To quantify the concurrent accuracy and the test-retest reliability of a Kinect V2-based upper limb functional assessment system. r correlation were used to evaluate the validity of the angles at the points of target achieved (PTA) and the range of motion (ROM). The intersession CMC and RMSE and the intraclass correlation coefficient (ICC) were used to assess the test-retest reliability of Kinect V2.  Ten healthy males performed a series of upper limb movements, which were measured concurrently with Kinect V2 and the Vicon motion capture system (gold standard). Each participant attended two testing sessions, seven days apart. Four tasks were performed including hand to contralateral shoulder, hand to mouth, combing hair, and hand to back pocket. Upper limb kinematics were calculated using our developed kinematic model and the UWA model for Kinect V2 and Vicon. The interdevice coefficient of multiple correlation (CMC) and the root mean squared error (RMSE) were used to evaluate the validity of the kinematic waveforms. Mean absolute bias and Pearson's  Both validity and reliability are found to be task-dependent and plane-dependent. Kinect V2 had good accuracy in measuring shoulder and elbow flexion/extension angular waveforms (CMC > 0.87), moderate accuracy of measuring shoulder adduction/abduction angular waveforms (CMC = 0.69-0.82), and poor accuracy of measuring shoulder internal/external angles (CMC < 0.6). We also found high test-retest reliability of Kinect V2 in most of the upper limb angular waveforms (CMC = 0.75-0.99), angles at the PTA (ICC = 0.65-0.91), and the ROM (ICC = 0.68-0.96).  Kinect V2 has great potential as a low-cost, easy implemented device for assessing upper limb angular waveforms when performing functional tasks. The system is suitable for assessing relative within-person change in upper limb motions over time, such as disease progression or improvement due to intervention. Three-dimensional 3D) upper limb functional movements such as reaching, pushing/pulling, and throwing have been studied in many areas including motor control , 2, neurD upper lMicrosoft Kinect is a low-cost markerless motion capture system, which estimates the 3D location of body joints based on 2D images with depth information using machine learning algorithms . Kinect Attempts have been made at using Kinect in upper limb assessment –19. DiffEstablishing the accuracy and reliability inherent in the Microsoft Kinect system is required before using it for upper limb assessment. The accuracy of the Kinect system in measuring lower limb kinematics has been evaluated using marker-based measurements as the gold standard , 20, 21.Ten healthy male university students  with no upper limb injuries or medication use that would have influenced their upper limb functions volunteered to participate. Participants were informed about the basic procedure of the experiment before the test. The experimental protocol was approved by the Research Academy of Grand Health's Ethics Committee at Ningbo University.This study used a concurrent validity, test-retest reliability design. The study was conducted at the biomechanics laboratory of Ningbo University. Upper limb kinematics were recorded concurrently by a Kinect V2 system with a sampling frequency of 30 Hz and a 3D motion capture system with eight infrared high-speed cameras  with a sampling frequency of 100 Hz. Prior to data collection, Kinect V2 was placed on a tripod at 0.8 meters above the floor. Subjects stood at 2 meters from the camera according to the recommendation .Task one is hand to the contralateral shoulder, which represents all activities near contralateral shoulder such as washing axilla or zip up a jacket. Subjects started with the arm in the anatomical position with their hand beside their body in a relaxed neutral position and end up with the hand touching the contralateral shoulder see Task two is hand to mouth or drinking, which represents activities such as eating and reaching the face. It begins with the same starting point and ends when the hand reached the subject's mouth see Task three is combing hair, which represents activities such as reaching the (back of the) head and washing hair. Subjects were instructed to move their hand to the back of their head see Task four is hand to back pocket, which represents reaching the back and perineal care. It begins with the same starting point and ends when the hand is placed on the back pocket see Each participant attended two testing sessions, seven days apart. For each session, reflective markers were attached to the anatomical landmarks of the participants according to the UWA upper limb model . First, At least five trials were collected for each task.The Vicon system tracked and stored the spatial trajectories of the reflective markers attached to the subjects. The UWA upper limb marker set was employed in this study , which iThe 3D coordinates of the anatomical landmarks identified from the skeletal model of the Kinect V2 system during functional tasks were also recorded. Local segment coordinates including torso and upper arm were established, and each of them was based on the global coordinate . Then, or correlation between the two systems were used to evaluate the concurrent validity of the Kinect-based system in assessing the joint angles at the point of target achieved (PTA) and the range of motion (ROM). Paired Student t-tests were used to compare the results of the angles at the PTA and the ROM with the significant level of 0.01. The concurrent validity was presented using data from session one.The concurrent validity of the Kinect V2 system for assessing the upper limb functional movement waveforms was carried out using the coefficient of multiple correlation (CMC)  and the k3,) coefficient with the absolute measure. The ICC and Pearson's r correlation as well as the descriptive statistics were performed using SPSS 22.0. The CMC and RMSE were analyzed using Matlab 2017a.The CMC and RMSE between the waveforms from session one and session two measured by the Kinect V2 system were used to assess the relative and absolute test-retest reliability. The reliability of the Kinect V2 system in assessing the joint angles at the PTA and the ROM from selected functional tasks was also carried out using the intraclass correlation  were observed for all investigated angles at PTA in all tasks except for moderate relative agreement of the shoulder internal/external rotation in the hand to back pocket task and elbow flexion/extension in the hand to contralateral shoulder task  and poor agreement of the shoulder and elbow flexion/extension angle in the combing hair task . For the ROM, excellent relative agreements were observed for shoulder flexion/extension and shoulder adduction/abduction angles in all tasks (r = 0.91-0.99) except for the combing hair task ; excellent agreements were found for shoulder internal/external rotation angles only in the shoulder to contralateral shoulder task and the combing hair task (r = 0.74 and 0.77), respectively; poor to moderate agreements were found for elbow flexion/extension angles in all tasks (r < 0.65) except for the hand to back pocket task, which showed excellent agreement (r = 0.97).Mean (SD) values for joint angles at the PTA and the ROM estimated by the Kinect V2 and Vicon systems are provided in There is a clear tendency that the Kinect V2 system overestimated shoulder flexion/extension angles and underestimated elbow flexion/extension angles in all tasks. According to the mean absolute bias of the angles at the PTA between the Kinect V2 and Vicon systems see , K-V, thThe test-retest reliability of the Kinect V2 system in assessing the upper limb angular waveforms, the angles at the PTA, and the corresponding ROM is presented in Results for the test-retest reliability of the Kinect V2 system in assessing the angles at the PTA and the ROM are also presented in This study tested the concurrent validity and the test-retest reliability of upper limb functional assessment using Kinect V2. We found that both validity and reliability are task-dependent and plane-dependent.The Kinect V2 system had good accuracy in measuring shoulder and elbow flexion/extension angles, moderate accuracy of measuring shoulder adduction/abduction angles, and poor accuracy of measuring shoulder internal/external angles. We also found high test-retest reliability of the Kinect V2 system in most of the upper limb angular waveforms, angles at the PTA, and the corresponding ROM. However, there are also some deviations both between the Kinect V2 system and the Vicon system (gold standard) and between two test sessions.The angular trajectories from the Kinect V2 and Vicon have similar waveform patterns, especially for the flexion/extension angular waveforms under all functional tasks (CMC = 0.81-0.94). Nevertheless, the RMSEs between the kinematic patterns measured by the Kinect V2 system and Vicon system are not consistent with the angular waveform agreements.Generally, the mean deviations between joint angles assessed by the Kinect V2 system and the Vicon system are greater for tasks or planes with a larger range of motions. The shoulder and elbow angular waveforms in the sagittal plane measured by the Kinect V2 system highly agreed with the reference angles, and the RMSEs of the Kinect V2 system in measuring sagittal plane kinematics are generally greater or comparable than angles in the frontal plane and transverse plane in comparison with those from the Vicon system. Similarly, the accuracy of the Kinect V2 system in assessing angles at the PTA and the ROM are task-dependent and plane-dependent. The angles at the PTA showed good relative agreement for almost all motions investigated except for motions in the combing hair task, which have a large range of motion.Our finding agrees with the results of , which fr values >0.9 for the majority of measurements for trunk and lower limb motions during lateral reach, forward reach, and single leg balance. In contrast, the Kinect V2 system showed lower concurrent validity in assessing upper limb motions in our study with the Pearson r values of 0.7-0.99 for most upper limb motions at the PTA and the ROM.The measurement performance of the Kinect V2 system is highly varied on tasks, joints, and planes of movement. According to Clark et al.'s study , 21, theThe abovementioned findings can be explained by the underlying real-time human pose recognition algorithm and the nature of the single-depth camera. The Kinect system estimates the 3D location of body joints based on 2D images with depth information using machine learning algorithms. The final set of confidence-weighted 3D joint proposals is based on a global optimization algorithm using training data, which represents postures in an entertainment environment . TherefoWe found a high degree of reproducibility in almost all of the upper limb angular waveforms, angles at the PTA, and the ROM measured by the Kinect V2 system during all functional tasks across the two testing sessions. Our finding suggests that the Kinect V2 system can reliably assess 3D shoulder motions and elbow flexion/extension for individuals carrying out such functional tasks. The repeatability of upper limb motions in the frontal and transverse planes was lower than that in the sagittal plane. However, there were still some between-session deviations in the starting point and ending point of the upper limb. Greater standardization of both the starting and ending points for the tasks would be required to improve the repeatability of the starting and ending points of this type of motion. The test-retest reliability of the trunk motion is high (ICC > 0.73) during lateral reach and forward reach in Clark et al.'s research , which iThe combing hair task is not an ideal upper limb functional task when using the Kinect V2 system as the outcome measure tool. During the combing hair task, the Kinect V2 system had the worst accuracy and reliability in assessing the angular waveforms, the angles at the PTA, and the corresponding ROM. The greatest deviations of the angular waveforms for the Kinect V2 system were identified for all investigated motions in the combing hair task. The Kinect V2 system also cannot measure shoulder and elbow flexion/extension angles at the PTA and the ROMs of all investigated motions in the combing hair task with low test-retest reliability (ICC = 0.27-0.65). During the task, the absolute test-retest reliability of shoulder F/E is the worst with the between-session RMSE of 18.91°. Thus, it is better not to use the same functional task like combing hair in the assessment of upper limb functions.Our results show that the Kinect V2 system can reliably measure upper limb motions. Kinect V2 has good relative agreements of angular waveforms and can accurately measure a few of the shoulder and elbow joint angles during the functional tasks. Although the measurement of upper limb kinematics may be not as accurate as the Vicon system, Kinect V2 is useable to track relative within-person changes in movements over time (such as the worsening of movement symptoms with disease progression or improvement due to intervention).In current clinical practice, clinical scales have been widely used to assess upper limb functions, which are subjective and labour-intensive. The development of a video tracking system based on low-cost markerless cameras would free clinicians from the necessity of dedicated (and expensive) motion capture laboratories and would allow for in situ data collection. This markerless system would have a significant impact in the fields of clinical practice. Unlike clinical scales, the markerless motion analysis technique is objective, which helps to quantify massive kinematic parameters and turn data into a knowledge-based data warehouse in a standard way. The whole process makes it possible for identifying and explaining relationships between different motion patterns or different population groups through data mining.The experiment had a limited sample size . The system has not yet been used in other populations or other environments such as upper limb disordered patients in clinical settings, elderly people at home or in the clinical environment, and people working in the workplace. The finding from this study may have differed if the assessment was undertaken in a clinical population. Upper limb motions of healthy people often have low variability. The low variability increases heterogeneity and would potentially lead to higher reliability estimates and stronger correlations between Kinect V2 and the reference. In the future, tests with larger sample size, including patients, will be conducted.Further work is also concerned to improve the accuracy of the Kinect system in measuring upper limb joint angles. There have been several recent studies in estimating body landmarks and motions using the Kinect sensor , 28. It The Kinect V2-based upper limb functional assessment system developed in this research has good test-retest reliability in assessing the upper limb angular waveforms and the angles at the point of target achieved except for the combing hair task. The Kinect V2-based system can accurately assess shoulder flexion/extension, elbow flexion/extension, and shoulder adduction/abduction in some upper limb functional tasks. The Kinect V2 sensor has great potential as a low-cost, easily implemented device for assessing upper limb angular waveforms when performing functional tasks. Our system is suitable for assessing relative within-person change in upper limb motions over time, such as disease progression or improvement due to intervention.


