were collected on questions and statements about the use of OM for upper limb assessment in neurorehabilitation. At least 69% consensus was required for each statement to be included as a recommendation for the current synthesis .Core set (3-star rating): OM that demonstrated strong evidence for validity, reliability, responsiveness and clinical utility AND were recommended by at least two sources.Extended set (2-star rating): OM that demonstrated strong evidence for validity, reliability, responsiveness and clinical utility OR were recommended by at least two sources.Supplementary set (1-star rating): OM that showed some evidence for validity, reliability, responsiveness and clinical utility OR were recommended by at least one of the sources.Data from all three sources  were systematically extracted and combined to form specific sets of recommended OM Fig. . The extThe core (3-star) and extended (2-star) sets of CAULIN recommended OM represent OM that are psychometrically sound, have suitable clinical utility and have a solid support base in the clinical and research community. The core 3-star OM should, however, always be considered as a first choice for all clinical trials and implementation protocols in clinical settings. The 1-star rated OM represent those with good potential, but where the psychometric properties, clinical utility or expert consensus is not fully established. These measures could be used where appropriate or for research purposes. For example, additional specific OM might be needed when investigating specific treatments, such as robot-assisted therapy or home-based therapy, or when patients present with specific problems or treatment goals.Two of the three sources, i.e., clinical practice guidelines and expert consensus, generated data on recommended procedures for assessment of upper limb functioning. Based on available information, data was extracted and categorized according to three characteristics: time spent in assessment; frequency and timing of assessments; person who should conduct the assessments. Due to limited data available, rating of recommendations as done with OM selection couldn’t be applied to assessment procedures, instead data synthesis consisted of summarizing and categorizing the evidence.The synthesized results for the CAULIN recommendations on specific OM are shown in Table core set (3-star rating) of OM for clinical practice consists of Fugl-Meyer Assessment of Upper Extremity (FMA-UE) and Action Research Arm Test (ARAT). These were the only two measures presenting good psychometric properties while being recommended in at least two of the three data sources .The recommended extended set (2-star rating) adds six more OM recommended for clinical practice and/or clinical research. Kinematic measures assessing movement quality and execution are recommended at body function level, although there isn’t sufficient information available in the examined sources to specify which kinematic variable(s) should be used . Recommended OM to assess at activity level add four capacity measures with each a slightly different focus: Box and Block Test , Chedoke Arm Hand Activity Inventory , Wolf Motor Function Test , Nine Hole Peg Test ; and the ABILHAND .The supplementary set (1-star rating) includes additional OM that can be used for specific research purposes. On body function level, the Motricity Index (MI), Chedoke-McMaster Stroke Assessment (CMSA) and Stroke Rehabilitation Assessment Movement (STREAM) are added. On activity level, the Frenchay Arm Test (FAT) and Motor Assessment Scale (MAS) are additional recommended OM to measure functional ability (activity capacity), as well as monitoring the amount of actual arm use in routine daily life (activity performance) through the use of body-worn movement sensors .The Assessments should be conducted at regular intervals during rehabilitation at a minimum of four time points .Global measures should be applied within 24 h of hospital admission and upper limb specific measures within 1 week.During a rehabilitation program, assessment should be made at baseline (beginning of the program), interim (during the program), final (end of the program), and follow-up (a set period of time after completion of the program).Patients should always be assessed prior to discharge or transfer in order to support appropriate follow-up.OM should be administered separately from treatment, last no longer than three hours and be conducted by healthcare professionals who are trained to use them.Although the extent of information available is limited on when and by whom assessments should be conducted, we have summarized the available evidence on assessment procedures from the three published data sources Table , as follcore set (including 2 OM), extended set (adding 6 OM), and supplementary set (extending by 6 OM).By combining existing evidence on OM from literature reviews, a systematic overview of national clinical practice guidelines across Europe and beyond, and expert consensus on a pan-European level, we compiled uniform and agreed evidence-based recommendations for Clinical Assessment of Upper Limb In Neurorehabilitation (CAULIN). As