Evaluation of an internet-based speech-in-noise screening test for school-age children

Objective: To evaluate a Dutch online speech-in-noise screening test (in Dutch: “Kinderhoortest”) in normal-hearing school-age children. Sub-aims were to study test–retest reliability, and the effects of presentation type and age on test results. Design: An observational cross-sectional study at school. Speech reception thresholds (SRTs) were obtained through the online test in a training condition, and two test conditions: on a desktop computer and smartphone. The order of the test conditions was counterbalanced. Study sample: Ninety-four children participated (5–12 years), of which 75 children were normal-hearing (≤25?dB HL at 0.5?kHz,?≤20?dB HL at 1–4?kHz). Results: There was a significant effect for test order for the two test conditions (first or second test), but not for presentation type (desktop computer or smartphone) (repeated measures analyses, F(1,75)?=?12.48, p?F(1,75)?=?0.01, p?=?0.982). SRT significantly improved by age year (first test: 0.25?dB SNR, 95% CI: –0.43 to –0.08, p?=?0.004. Second test: 0.29?dB SNR, 95% CI: –0.46 to –0.11; p?=?0.002). Conclusions: The online test shows potential for routine-hearing screening of school-age children, and can be presented on either a desktop computer or smartphone. The test should be evaluated further in order to establish sensitivity and specificity for hearing loss in children.     

Temporal maturation of neutralizing antibodies in COVID-19 convalescent individuals improves potency and breadth to circulating SARS-CoV-2 variants

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Dendritic cell maturation in active immunotherapy strategies

Dendritic cells (DCs) loaded with tumour antigen have become the centrepiece of clinical trials testing active immunotherapy strategies. Important variables include the source of DCs, the choice of antigens, the method of antigen loading and the route and timing of administration. Recently, the requirement for and the method of, DC maturation have been receiving particular attention. This is due to observations from in vitro studies and animal models demonstrating that mature DCs induce more potent antigen-specific T-cells responses than immature DCs. Furthermore, preliminary observations in human studies suggest that immature DCs might actually downregulate antigen-specific T-cell responses but mature DCs may augment them. Current studies are addressing how to define DC maturation, whether the variety of methods for maturation result in DCs with similar T-cell stimulatory capacity, how to maintain the maturational status and whether maturation in vitro before immunisation, or in vivo, after immunisation, results in better DC function.     

Molecular cloning and identification of mouse epididymis-specific gene mHong1, the homologue of rat HongrES1

Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHong1. The mHong1 gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHong1 mRNA shows similarity with HongrES1 in the expression patterns: (i) specific expression in epididymal tissue, especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHong1 with high specificity and sensitivity was raised. Like HongrES1, the mHong1 protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHong1 protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHong1 is localized to the equatorial segment, which is different from that of HongrES1. The mHong1 protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHong1 is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHong1.     

Automated image analysis of placental villi and syncytial knots in histological sections

IntroductionDelayed villous maturation and accelerated villous maturation diagnosed in histologic sections are morphologic manifestations of pathophysiological conditions. The inter-observer agreement among pathologists in assessing these conditions is moderate at best. We investigated whether automated image analysis of placental villi and syncytial knots could improve standardization in diagnosing these conditions.MethodsPlacentas of antepartum fetal death at or near term were diagnosed as normal, delayed or accelerated villous maturation. Histologic sections of 5 cases per group were photographed at ×10 magnification. Automated image analysis of villi and syncytial knots was performed, using ImageJ public domain software. Analysis of hundreds of histologic images was carried out within minutes on a personal computer, using macro commands.ResultsCompared to normal placentas, villi from delayed maturation were larger and fewer, with fewer and smaller syncytial knots. Villi from accelerated maturation were smaller. The data were further analyzed according to horizontal placental zones and groups of villous size.DiscussionNormal placentas can be discriminated from placentas of delayed or accelerated villous maturation using automated image analysis. Automated image analysis of villi and syncytial knots is not equivalent to interpretation by the human eye. Each method has advantages and disadvantages in assessing the 2-dimensional histologic sections representing the complex, 3-dimensional villous tree. Image analysis of placentas provides quantitative data that might help in standardizing and grading of placentas for diagnostic and research purposes.     