Here we review recent advances in our understanding of TNFR2 signaling in T cells and discuss how these can reconcile seemingly conflicting observations when manipulating TNF and TNFRs. As TNFR2 emerges as a new and attractive target we furthermore pinpoint strategies and potential pitfalls for therapeutic targeting of TNFR2 for cancer treatment and immune tolerance after allogeneic hematopoietic cell transplantation.An intricate network of molecular and cellular actors orchestrates the delicate balance between effector immune responses and immune tolerance. The pleiotropic cytokine tumor necrosis factor-alpha (TNF) proves as a pivotal protagonist promoting but also suppressing immune responses. These opposite actions are accomplished through specialist cell types responding to TNF via TNF receptors TNFR1 and TNFR2. Recent findings highlight the importance of TNFR2 as a key regulator of activated natural FoxP3 Tumor necrosis factor-alpha (TNF) regulates innate as well as adaptive immune processes and controls tissue homeostasis in various ways. TNF reached prominence as a prototypic proinflammatory cytokine, however, more recently, the TNF-TNF receptor system gained attention for its immunomodulatory and even anti-inflammatory functions. Here, we review important activities of TNF and its receptors crucial for T cell and Treg function under pathologic conditions such as acute graft-vs.-host disease (GvHD). The implications of the molecular basis of TNF receptor signaling are then discussed for the rational development of therapeutic TNFR-receptor-targeting reagents for clinical applications.TNF is a single spanning type II transmembrane protein and the name giving member of the TNF superfamily (TNFSF) . TNF andTNFR1 and TNFR2 can be assigned to two different subgroups of the TNFRSF. TNFR1 belongs to the TNFRSF death receptor subgroup. Death receptors are characterized by a cytoplasmic protein-protein interaction domain called death domain (DD), which enables these receptors to trigger cytotoxic signaling . AlthougInterestingly, TNFR2 recruits very efficiently TRAF2-cIAP1/2 complexes . Indeed,5 molecules per cell in tumor cell lines  mice enhanced diabetes onset while reducing CD4 effects . Moreoveactivity . Again iactivity . Accordiactivity . Notablyactivity . Alreadyactivity –57. Furtactivity . Likewisin vitro .+ expression marks the most suppressive subset of Tregs  or significant but slowly progressing (p65 deficient Tregs) lymphoproliferative disease . Employing a TNFR2-selective nonameric variant of murine TNF, several groups found that exogenous TNFR2 stimulation suffices to expand Treg numbers in mice . In. Inin vi in mice . TNFR2-prom GvHD .+ effector Tregs with high TNFR2 expression  display increased Treg numbers in the peripheral blood and bone marrow , which creatment .The goal of TNFR2 targeting clearly depends on the considered disorder. While inhibiting TNFR2 activities in Tregs or even destroying Tregs may be the goal in cancer, stimulating TNFR2 may be the aim to treat inflammatory conditions or inflammation-associated cancer to improve immune suppression by Tregs.in vitro on non-hematopoietic cells. In vivo, however, the situation might be complicated by the presence of immune cells and immune cell-associated expression of Fcγ-receptors (FcγRs). Various preclinical in vivo studies demonstrated that FcγR-binding can act as an all-dominant factor that determines the agonistic activity of TNFRSF receptor-specific antibodies and even converts antagonistic antibodies into strong TNFRSF receptor agonists  interacts with TNFR2 and TNFR1; second, limited ability of soluble TNF trimers to stimulate TNFR2 signaling. The challenge of TNFR2 selectivity had been solved early, by various groups identifying mutations conferring selectivity for one of each of the two TNF receptors. Furthermore, oligomerization of soluble TNF trimers potentiates their ability to stimulate TNFR2 . This kn variant .in vivo could rather deplete Tregs instead of promoting Treg expansion. Therefore, antibodies with a high intrinsic, FcγR binding-independent agonistic activity or Fc domain-mutated antibodies preferentially binding to inhibitory FcγRs may account for the best strategy to achieve TNFR2 activation with agonistic antibodies in vivo.Besides recombinant soluble TNFSF ligand variants, agonistic antibodies are the classical approach to activate receptors of the TNFRSF (in vivo is the use of non-competitive antibodies modifying the receptor response to soluble ligand trimers. It has been found that some non-competitive and per se non-agonistic antibodies against TNFRSF receptors can strongly enhance receptor activation upon soluble ligand binding presumably via aggregation of otherwise poorly active trimeric ligand-receptor complexes (An interesting option to achieve TNFR2 activation omplexes , 115. Thomplexes . Clinica+ T cells and CD8+ cytotoxic T lymphocytes (The pathophysiologic sequelae of acute GvHD follows a spatiotemporally orchestrated pattern of disease initiation and an ensuing effector phase –120. TNFphocytes , 124–126phocytes –129. Howphocytes , 131. Upphocytes . Fn14, aphocytes . This maphocytes .In patients, systemic TNF release of >100 pg/mL in the first 3 months after allo-HCT strongly correlated with acute GvHD, veno-occlusive disease, endothelial leakage syndrome, and interstitial pneumonitis , 129. AlThe detrimental effects of TNF on GvHD pathogenesis provided a clear rationale to test TNF-inhibitors in allo-HCT. Indeed, TNF blockade prevented acute GvHD in most mouse models but may also affect graft-vs.-leukemia activity as transplantation of TNFR1 deficient donor CD8 T cells resulted in an increased leukemia relapse after allo-HCT , 140. BaBlocking TNF does not only inhibit the primarily TNFR1-mediated proinflammatory TNF activities but also the predominantly TNFR2-mediated protective effects. The ambivalence of therapeutically targeting TNF is emphasized by the experience with TNF inhibitors in the treatment of autoimmune diseases. Clearly, TNF blockers have been a game-changer for the treatment of inflammatory diseases such as rheumatoid arthritis and colitis showing high response rates in many patients making them the commercially most successful biologicals on the market. However, many patients do not respond to TNF inhibitors and TNF blockers may even exacerbate inflammation in other diseases, e.g., heart failure or multiple sclerosis , 147. Thin vivo expansion of Tregs could prevent acute GvHD (As pointed out above, an important mechanism explaining these opposing outcomes of TNF-inhibition is the dichotomy of TNFR1- and TNFR2-mediated effects. Therefore, directly addressing TNFR1 or TNFR2 as therapeutic targets through TNFR1 antagonists or TNFR2 agonists appears as an attractive strategy to improve current clinical practice of GvHD treatment. So far, this strategy has been tested in preclinical mouse models employing TNFR2-selective agonists. TNFR2-mediated ute GvHD . Notablyute GvHD , 149. Rein vitro or, importantly, to enhance Treg activity by expanding TNFR2+ Tregs in patients before allo-HCT to prevent GvHD. This attractive approach promises to reduce the risk for GvHD while allowing for alloimmune responses against remaining leukemia cells or to allow for efficient immune control of opportunistic infections. More caution will be warranted to employ TNFR2-agonists at the time of donor lymphocyte infusion or at the onset of GvHD. Clearly, the stimulatory effects of TNFR2 on Tcons require careful assessment in preclinical in vivo models before TNFR2 agonists will enter clinical trials.Conclusively, therapeutically targeting of TNFR2 in patients appears as a highly promising approach to either propagate donor Tregs All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.Through the department of technology transfer of Würzburg University, HW and AB have filed patent applications regarding the development and generation of TNFR superfamily-addressing reagents.


