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Objective: The objective of this study was to develop a nomogram to predict long-term facial nerve (FN) function after vestibular schwannoma (VS) resection., Methods: A retrospective cohort study of two tertiary academic skull base referral centers was performed. Consecutive adults > 18 years of age with sporadic unilateral VS who underwent resection between September 2016 and May 2021 were included. FN function in the immediate postoperative period and at the most recent evaluation was measured., Results: A total of 306 patients (mean age 49 years, 63% female) were included, with a mean follow-up of 18 months. The mean maximum tumor diameter was 19 mm (range 1-50 mm), and 80 (26.1%) tumors were > 25 mm. Overall, 85% of patients showed good immediate postoperative FN function (House-Brackmann [HB] grade I or II) and 89% maintained good FN function at > 12 months of follow-up. An intraoperative FN electromyographic (EMG) response ≥ 100 µV to 0.05 mA of stimulation (OR 18.6, p < 0.001) was the strongest predictor of good HB grade in the immediate postoperative period. EMG response ≥ 100 µV (OR 5.70, p < 0.001), tumor size ≤ 25 mm (OR 3.09, p < 0.05), and better immediate postoperative HB grade (OR 1.48, p = 0.005) predicted good long-term FN function on multivariable analysis. A point-of-care nomogram based on these data predicted long-term FN function with a sensitivity of 89% and specificity of 69%., Conclusions: Better immediate postoperative HB grade, intraoperative FN EMG response ≥ 100 µV, and tumor size ≤ 25 mm strongly predicted good long-term FN function after VS resection. A point-of-care nomogram based on these variables could serve as a useful tool for postoperative counseling and prognosis of long-term FN recovery.

Intrinsically stretchable electronics with skin-like mechanical properties have been identified as a promising platform for emerging applications ranging from continuous physiological monitoring to real-time analysis of health conditions, to closed-loop delivery of autonomous medical treatment 1-7 . However, current technologies could only reach electrical performance at amorphous-silicon level (that is, charge-carrier mobility of about 1 cm 2  V -1  s -1 ), low integration scale (for example, 54 transistors per circuit) and limited functionalities 8-11 . Here we report high-density, intrinsically stretchable transistors and integrated circuits with high driving ability, high operation speed and large-scale integration. They were enabled by a combination of innovations in materials, fabrication process design, device engineering and circuit design. Our intrinsically stretchable transistors exhibit an average field-effect mobility of more than 20 cm 2  V -1  s -1 under 100% strain, a device density of 100,000 transistors per cm 2 , including interconnects and a high drive current of around 2 μA μm -1 at a supply voltage of 5 V. Notably, these achieved parameters are on par with state-of-the-art flexible transistors based on metal-oxide, carbon nanotube and polycrystalline silicon materials on plastic substrates 12-14 . Furthermore, we realize a large-scale integrated circuit with more than 1,000 transistors and a stage-switching frequency greater than 1 MHz, for the first time, to our knowledge, in intrinsically stretchable electronics. Moreover, we demonstrate a high-throughput braille recognition system that surpasses human skin sensing ability, enabled by an active-matrix tactile sensor array with a record-high density of 2,500 units per cm 2 , and a light-emitting diode display with a high refreshing speed of 60 Hz and excellent mechanical robustness. The above advancements in device performance have substantially enhanced the abilities of skin-like electronics., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: The influence of apnea- and hypopnea-predominance on hypoglossal nerve stimulation therapy outcomes (HGNS) is still poorly defined. We assessed the significance of apnea- and hypopnea-predominance in HGNS outcomes., Study Design: Case series with chart review., Setting: Single-institution tertiary care center., Methods: A total of 216 subjects were included, all of which had undergone drug-induced sleep endoscopy (DISE) and HGNS implantation. Demographic and polysomnographic data were collected. The 4% apnea-hypopnea criteria were used to calculate apnea-hypopnea index (AHI). Central apneas were omitted. Univariate logistic and linear regression were used to study the association between these data and apnea-predominance and hypopnea-predominance. Kruskal-Wallis rank sum test was used to compare medians between groups for DISE collapse patterns., Results: Sixty-three patients were apnea-predominant, and 153 patients were hypopnea-predominant. These 2 groups were similar demographically (p > .20). There was no significant difference in HGNS outcomes between the groups assessed using Sher20 criteria at the 1-year mark using all-night, single-setting polysomnography or home sleep studies. Apnea index (AI)/AHI and reduction in AHI from preoperative to titration were significantly associated (p = .046). The median preoperative hypopnea index was significantly lower (p = .033) in subjects with no oropharyngeal collapse than patients with partial or complete oropharyngeal collapse. There were no significant relationships between AI/AHI and the different degrees of collapse at the velopharynx, oropharynx, tongue base, or epiglottis., Conclusions: In line with CPAP, tonsillectomy, and mandibular advancement therapy studies, we found there was largely no significant difference in DISE anatomy or in HGNS treatment outcomes between apnea- and hypopnea-predominant individuals., (© 2023 The Authors. Otolaryngology-Head and Neck Surgery published by Wiley Periodicals LLC on behalf of American Academy of Otolaryngology-Head and Neck Surgery Foundation.)

