For comparison, these markers were also evaluated in sex-and-age-matched children born at term with childhood asthma (n = 28). Relative telomere length (RTL) was measured in whole blood with qPCR and serum YKL-40 with ELISA, and both were studied in relation to gas exchange and the regional ventilation/perfusion ratio using three-dimensional V/Q-scintigraphy  in children with BPD. Higher levels of YKL-40 were associated with shorter leukocyte RTL , but were not associated with a lower degree of matching between ventilation and perfusion within the lung. Serum YKL-40 levels were significantly higher in children with BPD compared to children with asthma . High levels of YKL-40 and short RTLs were associated to the need for ventilatory support more than 1 month in the neonatal period (p < 0.01). The link between enhanced telomere shortening in childhood and structural remodeling of the lung, as observed in children with former BPD but not in children with asthma at the age of 10 years, suggests altered lung development related to prematurity and early life inflammatory exposure. In conclusion, relative telomere length and YKL-40 may serve as biomarkers of altered lung development as a result of early-life inflammation in children with a history of prematurity.Extremely preterm infants are born with immature lungs and are exposed to an inflammatory environment as a result of oxidative stress. This may lead to airway remodeling, cellular aging and the development of bronchopulmonary dysplasia (BPD). Reliable markers that predict the long-term consequences of BPD in infancy are still lacking. We analyzed two biomarkers of cellular aging and lung function, telomere length and YKL-40, respectively, at 10 years of age in children born preterm with a history of BPD ( Bronchopulmonary dysplasia (BPD) is the most common severe complication affecting children born extremely preterm. It is a chronic lung disease associated with early mechanical ventilation, supplemental oxygen, and inflammation that may lead to impaired lung growth and long-term effects, such as reduced lung capacity, pulmonary hypertension, emphysema, and neuro-cognitive problems ,2,3,4. WThe cause of BPD is multifactorial and the development of the disease is driven by inflammation . ExtremeTelomeres are non-coding DNA sequences that stabilize chromosomal ends, and telomere length is a marker of cellular aging . OxidatiThe chitinase-like protein YKL-40, also called chitinase-3-like protein 1 (CHI3L1), is a potential serum biomarker associated with airway remodeling and vascular smooth muscle proliferation and migration . ElevateMarkers of lung tissue damage in relation to prematurity are scarce. Our group recently showed elevated levels of YKL-40 in serum when comparing school-aged children with bronchopulmonary dysplasia to children with allergic asthma, and this could be a possible marker of remodeling and inflammation following BPD . In the The long-term consequences of BPD may include impaired lung function, particularly involving the small airways, but this is difficult to evaluate with standard methods such as dynamic and static spirometry. Single photon emission computer tomography (SPECT) is a technique used to quantify functional loss by measuring how ventilation and perfusion in different regions of the lung match. It has previously been shown that lung volume with good V/Q matching is lower in children with BPD ,21 than We hypothesized that children born with immature lungs exposed to an environment leading to inflammation in the neonatal period will develop structural tissue damage and signs of cellular aging later in life. Therefore, we evaluated YKL-40 and relative telomere length (RTL) at ten years of age in a cohort of extremely preterm-born children with BPD.Patients included in this study were part of a larger follow-up study of preterm children with BPD, the Premature follow-up with Lung function Mannitol and Methacholine (PULMM) study. Detailed information about the clinical characteristics, lung function (dynamic and static spirometry), inflammation , and neonatal lung morbidity has previously been reported  and descIn the present cross-sectional study, a cohort of twenty-nine preterm-born 10-year-old children from the original PULMM cohort was included based on the availability of blood samples, No sample size calculations were performed, since this was a predefined study cohort. Parental written informed consent was obtained. The Regional Ethical Review Board in Stockholm approved the PULMM study  with an amendment for telomere analyses  and for SPECT study examination .−ΔCt method, where ΔCt = CtTEL − CtHBB. Relative telomere length (RTL) values were obtained by dividing the sample T/S values with the T/S value of the reference sample (CCRF-CEM DNA), which was included in each run.DNA was extracted from whole blood, and relative telomere length (RTL) was determined by quantitative PCR, as previously described ,25. In sThe serum level of YKL-40 was analyzed by ELISA, as previously described ,18,26. B99m-MAA; Mallinckrodt Medical, Petten, The Netherlands) followed by inhalation of Technegas aerosol  to record ventilation during normal tidal breathing, keeping the child lying in the same position. Ventilation perfusion ratios (V/Q ratios) were calculated within the lung at regional levels. A V/Q ratio between 0.6 and 1.4 was defined as matched. Areas with V/Q > 1.4 were defined as mismatched and regions with V/Q < 0.6 as reverse mismatched. In mismatched areas, perfusion defects contributed to the abnormalities to a greater extent as opposed to regions with reverse mismatch, where reduced ventilation was more prominent [Twenty-five children out of the 29 children in the BPD group were examined with three-dimensional single photon emission computed tomography ventilation perfusion scintigraphy  using a protocol described in previous studies ,27. Perfrominent .n = 29), the residuals were analyzed to identify deviations from normality with no relevant irregularities found. Pearson correlation coefficients were used for the correlation analysis. Correlations adjusted for sex, age at the time of blood sampling and for gestational age at birth were estimated by regressing the YKL-40 and RTL on age and sex and estimating the correlations among the residuals. An attenuation analysis was done to investigate the degree to which neonatal lung morbidity explained the relationship between YKL-40 and RTL. The morbidity variables , days on ventilator, surfactant administration, patent ductus arteriosus, retinopathy of prematurity and severity of bronchopulmonary dysplasia (BPD)) were added to the model both individually and cumulatively. Data with a normal distribution are reported as means and standard deviations, and data with non-normal distribution are reported as medians and interquartile ranges. The statistical significance was set to p < 0.05. Analyses were performed using STATA Version 15  and IBM SPSS Statistics version 27 .The relationship between YKL-40 and RTL was estimated using an ordinary least-squares linear regression. As the sample size was small  in the BPD group (children born preterm), as shown in There was a statistically significant correlation between shorter RTLs and higher levels of serum YKL-40  and shorter RTL , shown in The association between RTL and YKL-40 was not affected by neonatal lung morbidity . However, more severe respiratory morbidity in the neonatal period, here defined as duration of ventilatory support for more than one month , was associated with higher YKL-40 , as shown in the No associations were found between the percentage of lung volume with matched V/Q ratio and levels of YKL-40 or short RTLs. We could not find any difference when comparing the group with good ventilation perfusion matching  and those individuals with a higher degree of abnormalities in ventilation and perfusion distribution and matching (matched V/Q ratio of less than 70% within the lung). The V/Q mismatch was not associated with high values of YKL-40 or short RTLs, as shown in To our knowledge, this is the first time that an association between cellular aging and signs of structural lung disease has been described in children. Children born preterm with a history of BPD were found to have impaired lung function with higher levels of YKL-40 related to neonatal respiratory morbidity and inflammation, which was indicative of structural lung disease, at ten years of age. In that same cohort, the relative telomere length was similar to that of their term-born peers, but short telomeres were associated with impaired lung function irrespective of prematurity ,19,23. IThe long-term reduced lung capacity consequences of children born preterm have been well studied, but we still do not understand the exact mechanism by which this lung morbidity occurs . NordlunLungs develop postnatally, and markers of lung tissue remodeling are needed to individualize treatment and optimize the development of immature lungs. A short telomere length is a marker of obstructive lung disease in adults , but thiYKL-40 is a marker of airway remodeling  and was YKL-40 is not a good predictor for future development of asthma in pre-school children with wheeze ,36, and,This study has several limitations due to its cross-sectional design and because it was not powered or designed to examine the relationship between the structural tissue damage protein YKL-40 and RTL. The lack of a healthy control group and the small sample size meant that we had to interpret any results with caution. Despite these limitations, we show a novel association between blood telomere length and serum YKL-40 in relation to neonatal lung disease and impaired lung function in a well-characterized cohort of extremely preterm-born children. Understanding the consequences of early life inflammation contributes to the tailoring of treatment with the aim of enhancing organ development postnatally without tissue damage and thereby avoiding the long-term consequences of chronic lung disease. Further studies are needed to elucidate whether YKL-40 and telomere length can be used as biomarkers of altered lung development and be used to predict the long-term consequences of BPD.The link between enhanced telomere shortening in childhood and structural remodeling of the lungs seen in children with former BPD suggests altered lung development related to prematurity and early life inflammatory exposure.