To summarize the results of prenatal diagnoses and pregnancy outcomes of fetuses with thickened nuchal fold (TNF) in the second trimester.From 2009 to 2016, we studied 72 pregnant women with fetal nuchal fold (NF) measurements over 5 mm at 14 to 19 + 6 weeks or 6 mm at 20 to 28 weeks of gestation who received prenatal diagnosis. Karyotypes were first used to detect common chromosomal diseases, and then chromosome microarray analysis (CMA) was performed if karyotypes were normal. Prognoses were followed up by documentation in the hospital or over the telephone.In total, 12 fetuses with chromosomal defects, including 5 pathogenic copy number variants (CNVs) were detected. The risk of chromosomal defects when a TNF was associated with structural malformations (SMs) (35.5%) was much greater than that of an isolated TNF (3.7%) and a TNF associated with soft markers (0%). The rate of SMs when the NF measured ≥10 mm was greater than that NF measured 5 to 7.9 mm or 8 to 9.9 mm. Totally 27 fetuses had adverse pregnancy outcome.A TNF is not only associated with a high risk of trisomy 21 but also with other chromosomal abnormalities, including pathogenic CNVs. The rates of SMs and adverse outcomes increase when the NF thickness increases. Although nuchal translucence (NT) is commonly used as a first-trimester screening test, pregnant women start prenatal care visits at later stages in many developing countries or districts, and chromosomal defect screening, especially by NT measurement, is not performed. Thus, second-trimester ultrasound markers for assessment of risk, especially NF, are very important.The measurement of nuchal fold (NF) thickness during the second trimester is considered to be one of the most sensitive and specific isolated ultrasound marker for the identification of suspected cases of trisomy 21.Despite the large amount of available data regarding the relationship between a TNF and Down syndrome, few studies have examined the relationships between a TNF and other chromosomal defects and even their outcomes. Parents with fetus with a TNF are eager to learn more about those when they are undergoing clinical consultation.The objective of this retrospective study was to summarize the results of the prenatal diagnoses and pregnancy outcomes of fetuses with a TNF in the second trimester in a tertiary hospital in Southern China.22.1+6 weeks of gestation,4 or 6 mm at 18 to 28 weeks of gestation who received a prenatal diagnosis in the Prenatal Diagnostic Center, Guangzhou Women and Children Medical Center during the period 2009 to 2016 were enrolled. The study included all offspring of women who underwent fetal ultrasound at our center who were diagnosed with TNF. The inclusion criteria were as follows: single pregnancy; healthy pregnant women with detailed medical history who were willing to follow up; consistent fetal gestational weeks and gestational weeks according to ultrasound prediction.This was a retrospective single-center case series study. 72 fetuses with an NF≥5 mm at 14 to 17Patients who received amniocentesis or cordocentesis underwent a chromosome karyotype analysis, and if these cytogenetic analyses showed a normal result, a chromosome microarray analysis (CMA) was performed. Some cases associated with structural malformations (SMs) underwent a direct CMA without cytogenetic analyses Fig. . The stu,9 To increase the measurement accuracy, each patient underwent 2 independent measurements by 2 sonographers. To decrease intra-observer variability, the mean NF measurement obtained from these images was recorded. Fetuses with an NF measurement greater than 5 mm at 14 to 17+6 weeks of gestation or 6 mm at 18 to 28 weeks of gestation were included in the study, regardless of whether they were given an NT test before.All NF measurements were performed in the transverse plane of the fetal head, slightly removed from the biparietal diameter, which includes the cerebellum, occipital bone, and cavum septum pellucidum Fig. . The NF Based on whether a TNF was observed alone or with other defects, the fetuses were divided into 3 groups: Group A—isolated TNF, included 27 fetuses; Group B—TNF associated with SMs, included 31 fetuses; and Group C—TNF associated with ultrasonographic soft markers, included 14 fetuses.Besides, Fetuses were divided into 3 groups according to the degree of NFT: Group I—NF measuring 5 to 7.9 mm, included 44 fetuses, Group II—NF measuring 8 to 9.9 mm, included 11 fetuses, and Group III—NF measuring ≥10 mm, included 17 fetuses.2.2Genomic DNA was directly extracted from the uncultured amniotic fluid and cord blood samples using QIAamp DNA Blood Mini kits , according to the manufacturer's protocol. DNA was quantified using a NanoDrop spectrophotometer, according to the manufacturer's instructions, and the DNA quality was checked by agarose gel electrophoresis. Only cases that passed every step of the quality control process underwent microarray analysis.,11 the CNVs identified using CMA were classified as pathogenic CNVs, variants of unknown significance (VOUS) or benign CNVs. Alterations coinciding with known polymorphic CNVs were interpreted as benign. A CNV was considered pathogenic when its pathogenicity was confirmed by previously published literature, it contained a pathogenic phenotype-genotype-related region listed in the databases mentioned above, or evidence suggested that the dosage sensitivity resulted in a clinical phenotype. To identify possible candidate genes within the altered chromosomal regions, disease-associated and biological analyses were performed.A genome-wide, high-resolution SNP array, CytoScan HD , which includes both SNPs and oligonucleotide probes, was used. Procedures for DNA digestion, ligation, polymerase chain reaction (PCR) amplification, fragmentation, labeling and hybridization of the arrays were performed according to the manufacturer's protocols. The reporting threshold for copy number variants (CNVs) was set at 100 kb, with a marker count of ≥50. The results were analyzed using Chromosome Analysis Suite software and categorized using publicly available CNV databases and by investigating the gene content and published literature. The publicly available databases included the Database of Genomic Variants (DGV), DECIPHER database, International Standards for Cytogenomic Arrays (ISCA), Online Mendelian Inheritance in Man (OMIM), and the University of California Santa Cruz Genome Browser Database (UCSC). According to the literature,We summarized all types of detected chromosomal defects.2.3The prognoses of all cases were followed up by documentation in our hospital or over the telephone. Fetuses with normal fetal ultrasound findings during pregnancy, except for a TNF, and with normal physical examinations at least 1 year after birth were assumed to be normal. Adverse pregnancy outcomes included chromosomal defects, termination of pregnancy because of SMs, premature delivery, still births, and perinatal or infant death.2.4P value <.05 was considered significant. Descriptive statistics  are used for to present the results.All statistical analyses were performed using SPSS 20.0. The differences among groups were analyzed using χ2 tests. A 3The median age of the pregnant women in our study was 29 range 12–41) years old, and the procedure was performed at a median of 24 (range 16–35) weeks. Of the women who experienced adverse outcomes, 12 were carrying fetuses (16.7%) with clinically significant chromosomal abnormalities, (Table 2–41 yearP <.05)  of chromosome mosaicism: 46,X,idic(X) q22)[71] /45,X[29]. Group B . No statistically significant differences were observed among the 3 groups with regard to the positive rate of chromosomal defects (P >.05)  with chromosomal defects including 2 cases of trisomy 21, 1 case of trisomy 13, 1 case with a derivative chromosome 1 and 1 case of 22q11 microduplication syndrome. In Group I, 13 cases (29.5%) were associated with SMs, of which 11 patients chose termination, and 1 fetus died due to premature delivery. Group II comprised 2 cases (18.2%) with chromosomal defects, including 1 case with chromosome rearrangement, 46,X,idic(X) (q22)[71]/45,X[29] and 1 case with a pathogenic 17p12 microdeletion. In Group II, 5 cases (45.5%) were associated with SMs, of which 4 patients chose termination. Group III comprised 5 cases (29.4%) with chromosomal defects including 2 cases of Turner Syndrome, 1 case of a pathogenic 4q35.2 microdeletion, 1 case of both a pathogenic 9p24.3-p23 microdeletion and a pathogenic 11p15.5-p15.2 microduplication, and 1 case of 16p11.2 microduplication syndrome. In Group III, 13 cases (76.4%) were associated with SMs, of which 10 patients chose termination. The rates of SMs and adverse outcomes increased when NFT increased, especially when the NF measured greater than 10 mm  and segmental chromosome abnormalities. Similar to increased NT, a TNF as an ultrasound soft marker is highly related to trisomy 21. But the relationships between TNF and pathogenetic CNVs were never reported before. In our retrospective study, 72 fetuses with TNF were investigated by karyotype and/or CMA, and the results showed trisomy 21 accounting for 2.8%, Turner syndrome accounting for 2.8%, trisomy 13 accounting for 1.4%, pathogenetic CNVs accounting for 6.9% and other chromosome aberrant accounting for 2.8%. Using NF alone with a commonly used threshold of 5 or 6 mm, reported detection rates for trisomy 21 vary widely, from 4% to 35%.–16 The detection rate for trisomy 21 in our study was lower than the rate reported before, which is likely due to the prevalence of the NT test and Non-Invasive Prenatal Testing (NIPT) that allowed the early diagnosis and termination of trisomy 21 in the first trimester, resulting in fewer cases in the second trimester. It is obvious that regardless of aneuploidy or chromosomal structural aberrations, most of these TNF cases were associated with various structural abnormalities. Except for 1 isolated TNF case associated with chromosomal defects  (q22)[71] /45,X[29]) and another 1 case developing serious bilateral pleural effusion in the 3rd trimester, Other fetuses without SMs were born alive and with normal physical examinations at least 1 year after birth.The physiological cause(s) of TNF is still poorly understood. In a recent study that included a large number of subjects, a correlation was found between NT and NF measurements. Our study shows that the pathogenic CNVs rate in fetuses with a TNF associated with SMs is 16.1%, which is higher than that observed in Bornstein's study. This difference likely occurred because specific SMs that can cause a TNF have a higher risk of being associated with pathogenic CNVs. It is reasonable to use a CMA as a first-line test for fetuses with structural abnormalities, as the guidelines recommend.,18We also noted that the 5 cases with pathogenetic CNVs in our study were associated with SMs, including central nervous system (CNS), cardiovascular system and urinary system abnormalities as well as fetal hydrops and cystic hygroma. Bornstein et al recently reported the prevalence of pathogenic CNVs in 1980 high-risk and low-risk patients who had undergone prenatal diagnosis by CMA. As expected, pathogenic CNVs