Metal-octaaminophthalocyanine (MOAPc)-based 2D conductive metal-organic frameworks (cMOFs) have shown great potential in several applications, including sensing, energy storage, and electrocatalysis, due to their bimetallic characteristics. Here, we report a detailed metal substitution study on a family of isostructural cMOFs with Co 2+ , Ni 2+ , and Cu 2+ as both the metal nodes and the metal centers in the MOAPc ligands. We observed that different metal nodes had variations in the reaction kinetics, particle sizes, and crystallinities. Importantly, the electronic structure and conductivity were found to be dependent on both types of metal sites in the 2D cMOFs. Ni-NiOAPc was found to be the most conductive one among the nine possible combinations with a conductivity of 54 ± 4.8 mS/cm. DFT calculations revealed that monolayer Ni-NiOAPc has neither the smallest bandgap nor the highest charge carrier mobility. Hence its highest conductivity stems from its high crystallinity. Collectively, these results provide structure property relationships for MOAPc-based cMOFs with amino coordination units.

Objective: To examine if age, sex, body mass index (BMI), neck circumference, or apnea-hypopnea index can predict whether a patient will demonstrate velopharyngeal complete circumferential (CC) collapse on drug-induced sleep endoscopy (DISE)., Study Design: Single-center retrospective review at The Ohio State Wexner Medical Center of 289 patients between March 2014 and June 2020., Setting: Quaternary care hospital., Methods: Patient characteristic and DISE information was extracted from charts and summarized with mean and standard deviation for continuous variables and count and percentage for categorical. CC collapse and patient characteristic associations were explored: 2-sample t test for continuous and chi-square test for categorical. Classification and regression tree (CART) analysis with 3-fold cross-validation was employed to search for the best CC collapse predictors., Results: Male and female BMI and female neck circumference were correlated to velopharyngeal CC collapse, with BMI more strongly correlated. CART analysis for males showed that a BMI ≤34.8 kg/m 2 is associated with an 89.4% chance of not demonstrating velopharyngeal CC collapse vs 48% for BMI >34.8 (area under the curve [AUC] = 0.705; AUC >0.7 is acceptable). For females, the CART analysis showed that a BMI ≤36.4 is associated with a 98.4% of not demonstrating velopharyngeal CC collapse vs 30.8% for BMI >36.4 (AUC = 0.73). For females, a neck circumference ≤38.05 cm is associated with a 100% chance of not demonstrating velopharyngeal CC collapse vs 18.4% for >38.05 cm (AUC = 0.72)., Conclusion: The BMI values for males and females and the female neck circumference values established by the CART model may accurately predict DISE anatomy and possible candidacy for hypoglossal nerve stimulation.