Vaginal brachytherapy is currently recommended as adjuvant treatment in patients with high-intermediate risk endometrial cancer to maximize local control and has only mild side effects and no or limited impact on quality of life. However, there is still considerable overtreatment and also some undertreatment, which may be reduced by tailoring adjuvant treatment to the patients’ risk of recurrence based on molecular tumor characteristics.To compare the rates of vaginal recurrence in women with high-intermediate risk endometrial cancer, treated after surgery with molecular-integrated risk profile-based recommendations for either observation, vaginal brachytherapy or external pelvic beam radiotherapy or with standard adjuvant vaginal brachytherapyAdjuvant treatment based on a molecular-integrated risk profile provides similar local control and recurrence-free survival as current standard adjuvant brachytherapy in patients with high-intermediate risk endometrial cancer, while sparing many patients the morbidity of adjuvant treatment and reducing healthcare costs.A multicenter, international phase III randomized trial (2:1) of molecular-integrated risk profile-based adjuvant treatment  or adjuvant vaginal brachytherapy (standard arm).Women aged 18 years and over with a histological diagnosis of high-intermediate risk endometrioid endometrial cancer after total abdominal or laparoscopic hysterectomy and bilateral salpingo-oophorectomy. High-intermediate risk factors are defined as: (i) International Federation of Gynecology and Obstetrics stage IA (with invasion) and grade 3; (ii) stage IB grade 1 or 2 with age ≥60 and/or lymph-vascular space invasion; (iii) stage IB, grade 3 without lymph-vascular space invasion; or (iv) stage II (microscopic and grade 1).The primary endpoint is vaginal recurrence. Secondary endpoints are recurrence-free and overall survival; pelvic and distant recurrence; 5-year vaginal control (including treatment for relapse); adverse events and patient-reported symptoms and quality of life; and endometrial cancer-related healthcare costs.500 eligible and evaluable patients.Estimated date for completing accrual will be late 2021. Estimated date for presentation of (first) results is expected in 2023.NCT03469674) and ISRCTN (11659025).The trial is registered at clinicaltrials.gov ( PORTEC-4a is the first trial to introduce molecular factors in the adjuvant treatment of endometrial cancer.Randomization between standard or individualized treatment based on the molecular risk profile.PORTEC-4a will show if omitting treatment in cases of favorable molecular profiles is safe and cost-effective.The cornerstone of treatment for endometrial cancer is surgery, consisting of a total abdominal or laparoscopic hysterectomy and bilateral salpingo-oophorectomy. Based on clinicopathologic risk factors such as age, stage, histological type, grade, depth of myometrial invasion, and presence of lymph-vascular space invasion, patients are considered to be at low- to low-intermediate risk (approximately 50% of patients), high-intermediate risk (30%–35%), or high risk 15%–20%) for disease recurrence .1 Adjuva5%–20% foMultiple randomized trials have established that in stage I endometrial cancer with risk factors, adjuvant external pelvic radiotherapy provides a significant reduction of vaginal and pelvic recurrence. However no overall survival advantage was observed and pelvic radiotherapy is associated with treatment-related toxicities, predominantly gastrointestinal.POLE-mutant subgroup is characterized by mutations in the exonuclease domain of DNA polymerase epsilon and is associated with an excellent prognosis. The hypermutated subgroup with microsatellite instability (or mismatch-repair deficiency) and the copy-number-low group are both associated with an intermediate (stage-dependent) prognosis. The copy-number-high group, with highly frequent TP53-mutations, has an unfavorable prognosis.CTNNB1-exon 3 mutations.TP53-mutations, high tumor grade, and lymph-vascular space invasion.CTNNB1-exon 3 mutations are prognostic for higher risk of recurrence, especially in the copy-number-low group.The comprehensive genomic analysis of endometrial cancer by The Cancer Genome Atlas (TCGA) has led to important novel insights in the genomic landscape of endometrial cancer. The TCGA defined four molecular subgroups based on mutational burden and copy number alterations. The ultramutated POLE-mutations and CTNBB1-exon 3 status are determined by DNA sequencing. In a small subgroup (3%–6%) of cases the tumor will have more than one classifying feature .POLE-mutation irrespective of other classifiers; intermediate if both mismatch-repair deficiency and p53 abnormal staining, but unfavorable if also substantial lymph-vascular space invasion or L1-CAM overexpression is found. In any other case the least favorable factor will determine the risk profile  to the experimental arm with molecular-integrated risk profile-based adjuvant treatment: observation in case of a favorable risk profile, vaginal brachytherapy in case of intermediate risk, and pelvic radiotherapy in case of an unfavorable risk profile; or to the standard arm with adjuvant vaginal brachytherapy. After informed consent and randomization, histopathological slides and a representative tumor tissue block are sent for expert gynecopathology review and determination of the molecular-integrated risk profile. Microscopy is used to confirm histological type, grade and invasion, and, especially, evaluation and semi-quantification of lymph-vascular space invasion. Immunohistochemistry is needed to determine L1-CAM, p53, and mismatch-repair protein expression . Pathogenic file see .14In each participating country, at least one pathology laboratory has been validated for review and determination of the molecular-integrated risk profile.Based on previous analysis it is expected that approximately 50%–55% of patients with high-intermediate risk endometrial cancer have a favorable molecular profile.Currently, a total of 22 sites in Belgium, Czech Republic, Germany, France, Ireland, and The Netherlands are open for inclusion and another eight are preparing for participation. The study is supported by the Dutch Cancer Society (UL2011-5336 and UL20).Patients aged 18 years or older with a WHO performance status of 0–2 with a histological diagnosis of endometrioid endometrial cancer with high-intermediate risk features , grade 3 >60 yearsStage IB, grade 1 or 2 and age Stage IB, grade 1 or 2 with documented lymph-vascular space invasionStage IB, grade 3 without lymph-vascular space invasionStage II (microscopic), grade 1Treatment with curative intentAny diagnosis of malignancy <5 years ago (except non-melanoma skin cancer)Previous pelvic radiotherapyWHO performance status 3–4Expected interval between surgery and start of adjuvant treatment exceeding 8 weeks*International Federation of Gynecology and Obstetrics stagePatients diagnosed with malignancy (except for non-melanoma skin cancer) in the past 5 years and/or previous pelvic radiotherapy are excluded. In addition, patients are not eligible if the interval between date of surgery and start of radiotherapy is expected to exceed 8 weeks.The primary endpoint is vaginal recurrence. Secondary endpoints are 5-year recurrence-free and overall survival; vaginal control (including treatment for relapse); pelvic and distant recurrence; adverse events; patient-reported symptoms and health-related quality of life; and endometrial cancer-related healthcare costs.Translational research is an integral part of this molecular-integrated risk profile-based trial. Therefore, a tumor tissue block of all patients is collected and stored in the dedicated tissue bank at Leiden University Medical Center for further analysis.The study is an open-label phase III trial with (2:1) randomized allocation to either molecular-integrated risk profile-directed adjuvant treatment  or adjuvant vaginal brachytherapy (standard arm). Central randomization is done by a web-based randomization application with stratification for participating center, tumor grade (1 vs 2 vs 3), and type of surgery (lymphadenectomy yes or no) using a biased coin minimization procedure. The trial number and result of randomization are immediately obtained via the randomization system and confirmed by email.It is hypothesized that the experimental treatment strategy leads to less overtreatment, which would yield less morbidity and a better quality of life. As treatment will be deescalated in a substantial part of the patients in the experimental arm, a non-inferiority design was chosen to estimate vaginal recurrence in both groups with sufficient precision and to exclude a clinically relevant difference with an equivalence margin of 7%. Other first recurrences (pelvic and distant) and death are considered competing risks. Based on data from the PORTEC-2 trial and the molecular analysis from the pooled PORTEC-1 and 2 trials, the expected 5-year rates of vaginal recurrence are 2.0% in the vaginal brachytherapy arm and 4.625% in the molecular profile arm. The null hypothesis states that the 5-year cumulative incidence of vaginal recurrence in the molecular profile-directed treatment arm exceeds that of the standard vaginal brachytherapy arm by more than an equivalence margin of 7%: the null hypothesis will be rejected if the upper bound of the one-sided 95% Wald confidence interval of the difference (molecular arm minus standard arm) does not exceed the equivalence margin. The inclusion period was estimated to be 4 years, with a follow-up period of 3 years after inclusion of the last patient. For a total of 500 evaluable patients, with 167 patients in the standard arm and 334 in the molecular arm, a power of 84.4% is achieved. An additional power calculation was performed for a subgroup analysis among all patients with a favorable risk profile to determine the difference in vaginal recurrence between the experimental group (no adjuvant treatment) and the standard treatment group (brachytherapy). These groups will be compared using a non-inferiority design with an equivalence margin of 8.5%. The power will depend on the actual difference between the arms. For example, if the 5-year cumulative incidence of vaginal recurrence would be 4% in the experimental arm and 1.5% in the standard arm, the power will be 89.7%: for 5% vs 1.5% this would be 75%. The main aim of this analysis is to estimate the difference in vaginal recurrence in the favorable subgroups with sufficient precision (SE of the difference below 2.4%).The pilot phase (first 50 patients) of the PORTEC-4a trial has been completed and showed that the trial design is acceptable for patients and feasible in terms of the logistics for determining the molecular profile within 2 weeks.All analyses concerning treatment effects will be performed according to the intention-to-treat principle. Time-to-event analyzes will be performed with date of randomization as a starting point. Formal tests for the differences in relapse and survival rates between the two arms will be done with the Kaplan–Meier method, the log-rank test, and Cox regression analysis. The competing risk method will be used, with competing risks of death, pelvic recurrence, and distant recurrence for analysis of vaginal recurrence, and competing risk of death for analyses of pelvic and distant recurrence. Analysis of toxicity will be based on treatment received. P-values<0.05 will be considered statistically significant.t-tests with, if appropriate, multiple imputation of missing data.For the cost-utility analysis, endometrial cancer-related healthcare costs will be considered. This includes in both arms of the trial the costs of: primary treatment, care associated with adverse events, and first treatment for relapse and/or metastasis. Healthcare use across the follow-up period will be converted to costs using standard prices, discounted over time. The difference in quality-adjusted life years and costs between the two arms will be compared according to the intention-to-treat principle, using Health-related quality of life analysis will be performed for patients with a valid baseline measurement and at least one follow-up measurement. To evaluate the differences between the treatment groups with respect to the effect of treatment burden on life quality during and up to 5 years after treatment, the repeated measures of the scales of the quality of life questionnaires will be analyzed using mixed ANOVA models. Single items will be analyzed using logistic regression at different timepoints.Several randomized trials, such as the GOG-99, ASTEC, and PORTEC-1 and −2 trials, have established recommendations for the adjuvant treatment of endometrial cancer based on clinicopathological risk factors.POLE sequencing methods are being developed.The acceptance and implementation of the molecular-integrated risk profile in standard care will be facilitated if the cost-utility analysis shows that molecular-integrated risk profile-tailored adjuvant treatment leads to similar or even reduced healthcare costs. Even though the costs in the experimental group will be higher due to determination of the molecular-integrated risk profile by next-generation sequencing, about 50%–55% of the patients with a favorable profile will receive no adjuvant treatment which will substantially reduce costs. However, the costs of any additional recurrence in the experimental arm may partially counterbalance this effect. In addition, it is expected that a substantial reduction in the cost of the molecular profile will be realized as cheaper targeted During the trial both national and international pathology laboratories are being validated for determination of the molecular-integrated risk profile, leading to early and rapid dissemination and optimization of the molecular techniques and logistical processes. If cost-utility is confirmed, it is expected that molecular-integrated risk profile-directed adjuvant treatment will rapidly be accepted as the standard approach in clinical practice and used in future studies.