were much more common in cases of structural fetal abnormalities, with pathogenic CNVs rates of 5.9% for high-risk patients with fetal anomalies. Patients with CNVs matching this variant may have phenotypes such as intellectual disabilities, global developmental and motor delays, delayed speech and language development, generalized hypotonia, seizures, microcephaly, non-immune hydrops, abnormal facial shape, and others but not phenotypes such as short nasal bone, right hydronephrosis associated with ureteral dilatation, or gastric alveolar enlargement, as observed in fetus P6271. The CMA of case P6425 showed a 1.56 Mb deletion in 4q35.2 that overlapped with the deleted region in DECIPHER patient 286230. According to the DECIPHER database, patients with CNVs matching this variant experience symptoms such as intellectual disability, global developmental delays, delayed speech and language development, micrognathia, seizures, cleft palate, microcephaly, and muscular hypotonia. The 4q35.2 deletion contained 2 OMIM genes, including the FRG1  and FGR2 genes . Both FRG1 and FGR2 are associated with facioscapulohumeral muscular dystrophy , which is an autosomal dominant, inherited form of muscular dystrophy. FSHD is characterized by facial muscle weakness, shoulder weakness, hearing loss, abnormal heart rhythm, and loss of strength in abdominal muscles and typically has an onset in the first or second decade of life but can present later in life. These additionally identified pathogenic CNVs have different degrees of penetrance and expressivity, and based on the likelihood of a phenotype appearing and the existence of SMs, the parents decided to terminate the pregnancy. CMA is becoming the standard test for the prenatal evaluation of genomic imbalances, and there is likely to be an increase in the detection of unexpected diseases, especially for non-structural abnormalities, such as intellectual disability or late-onset disorders with unpredictable age of onset and severity. There is challenge for physicians and genetic counselors to address with patients, especially when a TNF is isolated without a specific structural abnormality. Thus, the development of consensus recommendations that are consistent with legal regulations and cultural beliefs are needed in the future to manage such incidental findings.We observed de novo imbalanced chromosomal regions in fetus A14285. CMA revealed a 9.41 Mb deletion in chromosome 9p24.3-p23 and a 14.76 Mb duplication in 11p15.5-p15.2. Thus, parental karyotype analysis was performed; the cytogenetic result showed that the paternal karyotype was 46, XY, t, and balanced chromosomal translocations can explain the CNV result. Additionally, we found that the CMA results of fetuses P6271 and P6425 did not match their phenotypes on ultrasound examination. The CMA of case P6271 showed a 17p12 deletion inherited from the non-phenotypic father, which is classified as pathogenic in the DECIPHER and ISCA databases. It overlaps with hereditary liability to pressure palsies (HNPP) syndrome in the DECIPHER database, which causes abnormalities in motor neurons and motor conduction block. This region includes 3 OMIM genes, among which the PMP22 gene (OMIM: 601097) results in morbidity. The PMP22 gene encodes a 22-kDa protein that comprises 2 to 5% of peripheral nervous system myelin and is expressed in the compact portion of essentially all myelinated fibers in the peripheral nervous system.,23 however, the relationship between NF measurements and chromosomal defects have not been reported. Our study shows that an increase in NF measurement correlates with an increased risk of chromosomal defects, especially when the NF measurement is ≥10 mm; however, no statistically significant differences were found, likely due to the small sample size. In our study, 14 pregnant women chose termination because of various SMs, and 1 fetus died of premature delivery at 28 weeks of gestation despite normal CMA results. Case A11076 was found to have an isolated TNF in the second trimester with a normal CMA but developed serious bilateral pleural effusion, and the decision was made to terminate the pregnancy. If the diagnosis of a TNF is suspected after prenatal ultrasonography, careful sonographic evaluation of fetal anatomy to exclude additional malformations is recommended, and a series of ultrasounds may be necessary.In our study, the rate of TNF cases associated with various SMs was high, up to 43%. As the NFT increased, the rate of SMs increased from 29.5% (NF measurement of 5–7.9 mm) to 76.4% (NF measurement ≥10 mm). The most commonly affected systems were the CNS and congenital heart disease (CHD) (Chart 1). Studies have shown that an increased NT measurement is correlated with a higher risk of chromosomal defects;The Case series are susceptible to selection bias and our case series study reports on a series of patients with a TNF from a particular population (such as our clinic). Therefore, our study cases may not appropriately represent the wider population and we acknowledge this as limitation of the study.5A TNF is associated with a high risk of trisomy 21 and other clinically significant chromosomal abnormalities, including pathogenic CNVs, especially when the TNF is associated with structural abnormalities. CMA is recommended for prenatal diagnosis in TNF cases with one or more fetal structural abnormalities. The rate of structural abnormalities and adverse outcomes increases when the NF increases; especially when the thickness reaches 10 mm. Detailed ultrasound examination is recommended once a TNF is identified.Lushan Li, Fang Fu contributed equally to this work.Conceptualization: Lushan Li, Fang Fu.Data curation: Lushan Li.Formal analysis: Lushan Li.Funding acquisition: Can Liao.Investigation: Lushan Li, Zequn liu.Methodology: Ru Li.Project administration: Fang Fu.Resources: Lushan Li.Software: Zequn liu.Supervision: Fang Fu, Can Liao.Writing – original draft: Lushan Li.Writing – review & editing: Lushan Li.Lushan Li orcid: 0000-0001-9450-6376.


Short jute fiber-reinforced acetylated lignin-based thermoplastic polyurethane (JF reinforced ASKLTPU) was prepared and characterized as a short-fiber-reinforced elastomer with carbon-neutrality and biodegradability. The acetylated softwood kraft lignin-based thermoplastic polyurethane (ASKLTPU) was prepared with polyethylene glycol (PEG) as a soft segment. Short jute fiber was modified using low-temperature pyrolysis up to the temperatures of 200, 250, and 300 °C in order to remove non-cellulosic compounds of jute fibers for enhancing interfacial bonding and reducing hydrophilicity with the ASKLTPU matrix. JF-reinforced ASKLTPUs with fiber content from 5 to 30 wt % were prepared using a melt mixing method followed by hot-press molding at 160 °C. The JF-reinforced ASKLTPUs were characterized for their mechanical properties, dynamic mechanical properties, thermal transition behavior, thermal stability, water absorption, and fungal degradability. The increased interfacial bonding between JF and ASKLTPU using low-temperature pyrolysis was observed using scanning electron microscopy (SEM) and also proved via interfacial shear strength measured using a single-fiber pull-out test. The mechanical properties, thermal properties, and water absorption aspects of JF-reinforced ASKLTPU were affected by increased interfacial bonding and reduced hydrophilicity from low-temperature pyrolysis. In the case of the degradation test, the PEG component of ASKLPTU matrix highly affects degradation and deterioration. Lignin is a kind of complex heterogeneous polymer with aromatic and aliphatic moieties and is the second most abundant natural biopolymers on earth ,2. LigniJute fiber is a kind of cellulosic fiber and often used for rope, yarn, cordage, textiles, paper products, and fiber reinforcement for composites . As natuPyrolysis means the thermal decomposition or degradation of the biomass in the absence of oxygen and is used for fuels or other high-value products . The dryThermoplastic polyurethane is a kind of thermoplastic elastomer with the structure of rubbery soft segments and separated hard domains . Short fIn the previous work, acetylated softwood kraft lignin-based thermoplastic polyurethane (ASKLTPU) were prepared and characterized . ASKLTPU®) was purchased from MeadWestvaco Co.  and kept in the desiccator without moisture. Raw jute fibers , approximately 50–100 μm in diameter, were cut into 65 mm segments and kept at 15.8 ± 0.2 °C and 26 ± 2% RH (relative humidity). Acetic anhydride (99.0%), polyethylene glycol , 4,4-methylene diphenyl diisocyanate , and tetrahydrofuran  were purchased and used. Sabouraud dextrose agar (model No: 210950) purchased from Becton, Dickinson and Company  was used as a medium and Aspergillus awamori fungi (ATCC 6970) from Korean Collection for Type Cultures  were used for the degradation test.Softwood kraft lignin (SKL) (Indulin ATv/v). The resultant solid, acetylated softwood kraft lignin (ASKL) was washed using deionized water, followed by vacuum drying at 105 °C for 24 h. The degree of acetylation of prepared ASKL was approximately 90% according to the previous work [SKL was acetylated according to the procedure described in the previous work ,57. A toous work .ASKL-based thermoplastic polyurethane (ASKLTPU30), whose ASKL content was 30 wt %, was prepared according to the previous works . About 5As a modification method, low-temperature pyrolysis of jute fiber was performed according to the previous work . Jute fiJF-reinforced ASKLTPU was prepared using a melt-mixing method followed by hot-press molding. RJF and PJFs (JF) as fiber components and ASKLTPU30 as a matrix component were vacuum-dried in 105 °C for 24 h before use. The fibers were chopped to approximately 10 mm lengths and ASKLTPU30 were mixed using a table-type kneader  at 160 °C, followed by a hot-press method using two post-manual hydraulic presses  at 160 °C. The fiber contents of the reinforced ASKLTPU were 5, 10, 20, and 30 wt %. JF reinforced ASKLTPU was labelled PJF200/ASKLTPU30 10/90, whose composition was 10 wt % of PJF200 and 90 wt % of ASKLTPU30. For comparison, ASKLTPU30P and ASKLTPU30 without fiber reinforcement were prepared using the same procedure.