Background: There is increasing evidence that students are completing medical school with insufficient surgical education. Near-peer tutoring and flipped classroom formatting may be used to enhance learning while simultaneously relieving faculty burden of teaching. Here, we qualitatively evaluate a 3-month course that integrates the use of near-peer teaching and flipped classroom formatting, with the goal of increasing first-year medical students' self-perceived confidence in performing basic sutures and knot-ties as well as interest in surgery., Methods: Twenty-one first-year medical students participated in a suturing and knot-tying course led by senior medical students. The course consisted of 2-h sessions held every 2 weeks for a total of five sessions. Students were sent publicly available videos prior to each session by which to learn the upcoming techniques and received live feedback from instructors during sessions. Questionnaires were completed pre-course and post-course., Results: Compared to pre-course ratings, post-course ratings of self-perceived confidence to perform various knot-ties and sutures all increased significantly ( p  < 0.05). All students stated that the course strengthened their desire to pursue a career in surgery. Student feedback of the course was overall positive., Conclusions: Near-peer teaching can be used in conjunction with flipped classroom to increase first-year medical students' self-perceived confidence in surgical suturing and knot-tying as well as interest in surgery. This curriculum may serve as an outline for student-led courses at other institutions., Competing Interests: Conflict of InterestThe authors declare no competing interests., (© The Author(s) under exclusive licence to International Association of Medical Science Educators 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

The nonstructural protein 1 (NS1) of several flaviviruses, including West Nile, dengue, and yellow fever viruses, is capable of inducing variable degrees of protection against flavivirus infection in animal models. However, the immunogenicity of NS1 protein of Zika virus (ZIKV) is less understood. Here, we determined the efficacy of ZIKV NS1-based vaccine candidates using two delivery platforms, methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV) and a DNA vaccine. We first show that expression of ZIKV NS1 could be significantly enhanced by optimizing the signal peptide. A single dose of mtdVSV-NS1-based vaccine or two doses of DNA vaccine induced high levels of NS1-specfic antibody and T cell immune responses but provided only partial protection against ZIKV viremia in BALB/c mice. In Ifnar1 -/- mice, neither NS1-based vaccine provided protection against a lethal high dose (10 5 PFU) ZIKV challenge, but mtdVSV-NS1-based vaccine prevented deaths from a low dose (10 3 PFU) challenge, though they experienced viremia and body weight loss. We conclude that ZIKV NS1 alone conferred substantial, but not complete, protection against ZIKV infection. Nevertheless, these results highlight the value of ZIKV NS1 for vaccine development. IMPORTANCE Most Zika virus (ZIKV) vaccine research has focused on the E or prM-E proteins and the induction of high levels of neutralizing antibodies. However, these ZIKV neutralizing antibodies cross-react with other flaviviruses, which may aggravate the disease via an antibody-dependent enhancement (ADE) mechanism. ZIKV NS1 protein may be an alternative antigen for vaccine development, since antibodies to NS1 do not bind to the virion, thereby eliminating the risk of ADE. Here, we show that recombinant VSV and DNA vaccines expressing NS1, alone, confer partial protection against ZIKV infection in both immunocompetent and immunodeficient mice, highlighting the value of NS1 as a potential vaccine candidate., (Copyright © 2020 American Society for Microbiology.)

Platelet-leukocyte aggregates (PLAs) are associated with increased thrombosis risk. The influence of PLA formation is especially important for cancer patients, since thrombosis accounts for approximately 10% of cancer-associated deaths. Our objective was to characterize and quantify PLAs in whole blood samples from lung cancer patients compared to healthy volunteers with the intent to analyze PLA formation in the context of lung cancer-associated thrombosis. Consenting lung cancer patients (57) and healthy volunteers (56) were enrolled at the Dana Cancer Center at the University of Toledo Health Science Campus. Peripheral blood samples were analyzed by flow cytometry. Patient medical history was reviewed through electronic medical records. Most importantly, we found lung cancer patients to have higher percentages of platelet-T cell aggregates (PTCAs) than healthy volunteers among both CD4+ T lymphocyte and CD8+ T lymphocyte populations. Our findings demonstrate that characterization of PTCAs may have clinical utility in differentiating lung cancer patients from healthy volunteers and stratifying lung cancer patients by history of thrombosis., Competing Interests: The authors have declared that no competing interests exist.