Several post-translational protein modifications lie predominantly within regions of disorder: the biased localization has been proposed to expand the binding versatility of disordered regions. However, investigating a representative dataset of 500 human N-glycoproteins, we observed the sites of N-linked glycosylations or N-glycosites, to be predominantly present in the regions of predicted order. When compared with disordered stretches, ordered regions were not found to be enriched for asparagines, serines and threonines, residues that constitute the sequon signature for conjugation of N-glycans. We then investigated the basis of mutual exclusivity between disorder and N-glycosites on the basis of amino acid distribution: when compared with control ordered residue stretches without any N-glycosites, residue neighborhoods surrounding N-glycosites showed a depletion of bulky, hydrophobic and disorder-promoting amino acids and an enrichment for flexible and accessible residues that are frequently found in coiled structures. When compared with control disordered residue stretches without any N-glycosites, N-glycosite neighborhoods were depleted of charged, polar, hydrophobic and flexible residues and enriched for aromatic, accessible and order-promoting residues with a tendency to be part of coiled and β structures. N-glycosite neighborhoods also showed greater phylogenetic conservation among amniotes, compared with control ordered regions, which in turn were more conserved than disordered control regions. Our results lead us to propose that unique primary structural compositions and differential propensities for evolvability allowed for the mutual spatial exclusion of N-glycosite neighborhoods and disordered stretches. N-linked glycosylation begins with the assembly of an oligosaccharide on dolichol pyrophosphate and the subsequent transfer of the oligosaccharide to the asparagine residues of polypeptides in the lumen of the endoplasmic reticulum; the oligosaccharide is further remodeled in the Golgi complex. Several proteins that end up in the extracellular milieu or as transmembrane proteins are N-linked glycoconjugates. The establishment of organismal morphologies has been sought to be understood through the interactions of a highly conserved set of proteins known as the developmental genetic toolkit2. Most toolkit proteins, which are involved in tissue-scale processes, such as cell-cell and cell-matrix adhesion, diffusion-driven signaling and cell movement, are extracellular- or membrane-bound glycoproteins4. The fundamental role of such toolkit proteins in mechanisms of organ- and organismal-development across diverse clades suggests evolutionary constraints on their structures and folds, while they may have continued to evolve to perform newer functions as organisms occupied and constructed newer niches.One of the common co- and post-translational modifications of polypeptides is the conjugation of branched glycosylations to asparagines (known as N-linked glycosylations)7. Other proteins, while not being entirely disordered can possess variable lengths of disorder. The inherently flexible nature of disordered regions enhances the proteins’ repertoire of interacting or binding partners, and also plays an important role in the catalytic functions of the proteins9. Multiple cytoplasmic and nuclear proteins  are highly disordered, although extracellular proteins have been found to also have disordered stretches13. In fact, more than 20% of extracellular proteins consist of more than 50% average disordered residues, with approximately 57% extracellular proteins containing at least one single disordered stretch of 30 continuous residues14. Several post-translational modifications (PTMs) have been reported to be located within disordered regions e.g., phosphorylations of serines and threonines are found within unstructured regions both in eukaryotes17 and in prokaryotes18. Serine/threonine phosphorylations within disordered stretches play crucial roles in the interactions between the proteins and their ligands. They can stabilize the tertiary structural organization of the disordered region in order to enhance its binding to the protein’s cognate ligand(s)19 besides further stabilizing the bound state of the protein-ligand complex20. In addition to phosphorylation, O-linked glycosylations have also been predicted to be conjugated to the disordered regions of proteins21 and mediate resistance to proteolysis leading to their evolutionary selection22.A large number of eukaryotic proteins show flexible tertiary structures and are known as intrinsically disordered proteins (IDPs)23. Intermediate steps in N-glycan biosynthesis act as checkpoints to ensure that only correctly folded proteins traffic along the ER-Golgi axis24. Thermodynamic and molecular dynamics studies show that glycans enhance the stability of the tertiary structure of proteins26. Keeping the above observations in mind, we asked whether the sites of N-glycan conjugation (N-glycosites) showed any bias in their locations vis-a-vis (dis)ordered regions of N-glycoproteins. Investigating a dataset of 500 representative human N-glycoproteins , wherein we mapped and predicted ordered and disordered regions, we found N-glycosites to be enriched predominantly within ordered regions relative to disordered stretches. We present biochemical (proximate) and evolutionary  reasons for this enrichment and the mutual exclusion of N-glycosites and disordered regions.An emerging body of literature links the contribution of N-linked glycosylation to the structure, tertiary fold and stabilization of proteins27. The sequences with annotated N-glycosites and predicted disordered regions are given in Supplementary File 28. In addition, another list of proteins that have been experimentally demonstrated to have disorder was curated from the database DISPROT30 and annotated for both their N-glycosites and disordered regions , which is a meta-predictor based on the following predictors of disorder: POODLE-I, IPDA, IUPRED-I, DISPRO, POODLE-S, IUPRED-S, SPRITZ-I, PRDOS, RONN, DISOPRED2, DISEMBL AND SPRITZ-S31. GOanna performs a BLAST search against protein sequences that have a GO number. Gene ontological diversity was measured for the 500-protein sequence set and the complete sequence set of human proteins with experimentally elucidated N-linked glycoconjugation. The GO details of both sets are given in Supplementary File For prediction of gene ontology terms, sequences of the selected proteins were scanned with the GOanna server32. Maximum likelihood was used to construct the phylogenetic tree of the proteins as it is used for the analysis of sequences of diverse origins, while maximum parsimony method was used to construct the phylogenetic tree of the proteins which have similar functions with a comparatively higher sequence homology than the former set of sequences. The phylogenetic tree was viewed using the program FigTree (http://tree.bio.ed.ac.uk/software/figtree/). The subsets of proteins used for tree construction were constituted based on results obtained from GO analysis. Supplementary Fig. The protein sequences were aligned using ClustalΩ and a phylogenetic tree was constructed using 1000 bootstrap value using MEGA5Pan troglodytes, Mus musculus and Gallus gallus were obtained from Ensembl and aligned with human proteins using ClustalΩ (Supplementary File http://molbiol.edu.ru/eng/index.html) 33. Bootstrap iterations were kept at 10,000, significance value was kept at 0.05 and Bonferroni’s multiple-comparison correction was applied.The comparison of frequency of occurrence of amino acids within N-glycosite neighborhoods, N-glycosite-less ordered and disordered regions was performed using Composition Profiler (http://lichtargelab.org/software/uet)34. The evolutionary trace of residues that varies among evolutionarily distant protein homologs is ranked higher than for residues that vary among evolutionarily proximal protein homologs35. The program searches for all homologous sequences within the Uniref90 database  up to 500 sequences.The mean of residue ranks was computed and compared between residues from the disordered control regions, ordered control regions and residues flanking N-glycosite neighborhoods order and N-glycosite location. N-linked glycoconjugated asparagines (or N-glycosites) were found to be significantly enriched within ordered regions.  of N-glycosites from (and within) disordered stretches is a characteristic of one or more phylogenetically conserved protein families, we classified our protein set on the basis of gene ontologies. We observed that our analyzed set of N-glycoproteins perform a diverse range of predicted biological functions , followed in turn by a serine (Ser) or threonine (Thr) S/T)37. Since asparagine, serine and threonine are polar in nature, we estimated the abundance of these three amino acids within ordered and disordered segments of our protein dataset. The percentage of asparagine was found to be insignificantly different between disordered and ordered regions of N-glycoproteins. Serine and threonine were surprisingly found to be significantly enriched within disordered- rather than ordered- stretches S/T sequon within ordered and disordered regions. Despite an unbiased distribution of asparagine between ordered and disordered regions, and relative enrichment of serine and threonine within the disordered region, the probability of constitution of the minimal signature required for N-glycan conjugation (N(X-P)S/T) was found to be significantly higher within ordered regions Fig. .39. We analyzed the identity of X in the sequons that are present in predicted regions of order and disorder: we found higher proportion of hydrophobic and bulky residues as X in the sequons inhabiting the ordered regions compared with the disordered region S/T). Empirical and bioinformatic analysis with model and natural proteins, respectively, have suggested that the identity of “X” in the sequon may determine the extent of its glycosylation: bulky, hydrophobic or acidic amino acids may reduce the efficiency of glycosylation. Conversely, small, hydrophilic or basic amino acids being present as X may increase the propensity of the sequon to be glycosylated40, charge, hydrophobicity41, flexibility42, surface exposure43, normalized frequency of