such, CAULIN provides evidence-based recommendations for upper limb assessment of patients with neurological conditions, primarily stroke, before, during and after therapy , to be used primarily in clinical applications, but also in clinical research. Furthermore, CAULIN defines the recommended time frame of applying structured assessment at four specific instances . The CAULIN recommendations defined OM at three levels: The core set recommends FMA-UE and ARAT to be included as the core (3-star) assessments of upper limb function and activity capacity in clinical practice. This is in agreement with the consensus-based recommendations of the Stroke Recovery and Rehabilitation Roundtable (SRRR)  and the The extended (2-star) set includes a mix of performance-based and patient-reported OM (PROM), addressing both capacity and perceived performance of the arm in daily life. These assessments can be used as complementary assessments, depending on patient-specific treatment goals or needs in clinical practice, or on specific research objectives in clinical research. For example, while the BBT and NHPT are easy to implement into clinical practice , they will provide summary information on task outcome. On the other hand, some of the other recommended OM are more comprehensive and will take more time and training, while adding valuable information on task execution and strategies used by the patient. The CAULIN recommendations, however, emphasize that the core OM (FMA-UE and ARAT) should be prioritized over the extended and supplementary OM sets.In addition, the CAULIN extended (2-star) set recommends kinematic measures for assessment of movement quality on body function level. This extends the information gained through clinical assessments about task execution with more detailed information about its underlying aspects, for example movement smoothness . This reThe use of PROM is recommended in each of the three CAULIN sources for perceived activity performance, with the ABILHAND as the only specific tool demonstrating sufficiently strong psychometric properties and clinical utility. In line with the overall aim of rehabilitation, perceived performance measures add valuable and necessary information about a person’s experienced limitations of upper limb use in daily life . In the The evidence-base for assessments included in the supplementary (1-star) set is smaller compared to recommended 3- and 2-star OM. The additional OM in the supplementary set will primarily be applicable for clinical research or in specific contexts of clinical practice, depending on research questions or patient-specific treatment goals, as each of these assessments have their particular focus and advantages. One of the OM included in the supplementary set is sensor-based assessment of actual arm use in daily life. This adds the perspective of actual performance in ecologically valid real-life settings to that of capacity measures, assessing the maximum score in a controlled setting, which are known to be incongruent . AlthougThe use of goal attainment OM were mentioned in all three CAULIN sources, although no specific OM could be identified. Remarkably, a Cochrane review reported that only 3 out of 39 studies, investigating the effect of goal-setting on psychosocial outcomes during rehabilitation of people with acquired disability, used a goal attainment evaluation . Goal AtIn terms of assessment procedures, the current synthesis derived the following recommendation: administration of upper limb specific OM should be done by trained healthcare professionals within 1 week of admission to rehabilitation and repeated prior to discharge or transfer, with specific time points during rehabilitation . Despite the recognized importance of structured administration procedures, more specific recommendations couldn’t be derived from the current synthesis. When considering only consensus-based evidence, specific advice on time points for clinical assessment has recently been proposed, with maximal 7 assessments across 12 months: within 3 days (OM at body functions level only), at day 7, at weeks 2, 4, 12, at 6 months, followed by every 6th month . These pThe current work has identified uniform and agreed OM for clinical assessment of upper limb with pan-European coverage, integrating evidence on psychometric properties with clinical practice guidelines and with evidence-based consensus among clinicians, researchers and engineers, considering also clinical utility in aspects such as language availability, affordability and practical applicability. Despite these strengths, several limitations and considerations should be noted.Kinematic measures and sensor-based actual arm use are included in the current recommendations to quantify movement quality and arm/hand use on body function and activity level, even though clinical applicability isn’t well-established yet. Such technology-supported assessments are increasingly used in research , 33, butPotential cultural differences that can influence the validity of task-based assessments  haven’t been directly addressed, even though language availability of OM has been considered. For example, for the FMA-UE