Semiconducting single-walled carbon nanotubes (swCNTs) are a promising class of materials for emerging applications. In particular, they are demonstrated to possess excellent biosensing capabilities, and are poised to address existing challenges in sensor reliability, sensitivity, and selectivity. This work focuses on swCNT field-effect transistors (FETs) employing rubbery double-layer capacitive dielectric poly(vinylidene fluoride-co-hexafluoropropylene). These devices exhibit small device-to-device variation as well as high current output at low voltages (<0.5 V), making them compatible with most physiological liquids. Using this platform, the swCNT devices are directly exposed to aqueous solutions containing different solutes to characterize their effects on FET current-voltage (FET I-V) characteristics. Clear deviation from ideal characteristics is observed when swCNTs are directly contacted by water. Such changes are attributed to strong interactions between water molecules and sp 2 -hybridized carbon structures. Selective response to Hg 2+ is discussed along with reversible pH effect using two distinct device geometries. Additionally, the influence of aqueous ammonium/ammonia in direct contact with the swCNTs is investigated. Understanding the FET I-V characteristics of low-voltage swCNT FETs may provide insights for future development of stable, reliable, and selective biosensor systems., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Supramolecular polymer sorting is a promising approach to separating single-walled carbon nanotubes (CNTs) by electronic type. Unlike conjugated polymers, they can be easily removed from the CNTs after sorting by breaking the supramolecular bonds, allowing for isolation of electronically pristine CNTs as well as facile recycling of the sorting polymer. However, little is understood about how supramolecular polymer properties affect CNT sorting. Herein, chain stoppers are used to engineer the conformation of a supramolecular sorting polymer, thereby elucidating the relationship between sorting efficacy and polymer conformation. Through NMR and UV-vis spectroscopy, small-angle X-ray scattering (SAXS), and thermodynamic modeling, it is shown that this supramolecular polymer exhibits ring-chain equilibrium, and that this equilibrium can be skewed toward chains by the addition of chain stoppers. Furthermore, by controlling the stopper-monomer ratio, the sorting yield can be doubled from 7% to 14% without compromising the semiconducting purity (>99%) or properties of sorted CNTs., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Internal N 6 -methyladenosine (m 6 A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m 6 A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m 6 A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m 6 A methylated and that viral m 6 A methylation promotes HMPV replication and gene expression. Inactivating m 6 A addition sites with synonymous mutations or demethylase resulted in m 6 A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m 6 A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m 6 A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m 6 A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m 6 A can serve as a target to attenuate HMPV for vaccine purposes.

Deoxynivalenol (DON) is one of the mycotoxins most frequently encountering in cereal-based foods throughout the world. Saccharomyces cerevisiae was used to alleviate porcine jejunal epithelia cell (IPEC-J2) injury induced by DON in this study. The results indicated that cell viability and proliferation rates were significantly decreased when DON concentrations were increased from 0 to 64 µM after 24 h incubation (p < 0.05). The longer incubation time and higher DON concentrations would cause more serious effects on cell viability. S. cerevisiae could significantly degrade DON and decrease lactic dehydrogenase (LDH) release in the cells induced by DON (p < 0.05). DON (4 µM) could increase necrotic and apoptotic cell rates as well as decrease viable cell rates, compared with the control group (p < 0.05). However, S. cerevisiae addition in the DON group could decrease necrotic, late apoptotic and early apoptotic cell rates by 38.05%, 46.37% and 44.78% respectively, increase viable cell rates by 2.35%, compared with the single DON group (p < 0.05). In addition, S. cerevisiae addition could up-regulate mRNA abundances of IL-6, IL-8 and IL-10 in IPEC-J2 cells (p < 0.05), but down-regulate mRNA abundances of tight junction proteins (TJP-1) and occludin by 36.13% and 50.18% at 1 µM of DON (p < 0.05). It could be concluded that S. cerevisiae was able to alleviate IPEC-J2 cell damage exposed to DON.

Wearable and skin electronics benefit from mechanically soft and stretchable materials to conform to curved and dynamic surfaces, thereby enabling seamless integration with the human body. However, such materials are challenging to process using traditional microelectronics techniques. Here, stretchable transistor arrays are patterned exclusively from solution by inkjet printing of polymers and carbon nanotubes. The additive, non-contact and maskless nature of inkjet printing provides a simple, inexpensive and scalable route for stacking and patterning these chemically-sensitive materials over large areas. The transistors, which are stable at ambient conditions, display mobilities as high as 30 cm 2  V -1 s -1 and currents per channel width of 0.2 mA cm -1 at operation voltages as low as 1 V, owing to the ionic character of their printed gate dielectric. Furthermore, these transistors with double-layer capacitive dielectric can mimic the synaptic behavior of neurons, making them interesting for conformal brain-machine interfaces and other wearable bioelectronics.