−1 at a resolution of 0.5 cm−1 with 32 scans.Spectroscopic analysis of PJF-reinforced ASKLTPU was performed using attenuated total reflectance—Fourier transform infrared spectroscopy (ATR-FTIR)  technique. The ATR-FTIR measurements were performed at room temperature in the range of 4000–650 cmA field emission scanning electron microscope (FE-SEM)  was used to observe the tensile fracture surfaces of PJF-reinforced ASKLTPU. The reinforced ASKLTPU were coated with a thin layer of platinum by sputter coater and visualized with a voltage of 2 kV.F is the load required to debond the fiber from the matrix, D is the diameter of the JF, and L is the embedded length of fiber into the matrix. At least five specimens were measured for each JF/ASKLTPU sample.A single-fiber pull-out test was performed to measure interfacial shear strength (IFSS) between JF and ASKLTPU30 ,59,60. TThe mechanical properties of PJF-reinforced ASKLTPU was analyzed via a tensile test using a universal testing machine (UTM) with an XLC series load cell . The tensile test was performed according to ASTM D 638-10 guidelines with ASTM D638 type V (dumbbell-shape) specimens, while some testing settings were modified due to characteristics of the samples . The gagAn optical microscope  was used to measure the length and the diameter of JFs in JF-reinforced ASKLTPU for a further modelling study of mechanical properties regarding the prediction of Young’s modulus. At least 120 specimens were observed to measure the frequency distributions.2. The force amplitude was 3 N and the displacement amplitude was 20 μm, while the strain frequency was 1 Hz.Dynamic mechanical thermal analysis (DMA) of PJF-reinforced ASKLTPU was performed using dynamic mechanical thermal analyzer . The samples were disk-shaped with a 6 mm diameter and about 2 mm in height. The storage and loss moduli were scanned using the compression mode in the temperature range from −50 to 200 °C at a heating rate of 3 °C/min with a purge of dry N2. The first cooling cycle and second heating cycle were investigated for thermal transition behavior. The thermal transition behaviors of JF-reinforced ASKLTPU were investigated using a differential scanning calorimeter . The analysis was performed in the range of −80–200 °C at heating and cooling rates of 10 °C/min with a purge of dry NX-ray diffraction (XRD) analysis was performed in order to check the crystallinity of JF-reinforced ASKLTPU using X-ray Diffractometer  with CuKα radiation (λ = 0.15406 nm) generated at 40 mA and 40 kV in the 2θ range 3–50° with a step size of 0.02.The thermogravimetric analyzer (TGA)  was used to investigate the thermal stability of JF-reinforced ASKLTPU30. Samples were analyzed in the range of 20–700 °C with filled nitrogen gas and the heating rate was 10 °C/min.Water absorption tests of ASKLTPUs and JF-reinforced ASKLTPUs was conducted using a modified long-time immersion method based on the ASTM D570-98 standard . The samAspergillus awamori fungi were seeded. The fungi in the medium were allowed to grow for 96 h at 25 °C and 80% RH. Afterwards, ASKLTPU and PJF-reinforced ASKLTPU samples in the form of the specimen for tensile test (ASTM D638 Type V) were placed on the middle of the medium coated surfaces in contact , a peak around 1600 cm−1 (N–H bending), and a peak around 1550–1500 cm−1 (Amide II) were derived from urethane bonds. The peaks that appeared at 2950–2850 cm−1 were assigned to CH2 stretching of mainly PEG components. The peaks of carbonyl groups derived from both the acetate groups of ASKL and the urethane bonds appeared at around 1760, 1726, and 1697 cm−1. Due to the abundance of carbonyl groups, a relatively intensive peak appeared at 1726 cm−1, which as assigned to non-bonded carbonyl stretching. Interestingly, a relatively weak peak at 1697 cm−1 (ASKLTPU), assigned to H-bonded carbonyl stretching, slightly shifted to 1696 and 1695 cm−1 via the incorporation of fiber or low-temperature pyrolysis. Despite the relatively weak intensity of the peaks, the phenomenon implied the potential formation and increase of hydrogen bonding between JF and ASKLTPU via fiber incorporation and the low-temperature pyrolysis [The chemical nature of ASKLTPU30P and JF-reinforced ASKLTPU was investigated using FTIR spectroscopy analysis, as shown in yrolysis ,68. The In order to investigate interfacial bonding between JF and ASKLTPU, the interfacial shear strength (IFSS) was obtained from a single-fiber pull-out test . The intThe tensile strength ranged between 3.1 and 5.6 MPa. The tensile strength increased as the fiber content increased due to the fiber-reinforcement effect of PJFs. Exceptionally, there was a significant decrease in tensile strength on PJF300/ASKLTPU30 30/70. It might be caused by the weakness of PJF300 discussed below, which might strongly affect the tensile properties of the composite when the fiber content reached 30 wt %. At a fiber content of 5 wt %, there was no significant change in tensile strength except PJF200/ASKLTPU30 5/95 because properties of the matrix predominantly determined the properties of composites due to a relatively low reinforcement content. The low-temperature pyrolysis of jute fiber also affected the tensile strength of JF-reinforced ASKLTPU. The low-temperature pyrolysis was expected to enhance the strength of JF-reinforced ASKLTPU due to the stronger interfacial bonding shown in previous morphological aspects; however, the tensile strength of JF reinforced ASKLTPU decreased as the maximum pyrolysis temperature of PJF increased. This might be due to the decrease of tensile strength of PJFs as the maximum pyrolysis temperature increased. The tensile strength of RJF, PJF200, PJF250, and PJF300 were 0.973, 0.877, 0.461, and 0.119 N/tex, respectively, in previous work [Young’s modulus ranged between 1.70 and 19.8 MPa. It increased significantly as the fiber content increased, while it decreased as the maximum pyrolysis temperature increased. Further discussions of Young’s modulus regarding the fiber aspect ratio frequency dispersion and prediction from related model will be discussed in the following section. The strain at break ranged between 170 and 2270%. It increased significantly as the fiber content decreased, and it increased as the maximum pyrolysis temperature increased. The increase of Young’s modulus and decrease of strain at break with fiber content increase was due to the reinforcement effect of stiff PJFs as the fiber component with high modulus and low elongation (jute fiber: 2.3% ). The grTensile toughness of the material was measured from the area under the stress-strain curve and is typically influenced largely by ductility. The tensile toughness of JF-reinforced ASKLTPU also showed similar tendency aspects to that of strain at break, which indicated the ductility of JF-reinforced ASKLTPU.The offset yield strength of JF-reinforced ASKLTPU was used to compare the required strength for initiation of appreciable deformation. The offset at 0.2% strain was used for measuring the offset yield strength in order to measure the strength sensitively. The offset yield strength showed a similar trend to the tensile strength or Young’s modulus because the stress at low strain was significantly affected by Young’s modulus. As the fiber content increased, the offset yield strength significantly increased, except at a low fiber content of 5 wt %. The offset yield strength of PJF200- and RJF-reinforced ASKLTPU were similar, while that of PJF250- and PJF300-reinforced ASKLTPU showed a lower yield strength.In summary, the tensile strength, Young’s modulus, and offset yield strength of JF-reinforced ASKLTPU composites increased as the fiber content increased and the maximum pyrolysis temperature of PJF decreased with the costs of decreases in ductility and tensile toughness. Although increase in the fiber-matrix interfacial bonding was expected due to the low-temperature pyrolysis of jute fibers, the weakness of PJF itself affect negatively on strength of the JF-reinforced ASKLTPU. However, PJF200-reinforced ASKLTPU showed the highest tensile strength due to the slightly weaker strength of PJF200 and an increase in interfacial bonding. From the investigations, the mechanical properties of JF-reinforced ASKLTPU composites could be tunable for possible applications by changing the fiber content and maximum pyrolysis temperature of PJF.The variations of length, diameter, and aspect ratio of JFs in JF-reinforced ASKLTPU with 10 wt % of fiber content was measured in order to investigate the effect of low-temperature pyrolysis of jute fibers on the JF-reinforced ASKLTPU . The lenThere are mathematical models for the prediction of mechanical properties including Young’s modulus of reinforced composites to anticipate properties or applications of the composite materials. The rule of mixture (ROM) ,71, HalpThe rule of mixture is basic and the simplest model. It is calculated based on the volume fraction of each component of the composite:upperE means the upper bound of the modulus, which corresponds to longitudinal loading, while lowerE means the lower bound of the modulus corresponding to transverse loading. E and v are the modulus and volume fraction of each component, respectively, while f and m are designated to the fiber and matrix component, respectively [ectively ,71.p is a composite property like modulus, ζ is the filler geometry reinforcement parameter, v is the volume fraction; r, m, and c are designated to reinforcement, matrix, and composite, respectively.Halpin-Tsai’s equation is based on “self-consistent micromechanics solutions” developed by Hill ,46 and uζ, is 2(l/d) for 11E  and 2 for 22E (transverse), while l, d, and l/d are designated for length, diameter, and aspect ratio of the fiber, respectively. In the case of randomly-oriented  discontinuous fiber composites or lamina, the predicted Young’s modulus was calculated using:In the case of fiber, the filler geometry reinforcement parameter, randomE from the Halpin-Tsai equation could be used for a prediction study of JF-reinforced ASKLTPU with the measured aspect ratios of RJF and PJFs as the reinforcement [The orcement ,72.oη is the orientation efficiency factor and Lη is the fiber length efficiency factor. The fiber length efficiency factor Lη is calculated using:f, Gm, R,l, A and fr are the length of the fiber, cross-sectional area of fiber, shear modulus of the matrix, the mean separation of the fibers normal to their length, and radius of fiber, respectively. R is often calculated from equations using the assumption of hexagonally packed fibers or square-packed fibers [The Cox-Krenchel approach is based on the equation derived from Cox’s “shear-lag” analysis regarding the effect of orientation of the fiber  and the d fibers ,73,74,75oη is given using:na is the fraction of fibers with orientation angle nΦ with respect to the reference axis [The orientation efficiency factor nce axis ,74.oη was set to 0.375 for random in-plane orientation (2-D random) [In the case of JF-reinforced ASKLTPU,  random) ,41 and tYoung’s modulus of JF-reinforced ASKLTPU modelled using Halpin-Tsai, Cox-Krenchel, and ROM was calculated in order to predict Young’s modulus and compare experimental results . ROM forM′), loss modulus (M″), loss tangent (tanδ), and specific values including the glass transition temperature (Tg) from the compression mode of DMA are shown in The dynamic mechanical thermal analysis of JF-reinforced ASKLTPU was performed to investigate the stiffness and the damping factor of the material under dynamic conditions as a function of temperature. PJF250-reinforced ASKLTPU with 10, 20, and 30 wt % of fiber loading was selected to measure and compare with ASKLTPU30P and RJF-reinforced ASKLTPU. The storage modulus (As shown in Tg) from the peak of loss tangent and from the peak of loss modulus are shown. Although the higher Tg from the loss tangent is also valid and widely used for determining Tg, the Tg from the loss modulus was more appropriate for determining Tg in order to denote the initial drop of storage modulus, especially on JF-reinforced ASKLTPU with a broad range of thermal transition region [Tg shifted toward a higher temperature