official transcultural adaptations and validations are available –38. AlsoFurthermore, the current synthesis shows that the majority of information about upper limb assessment deals with stroke. Nevertheless, wherever available, the CAULIN recommendations have used information on upper limb assessment from other populations. Based on available clinical practice guidelines, most information besides stroke was available from TBI, followed by SCI and MS . For MS,The current work showed that PROM, goal attainment OM and sensor-based assessment of actual arm use in daily life are important concepts to include in upper limb assessment, although concrete recommendations based on consensus, clinical utility and psychometric properties, can’t be provided at this point. More research is needed to establish specific measures and/or methods. In addition, technological development is required to mature measurement systems and methods for use in clinical practice or research. This is also valid for kinematic measures of movement quality, even though a basic application could be specified in the extended set of CAULIN recommendations. Increased availability of assessment of movement and task performance on ratio-level, considering such developments in the (near) future, enables better detection of underlying, detailed changes. This will add valuable information for prognosis of recovery and corresponding treatment planning on individual level, which can benefit the rehabilitation process .It is, however, conceivable that any new or additional OM will meet the selection criteria as defined for the CAULIN recommendations at some point. Moreover, some of those new or additional measures could potentially outperform some of the OM in the current selection, especially those with subjective (by the tester) and ordinal-level scoring involved. This entails that the recommendations should be updated in the future on a regular basis, to incorporate additional measures and revisit the selection of recommended OM, when these become available. Nevertheless, this means that the current CAULIN recommendations are limited to OM currently available in clinical practice.The CAULIN recommendations for OM and assessment procedures provide a clear, simple, evidence-based three-level structure for upper limb assessment in neurological rehabilitation. OM in all three levels have proven psychometric properties as well as evidence derived from systematic reviews and expert consensus. The three levels are: (1) Core set: OM that should be applied routinely in clinical practice with neurological patients undergoing conventional or technology-enhanced upper limb rehabilitation; (2) Extended set: OM that may be useful in clinical practice but are recommended as standard for research, and (3) Supplementary set: OM for specific research purposes. The CAULIN recommendations provide a comprehensive framework, in the context of currently available OM, within which to investigate the effectiveness of (technology-supported) interventions and better understand which patients benefit from which training approach. This will facilitate treatment planning in clinical practice on patient-specific basis. Widespread adoption and sustained use of the recommendations will increase opportunities for data pooling and meta-analysis, critical for the advancement of neurological rehabilitation.


Significant efforts in the past decades to teach evidence-based practice (EBP) implementation has emphasized increasing knowledge of EBP and developing interventions to support adoption to practice. These efforts have resulted in only limited sustained improvements in the daily use of evidence-based interventions in clinical practice in most health professions. Many new interventions with limited evidence of effectiveness are readily adopted each year—indicating openness to change is not the problem. The selection of an intervention is the outcome of an elaborate and complex cognitive process, which is shaped by how they represent the problem in their mind and is mostly invisible processes to others. Therefore, the complex thinking process that support appropriate adoption of interventions should be taught more explicitly. Making the process visible to clinicians increases the acquisition of the skills required to judiciously select one intervention over others. The purpose of this paper is to provide a review of the selection process and the critical analysis that is required to appropriately decide to trial or not trial new intervention strategies with patients. All health professions have mandated the use of evidence-based practice (EBP) as a tenet of ethical practice  as it isClinicalTrials.gov) and manualized interventions are two strategies developed to address the generalizability difficulties of clinical trials. These strategies support the clinician in assessing generalizability by providing rich information on the participants, settings, and characteristics of the interventions used [Second, we agree that some EBPs, such as handwashing for reducing infection, need to be implemented in all settings. However, not all interventions should be assumed to be the correct