occurrence in β structures and coils44, propensities to contribute to order45, and bulkiness40.We began by annotating five residues upstream- and downstream of the glycoconjugated asparagines as N-glycosite neighborhoods of proteins in our dataset. Additionally 11-residue stretches that did not contain N-glycosites were also identified in the disordered and ordered regions and were defined as disordered and ordered control regions respectively. We estimated the relative frequencies of amino acids between these three 11-residue subsets across our 500-protein dataset and estimated the significance in enrichment or deletion of residue properties such as aromaticity, polarityWe first compared the set of ordered control regions with disordered control regions  together, based on the above properties revealed two broad signatures , mouse (Mus musculus) and chicken  in order to assay a possible divergence in the extent of conservation between residues surrounding the N-glycosites (in the ordered regions) and residues in the disordered regions  were compared, disordered sequences exhibited greater entropy. In contrast, N-glycosite neighborhoods showed lower mean entropy than full-length protein sequences suggesting residues comprising N-glycosite neighborhood show higher, and those comprising disordered regions, lower, evolutionary conservation among amniote N-glycoproteins. Previous studies have shown that the association between residue conservation and residue disorder is context-specific49. Our observations indicate that N-glycosites represent high-conservation islands within ordered regions which themselves are more conserved within the amniotic clade with respect to disordered regions.We then asked if N-glycosite neighborhoods represented regions of higher evolutionary conservation within ordered parts of protein primary structures. We aligned the human N-glycoprotein sequences with their orthologs from chimpanzee 53. The triose core of N-glycoproteins enhances both the kinetics and stability of tertiary glycoprotein folds54. In fact, N-glycosylations seems to destabilize the unfolded state more than stabilize the folded state of proteins26. Our results suggest that not just N-glycans but the residue neigborhoods surrounding them may help perform these functions.We observed that while order-promoting residues are equivalently distributed between N-linked glycosites and control ordered regions, the former are relatively further depleted of disorder-promoting residues compared to the latter. N-glycans have been shown through mounting theoretical22. Therefore, N-glycosite neighborhoods are uniquely placed in terms of their biochemical properties separating them from the hydrophobic ordered core and the disordered stretches of N-linked glycoproteins.Disordered regions are known to contain variable amino acid sequences and we observe that they show relatively lower phylogenetic conservation among amniotes. However, some amino acid residues which have longer side chains than others, e.g., asparagine, proline, glutamate, methionine, serine, arginine, and glutamine are significantly enriched in the disordered regions. The long side-chains of these amino acids may have some significant function in the disordered region which could be related to their ability to enhance flexibility, exposure and disorder. The abundance of pyrrolidine ring-containing proline allows it to introduce kinks or turns in the protein structure and contributes to its propensity within interdomain linkers. It is well known that proline content is generally higher within disordered stretchesBased on our observations of evolutionary trace and amniote phylogeny, N-glycosite neighborhoods show residue conservation but do not seem to represent hotspots for the phylogenetic clustering of amino acids that determine function. This however, does not imply that N-linked glycans or even N-glycosites do not determine protein functions. Far from it, both are crucial to fold of the protein and the function performed by it. Additionally, our results imply that disordered stretches may represent hotspots for the further accumulation of function-determining residues, which may regulate the evolvability of such N-glycoproteins.55. Unfolded proteins are N-glycosylated within the ER lumen and bear 3 Glc residues at the terminal end of their A-branch. N-linked glycans being bulky hydrophilic polymers can increase the thermodynamic stability of proteins, enhance their folding and allow them to evade the enzyme UGGT that would otherwise deploy unfolded proteins for proteolytic degradation56. However, this regulation by N-glycans of protein folding does not necessarily bias the hypothetical localization of the N-glycosites within the ordered (or disordered) regions. We would in the future, perform computational studies to examine the effect on the dynamics of disordered regions by the hypothetical presence of one or more N-glycan(s).The N-linked glycosylation of proteins also tends to act as an important quality control check point for the ability of the protein to fold itself correctly57, wherein structural properties such as secondary structure were observed to contribute further to the accuracy of prediction of whether a potential N-glycosite is conjugated with a glycan structure, than local contact order. We observed, for example, that residues that are part of N-glycosite neighborhoods show greater propensity to be part of coiled structures. Whereas local contact order may not by itself be able to distinguish successfully between N-glycan-conjugated and -unconjugated glycosites according to Chuang and coworkers57, it may contribute to constraining the localization of conjugated N-glycosites within the ordered stretches of the protein.Our results are also consistent with an earlier approach that sought to develop algorithms to predict sites of N-linked glycosylation based on structure and pattern3. The determinacy of their developmental roles is a function of their stability and N-glycosylation may have played an important role in the latter. Our study qualifies this by proposing that the localization of N-linked glycosylations allowed these proteins to also have unstructured interfaces in order that they interact with the various components in the extracellular milieu without compromising the global structured fold of the proteins. Our phylogenetic analysis suggests our N-glycoprotein set consists of proteins with diverse functions. However, it also shows that at least for some of the ontological categories, proteins, which shared some common function also were phylogenetically related. This clustering may help to choose specific representatives of clustered protein sets for further molecular dynamical studies. Such studies will help us better understand the role of disordered regions and N-glycosites in the evolution of protein function at the molecular level.Our results are specific to N-glycoproteins, which are part of the extracellular milieu. Many of them being transmembrane- and secreted diffusible- proteins, function across multicellular tissue spatial scales and contribute to the developmental mechanisms by mediating discrete biophysical and biochemical functionsSupplementary Figure File.Supplementary File 1.Supplementary File 3.Supplementary File 6.Supplementary File 8.Supplementary File 2.Supplementary File 4.Supplementary File 5.Supplementary File 7.Supplementary File 9.


We report the clinical features of 3 patients in France who had parotitis  as a clinical manifestation of confirmed coronavirus disease. Results from magnetic resonance imaging support the occurrence of intraparotid lymphadenitis, leading to a parotitis-like clinical picture. The worldwide spread of coronavirus disease (COVID-19) is associated with the emergence of many clinical pictures of the disease. Patients might have nose and throat symptoms, such as loss of smell and taste , which indicated intraparotid lymphadenitis. In all three cases, we observed multiple unilateral or bilateral intraglandular lymph nodes in the deep and surface layers, in a relatively normal-sized gland. We preserved the lymph node architecture by using a preserved fatty hilum. We observed no juxtaglandular fat infiltration or thickening of the fascia. We also observed no intraglandular linear bands or cysts on the MRI . The 3 patients received 10–14 days of paracetamol (1 g 3–4×/d) for their COVID-19. The parotitis resolved over the next few days after diagnosis. The 3 patients had persistent loss of smell after the resolution of their general and parotitis-like symptoms.The occurrence of acute parotitis related to COVID-19 has been suggested in a recent case report , leading to saliva retention and parotid tissue inflammation. The lack of saliva might be associated with sticky saliva and taste impairment. Intraparotid adenitis differs from primary diffuse parotitis, which was recently reported in an unique case of COVID-19  complicate the characterization of parotitis. Thus, the main limitation of our report is the lack of functional examinations of the parotid during the clinical course of the disease. The assessment of the functioning of the saliva secretion and the detection of severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) in the saliva could provide further information about SARS-CoV-2 transmission through saliva.Future studies are needed to characterize the parotid manifestations in COVID-19 patients. Although the findings of this study support the hypothesis that intraparotid lymphadenitis is a causal factor, the direct spread of SARS-CoV-2 into the parotid tissue might be theoretically possible regarding the presence of angiotensin converting enzyme 2 (the virus receptor) in the parotid tissue and the potential risk for excretion of virions through the saliva (In conclusion, parotid inflammation might be encountered in COVID-19 patients and could be related to intraparotid lymphadenitis. Even in persons vaccinated against mumps, testing for viruses that cause a parotitis-like illness is important, including rubella virus, influenza virus, and SARS-CoV-2. Additional studies to characterize parotid manifestations in COVID-19 patients will help determine diagnosis and treatment.Additional information about parotitis-like symptoms associated with COVID-19, France, March–April 2020.