via the incorporation of fiber.The onset of the decrease of storage modulus or the peak of loss modulus and loss tangent around 0 °C indicated the glass transition of the compatible soft and hard segment mixtures of the ASKLTPU matrix . In Tabln region . The Tg In the case of the loss tangent of ASKLTPU and JF-reinforced ASKLTPU as an index of energy dissipation, the maximum loss tangent value decreased and the width of loss tangent peak broadened as the fiber content increased . The pheIn addition, the onset of the decrease or the peak of the storage modulus and loss tangent appearing over the temperature of 150 °C indicated the transition of a microphase-separated hard domain of thermoplastic polyurethane matrix . Due to Tg from DSC and Tg from DMA (from −11.7 to −2.3 °C) because the glass transition is a kind of kinetic transition and could differ with the measuring conditions and techniques; Tg of DSC was measured from heat capacity while that of DMA was measured from relaxation [Tg from DSC and Tg from DMA had a similar trend of change due to fiber loading and low-temperature pyrolysis. Both Tgs became higher via the incorporation of fiber, and the tendency of increasing Tg was observed as the fiber content of JF-reinforced ASKLTPU increased. Low-temperature pyrolysis of jute fibers also slightly increased the Tg of JF-reinforced ASKLTPU. The increase of Tg might have occurred from the reduced mobility of the matrix via the reinforcement effect of fibers. A similar trend has been shown in other studies about elastomers reinforced by short-fiber- or nano-reinforcement [Tg from an increase of reinforcement loading or modification of reinforcement.The thermal transition behavior of JF-reinforced ASKLTPU was investigated using differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMA) above. laxation . Neverthorcement ,69,76,82orcement ,80 or a orcement ,42,46 ofThere was no significant thermal transitions except the glass transition in DSC curves. The transition of the microphase-separated hard domain of thermoplastic polyurethane matrix, which is shown in DMA results, was too weak to analyze like the previous work . No signT−1%, T−5%, and T−10% indicated the temperatures at which 1, 5, and 10% of weight decreased. Td indicated the maximum decomposition temperature while Tpeak indicated the temperature at which the peak in DTG curves occurred, and the symbol ‘~’ identifies the shoulder peaks.Thermogravimetric analysis (TGA) is often used in order to rank the relative thermal stability of a group of samples and to study degradation behavior of the samples . Figure The DTG curve of ASKLTPU30P showed two thermal decomposition steps. The first step around 348 °C indicated the degradation of hard segment based on ASKL and decomposition of MDI with the breaking urethane bonds, while the second consecutive steps around 400 °C indicated the chain-scission of the soft segment based on PEG1000 ,42,56,83The DTG curves of RJF/TPU30 10/90 and PJF250/TPU30 10/90 showed one wide peak. It was estimated that the main thermal decomposition of jute fibers, the cellulose component at around 354 °C, overlapped with that of ASKLTPU30P. Therefore, it was shown as one wide thermal decomposition; however, in reality, there were different thermal decompositions in the peak. In the case of PJF250/TPU30 15/85, the larger portion of the PJF200 component contributed more to the thermal profile of the JF-reinforced ASKLTPU, so its DTG peak occurred at 366.9 °C and 400.5 °C.T−1%, T−5%, T−10%, and residual weight indicated the JF-reinforced ASKLTPU had better thermal stability considering the sum of the components’ weight fraction solely. This might have resulted from the modified fiber-matrix interface or structural stability. Overall, the low-temperature pyrolysis of the jute fiber enhanced the thermal stability of the JF-reinforced ASKLTPU.As shown in The water absorption behavior of ASKLTPU and JF-reinforced ASKLTPU was tested and evaluated for potential applications . As showAspergillus fungi, whose spp. produces esterase or lignin peroxide with the potential of lignin degrading ability [The degradation test of ASKLTPU and JF-reinforced ASKLTPU was performed with an acceleration of  ability ,87,88. TAspergillus fungi. The characteristic degradation and deterioration behavior was shown because the PEG component as a soft segment of ASKLPTU matrix degraded and deteriorated first during the degradation test.Pyrolyzed jute fiber-reinforced acetylated lignin-based thermoplastic polyurethane with a fiber content from 5 to 30 wt % was prepared using a melt-mixing method followed by hot-press molding and characterized as short-fiber-reinforced elastomers with carbon-neutrality and biodegradability. The increase of interfacial bonding between PJF and ASKLTPU was observed using scanning electron microscopy and a single-fiber pull-out test. PJFs showed good wetting aspects to ASKLTPU and higher IFSS between ASKLTPU than that of RJF. In the case of tensile tests, tunable mechanical properties of JF-reinforced ASKLTPU were obtained. The strength and modulus of JF-reinforced ASKLTPU composites increased as the fiber content increased and the maximum pyrolysis temperature of PJF-decreased with a decrease in ductility and tensile toughness. The weakness of PJF itself negatively affected the strength of the JF-reinforced ASKLTPU in spite of the increase in the fiber-matrix interfacial bonding via low-temperature pyrolysis of jute fibers. Young’s modulus predicted using Halpin-Tsai model and Cox-Krenchel models were comparable to experimental results; however, the difference between the prediction and experimental results should be considered. Among the thermal transition temperature of JF-reinforced ASKLTPU, the glass transition temperature of the soft segment of the ASKLTPU matrix increased via fiber incorporation and low-temperature pyrolysis. The thermal stability of JF-reinforced ASKLTPU increased via low-temperature pyrolysis. The water adsorption behavior and degradation characteristics of JF-reinforced ASKLTPU was also investigated via a water adsorption test and degradation test using In conclusion, the low-temperature pyrolysis of jute fibers affected the properties of JF-reinforced ASKLTPU via increasing interfacial bonding and reducing inherent hydrophilicity of natural fibers. The properties of PJF-reinforced ASKLTPU indicated potential utilization as a bio-based short-fiber-reinforced elastomers.


TNF and its syntenic genes to chicken chromosome 16 within the major histocompatibility complex (MHC) region. This mapping position suggests that avian TNF has a role in regulating immune response. To test its possible interaction with leptin within the immune system and beyond, we compared the transcription patterns of TNF, leptin and their cognate receptors obtained by meta-analysis of GenBank RNA-seq data. While expression of leptin and its receptor (LEPR) were detected in the brain and digestive tract, TNF and its receptor mRNAs were primarily found in viral-infected and LPS-treated leukocytes. We confirmed leptin expression in the duodenum by immunohistochemistry staining. Altogether, we suggest that whereas leptin and TNF interact as adipokines in mammals, in birds, they have distinct roles. Thus, the interaction between leptin and TNF may be unique to mammals.In mammals, leptin and tumor-necrosis factor (TNF) are prominent interacting adipokines mediating appetite control and insulin sensitivity. While TNF pleiotropically functions in immune defense and cell survival, leptin is largely confined to signaling energy stores in adipocytes. Knowledge about the function of avian leptin and TNF is limited and they are absent or lowly expressed in adipose, respectively. Employing radiation-hybrid mapping and FISH-TSA, we mapped  In mammals, leptin is a key adipokine that works in concert with other adipokines such as TNF  to regulate energy homeostasis ,2. TNF hTNF and leptin, erroneous sequences/annotations submitted to GenBank  led to erroneous publications. This was much more pronounced for leptin , but al Kaspers , also re Kaspers ).TNF receptor genes .  as OBR; , and oth as OBR; ,19,20,21TNF and leptin were recently identified in several avian species including chickens ,26,27,28TNF and leptin genes were published. The first publication of both genes [TNF, an additional difference between the two versions is the length of a glycine stretch close the C’-terminal end, which is hard to resolve due to the low coverage with RNA-seq data from this GC-rich part of the gene. These difficult regions were characterized by optimized PCR amplification methods and Sanger sequencing [TNF is of high importance since it includes its predicted transmembrane domain of TNF. The 5′ extended sequence of leptin includes the expected signal peptide. However, this sequence is longer than that of orthologous leptins.Two similar versions of genuine chicken th genes ,15 lacketh genes ,28. For quencing . The 5′ TNF gene maps to chromosome 6p21.3, contains four exons and spans about 3 Kilobases. The last exon shares similarity with lymphotoxin alpha  [TNF maps within the MHC locus between lymphotoxin α and β genes. These three genes share some redundant activities, in addition to their unique functions [The human s TNF-β) . These gs TNF-β) . Except s TNF-β) ,33. Thess TNF-β) ,35. In munctions . Lymphotunctions ,12.TNF is expressed primarily in embryonic and adult spleens, as well as in monocytes and macrophages [TNF is induced by lipopolysaccharide (LPS) as a model for gram negative infections [TNF profile of mRNA expression resembles that in mammals, except for low abundance in the adipose tissue [TNF onto the chicken genome is crucial proof for its correct identification. This proof is especially important due to the difference between the avian and mammalian expression in the adipose tissue; the relatively low sequence similarity between chicken TNF and its mammalian and reptilian orthologs ; and the erroneous annotation of chicken TNF sequence in GenBank. Thus, the primary aims of this work were to map and compare the chromosomal position of avian TNF with that of the mammalian TNF and to test if its mRNA expression profiling supports its possible interaction with leptin within the immune system and beyond.We have previously shown that the chicken rophages ,14. In tfections ,25,27,28fections . Unlike e tissue . ChromosTNF within the chicken genome, we employed the well-established RH panel in Wg3hCl2 cells, prepared by fusing chicken embryonic diploid fibroblasts with hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient hamster cells [TNF close to CSNK2B, between SEQ0111 and GCT2022 markers of the MHC cluster Y on the q arm of microchromosome GGA16 [TRIM7.2, BRD2, TAP2 and ABHD16A, which are located within the MHC cluster on GGA16 in the current genome assembly  [As a first indication for the location of chicken er cells . The aname GGA16 . This loassembly . The CSN cornix) .TNF on chicken GGA16, we used FISH-TSA on metaphase spreads of mitotic chromosomes. cDNA probes were prepared for TNF and for BRD2, TRIM7.2, CSNK2B and ABHD16A genes. Using dual-color labeling technique, the analysis showed that the three