action in all settings with all clients. It is important to note that some EBPs can be adopted as written, some may need to be modified in a specific context or with a specific patient, and some will not be appropriate for adoption at all. Appropriate implementation of any given intervention is therefore dependent on many factors, including the confirmation that the clients are members of the same population as the sample from the intervention study being considered for adoption to practice. Frequently, the descriptions of whom the EBPs is directed provides insufficient information to determine if that the client is a member of the population ,15. Impoons used ,19,20. Hons used . Most inons used . In otheLastly, significant efforts and interventions have been applied in the last twenty years to increase EBP implementation rates. In fact, the main purpose of the field of implementation science is to identify the methods and strategies that facilitate evidence-based practice and research by clinicians ,24. ImpoCognitive mapping has been used for over a half century to understand how learning changes a person’s cognitive structure of knowledge . InitialCognitive mapping provides two avenues for improving evidence-based practices in healthcare. First, evidence on the effectiveness of cognitive mapping suggests that it is an effective tool to support organizing new knowledge into a coherent representation and integration with prior knowledge  and the https://carethatfits.org/ (accessed on 14 February 2021)), and sufficient evidence to develop EBP activities. Critically, many of the intervention decisions are made in uncertain conditions [We agree that the development of various EBP curricula has led to better skills and knowledge of how to appraise and synthesize evidence to support local practice. These curricula have taken a complex, multistep process and made it easier by directly teaching the specific action steps that the clinicians are expected to do . Further, appraisal systems and tools  decrease cognitive load by providing cues or reminders on questions that need to be answered. More elaborate systems, like GRADE, even provide decision trees to decrease the chance of making an error. Importantly, the EBP process, appraisal systems, and decision-support tools have been demonstrated to improve the positive impact on the quality of the care and clinical outcomes as they increase the likelihood of selecting and enacting the correct decision . HoweverSince the Renaissance, science and medicine were rooted in the assumption that if problems are divided into smaller and simpler units, they become easier to solve . These sEvidence on what humans do when presented with a novel problem suggests that heuristic reasoning is used in time limited situations , as mostIn everyday life, adaptive response is the outcome of rapid decisions based on numerous different interrelated variables ,73. In aIdeally, each individual clinician relies on their own causal model to select the interventions to use with a specific client . Causal Linear thinking leads to erroneous causal assumptions, as it relies on the readily available data points , has difficulty accounting for feedback, and has difficulty accounting for other variables that do not fit in the linear model  ,42. HistThe decision of selecting one intervention over others arises out of the intersection of two different critical analysis processes: (1) the personal causal model of the problem and (2) the analysis of interventions  is to tentatively define the concept or problem and then critically reflect and analyze what they think they know, believe, and value. At this stage, they use their intuition and tacit knowledge to identify variables that they think may be involved. Consistent with the brainstorming stage of problem-solving frameworks, variables are placed on the map without regard to strength or plausibility of its relationship, as the goal is to bring to consciousness the variables that may influence thinking at later stages. In the second stage, they are explicitly taught to seek and read the literature and talk with peers in order to identify other variables that are theorized or empirically known to be influencing the problem. They add the newly identified variables and add linking verbs to identity relationships between the variables . At this stage, they also indicate the strength and direction of the relationship  . At thisMaking thinking visible—evidence-informed causal model.