Summary Members of the tetraspanin superfamily of proteins are implicated in a variety of complex cell processes including cell fusion. However, the contribution of individual tetraspanins to these processes has proved difficult to define. Here we report the use of recombinant extracellular regions of tetraspanins to investigate the role of specific members of this family in the fusion of monocytes to form multinucleated giant cells (MGC). In contrast to their positive requirement in sperm–egg fusion, previous studies using antibodies and knockout mice have indicated a negative regulatory role for tetraspanins CD9 and CD81 in this process. In an in vitro model of fusion using human monocytes, we have confirmed observations that antibodies to CD9 and CD81 enhance MGC formation; however, in contrast to previous investigations, we found that all members of a panel of antibodies to CD63 inhibited fusion. Moreover, recombinant proteins corresponding to the large extracellular domains (EC2s) of CD63 and CD9 inhibited MGC formation, whereas the EC2s of CD81 and CD151 had no effect. The potent inhibition of fusion and binding of labelled CD63 EC2 to monocytes under fusogenic conditions suggest a direct interaction with a membrane component required for fusion. Our findings indicate that the tetraspanins CD9, CD63 and CD81 are all involved in MGC formation, but play distinct roles.

Objective: To develop a screening test for fetal trisomy 13, 18, and 21 using cell-free DNA from maternal blood with an automated workflow using the Ion Proton sequencing platform. Methods: An automated next-generation sequencing workflow was developed using the Ion Proton sequencing platform and software developed for straightforward bioinformatic analysis. An algorithm was developed using 239 samples to determine the likelihood of trisomy, using DNA fragment counts and a fetal fraction validity check; the results were compared with those from invasive diagnostic procedures. A further 111 samples were used to assess the tests' sensitivity (detection rate) and specificity (1 minus false-positive rate). Results: The 110 of a possible 111 valid samples used to verify the IONA® test gave 100% sensitivity and specificity, compared with invasive diagnostic procedures; one failed the fetal fraction validity check giving a sample failure rate of 0.29% across all 350 analysed samples. Conclusion: The data indicate that the IONA test provides a robust, accurate automated workflow suitable for use on maternal blood samples to screen for trisomies 13, 18, and 21. The test has the potential to reduce the number of unnecessary invasive procedures performed and facilitate testing by screening laboratories.

BACKGROUND: In singleton pregnancies, studies investigating cellfree DNA in maternal blood have consistently reported high detection rate and low false-positive rate for the 3 common fetal trisomies (trisomies 21, 18, and 13). The potential advantages of noninvasive prenatal testing in twin pregnancies are even greater than in singletons, in particular lower need for invasive testing and consequent fetal loss rate. However, several organizations do not recommend cell-free DNA in twin pregnancies and call for larger prospective studies. OBJECTIVE: In response to this, we undertook a large prospective multicenter study to establish the screening performance of cell-free DNA for the 3 common trisomies in twin pregnancies. Moreover, we combined our data with that reported in published studies to obtain the best estimate of screening performance. STUDY DESIGN: This was a prospective multicenter blinded study evaluating the screening performance of cell-free DNA in maternal plasma for the detection of fetal trisomies in twin pregnancies. The study took place in 6 fetal medicine centers in England, United Kingdom. The primary outcome was the screening performance and test failure rate of cell-free DNA using next generation sequencing (the IONA test). Maternal blood was taken at the time of (or after) a conventional screening test. Data were collected at enrolment, at any relevant invasive testing throughout pregnancy, and after delivery until the time of hospital discharge. Prospective detailed outcome ascertainment was undertaken on all newborns. The study was undertaken and reported according to the Standards for Reporting of Diagnostic Accuracy Studies. A pooled analysis was also undertaken using our data and those in the studies identified by a literature search (MEDLINE, Embase, CENTRAL, Cochrane Library, and ClinicalTrials.gov) on June 6, 2020. RESULTS: A total of 1003 women with twin pregnancies were recruited, and complete data with follow-up and reference data were available for 961 (95.8%); 276 were monochorionic and 685 were dichorionic. The failure rate was 0.31%. The mean fetal fraction was 12.2% (range, 3%e36%); all 9 samples with a 3% fetal fraction provided a valid result. There were no false-positive or false-negative results for trisomy 21 or trisomy 13, whereas there was 1 false-negative and 1 false-positive result for trisomy 18. The IONA test had a detection rate of 100% for trisomy 21 (n¼13; 95% confidence interval, 75e100), 0% for trisomy 18 (n¼1; 95% confidence interval, 0e98), and 100% for trisomy 13 (n¼1; 95% confidence interval, 3e100). The corresponding false-positive rates were 0% (95% confidence interval, 0e0.39), 0.10% (95% confidence interval, 0e0.58), and 0% (95% confidence interval, 0e0.39), respectively. By combining data from our study with the 11 studies identified by literature search, the detection rate for trisomy 21 was 95% (n¼74; 95% confidence interval, 90e99) and the false-positive rate was 0.09% (n¼5598; 95% confidence interval, 0.03e0.19). The corresponding values for tri...

