The involvement of Eph–Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements

AbstractIn Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph–ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements

Ultrasensitive biosensing platform for Mycobacterium tuberculosis detection based on functionalized graphene devices

Tuberculosis (TB) has high morbidity as a chronic infectious disease transmitted mainly through the respiratory tract. However, the conventional diagnosis methods for TB are time-consuming and require specialists, making the diagnosis of TB with point-of-care (POC) detection difficult. Here, we developed a graphene-based field-effect transistor (GFET) biosensor for detecting the MPT64 protein of Mycobacterium tuberculosis with high sensitivity as a POC detection platform for TB. For effective conjugation of antibodies, the graphene channels of the GFET were functionalized by immobilizing 1,5-diaminonaphthalene (1,5-DAN) and glutaraldehyde linker molecules onto the graphene surface. The successful immobilization of linker molecules with spatial uniformity on the graphene surface and subsequent antibody conjugation were confirmed by Raman spectroscopy and X-ray photoelectron spectroscopy. The GFET functionalized with MPT64 antibodies showed MPT64 detection with a detection limit of 1 fg/mL in real-time, indicating that the GFET biosensor is highly sensitive. Compared to rapid detection tests (RDT) and enzyme-linked immunosorbent assays, the GFET biosensor platform developed in this study showed much higher sensitivity but much smaller dynamic range. Due to its high sensitivity, the GFET biosensor platform can bridge the gap between time-consuming molecular diagnostics and low-sensitivity RDT, potentially aiding in early detection or management of relapses in infectious diseases

Identification of Binding Proteins in Xenopus laevis by MALDI-TOF/TOF Mass Spectrometry

MALDI-TOF/TOF MS has been widely used for the identification of proteins in the proteomics field. Here we tried to identify novel proteins during vertebrate development by this MS and targeted the protein binding to Xenopus β-arrestin 2 (xβarr2). First, we prepared total lysate from dorsal marginal zone (DMZ) tissues of Xenopus gastrula embryos. To isolate proteins from DMZ lysate, which bind with xβarr2, we performed GST pulldown assay with GST-fused xβarr2 protein and then separated the proteins by SDS-PAGE. Finally, the bound proteins were analyzed in the tandem MS (TOF/TOF) mode to generate fragment ions for determination of their amino acid sequence. As the result, we identified Xenopus β-tubulin (xβTub) as a binding partner of xβarr2. To verify this discovering method, we performed several in vivo and in vitro experiments. Whole mount in situ hybridization showed that xβTub had similar expression pattern to that of xβarr2 during Xenopus gastrulation. Moreover, forced expression of xβTub caused severe gastrulation cell movements, which is previously shown in xβarr2 overexpression. Protein-protein interaction of xβarr2 and xβTub was also verified by immunoprecipitation, suggesting that we have demonstrated that discovering binding partner of a certain protein can be successfully done by MALDI-TOF/TOF MS in Xenopus embryonic system

Mass Spectrometry-Based Metabolomic and Lipidomic Analyses of the Effects of Dietary Platycodon grandiflorum on Liver and Serum of Obese Mice under a High-Fat Diet

We aimed to identify metabolites involved in the anti-obesity effects of Platycodon grandiflorum (PG) in high-fat diet (HFD)-fed mice using mass spectrometry (MS)-based metabolomic techniques. C57BL/6J mice were divided into four groups: normal diet (ND)-fed mice, HFD-fed mice, HFD with 1% PG extract-fed mice (HPGL), and HFD with 5% PG extract-fed mice (HPGH). After 8 weeks, the HFD group gained more weight than the ND group, while dietary 5% PG extract attenuated this change. The partial least squares discriminant analysis (PLS-DA) score plots showed a clear distinction between experimental groups in serum and liver markers. We also identified 10 and 32 metabolites in the serum and liver, respectively, as potential biomarkers that could explain the effect of high-dose PG added to HFD-fed mice, which were strongly involved in amino acid metabolism (glycine, serine, threonine, methionine, glutamate, phenylalanine, ornithine, lysine, and tyrosine), TCA cycle (fumarate and succinate), lipid metabolism (linoleic and oleic acid methyl esters, oleamide, and cholesterol), purine/pyrimidine metabolism (uracil and hypoxanthine), carbohydrate metabolism (maltose), and glycerophospholipid metabolism (phosphatidylcholines, phosphatidylethanolamines, lysophosphatidylcholines, and lysophosphatidylethanolamines). We suggest that further studies on these metabolites could help us gain a better understanding of both HFD-induced obesity and the effects of PG