Cystic partially differentiated nephroblastoma in an adult: a case imitating the process of normal nephrogenesis along with corresponding WT1 expression

Cystic partially differentiated nephroblastoma (CPDN) is extremely rare in adults. Only 2 cases have been documented in the English literature. Herein, we present a third case of CPDN with unique morphological and immunohistochemical features. A 45-year-old man had a multicystic right renal mass, with a maximum diameter of 3 cm on magnetic resonance imaging. Being unable to rule out malignancy, partial nephrectomy was performed. The surgically resected specimen contained a multicystic mass, 3 × 3 × 2.5 cm in size, without an expansile solid nodule. Histopathological examination revealed nephroblastomatous elements without identifiable blastema; transition from cap-mesenchyme-like cells to an immature glomerulus was observed and maturing tubules and a glomerulus were present. Despite the lack of a blastema, the diagnosis of CPDN was the most appropriate. Immunohistochemical WT1 expression imitated the pattern of ongoing normal nephrogenesis. Therefore, we believe that the blastema disappeared because of maturation.     

Major Hepatectomy Induces Phenotypic Changes in Circulating Dendritic Cells and Monocytes

Introduction  Patients undergoing major hepatectomy are at increased risk for post-operative morbidity and mortality, and changes in the phenotype of effector cells may predispose these patients to infectious sequelae. Methods  To better understand post-hepatectomy immune responses, peripheral blood from 15 hepatectomy patients was drawn immediately before and after liver resection and on post-operative days 1, 3, and 5. Circulating monocytes and dendritic cells were analyzed by flow cytometry for quantity, phenotype, activation status, human leukocyte antigen DR (HLA-DR) expression, and toll-like receptor-2 and -4 expression. Results  Major hepatectomy increased the numbers of activated CD16^bright blood monocytes and the percentage of activated dendritic cells, although monocyte HLA-DR expression was reduced. These results may represent both dysfunctional antigen presentation and pending anergy, as well as cellular priming of immune effector cells. Better understanding of the alterations in innate immunity induced by hepatectomy may identify strategies to reduce infectious outcomes.     

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as potential substrates for proteolytic maturation

The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intrahexameric and interhexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the oligomerization state of CA at lattice edges and found that CA forms partial hexamers. These structures reveal the network of interactions formed by CA-SP1 at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, partial hexamers exist at lattice edges, these are in a helix-coil dynamic equilibrium, and partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation.