®®® (Available online: https://cmap.ihmc.us/ (accessed on 14 February 2021)) allows for students to actively organize and construct their own maps, which also allow them to connect maps together, embed resources, and share maps with others. For clinical problems, causal maps provide four specific benefits. First, it makes searching for evidence feasible and more efficient as the variables can become search terms. Second, the factors can be used to identify interventions that are theorized to affect the clinical presentation and the outcome of interest. Third, a causal model in which interventions have been added will contain alternative options for achieving an outcome. Interventions connected to different variables can be inserted into the clinical question . In other words, the variables and concepts included on a cognitive map provide starting points for the formal search strategies found in EBP textbooks, which can be a very difficult and frustrating experience for people with limited knowledge of a phenomena. As illustrated in the figures, cognitive maps are able to cogently translate information and complex relationships. Many interventions addressing behavioral change are complex, consisting of various components that have been combined as they theoretically cause change to important variables affecting the clinical presentation . InterveComponents, or “ingredients” in intervention theory, include anything that is done by the clinician to a client, including, for example, medications, assistive devices, motivational speech, ultrasound, manual touch, instruction, feedback, and physical cues. Ingredients target small aspects of the clinical outcome. In practice, interventions contain both active ingredients that are required to enact change in outcome, and inert ingredients, which have no effect on the targeted outcome ,93. IdenAn example from the clinic on how fidelity tools were used to examine practice is the history of constraint-induced movement therapy (CIMT). CIMT is an intervention that addresses function of a hemiplegic upper extremity. Early protocols called for constraining the uninvolved upper extremity for 90% of the waking hours and intensive therapy for six hours a day, for 10 days over two weeks . Howevertarget of the treatment  that is altered by the intervention; the ingredients that produce the change; and the mechanism that the ingredients cause change in the target of the treatment [One issue with many EBPs is that the descriptions of the interventions lack a clear explanation of parameters  for any specific activity or component ,101. Thireatment ,94,102. reatment ,103  and the need for better client outcomes—in other words, what it typically achieved is less than the potential ,94. Thishttps://www.tomatis.com/en (accessed on 12 February 2021)), (Available online: https://soundsory.com/samonas-sound-therapy/ (accessed on 12 February 2021)), Listening Program (Available online: https://advancedbrain.com/about-tlp/) (accessed on 12 February 2021)), Therapeutic Listening ), etc.) [By the mid-1990s, auditory integration training became a worldwide popular intervention for people with autism. It was theorized to improve many personal factors such as attention span, eye contact, tantrums, etc. Due to its prevalent use, multiple efficacy studies were completed and showed that the intervention had no effect . However), etc.) . Importa), etc.) .In order to select the best intervention, it is essential that the clinician use an evidence-informed causal model and has appropriately integrated information on many different variables. Evidence supports that making the complex thinking process visible supports deep learning and improves the causal reasoning, which is used to predict the best intervention option. Expanding our instructional methods to include direct instruction (making thinking visible) has the potential to improve the accuracy of decisions, and therefore, client outcomes, as it supports the development of underlying thinking habits of clinicians. Importantly, intervention selection and EBP are both highly influenced by the clinician’s causal model. Therefore, improving the quality and habits of thinking has the potential to increase the likelihood of selecting the “best” interventions with our clients. Specifically, teaching and helping students to establish the habit of developing an evidence-informed causal model has the potential to improve heuristic decision-making as evidence suggests that one’s causal model highly influences heuristic decision-making. Using cognitive support tools such as the intervention rubric  also has


Church of the Annunciation  were characterized, for the first time, both at the elemental and molecular levels, by means of portable X-ray fluorescence (XRF) and Raman spectroscopy, to achieve information on the “state of health” of the whole structure. The understanding of their degradation mechanisms and the identification of consequent degradation patterns can define the environmental factors responsible for interpreting the potential pathological forms that can impact the general building vulnerability. In this sense, the results obtained in this article provide relevant information to identify and address both the characterization of building materials and the fundamental causes of their deterioration. At the same time, if coupled with the attempt to supply a chronological order of the major restoration interventions carried out on the investigated site, they provide new insights to calibrate the models for building vulnerability studies.Plasters and mortars of the  The importance of multi-technique characterization of materials  is linkeIn the variety of techniques employed in different research areas of archaeometry ,3,4,5, XRaman spectroscopy is another analytical technique widely used in archaeometry ,8, offerFor both methodologies, the development of mobile instrumentation is becoming available to the community to analyze art objects directly in situ ,10,11,12A new and relatively modern approach in archaeometry consists of evaluating the seismic vulnerability of buildings. Ever-increasing attention towards cultural heritage safeguarding, especially in seismic areas, has been paid in order to avoid damage and/or collapse of historical buildings, with consequent loss of priceless heritage and human lives ,14. In tChurch of the Annunciation (hereinafter referred to as “Church”) at Tortorici. The obtained results represent a first step for the definition of the best pathway for the vulnerability assessment and usability of the investigated site, and provide, at the same time, useful information for planning future restoration strategies to be applied.The main body of this article comprises, therefore, the use of portable XRF and Raman spectroscopy to define the nature of the materials used during the realization of the Church of the Annunciation is a masonry Roman Catholic Church located in the center of Tortorici, a municipality in the Metropolitan City of Messina . The Church, characterized by the specularity of the two façades as a unique and distinctive feature, is also known as “Batia” because the Abbey of the Poor Clares, a contemplative Order of nuns in the Catholic Church, was annexed to it.The The investigated Church belongs to a period of architectural transition in Sicily, between the XVIII and XIX centuries, where Baroque and neoclassical motifs were combined in the main Sicilian cities. Many of the religious buildings built in this period were destroyed or not completed, due to earthquakes, limited financial resources, and changes in taste by developers or designers. In the light of this, the investigated Church can be considered as a structure of significant level in the context of this peculiar rural and local architecture, up to now not well known or studied.It is worth noting that only incomplete and inaccurate information regarding the Church’s role in local society are available. What is certainly known is that the Church served religious functions until 1963, when it was closed to the public due to its highly deteriorated state. Since then, the Church fell into a state of disrepair. Only at the end of the 20th century, the local authorities undertook a significant restructuring. Actually, the Church shows several extended fractures, of dubious origin, on its internal walls and no longer plays a role in the religious community, having become the center of artistic and cultural events in Tortorici.A geolocation map of the city of Tortorici, with the investigated Church, are reported in A total of 16 points, representative of different pigmented plasters, preparatory ground, crystalline deposit of salts (efflorescence), and mortars of the interior of the Church, were investigated.The exact positions of the investigated points within the Church are indicated in The investigation was performed in situ by using portable XRF and Raman instrumentations.2) as detector. For each point, two sequential tests were performed, the first with operating conditions of 40 kV and 7 µA and the second with 15 kV and 5 µA, for a total spectrum collection time of 120 s. The instrument was controlled by a Hewlett-Packard iPAQ Pocket PC , which was also used for the data storage. The calibration was performed using a soil light element analysis program (LEAP) II and was verified using alloy certified reference materials produced by Analytical Reference Materials International. When Cu is present, the Lα line of Ta is superimposed to the Kα line of Cu.XRF measurements were carried out using a portable XRF Alpha 4000 analyzer , which allows the detection of chemical elements with an atomic number (Z) between phosphorus and lead. It is equipped with a Ta anode X-ray tube as source and a Si PIN diode  spectrometer with an excitation wavelength of 785 nm (diode laser), a maximum laser power of 280 mW at the excitation port, and a charge coupled device (CCD) detector . The laser output power was continuously adjusted by maximizing the signal-to-noise ratio while minimizing the integration time. The laser spot size was 85 μm at a working distance of 5.90 mm. The maximum power at the samples was about 55 mW. All the spectra were registered in the wavenumber range of 60–3150 cmThe abbreviations of minerals reported in the present study follow the list approved by the International Mineralogical Association (IMA) Commission on New Minerals, Nomenclature and Classification (CNMNC) .