Modern means of transport will play a significant role in the smart city. In the automotive industry, high-strength steels such as Docol are employed more often. This kind of material is relatively not very well weldable. The main reason is related to the Heat Affect Zone, the region in which cracks occur. Another disadvantage is connected with differences in values of ultimate strength of parent and weld material. The differences can be diminished using the correct welding process, which employs nickel and molybdenum electrode wires at much lower sulfur content. The weld metal deposit contains mainly martensite and bainite with coarse ferrite, while the parent material contains mainly martensite and rather fine ferrite. New technology, micro-jet cooling after the joining process enables to obtain the microstructure of weld metal deposit at acceptable parameters. Welding with micro-jet cooling could be treated as a very promising welding Docol steels process with high industrial application. Results of non-destructive inspections on macro samples corresponded with further destructive test results . This article aims to verify fatigue behavior of Docol 1200 M steel after welding supported by the cooling using the micro-jet technique. For the first time, micro-jet cooling was used to weld this kind of steel to check the mechanical properties of the joint, especially to determine the fatigue limit. This study is formulated as follows: investigating fatigue resistance of the Docol 1200 M weld manufactured at the cooling process with micro-jets. The joints were produced in the MAG  technology modified by micro-jet cooling. The results collected in the fatigue test were processed in the form of the Wöhler’s S–N diagram following the fatigue limit of the weld examined. All data have indicated the possibility of obtaining a new method of welded joints with high fatigue limit minimum of 480 MPa. It could be important to achieve a tensile strength of 700 MPa while maintaining the best relative elongation at the level of the base material. The progress in the automotive industry is created by the application of modern materials and types of joining as well as new components and constructions ,2,3,4. Ihigh ultimate tensile strength, up to 1700 MPa,high yield stress, up to 1350 MPa,elongation, up to 6%.Docol Advanced High-Strength Steel (AHSS) are obtained in the technological process such as: hot and cold rolled as well as hot-dip and electro galvanized. That material range includes thin sheet steel with a thickness range from 0.4 mm to 16 mm. Docol steels have become particularly attractive in the automotive industry for three important reasons :high ultThe application of Docol steels in the automotive branch of industry results from reducing the thickness of the car body at also acceptable or more attractive mechanical parameters in comparison to typical structural materials. For the manufacturing of vehicles, attempts are being made to use high-strength martensitic steels from the Docol group ,14,15,16For instance, Reference  presentsIn Reference  the authIn summary, it can be concluded that the modern high-strength steel plates have an excellent combination of strength, fracture toughness, and resistance on impact based on micro-alloying and complex microstructure . RetainiThis research aimed to investigate the mechanical properties of the high-strength steel Docol 1200 M after MAG welding with micro-jet cooling. The utilitarian goal of the study is to develop a welding method for these steels. The new technology will be based on classic MIG and TIG  processes, modernized through the use of cooling attachments, affecting the structure of the connections obtained. It was assumed that the selection of welding and micro-second cooling parameters would allow for crack-free joints with a significant amount of fine-grained martensite, which translates into high-strength properties of the welds.The newly developed technology for the analyzed steels will allow its use in the automotive industry and should contribute to a wider use of the discussed steels in practice.These steels are relatively not very well weldable because of the cracks can occur in the weld and Heat Affected Zone ,22,23,24Selecting the steel type to the application is conducted based on a range of the material type from Docol 900 M up to Docol 1500 M at details of chemical composition  and mech2 molecule (recombination). Hydrogen accumulation in the metal leads to internal pressure, causing an increase of stress, which in turn causes Hydrogen Induced Cracking (HIC) [During AHSS steels welding, the regime determined by thermodynamic analysis of the process should be reached. The linear energy up to 4 kJ/cm is required to refine the ferrite and reduce stress due to welding process ,24,25. Png (HIC) ,30,31. Hng (HIC) . Hydrogeng (HIC) ,8. The rng (HIC) . When weng (HIC) .As can be noticed in ,22,23, fA Docol 1200 M welded (BW) butt joints at a thickness of 2 mm was manufactured. The requirements of EN 15614-1 standard were used for the MAG welding method at the low position (PA). The single-stitch welding and a finished 3 mm thick weld are illustrated in 2 . All of the samples were welded with an electrode wire EN ISO 16834-A G 89 6 M21 Mn4Ni2CrMo−UNION X90  at the chemical composition presented in [It was decided to produce welds at MAG  process using the gas mixture as of: Ar + 18% COVisual tests (VT) of welded joints were conducted with the eye fitted with a magnifying glass at 3× magnification—the test was performed according to the requirements of PN-EN ISO 17,638 standard and the assessment criteria according to EN ISO 5817 standard,Magnetic-powder tests (MT) were carried out in accordance with the PN-EN ISO 17,638 standard and the assessment criteria according to EN ISO 5817 standard using a magnetic flaw detector device type REM-230 .The scope of research included non-destructive testing (NDT):Examinations of the microstructure of the samples digested with the Adler reagent using a light microscope (LM),Hardness measurement was done focusing on guidelines of the PN-EN ISO 9015-1:2011  and PN-ETensile test was carried out basing on the regimes of the PN-EN ISO 6892-1:2020 standard . The expFatigue test collecting specimen designing was performed at rules of the ASTM E468-18 standard .Evaluation of microstructure and mechanical properties:The article presents the possibility of welding a thin-walled structure (3 mm) made of high-strength Docol 1200 M steel. These steels are difficult to weld ,40, as cquantity of cooling nozzles: 1,form of cooling medium: Ar or He,pressure of the cooling medium: 0.6−0.7 MPa,diameter of micro stream: 60−70 µm,distance of the micro-jet nozzle from the welded surface: 20 mm.To further enhance the mechanical properties of joints after MAG welding, an addition of micro-jet cooling technology was selected. For the steel structure welding process, the following micro-jet cooling parameters were implemented:After the welding with micro-jet cooling, the following non-destructive tests (NDT) were carried out: visual (VT), magnetic-particle (MT), and radiographic. The gap between the welded elements arm-gap  was checResults of non-destructive tests exhibited no cracks occurrence in the weld manufactured at the micro-jet cooling technique .The table data shows that the gap between elements should be equal to 1.5 mm and micro-jet cooling should not be too intensive . For the 2 mm gap, no cracks were observed, only for low-intensity helium micro-jet cooling (sample S2b) . It was strength of Docol 1200 M steel with the use of argon micro-jet cooling,strength of Docol 1200 M steel with the use of helium micro-jet cooling.After the hardness assessment, the strength tests of the welded elements were carried out and the 3369 INSTRON testing machine  was selected for that purpose. Two analyses were performed:Micro-jet gas pressure (Ar or He) was equal to 0.7 MPa, micro-jet stream diameter was 60 µm. Each test was repeated 3 times . Tensile strength and fatigue tests were performed as the main research to check the quality of the joint. The results are presented in As captured in the tensile test, the Docol 1200 M steel is a very attractive structural material because of the value of proportional limit taking 771 MPa, ultimate tensile strength exceeding 1200 MPa, and elongation that reaches min. 8% a. These Application of micro-jet cooling during the welding process improved YS and UTS values. The average YS value of MAG welded joint without micro-jet cooling was 495 MPa and the average UTS value was 723 MPa. They increased respectively to the values 551 MPa (YS) and 751 MPa (UTS) for argon micro-jet cooling, and respectively to the values 534 MPa (YS) and 762 MPa (UTS) for helium micro-jet cooling. It has been confirmed from NDT observations that helium micro-jet cooling gives better results. Next, the microstructure analysis was carried out. A typical microstructure of weld is presented in Fatigue tests were employed to follow the behavior of the micro-cooling weld joining of the Docol 1200 M under cyclic loading ,42. All The fatigue tests have reflected the weld response on cyclic loading up to fracture  and 700 At 500 MPa and 550 MPa, the region degradation occurred at relatively little plastic deformation a,b, compFatigue of the micro-jet cooling weld is also represented by the fracture at the following maximum values of stress: 700 MPa, 650 MPa, 600 MPa, 550 MPa, and 500 MPa, taking the relationship presented in Two micro-jet gases were used, of which micro-jet cooling helium gave better results , which was confirmed by non-destructive tests .Tensile strength and fatigue tests were performed as the main research to check the quality of the joint. It was noticed that the joints with better plastic properties were obtained when micro-jet cooling was additionally used for MAG welding. Relative elongation increased from 8% to 11% when micro-jet cooling was used .The yield stress of the joint without micro-jet cooling was below the required value of 500 MPa, while welding with micro-jet cooling resulted in a yield stress value of 520 MPa both when helium and argon were used in micro-jet cooling. Similarly, a higher tensile strength was obtained thanks to the use of micro-jet cooling.Yield stress and tensile strength values were at a similar level of 750 MPa when helium or argon was used for micro-jet cooling. The joint after welding with He micro-jet cooling had slightly higher elongation (11%) in comparison with Ar micro-jet cooling (10%); therefore, helium was selected as the micro-jet gas for fatigue tests. Without micro-jet cooling, tensile strength was obtained at a lower level of 720 MPa .The obtained results show that:Generally, the fatigue tests confirmed that the joints are of high quality. The Wöhler curve has been established as a key characteristic for determining the quality of the weld examined. The fatigue strength was obtained at a satisfactory level of 480 MPa . TherefoThe welded joints were made of the difficult-to-weld material Docol 1200 M. The most favorable welding conditions and parameters were determined. The possibility of welding Docol MAG steel with micro-jet cooling has been tested. The most important parameters of micro-jet cooling have