Human noroviruses (HuNoVs) are responsible for more than 95% of the non-bacterial acute gastroenteritis epidemics in the world. The CDC estimates that every year 21 million individuals suffer from HuNoV-induced gastroenteritis in the United States. Currently, there is no FDA-approved vaccine for HuNoVs. Development of an effective vaccine has been hampered by the lack of an efficient cell culture system for HuNoVs and a suitable small animal model for pathogenesis study. In this study, we developed lactic acid bacteria (LAB) as a vector to deliver HuNoV antigen. A LAB strain ( Lactococcus lactis ) carrying VP1 gene of a HuNoV GII.4 virus (LAB-VP1) was constructed. It was found that HuNoV VP1 protein was highly expressed by LAB vector and was secreted into media supernatants. To test whether LAB-based HuNoV vaccine candidate is immunogenic, 4-day-old gnotobiotic piglets were orally inoculated with various doses of LAB-VP1. It was found that LABs were persistent in the small intestine of piglets and shed in pig feces for at least 25 days post inoculation. LAB DNA and VP1 were detected in mesenteric lymph nodes and spleen tissue in LAB-VP1 inoculated groups. HuNoV-specific IgG and IgA were detectable in serum and feces respectively at day 13 post-inoculation, and further increased at later time points. After being challenged with HuNoV GII.4 strain, a large amount of HuNoV antigens were observed in the duodenum, jejunum, and ileum sections of the intestine in the LAB control group. In contrast, significantly less, or no, HuNoV antigens were detected in the LAB-VP1 immunized groups. Collectively, these results demonstrate that a LAB-based HuNoV vaccine induces protective immunity in gnotobiotic piglets.

In this study, the complete mitochondrial genome of band-rumped Storm-petrel, Hydrobates castro, was determined through sequencing of PCR fragments. The complete mitochondrial genome of H. castro was 17,065 bp in length and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The overall nucleotide composition is: 30.4% A, 24.5% T, 30.9% C, and 14.2% G, with a total G + C content of 45.1%. By phylogenetic analysis using Bayes method, H. castro showed the closest relationship with the white-faced storm petrel ( Pelagodroma marina )., Competing Interests: No potential conflict of interest was reported by the authors., (© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

High-throughput and low-temperature processing of high-performance nanomaterial inks is an important technical challenge for large-area, flexible printed electronics. In this report, we demonstrate nitrocellulose as an exothermic binder for photonic annealing of conductive graphene inks, leveraging the rapid decomposition kinetics and built-in energy of nitrocellulose to enable versatile process integration. This strategy results in superlative electrical properties that are comparable to extended thermal annealing at 350 °C, using a pulsed light process that is compatible with thermally sensitive substrates. The resulting porous microstructure and broad liquid-phase patterning compatibility are exploited for printed graphene microsupercapacitors on paper-based substrates.

Selective extraction of semiconducting carbon nanotubes is a key step in the production of high-performance, solution-processed electronics. Here, we describe the ability of a supramolecular sorting polymer to selectively disperse semiconducting carbon nanotubes from five commercial sources with diameters ranging from 0.7 to 2.2 nm. The sorting purity of the largest-diameter nanotubes (1.4 to 2.2 nm; from Tuball) was confirmed by short channel measurements to be 97.5%. Removing the sorting polymer by acid-induced disassembly increased the transistor mobility by 94 and 24% for medium-diameter and large-diameter carbon nanotubes, respectively. Among the tested single-walled nanotube sources, the highest transistor performance of 61 cm 2 /V·s and on/off ratio >10 4 were realized with arc discharge carbon nanotubes with a diameter range from 1.2 to 1.7 nm. The length and quality of nanotubes sorted from different sources is compared using measurements from atomic force microscopy and Raman spectroscopy. The transistor mobility is found to correlate with the G/D ratio extracted from the Raman spectra.