−1, along with other contributions at ~162, ~282, ~712, and ~1435 cm−1, all associated to the presence of calcite, the most stable polymorph of calcium carbonate (CaCO3) [As far as the preparation ground is concerned, identified as point PG, the Raman spectrum collected on it  revealed (CaCO3) ,18,19.Going on, −1, associated to white lead (2PbCO3·Pb(OH)2), and, in the 250–650 cm−1 range, different peaks compatible with the mentioned iron oxides [A first inspection of n oxides .3O4 and other iron oxides as black pigments. This can justify the high concentrations of Pb and Fe detected by XRF in the corresponding area. Moreover, as well known, white lead is unstable in the presence of sulfurous compounds and gases and reacts with them to form black lead (II) sulfide (PbS) [From the results, the use of white lead as the main component to prepare the gray pigmented plaster  can be hde (PbS) . This co−1. In particular, these signals are characteristic of sp2 carbon materials and have been traditionally assigned to the D (disorder) and G (graphitic) bands of amorphous carbon [Furthermore, the presence of a carbon-based compound was identified in the Raman spectrum of P1 through the observation of two strong contributions located at ~1300 and ~1600 cms carbon , respect−1 suggests the presence of a preparation ground made of calcium carbonate (CaCO3). However, the absence of the other characteristic peaks of calcite  at ~162, ~282, ~712, and ~1435 cm−1 prevents us from defining with certainty the mineralogical nature of the carbonate [−1 [The interpretation of the Raman spectrum of P2 d is morearbonate ,18,19. Inate [−1 .3) has been presumably employed to prepare the dark gray pigmented plaster. In addition, the light gray pigmented plaster can be described as a mixture of iron oxides, lead white, and a binder made, likely, of calcium carbonate (CaCO3).Based on the aforementioned considerations, a mixture of carbon-based pigment with a binder made of calcium carbonate (CaCO−1) [−1 range that can be assigned to some iron oxides and hydroxides [2O3) and goethite (αFeOOH) are the most frequent, and of white pigments  [The XRF spectrum collected on point P3 almost completely matched that of sample P4 c, indica−1) ,18,19, tdroxides ,25,26,27droxides , ochres olomite) ,29. In o−1, interpreted as the characteristic pattern of carbonaceous and carbon-based materials as previously discussed. As explained before for the gray pigmented plasters, the bands related to the carbon-based materials can be ascribed to the presence of carbon nanoparticles due to the candle soot. Therefore, it can be concluded that the yellow pigmented plasters are the product of a mixture of yellow ochre with a binder made, presumably, of calcite.In addition, the Raman measurements show, for both investigated points, the two strong bands located at ~1300 and ~1600 cm−1, can be ascribed to manganese and iron oxides–hydroxides [−1 indicates the presence of the preparation ground made likely of calcium carbonate (CaCO3). In addition, even in this case, a carbonaceous/carbon-based material can be distinguished by its contributions at ~1300 and ~1600 cm−1.In particular, XRF results b indicatdroxides ,31, in adroxides . Finally−1) [−1, ascribable to the presence of the preparation ground likely made of calcium carbonate (CaCO3). Moreover, peaks in the 350–700 cm−1 range can be assigned to some iron oxides and hydroxides, suggesting the application of red ochre [−1) are weaker than the other samples analyzed.On one hand, XRF results b indicat−1) ,34 togeted ochre ,32. HemaAccording to the aforementioned considerations, the investigated point was reasonably realized by mixing red ochre with gypsum.−1) [−1 indicates the presence of the preparation ground likely made of calcium carbonate (CaCO3). Moreover, peaks in the 160–700 cm−1 range can be assigned to some iron and manganese oxides and hydroxides [−1) are very low compared to the other samples.The XRF spectrum b shows t−1) ,35. Furtdroxides ,29,30,31Based on the aforementioned considerations, the pink pigmented plaster can be considered as the product of a mixture of a red earth with a preparation ground made of gypsum, similarly to what has been observed for the point P6.−1, which can be assigned to the presence of an ultramarine blue pigment [3) from preparation ground, respectively.In particular, XRF spectra of points P8 c and P9  pigment ,36 and t8(AlSiO4)62, which is responsible for its overall blue hue. Inclusions of several other minerals are also common, including pyrite (FeS2), calcite (CaCO3), diopside (CaMgSi2O6), forsterite (MgSiO4), and wollastonite (CaSiO3), in varying amounts [2+, K+, or Na+), anions , and neutral species (H2O) are entrapped within these cages [There is much confusion in the literature between ultramarine blue (a pigment obtained from ground lapis lazuli), lapis lazuli , and lazurite  . Lapis l amounts . Lazurit amounts . The sodse cages .3−) is mainly responsible for the blue color, but contributions from disulfur (S2−) and tetrasulfur (S4−) radicals can shift the color towards yellow or red, respectively [Lazurite’s blue color is attributed to sulfur polyanion radicals trapped in the β-cage structure. Variations in color appear to be related to ratios between various sulfur species: the trisulfur radical . In particular, for points P8 and P9 the finding of the synthetic ultramarine blue allowed us to hypothesize that such first phase belongs to an age later than the first two decades of 1800, which, as a matter of fact, could be considered as the original wall painting of the investigated Church. On the contrary, plasters with a preparation ground made of gypsum (P6 and P7) belong to a late phase of