been thoroughly checked. The most suitable micro-jet gas was selected. Non-destructive and destructive tests were carried out to assess the quality of the joint. The microscope (LM) observations, tensile and hardness tests, as well as fatigue approaches were carried out. Fatigue tests were treated as the most important destructive experiment in the assessment of the quality of Docol welds. The results from the tensile test indicated that the Docol 1200M steel is sensitive to the micro-jet welding processes, expressing half-smaller values of mechanical parameters than the parent material, not including elongation. Independently of the material state examined, the value of this parameter was very similar. The weld behavior under fatigue had a range of significant differences in the number of cycles to fracture at the stress value from 700 MPa up to the fatigue limit of 480 MPa. This was also reflected by a gradual fracturing of the steel at the smaller values of stress.the micro-jet cooling technique allows producing the weld for Docol 1200 M steel with a martensitic microstructure similar to the parent material;mechanical properties of the weld supported by the micro-jet cooling have expressed 50% lowering and maintaining elongation value in comparison to the steel in the as-received state;tensile curve of the weld with respect to the welding process used has received more regular shape, which is requested by engineers for designing and research for modeling;fatigue limit of the weld tested was very beneficial because of the 480 MPa that was determined. This value can be directly employed in designing, calculations, and modeling as fundamental data for assessing the technical state of a component;the results have confirmed that the elaborated joining technology allows obtaining the assumed parameters of the joint with respect to high plastic properties (elongation = 10%), comparable to the parent material. Publications indicate that, so far, high and repeatable parameters of the joint have not yet been obtained without micro-jet cooling.Results from non-destructive and destructive tests on the weld joint manufactured at the micro-jet cooling have enabled us to formulate the following concluding remarks:


The normalized compound motor action potentials of the vastus medialis (VM) and biceps femoris (BF) were improved in the EEGR group (p < 0.05). However, the normalized real-time peak amplitude and total, mean area under the curve of VM were decreased during rehabilitation in the EEGR group (p < 0.05). No significant differences were found between operated and non-operated knees in the EEGR group. Five-day EEGR-assisted rehabilitation induced strengthening in the knee flexors and the muscular reactivation of the BF and VM after TKA, while reducing the real-time use of the VM. This observation may suggest the feasibility of this technique: EEGR modulated the neuronal system of the patients rather than training their muscles. However, because the study was underpowered, all of the findings should be interpreted with the utmost caution.Deafferentation and weight offloading can increase brain and spinal motor neuron excitability, respectively. End-effector gait robots (EEGRs) can blend these effects with stereotyped movement-induced neuroplasticity. The authors aimed to evaluate the usefulness of EEGRs as a postoperative neuro-muscular rehabilitation tool. This prospective randomized controlled trial included patients who had undergone unilateral total knee arthroplasty (TKA). Patients were randomly allocated into two groups: one using a 200-step rehabilitation program in an EEGR or the other using a walker on a floor (WF) three times a day for five weekdays. The two groups were compared by electrophysiological and biomechanical methods. Since there were no more enrollments due to funding issues, interim analysis was performed. Twelve patients were assigned to the EEGR group and eight patients were assigned to the WF group. Although the muscle volume of the quadriceps and hamstring did not differ between the two groups, the normalized peak torque of the operated knee flexors (11.28 ± 16.04 Nm/kg) was improved in the EEGR group compared to that of the operated knee flexors in the WF group (4.25 ± 14.26 Nm/kg) ( Several types of gait robots have been investigated for use in stroke patients and patients with spinal cord injuries , 2. ThesSimilarly, cortical plasticity for movement in a specific body part can also be induced in those without central nervous system injuries. The cortical plasticity of kinematic movements was observed in 20 healthy individuals shortly after repetitive thumb exercises for 5–30 minutes , and theThe idea that deafferentation showed increased peripheral motor neuron excitability was first introduced in an animal experiment in 1956 , 11. ThaFurthermore, bearing weight on the legs can affect α-motor neuron excitability in the spinal cord during ambulation, as the activation of motor neuron excitability depends on the amount of weight the legs are bearing –17. ThatIn contrast to exo-skeleton type robots (EXSRs), which only control weight bearing, end-effector gait robots (EEGRs) can provide deafferentation using an end effector with a guidance force of 100% in addition to weight bearing control . TherefoChanges in neuromuscular control before and after total knee arthroplasty (TKA) have been reported on a local/muscle level as well as on larger levels that attempt to describe the overall control strategy for patients undergoing TKA. Many patients who cannot walk after TKA have difficulty bearing their full body weight on their legs due to pain, poor muscular activation, or muscle atrophy –22; it cet al. reported that maximal voluntary activation of the quadriceps femoris muscles spontaneously and partially recovered within 25 months after TKA , 583.58 ± 266.47 cm2/second, respectively) were significantly smaller than those  of the operated knees in the WF group (p < 0.05). Similar results were observed for the simultaneously recorded normalized vastus medialis peak compound motor action potential amplitude and the normalized vastus medialis total and mean area under the curve of the non-operated knees in the EEGR group  compared to those of the operated knees in the WF group (p < 0.05). However, there were no significant differences between the operated and the non-operated knees in the EEGR group. Meanwhile, in the biceps femoris, these variables did not differ significantly among the three groups  the normalized peak torque of the flexors and the normalized EMG activities of the vastus medialis and biceps femoris were significantly higher in the EEGR group than in the WF group; (2) these improvements in the EEGR group were not accompanied by increases in the maximal ratio of muscular recruitment or in cross-sectional areas / volumes of the same muscles; (3) the nonnormalized motor evoked potential amplitudes of the vastus medialis and biceps femoris were significantly increased by EEGR; (4) the nonnormalized H-reflex amplitudes of the vastus medialis and biceps femoris was significantly increased in the EEGR group but were reduced in the WF group; (5) the real-time normalized EMG activities of the vastus medialis were decreased in the EEGR group; and (6) these changes were not observed between the operated and non-operated knee in the EEGR group. However, recruitment stopped due to a lack of funding, and therefore, all of the findings should be interpreted with the utmost caution. Due to under-enrollment, it would not be possible to determine whether a non-significant result was from a lack of sufficient enrollment or a scientific discovery into an underlying principle. Although the current trial might have been overpowered in the beginning due to the deviated standardized difference that was calculated using the high knee extensor peak torque value in young male volunteers , it is uThese findings showed that EEGR-assisted rehabilitation for 5 days significantly affected the peak torque and EMG activities of the flexors and extensors of both the operated and the non-operated knees. These results are in agreement with findings in 10 healthy volunteers, in that EEGR-assisted training for 5 minutes with deafferentation and weight offloading resulted in prompt peak torque improvement in the knee flexors and extensors . AlthougIn this study, interval changes in the cross-sectional areas were assessed at three levels to reconstruct the three- dimensional volumes of the operated and the non-operated knee flexors (quadriceps femoris) and extensors , as described in the Materials and Methods section. Although peak torque and EMG activities are dependent on muscle volume, the improved peak torque and voluntary muscular reactivation in the EEGR group were not accompanied by increases in area or volume. These findings indicate that factors at the supra-muscular level, not at the muscular level, may be involved in these improvements. However, it should be concerned that muscle volume and muscle strength are often nonlinearly related. Based on the finding that the cross-sectional area at the mid-thigh level is strongly related to the total volume of skeletal muscle in the body , the autBrain plasticity can be rapidly induced by stereotyped movements , 5. EEGRAs we mentioned above, EEGRs may take advantage of inducing deafferentation-induced brain plasticity , 49 and In the current trial, the nonnormalized spinal motor neuron excitability of the vastus medialis and biceps femoris in both the operated and non-operated knees in the EEGR group was significantly increased, but was reduced in the operated knees of the WF group. EMG activities of the lower legs can be observed during passive ambulation, even in complete paraplegic patients . The H-r®-assisted treadmill ambulation than during treadmill ambulation alone [®-assisted training increased the muscular activation in the lower extremities more than passive engagement [Methodologically, the combination of the 100% guidance force and weight offloading might have induced synergistic or additive effects on neuroplasticity. However, no significant differences were observed in the normalized values. Therefore, in the current trial, it was not easy to clarify whether EEGR contributed synergistic or additive effects to neuroplasticity. Meanwhile, the EXSR did not significantly induce neuromodulation; Beta and mu rhythms in the brain cortex were more inhibited during Lokomaton alone . Moreovegagement . In the The maximal rate of torque development is defined as the speed of the recruitment of peripheral motor units during the force-time curve , so that® enhanced recruitment of the vastus medialis, vastus lateralis, and biceps femoris, but the amount of body weight support was uncontrolled [®, a famous EXSR, increased EMG activities in the thigh muscles in healthy individuals [®, another EXSR, yielded results contrasting those observed in this trial, including the increased activation of the vastus medialis and vastus lateralis over all gait cycles [The current trial showed that the normalized mean, total area under the curve, and the normalized peak of the compound motor action potential amplitude of the vastus medialis were significantly lower in the operated and the non-operated knees of the