Potential Usefulness of Streptococcus pneumoniae Extracellular Membrane Vesicles as Antibacterial Vaccines

The secretion of extracellular membrane vesicles (EMVs) is a common phenomenon that occurs in archaea, bacteria, and mammalian cells. The EMVs of bacteria play important roles in their virulence, biogenesis mechanisms, and host cell interactions. Bacterial EMVs have recently become the focus of attention because of their potential as highly effective vaccines that cause few side effects. Here, we isolated the EMVs of Streptococcus pneumoniae and examined their potential as new vaccine candidates. Although the S. pneumoniae bacteria were highly pathogenic in a mouse model, the EMVs purified from these bacteria showed low pathological activity both in cell culture and in mice. When mice were injected intraperitoneally with S. pneumoniae EMVs and then challenged, they were protected from both the homologous strain and another pathogenic serotype of S. pneumoniae. We also identified a number of proteins that may have immunogenic activity and may be responsible for the immune responses by the hosts. These results suggest that S. pneumoniae EMVs or their individual immunogenic antigens may be useful as new vaccine agents

Computational Method-Based Optimization of Carbon Nanotube Thin-Film Immunosensor for Rapid Detection of SARS-CoV-2 Virus

The recent global spread of COVID-19 stresses the importance of developing diagnostic testing that is rapid and does not require specialized laboratories. In this regard, nanomaterial thin-film-based immunosensors fabricated via solution processing are promising, potentially due to their mass manufacturability, on-site detection, and high sensitivity that enable direct detection of virus without the need for molecular amplification. However, thus far, thin-film-based biosensors have been fabricated without properly analyzing how the thin-film properties are correlated with the biosensor performance, limiting the understanding of property-performance relationships and the optimization process. Herein, the correlations between various thin-film properties and the sensitivity of carbon nanotube thin-film-based immunosensors are systematically analyzed, through which optimal sensitivity is attained. Sensitivities toward SARS-CoV-2 nucleocapsid protein in buffer solution and in the lysed virus are 0.024 [fg/mL](-1) and 0.048 [copies/mL](-1), respectively, which are sufficient for diagnosing patients in the early stages of COVID-19. The technique, therefore, can potentially elucidate complex relationships between properties and performance of biosensors, thereby enabling systematic optimization to further advance the applicability of biosensors for accurate and rapid point-of-care (POC) diagnosis.N

MicroRNA expression profiling in recurrent anaplastic oligodendroglioma treated with postoperative radiotherapy

In anaplastic oligodendroglioma (AO), genetic alternation was associated with clinical outcome. We explored radiation-associated up or downregulation of microRNAs (miRNAs) in AO by comparing miRNA expression profiles in newly-diagnosed AO to recurrent AO treated with postoperative radiotherapy. We identified that 23 miRNAs were upregulated and 42 miRNAs were downregulated in recurrent AO compared with newly-diagnosed AO. Especially, the expression of MiR-124, miR-128, miR-139-5p, miR-153, miR-210, miR-582-5p, and miR-96 were highly increased in the patient with recurrent AO. In contrast, MiR-1, miR-1180, miR-133b, miR-135b, miR-1539, miR-193a-5p, miR-196a, miR-196b, miR-200b, miR-21*, miR-221*, miR-224, miR-24-1*, miR-31, miR-32*, miR-34a*, miR-34c-5p, miR-455-5p, miR-503, and miR-631 showed extremely decreased expression in the patient with recurrent AO. Our results provide meaningful data for miRNAs as molecular diagnosis and novel therapeutic targets in recurrent AO treated with radiotherapy