wall painting, which may be connected to the major restructuring interventions carried out during the XX century [All investigated pigmented plasters had an essential role in determining at least two different manufacturing phases. More in detail, pigmented plasters, with the exception of P6 and P7, belong to the first, and are characterized by a preparation ground made likely of calcium carbonate 2·4H2O) [−1 corresponds to the arcanite (K2SO4) mineralogical phase [On one hand, XRF results a indicat−1 ,18,19; t)2·4H2O) ; the peaal phase .Calcium carbonate, or calcite, individuated in the efflorescence is the product of a secondary recrystallization. Indeed, calcium carbonate is the main component of lime mortars, and it can, however, dissolve and recrystallize under specific conditions. A common cause of degradation occurs when the carbon dioxide, from the atmosphere, slowly penetrates the mortars and converts the calcium hydroxide and cementing phases in calcium carbonate. This phenomenon is called carbonation and its effects are important in relation to the durability of mortars themselves .One of the most commonly occurring alkali sulfates in mortars and cement is arcanite. During normal cement hydration reactions with water, these alkali sulfates can contribute to set control. However, hydration of alkali sulfates during cement storage can lead to lumping and flowability problems ,46. AlkaThe information obtained from the analysis of the alteration products is sufficient to identify the development of alteration pathologies that afflict the entire building. Among these pathologies, the rising dump is the most recognized.As far as mortars are concerned, XRF results a indicat−1, suggesting the presence of calcium carbonate (CaCO3) [−1. Interestingly, all the contributions falling in the 150–1000 cm−1 range can be assigned to the presence of cement and clay minerals. In this range there is significant overlap between the mineral phases with a large fluorescence effect as suggested by [44- tetrahedral units. In our case, it is possible to distinguish the symmetric (ν2) and antisymmetric (ν4) bending of the O-Si-O bonds, centered at ~448 and ~607 cm−1, respectively, together with the symmetric (ν1) and antisymmetric (ν3) stretching of Si–O bonds at ~777 and ~935 cm−1 [3S, C2S, C3A, and C4AF . The bands in the 200–400 cm−1 region probably belong to Ca–O vibrations. Furthermore, the bands in the region between 525 and 550 cm−1 can be assigned to O–Si–O bending modes of C3S/C2S. A broad feature near 730 cm−1 is composed of vibration of C4AF at 737 cm−1 and of C3A at 754 cm−1. The stretching vibration bands of Si–O characteristics of C3S and β-C2S phases can be seen in the Raman spectrum in the 800–900 cm−1 region. Finally, the band at ~854 cm−1 can be assigned to C2S/C3S [Going on, all Raman spectra b show cl (CaCO3) ,18,19. Eested by . Silicat C2S/C3S .From the geological setting surrounding the city of Tortorici, it is known that the lithology of different formations is mainly composed of phyllosilicates , quartz, albite, calcite, and minor amounts of K-feldspar , displayAccording to ,52, in oAs final remark, it is worth highlighting that the methodology employed can be helpful in view of possible subsequent investigations and modeling stages. Moreover, the obtained results furnished preliminary information useful in view of the vulnerability assessment of the building.Church of the Annunciation  were characterized by means of portable XRF and Raman instrumentations.In this paper, plasters and mortars of the In particular, concerning the pigmented plasters, two different typologies were distinguished based on the presence of calcium carbonate and gypsum, respectively. According to this classification, two different manufacturing phases were revealed. The first, belonging to an age later than the first two decades of 1800, was considered as the original wall painting of the investigated Church, while the second to a late phase, which may be connected to the major restructuring interventions carried out during the XX century.Going on, information obtained from the analysis of joint mortars revealed a general homogeneity in composition. The identification of minerals connected to calcium silicate hydrates or calcium aluminate hydrates suggests the use of heated local raw materials in the preparation of the investigated mortars.Nonetheless, further study of, and research into, this site is needed, and it will be developed in the future. In particular, (i) infrared thermography is planned to be used to detect subsurface features, such as defects and anomalies, based on temperature differences and to map moisture distribution and detect areas of moisture-related deterioration in structures. (ii) A more detailed sampling campaign of mortars needs to be planned, also by micro-coring of masonry walls to identify the state of health of the mortars themselves and their related potential degradation products. (iii) Finally, 2D and 3D digital models of the monument using digital photogrammetry need to be created in order to map the different restoration phases and create tools for the usability of the studied site.