EEGR group than in the operated knees in the WF group, as determined by real-time monitoring of EMG activities. This result might be attributed to the fact that the deafferentation and the weight offloading of EEGR made muscles highly ready for being recruited to perform the same rehabilitation (200 steps) through the enhanced brain and spinal motor neuron excitability. However, an EEGR trial using the Haptic Walkerntrolled . In contividuals , 62. Howividuals . The G-Et cycles . Howevert cycles , 64. Thet cycles , 66. UnlThe current findings should be interpreted with caution due to the fact that it is an interim, per-protocol analysis  with sinWalking speed can affect the EMG activities of the leg muscles . As ambuAlthough the ground reaction forces in end-effectors change under various gait conditions , these f®) is approximately 300,000 USD, and one of the most popular commercialized EXSR (Lokomat®) costs 330,000 USD. However, traditional physiotherapy, which involved infrared radiation and continuous passive ROM exercises in the current trial, generally costs only 1,000 USD per patient for 1 month in the authors’ country.In practical terms of healthcare costs, whether the extra cost of using the robot justifies the authors’ proposal should be taken into consideration; the price of the current EEGR Click here for additional data file.S2 Tablep-value by two-way repeated measures analysis of variance or paired T-test.WF training with walkers on a floor; EEGR training with end-effector gait robot, EMG electromyography, VM vastus medialis, BF Biceps femoris; AUC area under the curve, (DOCX)Click here for additional data file.S3 Tablep-value by two-way repeated measures analysis of variance or paired T-test.WF training with walkers on a floor; EEGR training with end-effector gait robot; MEP motor evoked potential; BF biceps femoris; VM vastus medialis, H-reflex Hoffmann-reflex, (DOCX)Click here for additional data file.S4 Tablep-value by Mann-Whitney U tests or paired T-test.EMG electromyography; WF training with walkers on a floor; EEGR training with end-effector gait robot; VM vastus medialis, BF Biceps femoris, CMAP compound motor action potential; AUC area under the curve, (DOCX)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


The mitotic activity index is a key prognostic measure in tumour grading. Microscopy based detection of mitotic nuclei is a significant overhead and necessitates automation. This work proposes deep CNN based multi-phase mitosis detection framework “MP-MitDet” for mitotic nuclei identification in breast cancer histopathological images. The workflow constitutes: (1) label-refiner, (2) tissue-level mitotic region selection, (3) blob analysis, and (4) cell-level refinement. We developed an automatic label-refiner to represent weak labels with semi-sematic information for training of deep CNNs. A deep instance-based detection and segmentation model is used to explore probable mitotic regions on tissue patches. More probable regions are screened based on blob area and then analysed at cell-level by developing a custom CNN classifier “MitosRes-CNN” to filter false mitoses. The performance of the proposed “MitosRes-CNN” is compared with the state-of-the-art CNNs that are adapted to cell-level discrimination through cross-domain transfer learning and by adding task-specific layers. The performance of the proposed framework shows good discrimination ability in terms of F-score (0.75), recall (0.76), precision (0.71) and area under the precision-recall curve (0.78) on challenging TUPAC16 dataset. Promising results suggest good generalization of the proposed framework that can learn characteristic features from heterogenous mitotic nuclei. It most commonly occurs due to irregular cell division of breast tissue such as milk duct or lobules, resulting in the formation of tumour. Mitotic activity index (number of dividing cells under 2 mm2 tissue area) is the key factor for determining tumour size, proliferation rate, and aggressiveness2. Due to the imperative importance of mitosis count, it is considered as a prognostic measure in Bloom-Richardson’s grading system3. In a routine histopathology workflow, a pathologist analyses the biopsy sample of the affected region under a microscope and counts the number of mitotic nuclei in 10 High Power Field (HPF) areas. However, manual analysis is tiresome and time-consuming as there are, low density of mitotic nuclei per HPF surrounded by an overwhelming number of other cellular components4. Moreover, it is highly dependent upon the experience of a pathologist and may suffer from inter- and intra-observer variability due to the element of human subjectivity5. The development of an automated mitosis detection system is thus required to reduce the burden on pathologists and to improve the objectivity of their decisions by providing an additional opinion.Breast cancer is the second most commonly diagnosed cancer, which has affected women’s lives globally8. Deep CNNs are a type of representative learning algorithms that automatically extract the relevant information from raw images without putting effort into manual designing of feature descriptors9. CNN based models have been successfully applied in several histopathology problems with success, for instance: categorization of breast tissue10 into normal, benign, in situ and invasive carcinoma, detection of cancer metastasis11, quantification of lymphocytes12, demarcation of tumour region13, segmentation of cell nuclei and many others14.With the advent of digital pathology, many computational algorithms have been developed for the automation of pathological workflow. Recent advances in deep convolutional neural networks (CNNs) and their commendable performance on image classification, detection, and segmentation have accelerated their use in medical imaging problems17. Automated detection of mitotic nuclei is challenging due to their atypical configuration and difference in the texture of cells in different morphological phases Exploitation of deep segmentation model using transfer learning (TL) to improve the labels of weakly annotated mitoses by learning morphological information from small patient dataset.(ii)Exploitation of instance-based detection and segmentation model using multi-objective loss to reduce the class imbalance with minimum loss of mitosis.(iii)Concept of weight transfer and cross-domain TL are exploited to adapt the deep architectures on small dataset.(iv)Custom-made deep CNN “MitosRes-CNN” is proposed that is robust against the mitotic appearance variability.(v)Comparative analysis of the proposed “MitosRes-CNN” is provided with diverse categories of state-of-the-art CNN architectures for mitosis detection problem.To address all of the above mentioned challenges, in this study we have proposed a new learning framework for weakly labelled dataset. In this regard, a deep CNN based multi-phase mitosis detection framework “MP-MitDet” is developed that analyses the Haematoxylin and Eosin (H&E) stained breast cancer histopathology images  proposed use of state-of-the-art Wide Residual Networks for pixel-wise classification of mitosis on breast histology images. Post-processing was applied to the output to remove the noise and less probable mitoses, whereas the final decision was taken by combining the output of several networks using the majority vote26.There are a few techniques that have used end-to-end pixel-wise classification scheme for mitosis detection. In earlier studies, Ciresan et al. (2013) developed a max-pooling based Fully Convolutional Net (FCN) that performed pixel-wise classification. The developed model was applied to the unseen data in a sliding window manner to locate the mitoses27. In the first phase of Chen et al.’s technique, an FCN was used to locate the candidate mitoses. In contrast, in the second phase, a fine discrimination model was developed that utilized the cross-domain knowledge to remove false positives. Wahab et al. (2017) proposed a two-phase classification approach to deal with the class imbalance problem. Initially, the dataset for classification was created by performing global binary thresholding on blue ratio images28. In the first stage of the proposed technique, CNN was trained on the original dataset to identify the hard examples. Whereas in the second stage, the dataset was improved by performing the under-sampling of the negative examples using histogram-based k-means clustering on blue ratio images and augmented the dataset by including hard negative examples. In another study, Wahab et al. (2019) proposed TL based efficient mitosis classifier29. They initially used customized pre-trained FCN for the mitosis detection, whereas in the second phase, predictions of the first phase were refined by assigning the output to another CNN that was hybrid of AlexNet and custom layers. The mitosis detection technique that got first place in the TUPAC16 challenge also adopted a two-stage detection approach to get an improved F-score30.Due to the intricate nature of mitosis detection problem, most of the techniques address it by employing multiple detection and classification models. ICPR14 dataset, instead of providing the morphological information of the mitosis, only provided centroid annotation, thus increasing the difficulty level of detection. Chen et al. (2016) overcame the challenging nature of the dataset by adopting a deep cascaded CNN based approach and this technique won the ICPR14 challenge31 have shown good performance for object detection problems in computer vision. Therefore, Li et al. (2018) exploited the region information of the mitosis using VGG16 backboned faster R-CNN to filter out the probable mitotic regions that are further refined by assigning the predictions to another deep network to remove false positives32. Likewise, MitosisNet proposed by Alom et al. (2020) also consisted of multiple deep learning models, including segmentation, detection, and classification models for the final decision of the mitosis regions33. Similarly, Mehmood et al. handle the complex nature of mitosis by initially identifying probable mitotic regions through R-CNN. They isolated non-mitotic regions from the selected regions by assigning the initial phase results to the ensemble of ResNet5034 and DenseNet20135. The proposed approach achieved 0.858 and 0.691 F-score for the ICPR 2012 and ICPR 2014 dataset, respectively.Region based CNNs (R-CNNs)36. Initially, the detection model was trained on a small size labelled dataset. This trained model was further used to filter out the mitosis samples from unlabelled WSIs. In this way, a large dataset was built, used for the final training of the detection model.Mitosis labelling is tedious, and it is not easy to rigorously annotate them on the Whole Slide Images (WSIs). To overcome this limitation, Akram et al., proposed a semi-supervised learning model for WSIs that shows promising results on the TUPAC16 dataset15. The defined scheme uses concentric circular labels to represent the mitotic region and proposed a concentric loss function that only considers the region inside the circle whereby it excludes the chance of non-mitotic region overlap with the mitotic region. Similarly, Sebai et al. (2020) adapted the semantic segmentation model for