ObjectiveTo evaluate the clinical accuracy of the IONA® test for aneuploidy screening.MethodsThis was a multicenter blinded study in which plasma samples from pregnant women at increased risk of trisomy 21 underwent cell‐free DNA analysis utilizing the IONA test. For each sample, the IONA software generated a likelihood ratio and a maternal age‐adjusted probability risk score for trisomies 21, 18 and 13. All results from the IONA test were compared against accepted diagnostic karyotyping.ResultsA total of 442 maternal samples were obtained, of which 437 had test results available for analysis and assessment of clinical accuracy. The IONA test had a detection rate of 100% for trisomies 21 (n = 43; 95% CI, 87.98–100%), 18 (n = 10; 95% CI, 58.72–100%) and 13 (n = 5; 95% CI, 35.88–100%) with cut‐offs applied to likelihood ratio (cut‐off > 1 considered high risk for trisomy) and probability risk score incorporating adjustment for maternal age (cut‐off ≥ 1/150 considered high risk for trisomy). The false‐positive rate (FPR) was 0% for trisomies 18 and 13 with both analysis outputs. For trisomy 21, a FPR of 0.3% was observed for the likelihood ratio, but became 0% with adjustment for maternal age.ConclusionThis study indicates that the IONA test is suitable for trisomy screening in a high‐risk screening population. The result‐interpretation feature of the IONA software should facilitate wider implementation, particularly in local laboratories, and should be a useful addition to the current screening methods for trisomies 21, 18 and 13. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

The Arabidopsis sensitive-to-freezing8 (sfr8) mutant exhibits reduced cell-wall (CW) fucose levels and compromised freezing tolerance. To examine whether CW fucosylation affects the response to desiccation also, we tested the effect of leaf excision in sfr8 and the allelic mutant mur1-1. Leaf water loss was strikingly higher than wild type in these, but not other, fucosylation mutants. We hypothesised that reduced fucosylation in guard cell (GC) walls might limit stomatal closure through altering mechanical properties. Multifrequency atomic force microscopy (AFM) measurements revealed a reduced elastic modulus (E’), representing reduced stiffness, in sfr8 GC walls. Interestingly, however, we discovered a compensatory mechanism whereby a concomitant reduction in the storage modulus (E’’) maintained a wild type viscoelastic time response (tau) in sfr8. Stomata in intact leaf discs of sfr8 responded normally to a closure stimulus, ABA, suggesting the time response may relate more to closure properties than stiffness does. sfr8 stomatal pore complexes were larger than wild type and GCs lacked a fully developed cuticular ledge, both potential contributors to the greater leaf water loss in sfr8. We present data that indicate fucosylation-dependent dimerisation of the CW pectic domain rhamnogalacturonan-II may be essential for normal cuticular ledge development and leaf water retention.