The polyprotein Gag is the main structural component of HIV-1, consisting of the MA (matrix), CA (capsid), NC (nucleocapsid), and p6 domains as well as the spacer peptides SP1 and SP2 (1). Gag is produced in infected host cells and trafficked to the plasma membrane, where it assembles into a hexagonal lattice via its CA domain and recruits other viral proteins and the viral RNA genome (1, 2). Assembly of the curved Gag lattice is commensurate with membrane bending at the site of assembly, after which recruitment of Endosomal Sorting Complex Required for Transport III (ESCRT-III) components by the p6 domain of Gag induces membrane scission and release of the immature virus particle (2). The hexagonal Gag lattice accommodates curvature in the growing bud by incorporating vacancy defects (3). The activity of ESCRT-III is timed such that the final immature lattice is incomplete, giving rise to an additional large gap in the lattice, resulting in a truncated spherical shape (4–6).During or after budding, the viral protease is activated and cleaves this immature Gag lattice into its component domains, which leads to structural rearrangement within the virus particle (2). The released CA domains assemble to form a closed, conical capsid around the condensed ribonucleoprotein (RNP) complex of the mature virus (1, 7). Maturation is required for the virion to become infectious (1).Within the immature virus particle, the N-terminal domain of CA (CA_NTD) forms trimeric interactions linking three Gag hexamers while the C-terminal domain of CA (CA_CTD) forms dimeric interactions mediated by helix 9 of CA, linking two Gag hexamers together (8). The CA_CTD additionally forms intrahexamer interactions around the sixfold axis of the hexamer (8, 9). Amphipathic helices formed by the C-terminal residues of CA_CTD and the N-terminal residues of SP1 junction assemble into a six-helix bundle (6HB), thereby imposing hexagonal order on the CA domains, via classical knobs-in-holes packing mediated by exposed hydrophobic side chains, as also seen in coiled coils (9, 10). In combination, these relatively weak interactions give rise to a very dynamic, reversible assembly process that prevents the assembling lattice from becoming trapped in kinetically unfavorable states (11). This robust assembly behavior is consistent with icosahedral viruses (12–15). It is not surprising, therefore, that the energetics of Gag assembly are tightly controlled and highly dependent on scaffolding effects from the viral RNA and the membrane-interacting MA domain of Gag in order to ensure productive viral assembly (11, 16). Analysis of the diffusion pattern of fluorescently labeled Gag supports the notion that Gag is trafficked to the site of assembly as low-order multimers, although it is still unclear whether these are Gag dimers, trimers, or other multimeric forms of Gag (16, 17).The primary assembly unit of the Gag lattice remains largely unknown. We can identify two hypothetical ways in which the lattice could assemble. First, the lattice could grow by addition of Gag hexamers (or sets of six component monomers), such that the CA-SP1 junction is assembled within a hexameric 6HB at all positions in the lattice. In this case, interfaces between hexamers would be unoccupied at the edge of the lattice. From a purely energetic perspective, this appears most reasonable. Second, the lattice could form via addition of Gag dimers or Gag trimers (or equivalently from sets of either two or three component monomers). This would maintain, for example, the dimeric CA-CA interhexamer interactions but leave incomplete hexamers at the lattice edges, including unoccupied hexamer-forming interfaces along the CA-SP1 bundle. It additionally remains unclear whether the unoccupied Gag-Gag interfaces at the lattice edges are simply exposed, or whether they are stabilized by alternative conformations of individual domains or proteins, or by other binding partners. Understanding the structure of the edge of the immature Gag lattice therefore has implications for understanding the mechanism of virus assembly.Viral assembly, budding, and maturation are tightly linked, and disrupting the kinetics of any of these processes can give rise to defects in maturation and formation of noninfectious viral particles (1, 18, 19). The rate-limiting proteolytic cleavage site in the maturation process resides within the CA-SP1 6HB (20). Unfolding of the helical bundle is required to allow proteolytic cleavage to proceed (21–23), but the exact mechanism for protease access to this site is not known. The spatial localization of proteolytic processing within the context of the immature Gag lattice is relevant: Does the protease act on Gag within the lattice, or does it act on the edges of the Gag lattice, causing a cascade of lattice disruption? At the lattice edge, is the substrate for the protease with a 6HB or within an incomplete hexamer? Understanding the structure of the edge of the immature Gag lattice therefore has implications for understanding the mechanism of virus maturation.High-resolution immature Gag structures have previously been determined directly from purified viruses by cryo-electron tomography (cryo-ET) and subtomogram averaging (10). These structures represent an average hexamer within the immature lattice, with a full complement of six Gag hexamer neighbors. Here, we have applied subtomogram classification and averaging approaches to an existing immature virus dataset (10) in order to determine the structures of Gag assemblies at lattice edges. We also applied atomistic molecular dynamics simulations to assess the roles of the different CA-CA interactions in immature lattice stabilization and predict the properties of the structures we observe at lattice edges. Together, our results suggest that the basic unit of immature HIV-1 assembly is a Gag dimer and partial CA-SP1 helical bundles are present at the edges of the assembled lattice and may be substrates for initiation of maturation.     