the mitosis detection problem53. They handled the issue of weak labels by integrating two deep networks in an end to end manner. They separately trained both networks on weak and pixel-level labels in a simultaneous way. The final decision was made by combining the prediction of both the models. Most of the techniques discussed above are based on state-of-the-art pre-trained CNN models without custom modifications that were previously reported in literature.Training of a deep learning model with a weakly annotated dataset is challenging. This problem is addressed by Li et al. (2019), who introduced a new learning scheme to identify the mitotic nuclei using semantic segmentation based FCN. Their approach achieved state-of-the-art results on the MITOS14 dataset, AMIDA13 dataset, and TUPAC16 dataset with an F-score of 0.562, 0.673, and 0.669, respectivelyThis work suggests a multi-phase deep CNN based mitosis detection framework (MP-MitDet) for H&E stained breast cancer histopathological images. The workflow is decomposed into 4 phases: (1) refinement of weakly labelled mitosis dataset, 2) mitotic region selection at tissue-level, (3) blob analysis, and (4) enhancement of mitosis detection results at cell-level. The overall workflow of the proposed detection model is shown in Fig.  mitotic 21. This dataset consisted of specified regions of breast biopsies corresponding to 10 HPF selected by the pathologists from WSIs. The provided images were collected from three different centres of Netherlands and were scanned by two different scanners under 40 × magnification. The dataset consisted of 656 images collected from 73 patients. This challenge provided a maximum number of patient samples to date for mitosis detection problem, whereby it provided 50 new patients and included 23 patients’ samples from the AMIDA13 challenge. This dataset is challenging as it provided only centroid labels of the mitoses. Provided mitoses were annotated rigorously by two pathologists. In addition to the TUPAC16 dataset, patient samples from two other previous challenges: MITOS12 and MITOS14 were also included in the training to augment the small number of patient samples and to improve the learning of deep CNN models 01937 stu38 technique to mitigate the variations in staining colours. It is expressed in Eq. .6\djth anchor. Likewise, jth anchor. Bounding box regression branch returns the real values therefore the difference between ground-truth spatial coordinates and predicted coordinates is minimized by computing smooth L1 loss  of each region proposal with ground truth and retains only those regions that overlap by at least 70%. These region proposals are mapped to the feature map using ROI align that resizes them using bi-linear interpolation and assigns them to the detection and segmentation head for the classification of the selected regions.In this study, mitotic region selection is considered as a binary class detection problem. For the training of detection module (Mask R-CNN), histopathological images are labelled in a COCO format42. Deep NNs usually require a massive amount of data for training and suffer from overfitting on insufficient data. TL addresses this problem by allowing to reuse the knowledge of the pre-trained network to a new task and has shown remarkable results when labelled data is insufficient in the target domain. TL can be employed by using pre-trained architecture as a fixed feature extractor, freezing lower layers and fine-tuning higher layers, or leveraging parameter space of pre-trained architecture to the target domain43.Mask R-CNN is trained on 3904 images of train dataset Table  by explo44. So, we define the source domain as In this work, the backbone architecture of Mask R-CNN is pre-trained on 1 million natural images from ImageNet, whereas the detection and segmentation head is pre-trained on the COCO datasetIn this phase, Mask R-CNN predictions are filtered out at threshold cut-off of 0.5 and all regions that are predicted with more than 50% confidence as mitotic regions are regressed to patient’s tissue patches Fig. b. CandidIn the last phase, refinement of the selected mitotic regions is performed to reduce the false positives Fig. c. TherefHistopathological images exhibit colour appearance multiplicity due to variation in staining protocols across labs and image acquisition under different scanners. Different image variations are applied on-the-fly during the classifier’s training to make it robust towards unseen patient examples. Augmentation strategies include horizontal and vertical flip, rotation, and colour jitters. Mitotic examples are augmented by extracting patches at various positions to avoid the overfitting. Furthermore, during training, the effect of imbalance is reduced by controlling the proportion of minority and majority class by fetching more positive examples based on the ratio of negative and positive examples. Validation and test sets are drawn from patients without augmentation to emulate the real-world scenarios.34 [Eq.  in combeLU (Eq. ) is usedc shows the feature map depth,  represents spatial coordinates of kernel and  shows the receptive field occupied by kernel. In Eq.  to incoink Eq.  & 12) to to\documDropout with a 50% probability is used for fully connected layers to limit the overfitting. SoftMax is used at the end of a fully connected layer to compute the probability of each sample belonging to mitosis or non-mitosis. Details of the proposed architecture are mentioned in Table In the above Eq. , W speci9. Deep networks learn features in a hierarchical manner, including generic and high-level features specific to the problem. This distinct characteristic allows us to reuse the pre-trained architectures for diverse tasks. Domain adaptation is a type of TL that allows reusing the already pre-trained architecture to a new problem. In this special type of TL, both target and source domains belong to different data distribution but are assigned to the same task. The advantage of this TL is gaining many low and intermediate level features that are shared among the diverse categories of images.Deep CNNs have shown exemplary performance in diverse image classification tasksIn this study, we have exploited the concept of supervised cross-domain feature space adaptation and leveraged the knowledge of state-of-the-art deep CNNs models that are pre-trained on the ImageNet dataset for mitosis recognition problem. In this case, source entclass1pt{minimaClassical classification algorithms, including Naive Bayes, Logistic regression, SVM, Decision tree, Random Forest, and XGBoost, are also evaluated for mitosis detection problem. These classifiers were trained on the original feature space as well as on the HOG and LBP extracted feature space.48. According to it, mitosis in the telophase phase is under duplication; therefore, it is counted once, and all the mitoses that lie within 30 pixels of ground truth are counted as true mitoses.The performance of the proposed Mitosis detection framework is evaluated using F-score, Recall, Precision, and Precision-Recall (PR) curve Eqs. –18. MitoPatches of size 512 × 512 pixels with 15% overlap were extracted for label refinement and mitosis detection at tissue-level stage for the training of Mask R-CNN. Whereas patches of 80 × 80 pixels were generated for the training of classification models. Input images for classification were resized to 120 × 120 and 224 × 224 pixels for custom and state-of-the-art CNN, respectively, using bilinear interpolation.Custom and pre-trained CNN architectures were fine-tuned using cross-entropy loss function by setting batch size equal to 16 and 6, respectively, assigning a learning rate of 1e−3, and weight decay of 1e−5 for 250 epochs. During training the model with best weight state was saved based on the validation dataset. Mask R-CNN was trained for 30,000 epochs using batch size 2, and the learning rate equal to 0.00025. The number of region proposals was set to be 128 for assigning mitosis and non-mitosis examples to ROI head for classification. Mask R-CNN and classification based CNN were optimized using SGD with warmuprestart and cosine annealing optimization strategy, respectively.This study aims to develop a mitosis detection framework that can effectively learn the mitosis representation from the weakly labelled (centroid annotation) histopathological images. In this study, 61, 14, and 17 patients’ datasets Table  were useThe performance of the proposed detection module is evaluated for the maximum number of true mitoses against the small fraction of false positives (non-mitosis). Discrimination power is assessed on an independent test set, which was kept separate during the training and validation Table . The genTUPAC16 dataset is provided only with centroid information of the mitotic nuclei, which is not sufficient for the training of supervised machine learning models, therefore, we used label-refiner to automatically annotate mitoses on tissue patches corresponding to 10 HFP area  and complex in the pattern, therefore, a complex architecture with a large number of filters can lead to overfitting on the training dataset. The exploitation of multiple transformation in a simple way with the idea of the effective receptive field and residual learning in the proposed CNN helps in learning abstractions at different levels while maintaining good generalization. Additionally, the comparison of the proposed MitosRes-CNN with state-of-the-art CNN architectures Tables  and 8 suThis study also demonstrated that the state-of-the-art CNN architectures designed for non-medical tasks could be effectively adapted for mitosis detection by adding task-specific layers, selecting appropriate hyperparameters, and finetuning them via cross-domain TL. We have further shown that the exploitation of the learning experiences of different deep CNN architectures through decision score based ensemble can result in the significant gain in F-score, precision, and recall.30 who also performed detection at two levels. The aforementioned approaches passed the segmented output to the classifier in the first phase to identify the hard negative examples. In the second step, easy-to-classify negatives examples are undersampled in the training data, while hard examples are augmented. Contrary to this, in our proposed “MP-MitDet” approach, we have performed cell-level classification for output of the detection phase without additionally mining the hard instances. The promising performance of the proposed “MP-MitDet” approach on the TUPAC16 dataset in terms of F-score, recall, and precision compared with the existing techniques  with low density of false mitosis examples. The challenges of heterogeneous mitotic morphologies and inter-class resemblance are resolved by developing custom CNN “MitosRes-CNN” that enhance the results by significantly reducing mitosis-like-hard example with a significant precision  on the test dataset. Promising performance of the proposed “MP-MitDet” on different variations of dataset and its comparison with existing techniques suggests its good generalization and potential to learn characteristic features of mitosis. In future, it can be adapted to reduce the decision space of the pathologist by suggesting the probable mitotic regions.