chicken genes, TNF, ABHD16A and CSNK2B were co-localized with either BRD2 or TRIM7.2 on a single microchromosome . C4 is closely associated with TRIM 7.2, BRD2 and TAP2 in the current GGA16 assembly.This mapping characterization summarized in TNF and leptin interact with each other to control immune functions and endocrine activities of the adipose tissue [TNF and their cognate receptors in tissues and immune cell lines by meta-analyzing the available RNA-seq data in the Sequence Read Archive (SRA) in NCBI. Sequence reads were counted using the SRA BLAST tool and sequences of chicken leptin, LEPR, TNF, TNFR1 and TNFR2 as baits ; no induction of TNFR2; and no or low expression of leptin and LEPR (<1 RPKM), respectively . Since the data was obtained from pools of RNA samples from three birds of each treated group [LEPR are neither important for immune response nor to the degree of sensitivity of chicken spleens to viral infection. On the other hand, these results supported a role for TNF and TNFR1 mRNAs in the infected spleens. Yet, an additional BLASTN search was performed on a dataset from primary cell lines of macrophages and dendritic cells derived from bone marrow and from heterophil cells isolated from blood , respectively. For chicken TNF, this analysis confirmed previous indications suggesting TNF implication in the immune response [Analysis of RNA-seq dataset from spleens of chickens four days after intranasal infection with bursal disease virus (IBDV) showed a significant induction of ectively A. Anotheed group , statistom blood C. The ceresponse ,14. For response , the chiTNF and their cognate receptor expression patterns [TNF was prominently expressed only in the bursa (above 20 RPKM), and it was also detected at a low level (<1 RPKM) in the spleen, adrenal, heart and lung. Similarly-low TNF mRNA levels were also observed in caecal tonsil, ileum and duodenum, which likely represent the high activity of these tissues in innate immune response through recruitment of immune cells [TNF receptors, TNFR 1 and 2 seem to be ubiquitously expressed. This pattern confirmed the primary role of TNF in the immune system and suggested that, like in mammals, avian TNF affects survival of most cell types.We extended our previous profiling of leptin, patterns ,15,28, une cells ,47. InteLEPR in brain tissues of red junglefowl  and at lower levels also in the ileum and pancreas . This expression pattern suggests a role for chicken leptin in the digestive tract.In addition to the known expression of the chicken leptin and erebrum; ,28), expIn mammals, expression of leptin in the stomach is well known. Therefore, our finding suggested that this site of leptin expression represents an important role.TNF mRNA [The surprising finding of chicken leptin mRNA in tissues belonging to the digestive tissue indicates a possible role of leptin in chickens. To estimate if, like in mammals ,49,50, cTNF mRNA . The speTNF mRNA . While lTNF gene together with additional four syntenic genes in mammals  to chicken chromosome 16q, within the MHC locus and demonstrated that these genes form a syntenic block also in chickens consistent with their co-localization in mammals and several other vertebrates [We have mapped the chicken tebrates . This matebrates .TNF. Interestingly, this position seems to be part of the GC-rich region observed within the GGA16 chromosome by a low DAPI staining [TNF gene is likely to be shared by its neighboring genes and their intragenic sequences. Therefore, it is possible that the two closest neighboring genes of TNF in other vertebrates (lymphotoxin A and B), missing in chickens, would also be found; as happened in similar scenarios e.g., the leptin and RBM28 genes in the centromeric GC-rich region of GGA1 [The use of two different approaches for the mapping: FISH and RH-mapping strongly confirmed the genomic allocation of staining . Thus, t of GGA1 .TNF, TNFR1 and TNFR2 mRNA expression in RNA-seq datasets in the SRA extended a previous study of TNF [TNF and TNFR1 are induced in spleen and in immune cells upon virus infection and LPS treatment. The comparative mRNA expression analysis of TNF and its receptors shown here also suggested that TNF in the spleen and in immune cells has paracrine/autocrine mode of action in addition to its well established endocrine action. Unlike TNFR2, TNFR1 has a death domain, for executing TNF’s cytotoxicity and has high affinity to both the soluble and membrane bound TNF [TNFR1 mRNA levels were more responsive to viral and LPS treatments than those of TNFR2 is compatible with TNFR1 functions. Strikingly, in the same datasets, leptin mRNA was absent in the spleen and in immune cells under all of the experimental conditions. LEPR mRNA was lowly expressed and did not respond to treatments or to selective breeding toward resistance or sensitivity to infection. Since the role of the mammalian leptin is to communicate energy status to the immune system [Our meta-analysis of y of TNF ,14, indiound TNF . Our fine system , this obe system ,27,28,29TNFR1 and 2 mRNA expression in a variety of chicken tissues showed a wide range of expression of both receptors but with more abundant expression of TNFR1 in most of the tissues. This characterization suggested a further role of chicken TNF not only in the immune system, but also in general cell survival, which is among its classical roles in mammals [TNFR2 is predominately expressed in cells of the immune system and endothelial cells [Profiling  mammals . Since ial cells , it is lThe most surprising result in the current study was our finding of leptin and LEPR mRNA in the digestive system, opening a new frontier for chicken leptin research. In mammals, gastric leptin was discovered in 1998 by Bado et al.  and has The immunostaining of leptin in the duodenum and the finding of its presence in mucosal cells (enterocytes and goblet cells), provided a critical proof that the low expression of leptin mRNA (0.3 RPKM) in J-line duodenum is significant. Our finding of leptin in the digestive system raised the possibility that chicken leptin may operate in short term regulation of appetite based on the digestive activity. This may take place either via the afferent vagus nerve or through the blood circulation or both. However, more work is needed to evaluate this possibility.PCR amplifications were carried out for each marker  in 15 μLPrimary line of chicken CEF cells, were prepared from 10-day old pooled chick embryos  of an inbred White Leghorn strain  as previABHD16A sequence) and conditions previously described [Total RNA was isolated from chicken DF-1 cells  using TRescribed . The PCRDouble color FISH-TSA protocol was adapted from Krylov et al.  with minFISH-TSA images were captured with camera DFC 7000T ) coupled to an epifluorescence microscope Leica DM6B equipped with a set of three narrowband fluorescent filters. Digital pseudocolored images . Sequence reads were assembled with CLC genomics workbench, Qiagen, Bustehrad, Czech republic and with Lasergene SeqMan .Deducing the sequence of the chicken ortholog of http://www.hylinena.com/userdocs/products/lohmann_brown_lite_commercials_2011.pdf), as detailed previously [Commercial females of the Leghorn breed (layers) were purchased from commercial husbandries  at the age of 1 d and grown at the Volcani Center according to recommended husbandry and feeding conditions (NRC 1994) with free access to food and water. Maintenance conditions and feeding formulas were according to the Lohmann guideline (eviously . At the A custom-made antibody directed against chicken leptin-peptide (amino acid sequence: PPRAEKLRADARSLSRTLSARLGD) was prepared in rabbits and affinity purified by HY Laboratories LTD, Rehovot Israel. Anti alpha tubulin antibody was purchased from ABCAM .Protein concentration was measured by Bradford assay . Total proteins (20 mg) were subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane.Chicken duodenum was fixed in buffered formalin for ~48 h. Fixed specimens were dehydrated through graded ethanol concentrations, cleared in xylene and embedded into paraffin. Five micrometers longitudinal sections were prepared and mounted onto positively charged glass slides. For immunohistochemical staining sections were deparaffinized in xylene and rehydrated through descending ethanol concentrations. Rehydrated sections were incubated with rabbit polyclonal antibodies to chicken leptin diluted 1:600 in TBST  for 1 h at room temperature. After washing in TBST (5 times for 2 min) sections were incubated with anti-rabbit ZytoChem Plus HRP-Polymer  for 30 min. Then sections were washed in TBST and peroxidase activity was revealed by incubation in a mixture of 0.05% diaminobenzidine hydrochloride  with 0.03% hydrogen peroxide in 0.1 M Tris-HCl, 10 mM imidazole (pH 7.5). Sections were slightly stained with Gill’s hematoxylin, dehydrated, cleared in xylene and mounted in DPX (Sigma-Aldrich). Digital microphotographs were prepared using SPOT InSight CMOS camera attached to BX51 microscope . This analysis was conducted by Smart Assays, Ness Ziona, Israel.p ≤ 0.05); means ± SEs are reported.Statistical analyses of the qRT-PCR analysis and leptin bioassay were performed by one-way ANOVA and Tukey-Kramer honestly significant difference test (


The first document dates back to 1926 and the last one belongs to 1945. The type of papers for official ones were white or pale brown papers and simple ones were yellow or green-brown papers like Chinese Khanbaligh papers. Documents show that on behalf of government, health office (Edare-Sehiieh) directly in Shiraz (center of Fars Province) and also by the help of mobile physicians in towns, informed people the importance of vaccination. One of the oldest documents dates back to 1926  for the villages around Shiraz. There was not any approved salary for these vaccination officers yet. It was asked from local reeves and doyens to pay them. The second document belongs to Nov 1932  Kazeroon) in Shiraz for smallpox vaccination. It was a public announcement. Less than 2 mounts later, he wrote 2 letters  for non-cooperation with him. Only 2 months later, this officer wrote another letter  and then Amir Kabir, prime minister of Iran 1807–1852) ..6). Our