Growth hormone replacement improved oocyte quality in a patient with hypopituitarism: A study of follicular fluid

BackgroundGrowth hormone (GH) is known to be involved in ovarian folliculogenesis and oocyte maturation. In patients with poor ovarian response without growth hormone deficiency (GHD), adjuvant GH treatment improves in-vitro fertilization (IVF) results. Improvement of oocyte quality in IVF by GH replacement was reported in only a few patients with GHD. We report on a new case with study of follicular fluid.MethodsA 29-year-old patient with hypopituitarism was referred to our infertility center. She was undergoing hormonal replacement for hypogonadotropic hypogonadism and diabetes insipidus, and did not consider at first GH replacement. Four IVF procedures were performed between 2011 and 2014. Growth hormone replacement (somatotropin 1.1 mg/day) was initiated before the fourth IVF procedure and unmasked central hypothyroidism; levothyroxine (75 mg/day) was introduced. It took 10 months to reach the treatment objectives for insulin-like growth factor 1 (IGF1), free triiodothyronine (fT3) and free thyroxine (fT4). GH, IGF1 and thyroid hormones were measured in the blood and follicular fluid before and after GH and thyroid hormone replacement. Oocyte and embryo quality were also compared.ResultsThe first 3 IVF procedures were performed without GH replacement. 62% to 100% of mature oocytes presented one or more morphologic abnormalities: diffuse cytoplasmic granularity, large perivitelline space with fragments, fragmentation of the first polar body, ovoid shape, or difficult denudation. Embryo quality was moderate to poor (grade B to D), and no pregnancy was obtained after embryo transfer. After GH replacement, hormones levels increased in follicular fluid: GH [7.68 vs. 1.39 mIU/L], IGF1 [109 vs. < 25 ng/mL], fT3 [3.7 vs. 2.5 pmol/L] and fT4 [1.45 vs. 0.84 ng/mL]. Concomitantly, there was dramatic improvement in oocyte quality (no abnormal morphologies) and embryo quality (grade A), allowing an embryo transfer with successful pregnancy.ConclusionsThis is the first report illustrating changes in hormonal levels in follicular fluid and the beneficial effect of GH replacement on oocyte and embryo quality during an IVF procedure in a patient with hypopituitarism. These results suggest that GH replacement is beneficial for oocyte quality in patients with GHD.     

Long event-free survival after anti-BCMA CAR-T cell treatment for relapsed and refractory multiple myeloma patients: Two case reports

Introduction:Chimeric antigen receptor T (CAR-T) cells targeting B-cell maturation antigen (BCMA) have been used in the treatment of relapsed and refractory multiple myeloma (RRMM). The response rate and the depth of responses induced by anti-BCMA CAR-T cells are impressive. However, despite this, remissions are not sustained, and the majority of patients eventually relapse.Patient concerns:Two patients with multiple myeloma (MM) were selected to enroll in a phase I study involving anti-BCMA CAR-T cells (ChiCTR-OPC-16009113) because they did not have the good effect after traditional treatment. One is a 48-year-old male patient who received a diagnosis of IgG lambda MM in June 2015, he has received 4 cycles of cyclophosphamide, bortezomib, and dexamethasone (CyBorD) and obtained a complete response (CR). Approximately 11 months later, the disease progressed. Subsequent treatment included regimens incorporating liposomal doxorubicin, bortezomib, and dexamethasone (3 cycles); the response was poor, and the disease kept progressing. Another 65-year-old female patient received a diagnosis of IgG lambda MM in September 2016, she has received induction therapy with 1 cycle of bortezomib and dexamethasone (VD) and 4 cycles of lenalidomide and dexamethasone, the response was poor.Diagnosis:Both patients were diagnosed with RRMM according to the International Myeloma Working Group criteria.Interventions:Both patients received infusions of anti-BCMA CAR-T cells following an induction chemotherapy regimen of cyclophosphamide and fludarabine.Outcomes:Both of them achieved a stringent CR at the 30th day with minimal residual disease-negative bone marrow by flow cytometry and serum monoclonal protein was undetectable at 4 and 10 months after cell transfusion. The CR has persisted in the 2 patients for >36 months.Conclusions:Our findings demonstrate the anti-BCMA CAR-T cell treatment is a feasible therapeutic option for patients with RRMM. Fewer early lines of treatment may be beneficial to maintain the efficacy of CAR-T cells.Trial registration:ChiCTR-OPC-16009113.