Understanding the law of soil microbial degradation of nitrogen is of great practical significance for the remediation of soil nitrogen pollution. From fluvo-aquic soil of Hebei plain, 8 kinds of bacteria,including nitrification bacteria, ammonification bacteria, heterotrophic nitrification to aerobic denitrification bacteria were isolated and analyzed by 16S rDNA identification, and the most suitable strains were selected to prepare bacterial agents with optimized carrier materials. The effects of the screened strains of bacteria or antagonistic fungus to Chinese cabbage in the process of drip irrigation on nitrogen and PLFA (Phospholipid Fatty Acids) values in the soils were discussed. The results showed that by using diatomite as a carrier, the ammonia nitrogen degradation rate was 38% and the synthetic rate of nitrate was 205% with ammonibacteria, the synthetic rate of ammonia nitrogen was 1711% with ammonibacteria, the degradation rate of nitrate nitrogen was 367% with heterotrophic nitrification to aerobic denitrification bacteria. In drip irrigation experiment, total nitrogen and ammonia nitrogen in soil were better degraded and nitrification was stronger in the soil, meanwhile, PLFA in the soil increased and content of microbes reached the peak in shorter time, the soil biological environment was better after irrigation with screened bacteria.

Intense pulsed light (IPL) annealing of graphene inks is demonstrated for rapid post-processing of inkjet-printed patterns on various substrates. A conductivity of ≈25,000 S m(-1) is achieved following a single printing pass using a concentrated ink containing 20 mg mL(-1) graphene, establishing this strategy as a practical and effective approach for the versatile and high-performance integration of graphene in printed and flexible electronics., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To study the clinical features, diagnosis and therapy of hydroa vacciniforme-like cutaneous T cell lymphoma., Methods: The clinical presentations and the findings of laboratory examinations and skin biopsy of affected tissue in a child with hydroa vacciniforme-like cutaneous T cell lymphoma were retrospectively reviewed., Results: The child manifested as rash, fever and lymph node intumesce. Rash was pantomorphia, including edematous erythema, vesicles, crusts, necrosis and depressed scar, and it was mild in winter and severe in summer, mainly involving in the face and extremities. Epstein-Barre virus (EBV)-IgM was positive. Histopathological findings revealed focal lymphocyte invasion in subcutaneous panniculus adiposus, mainly surrounding the blood vessels. Immunohistochemistry showed CD3 (+), CD43 (+), CD20 (-), pax-5 (-), TIA (+), CD5 (+), CD8 (+), Granmye (+) and CD4 (-). The clinical symptoms were improved after glucocorticoid treatment in this child., Conclusions: Hydroa vacciniforme-like cutaneous T cell lymphoma has special clinical manifestations. This disorder may be definitely diagnosed by skin biopsy of affected tissue and immunohistochemistry assay. Glucocorticoid treatment is effective. EBV infection may be related to the development of this disorder.

X-linked spondyloepiphyseal dysplasia tarda (SEDL) is a rare osteochondrodysplasia caused by the mutation of SEDL gene, which mainly involves vertebral bodies and hips. To explore the effect of the novel splicing mutation (IVS2 -2A-->C) of SEDL gene on mRNA processing in a large Chinese family with X-linked spondyloepiphyseal dysplasia tarda and to elucidate the molecular base of SEDL, total RNA was isolated from EDTA blood samples of patients and controls. RT-PCR was performed on total RNA. cDNA was analyzed by bi-directionally direct sequencing of PCR products and Polyacrylamide gel electrophoresis (PAGE). Sequencing analysis revealed that there were two kinds of cDNA in patients. One is that exon 2 directly spliced exon 4, that is, exon 3 absence from the mature mRNA; and the other is that exon 1 directly spliced exon 4, meaning both exon 2 and 3 being spliced out completely. Meanwhile one kind of cDNA that exon 1 directly spliced exon 3 was found in normal controls. By PAGE, RT-PCR amplified products, 679bp and 537bp, were detected in normal controls, while 567bp and 425bp fragments were found in affected individuals. Our data show that the mutation of the splice-acceptor site in intron 2 causes exon 3 entirely exclusion from the mature RNA transcripts in affected individuals. As the translation start site of the SEDL gene locates on exon 3, the splicing defect causes affected individuals failure to produce sedlin, which elucidates the causative role of SEDL gene in the pathogenesis of SEDL. The absence of exon 2 indicates that there is alternative splicing in SEDL gene. The alternative splicing was also found in normal controls, which demonstrated that the alternative splicing might not be related to the phenotype of SEDL. Because the alternative splicing of exon 2 occurred in the 5'UTR, it is not clear whether it affects the gene expression.