Heart failure (HF) with preserved ejection fraction (HFpEF) comprises nearly half of prevalent HF, yet is challenging to curate in a large database of electronic medical records (EMR) since it requires both accurate HF diagnosis and left ventricular ejection fraction (EF) values to be consistently ≥50%.We used the national Veterans Affairs EMR to curate a cohort of HFpEF patients from 2002 to 2014. EF values were extracted from clinical documents utilizing natural language processing and an iterative approach was used to refine the algorithm for verification of clinical HFpEF. The final algorithm utilized the following inclusion criteria: any International Classification of Diseases-9 (ICD-9) code of HF (428.xx); all recorded EF ≥50%; and either B-type natriuretic peptide (BNP) or aminoterminal pro-BNP (NT-proBNP) values recorded OR diuretic use within one month of diagnosis of HF. Validation of the algorithm was performed by 3 independent reviewers doing manual chart review of 100 HFpEF cases and 100 controls.We established a HFpEF cohort of 80,248 patients . Mean age was 72 years; 96% were males and 12% were African-Americans. Validation analysis of the HFpEF algorithm had a sensitivity of 88%, specificity of 96%, positive predictive value of 96%, and a negative predictive value of 87% to identify HFpEF cases.We developed a sensitive, highly specific algorithm for detecting HFpEF in a large national database. This approach may be applicable to other large EMR databases to identify HFpEF patients. Heart failure with preserved ejection fraction (HFpEF) is increasing in prevalence , and alrThe study was approved by the VA Boston Healthcare System Human Subjects Subcommittee IRB #2868). We used the national VA database to develop a HFpEF cohort utilizing EMR data recorded between 1/1/2002 to 12/31/2014. The VA uses Veterans Information Systems and Technology Architecture (VistA), which is a national integrated healthcare system with a comprehensive EMR across all the VA healthcare facilities. The VA has provided healthcare to millions of Veterans over the last two decades in more than 1700 hospitals, clinics, and nursing homes, with the majority of care recorded through VistA. A NLP tool which has been well established in VA database , 6, was 8. We useInternational Classification of Disease-Ninth Revision (ICD-9) codes diagnosis of HF (any 428.xx), (2) either B-type natriuretic peptide (BNP) or aminoterminal pro-BNP (NT-proBNP) values recorded OR diuretic usage within one month of HF diagnosis, (3) availability of echocardiogram in our system, and (4) having all recorded EF values ≥50%. We excluded patients with constrictive pericarditis (ICD9 code 425.1) and hypertrophic cardiomyopathy (ICD9 code 423.2) and any previous history of HF. We specified the criteria of all EF values ≥50% to ensure a cohort of definite HFpEF through the period of study. As shown in fig. HFpEF algorithm inclusion crtieria consisted of 1)  InternatFigure We used clinical trials definition to validate definite and probable HF cases . DefinitSymptoms of HF included non-exertional and exertional dyspnea, orthopnea, paroxysmal nocturnal dyspnea, fatigue, decreased exercise capacity, and rapid increase in weight, edema, or abdominal girth. Signs included rapid weight loss on diuretics, elevation of jugular venous pressure, edema, elevation of BNP/NT-proBNP, pulmonary edema/rales, any radiologic evidence of HF , hemodynamic measurements showing elevated filling pressures, and presence of ascites or hepatomegaly.Cases for chart review were randomly selected from the algorithm derived HFpEF cohort. Controls were randomly selected from a cohort who had a procedure code for echocardiogram in the medical records between 2002 and 2014 but no ICD-9 code for HF. Three independent reviewers  completed the chart review of 100 cases and 100 controls in a blinded fashion, with each chart reviewed by two reviewers. Any discrepancies between the reviewers were addressed by further discussion and a consensus was achieved.After manually reviewing the medical records for the 200 randomly selected patients, sensitivity, specificity, positive and negative predictive values were calculated to determine the ability of the algorithm to identify cases and controls relative to identification through manual chart review, which was considered the gold standard , 9. Our Comparison between algorithm-defined and manually reviewed cases and controls is shown in Table Table In a national cohort of VA patients, we demonstrated the feasibility of using NLP and EMR data to curate a HFpEF cohort with high specificity. Combining structured elements  captured from EMR with a NLP tool to capture LVEF from unstructured data, our algorithm was able to rapidly and accurately curate HFpEF from a large EMR database. Our approach led to the creation of a cohort of veterans with HFpEF which allows for investigation of clinical predictors and outcomes in HFpEF.Since HFpEF diagnosis requires clinical symptoms and signs inclusive of laboratory data and radiographic images and documentation of EF, correctly identifying HFpEF patients in a large cohort could be extremely challenging. This is a major limitation for population-based HFpEF research. The recent American College of Cardiology (ACC)/American Heart Association (AHA) guidelines suggested various cutpoints of EF values to diagnose different types of HF; including HFpEF with EF > 50%, heart failure with reduced ejection fraction (HFrEF) with EF < 40%, HF with intermediate EF with EF 40–50%, and HF with recovered EF whose values were < 40% in past but has now become > 50% . To our Similar approaches have been used to curate HF cohorts from EMR databases. For example, Bielinski and colleagues developed a tool to identify HF cases differentiated as HFpEF and HFrEF in an EMR database . In thisSimilar to ours, other algorithms have been developed for various other clinical variables. Electronic Medical Records and Genomics (eMERGE) ePhenotyping algorithms were developed using a similar iterative approach, where each clinical disorder algorithm goes through a series of iterations until the algorithm performs to a desired criteria. These algorithms have been successfully utilized to define phenotypes in various clinical disorders including cataract , dementiOur cohort is similar to other published epidemiological and clinical trial cohorts like Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist (TOPCAT) , OrganizThere were limitations in our algorithm. We may not have included all patients with HFpEF as we required stringent inclusion criteria, but this approach allowed us to more confidently identify true HFpEF patients. We did not have detailed echocardiographic parameters except EF values to characterize diastolic HF and its severity, although recent ACC/AHA guidelines  do not rIn conclusion, using a combination of structured, clinical parameters, and unstructured data, we have curated a well-refined algorithm to identify HFpEF patients to facilitate population-based research in HFpEF.


GLA). FD is still an underdiagnosed disorder worldwide. Moreover, there is delay between symptom onset and Fabry diagnosis of at least 10 years. Family screening offers an important benefit for detection of new patients. The aim of this work is to present the approach along with the results of a targeted genetic strategy for pedigree analysis for FD in Argentina.Fabry disease (FD) is an X‐linked disorder of glycosphingolipid catabolism caused by a deficiency of the lysosomal enzyme alpha‐galactosidase A (By this strategy as soon as a new index Fabry patient is diagnosed, the pedigree group contacts the physician and a meeting is arranged with the physician and the family to build the family tree.Pedigree analysis was carried out for full in 31 families. In the work period, we have tested 1,462 relatives, and 501 were diagnosed FD. The proportion of positive detection was 33%.The targeted family screening approach is successful to detect undiagnosed Fabry patients. By this approach, the highest ratio index to pedigree ever reported for FD pedigree analysis of 1:15 was obtained. This enzymatic defect leads to a chronic and progressive systemic accumulation of glycosphingolipids, mainly globotriaosylceramide, in lysosomes of cells of different tissues. Clinical presentation in classical Fabry hemizygous males may present during childhood or adolescence with acroparesthesia, anhidrosis, angiokeratoma, cornea verticillata, gastrointestinal symptoms, and microalbuminuria. At a later age, progressive renal failure, hypertrophic cardiomyopathy and cerebrovascular disease can occur, reducing life expectancy  (OMIM 301500) is an X‐linked disorder of glycosphingolipid catabolism caused by a deficiency of the lysosomal enzyme alpha‐galactosidase A  and with the Helsinki Declaration of 1975, as revised in 2013. All patients provided written informed consent to participate in this study.3GLA activity determination was carried out on dried blood filter paper or leukocytes, according to the method of Chamoles, Blanco, and Gaggioli (The Gaggioli  and descGaggioli .4GLA gene (NG_007119.1) was done using DNA isolated from EDTA blood samples. Polymerase chain reaction amplification of specific exon by the use of specific primers was carried out. The amplicons were purified and then sequenced in both directions in a DNA Sequencing device .Mutation analysis of In the period we have worked with 36 different Fabry families. Each family has a different mutation. Type of mutation detected is represented in Table The first part of the work consisted of analyzing if the mutation is inherited or de novo. In 5 of the families (14%), the mutation was de novo. In this 5 families, pedigree analysis was carried only with the descendants of the index case. In only 1 family (N° 19) there were descendants, we studied 3 daughters from the index case who were heterozygotes.So, pedigree analysis was carried out for full in 31 families in which the mutation was inherited by the index case. In the work period, we have tested 1,462 relatives, and 501 were diagnosed FD (Table The use of a targeted genetic approach versus at random analysis. Pedigree analysis in this work was done generation by generation in a stepwise mode, analyzing each time the risk of each patient of having inherited the mutation. We have analyzed only at risk cases. In this way we avoided testing relatives who have no risk at all such as children of normal parents or sons from affected males.Pedigree tree: The social worker in the pedigree analysis team had a central role. Her work is to contact the relatives who have a greater knowledge of the family. It let us to build a more complete and expanded tree.Distances: we could contact relatives living far away from the index case in our vast country. For example, in family 4, there were relatives living 3,000 kilometers far away. In other families, there are relatives living in 3 or more different provinces.Assistance to the index case physician: In most of the cases, physicians of the index cases are nephrologists or cardiologists. They do not have experience in genetic diseases and how to carry out pedigree analysis and genetic counseling. Moreover, generally, they do not have enough time in their consultant to work with the family. The assistance of the pedigree team, who works in close contact with him, gives him the possibility to have the whole family diagnosed.We obtained a high proportion of positive cases detection. The reasons are numerous:There is no vast information on pedigree analysis in FD. Few experiences of pedigree analysis are reported in literature, with variable ratios between 1:2 and 1:13. In this work, to our knowledge we are reporting the highest ratio of pedigree analysis for FD of 1:15. The ratio of 1:13 was reported in a work with only 2 families (Maron et al., In conclusion, the targeted family screening approach is successful to detect undiagnose Fabry patients. By this approach, the highest ratio ever reported of 1:15 was obtained.PR and MF had received grants and consultant fees from Shire. RN, RF and RC had no conflict of interest. BM and YC are employees of Shire.