Objective: X linked spondyloepiphyseal dysplasia tarda (SEDL) is heritable osteochondrondysplasia characterized in affected males by disproportional short stature with short neck and trunk resulting from a growth defect of the vertebral bodies, accompanied by barrel chest and degenerative osteoarthropathy of hip joints. This progressive skeletal dysplasia is caused by the SEDL gene located approximately 100 kb centromeric of DXS16 at Xp22. The disorder usually manifests in late childhood without systemic complications, and generally female carriers of SEDL are asymptomatic. So the diagnosis of potential carriers and presymptomatic patients is almost impossible. This study aimed to establish methods of gene diagnosis for finding out potential carriers and presymptomatic patients., Methods: The blood samples were collected from 21 individuals in a large Chinese pedigree with SEDL. Microsatellite marker DXS16 was selected for linkage analysis. In order to confirm the allele of DXS16 linked to the pathogenic SEDL gene, polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) were used to examine the variability of the lengths of DXS16, and linkage analysis was performed for the diagnosis of potential carriers and presymptomatic patients. Then the pathogenic mutation of the SEDL gene in the family was identified by bi-directionally direct sequencing of PCR products amplified for each of the four coding exons as well as their exon/intron boundaries. The potential carriers and presymptomatic patients were also diagnosed in this way., Results: Six young individuals (IV(14), IV(19), IV(21), IV(23), V(4), V(7))who wanted to know whether they were carriers or presymptomatic patients were diagnosed by linkage analysis. Four females of them (IV(14), IV(19), IV(21), V(7)) were determined being carriers because they carry the allele of DXS16 which links the pathogenic SEDL gene, and the other two (IV(23), V(4)) being normal individuals for their alleles of DXS16 linked with wild SEDL gene. DNA sequencing identified that the pathogenic mutation of SEDL gene in the family, which was a nucleotide substitution of the splice-acceptor site in intron 2, IVS2 -2 A-->C. This is a novel mutation in the SEDL gene. Four female individuals (IV(14), IV(19), IV(21), V(7)) carried the mutation; individuals IV(23) and V(4) carried the wild SEDL gene. The results of diagnosis of linkage analysis coincide completely with that of DNA sequencing., Conclusion: Linkage analysis is a simple, rapid and inexpensive gene diagnosis method for SEDL and its accuracy was the same as DNA sequencing. Each of linkage analysis and DNA sequencing can be used to diagnose SEDL, which is very helpful for finding potential carriers and presymptomatic patients.

Objective: To identify the mutation of spondyloepiphyseal dysplasia tarda (SEDL) gene in a large Chinese family with X-linked spondyloepiphyseal dysplasia tarda and to make a discussion on the pathogenesis of SEDL at the molecular level., Methods: In two patients, four exons comprising the SEDL open reading frame as well as their exon/intron boundaries were analyzed by bi-directional direct sequencing of PCR products. The sequencing results were compared against the normal sequences in GenBank to find the mutation. Then the mutation was identified in other members of the family., Results: A nucleotide substitution of the splice acceptor in SEDL intron 2, IVS2 -2A-->C,was detected in two affected individuals (IV(15) V(3)) in the Chinese family with SEDL, but no sequence change occurring on exons 3-6 was detected. The transversion was also identified in four heterozygous carriers. The mutation was not found in two unaffected male individuals and fifteen normal controls. Furthermore, four potential carriers were identified in the family., Conclusion: The mutation IVS2 -2A-->C of SEDL gene was firstly determined in the world. The change of the splice acceptor in SEDL intron 2 may cause skipping of exon 3 which is responsible for the disease. Molecular diagnosis can be made by detecting the mutation.